Introduction to

Microscopy by Means
of Light, Electrons,
X Rays, or Acoustics
SECOND EDITION
Acoustic micrograph of a composite of boron and glass fibers in an
epoxy matrix. Boron fibers are seen in cross-sectional view and the
smaller glass fibers are in both the longitudinal and cross-sectional
view. The photomicrograph was taken with 400-megahertz acoustic
radiation. (Courtesy of D. A. Downs, A. El-Shiekh, M. H. Mohamed,
P. A. Tucker, and J. C. Russ of North Carolina State University.) (See
Chapter 18.)
Introduction to
Microscopy by Means
of Light, Electrons,
X Rays, or Acoustics
SECOND EDITION

Theodore George Rochow and

Paul Arthur Tucker
North Carolina State University at Raleigh
Raleigh, North Carolina

SPRINGER SCIENCE+BUSINESS MEDIA, LLC

 L1brarv of Congress Catalog1ng-1n-Publ1cat1on Data

Rochow, Theodore George.

Introduct1on ta m1croscopy by means of 11ght, electrons, X rays,
ar acoust1cs 1 Theodore George Rochow and Paul Arthur Tucker. -- 2nd
ed.
p. cm.
Rev. ed. of: An 1ntroduct1on ta m1croscopy by means of 11ght,
electrons, X rays, ar ultrasound. c1978.
Includes b1b11ograph1cal references and 1ndex.
ISBN 978-1-4899-1515-3 ISBN 978-1-4899-1513-9 (eBook)
DOI 10.1007/978-1-4899-1513-9
1. M1croscopy. I. Tucker, Paul Arthur. II. Rochow, Theodore
George. Introduct1on to m1croscopy by means of 11ght, electrons, X
rays, ar ultrasound. III. T1tle.
[DNLM· 1. M1croscopy. 2. X Rays. 3. Acoust1cs. OH 205.2 R6811
1994]
OH205.2.R63 1994
502 · . 8 · 2--dc20
DNLM/DLC
for L1brary of Congress 94-15345
CIP

ISBN 978-1-4899-1515-3

© 1994, 1978 Springer Science+Business Media New York

Originally published by Plenurn Press, New York in 1994
Softcover reprint ofthe hardcover 2nd edition 1994

All rights reserved

No part of this book may be reproduced, stored in a retrieval system, or transmitted in any
form or by any means, electronic, mechanical, photocopying, microfilming, recording, or
otherwise, without written permission from the Publisher
 To the North Carolina
State University at Raleigh
Preface

Following three printings of the First Edition (1978), the publisher has
asked for a Second Edition to bring the contents up to date. In doing so the
authors aim to show how the newer microscopies are related to the older
types with respect to theoretical resolving power (what you pay for) and
resolution (what you get).
The book is an introduction to students, technicians, technologists,
and scientists in biology, medicine, science, and engineering. It should be
useful in academic and industrial research, consulting, and forensics; how-
ever, the book is not intended to be encyclopedic.
The authors are greatly indebted to the College of Textiles of North
Carolina State University at Raleigh for support from the administration
there for typing, word processing, stationery, mailing, drafting diagrams,
and general assistance. We personally thank Joann Fish for word process-
ing, Teresa M. Langley and Grace Parnell for typing services, Mark Bowen
for drawing graphs and diagrams, Chuck Gardner for photographic ser-
vices, Deepak Bhattavahalli for his work with the proofs, and all the other
people who have given us their assistance.
The authors wish to acknowledge the many valuable suggestions
given by Eugene G. Rochow and the significant editorial contributions
made by Elizabeth Cook Rochow.

Theodore G. Rochow
Paul A. Tucker
Raleigh, North Carolina

vii
Contents

1. A Brief History of Microscopy

1.1. Introduction............................................ 1
1.2. Correcting for Aberrations............................... 6
1.3. Condensers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
1.4. Dark-Field Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.5. Polarized Light Microscopy.............................. 9
1.6. Near-Field Scanning Light Microscopes . . . . . . . . . . . . . . . . . . . 11
1.7. Light Microscope Manufacturers.......................... 11
1.8. Transmission Electron Microscopes....................... 12
1.9. Scanning Electron Microscopes........................... 15
1.10. Electron-Probe Microanalyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.11. Field-Emission Microscopes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.12. Scanning Tunneling Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . 16
1.13. Scanning Acoustic Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.14. Atomic Force Microscopes............................... 18
1.15. X-Ray Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
1.16. X-Ray Laser Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
1.17. Microscopy Society of America........................... 19
1.18. Summary.............................................. 20

2. Definitions, Attributes of Visibility, and General Principles

2.1. Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
2.2. Attributes of Visibility.. . .. . .. .. .. . .. .. . .. . .. .. . . . .. . .. . . 25
2.2.1. Correcting for Aberrations........................ 25
2.2.2. Sample Quantity and Quality . . . . . . . . . . . . . . . . . . . . . 25
2.2.3. Focus Depth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2.4. Focus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26
2.2.5. Illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2.2.6. Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

ix
X Contents

2.2.7. Anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2.8. Magnification.................................... 31
2.2.9. Stereoscopy..................................... 32
2.3. General Principles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.1. Specimen Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.3.2. Specimen Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
2.3.3. Specimen Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.4. Experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.5. Specimen Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.6. Specimen Behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
2.3.7. Photomicrography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.3.8. Video. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.4. Summary............................................... 35

3. Simple and Compound Light Microscopes

3.1. Limits of Resolution by the Eye........................... 37
3.2. Simple Microscopes: One-Lens Systems.................... 38
3.3. Compound Microscopes: Two or More Lens Systems . . . . . . . 40
3.4. Stereocompound Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
3.4.1. Illuminating with Stereomicroscopes............... 48
3.4.2. Preparing the Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5. Biological Microscopes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5.1. Objectives....................................... 50
3.5.2. Eyepieces....................................... 51
3.5.3. Condensers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
3.5.4. Illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
3.5.5. Ultraviolet and Infrared Light . . . . . . . . . . . . . . . . . . . . . 58
3.5.6. Anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.5.7. Magnification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
3.5.8. Photomicrography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
3.6. Summary............................................... 59

4. Compound Microscopes Using Reflected Light

4.1. Studying Surfaces by Reflected Light...................... 61
4.2. Resolving Power . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
4.3. Contrast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4.4. Correcting for Aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.5. Specimen Cleanliness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.6. Focus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
4.7. Illumination............................................ 68
4.8. Radiation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.9. Magnification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
4.10. Field of View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
Contents xi

4.11 Glare ................................................. . 78

4.12. Depth ................................................ . 79
4.13. Working Distance ...................................... . 80
4.14. Structure .............................................. . 81
4.15. Morphology ........................................... . 82
4.16. Information about the Specimen ......................... . 83
4.17. Experimentation ....................................... . 83
4.18. Specimen Behavior ..................................... . 83
4.19. Specimen Preparation .................................. . 86
4.20. Photomicrographic Techniques .......................... . 87
4.21. Summary ............................................. . 87

5. Microscopy with Polarized Light

5.1. Overhead Projections.................................... 89
5.2. Anisotropy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
5.3. Numerical Aperture and Interference Figures . . . . . . . . . . . . . . 94
5.4. Resolution: Specimen Interaction and Polarized Light . . . . . . . 95
5.5. Contrast: Michel-Levy Interference Chart. . . . . . . . . . . . . . . . . . 97
5.5.1. Personal Interpretation of Interference Colors....... 99
5.5.2. Retardation Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
5.5.3. Specimen Thickness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
5.6. Correcting for Aberrations Due to Strain . . . . . . . . . . . . . . . . . . 101
5.7. Cleanliness: Freedom from Interference Films.............. 102
5.8. Focus. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.9. Illumination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
5.10. Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.11. Magnification.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104
5.12. Field of View of an Interference Figure.................... 105
5.13. Glare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
5.14. Depth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106
5.15. Working Distance....................................... 106
5.16. Specimen Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.17. Specimen Morphology................................... 107
5.18. Information about the Specimen . . . . . . . . . . . . . . . . . . . . . . . . . . 107
5.19. Experimentation........................................ 107
5.20. Specimen Behavior. . . . . . . . . . . . . . .. . . . . . .. . . . . . . . . . . .. . . . 108
5.21. Specimen Preparation................................... 108
5.22. Photography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
5.23. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109

6. Microscopical Properties of Fibers

6.1. Introduction............................................ 113
6.2. Fiber Morphology.. .. . .. .. .. . .. .. .. . .. . .. .. .. .. .. . .. .. .. 113
xii Contents

6.3. Anisotropy in Fibers. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135

6.4. Molecular Orientation and Organization . . . . . . . . . . . . . . . . . . . 137
6.5. Transparent Sheets, Foils, and Films....................... 141
6.6. Fiber Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
6.7. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144

7. Microscopical Properties of Crystals

7.1. Structural Classifications.......... . . . . . . . . . . . . . . . . . . . . . . . 145
7.2. Morphology............................................ 145
7.3. Miller Indices........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
7.4. Isomorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
7.5. Skeletal Morphology..................................... 150
7.6. Isotropic Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
7.7. Uniaxial Crystals. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
7.8. Biaxial Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
7.9. Optical Properties of the Liquid Crystalline or
Mesomorphic State . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 159
7.10. Thermotropic, Mesomorphic, Single Compounds........... 163
7.11. Morphology Types...................................... 165
7.11.1. Homeotropic Textures............................ 165
7.11.2. Focal Conic Textures............................. 165
7.11.3. Other Smectic Textures . . . . . . . . . . . . . . . . . . . . . . . . . . . 167
7.11.4. Nematic Textures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168
7.11.5. Cholesteric Textures.............................. 168
7.12. Lyotropic Phases........................................ 169
7.13. Liquid Crystalline Polymers.............................. 172
7.14. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

8. Confocal Scanning Light Microscopy

8.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
8.2. Scanning Modes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
8.2.1. Nipkow Disk Scanning. . . . . . . . . . . . . . . . . . . . . . . . . . . 180
8.2.2. Beam Scanning.................................. 181
8.2.3. Stage Scanning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
8.3. Illumination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
8.4. Comparing Three Types of Confocal Microscopy . . . . . . . . . . . 183
8.5. Conventional, Confocal, and Axial Resolutions . . . . . . . . . . . . . 184
8.6. Data Acquisition and Processing . . . . . . . . . . . . . . . . . . . . . . . . . . 185
8.7. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Contents xiii

9. Micrography
9.1. Micrograph: Image Produced by Light, Electrons,
or X Rays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
9.2. Experience: Records of Negatives....... . . . . . . . . . . . . . . . . . . 191
9.3. Imagination............................................ 191
9.4. Resolving Power........................................ 191
9.5. Resolution with Photomacrographic Lenses. . . . . . . . . . . . . . . . 191
9.6. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195

10. Contrast: Phase, Amplitude, and Color

10.1. Contrast: Colorless and Color ........................... . 199
10.2. Interference: Destructive and Constructive ................ . 199
10.3. Phase-Amplitude Contrast .............................. . 202
10.4. Phase-Amplitude Contrast in Determining
Refractive Index ....................................... . 204
10.5. Variable Phase-Amplitude Microscopy ................... . 207
10.6. Modulation-Contrast Microscopy ........................ . 210
10.7. Dispersion Staining .................................... . 212
10.8. Special Accessories ..................................... . 215
10.9. Schlieren Microscope ................................... . 216
10.10. Summary ............................................. . 218

11. Interference Microscopy

11.1. Interference of Two Whole Beams . . . . . . . . . . . . . . . . . . . . . . . . 221
11.2. Types of Interference Microscopes........................ 222
11.2.1. Single Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
11.2.2. Mach-Zehnder Systems . . . . . . . . . . . . . . . . . . . . . . . . . . 227
11.3. Applications to Highly Birefringent Specimens... . . . . . . . . . . 230
11.4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230

12. Microscopical Stages

12.1. Introduction............................................ 233
12.2. Micromanipulators. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 237
12.3. Heatable Stages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
12.3.1. Hot Stages with Long Working Distances.......... 240
12.3.2. Hot Stages with Short Working Distances.......... 243
12.3.3. Hot-Wire Stages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
xiv Contents

12.4. Very Hot Stages......................................... 248

12.5. Cold Stages. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
12.6. Thermal Stages: Hot and Cold.. . . . . . . . . . . . . . . . . . . . . . . .. .. 249
12.7. Other Special Cells and Cuvettes.......................... 251
12.8. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

13. Fourier Transform Infrared Microscopy

13.1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
13.2. Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
13.3. FT-IR Microscopes .................................. :. . . 260
13.4. Specimen Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 261
13.5. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263

14. Transmission Electron Microscopy and

Electron Diffraction
14.1. Electron Microscopes ................................... . 265
14.2. Electron Lenses ........................................ . 268
14.3. Resolving Power ....................................... . 270
14.4. Resolution ............................................. . 271
14.5. Contrast ............................................... . 272
14.6. Aberrations ............................................ . 273
14.7. Oeanliness ............................................ . 274
14.8. Focus Depth ........................................... . 275
14.9. Focus ................................................. . 275
14.10. mumination ........................................... . 275
14.11. Anisotropy ............................................ . 276
14.12. Useful Magnification ................................... . 276
14.13. Field of View .......................................... . 277
14.14. Artifacts ............................................... . 277
14.15. Depth Cues ............................................ . 278
14.16. Specimen Thickness .................................... . 280
14.17. Field Depth ............................................ . 280
14.18. Specimen Structure ..................................... . 280
14.19. Spedmen Morphology .................................. . 281
14.20. Specimen Information .................................. . 281
14.21. Experimentation ....................................... . 282
14.22. Specimen Preparation ................................... . 282
14.23. Electron Micrography ................................... . 285
14.24. Electron Diffraction ..................................... . 288
14.25. Summary.............................................. . 294
Contents XV

15. Scanning Electron Microscopy and Compositional Analysis

15.1. Introduction ........................................... . 297
15.2. Magnification and Resolving Power ...................... . 300
15.3. Resolution ............................................ . 301
15.4. Contrast .............................................. . 302
15.5. Aberrations ........................................... . 305
15.6. Cleanliness ............................................ . 305
15.7. Focus Depth ........................................... . 307
15.8. Focusing .............................................. . 307
15.9. Illumination ........................................... . 308
15.10. Radiation ............................................. . 309
15.11 Useful Magnification ................................... . 315
15.12. Field of View .......................................... . 316
15.13. Noise ................................................. . 316
15.14. Depth Cues ........................................... . 317
15.15. Working Distance ...................................... . 318
15.16. Field Depth ........................................... . 318
15.17. Structure .............................................. . 318
15.18. Morphology ........................................... . 321
15.19. Information ........................................... . 321
15.20. Dynamic Experimentation .............................. . 322
15.21. Specimen Behavior ..................................... . 323
15.22. Specimen Preparation .................................. . 324
15.23. Photomicrography ..................................... . 325
15.24. Summary ............................................. . 326

16. Emission Microscopies

16.1. Introduction............................................ 329
16.2. Field-Emission Microscopes . .. . . . . . . . . . . . . . . . . . . .. . . . . . . . 329
16.3. Attributes Contributing to Visibility by Field-Emission
Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
16.3.1. Thought, Memory, and Imagination . . . . . . . . . . . . . . 333
16.3.2. Resolving Power. . . . . . . . . . .. . . . . . . . . . . . .. . . . . . . . 333
16.3.3. Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
16.3.4. Contrast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
16.3.5. Aberrations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
16.3.6. Cleanliness. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
16.3.7. Focus Depth. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
16.3.8. Illumination. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
16.3.9. Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 335
16.3.10. Magnification................................... 336
16.3.11. Field of View................................... 336
xvi Contents

16.3.12. Artifacts. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336

16.3.13. Working Distance............................... 337
16.3.14. Field Depth..................................... 337
16.3.15. Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 337
16.3.16. Morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
16.3.17. Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
16.3.18. Experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
16.3.19. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 338
16.3.20. Specimen Preparation. . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
16.4. Scanning-Tunneling and Atomic Force Microscopies . . . . . . . . 339
16.4.1. Scanning-Tunneling Microscopy.................. 340
16.4.2. Scanning Near-Field Light (Optical) Microscopy . . . . 344
16.5. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

17. X-Ray Microscopy

17.1. X Rays. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
17.2. Condenser Lenses....................................... 352
17.3. Recent Developments.................................... 354
17.4. Summary. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

18. Acoustic Microscopy

18.1. Ultrasound Waves from Microspecimens . . . . . . . . . . . . . . . . . . 361
18.2. Acoustic Microscopes: SLAMs versus SAMs . . . . . . . . . . . . . . . 361
18.2.1. Theoretical Resolving Power of Acoustic
Microscopes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 366
18.2.2. Practical Resolution of Acoustic Microscopes. . . . . . . 367
18.2.3. Contrast in Acoustic Images. . . . . . . . . . . . . . . . . . . . . . 368
18.2.4. Aplanatic Lenses in SAMs . . . . . . . . . . . . . . . . . . . . . . . 368
18.2.5. Cleanliness in Acoustic Microscopes. . . . . . . . . . . . . . . 368
18.2.6. Focus Depth in SAMs . . . . . . . . . . . . . . . . . . . . . . . . . . . 368
18.2.7. Focusing SAMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
18.2.8. Acoustic Radiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369
18.2.9. Magnification. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
18.2.10. Field of View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 370
18.2.11. Stray Acoustic Radiation......................... 370
18.2.12. Three-Dimensional Aspect of SLAMs............... 370
18.2.13. Specimen Thickness............................. 371
18.2.14. Working Distance............................... 371
18.2.15. Specimen Structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373
18.2.16. Anisotropy..................................... 373
18.2.17. Specimen Morphology.. . . . . . . . . . . . . . . . . . . . . . . . . . 374
18.2.18. Information about Acoustical Images. . . . . . . . . . . . . . 374
Contents xvii

18.2.19. Experimentation............ . . . . . . . . . . . . . . . . . . . . 374

18.2.20. Specimen Behavior. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
18.2.21. Specimen Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
18.2.22. Photomicrography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375
18.3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 376

19. Image Collection, Analysis, and Reconstruction by Computer

19.1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
19.2. Microscopes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380
19.3. Video Systems.......................................... 381
19.4. Computers and Software . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
19.5. Display and Networks................................... 382
19.6. Contrast, Look-Up Tables, and Color...................... 384
19.7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384

20. Specimen Preparation

20.1. Introduction ........................................... . 387
20.2. Preparing Fiber, Fur, and Hair .......................... . 392
20.3. Microtomy ............................................ . 395
20.4. Ultramicrotomy........................................ . 396
20.5. Replication ............................................ . 397
20.6. Fracture Surfaces ...................................... . 398
20.7. Thin Sections of Hard Materials ......................... . 400
20.8. Transparent Particulate Specimens ....................... . 400
20.9. Monomeric Embedding Media ......................... .. 400
20.10. Preparing for Reflected ffiumination ..................... . 405
20.11 Preparing Metals and Other Opaque Materials ............ . 405
20.12. Summary ............................................. . 410

References.................................................... 413

Author Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 437

Subject Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443

Introduction to
Microsco py by Means
of Light, Electrons,
X Rays, or Acoustics
SECOND EDITION
A Brief History of Microscopy

1.1. INTRODUCTION

All kinds of microscopy have a common beginning in mankind's

intellectual goal to see better. Visible light was the first medium, and
visibility was limited to the unaided eye until the first century A.D., when
Seneca discovered(!) that by looking through a clear spherical flask filled
with clear water "letters however small and dim are comparatively large
and distinct."<2) This idea led to simple magnifiers, whether a single large
lens to accommodate both eyes together or two lenses of a smaller diam-
eter to accommodate each eye separately.
· During the next dozen centuries, spherical segments of clear minerals
were set in frames as eyeglasses to help older people see better. In about
the year 1300, clear silicate glass was made in Italy, and superstition
prohibiting its use in eyeglasses for far-sightedness was overcome.<3-s) By
the sixteenth century concave lenses were also for sale to help near-sighted
people. The availability of both convex and concave lenses led the Dutch
to the linear combination of the two as a crude compound microscope. Just
who invented the Dutch microscope is still not known; credit generally is
given to three Dutch spectacle makers around the turn of the seventeenth
century.<6.6•) The principle of a concave lens serving to amplify the objective
is still used today to provide a flat field in some instruments for projection
and photomicrography.
In 1611 the Dutch microscope came to the attention of the German
mathematician and astronomer Johann Kepler (1571-1630). Kepler was
quick to realize that enlargement could also be obtained by combining a
convex (instead of concave) ocular with the convex objective but that the
image would appear inverted rather than upright. The Kepler ocular
serves to enlarge the real image of the objective, which is the principle of
the modern compound light microscope.< 2) Kepler also explained the diop-

1
 2 Chapter 1

tries of the eye and showed how convex spectacle lenses correct for hyper-
opia, while concave lenses correct for myopia. By 1619 Cornelius Drebbel
in London had made a compound microscope with a biconvex eye lens
and a plano-convex objective the size of a small cherry, stopped down by
a diaphragm opening the size of a thick needle. The microscope produced
an inverted image.(7) In May 1624, Galileo received a Drebbel microscope;
he demonstrated how it worked and indicated that he had made similar
but better "occhiali" (eyeglasses). In the same year Galileo ground lenses,
mounted them into microscopes, and sent them to friends.< 7•7a,BJ Galileo
exhibited his cleverness when demonstrating his telescope, and some ob-
servers doubted that what they saw was real. He simply turned the tele-
scope toward something very familiar nearby and invited the doubters to
take a look. Thus he demonstrated that telescopy, like microscopy, is the
interpretive use of the instrument by the observer.
The word microscope was coined by Giovanni Faber on April 13,
1625.<6>It implies subjectively seeing (resolving) small things or their parts.
However public dictionaries to this day define microscope objectively as
a powerful magnifier. This definitely was the reason for using a micro-
scope in the days of Robert Hooke (1635-1703). Hooke owned a micro-
scope (made by his friend Christopher Cock) with an objective lens and an
lJcular lens. Hooke also possessed a third (field) lens, which he called his
middle glass. He experimented with the relative placement of these lenses
for more magnification while viewing such materials as cork. His skill as
an artist is illustrated in Figure 1.1. Indeed one of Hooke's drawings, Eyes
and Head of a Grey Drone Fly, is included in the 1991 exhibition of sixteenth-
and seventeenth-century art The Age of the Marvelous. <8>
Robert Boyle was particularly impressed with Hooke's ability and
recommended him, at the age of 27, to be life-time curator of experiments
for the Royal Society. Consequently King Charles II requested a "hand-
some book" of Hooke's reports. In his resulting book Micrographia,< 9>
Hooke also described how to make a simple (single-lens) microscope by
means of a very small spherical bead:
Draw a thread of glass, run it into a bead in the flame, and then snap
off the apex. Grind that region flat with jeweler's abrasives. Fit the tiny
lens in a hole made in a flat piece of metal. Impale the specimen or
hold it with wax (or place a drop of a liquid specimen) on a spike
hand-made into a screw for focusing the lens.<9•9a>

As with all simple microscopes, the object is placed very close to the lens.
Likewise the eye must be placed close to the lens to see the whole field.
Antony van Leeuwenhoek (1632-1723), the Dutch draper, was 40
years old before he began making and using simple microscopes (for 50
A Brief History of Microscopy 3

FIGURE 1.1. Hooke's drawings of cork showing the cells, a word he used because the tiny
spaces reminded him of monks' cells.<8>

more years). He followed Hooke's directions<9> and made more than 400
microscopes, of which only nine remain. These range in numerical aper-
tures from 0.11--0.37 (69x-266x). The one at the Museum of Science at the
Utrecht University is the best, and it has been evaluated recently.< 10>
These two microscopical giants of their time, Hooke and Leeuwen-
hoek, differed personally in attitude. The naive Dutchman, with his simple
microscope, persisted in searching for microscopic objects, such as "ani-
malcules." For two-and-a-half centuries however, there have been those
who doubted that Leeuwenhoek could have discovered bacteria and other
microorganisms-and with only a single-lens microscope held in his hand.
However recent work, especially that of Brian J. Ford and his colleagues,
4 Chapter 1

using Leeuwenhoek's surviving microscopes and modem versions of sim-

ple microscopes, has completely substantiated Leeuwenhoek's work and
results.<11- 13> Why did Leeuwenhoek succeed in resolving such small di-
mensions? The main reason seems to be that he held specimen, lens, and
his eye all very close together. This rule remains today for all simple
microscopes (and magnifying glasses).
Hooke preferred to use a compound microscope exclusively, which
resembled Galileo' s telescope, with objective and eyepiece considerably
further apart<8> than today' s standard tube length. Moreover Hooke was
convinced that more and more magnification was required to see more
detail than was already known. For example he tried to examine green
"tarnished" water with his long compound microscope to see "whether
this was like moss-yet so ill and imperfect are our microscopes, that I
could not certainly discriminate any."<9> The main reason for the difficulty
was that his compound microscope magnified aberrations along with the
images.
Throughout the seventeenth century experiments in empirically ar-
ranging two, three, or more lenses in a tube were continued, but the salient
advance for us today was the advent of the Huygens eyepiece, invented by
the Dutch physicist and astronomer Christiaan Huygens (1629-1695) and
his brother. The Huygens eyepiece has two planoconvex lenses, each with
the convex side facing the objective. The lower (field) lens modifies the real
image from the objective to give a brighter but smaller image, which is then
examined by the upper lens. The Huygens design is still the principal one
for microscopical eyepieces of magnifications of lOx and less. Reticles of
linear, areal, or angular scales are used now in such eyepieces for mi-
crometry, as they were in Huygens' day.
The hand-held crude wooden or iron microscopes of the seventeenth
century evolved into elegant eighteenth-century table microscopes, made
chiefly of brass. The importance of the evolution of microscopical stands
centers on the illumination of the object: Mirrors were developed to trans-
mit light through transparent objects, while others were made to reflect
light from opaque objects. Some stands have mirrors to direct a beam of
light through transparent objects; other stands have mirrors that direct a
beam of light on opaque objects so that it may be reflected to the objective.
The problems of viewing through compound microscopes persisted
into the nineteenth century. Some microscopists and manufacturers pre-
ferred the simple microscope with only a one-lens system that was achro-
matically corrected. Robert Brown, the great botanist, not only preferred
the microscope without an eyepiece but also without a condenser. Instead
he used a double-sided mirror mounted on a rotating collar to increase the
A Brief History of Microscopy 5

variation of illumination in both kind and extent, as illustrated in Figure

1.2. He traveled widely, exploring Australia and Tasmania, and returned
with over 4000 species to identify. In his reports on this work in 1827 and
1829, he mentioned the "active molecules," the phenomenon now known
as Brownian movement (motion.)<14l
Brown's conclusion about active molecules, like Leeuwenhoek's
about animalcules, has been sharply criticized, but both have been sub-
stantiated and publicized by Brian J. Ford.<11- 14l Besides, Brown himself
asked George Dolland (1774-1853) to use his uncle's fine triple achromatic
objective lens (1764) to confirm the Brownian phenomenon. He did. Ford
writes, "There is a belief that nowadays we can do things which of course
could not have been possible in those unrefined, far off, and primitive
times. Yet as I have shown elsewhere, the images that can be obtained even
with a Leeuwenhoek lens are not so different from those you would obtain
with the best optical microscope available today."<14l Microscopy is the
interpretive use of the microscope.

FIGURE 1.2. The microscope with which Robert Brown observed active molecules, now
called Brownian movement (or motion).<14l Courtesy of McCrone Research Institute.
 6 Chapter 1

1.2. CORRECTING FOR ABERRATIONS

During the seventeenth century there was little optical improvement

because the causes of aberrations and artifacts were not understood. How-
ever, at the tum of the eighteenth century, David Gregory (1661-1708), a
friend of Sir Isaac Newton (1642-1727), noted something that had escaped
the master: Different kinds of glass spread out the colors of the spectrum
to different extents.<4>Gregory suggested that a proper combination of two
kinds of glass might produce no spectrum at all. The first commercial
achromatic objectives appeared in 1824 and were of French manufacture.
The early ones had a smaller numerical aperture than the corresponding
uncorrected objectives,<2>so these produced less brilliant images and were
at first unpopular. However achromatic objectives of higher aperture were
perfected rapidly during the nineteenth century.<1s.-21 >A vivid example of
this progress is evident from comparing Figures 1.3a and 1.3b.<7l
In Figure 1.3b, use of the achromatic objective of 1850 has increased
the resolution in Figure 1.3a, but contrast has decreased and glare has
increased. During the next hundred years, resolution was further im-
proved by means of the apochromatic objective. With improvements in
illuminating systems and other parts of the light microscope, contrast was
increased, glare decreased, and resolution brought to very nearly theoret-
icallimits.
Meanwhile there was corresponding improvement in oculars. In 1782,
Jesse Ramsden (1735--1800), an English telescope maker, produced an ocu-
lar of new design. Like the Huygens' eyepiece, Ramsden's contained the
two planoconvex lenses, but the convex sides were turned toward each
other, and they were close together for better correction of spherical ab-
errations. Both lenses were mounted so that they were above the focal
plane of the objective's real image, where the cross hair or micrometer scale
normally is placed; thus the ocular could magnify both. The Ramsden type
of eyepiece is used today for most work requiring eyepiece magnification
of about 12x or greater.<I8l
By the beginning of the twentieth century, microscopists were setting
up standards for measuring resolving power,<22>employing such natural
structures as the striated scales of butterflies, followed by diatoms and then
by artificial rulingsP> Thus it was discovered that resolution increased
with angular aperture in the objective and condenser.
One of the chief investigators, Giovanni Battista Amici, combined the
qualities of theorist, practical optician, and microscopist. Among his im-
portant innovations was the use of a single hemispherical front lens in the
microscopical objective (in 1844), a principle that still prevails. In 1830
Joseph J. Lister (1786-1869), father of the famous surgeon, discovered the
FIGURE 1.3. (a) Gnat's wing as seen with an uncorrected Cuff objective, ca. 1750. Photo-
micrograph courtesy of Dr. Maria Rooseboom, director of the National Museum of the
History of Science, Leiden, the Netherlands. (b) Gnat's wing as seen with an achromatic
Oberhauser objective, ca. 1850. Photomicrograph courtesy of Dr. Maria Rooseboom, director
of the National Museum for the History of Science, Leiden, the Netherlands.
 8 Chapter 1

two aplanatic foci of achromatic doublets. He worked on the problem of

the increasing influence of cover-glass thickness when using objectives of
increasing aperture. Different solutions to the problem were found. In
1837, Andrew Ross made objectives with movable lens separations to
correct for varying thicknesses of cover glasses. A decade later Amici
showed that immersing the objective in a liquid of appropriate refractive
index could eliminate problems in the cover glass. <7l
Until the early nineteenth century, it was thought that perfecting
lenses alone would lead to the resolution of infinitely small structures.
Then, in 1834, the astronomer George B. Airy (1801-1892) showed that
light from a star would never be focused at a single point but only in a disk.
Therefore in microscopy there must also be a limiting angular distance
between self-luminous objects to make them appear separate. This angle
would depend on the radius of the lens and the wavelength of light. In
1873, Ernest Abbe (1840-1905) came to essentially the same conclusion for
periodic structures, thereby giving practical meaning to the concept of
numerical aperture. Abbe worked with the Carl Zeiss Company in Jena in
1846; his many inventions include apochromatic objectives (1885) and their
compensating eyepieces. Along with Otto Schott, he succeeded in greatly
improving the quality of optical glass. In 1857 the British Royal Micro-
scopical Society introduced a standard size and pitch of screw thread for
objectives but met with difficulty in perfecting taps to be supplied to
manufacturers. In an effort to perfect the standard, the American Micro-
scopical Society worked with the Royal Microscopical Society and even-
tually arrived at a joint standard. Today machining practice is also stand-
ardized. Virtually every manufacturer in the world now produces
objectives and microscopes fitted with "Society" threads, and to this extent
interchangeability is ensured.
Standards for eyepieces and substages have not been universally ac-
cepted; however there is a set of standards for these and other parts under
the supervision of the British Standards Institution.<23> In the United States
interchangeability of microscopical parts is within the scope of the Amer-
ican Society for Testing and Materials.<24>

1.3. CONDENSERS

Until about 1880 practically all microscopes using visible light trans-
mitted through the specimen were without condensers, which were later
introduced for two reasons: the growing importance of bacteriology and
the availability of the Abbe condenser produced by Zeiss.<21> As bacteriolo-
gists demanded more and more resolving power, they were demanding
A Brief History of Microscopy 9

more and more numerical aperture in the illuminating cone of light, even
at the expense of some contrast.
E. M. Nelson was among those who advocated using the condenser to
focus light onto the specimen itself-a process known as Nelsonian illu-
mination. While this use of the condenser produces the brightest field, it
is only as uniform as the source. Thus an incandescent filament as the light
source focuses unevenly (the image of a coil filament). Such illumination
can also be glary. In 1893, August Kohler published a paper advocating
focusing the light source, such as an incandescent filament, in the entrance-
plane of the condenser (on the iris diaphragm if it has one). In such a case
manipulating the diaphragm also controls the size of the illuminated field,
according to the numerical aperture of the objective in use, thus excluding
glare.(21 l

1.4. DARK-FIELD MICROSCOPY

It is reported(12l that Leeuwenhoek sometimes used dark-field illu-

mination sometimes, but this is not certain. Two centuries after Leeu-
wenhoek, Lister (in 1830) used dark-field microscopy to take advantage of
light scattered by surfaces and other discontinuities.(!) Special dark-field
condensers for higher powered objectives were made available by Francis
H. Wenham (1823-1908),(2) who used a paraboloid condenser and central
stop to produce a hollow cone of light directed outside the angular cone
of the objective.

1.5. POLARIZED LIGHT MICROSCOPY

The double refraction of calcite was described by Erasmus Bartholin

(1626-1697) in 1669, and Etienne L. Malus (1725-1812) coined the term
polarized light. In 1828, William Nicol (1768-1851) used calcite to make his
famous polarizing and analyzing prisms. Other designs followed, but all
plane-polarizing calcite prisms became known as nicols. Polarizer plus
analyzer are used as a pair in a polarimeter to study the optical activity of
liquids, or in a polariscope to study the optical properties of solids. Louis
Pasteur (1822-1895) used the pair with his microscope on tartaric acids and
their salts to discover a whole new class of chemical isomers.(25•26l
The Englishman Henry Clifton Sorby (1826-1908) employed polar-
ized light microscopically to study thinned sections of limestones and
other transparent rocks and identify their mineral constituents.(27•27al Sorby
and Pasteur represent a group of scientists in the golden nineteenth cen-
 10 Chapter 1

tury whose microscopical research went far beyond detailed description of

specimens. They postulated mechanisms, drew broad scientific theories,
and pointed the way to a variety of technologies.(26,26•>
By the twentieth century the light microscope had reached the limit of
highly developed theory for resolving power and contrast not only for
visible light but also for the ultraviolet and infrared regions. By the 1930s
light microscopy was playing a part in every science and technology. Great
professors, such as Ernest Abbe in Germany, had worked with outstand-
ing producers of optical glass, namely, Schott, and of microscopes, namely,
Zeiss.(21> Abbe's experiments with phase differences and amplitude con-
trasts in microscopical images were followed by Frits Zernike' s phase-
amplitude microscope, for which he won the Nobel prize in 1953.(28>
The development, production, and use of interference microscopes
followed a similar pattern. From Newton's general observations of inter-
ference phenomena in the seventeenth century, Thomas Young developed
the fundamental theory of interference in 1801. During the first quarter of
the twentieth century, Albert Michelson (1852-1931) developed an inter-
ferometer for reflecting objects, and Ernest Mach and Zender developed an
interferometer for examining transparent objects. By the middle of the
century, several companies offered interference microscopes.(29--32>
In 1932, Edwin H. Land (1909-1991)( 33> invented Polaroid® polarizing
film, which has supplanted calcite as polarizer and analyzer in modern
polarized-light microscopes, used in petrography, chemistry, resinog-
raphy, metallography, and nowadays in biology.
At the turn of the century and well into the twentieth century, pet-
rographers and chemists were also changing their microscopes to examine
specimens.(18> The condenser occasionally needed a central, eccentric, or
colored stop. This requirement called for a substage rack. As the Nicol
polarizer became widely used, so did the condenser need to accept it
mechanically. Later, the advent of the Land Polaroid® polar simplified the
mechanics of the substage, and by the same token, there was more room
for filters.
Among Land's many inventions(33> are Polaroid® self-processing pho-
tosensitive film and the Polaroid® camera to use it. These have influenced
not only light microscopy but all other microscopies and indeed all of
photography. For example Land and his associates published a paper in
1949(34> on combining the Polaroid® photographic system with a Zeiss
reflective objective. Another reflective objective was used as condenser,
and the entire microscopical system functioned in the strictly ultraviolet
region. Moreover these particular objectives are apochromatic for three
different wavelengths strictly in the ultraviolet region. These investigators
made three separate Polaroid® transparencies, employing the three sep-
A Brief History of Microscopy 11

arate (invisible) wavelengths in the ultraviolet region, then they substi-

tuted three beams of different wavelengths in the visible range. Thus they
obtained color-contrast and higher resolution by employing apochromatic,
strictly reflective lenses. <34>
These results gave Robert C. Gore the idea of employing the Zeiss
reflective microscopical lenses in the infrared region (1949) to perform
infrared spectrometry on microscopic samples.(35J In the 1950s, the Perkin-
Elmer Company, pioneer builder of infrared spectrometers, added a mi-
croscope to their single-beam infrared (IR) spectrometer. This in tum led
to Fourier transform-infrared (FT-IR) microscopy as described by John A.
Reffner. (36,36aJ

1.6. NEAR-FIELD SCANNING LIGHT MICROSCOPES

These light microscopes without lenses were invented at Cornell Uni-

versity by Professor Michael Isaacson and his associates, who realized that
the resolving power of lenses is not only limited by the wavelength(s) of
the emanation, but also by the aberrations imposed on an image formed
relatively far from the object-that is, in the far field. Therefore, instead of
focusing light with a lens, Isaacson passed light through a very small hole
and through a sample so close to the hole that the light beam had no chance
of spreading out. Using yellow green light (y = 550 nm), Isaacson's team
obtained a resolution of about 40 nm with this microscope, so the team is
not resolving atoms (as with a scanning tunneling microscope) but using
light. The limit to resolution is not so much the wavelength of the light but
the fact that the amount of sample is so small as to be undetectable. Aaron
Lewis at Hebrew University in Jerusalem and Raoul Koppelman at the
University of Michigan plug the 50-nm hole with a tiny crystal of anthra-
cene because it fluoresces in ultraviolet light, yielding excitons that pass
easily through the tip and tum into visible light (fluoresce). The result is
a much brighter image, say, of a biological cell, from a near-field light
microscope. <37>

1.7. LIGHT MICROSCOPE MANUFACTURERS

Figure 1.4 shows the development of the light microscope industry

from 1970-1992. In particular the Karl Zeiss Company of Jena, East Ger-
many, and the Carl Zeiss Company of West Germany were recently re-
united. <38> The Leitz Company of West Germany and the Wild Company of
Switzerland became Leica,<39> then Leica was joined by the microscopical
12 Chapter 1

CARL ZEISS (W. GERMANY) ===============--

KARL ZEISS JENA (E. GERMANY)
ZEISS

LEITZ ('H. GERMANY) ~~ LEICA

WILD(SWITZERLAND) ~------
I

BAUSCH & LOMB (USA)

AMERICANOPTICAL:>USA)
AMERICAN OPTICAL
I
CAMBRIDGE ( E N G L A N D ) - - - - - - - - - CAMBRIDGE

REICHERT (AUSTRIA)
JUNG ('N. GERMANY)

NIKON (JAPAN) NIKON

OLYMPUS (JAPAN) <X..YMPUS
COOKE, TROUGHTON & SIMMS (ENGLAND)
JAMES SWIFT (ENGLAND) JAMES SWIFT

FIGURE 1.4. Manufacturers of Light Microscopes. Courtesy of Don Felty and Robert Martin.

part of Bausch and Lomb in the United States. The American Optical
Company merged with Reichert of Austria and Jung of West Germany.
That conglomerate joined the Cambridge Company, which merged with
Leica. <39>Cooke, Troughton, and Simms of England joined with James Swift
of England. Nikon<40> and Olympus,<41> both of Japan, have expanded into
the United States.

1.8. TRANSMISSION ELECTRON MICROSCOPES

Two events in the 1920s brought about the development of the elec-
tron microscope. One was the realization from the de Broglie theory (1924)
that particles have wave properties and very short wavelengths (e.g., 0.05
A) are associated with an electron beam of high energy. The other event
was the demonstration by Busch in 1926-1927 that a suitably shaped
magnetic field could be used as a lens to create electron microscopes.<42•43>
Busch and Ernst Ruska initiated studies of electromagnetic lenses in 1928-
1929 and published a description of an electron microscope in 1932. In
1934, Ruska described the construction of his type of electron microscope,
which surpassed for the first time the resolution of light microscopes. In
A Brief History of Microscopy 13

1938, Ruska and von Borries designed and built a practical microscope for
the Siemens and Halske Company, but World War II prevented its sale and
use outside of Germany. In 1986, the Nobel prize in physics was awarded
jointly to Ruska for his pioneering work and to Heinrich Rorer and Gerd
Binnig for the subsequent development of the scanning tunneling electron
microscope (see Section 1.12).
Independently at the University of Toronto in Canada, under the
supervision of E. F. Burton,C44> A. Prebus and James Hillier built an elec-
tromagnetic electron microscope, which they described in 1939. A similar
instrument built in the United States was described by Cecil E. Hall.<42>In
1934, Ladislaus L. Marton built an electron microscope in Brussels with
which he took the first electron micrographs of biological objects, such as
bacteria. With James Hillier and Vance at the Radio Corporation of Amer-
ica, Marton helped build an electron microscope (1940) under the direction
of Vladimir K. Zworykin, the inventor of the television picture tube. The
first RCA commercial model of this electron microscope went to the Amer-
ican Cyanamid Research Laboratories in Stamford, Connecticut, on De-
cember 9, 1940.<4Sl
In 1940, the Columbian Carbon Company built an electron micro-
scope at the University of Toronto under the terms of a Research Fellow-
ship for William A. Ladd, another of Burton's students. The microscope
was moved to the Columbian Carbon Research Laboratories in 1941,
where pioneering industrial research was performed. <46•47J
In England, Metropolitan Vickers produced a prototype electron mi-
croscope in 1939, which was developed into a series of improved mod-
els.<48l The Phillips commercial electron microscope was being developed
in the Netherlands at the same time. In the 1940s, companies in France,
Germany, Japan, and other countries developed and produced this type of
microscope, now known as the scanning transmission electron microscope
(STEM).
The word transmission suggests one of the most important practical
problems: obtaining specimens thin enough to allow sufficient electrons'
transmission to affect the photographic material satisfactorily without af-
fecting the specimen detrimentally (by heat absorption of electrons).<48l In
the vast science of biology, the development of practical electron micro-
scopy depended primarily on a corresponding improvement in microt-
omy, so that sections of tissue could be sliced much more thinly than those
required in light microscopy. Differential stains also had to be developed
on the basis of differential electron absorption by elements of relatively
high atomic number rather than by differential light absorption (color).
Films, whether in the form of specimens, substrates, or replicas, must be
sufficiently thin. In the 1940s, in both biological and nonbiological sciences,
 14 Chapter 1

there were also problems in obtaining contrast; shadows were made by

preferentially evaporating a metal onto the specimen in a high vacuum.
Such problems were complicated by obtaining and maintaining a high
vacuum in the electron microscope itself. There were other important
mutual problems, such as maintenance, repair, resolution, magnifica-
tion,<49l and interpretation.<50l Whereas light microscopists had struggled
for centuries over interpreting images, including macroscopic ones, this
problem was newly introduced to electron microscopists in 1945 (see
Figure 1.5).<50> See Chapter 20 on specimen preparation.
In 1938 M. von Ardenne added scan coils to the STEM. Applications
were limited to specimens thin enough to transmit electrons to activate the
photographic medium.<51 l Historically the chief importance of these scan
coils may lie in the experience that led to the scanning (reflection) electron
microscope (SEM), whose great depth of focus allows quantitative evalua-
tion of specimens topography.

FIGURE 1.5. Two different orientations (180° apart) of the same photomacrograph of de-
pressions in wet sand; (left) apparent depressions; (right) apparent elevations.<50J Taker. to
demonstrate the importance of orientation in interpreting images.
A Brief History of Microscopy 15

1.9. SCANNING ELECTRON MICROSCOPES

Instead of transmitting the primary electrons to form an image, in

1942 Zworykin, Hillier, and R. L. Snyder of the Radio Corporation of
American used the secondary electrons reflected from the surface of a
specimen to produce an image of the topography. The limiting resolution
however was only 1!-lm,less than that of the light microscope (0.1!-lm). By
reducing the size of the scanning spot and making other improvements,
they increased the resolution to 0.05 llm. Further development of the SEM
was suspended during World War II.
In 1948, C. W. Oatley at the University of Cambridge became inter-
ested in the SEM. He and D. McMullen built one with a resolving power
of 0.05 llm. K. C. A. Smith (1956) made several technological improve-
ments, and T. E. Everhart and R. F. M. Thornley (1960) made use of a light
pipe to reduce noise. R. F. W. Pease (1963) and W. C. Nixon (1965) pro-
duced a prototype of Cambridge Scientific Instruments' Mark 1.<51,52>
In 1942, Zworykin, Hillier, and Snyder tried a cold field-emission
sharp cathode as the source of electrons in their experimental SEM to
reduce the size and improve the intensity of the source. Instability how-
ever forced these experimenters to return to the thermionic electron gun.
The cold-emission point source was finally improved by A. V. Crewe in
1969 to the point of successful application in the SEM. Another type of
electron gun, developed by A. N. Broers, employs a heated, pointed rod of
lanthanum hexaboride (LaB6 ) because it is brighter and lasts longer than a
tungsten filament. The requirement of a higher vacuum however prohibits
incorporating the LaB6 gun in some types of SEM.<42>
Advances in the sixties and seventies have involved contrast mecha-
nisms not available in other types of instruments. Better crystallographic
contrast was produced by crystal orientation, lattice orientation, and lattice
interactions with primary beams by D. G. Coates in 1967.<51 > Since 1969 the
TEM has been modified with features of the SEM, resulting in the scanning
transmission electron microscope (STEM).<51>
In a reflection microscope the contrast between features is often too
low, but it can be enhanced by processing the digital signal. Early pro-
cessing was done by nonlinear or differential amplification. Derivative
signal processing (differentiation) was introduced in 1970 and 1974. Image
storage circuits have been developed so that one can observe the image
and/ or operate on it off-line. Grain sizes, physical-chemical phases, and
other analytical features are emphasized by computer evaluation and
scanning electron microscopical images (CESEMI). In fact computer inter-
action with the SEM has yielded many benefits.
16 Chapter 1

1.10. ELECTRON PROBE MICROANALYZERS

A close relative of the SEM is the scanning electron probe microana-

·lyzer (EPMA). Instead of recording the scattered electrons, the microana-
lyzer records the emitted X rays and sorts them according to wavelength
with a Bragg spectrophotometer. A quantitative analysis is made of a
chemical element as the scanner picks up the distribution of the element.
At the same time, an enlarged image may be displayed if the X-ray mi-
crospectrometer is part of a scanning electron microscopical system. <51>

1.11. FIELD-EMISSION MICROSCOPES

In 1897, R. W. Wood described the phenomenon of the field emission

of electrons, the process of emitting electrons from an extremely small area
of a cathodic surface in the presence of a strong electric field. In 1936 E. W.
Muller (1911-1977) applied this principle to a negatively charged very fine
tip(< 1-J.Ull radius) of tungsten wire in the high vacuum of a cathode-ray
tube. In this field-electron microscope, Muller obtained a pattern on the
fluorescent screen that represented the array of atoms (see Figure 1.6).
In 1950, Miiller charged the acicular tip positively, introduced helium
into an extremely high vacuum, and formed He+ ions at the tip. Some of
the atoms hopped off the tip and activated the fluorescent screen to form
a pattern typical of the field-ion microscope.<53.54l

1.12. SCANNING TUNNELING MICROSCOPES

The history of microscopy has already taught that the limit of resolu-
tion of any microscope depends on the limiting wavelength of the particular
radiation used. Thus electron microscopes manifest their high resolving
power because the wavelengths of electron beams are so short, for ex-
ample, 0.5 nm (0.05 A) for an electron beam accelerated by 60,000 V.
However electron beams, as well as light, also manifest the particulate
nature of electrons. In 1973 Brian Josephson won a share of the Nobel Prize
in physics for explaining the phenomenon called tunneling: If two elec-
trically conducting surfaces are brought close enough together, the elec-
tron wave forms (clouds) surrounding their atoms overlap. If a small
voltage is applied between the conductors, electrons tunnel from one cloud
to another. This is true even though the voltage is much lower than
classical physics wov.ld require.
A Brief History of Microscopy 17

FIGURE 1.6. Field-ion micrograph of a platinum crystal by the late Professor Erwin W.
MiiUer<54> of the Pennsylvania State University.<2> Courtesy of Professor T. T. Tsong,<53>
Department of Physics, the Pennsylvania State University.

In the mid-1970s Heinrich Rohrer at IBM Research Laboratories in

Zurich turned his attention from phase transitions, critical phenomena,
and magnetic fields to electrical and mechanical layers of very thin oxides.
He and his new research assistant, Gerd Binnig, probably needed to vis-
ualize inhomogeneities on a much smaller scale than microns (micro-
meters) or even a hundred angstroms (10 nanometers). It seemed natural
in late 1978 to use the tunneling effect by making a sharp point and trying
to tunnel through a vacuum. If the tip's position could be controlled
accurately and scanned accurately over a surface, one would have an
imager (microscope) delivering a detailed atomic map. In mid-1979 Rohrer
and Binnig submitted their first patent disclosure on a scanning tunneling
microscope (STM). In 1986, Rohrer and Binnig received half of the Nobel
Prize in physics.<5>-57> The STM however does not work well with noncon-
ducting surfaces.
 18 Chapter 1

1.13. SCANNING ACOUSTIC MICROSCOPY

There are two types of acoustical microscopes, both based on the idea
of the Russian scientist S. Sokolov, who in 1936 proposed using short
wavelengths of ultrasonic energy instead of light to look directly inside an
opaque specimen. This idea was not put into actual practice until the 1970s,
when the manufacture of working models was begun by L. W. Kessler and
associates. They produced the scanning laser acoustic microscope
(SLAM).<55> Meanwhile C. F. Quate and associates<55•56> developed a mech-
anical scanning acoustic microscope (SAM); see Chapter 18.

1.14. ATOMIC FORCE MICROSCOPES

The atomic force microscope (AFM) was invented in about 1986 by

IBM Zurich's Binnig and Berger in collaboration with Calvin Quate at Stan-
ford University. The AFM (like the STM) scans a surface by moving a tip
along one straight line at a time, but the tip is in actual contact with the
material and senses the minute forces between atoms. The original AFM
used a diamond tip mounted on a tiny gold foil cantilever spring. (This
arrangement is like an old-fashioned phonograph arm fitted with a dia-
mond needle-but with only one millionth of the weight.) When the tiny
diamond tip goes over an electron "bump," the tip goes up, and the tip goes
down over a void. To measure these tiny deflections, inventors sandwiched
the diamond tip and its gold spring between the sample and an STM tip! As
the diamond of the AFM rises, there is more tunneling current; while there
is less tunneling current when the diamond goes down. The diamond tip,
only one atom or so wide, is obtained by smashing an inexpensive diamond
and selecting the right fragment by means of a light microscope.<57l
However the tunneling AFM has already been replaced by the optical-
lever AFM-at IBM Zurich Research Laboratory and independently at IBM
Research in Yorktown Heights, New York. The optical lever is a laser beam
bounced from a small mirror mounted on the diamond tip. "Light reflected
from the moving mirror is detected by a sensor; minute deflections are
amplified, or 'levered,' by the geometry of the system so that the unit can
detect a vertical change in a surface of only 0.1 angstrom" (0.01 nm).<6•>

1.15. X-RAY MICROSCOPY

X-ray microscopes were probably imagined soon after Wilhelm Ront-

gen discovered X rays in 1895. But the great penetrating power of X rays
A Brief History of Microscopy 19

was offset by the inability to refract or reflect X rays. Therefore images

were obtained, and still are obtained today, particularly by the medical
profession, by contacting photosensitive film with the object. The result is
simply a macroradiograph. However electron microscopes in the 1940s
could be adapted to provide a point source for X rays. In 1958, Martin C.
Botty and Fred G. Rowe patented an adapter so that the objective lens of
an electron microscope could focus its electrons onto a target to produce
X rays instead of its original function.< 58•59> Subsequently electromagnetic
lenses were employed to market point-projection microscopes.<60>
Another important advance has been to focus X rays by means of a
Fresnel zone plate with alternating transparent and opaque rings whose
spacing diminishes outward from the center. Fresnel zone plates had been
used to focus light, radio waves, sound, and even neutrons, and in 1960
Albert V. Baez at the Harvard-Smithsonian Astrophysical Observatory
proposed using Fresnel zone plates to focus X rays. Achieving resolution
of detail greater than that of light microscopy requires spacing
the zone plate for X rays about 1/20 the wavelength of light. This is being
done by procedures that bring us to the present day; see Chapter 17.

1.16. X-RAY LASER MICROSCOPES

Descended from the electron microscopes of the 1940s, X-ray laser

microscopes use a special mirrored surface to focus X rays beamed
through a specimen. The result is a sort of relief map based on the differ-
ential absorption of X rays. Although the X-ray laser microscope does not
have the resolving power of electron microscopes, it has the advantage of
not killing live specimens, such as living cells. Thus the X-ray laser micro-
scope greatly facilitates the study of the structure and function of biolog-
ical cells. <61>

1.17. MICROSCOPY SOCIETY OF AMERICA

It began in 1942 as the Electron Microscope Society of America

(EMSA) "to increase and to diffuse the knowledge of the science and
practice of electron microscopy."<49> In 1992, while EMSA celebrated its
fiftieth anniversary, the name was changed to the Microscopy Society of
American (MSA) so as to express the importance of "confocal, infrared,
fluorescence, x-ray, scanning, tunneling, atomic force, ion, and acoustic
microscopies, as well as highly sophisticated methods for chemical and
20 Chapter 1

structural analysis that utilize the interactions of photon, electron, or ion

beams with matter."*

1.18. SUMMARY

The earliest microscopists studied mostly biological objects. Hooke

(1635-1703) studied cork, for example, and coined the word cell (see Figure
1.1). Leeuwenhoek (1632-1723) studied many natural objects, including
stagnant water. He discovered microorganisms, which he termed beasties.
Robert Brown (1827-1829) reported "active molecules," which we now
term Brownian movement or motion. Around this time, Sorby (1826-1908)
used reflected light to study cast iron and its ores. He used polarized
transmitted light to study thinned sections of limestone, etc. Pasteur (1822-
1895), originally a chemist, used polarized light to discover a whole new
class of chemical isomers.
At the turn of the century, more and more mineralogists and petrog-
raphers were using polarized light with the petrographical microscope.
Meanwhile more and more chemists were using the polarized light (chem-
ical) microscope in research and development of chemicals, drugs, poly-
mers, plastics, textiles, paper, etc. Today the chemical microscope and
petrographic microscope have become about the same. Today's consol-
idated list of manufacturers is given in Figure 1.4.
Meanwhile, in the 1940s, the TEM was developed and commercially
produced. Its optics have been improved with regard to aberrations, illu-
mination, practical resolution, and contrast. Scanning electron microscopy
followed closely and pushed the resolving power further (see Figure 2.2).
The resolving power and contrast of the TEM is limited by the lenses;
therefore the SEM was developed, and the progress in resolving power has
been steadily increased from the 1940s to the present (see Figure 2.2).
Since the 1970s, STMs have pushed the resolving power toward the
level of atomic spacing (see Figure 2.2). The STMs include AFM, which
employs an extremely sharp diamond mounted on a gold foil spring that
goes up and down over atoms in the object. The tunneling AFM has been
succeeded by the optical-lever AFM, which uses a laser beam bounced
from a small mirror on the diamond tip, thereby reducing the resolution
to 0.1 A (0.01 nm).
While X rays cannot be refracted, they can be reflected and focused by
means of a Fresnel zone plate, thus achieving resolution of detail greater

•statement (1992) of the Microscopy Society of America, P.O. Box EM, Woods Hole, MA
02543.
A Brief History of Microscopy 21

than with the light microscope. The X-ray laser microscope employs a
special mirrored surfaced to focus X rays beamed through, say, a live
specimen without killing it.
Within its broad range of capabilities, light microscopy continues to
serve both biological and nonbiological sciences and technologies. With
the advent of reflective optics, both infrared and ultraviolet techniques are
included in the visible range. The fundamental value of light microscopies,
used alone or together with any of the high-tech microscopies, lies in the
use of polarized light to show structure (however complex) in either biolog-
ical or nonbiological material. <62>
 2

Definitions, Attributes
of Visibility, and
General Principles

2.1. DEFINITIONS

Microscopy is the interpretive use of microscopes.<1•1•·2) No matter what

kind of microscope, employing whatever medium, in whatever manner,
with whatever kind of specimen, microscopy also requires a primary ob-
server to interpret the image. Accordingly, microscopy can be interpreted
as science,<2•) art,<3•3•) or a game-what is it?
Interpretation requires both a sharp eye and an active brain_<l•) To-
gether they form the subjective, microscopical part of microscopy. The spec-
imen on or in a microscope is the objective, microscopic part of micro-
scopy.<!)
A microscope is an instrument that increases resolution over that of the
human eye alone (about 150 !!m between two points or lines). Resolution is
the distance between two specific points or parts of the object as viewed by
the eye, microscope, camera, or video. Actually resolution is the revelation
of the two points or lines on two individual receptors (rods and cones),<4•5)
separated by at least one other of these receptors situated on the retina of
the eye (see Figure 2.1). The rods are very sensitive at low levels but
respond only to white light. In the center of the retina is a small spot only
about 1.5 mm wide, called the fovea. Its center contains densely packed
cones that are sensitive to colors.<5•) Variation in the kind and extent of the
cones' color sensitivity among microscopical observers probably accounts
for variations in personal conclusions about color.<6)
Resolving power is the ability to distinguish two points of an object as
separate in an image (with their diffraction discs not overlapping more

23
24 Chapter 2

FIGURE 2.1. Diagrammatic section through

eyeball; M: ciliary muscles; L: lens, elastic; R:
retina.

than half their diameters).(I,Zl Figure 2.2<6al indicates the potential resolving
powers of various kinds of microscopes since 1850 and projected to the
year 2000. Data are based on Abbe's diffraction theory of resolution. Abbe
and the Zeiss Company were the first to design and make apochromatic
objectives (corrected for spherical aberration at two wavelengths of light
and for chromatic aberration at three wavelengths).(l)
Practical resolution needs adequate contrast; the Zemike phase-con-
trast method led the way in light microscopy at the tum of the twentieth

g
a:
~
ll..
(!)
z
~
~
w
a:

1850 1900 1950 2000

YEAR

FIGURE 2.2. Potential resolving powers of various kinds of microscopes.<6l

Definitions, Attributes of Visibility, and General Principles 25

century, as shown in Figure 2.2. In the middle of the twentieth century, the
TEM displayed practical resolving powers from 10-6-10- 10 m. The SEM and
the STEM offer advantages in both resolving power and contrast, as do the
scanning laser microscope (SLM) and the scanning near-field light micro-
scope (SNLM). (6l
Figure 2.2 shows where resolvabilities of various microscopes overlap
or join; actually light microscopy begins where the resolvability of the
unaided eye ends (ca. 150 J.tm). The degree of resolution may lie within:

A biological organism or nonbiological material; electron microscopies

begin somewhere around the limit (see Figure 2.2) of light micro-
scopy (ca. 10-6 m)
A biological organ or material part of the whole
A biological skin or membrane or material surface or interface; several
kinds of microscopies can be involved
A biological cell or polymer; a material polymer, monomer, or atom
itself. (7•8>

2.2. ATTRIBUTES OF VISIBILITY

2.2.1. Correcting for Aberrations

An aberration in any kind of microscope is the failure of an object's

point to be imaged as a point.<1> With a lens transmitting light, visible or
invisible, there are five kinds of aberrations: spherical, coma, astigmatism,
curvature of field, and distortion. The most important aberration is spher-
ical aberration; these are all discussed in Chapter 3. Electron microsco-
pical lenses possess aberrations, too, and these are discussed in Chapter
14.

2.2.2. Sample Quantity and Quality

When selecting the kind and extent of microscopy, the size of the
object and nature of the problem can be paramount. As Chapter 13 ex-
plains infrared spectrometry and infrared microscopy were combined in
the late 1940s when all-reflective objectives and condensers became avail-
able for samples as small as the milligram range and smaller.<9>Microman-
ipulators have been developed for use with many kinds of microscopies
(see Chapter 12).
26 Chapter 2

2.2.3. Focus Depth

With far-field microscopes (light/electron/Xray) focus depth in the

image space<1•2> refers to the other side of a particular lens with respect to
the field depth in the object, as shown in Figure 2.3. Although the terms
focus depth and field depth may be considered synonymous,<10> they are
discussed separately here, since field depth is objective while focus depth
is subjective with respect to the microscopist. When working visually it
may be an advantage to have an image equal to the great focus depth of
the human eye with its low numerical aperture (NA) of 0.002. With a
deeper image (see Figure 2.3), the observer does not have to adjust the
focusing controls as often; in such cases choose an objective of low NA.
On the other hand, great focus depth is a disadvantage in optical
sectioning when studying spatial arrangements in structures. In such stud-
ies a very thin field depth is required to observe sharp changes in the
object's contours with very small changes in focus. Hence select an ob-
jective with a high NA.<10>

2.2.4. Focus

In visual microscopy the eye (see Figure 2.1) should be focused so that
the ciliary muscles (M) controlling the thickness of the eye's lens (L) are
relaxed and the individual feels at ease when the object is in focus. <11>If the
microscopical image goes out of focus, refocus the objective rather than
accommodate it with the ciliary muscles.
Objectively focusing means turning mechanical wheels or electronic
knobs to move the object or objective or change the optics. Subjectively
focusing is a personal choice of the image by the direct observer, photo-

Depth of image in foc:us

~~~----.!------·

B A C Ia lc

FIGURE 2.3. Field depth and focus depth employing light as an example. Courtesy of
Microscope Publications.
Definitions, Attributes of Visibility, and General Principles 27

micrographer, or armchair microscopist. Focusing does not always mean

choosing the sharpest image; sometimes it is the process of over- or un-
dershooting to gain contrast, resolution, or some other attribute to visibil-
ity. Whatever else it may be, focusing boils down to each individual's
personal choice in the interpretation of images.

2.2.5. Illumination

Illumination is very important, and in light microscopy there are sev-

eral types, as illustrated in Figure 2.4.<12> There are two principal methods
of illumination by transmitted light: Nelson's method and Kohler's meth-

A1dal reflected

Axial traMmltted

I
FIGURE 2.4. Principal types of illumination. Directional beams lie on or within a main
cone.<12l
 28 Chapter 2

od. Nelson's "critical illumination" was devised in the days before elec-
tricity to focus the side of a burning oil wick in the plane of the specimen
by means of a substage condenser. <13>Nelson's method remains satisfactory
in these days of electricity and ribbon filaments for either light or electron
microscopies. However there is the problem of unevenness if a coil filament
is sharply focused in the plane of the specimen, as shown in Figure 2.5.<10>
Throwing the coil filament out of focus and/ or inserting a ground-glass
diffuser in the lamp's housing compromises the purposes of Nelsonian
illumination, which is to illuminate evenly the whole area of the specimen
in focus; yet Nelsonian illumination remains common in light microscopy.
In Kohler's illumination (see Figure 2.6), the source F is equipped with
its own auxilliary condenser lens and field diaphragm A2-A2• These may be
housed as a unit illuminator or as part of the microscope itself. The im-
portant point is to focus the field diaphragm's opening at A1-A1, the iris
diaphragm of the substage condenser (not on the specimen).<14>
Referring to Figure 2.6, the substage condenser is moved to bring the
image of the lamp's iris diaphragm A 2-A2 into the plane of the object F1•
Then the lamp's diaphragm A 2-A2 is opened just enough to fill the ob-
jective's field of the object at F1• Another advantage is that the lamp's
filament is out of focus, so that the field is evenly illuminated. The field
stop A 2-A2 also enables the observer to center the optical axis to coincide
with that of the objective. The substages of microscopes are generally
equipped with centering devices for the condenser system. When both F
and the object are in focus, the centering screws should be used to center
the field-stop image with the field of view at F1.<15>

lamp's condenser

FIGURE 2.5. Modem version of Nelson's method of critical illumination whereby the source
of illumination (f) is focused in the plane of the object as F1 by means of the lamp's condenser
and the substage condenser, whose aperture A is controlled by its iris diaphragm to equal the
field of view.<ili)
Definitions, Attributes of Visibility, and General Principles 29

coil filament

auxiliary condenser

FIGURE 2.6. Illumination system for transmitted light (Kohler illumination).(lS)

Routine procedure for Kohler's illumination involves the following

steps:
1. Focus on the specimen (see Figure 2.6).
2. Open the iris diaphragm AcA1 of the substage condenser.
3. Almost close the field diaphragm A2-A2 •
4. Focus the substage condenser until the field diaphragm A 2-A2 is
simultaneously in focus with the object at F1•
5. Open the field diaphragm A2-A2 so that all of the field is in view but
no more.
6. Recenter the substage condenser if necessary.
Caution: Do not open the field diaphragm A 2-A2 any farther to avoid
burning the retina or paralyzing the tiny muscles controlling the size of the
iris diaphragm. This precaution is especially important in photomicrog-
raphy when light sources of high intensity are likely to be used.
Kohler's method is available in the electron microscope by means of
automated controls that select the appropriate aperture and lens condi-
tions to illuminate homogeneously only the imaged area. An advantage in
electron microscopy is that Kohler's illumination protects the specimen
from heat damage(14l (see Chapter 14).

2.2.6. Radiation

Microscopy employs light, electrons, X rays, or acoustic energy in an

expanding number of analytical or synthetical fields. The present changes
30 Chapter 2

in the kinds and varieties of radiation have resulted from the interaction
of equipment, research, and application. During the first half of this cen-
tury, ultraviolet invisible light was used to gain fluorescence, resolution, or
contrast.<12> However electron microscopy surpassed the advantages of
ultraviolet (UV) light microscopy. Not all the effort expended on UV
microscopy was lost, since it was possible to take three UV photographs
and convert them to a color photograph of excellent contrast.<16> The same
reflective optics also led to advances in infrared microscopy<17l and thence
to FT-IR microscopy<9> (see Chapter 13).
With UV and infrared regions of light, there are of course fewer
options with illumination, and these are discussed in succeeding chapters.
With other types of radiation there are not many variations in kinds of
illumination. These are discussed in the chapters that follow.

2.2.7. Anisotropy

Anisotropy (re light) may originate in a structure in one or more man-

ifestations of influence by polarized light:
Molecular anisotropy resulting from the orientation of dipoles, usually
in long or flat molecules<18>
Birefringence(!> in units of all anisotropic crystals
Polarization patterns, such as the permanent cross seen in spherulites<1.2l
like starch grains
Flowed, streamed, or grown masses of long or flat units (not necessarily
crystalline) in a medium of different refractive index<tB,t9)
Regions of local strain anisotropy photoelastic effect in a normally
isotropic substance, such as inorganic or organic glass under
stress.
Many materials owe their anisotropy to two or more of the preceding
origins. Cotton fibers are a good example, as shown in Figure 2.7. All fibers
except inorganic glass manifest eight independently determinative prop-
erties based on their anisotropy. Therefore microscopical examination re-
veals both structure and morphology (see Chapter 6).
Crystals reveal even more optical properties than fibers, due to the
variations in crystallographic symmetry, as discussed in Chapter 7. Liq-
uid crystals are in a class by themselves with regard to their many opti-
cal properties based on anisotropy.<18> These are also discussed in Chapter
7.
Definitions, Attributes of Visibility, and General Principles 31

50JJm

FIGURE 2.7. Cotton fibers, mounted in mineral oil and shown between partially crossed
polars, revealing the convolution of fibers resulting from the complex structure. Taken in
class by Mrs. G. Berry with modest equipment, ca. 1975.

2.2.8. Magnification

Useful magnification is incidental to the resolving power (NA) of the

objective and the eye; the approximate relationship is

maximum NA (microscope) = 1.40

= 700x
minimum NA (eye) 0.002

which can also be stated as

.,.---l_im---:-it_o_f-:r_es_o_lu_t-io_n_(,_e"-ye....!.)_-:-
:- = 0.15 mm
=750x
limit of resolution (microscope) 0.0002 mm

The theoretical optimum magnification for light microscopy is therefore

about 750x. Useful magnification in practice may be somewhat higher to
assure that adjacent points in the object are imaged on separated receptors
 32 Chapter 2

(rods and cones) on the retina. Separated receptors mean that the two
nerve endings receiving images from different points of the object are not
adjacent; instead there must be at least one other nerve ending between the
two. The necessary magnification may be as high as 1000 times the NA of
the objective.(10l
The chief function of an eyepiece is to increase the incidental mag-
nification of the objective to a total magnification that is useful to the eye:

magnification (objective) x magnification (eyepiece)

= 1000 x NA (objective)(l 0l

2.2.9. Stereoscopy

Stereoscopy involves simultaneously seeing from slightly different an-

gles a slightly different image with one eye than with the other eye. The
brain combines the two images to give the impression of depth and so-
lidity. Achieving this effect with any kind of microscopy involves taking
two micrographs at slightly different angles and then viewing them with
a stereoscope. This simply holds the two illuminated micrographs, which
are then viewed through a pair of spectacles at the personal interpupillary
width. Of course stereoscopic microscopes are independent of dual micro-
graphs and holders.
With the electron microscope, two slightly different views are rou-
tinely taken by tilting or turning the specimen after taking the first micro-
graph. While the procedure for some of the newer kinds of microscopy
may not be well established, microscopical stereoscopy has been very
convincing, especially in consulting work.
In light microscopy, stereomicrographs are usually taken with a ster-
eoscopic microscope, sometimes called a binocular (like a field glass).
However, not all microscopes fitted with binocular eyepieces are stereo-
scopic. There must be a corresponding pair of objectives (at the angle of
close vision) or else a common objective broad enough to include two
separate effective views of the object.(20•21l

2.3. GENERAL PRINCIPLES

2.3.1. Specimen Structure

To the biologist structure is practically synonymous with morphology;

to the crystallographer(22.23> structure describes the kind of periodic ar-
Definitions, Attributes of Visibility, and General Principles 33

rangement of the units of organization, whereas morphology pertains to

the size and shape of the resultant architecture. Structure and morphology
are the two coordinates in Figure 7.V22l
Abbe's fundamental theory of limiting resolving power assumes that
the specimen has microscopic periodic structure. Diatoms are such struc-
tures, and these have been involved in the history of microscopical devel-
opment. For example the test diatom Pleurosigma angulatom (Quekett) is
composed of hexagonally dose-packed cells. If the resolving power of the
light microscope is not quite adequate, the pores appear to be hexagonally
shaped, as in Figure 2.8.<24> Other investigators<25•26l demonstrated that the
structure of the unit cell itself is too complicated to test the resolving power
of a light microscope. While the TEM has helped reveal the complicated
structure, scanning electron microscopy has also helped<27l (see Figure
15.20).

2.3.2. Specimen Morphology

The morphology (size and shape) of a specimen<1•2> affects its visibility:

The smaller the size and the more complex the shape of an object, the
greater the resolving power, contrast, correction of aberrations (and most
of the other attributes contributing to visibility) required to obtain a clear,

FIGURE 2.8. Pleurosigma angulatum, test diatom.

34 Chapter 2

useful image. A regular polygon with n number of sides is resolved only

if its diameter is n times the limit of resolution.<10l

2.3.3. Specimen Information

Information about the specimen is often of utmost importance. What

are we looking for? Why? Is the sample representative or nonrepresenta-
tive (exaggerated)? Is it of a type already on the market? Then information
on possible solutions to the problem can be gathered from technical lit-
erature.

2.3.4. Experimentation

Experimentation with equipment and varied conditions surrounding a

specimen may be necessary not only to improve visibility but also to justify
image interpretation. To obtain optimum resolution, contrast, cues to
depth, depth of focus, etc., one must experiment with objectives, oculars,
condensers, polarized light, different illumination, and all the rest. If all the
equipment is not available, one can improvise.
Of course keeping good, orderly records is also important for arriving
at satisfactory conclusions. Good records and easy retrieval save a lot of
future experimentation.

2.3.5. Specimen Preparation

Every sample should be examined immediately as received. If precau-

tions to preserve it are specified, these should be taken. If the sample is
cleaned or fractionated in any way, all fractions, including "dirt," should
be saved and labeled.
Some preparations are routine and need no deliberation, such as
mounting in an inert liquid of chosen refractive index to improve trans-
parency and visibility, and cutting or otherwise preparing a surface quick-
ly. More drastic and time-consuming preparations should be considered
carefully and used on only part of the sample (see Chapter 20).

2.3.6. Specimen Behavior

Behavior means change in the sample with time, temperature, weather

(natural or simulated), humidity or dryness, atmosphere (or vacuum), etc.
Definitions, Atbibutes of Visibility, and General Principles 35

Even during storage or shelf life, samples should be examined often for
change and to interpret these in terms of the problem.

2.3.7. Photomicrography

Photography at best is an illustration of the appearance of the specimen

under very specific conditions. However photography has its own advan-
tages and limitations of resolution, contrast, color, and emanation sensi-
tivity introduced by grain size or color sensitivity of the photographic film
or paper and by its development conditions. Hence photomicrography is a
separate discipline that combines the art of the photographer with the
science of microscopy. There are frequent references to photomicrog-
raphy(28> in later chapters.

2.3.8. Video

In television, too, the picture has its own resolution, contrast, and color
characteristics. These and other attributes are variable, and they contribute
to microscopical conclusions.

2.4. SUMMARY

Microscopy is the interpretive use of microscopes. Interpretation can be

science, art, or a guessing game. In any case interpretation requires a sharp
eye and an active brain, which together form the subjective microscopical
part of microscopy. The specimen is the objective microscopic part of micro-
scopy.
A microscope is any instrument that increases the resolution of the
human eye. Resolving power (what you pay for) is expressed theoretically as
the minimum distance between two adjacent, separate points in the object.
Resolution (what you get) is the actual perception of the two separate points
because their separate images fall on two receptors separated by at least
one other receptor on the viewer's retina. The potential resolving power of
today's light microscope is close to the theoretical value (0.2 f!m). The
potential resolving powers of the scanning laser microscope (SLM) and the
scanning near-field microscope (SNFM) lie between the highest for light
microscopes and the lower ranges of the TEM; the STM has the highest
potential resolving power of all. While resolution is the most important
attribute to visibility, contrast is almost as important: If two points are
36 Chapter 2

separated by a particular microscope, but their images are no brighter (or

darker) than the space in between, they will not be visible. Therefore most
of Chapter 2 is devoted to various ways of obtaining resolution with
adequate contrast.
The greatest problem in the development of light microscopy was
correcting for aberrations (the failure of an object's point to be imaged as a
point); at least some of these problems remain in electron microscopy.
Problems of sample quantity and quality are addressed by combining infra-
red light microscopy with infrared spectrometry to create FT-IR micro-
scopy. With far-field microscopes (light/ electron IX ray), depth of field and
depth of focus are considered separately, since the former is objective and the
latter is subjective. The more depth of field, the less resolving power, or vice
versa. In visual light microscopy, the eye should be relaxed (focused on
infinity). Focusing on the object should be done by the microscope, not the
eye.
There are many types of illumination in light microscopy, including
Nelson's critical illumination, where the source, an incandescent filament,
for example, is focused on the specimen by the substage condenser. In
Kohler's method the filament or other source is focused in the plane of the
iris of the substage condenser, and the image of the lamp's field diaphragm
is focused in the plane of the specimen, thus controlling centering and the
size of the evenly illuminated field. Up to 1990, electron microscopes were
illuminated by Nelson's method, but since then Kohler's method has been
introduced.
 3

Simple and Compound

Light Microscopes

3.1. LIMITS OF RESOLUTION BY THE EYE

The limiting resolution d, the distance between two points barely

resolved by the human eye, is called visual acuity. It varies directly with the
distanceD between the object and the eye. In Figure 3.1 the eye's viewing
angle Vis purposely exaggerated, and the distance D from object to eye is
not drawn to scale with respect to d, which separates the barely resolved
points P and P. Figure 3.1 demonstrates that when distance D' to the nearer
points P' and P' is exactly half the distance D, then the barely resolved
distance d' between P' and P' is exactly half of d. This direct relationship
persists as the object is brought closer and closer to the unaided eye, until
the object is brought to a certain minimal distance, approximately 250 mm
from the normal eye. At this distance of closest vision, the eye can resolve
two points that are about 0.15 mm apart. These limits are set by the NA =
0.002 for the eye with its iris diaphragm wide open. At this setting the eye's
lens is as thick as physiologically possible.(l)
A far-sighted person, whose eyes cannot accommodate objects much
closer than arm's length, requires either eyeglasses prescribed for hyper-
opia or a magnifying glass to achieve normal visual acuity. Following the
same principle, a person with normal eyesight can increase his/her visual
acuity by using a magnifying glass. The simple microscope, illustrated in
Figure 3.2, follows the same principle. The auxiliary lens bends the rays
from the object so that they fall within the eye's viewing angle and are
focused on the retina rather than beyond it.
Combining the focal length fin Figure 3.2<2) with distances D and D'
in Figure 3.1 permits calculation of the expression<3)

37
38 Chapter 3

I
p

d
I

i
I
I
'
l'
I
I I

- p-------------~'~-----------·: [j
------------------------------- [)" ----------------------- ------:!
FIGURE 3.1. The viewing angle. The relationship between the distance d between barely
resolved points P and their distanceD from the eye. Courtesy of Microscope Publications.<2>

1 1 1
T =o + v·
where D equals object distance from the lens and D' equals image distance
from the lens if the lens is assumed to be thin. Magnification M equals
PP I P' P' = DID' By eliminating D or D', the magnification equation is<3>
_ f _ (D' -f) _ _Q_ _ _ (___Q_)
M - (D -f) - f - f 1- - f+1
Real distances are positive, and virtual distances are negative. With this
equation we can estimate magnification from knowledge of only the object
distance from the lens and the magnification without actually measuring
the size of the formed image. If we examine this equation with the value of
D' = 25 em as the distance of distinct vision for most people and assumes f =
5 em, as in many simple lenses, magnification equals six times. A Leeu-
wenhoek lens off= 1 mm gives a magnification of 251 times (approximately
250, as often reported). Of course this lens does not have corrections for the
lens defects or aberrations, but this high magnification helps explain why it
was initially superior to early compound microscopes.

3.2. SIMPLE MICROSCOPES: ONE-LENS SYSTEMS

Simple microscopes have only one lens unit, as indicated in Figure

3.2a. These are inexpensive, have relatively large fields, and give upright
 p'
---

Lens

--
,.. i
, !
--- 2~0mm
P'··--·······-··············-·---················-···········- :
a

b
FIGURE 3.2. (a) A simple microscope.<1l Courtesy of Microscope Publications. (b) Some
magnifiers and simple microscopes for use in macroscopy: !-magnifying glasses; 2-simple
microscopes: pocket and desk sizes; 3-auxiliary lenses; 4-double Ioupe for eyeglasses; 5-
transparent base magnifier, with or without reticle; 6-vertical (table model) magnifier with
reticle. Courtesy of Bausch and Lomb_(Za,4l
40 Chapter 3

images. There are many uses for the simple microscope, as many as there
are kinds: magnifying glasses, magnifiers with reticules (many kinds),
pocket magnifiers with carrying cases, simple binoculars (nonprescription
eyeglasses), simple- and double-eyeglass loupes, for example<2a> (see Figure
3.2b). With all simple microscopes (as with compound microscopes), keep
the eye(s) close to the eyelens(es)! The tendency to violate this rule be-
comes greater the larger the lens is. Follow Leeuwenhoek!

3.3. COMPOUND MICROSCOPES: TWO OR MORE

LENS SYSTEMS

Compound microscopes are composed of two or more lens systems.

Some compound microscopes contain only the minimum: an objective
plus an eyepiece (see Figure 3.3). The objective forms a real image P' - P'
in the eyepiece plane.<1>The eyepiece is a magnifier that causes the images
of two separate points P and P to fall on separate receptors P" and P" on
the retina.
Figure 3.4 illustrates an uncomplicated compound microscope being
used by hand in micrometry. Even smaller models, scarcely larger than a
fountain pen, are available.<4>Portability is their main advantage, but low

FIGURE 3.3. Light path of two characteristic rays through

p p a compound microscope comprised of only objective and
eyepiece. Courtesy of Microscope PublicationsP>
Simple and Compound Light Microscopes 41

FIGURE 3.4. Hand compound microscope

being used in micrometry by reflected light for
opaque specimens. Courtesy of Helen Clapp.

NA = 0.06--0.10 is their main limitation (see Table 3.1). Moreover hand

microscopes cannot substitute for those with sturdy stands, so most mi-
croscopical work is done with compound microscopes mounted on con-
venient stands and equipped with accessories necessary for proper illu-
mination and precise focusing.( 5l
Field microscopes are also held in the hand, but they have a stage and
other facilities for transparent specimens, as indicated in Figure 3.5. The
model shown comes with three objectives: 4x, lOx, and 40x. It is intended

TABLE 3.1
Theoretical Resolving Powers of Light Microscopes
Approximate
Theoretical Limit
of Resolution (d)"
Focal Numerical Depth
Length Aperture Axial Oblique of Field
(mm) (NA) Maximum Useful Magnification (IA.ffi) (~-tm) (~-tm)

250 0.002 Eye alone, lx 150

25 0.10 Hand lens, lOx 10 42
32 0.10 Compound microscope, lOOx 5 2.5 25
16 0.25 Compound microscope, 250x 2 1 3.8
8 0.50 Compound microscope, 500x 1 0.5 0.86
4 0.95 Limit, air-immersion objective, lOOOx 0.52 0.26 0.083
3 1.38 Oil immersion (visible, 500 nm), 1500x 0.36 0.18
3 1.38 Oil immersion (UV, 270 nm), 2000x 0.20 0.09 0.04
"Based on Abbe's theory d = A./ (1-2 NA) disregards aberrations). This is about maximum obtainable
resolution and assumes adequate contrast. Any reduction of aperture of objective or condenser increases
d. Photography has its own limitations.
42 Chapter 3

FIGURE 3.5. Swift field microscope supplied with carrying case, three objectives (4x, lOx,
and 40x), and a battery-powered illumination; available with phase optics, objectives with
longer working distances, adaptors for photography.<6> Courtesy of Swift Instruments.<6>

for use in the home, field, factory, pilot plant, or at the scene of an accident
or crime.<2a> The main advantages are portability and handiness. The var-
ious limitations involve stability, versatility, kind and extent of illumina-
tion, lack of polarized light, etc., as discussed later in this chapter and in
Chapters 4 and 5.
Most light microscopical work is done with compound microscopes
mounted on convenient stands and equipped with accessories necessary
for proper illumination and precise focusing. Figure 3.6 illustrates a com-
pound microscope that may be suitable for students in introductory biol-
ogy or for anyone satisfied with its limitations and comparatively low
price. The microscope's sturdy stand has an up-to-date appearance and
built-in light source. It is binocular but not stereoscopic because both eyes
Simple and Compound Light Microscopes 43

FIGURE 3.6. Compound microscope

at a relatively modest price. Courtesy
of Edmund Scientific Co.!4>

receive the same view from the single objective in use. The several ob-
jectives are parfocal and satisfy the standards of the Deutsche Industrie
Normung (German Standardization Institute), as do all the other optical
components. The binocular eyepiece is at 45° to the vertical axis for per-
sonal comfort and satisfaction, and it is 360° rotatable for viewer con-
venience. The eyepieces are also adjustable for interpupillary distance. 4

3.4. STEREOCOMPOUND MICROSCOPES

Microscopical stereoscopic vision is obtained by means of two com-

pound microscopes, one for each eye, at about the ordinary interpupillary
angle for reading or writing. This aspect alone makes for comfort and ease.
The stereoscopic effect results from a slightly different angular view for
each of the eyes. Moreover the image is erect, and the depth of field in
focus is relatively long to make it easier to manipulate tiny tools when
operating on small objects.
Stereomicroscopes are essentially composed of two plain compound
microscopes, such as those shown in Figures 3.7a and 3.7b. Hence there are
two kinds of compound stereomicroscopes: the binobjective-binocular
 44 Chapter 3

(Greenough) type and the monobjective-binocular (common objective

[CMO]) type.(7) The binobjective-binocular type is really two compound
microscopes mounted at a slight angle to each other on a single stand. As
shown in Figure 3.7a, this binocular microscope has an objective and an
eyepiece for each eye, mounted at the interpupillary angle of close vision.
The natural stereo effect of the binocular system is enhanced by a pair of
erecting prisms, adjustable to individual interpupillary distance between
eyepieces. The paired objectives are slender, compact, and close together,
providing easy access to the specimen. Furthermore, aberrations are rela-
tively easy and inexpensive to correct because each of the two beams
enters its respective lens axially. (7)
Disadvantages of the binobjective-binocular system are
Intermediate images are inclined from the plane of the specimen stage
and tilted toward each other, so that only the central portions of the
two images are in simultaneous focus.

b
FIGURE 3.7. (a) Greenough stereomicroscope system: A diagram of the beam path.<7) (b) The
common main objective stereomicroscope system: A schematic diagram of the beam path.(3)
Simple and Compound Light Microscopes 45

Both images are tilted unless the specimen is tilted for only one
image.<7l
The other type of stereomicroscope has only one objective, but it is
large enough to yield different views to the two eyepieces, as shown in
Figure 3.7b. This type may be equipped with erecting prisms like the
Greenough type. The advantage of the CMO is that the intermediate image
planes are parallel to the plane of the object without tilting toward each
other. Therefore both images are in simultaneous and complete focus, and
consequently accessories are easily adapted. However since the two imag-
ing beams traverse the large common objective obliquely, corrections for
aberrations are relatively difficult and expensive to make.<7l
Either the monobjective or the binobjective type of stereomicroscope
can be equipped with old-style stepwise changes in objectives or with the
newer style zoom objectives with a lens system of continuously variable
magnification.(7) One built-in advantage to the stepwise system of ob-
jectives of fixed magnification is in micrometry. Once each objective has
been calibrated with a given micrometer eyepiece by means of a standard-
stage micrometer, the calibrations are fixed.<7l
The corresponding disadvantage of variable magnification in the
zoom objective is obvious, but it is partially overcome in a newer design
by click-stops on the rotating collar. The manufacturer of the design has
published information about its zoom stereomicroscopes relating NA, res-
olution, depth of field, and definite magnifications. Data are reproduced as
Figure 3.8 and Table 3.2<7l With reference to Chapter 2, NA is independent
of the eyepiece's magnification (lOx in Table 3.1).
The relatively great depth of field of either type of stereobinocular
microscope is a distinct advantage in examining surfaces that have suf-
fered fracture, cracking, corrosion, erosion, weathering, etching, sawing,
or abrading. It is also an advantage in examining, sampling, separating,
dissecting, selecting, and orienting biological materials, crystals, soils,
sands, powders, flours, spots, and specks, particularly on uneven surfaces.
Such an instrument may also be used to examine, at least in a pre-
liminary way, natural, synthetic, or artificial composites and their con-
stituents. The utmost advantage of the stereomicroscope lies in the realistic
three-dimensional image of any specimen whose third dimension is within
the relatively great depth of focus. A further advantage is the character-
istically large field. Ordinary kinds of illumination may be employed, and
since their effects are known, the image is easy to interpret. Also the image
is erect, and therefore manipulating the specimen is easy. All these advan-
tages come with a compound microscope, which is comparatively in-
expensive.
 46 Chapter3

J.lm•IO J.lm•IOOO Depthci fieldin 11m

~r---------------~=.:~~---=~~~
.oa
......~--------------~
.oa
.04
.011
.ell
m
.ell
.ell
f-10
f .II
!_.II
<t.ll
.14
.Ill \
\
.II
.17
.II
.II

I I 4 11111110 I I 4 11171110 I I 4 11171110 I I 411171110

L/mm•IO LlmmaiOO LlmmaiOOO
Resolution in lines /mm
FIGURE 3.8. Resolving power and field depth in stereomicroscopes. The solid curves drawn
specifically for total magnifications (as shown) obtained with the M5 stereomicroscope, are
applicable in principle for other stereomicroscopes made by Wild Heerbrugg. The dash-
dotted curve, representing the correlation between NA and resolving power expressed in
lines per millimeter, has general validity for reflected light observation. Courtesy of Wild
Heerbrugg Instruments.(7)

TABLE 3.2
Numerical Apertures of Various Optical Combinations
for the MS Stereomicroscope"
Magnification-changer positionb

Optical combination 50x 25x 12x 6x

Main objective alone 0.0800 0.0750 0.0445 0.0223
Main objective with 2.0x attachment objective 0.1650 0.1500 0.890 0.0446
Main objective with 1.5x attachment objective 0.1200 0.1125 0.0667 0.0334
Main objective with 0.5x attachment objective 0.0400 0.0375 0.0223 0.0112
Main objective with 0.3x attachment objective 0.0240 0.0225 0.0134 0.0067
"Table courtesy of Wild Heerbrugg Instruments, IncPl
~e magnification-changer position indicates total power when lOx eyepieces are used. It
should be noted that numerical apertures for each position are independent of the eyepiece
power.
Simple and Compound Light Microscopes 47

The chieflimitation of the stereomicroscope is its relatively low resolv-

ing power. There is no substage or condenser, and therefore the kinds, and
intensities of transmitted illumination are limited. For reflected light,
vertical bright-field illumination is very limited in its ability to gain con-
trast.
The most convenient laboratory stand for the stereomicroscope is
separable at the joint between the specimen stage and the base. After
separation the whole microscope may be placed directly on a large flat
object, thus avoiding destructive sampling. In this position ordinary in-
cident oblique illumination is usually employed. For transmitted light the
base carrying the mirror and the glass window is put into place. Double
hand rests are usually supplied for convenience in manipulating the spec-
imen and/ or instruments.(7) The complete microscopical head should be
removable to go onto other stands, one of which is a heavy stand with a
long crossarm, so that the microscope can be swung over an irregular
object or part of a large machine or other assembly (see Figure 3.9).

FIGURE 3.9. Leica SZ4 stereomicroscope on a Flex-Arm stand featuring two different
configurations: one, a rigid 12-in. (31-cm) arm section for applications that require only
horizontal movement; and two, a 13.33-in. (34-cm) counterbalanced section with an adjust-
able weight range of 4-21 lb (2-9 kg) is used for situations demanding both horizontal and
vertical movement. By permission of Leica.<8)
48 Chapter 3

3.4.1. Illuminating with Stereomicroscopes

Lighting a specimen by oblique illumination to use a stereomicro-

scope can be performed with an ordinary desk-type microscopical lamp
fitted with a field condenser and diaphragm. Lately a fiber-optic cable has
been offered instead;(8•9> the resulting beam is only about so from vertical
illumination.(9l Such illumination is useful, even necessary, for examining
metals, ores, rock, concrete, bones, etc. (see Figure 3.10).(8)
A great deal can be done under the stereomicroscope with unsophis-
ticated tools. For example R. B. Scott(10> simply taped an acrylic fiber onto
a microscopical slide under a stereomicroscope at about 60x. Then with a
hand-held razor blade, he made a cut, as illustrated in Figure 3.11, and
peeled off the skin, which could be examined separately. By making a
second cut (see Figure 3.11b), he peeled off a longitudinal section only 5-10
j..t.m thick and examined it between crossed polars (see Chapter 5). The
success in making these longitudinal sections may be attributed chiefly to
the layered structure of the fiber that allows it to be peeled, thin layer after
thin layer. The phenomenon is all the more remarkable considering that a
filament of only about 20 j..l.m in diameter was operated on under a limiting
resolving power of about 5 j..l.m (see Table 3.1). The attributes contributing
to visibility are often surprising.

FIGURE 3.10. Leica Fiber-Optic Illuminator, which has many potential uses, such as nearly
vertical illumination with a stereomicroscope.(9) Courtesy of Leica.
Simple and Compound Light Microscopes 49

FIGURE 3.11. Peeling a fiber to yield (a) skin and (b) longitudinal section.(IO)

3.4.2. Preparing the Specimen

For preliminary examination with a stereomicroscope, no preparation

is desired, much less required. The idea is to look and see exactly what has
been received. Anything done to the specimen will change it. Besides any
action will take time, which alone may cause the specimen to change.
If the specimen will not stand by itself, stick it in modeling clay or
flatten a bit of it to make a base. If the specimen is smooth enough for its
surface to be within the depth of focus of a low-power objective but not all
in focus with a higher power objective, rather than smoothing it (and
changing the surface), revert to a lower power objective and choose a
higher power eyepiece.
For the reasons just pointed out, no loss of numerical aperture or
resolution results from this practice. To expose the interior of the sample
or to prepare a fresh surface, try a simple appropriate method: If the
sample is soft enough, cut it with a knife or razor blade, if rubbery, use a
pair of scissors or shears. If hard, try breaking, sawing, or abrading it. If the
resulting surface is too rough or glary and refining the surface is in-
appropriate, wet it with an inert liquid and cover with a cover glass.

3.5. BIOLOGICAL MICROSCOPES

In Chapter 1 we saw that historically biology was the first science to

grow up with microscopy, particularly through the nineteenth century.
50 Chapter 3

This dual growth was quite separate from developments in petrography

and chemistry, where different requirements for the microscope were
being met. Thus the distinctive instrument called the biological microscope
appeared. It is a compound microscope, highly developed in its best mod-
els in accordance with most of the theoretical principles expressed in
Chapter 2. The objectives are manufactured and described primarily in
terms of NA. (Magnification in objectives is of secondary importance; it can
be bolstered if desired by means of the eyepiece.) Next to NA in importance
is the kind and degree of correction in the objective (achromatic versus
apochromatic). Competent biological microscopists also understand the
use and care of oil-immersion objectives as well as air-immersion ob-
jectives.
Relatively recently professional light microscopical stands were en-
larged. Thus, the lamp, its field condenser, and its field diaphragm are
aligned and enclosed, as illustrated in Figure 3.12a.<11> Figure 3.12b is a
diagram of the corresponding optical system<12l of the corresponding kind
of microscope. Figure 3.12a shows that one end of the tube of the micro-
scope carries the objectives on a rotatable nosepiece. The other end of the
tube is to contain the eyepiece. If instead there is a binocular (but not
stereoscopic) arrangement, it is, by means of prisms, to divide the rays
equivalently to form a whole image for each of the two eyes. If there is a
third tube, it is primarily for a photomicrographical attachment. The mi-
croscope is attached to a heavy housing to minimize vibrations.<12l As a
biological microscope, the specimen rests on a fixed stage, which usually
carries a (graduated) mechanical device for moving the specimen in x-y
directions. There is a hole in the center of the stage to pass the beam of light
from the source, through the condenser and specimen, into the objective.
Usually a rotatable nose piece carries three objectives of choice.<12>

3.5.1. Objectives

These are the most important optical part of a professional light micro-
scope. The principal attributes of an objective are (NA) and degree of
correction (achromatic, semiapochromatic [fluorite], and apochromatic).
Having chosen an objective of a given NA and degree of correction for
aberrations, any convenient increase in magnification, as in micrometry,
should be made by choice of eyepiece.
Chromatic aberration is due to the greater refraction of shorter wave-
lengths than longer ones; hence the focal length of a simple lens is shorter
for blue rays than for red ones. This dispersion causes color fringes in the
Simple and Compound Light Microscopes 51

image field of a lens with chromatic aberration. The phenomenon is dia-

grammed in Figure 3.13a, and the approximate compensation is suggested
in Figure 3.13b to form an achromatic objective. As indicated achromats are
corrected chromatically for red and blue light and also for spherical aberra-
tion for green light. Thus achromats are best used visually with a green
light filter and photomicrographically with the green filter and black and
white film.< 12>
By using mineral fluorite to some degree among lens elements, sphe-
rical aberration can be corrected for two colors, so that fluorite objectives
(semiapochromats) are better than achromats for photomicrography with
color film. The best (and most expensive) objectives are the apochro"llats,
which are composed of many elements, as indicated in Figure 3.13a.<12>

3.5.2. Eyepieces

There are two principal kinds of eyepieces: Huygenian (negative) and

Ramsden (positive). As shown in Figure 3.14a, the Huygenian eyepiece has
only the upper (eye) lens above the diaphragm, whereas the Ramsden type
(see Figure 3.14b) has both lenses above the diaphragm; incidentally this
is an easy way of distinguishing between the two different types, espe-
cially if there is no labeling. The simple Huygenian eyepiece is suited for
use with ordinary achromatic objectives (with focal length of 32, 16, 8, or
4 mm). The Ramsden eyepiece has both the eye lens and the field lens
above the diaphragm; the two lenses may be cemented together. Ramsden
eyepieces are generally corrected better than the Huygenian type. Some
Ramsden eyepieces are designed to compensate for residual aberrations in
fluorite and apochromatic objectives. Compensating eyepieces are also
used to advantage with fluorite objectives of focal length 4 mm or shorter.
Because Ramsden eyepieces have all their lenses above the diaphragm and
can be sharply focused on its plane, they can all be used with any kind of
pattern placed on the diaphragm: a calibrated linear scale, a grid of cross
lines, a pattern of angles, etc.; thus the Ramsden eyepiece is used in
quantitative linear or areal analysis and when comparing an object with
some other pattern or type.<12>

3.5.3. Condensers

Figure 3.15 shows three kinds of condensers, distinguished by the

degree of correction: Abbe, aplanatic, and aplanatic/achromatic.<12> Used
 52 Chapter 3

properly, the Abbe condenser is generally satisfactory with achromatic

objectives. With all oil-immersion objectives, all condensers require im-
mersion oil between the upper lens of the condenser and the underside of
the microscopical slide.<12>
Figure 3.16a shows two adjacent points that diffract a ray of a certain
wavelength in the dry object at point 0 thus subtending an angle a', which
happens to be less than half the angular aperture AA of the objective.
Therefore the ray a' can enter the objective and do its part toward resolving
the two particular points in the object.
In Figure 3.16b it is assumed that the object has been mounted in a

FIGURE 3.12. (a) "Biological" type of compound microscope.<12>Courtesy of Olympus Cor-

poration.<11> (b) Diagram pertaining to Olympus microscope shown in Figure 3.12a.< 12>Cour-
tesy of Olympus Corporation.<12>
Simple and Compound Light Microscopes 53

Uppt•cfcotalpi,Jm•
uf~l'{'l~ns

[Y[I'IE([

.s:
.c a c
'iO ~
c
~ ~
~ 2
2 ~
~ c
a ..
.1:
0 ~
~

rex .cll<•nHih
Lowcc fO<.>I pl.m~ o ohl"""""
Uppc-l rOl.ll pl.'ln~ n l.Uncff'n~ r

FIGURE 3.12. (Continued)

 RED - - - - - - - - -
REN----- ------ -
6LUE • • • • • • • • • • • • • • • • • • • • •
a
1 2

IUX.Il lllN A IOIIX , l. 40 A

b AP IIROMAI 1\I'OCI IROMAT

FIGURE 3.13. (a) Chromatic aberration: Failure of a simple lens to bring light of different
wavelengths to a common focus (1) can be compensat ed for in part by using an achromatic
lens (2).<12) Courtesy of Mortimer Abramowitz and the Olympus Corporation.<12l (b) A series
of apochromatic objectives showing the number, shapes, and kinds of segmentsY 2l Courtesy
of Mortimer Abramowitz and the Olympus Corporation.<12l
Simple and Compound Light Microscopes 55

PLANE OF REAL IMAGE

()8\~g!e~E~w~~mJ~
OF DIAPHRAGM
--

HUYGENIAN !NEGATIVE)

FIGURE 3.14. (a) Huygenian eyepiece in cross section. (b) Ramsden eyepiece in cross sec-
tion.<12l Courtesy of Mortimer Abramowitz and the Olympus Corporation.<12l

liquid of the same refractive index as that of the cover glass (ca. 1.55).
Therefore angle a" is less than a' (see Figure 3.16c), but on reentering air
angle a" becomes a' again. However if the space between the cover glass
and an oil-immersion objective in Figure 3.16c is filled with immersion oil (n
= 1.55), the narrower angle a" is maintained. Therefore if the AA of the
particular objective is totally filled, smaller units can be resolved with a
properly employed oil-immersion objective than those resolved with the
best air objective. However it is assumed that optical homogeneity is
maintained from object to the front lens of the oil-immersion objective and from
the top lens of the condenser to the underside of the object slide.*
•see the advantages of a water-immersion objective: M. Brenner, American Laboratory, pp. 14,
16-19, April, 1994.

FIGURE 3.15. Three types of light-microscopical, substage condensers, arranged in order of

c;legree of correction for aberration(s).<12l Courtesy of Mortimer Abramowitz and the Olym-
pus Corporation.(tl)
56 Chapter 3

air

0
(c)
FIGURE 3.16. Effective angular aperture of (a) dty, (b) covered, and (c) homogeneously
immersed specimens.<1>

3.5.4. Illumination

In Chapter 2 illumination is discussed in terms of Nelson's method

versus Kohler's. Nelson's method is satisfactory with a ribbon filament as
the electrical source. However most if not all light-microscopical lamps
employ electric light bulbs with coiled filaments, which by Nelson's meth- .
od produce an image of the coil! That is, the microscopical field is unevenly
illuminated. Throwing the image out of focus defeats the purpose of crit-
ical (focused) illumination. With Kohler's method the coil filament is im-
aged by means of the lamp's lens (see Figure 3.17) onto the back aperture

CENTER! G lAMP FIELD

SCREWS DIAPHRAGM

FIGURE 3.17. Microscopical illuminator. As a separate lamp the housing contains its con-
densor lens and field iris diaphragm.<12>Courtesy of Mortimer Abramowitz and the Olympus
Corporation. <12>
Simple and Compound Light Microscopes 57

(iris diaphragm) of the microscope's condenser; thus the condenser's lens

provides uniform illumination to the object. Moreover the condenser's
diaphragm limits the size of the illuminated area to exactly that of the
particular objective in use.(12)
Figure 3.18 summarizes the optical requirements of Kohler's illumina-
tion,(12) particularly regarding a housed lamp separate from the micro-
scope's housing, which is the case in classrooms and laboratories where
the set-up is repeatedly taken down and set up again. The relevant advan-

FILM PlfiNE

IMfiGE
FORMED BY
OBIECTIVE
UNTERMEDifiTE
IMAGE PLANE)

SPECIMEN

FIELD
FIGURE 3.18. Setup for Kohler's illu- DlfiPHRACM
mination. Two ray paths are traced
from the filament of the light bulb to
a point on a photomicrographical
film.! 12) Courtesy of Mortimer
Abramowitz and the Olympus Cor-
poration. (tZ)
58 Chapter3

tages of housing the lamp and microscope together are seen in Figure
3.18.(12,13)

3.5.5. Ultraviolet and Infrared Light

Ultraviolet light is used in biology to excite fluorescence in the visible

range.<19.20> For such radiation there are special objectives through which
the rays are guided by reflection. Reflecting objectives (see Figure 3.19) are
without chromatic aberrations throughout the entire spectrum of light,
including the UV region for fluorescence microscopy and the infrared
region for FT-IR microscropy<23> (see Chapter 13).

3.5.6. Anisotropy

Anisotropy may exist in biological specimens in any or all of the man-

ifestations previously described in Section 2.13. However equipment for
viewing these phenomena is not usually included in a strictly biological
microscope. The usual instrument has no polars, no rotatable stage, nor
any slot for retardation plates, and adding these to a biological microscope
is usually unsatisfactory even if possible. Besides the objectives and other
optical parts provided on biological microscopes are not necessarily strain
free, and they tend to interfere with polarization phenomena. Therefore
further discussion of anisotropy is reserved for Chapter 5, devoted to
polarizing microscopes.

3.5.7. Magnification

Magnification is still emphasized by biologists rather than resolving

power and other attributes contributing to visibility. This is evident when-
ever biologists persist in using magnification as the first criterion in de-

FIGURE 3.19. Reflecting objective.(!> Courtesy of Microscope

Publications.
Simple and Compound Light Microscopes 59

scribing any objective, e.g., 40x regardless of whether the objective has an
NA of 0.65, 0.75, or 0.95 or it is achromatic, apochromatic, or in between.<1>
Magnification has its place, especially in micrometry, but a distinction
must be made between useful and empty magnification.

3.5.8. Photomicrography

Nowadays photomicrography is so involved with microscopies of all

kinds that cameras either are built into or easily attached to the micro-
scope. The question is what type of film is used: 32-mm roll film, Polaroid®
type, or both? Manufacturers anticipate that the answer is both because the
professional microscopist has to be prepared to present positive micro-
graphs on occasion and also to take micrographs to be presented either as
slides for viewing on a screen or as enlargements for reports and publica-
tion. Thus it is wise for the microscopist to be on the mailing list of
photomicrographical research and development companies.<14.15•16>

3.6. SUMMARY

Visual acuity, which is the inherent resolving power of the human

eye, increases (up to a point) as the eye is brought closer to the object. The
limiting point is reached at a distance of about 25 em, where the eye's lens
is contracted as much as possible without strain. Since the limit of resolu-
tion of the eye is inversely proportional to its distance from the object, the
only way to increase resolution is to supplement the lens of the eye with
an external lens or lenses, thereby bringing the eye closer to the object than
the natural limit of 25 em.
The simplest device for accomplishing this is the common magnifying
glass, which has its limitations: It cannot be held steady, the focus point
keeps changing and is seldom optimum, and the image at the circumfer-
ence of the lens is greatly distorted. When the lens is corrected and sup-
ported and brought into proper alignment and focus, it is called a simple
microscope, and it is a very useful instrument. With it the limit of resolution
is improved from 150 f..tm (for the unaided eye) to 10 f..tm (for the simple
microscope of highest practicable NA).
The compound microscope allows increased NA and great improvement
in resolution by using two or more lens systems. An objective (usually a
multiple-lens system of short focal length) forms a tiny real (but inverted)
image in the focal plane of a second lens system called the eyepiece, which
magnifies that image to a point where it can be resolved on the retina of
60 Chapter 3

the eye. For optimum utility the lenses are corrected according to the
optical principles of Chapter 2 and mounted in a sturdy stand equipped
with a light source, a condenser, a suitable stage for the specimen, a system
of focusing, and a variety of accessories for micrometry, special illumina-
tion, micrography, and so on.
Compound microscopes vary in kind and extent of their contributions
toward optimum visibility. The two general types considered in this chap-
ter are the stereomicroscope and the biological microscope. Stereomicro-
scopes are very popular because they allow both eyes to be used to per-
ceive depth, and they are usually equipped to give an upright and hence
easily interpreted image. Two designs are available, the first with two
objectives, two eyepieces, and two body tubes inclined at a slight angle to
each other, so that the lines of sight converge on the object. This is the
classical Greenough type, constructed with dry objectives of long working
distance, so that almost any object can be handled, turned, treated, and
even dissected during observation. Since the left and right images are
formed separately in axial lens systems, the systems can be fully corrected.
One disadvantage is that both axes are inclined to the plane of the spec-
imen, so that at maximum resolution, only a portion of either field can be
in focus at one time. A greater disadvantage is that there is no substage
condenser, for the simple reason that it is impossible to shape a biaxial lens.
Hence there can be no critical illumination, and resolution is severely
limited. The other design of stereomicroscope can indeed have a substage
condenser because it uses only one large objective and splits the light from
that objective into two parts that (after passing through image-erecting
prisms) are observed through separate eyepieces in separate parallel body
tubes. Both images are in simultaneous focus, and various accessories for
different kinds of illumination are easily fitted.
The biological microscope is traditionally a nonpolarizing compound
microscope designed for examining transparent or translucent specimens
and demonstrating their parts and structures. The modem biological com-
pound microscope is housed in a single stable unit containing the illumi-
nating system designed for the Kohler type of precise illumination. The
more elaborate models are equipped for two eyepieces by means of erect-
ing prisms for one person using two eyes (or two persons using one eye
each). Since only one objective can be employed at a time, there can be no
stereoscopic effect. A third tube may be provided for photomicrography at
additional cost.
Chapter 3 explains the design and proper use of various objectives,
condensers, eyepieces, and accessories. Biologists now recognize the ad-
vantages of polarized light, and its use is becoming more common in
microscopes. This subject is included in Chapters 5-7.
 4

Compound Microscopes
Using Reflected Light

4.1. STUDYING SURFACES BY REFLECTED LIGHT

Microscopy by reflected light may be used for a number of reasons: to

look at a natural surface such as a leaf, feather, skin, shell, or fossil; to
compare surfaces after aging, usage, weathering, or other exposure; or to
prepare an inside surface for studying an opaque substance, such as bone,
metal, coal, ore mineral, ceramics material, or pigmented plastic. Micro-
scopy can answer such questions as how many layers are there in a
seashell, tree's growth, laminated paper, or board? What is the structure of
a sponge, tree cone, botanical cane, zoological organ, fossil, rock, ore
(Figure 4.1 ), brick, cement, or plastic filled with biological material (Figures
4.2-4.5)?(1-s) Indeed the specimen may be a particulate material, such as
seeds, tiny insects, sand, rock dust (Figure 4.6<1>), or small crystals. Such
specimens may be better embedded in a dark, pigmented resin for
reflected light than in a clear, colorless resin for transmitted light.
Experience, habit, or equipment may determine whether the light
should be reflected from the prepared surface of a thick specimen or
transmitted through a prepared thin section. Metallographers,<2•2"·3> ore-
dressing engineers,<4•5> and resinographers<6-8> are experienced in using
reflected light, while biologists are more experienced in using transmitted
light.<9> If the biologist has to use reflected light, he/she should gain
sufficient experience in solving microscopical problems.
Reflected illumination may reveal more or different information than
transmitted light. For example, anthracite coal may be petrographically
prepared thin enough to be examined classically by transmitted light.
However a thick section may be examined by reflected light as polished or

61
62 Chapter 4

50pm

FIGURE 4.1. Lead-zinc deposit, South Africa. Sphalerite (sp) replaced along its cleavage
planes by lead-zinc oxidation products (ox) and by galena (ga). Polished thick section,
reflected light. Courtesy of American Cyanamid Co.

as etched, with or without polarizing the light. Figure 4.7 shows the dif-
ferences under reflected light by varying the methods of examination.
With the increasing development of man-made composites of opaque
and transparent, hard and soft, large and small components, organic and
inorganic plastics, came greater use of reflected light on strictly transparent
specimens!10•11 > (Figure 4.8)<121.

4.2. RESOLVING POWER

The resolving power of microscopical objectives for use by reflected

light can be slightly higher than those intended for use with transmitted
light and a cover glass. No cover is necessary on a prepared surface
FIGURE 4.2. Wood flour mounted in black phenolic resin. Polished cross section, reflected
light. The fibers are straight in lengthwise section, and some cross sections occur in groups.<1l

FIGURE 4.3. Sugar cane (bagasse) mounted in black phenolic resin. Polished cross section,
reflected IightY>
FIGURE 4.4. Peanut shells mounted in black phenolic resin. Polished cross section, reflected
light, showing plates of short, interlocked fibers in longitudinal and transverse sections.(l)

50~tm

FIGURE 4.5. Cotton mounted in black phenolic resin. Polished cross section, illumination by
reflected light. Most of the fibers are shown in cross section. Oval cross sections are shown
with a slit in the center. Actually they are cross sections of collapsed cylindrical tubes. (l)
Compound Microscopes Using Reflected Light 65

FIGURE 4.6. Dust from Vermont chrysotile asbestos fiber. illumination by vertical reflected
light. Gray particles are chrysotile asbestos or massive serpentine. White particles are asso-
ciated magnetite. Matrix is Melmac® melamine-formaldehyde resin.(l)

examined by reflected light, so the refractive index n in the formula for

numerical aperture
NA = n sin a
is not restricted to 1.52 for glass or any other cover. Accordingly an im-
mersion objective of NA = 1.60 was made for use with monobromonaph-
thalene (n 0 = 1.66}(1 1> as the immersion liquid, with corresponding high
resolution. Unfortunately this kind of objective is no longer manufactured;
evidently there was insufficient demand for it, presumably because pro-
spective users did not understand its optical advantages.
The resolution of fine structures by reflected light begins with proper
objectives and their proper use.<13> If the objectives are corrected for use
without a cover glass, no cover should be used. Reflected-light objectives
are usually in short mechanical mounts for optical reasons and bear the
manufacturer's information code. It is essential to know the code and use
the information. If the objective is corrected for infinity, it is probably not
interchangeable with those of a microscope of any other make (or perhaps
other models of the same make). If an objective is corrected for use with a
66 Chapter 4

FIGURE 4.7. Four ways to view a field of polished anthracite coal by reflected light: (upper
left) between crossed polars; (upper right) unpolarized bright field; (lower left) unpolarized
dark field; (lower right) bright field after etching with chromic acid (for microscopy with
polarized light, see Chapter 5).

certain eyepiece or projection piece, use it. If an objective is to be immersed,

make sure that the immersion fluid has the proper refractive index.

4.3. CONTRAST

Contrast in images by reflected light is low because of many partial

reflections from various surfaces as the beam goes down through the
objective to the specimen and back through the objective, illuminating
reflector, and eyepiece. With highly reflecting specimen surfaces like those
of polished metals and ores, there is still plenty of contrast. However
poorly reflecting, highly scattering surfaces like those of paper, textiles,
and wood cause much glare.<11> Even so as shown in Figures 4.1-4.8,
Compound Microscopes Using Reflected Light 67

S0 11m

FIGURE 4.8. Wax on glass, cooled in about 20 seconds. Micrograph by reflected light.
Courtesy of Continental Oil Co.<12>

enough contrast and sharp resolution can be obtained from transparent

material by reflected light to provide valuable information to those who
will work for it.<12>

4.4. CORRECTING FOR ABERRATIONS

Corrections for aberrations in dry objectives of NA = 0.5 or more for use

by reflected light are designed with the assumption that no cover glass will
be used.<13> On the other hand, corrections for immersion objectives assume
that any cover should have the same refractive index as the immersion
fluid. Ordinary oil-immersion objectives assume that the oil will have n =
1.52, so ordinary glass covers can be used with oil-immersion objectives
when covering will help the visibility. For example, the surfaces of paper,
·textiles, and wood scatter much less light if covered with the proper
immersion fluid in conjunction with an immersion objective. If the spec-
imen is fibrous, particulate, or dusty, the particles may be held in place by
a proper cover slip and immersion liquid.
68 Chapter 4

4.5. SPECIMEN CLEANLINESS

Cleanliness of the specimen is a special attribute contributing to micro-

scopical visibility of a surface by reflected light. (SJ Dust, abrasives, films,
and even fingerprints will scatter light enough to add glare to the image
and confusion to the interpretation. Precautions should also be taken with
regard to the illuminating reflector inside the microscope, which is usually
directly under the eyepiece or projecting unit. It is especially important
that the tube always be covered to protect the illuminating surfaces from
dust and dirt, and they must be inspected and cleaned periodically.
Oeanliness in a broader sense includes protection of the specimen
from contamination and damage, care of the equipment, knowledge of
optical principles, and order in general. All aspects of cleanliness are
especially important in working with reflected light because so many
optical surfaces are involved twice, first in illumination and again in image
formation. Proper alignment of the parts and adjustment of diaphragm
apertures are important for the same reason.

4.6. FOCUS

Focus itself is perhaps not so important in working by reflected light

as by transmitted light, provided the specimen's surface is flat and
oriented exactly 90° to the axis of the microscope. Yet there are times when
a deep etch or relief polish and unidirectional oblique illumination are
desirable to produce shadows. In such cases selecting the plane of focus is
arbitrary and therefore critical.
Depth of focus is characteristically important in images formed by
reflected light because the tilt of the illuminator is critical. If the tilt is not
exactly 45° to the axis of the microscope, all of the image will not be aligned
90° to the axis. The eye has great depth of focus, so if the depth of focus
in the objective is great enough, all of the image may be in acceptable
visible focus. Yet in photomicrography the eye is not directly involved,
making the depth of focus in the objective critical for obtaining a good
photomicrograph.

4.7. ILLUMINATION

Illumination is the most determinative attribute contributing to micro-

scopical visibility by reflected light. As Figure 2.4 shows, reflected illu-
mination can be either dark field or bright field; it can be unidirectional,
Compound Microscopes Using Reflected Light 69

symmetrical, or in between. Unidirectional dark-field illumination can be

obtained from an ordinary reading lamp with a stereomicroscope (see
Chapter 3) or from a smaller lamp and an ordinary compound microscope.
Symmetrical dark fields with such microscopes can be obtained with the
Lieberkiihn type of concentric, concave mirror (Figure 4.9)(1 4); from a ring-
shaped lamp fitting around the objective, such as the old-fashioned wreath
of small incandescent lamps<13l; from the newer circular fluorescent lamp,
small enough to fit close to the objective; or from a circle of fibrous light
wires<14•14•l surrounding the objective. In any case with dark-field illumina-
tion, a mirrorlike surface appears dark because light is reflected away from
the objective, but a light-scattering surface like paper appears bright.
With stereoscopic or monobjectives of far enough working distance,
bright-field (vertical) illumination can be obtained simply by placing a
partially reflecting, clearly transmitting sheet (such as a thin, flat cover
glass) in front of the objective at 45° to its axis and to the flat surface of the
specimen, as shown in Figure 4.10<13); the photograph in Figure 4.11 was
taken this way. It shows a ball bearing of high-carbon steel retained in both
halves of its case-carburized steel race.

Adjustable

Transparent
dorkwell support
in filter carrier

FIGURE 4.9. The Lieberkiihn type of

illuminator.<14) Courtesy of Micro- Illuminating
scope Publications. light rays
70 Chapter 4

FIGURE 4.10. Vertical illumination by a transpar-

ent reflector between objective and specimen.<13l

For higher resolution the transparent reflector must be placed beyond

the objective. As shown diagrammatically in Figure 4.12, the central mirror
MM is for bright-field illumination of the specimen SP, which returns some
light to the transparent reflector MM, allowing light rays to pass through
the system to form an image. Light from the source goes through a series
of lenses and apertures then enters the side of the microscope. There the
light strikes the transpar~nt mirror MM, and is reflected into the objective,
which acts as a condenser. In the manner of Kohler's illumination, the field
iris diaphragm FF is focused in the plane of the object. The iris FF is opened
just enough to fit the field of the particular objective. Light is reflected from
the object through the objective, this time through the mirror MM to form

FIGURE 4.11. High-carbon-steel ball bear-

ing in its low-carbon, case-carburized race,
mounted in bakelite, sectioned, polished,
and etched. Illuminated as shown in Figure
4.10.
Compound Microscopes Using Reflected Light 71

OBJECTIVE

s p

FIGURE 4.12. Vertical bright-field Kohler illumination by reflected light.(15> Courtesy of

Microscope Publications.

a real bright-field image.(15> This image is enlarged by the eyepiece or

projection piece (neither is shown in Figure 4.12). By shifting FF and/ or
tilting MM, oblique bright-field illumination forms shadows on ups and
downs in the object.(1,13> Figure 4.2 illustrates the use of slightly oblique
reflected illumination in observing a transparent object.
Figure 4.13 shows a current model(15> of a microscopical stand fitted to
illuminate a specimen by reflection, thereby producing either bright-field
or dark-field illumination with its vertical illuminator (see Figure 4.14) and
dark-field (DF) and bright-field (BF) sliding adapters (see Figure 4.15).(15>
The sliding adapters DF and BF allow for quick and easy interchange.
Moreover by sliding out both of them and replacing the high dry objectives
with cover glass or oil-immersion objectives, the microscope is ready for
transmitted light. (15)

4.8. RADIATION

The radiation most used by reflecting microscopes continues to be

produced by an incandescent tungsten filament. Olympus microscopes
usually employ a 12-volt tungsten halogen lamp of 50 or 100 watts(15>; other
photomicroscopes employing incident light are shown as Figures 4.16-
72 Chapter 4

FIGURE 4.13. An incident-light microscope with vertical illuminator in place. Courtesy of

Olympus Corporation.<15l

4.18.<16l Figure 4.19 shows an inverted microscope, designed to hold a

polished, briquetted specimen<l7) with its plane perpendicular to the axis of
the microscope. The inversion of the plane-polished specimen is one of the
advantages of metallographs, which are nevertheless outmoded because
they are large, heavy, and limited in use.
Many minerals and other opaque crystalline materials are anisotropic,
which means that both reflectance and color vary with the vibrational
direction of polarized light with res~ect to the orientation of each crystal
Compound Microscopes Using Reflected Light 73

FIGURE 4.14. Vertical illuminator on Olympus microscope employing incident illumina-

tion.<15l Courtesy of Olympus Corporation.

grain. Anisotropic crystals also have positions of extinction between

crossed polars (see Chapter 5). These phenomena mean that polished
anisotropic crystalline grains present various kinds and degrees of visi-
bility as they are rotated between crossed polars. At the back aperture of
an objective with a high NA, an interference figure may be seen<18l (see
Figure 4.20). To observe such phenomena, a microscope must be fitted
with a polarizer, an analyzer, and an adequate source of illumination<14l
(see Chapter 5).

FIGURE 4.15. Bright-field and dark-

field semitransparent mirrors in mounts
for insertion in Olympus microscope.(IS)
Courtesy of Olympus Corporation.
 FIGURE 4.16. Microscope equip-
ped for photography by incident
and reflected light. Courtesy of
Nikon.(16>

.-:- . - .
FIGURE 4.17. Microscope equipped for photography by reflected light. Courtesy of Carl
Zeiss.(16>
Compound Microscopes Using Reflected Light 75

FIGURE 4.18. A metallographic microscope with reflected and transmitted illumination

with Nomarski interference contrast optics plus automatic camera. Courtesy of Olympus
Corp.<15>

4.9. MAGNIFICATION

Exact magnification is determined by reflected light in the same way as

by transmitted light<14>: by comparing the real or virtual image with the
graduations of a standard scale placed as the object on the microscopical
stage. However, when using reflected light, spaces between graduations
on the standard scale must be specularly reflecting (silvered) instead of
transparent (clear). Real images of the standard scale, such as on a ground-
glass projection screen or photomicrograph, are measured in enlargement
by a commensurately larger scale(" ruler"). Virtual images (those observed
directly in the eyepiece) are measured by a secondary micrometer (reticle)
in the eyepiece. Such a micrometer must be calibrated against the standard
scale placed as the object on the stage for the particular objective, tube
length, and eyepiece. During calibration make sure that unit graduations
at both ends of the scale have the same value (within your tolerance) as
76 Chapter 4

FIGURE 4.19. An inverted microscope arranged so that the briquetted specimen lies flat on
its plane, polished face. Also fitted with large and small cameras.<16l Courtesy of Nikon.

FIGURE 4.20. Figures obtained by reflected light with pyrite (isotropic, left) versus arseno-
pyrite (anisotropic, right), with an objective of high NA. Courtesy of American Cyanamid Co.
Compound Microscopes Using Reflected Light 77

those graduations in the middle of the scale. If not, return the scale to the
manufacturer for one that does. For very accurate work an institution
should have at least one stage micrometer that has been checked and
corrected by the National Bureau of Standards in Washington, D.C.
Designation of magnification is essential in all kinds of micrometry.
Figure 4.21 is typical of the measurement of layers on case-hardened
low-carbon steel. The scale at the measured magnification is on a negative
image of the specimen during printing.
Knowledge of the magnification is essential when estimating grain
size in metals and alloys.(19) Likewise magnification must be known when
measuring the microhardness of materials(20) (see Figure 4.22).

4.10. FIELD OF VIEW

The field of view should be large enough to tell the story, as in Figures
4.21 and 4.22. On the other hand a picture should be cropped of extraneous

0 100!-lm
'

FIGURE 4.21. Bright-field, vertical illumination of a case-hardened specimen of very low-

carbon iron.
78 Chapter 4

FIGURE 4.22. Hardness differences in a weld by means of indentation. Courtesy of A.

White.

or distracting portions if they are on the periphery. By the same token the
most important part of a picture should be placed in the center of the field
of view if that is feasible.

4.11. GLARE

Glare from reflected illumination is much greater than from trans-

mitted illumination because there is much more attenuation of the light as
it goes through the objective (or dark-field condenser) to the object and
back through the objective. Even in the least complex system, there is some
scattered light. The problem becomes worse as we add lenses, thick glass
prisms for illumination, polars, interference filters, etc. To see how much
glare there is, focus on the lighted specimen, then remove it, and with the
light still on, look again. You may see as much as one-third of the original
intensity due to scattered light. Removing the eyepiece and looking down
the tube may reveal specific reflections from metallic holders or frames for
the reflector, objective, polars, etc., suggesting that some jet black paint in
the right places may help remove glare.<13> Make sure the illumination is
centered and the field diaphragm has not been closed to frame the field of
view and exclude stray light (Kohler illumination).<13· 15>
The intensity of partially reflected light, such as from the glass-to-air,
interfaces of objectives, field lenses, and filters, can be reduced by coating
such interfaces with a thin film of a substance (such as magnesium fluor-
Compound Microscopes Using Reflected Light 79

ide) that is less refractive than the glass. The intensity of partially reflected
light at the illuminator may be increased by coating it with a substance
more highly refractive or more highly reflective (e.g., silver).<11> Reducing
the aperture behind the objective to cut out the most oblique, glare-pro-
ducing rays often restores acceptable contrast. Of course another objective
of lower NA will do the same thing but at lower objective magnification.<15>
Scattering at the surface of the object can also be reduced by covering it
with immersion fluid and using the proper immersion objective. On the
other hand, visibility may be better with dark-field illumination, since
scattered light rays are then purposely collected by the objective and
turned into the image, while specularly reflected rays are rejected.<11>
Some of the reflected light is polarized and can therefore be eliminated
by orienting a polar so that its direction of vibration is crossed with the
direction of vibration of most if not all of the polarized glare (see Chapter
5). The optimum effect is obtained by rotating the polar or specimen
empirically until the glare is minimized.

4.12. DEPTH

The best cue to depth with reflected light is attained by casting shadows
inside depressions and outside elevations, shallow as they may be. This
can be done with bright-field illumination by tilting the mirror. With
dark-field illumination shadows are created either by shutting off half of
the dark-field beam or by decentering the hollow mirror. Either way relief
polishing or deeply etching the specimen will lengthen the shadows.
Of course etching or relief polishing must not be so severe as to exceed
their field of depth of the particular objective. This requirement is usually
achieved when the specimen is first prepared as a smooth plane and then
etched or relief polished lightly and progressively until the desired effect
is obtained. Usually a flat surface attained by commercial planing or rol-
ling yields an image within the field depth limitations of the optical system
without further treatment. This is true of most samples of sheet metal,
wooden boards, laminated paper, and cardboard, with or without paint,
lacquer, varnish, or other finish. Of course there will always be some
specimens that are too rough for a particular objective's depth of focus. The
task then is to prepare a smoother surface that will remain significant and
representative.
Lack of field depth with high-power objectives on upright micro-
scopes using reflected light can also be a problem if the prepared surface
is not sufficiently flat, a partial shim under the low side or comer may be
all that is needed to bring all of the field into focus. Mechanical clamps are
80 Chapter 4

available, or can be made, for holding the surface against a hole whose
frame is parallel to the base. For low powers at least, an irregularly shaped
specimen may be propped up with a little modeling clay and a modicum
of patience. With inverted microscopes and metallographs (see Figure 4.23),
the specimen rests on its plane-polished surface against the stage, which is
perpendicular to the axis of the microscope.(11l

4.13. WORKING DISTANCE

Working distance between the specimen and objective for reflected light
is usually not a severe problem, and as a rule there is no cover glass (...0.18
mm thick or thicker), since the specimen is usually smooth and flat without
peaks to take up working distance. Considerable working distance is
needed in performing many kinds of operations and tests by reflected
light, so provision has to be made. For example, in Figure 4.24 space is
provided for a dental drill to remove, for a microchemical test, a certain
particle in a complex mixture of ore minerals and gangue in a polished
section.(Il Incidentally, the micromanipulator is used to control other tools,
such as a tiny magnet, dissecting needles, or syringes. Another kind of test
that requires considerable working distance is one for microhardness,(20>

FIGURE 4.23. The microscope is inverted

and illuminated with a tungsten lamp or a
xenon arc. Courtesy of Unitron Instruments.
Compound Microscopes Using Reflected Light 81

FIGURE 4.24. Dental drill and micromanipula-

tor. The chuck of the dental drill is held in a spe-
cial damp attached to a micromanipulator.<1l

either by scratching or indenting with a diamond. Measuring the width of

a scratch or indentation also requires considerable field depth.

4.14. STRUCTURE

Studying structure by reflected light is the very basis of metallog-

raphy,<2·21·213l ore mineralogy,<4•5> resinography,(7) wood technology,<10l and a
host of other technologies. In metals, alloys, ores, and some plastics, we
often have granular structures and sometimes fibers. In natural tissues,
such as woods, reeds, bark, and shells, we have characteristic cells and
fibers. The visible kinds of structure and their distribution are of funda-
mental importance. We must also consider the orientation of these struc-
tures, as shown in Figure 4.25.
Crystal structure is fundamental for visibility. Practically all metals,
alloys, minerals, and starches are crystalline; consequently there are cer-
tain properties and phenomena derived specifically from the crystalline
structure of such materials. For example, in Figure 4.25 the granular struc-
ture of a metal is brought out by etching, principally because of the dif-
ferential solubility of crystals in different orientations within the mass.
Such characteristic orientation of crystal grains is inherent in the crystalline
structure. Likewise, the slippage direction of the planes of atoms in a
82 Chapter4

FIGURE 4.25. Stainless steel 18Ni + 8Cr, cold-rolled, polished, and etched, showing one,
two, or three slip directions, depending on the specific orientation of each crystalline grain
with respect to the direction of cold working.

crystal of stressed metal varies with the crystallographic orientation of

each grain with respect to the direction of stress.
Although the potential visibility of structure lies within the specimen,
other attributes depend on the microscopy and microscopist. In Figure
4.25, structure visibility is aided by adequate resolution, contrast, shad-
ows, field of view, preparation, and photography.

4.15. MORPHOLOGY

Visibility of the internal features of the metal in Figure 4.25 is aided by

appropriate morphology: size and shape of the grains, distance between slip
lines (representing the slip plane in section), and thickness of intergranular
material. In a similar way morphology of various phases in heat-treated,
plain-carbon steels is made visible by reflected-light microscopy. This has
enabled metallographers to understand the principles of precipitation
hardening, summarized in Figure 4.26, which illustrates the metallog-
Compound Microscopes Using Reflected Light 83

raphy of heat treating 0.89% carbon steel at various rates of increasing and
decreasing temperatures. On the other hand, the precipitation hardness of
aluminum alloys was not understood by light microscopy because the
morphology of the precipitates was too fine. Nevertheless, experience with
steels and brasses led metallographers to imagine what to look for using
electron microscopy, and then their efforts were successful.

4.16. INFORMATION ABOUT THE SPECIMEN

Information about any specimen contributes to its visibility; in micro-

scopy by reflected light, there is hardly an exception. Usually sufficient
information can be obtained firsthand to enable the microscopist to decide
how to go about solving the present problem. If there is insufficient in-
formation, it is best to go to the primary literature about the material before
starting the investigation.
Figure 4.23 shows a microscope (one of several varieties) that allows
quick choice between reflected and transmitted illumination. Figure 4.27
shows a table model of a polarizing microscope that also offers this choice.
Whether reflected or transmitted light (or both) are used depends on the
descriptive information accompanying the sample, what microscopical
experiments are to be performed, the behavior of the specimen on the
microscope and in the room during the scheduled time, and how much
preparation is necessary.

4.17. EXPERIMENTATION

Microscopy involves experimentation as well as observation, and some

possible experiments are suggested in Figures 4.7, 4.21, 4.22, and 4.24.
Almost any experiment feasible by transmitted light can be performed by
reflected light, with the added advantage of having the specimen un-
covered so that the microscopist can pick, poke, scratch, cut, tum, or roll
it. A small magnet, a tiny "soldering iron," a thermocouple, or any other
active point can be applied. The specimen can be placed on a hot or cold
stage,<13l and it can be stretched, pushed, or twisted.

4.18. SPECIMEN BEHAVIOR

Specimen behavior during observation may be fortuitous, since the

specimen is uncovered and often exposed to a hot beam of intense light.
 ~
OF oc
All•tente (Stable)
-re,--- --
700

'!(<
• .' • BHN 293
Rc 31
BHN 170
'~- ~ Rc 5

600

BHN 401
Rc 42 500

400

f
....
 I
\ {
~'
fw>ltllile a::
(lftld:io)

i"
_ _Mi._ _ _ _
i
Ill

s·~
011
~
1----- - 50" MARTENSITE
f
flo
!'"'
1 - - - - -90" MARTENSITE
i
100

~:) a~~ ~~2

10 10 2 10 3 10 4 10 5
TIME -SECONDS

FIGURE 4.26. Isothermal transformation diagram of eutectoid carbon steel; C, 0.89% Mn, 0.29%. Austenitized at 1625° F. Grain
size: 4-5. Photomicrogra phs originally 2500x. Courtesy of U.S. Steel Research Laboratory. ~
 86 Chapter 4

FIGURE 4.27. D. W. Davis using an all-pur-

pose polarizing compound microscope,
used in industrial microscopy with either
reflected or transmitted illumination, bright
field or dark field. Courtesy of American
Cyanamid Co.

Nevertheless, any change in the appearance should be noticed, recorded,

and explained. The uncovered, freshly prepared specimen may have been
stored a long time in an ordinary or extraordinary atmosphere, dry or
humid, before or after etching, and it may have undergone change. Be-
havior differences should be noted just as though the exposure were
planned, as well it might have been.(10> Some reactions to look for are
efflorescence, deliquescence, oxidation, sulfiding, and polymerization.
The chief limitation of microscopical examination by reflected light is
the condition of the surface, so any change in that surface is significant. The
surface must be flat enough and smooth enough for whatever objective is
required to do the job, which is why a preliminary look at low power is so
important. Even if the problem is not solved then and there, we can get an
idea of how well the surface, as received, is suited to reflected illumination;
how much preparation will be needed to see more; and how the surface
has changed during storage and observation.

4.19. SPECIMEN PREPARATION

If the surface is dusty or dirty, clean it with a stream of air or some

inert liquid. The safest liquid is water and a little wetting agent, perhaps
using a brush, soft at first, then stiff if needed. If the surface has too many
Compound Microscopes Using Reflected Light 87

scratches or pits and it is hard enough, a fine abrasive (lJ.tm or less) may
improve the image, but any substantial abrasion will reveal the interior
rather than the exterior.
To expose the interior of a sufficiently solid, cohesive material, we can
cut, smooth, and polish it. This is the way most metals, many rocks, ores,
ceramics, cements, glasses, and plastics are prepared. If a specimen is
powdery, particulate, fibrous, thin, porous, or irregularly shaped, it should
be mounted by molding it in a block of resin. A molding press and mold
are available for compression-molding bakelite and similar resins and
provide a convenient method of mounting such a sample.<1>If compression
molding is not feasible, casting in a polymerizable liquid is recom-
mended.<22·23>
Etching is the key to the light-microscopical examination of all metals.
Its primary purpose is to remove metal that has been modified by pre-
paratory procedures. (t3,14.24l

4.20. PHOTOMICROGRAPHIC TECHNIQUES

Photomicrographic techniques by reflected light include those of general

photomicrography and metallography. A special problem may be poor
contrast between phases that do not differ much in reflectivity. Improved
contrast can be obtained by using high-contrast photosensitive materials
and under extreme conditions, a high-contrast developer.(1 6>

4.21. SUMMARY

Since many materials are opaque, their microscopical examination

must be accomplished by using reflected rather than transmitted light. For
example a metal cannot be examined by transmitted light no matter how
thin a section is prepared or how thin a foil is chosen. Reflected light offers
the only chance of learning something about the structure and morphology
of the metal. In other situations only the surface of a transparent or trans-
lucent material may be important, and surface examination by reflected
light may be the method. For such cases equipment and techniques for
microscopical observation by reflected light have been developed to just as
high degree as those for transmitted light.
The simplest optical arrangement for studying a specimen by reflected
light is a polished glass plate mounted between the objective and the
specimen at an angle of 45° to the axis of the microscope. Light from a
source off to the side, at right angles to that axis, is reflected onto the
 88 Chapter4

sample. Different components of the specimen's surface then reflect the

light in a different and characteristic manner through the glass plate and
into the optical system of the microscope, forming an image of that surface.
Obviously, this arrangement works only for objectives of low power and
long working distance. For more detailed study the 45° reflector must be
positioned behind the objective, within the microscope body. The reflector
may then be a mirror, a prism, or even a polarizing crystal positioned to
reflect light downward on the sample. One advantage of this arrangement
is that the objective automatically becomes condenser as well as objective
(and a better corrected condenser of higher NAthan most external ones),
so that excellent illumination is assured. Furthermore, in most instances a
cover glass is not necessary, so that an immersion liquid of suitable re-
fractive index can be used between the objective and the sample, thereby
improving resolution.
Light sources for reflective microscopy must be more intense than
those used in transmissive work, due to greater losses in the optical sys-
tem. Tungsten-halogen lamps, mercury or argon arcs, and even old-fash-
ioned carbon arcs are preferred. Illumination may be bright field or dark
field, according to various optical arrangements described in the text and
shown in diagrams. With sufficiently intense sources and appropriate
filters, advantage may be taken of some special spectral portion of the
illuminating radiation to produce differential fluorescence (or other ex-
aggerated response) within the sample components.
One drawback of reflective microscopes is that part of the light is
reflected or scattered by the many surfaces it meets on the way to and from
the sample, so there is likely to be extraneous light (called glare) that
reduces contrast and impairs visibility. The amount of glare can be re-
duced by scrupulously cleaning optical components, hunting down and
blackening any reflective metal parts in the microscope, using coated
lenses, and (if absolutely necessary) constricting apertures or installing
diaphragms to eliminate wide-angle reflected light. Since such light is
partially polarized, it can be reduced in intensity by installing an eyepiece
polar and rotating it for maximum effect.
Not only existing surfaces but also the interior structure and morphol-
ogy of such opaque materials as bones and teeth can be studied by
reflective microscopy. Suitable sections are sawed or cut, and the fresh
surface is smoothed and then polished until all scratches are invisible and
the surface features can be seen. Etching the polished surface of a metal is
essential for distinguishing the structural components and studying the
morphology. Etching also helps study plastics, polymers, minerals, and
ceramics. Reference to a complete list of etchants for metals is given, and
precautions for their use are included in ASTM Standard E407, Microetch-
ants for Metals and Alloys.<24)
 5

Microscopy with
Polarized Light

5.1. OVERHEAD PROJECTORS

The historic observation of double imaging by a highly birefringent

crystal, such as that of calcite (see Chapter 1), can easily be repeated on an
overhead projector. A small dot of black paper is pasted on the projector's
window near its center, then a cleavage rhombohedron of clear calcite is
placed over the dot. Two images of the dot appear on the projection screen,
which means that the incident beam is being divided into two beams that
do not interfere with one another because they are vibrating in different
(perpendicular) planes. When the calcite rhombohedron is rotated on
whatever face it happens to rest, one image of the dot is stationary while
the other image curiously traces a circle around the first image (see Figure
5.1). The fact that the so-called extraordinary image is displaced from the
ordinary image means that the extraordinary ray travels at a different
velocity than the ordinary ray. The fact that the extraordinary ray tra~es a
circle rather than a spot confirms that the difference in velocities varie.s with
the orientation of this crystalline species. The ordinary image is stationary
because its velocity is constant with all orientations of the crystal, and
accordingly the ordinary refractive index ro is constant. The various values
for the refractive index in the path of the extraordinary ray can be given
a general symbol, such as E', or specific symbols, such as £1, £21 £ 31 etc.
The overhead projector may be converted into a polariscope<t-3) by
placing two polarizing sheets (polars),<1> such as Polaroid®, over the illu-
minated window. The two polars are separated vertically by about 10 em
by means of two blocks, so that the specimen can be placed and turned by
hand between the polars. If the polars are crossed with respect to their
directions for vibration of light, the field will be black because none of the

89
90 Chapter 5

FIGURE 5.1. Single crystal placed over a single dot, showing double imagery: (1) The central
imaged dot is stationary, since it is represented by the ordinary ray (n1 =oo) inside calcite. (2)
The circular ring of imaged dots indicates that there is a circular path as the calcite rhombo-
hedron is rotated over the single object dot (n II =symbol, such as E').

polarized light from the first polar (polarizer)<1>is vibrating in the direction
for the light to vibrate through the crossed polar (analyzer).<1> An aniso-
tropic crystal, such as a calcite rhombohedron, will appear bright in all
positions of rotation except the four times in a revolution when the direc-
tion for ro or £' corresponds to the polarization direction of the polarizer or
analyzer, as indicated in Figure 5.2. In all other positions of rotation, the
crystal appears bright because there is a vector corresponding to the direc-
tion of vibration in the analyzer, so that some light appears.
The extinction displayed by calcite, shown in Figure 5.2, is called

FIGURE 5.2. Calcite rhombohedron in one of four analogous positions of extinction (dark-
ness) when vibration direction oo or £' in calcite corresponds to vibration direction PP in
polarizer or AA in analyzer. <4>
Microscopes with Polarized Light 91

symmetrical extinction because at extinction the obtuse angle a is bisected

by PP or AA, the direction of vibration in the polarizer or analyzer. Some
other species of crystal display parallel extinction, which occurs when PP
or AA corresponds to a prominent edge of the crystal; otherwise an aniso-
tropic crystal manifests oblique extinction. This and other optical prop-
erties in microscopic crystals call for microscopical examination by means
of a polarizing microscope. Such a microscope is employed in optical
crystallography,<4-7l mineralogy, petrography, chemistry, physics, and biol-
ogy.<8> Anisotropy is also observable in liquid crystals<9>; strained glasses<10>;
stressed plastic materials and models; partially crystallized resins and
polymers<10>; reflecting surfaces; flowing colloids; man-made filaments;
and biological fibers, cells, and tissues. <11•12l

5.2. ANISOTROPY

There are five kinds of anisotropy:

The optical anisotropy<1> of each single anisometric crystal indicates that
the bonding of the unit molecules, ions, radicals, or elements is different
in at least two of the crystallographic directions. Such crystals as those of
calcite, quartz, melamine, and sucrose display 10 or more individual op-
tical properties that can be detected and measured on these microscopic
crystals. Such properties are very useful in characterizing materials in
synthetical and analytical science and technology, as discussed in Chapter
7.
Multiples of anisotropic crystals have optical characteristics above and
beyond those of the individual crystals. The simplest multiples are twins
(see Figure 5.3)-two crystals sharing a single "composition" plane<4>but
also manifesting individual properties. Figure 5.4 shows twins and higher
multiples of subcrystals within crystals. Spherulites<1l have still higher
multiples of crystals, with each one contributing to a unit effect on polar-
ized light. Such spherulites are manifested for example by a single grain
of raw starch or of polyoxyethylene.
Molecular birefringence is manifested by long or flat molecules, espe-
cially by macromolecules (polymers). In long molecules the atomic dipoles
are primarily arranged in chains. By induction the dipoles are stronger
than if the atoms were widely separated. When polarized light is vibrating
lengthwise to the chain, the average strength of the dipoles is greater than
when light is vibrating crosswise. The optical properties of a system of
parallel long molecules are derived from those of the individual long
molecules. <9>
Light vibrating parallel to a molecular chain or the axis of a (fibrous)
92 Chapter 5

FIGURE 5.3. Twinned crystals (with separate

extinction directions). 4

FIGURE 5.4. Thin section of pegmatitic rock (granite) between crossed polars, showing
crystalline grains. The large grain Or-Or is a twin. The grains 01-01 and Mi are multiple
crystals. 01, oligoclase; Or, orthoclase; Mi, microcline; Mu, muscovite; Qu, quartz. Courtesy
of American Cyanamid Co.
Microscopes with Polarized Light 93

system of aligned long molecules travels more slowly than light vibrating
crosswise, that is, the refractive index for light vibrating lengthwise (n 11 )
is higher than for light vibrating crosswise (n.L). This is conventionally
called positive birefringence in fibers (see Chapter 6), and it is strong in
natural cellulose fibers and many man-made fibers. Of course side chains on
the molecule tend to reduce the strength of birefringence in the main chain
of the molecule and therefore that of a fiber made up of such branched
molecules<9) (see Chapter 6).
Flat molecules tend to slow down light that vibrates in their planes. If
these molecules are arranged with their planes parallel, as in a film or foil,
light vibrating in the plane of the arrangement has a refractive index
higher than the refractive index of light vibrating perpendicularly to the
planar arrangement. Symmetrically planar molecules (such as ring
configurations) are isotropic in their planes, so that films or foils made of
symmetrically planar molecules are isotropic when viewed perpendicu-
larly to the sheet. Although long flat molecules tend to be anisotropic in
their planes if they are oriented at random, the resultant sheet is still
isotropic in its plane. However, if the long flat molecules are oriented with
their lengths parallel and their planes parallel, their sheets tend to be
birefringent in their plane as well as perpendicular to their plane.<9)
If the long flat molecule also preferentially absorbs polarized light
strongly in one direction (generally lengthwise), the film made with such
molecules tends to be polarizing like Polaroid® polarizing film.
Form birefringence,<9 ) also called rod or plate birefringence,<4) is man-
ifested by a two-phase system whose mass of long or flat particles, not
necessarily anisotropic, has been oriented in a medium with a different re-
fractive index by a flowing, streaming, or growing process. The slower
vibration direction (higher refractive index) of the system is parallel to the
length of the rods or in the plane of the plates. This kind of anisotropy is
found in some fibers, films, and other colloidal systems. It is detected by
a change in degree of birefringence while the specimen is mounted in a
liquid (such as a standard of refractive index). Such a liquid changes the
refractive index of the continuous phase, thereby changing the difference
in indices between phases. Soaking in such a liquid may be sufficient to
eliminate all of the form birefringence, leaving only the true double re-
fraction. If soaking produces isotropy, all of the original anisotropy was
due to form birefringence.<4)
The photoelastic effect is the local anisotropy manifested as strain result-
ing from stress on normally isotropic materials, such as an inorganic or
organic glass<10) (see Figure 5.5).
Anisotropy observed in a material can provide more visibility in
microscopical images than ordinary unpolarized light as well as patterns of
94 Chapter 5

FIGURE 5.5 Interference pattern in the neck produced by pulling a fiber of nylon by hand.
Taken between crossed polars at 45" from position of extinction by a student.

light and dark, usually in colors. The need for both kinds of information
led to the development of the petrographical microscope, which has a va-
riety of accessories<11- 16> added to the ordinary compound microscope.
Chemists later found they needed the same information, so the chemical
microscope followed. In addition to petrographical and chemical micro-
scopes, the newer research instruments and universal microscopes for use
in all sciences also carry polarizing equipment.

5.3. NUMERICAL APERTURE AND INTERFERENCE FIGURES

The NA takes on special significance, particularly in the search for a

principal kind of pattern, the interference figure (see Figure 5.6), sometimes
called a directions image<4>because it is the pattern formed by summing the
directional effects of polarized light rays on the specimen within the limita-
tions of the angular aperture of the objective or its condenser, whichever is
the smaller. Thus when producing interference patterns, the value of NA
transcends that of the resolution of fine detail in a pictorial image. The
purpose is to obtain as large a solid cone of rays as is commensurate with
other attributes contributing to visibility. To obtain data from interference
figures that are compatible with conventional data conversion charts, some
authors<6> standardize on an NA of 0.85.
Microscopes with Polarized Light 95

FIGURE 5.6. Diagram of an interference figure; the

pattern is formed by the summation of effects by
the specimen on the polarized light rays coming
from all the directions included in the angular aper-
tures of objective and condenser.<4l

5.4. RESOLUTION: SPECIMEN INTERACTION AND POLARIZED

LIGHT

Resolution of the specimen's effects on polarized light depends on

producing plane-polarized light with the polarizer and analyzing effects
with the analyzer. 0 2l At present both polars<1l are usually made of polarizing
film, e.g., Polaroid®, sandwiched between two protective pieces of thin
glass. For transmitted light the polarizer P is generally placed in front of
the iris diaphragm of the substage condenser, as shown in Figure 5.7.<9>The
analyzer A is usually placed in a slide fitting in or out of the tube of the
microscope above the objective 0.
Resolution of the specimen's effects on polarized light means that
everything else in the usual optical path must have no effect: Objectives,
eyepieces, condensers, microscopical slides, and cover glasses must be
strain free. The test is to remove the specimen and with the light source
turned on and the polars crossed, look into the microscope. There should
be no light. If there is a spot of light that does not fill the field, turning one
optical element at a time may indicate which element is strained. If the spot
does not move, remove one element at a time to find which ones are
strained. <4> The manufacturer tries to supply all polarizing microscopes
96 Chapter 5

-- --------- Fb

B BERTRAI'{) LENS

A ANALYZER
SLOT L

OBJECTIVE

- - DIAPHRAGM OPEN D
POLARIZER P

FIGURE 5.7. The polarizing microscope as used for conoscopic observations.(9l Courtesy of
Microscope Publications.

with strain-free components, but sometimes objectives, eyepieces, and

condensers are switched in a laboratory. The objectives carry some symbol,
such as P, to indicate that they are for polarized light.
The eyepieces can all be of the Huygenian type because their
magnifications and corrections are usually high enough for a polarizing
microscope. There should be at least three eyepieces: one eyepiece
equipped with cross lines and a lug on the eyepiece to engage in a notch
in the tube of the microscope, so that cross lines are oriented w E to the
observer; another eyepiece fitted with a linear micrometer (to be calibrated
t
as explained in Chapter 4) for distances on interference figures, etc., as
discussed later. A third eyepiece should be clear of reticules for photo-
micrography or whenever no reticule is desired. However other reticules,
such as a protractor for estimating angles or cross-hatching for estimating
areas, may be kept on hand, ready to drop into the eyepiece E at the focal
plane F, in Figure 5.7.
The condenser C should have an easily removed upper lens, such as
one that flips out of the way and back into position. The iris diaphragm D
is essential. The polarizer P does not have to be removable; it should be
Microscopes with Polarized Light 97

rotatable but also have a catch of some sort to indicate when the vibration
direction of light through it is parallel to one of the cross lines.
Slot L in Figure 5.7 is designed to accommodate retardation plates
such as a first-order red plate, or a compensator, such as the quartz wedge
used to make Figure 5.8. The B in Figure 5.7 indicates a Bertrand lens, a
simple lens system that, with eyepiece E, constitutes a compound micro-
scope for looking at an enlarged interference figure at the back aperture of
objective 0. When not in use the Bertrand lens can be slid or flipped out
of the way.

5.5. CONTRAST: MICHEL-LEVY INTERFERENCE CHART

Contrast, color or neutral gray on a dark background, is an outstand-

ing attribute of viewing an anisotropic object between crossed or partially
crossed polars, as illustrated in Figure 2.7. The light (first-order gray or
high-order white) or colors (see Figure 5.8) is an interference phenomenon.
Figure 5.9 diagrams how interference comes from polarizer P and analyzer
A. The polarizer feeds plane-polarized light to the birefringent specimen S,
which splits the beam into two components.(19> The specimen is in a po-
sition of brightness; that is, it is oriented on the rotatable stage (RS in Figure
5.7). Thus its two kinds of rays are vibrating in different azimuths from
those of PandA, yet both bundles have to go through analyzer A. For some
wavelengths (colors) two wave trains will be in phase and reinforce each
other. For other wavelengths the two trains will be out of phase and
interfere with each other. The potential result is Newton's series of inter-
ference colors, depending on how much the slower component falls be-
hind the faster component. It is as though we have two runners, one faster

retardation

plane - polorozed
light

FIGURE 5.9. Diagram used to explain the functions of the polarizer and the analyzer.(4)
 98 Chapter 5

than the other. Assuming that their speeds are constant, how much the
slower runner falls behind the faster one depends on their difference in
speed and how far they run. If we know two of the three variables (dif-
ference in speed, distance traveled, and distance between the two), we can
determine the third variable.
The difference in light ray velocity is expressed by the difference in
refractive index n1-n2• The distance the rays travel is the thickness of the
specimen, expressed in micrometers (formerly microns). The distance the
slower light ray falls behind the faster one (retardation) is expressed in
nanometers (formerly millimicrons). These three variables can be charted
as a Newtonian series of interference colors, such as displayed by a quartz
wedge showing several orders of retardation colors.
The classical chart is the Michel-Levy scale of birefringence<4> (see
Figure 5.8). This chart was originally designed for petrographers' thin
sections of rock, with the standard thickness of professional sections, 30
iJ.m, in the middle of the ordinate. But the chart is also appropriate for
fibers, since they run commercially from 10-40 ,_..m and even greater for
thicker fibers used in carpets. The general equation for Figure 5.8 is

retardation (r) = thickness (t) x birefringence (L\n).(7)

The abscissa on the bottom is graduated in nanometers of retardation.
The scale is organized into orders; each order ends with red, about every
565 nm of retardation. The first order (up to L\, .. 565 nm) of course starts
with black (L\, = 0) and goes from dark gray to light gray to white of the
first order (1 °). Some people see dark gray as dark blue, which is just as
diagnostic as dark neutral gray. It is important to recognize grays and
white of the first order. Around L\, = 275 nm the specimen in positions
between crossed polars appears to be yellow, then at about 450 nm, it
appears orange, then red, and finally magenta. The second order contains
the most brilliant colors, from blues to greens, to yellows, to oranges, and
finally to red. The third-order colors are less brilliant (less saturated). In the
fourth order, colors are still less brilliant, i.e., nearly pastel. To many
people fourth-order blue seems faded. By the fifth order (see Figure 5.8)
blues are invisible to most people, and by the sixth order only pink (faint
red) can be seen. After the eighth order retardation is visible only as
high-order white, so the sequence of orders of retardation can no longer be
detected by color. We then need special accessories, some of which mea-
sure as many as 30 orders of retardation; more is said about them later in
this chapter.
The upper abscissa of the Michel-Levy chart is expressed in terms of
birefringence, n1-n2• The scale moves horizontally from 0.001-0.040 and
Microscopes with Polarized Light 99

then vertically down to 0.20. Usually the problem is to obtain the bi-
refringence from the thickness and retardation. Low orders of retardation
are determined ordinarily with the aid of retardation plates.

5.5.1. Personal Interpretation of Interference Colors

Why does interpretation of interference colors differ? Subjectively

people differ physiologically in their sensitivity to various colors, as ex-
plained by Land's retinex theory.(I 6•>
In the Michel-Levy chart of birefringence (see Figure 5.8), colors rep-
resent the complex admixture of rays varying in wavelength and intensity.
The total interference color observed for any value of retardation is the sum
of the intensities at various wavelengths along the ordinate axis. In other
words there are no pure colors. For example a straight line through 260 nm
of retardation from violet to red reveals enough of each color to render
first-order white. On the other hand a vertical line through the intensities
shown in Figure 5.10 at 1300 nm indicates third-order green because there
is so little of the other wavelengths (colors). Thus the more objective color
differences there are, the more they affect color differences among ob-
servers.
As the caption to Figure 5.8 explains, there is a slight discrepancy
between the subjective appearance of interference colors of the quartz
wedge to the human eye and the objective rendition of the colors. The
version of the Michel-Levy chart in Figure 5.8 was obtained by photo-
graphing the microscopical image of a standard quartz wedge, so sensi-
tivity to individual colors was based on the particular color photosensitive
film employed. Further discrepancies appeared during printing, and as a
result an artist's skill was added. Based on successful use of the first edition
of this book in the classroom, the chart is reproduced in this edition.

UNPOLARIZED POLARIZED
LIGHT LIGHT

FIGURE 5.10. Light partially polarized by reflection from a surface.

100 Chapter 5

5.5.2. Retardation Plates

Most polarizing microscopes are sold with two retardation plates as

standard equipment: a first-order (1 °) red plate and a quarter-wave (0.25A.)
plate. The 1o red plate is also called the sensitive-tint plate because just a
very little retardation added to, or subtracted from, that plate turns the
retardation color a blue or yellow, respectively. Adding retardation means
that the slower component of the specimen is parallel to that of the 1o red
plate. Subtraction means that the two slower components are crossed. The
vibration direction of the slower component is marked on the 1o red plate;
this provides a means of detecting the vibration direction of the slower
component as it emerges from the specimen.
Similarly the vibration direction of the slower component is marked on
the 0.25A. (1 o white) plate, and hence that plate can also be used to detect the
vibration direction of the slower component from a specimen with a first-
order polarization color. A specimen manifesting first-order white for ex-
ample would be dark gray or black (coqtpensated) when its slower compo-
nent is crossed with that of the 0.25A. plate and bright white or yellow when
the slower components are parallel. But if a white polarization color remains
white while the specimen is rotated between crossed polars and the 0.25A.
plate is in place (usually NW +-+ SE), the white is of high order.
Compensation is exactly matching retardation in the specimen to a stan-
dard retardation device. Criterion for matching involves observing dark-
ness when the specimen is in a position of brightness until the retardation
device renders the specimen black. Compensation would occur fortuitous-
ly with a 0.25A. plate if the specimen had a retardation of exactly 0.25A. and
the two slower components were crossed. Compensation also occurs for-
tuitously with a 1o red plate if the specimen has first-order retardation and
the two slower components are crossed. In either fortuitous situation, and
only then, the retardation plate can be termed a compensator.
A true compensator is a device of variable retardation, such as a
quartz wedge, say, of three orders. If the specimen, such as a fiber of nylon,
is rotated into the NW or SE quadrant, and the quartz wedge is slid into
its slot until the specimen becomes black, the compensating order and hue
may be determined. From these data the approximate retardation may be
estimated by using the Michel-Levy chart. The Senarmont compensator<17l
is more accurate because a graduated quartz wedge is slid over the first one
until compensation is reached.
Another type of compensator uses the principle of varying the direc-
tion of the incident light by tilting an anisotropic material of constant
thickness. One variety is called the Berek compensator.<17l
Microscopes with Polarized Light 101

5.5.3. Specimen Thickness

If the exact path length is not known, it must be measured. Many

old-fashioned and some new microscopes are equipped with fine-focusing
wheels graduated in units, such as microns. With such a microscope,
measure specimen thickness by using an objective of high NA (shallow
depth of focus) and focusing upward from the bottom of the object. After
reading the micrometer's scale with the bottom in focus, focus upward to
the top of the object, then read the scale again. Focusing is always done in
the same direction of knob-turning to take up any slack or backlash in the
focusing mechanism. The difference in the two readings must be multi-
plied by the refractive index of the specimen because top and bottom
images are displaced by that much. Some specimens can be measured for
thickness before mounting by means of a thickness micrometer.<18•19>
If vertical distance cannot be measured, distance must be measured
horizontally. If the specimen happens to be a cube, a cylinder, or a sphere,
diameter is measured horizontally. Otherwise the specimen has to be
turned on its side by micromanipulation<18> a spindle stage, or a universal
stage.<20>Horizontal measurements are made with an eyepiece micrometer,
calibrated for tube length and the particular objective in use by a certified
stage micrometer.<18>

5.6. CORRECTING FOR ABERRATIONS DUE TO STRAIN

It is apparent by now that the most important correction for aberrations

in the polarizing microscope is that of color. For visual work achromatic
objectives are adequate, but apochromats are preferred for photography.
The ordinary uncorrected condenser has such severe chromatic aberra-
tions that its colors may be misinterpreted as retardation colors. Centering
the illuminating beam and the condenser helps reduce aberrational color
in an ordinary condenser. At the same time the color temperature of
the illuminating beam should be controlled by using a daylight color
filter.
All optical glass in the polarizing microscope system must be strain
free. Objectives certified as strain free by the manufacturer are marked with
a symbol, such as P. All other objectives should be tested between crossed
polars. Eyepieces, condensers, slides and cover glasses, auxiliary lenses,
retardation plates, and compensators should also be tested for strain;
likewise windows, such as those in hot stages, should be tested for strain.
(If strained, try loosening the retaining screw ring.)
102 Chapter 5

Reflected light and all glare are partially polarized. One probable
source of these is from overhead light entering in, or reflected from, the
eyepiece. Once investigated, such aberrations are usually easy to correct.

5.7. CLEANLINESS: FREEDOM FROM INTERFERENCE FILMS

Cleanliness is especially important in working with the polarizing

microscope. Thin films of oil, air, or other matter may result in interference
colors that can be misinterpreted as polarization colors because they be-
long to Newton's series of interference colors (see Figure 5.8). Care must
be taken to leave no air spaces or bubbles between the slide and cover glass
because the appearance of the specimen in air may be misinterpreted for
that in a liquid, which has a much higher refractive index than air.

5.8. FOCUS

We have seen that the optical properties of an anisotropic material

vary with the direction of the incident light. Theoretically in determining
these properties only axial rays should illuminate the specimen. With
objectives of low NA and the top lens of the condenser flipped off, illu-
mination is close enough to ideal, and focus depth is adequate. With
high-aperture objectives however, the iris diaphragm of the condenser
should be closed as much as feasible, and again focus depth is a bonus. Of
course resolution is decreased, and if resolution is inadequate, fine detail
should be studied separately with the iris diaphragm open.<5l
When anisotropic material is studied between crossed polars in a
position of brightness (by rotating the stage), the material is bright against
a black background, as in dark-field illumination (but for a different rea-
son). In both instances the specimen appears to be self-luminous, like the
moon at night. Such images are seldom in sharp focus. The sharpest image
is obtained by first focusing with parallel polars and then crossing them to
obtain the polarization image. Since interference figures are not images,
the specimen need not be in sharp focus, and a thick specimen can usually
be in focus.

5.9. ILLUMINATION

Illumination needed to obtain the optical properties mentioned in Fig-

ure 5.10 is called orthoscopic.<5l Except that the light is polarized, ortho-
scopic illumination is the axial, bright-field transmitted illumination dis-
Microscopes with Polarized Light 103

cussed in Chapters 2 and 3. The image is the same with unpolarized or

polarized light. By crossing or partially crossing the polars, we obtain
polarization images as in Figure 5.6.
When an objective of moderate or high NA is filled with light by an
adequate condenser, as in Figure 5.7, illumination is appropriately called
conoscopic.15> Such illumination is primarily used for observing polarization
figures at the back aperture of the objective. Small but bright polarization
figures are viewed with the eyepiece out. Larger but less brilliant figures
are best seen with both the eyepiece E and Bertrand lens B in, as shown in
Figure 5.7.15>

FIGURE 5.11. Olympus polarizing microscope.l15> Courtesy of M. J. Abramowitz and the

Olympus Corporation.116>
104 Chapter 5

With either orthoscopic or conoscopic illumination, and with the pol-

ars crossed, incident light must be intense enough to see the effect that the
specimen is producing. Even if 100% efficient, a Nicol polarizer absorbs
half the light in order to polarize it. Common polarizing films absorb 65%
of the light reaching them. The analyzer absorbs at least another 50-65%
of the light that comes through the specimen, however transparent. So
with only 17-25% of the original light available, even if the specimen fills
the whole field (which it usually does not), the initial intensity of illumina-
tion has to be high.

5.10. RADIATION

Usually, a special6-V incandescent lamp of the type already described

is amply intense. Intensity is regulated by reducing the voltage as neces-
sary by means of a variable transformer, but the radiation so obtained will
be redder. A daylight filter stabilizes the color.
Something should be said here about determining the direction of
light vibration from the polarizer, which must be known to detect and
measure optical properties of anisotropic materials discussed in Chapters
6 and 7. To date manufacturers of polarizing microscopes have differed
about whether the ~ or the w - E cross line represents vibration direction
in the polarizer wh~n it clicks into place. To determine which is actually
the direction, use light reflected from a flat black surface, such as the black
enameled foot of a microscope stand. As shown in Figure 5.10, part of the
light reflected from a plane surface is polarized, and the vibration direction
lies in that surface at right angles to the reflected beam. So if the polarizer
(or analyzer, whichever is easily removable) is held in the hand, looked
through, and turned to bring the reflected beam to maximum darkness, the
vibration direction of the light from the polar is at right angles to the plane
of reflection.
Figure 5.12 also explains why in reflected-light microscopy (see Chap-
ter 4) turning an analyzer eliminates some of the glare coming from the
specimen and adds contrast to the image.

5.11. MAGNIFICATION

The total magnification of a polarization image or pattern should be no

greater than is useful (..1000 x NA).<4> Empty magnification<4> only de-
creases intensity of the image, which may already be low.
Microscopes with Polarized Light 105

Eyepiece

(Eyepieces with cross-hair (().

or micrometer should be
Inserted Into the right
eyepiece tube.)

Circular dovetail mount

Intermediate attachment
clamping screw

Toac
~I
Una cord

FIGURE 5.12. Diagram of the Olympus polarizing microscope.< 15l Eyepieces with a crosshair
or micrometer should be inserted into the right-hand eyepiece tube. Courtesy of the Olympus
Corporation.< 16l

5.12. FIELD OF VIEW OF AN INTERFERENCE FIGURE

The field of view of an interference figure should include only the one
crystal involved, lest the figure be confused by effects of surrounding
crystals. With small crystals, grains, or particles, a pinhole cap can be
inserted in place of the eyepiece (with the Bertrand lens also removed). The
smaller the hole in the cap (2 mm or less), the smaller the field of view. The
Wright cross-slit diaphragm is more convenient because the size and po-
sition of its opening can be adjusted to contain only the one selected crystal
observed while looking through the hinged eyepiece (replacing the regular
eyepiece). When the single crystal has been framed, the Wright eyepiece
can either be swung away or kept in place to work with the Bertrand lens.<4l

5.13. GLARE

With reflected illumination (see Chapter 4), there is some glare from
the specimen itself. Since a portion of any reflected light is polarized (see
106 Chapters

Figure 5.12), this much of it can be removed by rotating a properly placed

polar until glare is at a minimum. As indicated before the polar is also
effective on light scattered from other surfaces, including lenses in the
objective. In any case the lower the objective NA, the less glare. Achro-
matic objectives present less glare than apochromats of the same NA
because they have fewer air-glass interfaces.

5.14. DEPTH

As already illustrated in Figure 2.15, images of anisotropic specimens

obtained between crossed or partially crossed polars can be a depth cue.
The three-dimensional twists and spirals of cotton fibers present various
orientations to axial polarized light to vary the degree of birefringence.
Varying fiber depth changes the path length contributing to the fluctuating
retardation and consequently introduces a variety of polarization grays
and colors. Figures 5.4 and 5.5 (among others) also illustrate the fact that
polarization effects are cues to depth.
In anisotropic crystals, since retardation of one of the double rays
behind the other depends on the thickness of the specimen as well as the
extent of birefringence, it is important for the thickness to be within the
acceptable focusing power of the objective. As discussed in Section 5.8,
with a self-luminous object the focus cannot be so sharp as with a dark
object against a bright background. Therefore the field depth obtained with
crossed polars is not so important as that obtained with bright-field illu-
mination. It is important for the thickness of the specimen to be known or
constant. In petrography thin sections are standardized to a thickness of 30
J.l.lll (the middle of the range in Figure 5.8). Fortunately most fibers used for
paper, textiles, and cordage are also well within the range of Michel-Levy
chart.
Specimens for obtaining polarization figures may be even thicker than
those for imaging specimens between crossed polars because polarization
figures are images of the back aperture of the objective, not of the spec-
imen. In fact the depth of the interference figure need be no shallower than
the (long) depth of focus of the Bertrand lens (see Figure 5.7).

5.15. WORKING DISTANCE

The working distance of an objective used on a polarizing microscope

should be within the range of specimen thickness in Figure 5.8 plus the
cover glass thickness. <21>These are ordinary considerations for designers of
microscopical lenses. The practicing microscopist however should always
Microscopes with Polarized Light 107

be ready to use a hotstage<22> on a polarizing microscope, and he I she must

have adequate working distance above it. In general working distance is
precious, and the more of it the better.
Some extra-long working objectives are designed and built primarily
for universal stages.<20> These objectives are also very handy for use with
hot and cold stages, microdynamometers,<23> and other setups for dynamic
experiments<24> requiring objectives with extra-long working distances.

5.16. SPECIMEN STRUCTURE

Specimen structure is the most fundamental concern when employing

polarized light to study a material. An anisotropic material is sure to reveal
different information when it is restricted to unpolarized light. This attri-
bute was discussed in Section 5.2, and some of its practical aspects are
continued in Chapters 6 and 7.

5.17. SPECIMEN MORPHOLOGY

Specimen morphology is also important. If there is a choice of thickness,

choose one well within the limitations in Figure 5.8. If only thicker spec-
imens are available, think about preparation methods that will reduce
thickness but not change structure. If the sample is a powdery mixture
of different-sized particles, optimum-sized particles can probably be
screened out or otherwise separated.<24>
Crystals should always be examined as received under a microscope
even though they are likely to be aggregated, broken, or rounded. Recrys-
tallization should always be contemplated but employed after the pre-
liminary examination.

5.18. INFORMATION ABOUT THE SPECIMEN

Information about the specimen should include history that may have
brought on more or less anisotropy: stress, annealing, weathering, con-
gealing, or precipitation due to short shelf-life, etc.

5.19. EXPERIMENTATION

Experimentation has already been discussed in Sections 5.15, 5.17, and

5.18. For example, if information indicates a working hypothesis that will
help solve the problem microscopically, experiment!
 108 Chapter 5

5.20. SPECIMEN BEHAVIOR

Specimen behavior may be planned purposely for experimentation, but

it may also be observed incidentally or fortuitously. For example both
phases in a commercial sample of trisodium phosphate (TSP) were clear
when received. Between crossed polars one phase was isotropic, and the
other phase was anisotropic. While being examined under a reading lamp,
the anisotropic phase turned white (dehydrated into light-scattering pseu-
domorphs). This behavior called for experimentation and preparation to
confirm the identity of the two phases.

5.21. SPECIMEN PREPARATION

One of the most useful methods of preparing a sample for observation

with a polarizing microscope is recrystallization. Every microscopist
should know how to do it well and quicldy,<4l using only a few tiny crystals
and a single drop of water or other solvent. For recrystallization from
water, the impure or imperfect material is added to a drop of water on a
corner of a microscopical slide and dissolved by warming the slide over a
microheater<2l while stirring with a drawn-down tip of a slender glass rod.
When all is dissolved, the slide is allowed to cool until crystals begin to
form. If necessary, the slide can be warmed again to evaporate some of the
water until well-formed crystals appear. Placing a cover glass on the drop
will then retard further evaporation.

5.22. PHOTOGRAPHY

Photomicrography employing polarized light is somewhat demand-

ing with respect to the kind and extent of equipment. Resultant images are
usually colored by various and variable wavelengths of light. Accordingly
the condenser, objectives, eyepieces, and all other optical equipment
should be chromatically correct. These and later remarks pertain to Figures
5.11, 5.12, 5.13, and 5.14. Figure 5.13 depicts a microscope employing
transmitted polarized light, like Figure 5.11, but it is equipped with a
sliding Bertrand lens, which together with the eyepiece, permits the ob-
servation and photomicrography of interference figures. These and other
polarization colors require careful attention to the color sensitivity of pho-
tographic film.<2~29 l
Figure 5.14 depicts a microscope employing reflected polarized light
by means of a vertical illuminator.<14l Such a microscope can be employed
Microscopes with Polarized Light 109

FIGURE 5.13. Nikon microscope precisely designed for microscopy by transmitted, polar-
ized light, including photomicrography and television.( 14l Courtesy of Nikon Instrument
Group.0 4l

to differentiate between anisotropic and isotropic opaque, polished sur-

faces, such as arsenopyrite versus pyrite (see Figure 4.16).

5.23. SUMMARY

Light is unique among all other kinds of emanation: It can be polarized

to vibrate in only one direction. Consequently light microscopy is unique
among all other kinds of microscopies. By polarizing the incident light,
kind and extent of birefringence (double refraction) can be detected and
analyzed according to the three principal perpendicular directions in the
specimen. This structural information is then compared with the mor-
phological information in biology, crystallography, description or analysis
of natural versus man-made fibers, foils and films, stress and strain during
test and use of materials, etc.
110 Chapter 5

FIGURE 5.14. Polarizing microscope equipped for use with reflected light. Courtesy of
Nikon.<14>

Illumination with polarized light reveals five kinds of anisotropy:

1. Optical anisotropy of a single anisotropic crystal in at least two of
the three crystallographic directions. Such crystals as calcite, quartz, mel-
amine, and sucrose display 10 or more separate properties that can be
measured on microscopic crystals.
2. Multiples of anisotropic crystals, such as twins, grains of metal,
rock, ore, and spherulites (such as starch grains, characteristic of the plant
species).
3. Molecular birefringence as manifested by long or flat molecules,
especially as arranged in a film or foil.
4. Form birefringence, also called rod or plate birefringence, as man-
ifested in a two-phase system where long or flat particles are oriented in a
medium of different refractive index by a flowing, streaming, or growing
process.
Microscopes with Polarized Light 111

5. The photoelastic effect is the local anisotropy manifested as strain

resulting from stress on normal isotropic materials, such as inorganic or
organic glass fibers, films, or solids. Small units can represent big units;
indeed fibers can represent rods or girders. Films can represent sheets, and
particles can represent rooms or even buildings, with respect to both struc-
ture (architecture) and morphology (size and shape), as different as these
are.
 6

Microscopical Properties
of Fibers

6.1. INTRODUCTION

Fibers are unique units of biological tissues, mineral habits, or spin-

ning processes; examples include muscle and nerve fibers, wool, fur, hair,
cotton, linen, natural silk, natural and regenerated cellulose, asbestos, spun
silicate glass, and man-made polymeric fibers. Although fibers vary wide-
ly in chemical nature, they are physically alike, being much longer than
wide, very strong for their small cross sections, and anisotropic. The mi-
croscopically determinative properties of fibers are both morphological
and structural. The distinctive morphology of a fiber type includes sizes
and shapes in both longitudinal and cross-sectional views.

6.2. FIBER MORPHOLOGY

The determinative morphology of fibers was developed in the latter

nineteenth century and the first-half of the twentieth century by means of
light microscopy. Methods have been somewhat standardized, and deter-
minative light micrographs have been published.<1- 3l Transmission electron
microscopy brought more detail of the morphology of fibers< 3l; however,
scanning electron microscopy has brought even more contrast and depth
to the determinative micrographs of fibers.<4•5l Some examples are shown
in Figures 6.1--6.13.<5!
In both longitudinal and cross sections, Figures 6.1-3 show similari-
ties and slight differences in wool from merino sheep, blackface sheep, and
cashmere goats. Together however the three species show the very char-

113
114 Chapter 6

FIGURE 6.1. Scanning electron micrographs of wool (merino). Courtesy of Shirley Insti-
tute.<5l

FIGURE 6.2. Scanning electron micrographs of wool (blackface). Courtesy of Shirley In-
stitute. (S)
Microscopical Properties of Fibers 115

FIGURE 6.3. Scanning electron micrographs of a cashmere goat. Courtesy of Shirley ln-
stitute.<5>

acteristic exterior scales that hook on to one another during spinning to

make yarn.
The next three scanning electron micrographs have a botanical origin:
flax, cotton, and mercerized cotton (Figures 6.4-6).<5>Figure 6.4 shows cross
and longitudinal sections of flax fibers for making linen. The cross sections
are very irregular in size, depending on how many fibrils these contain.
The longitudinal views of flax include nodes, which are dislocations of the
fibrils. Figure 6.5 shows cotton fibers; their cross sections show tiny, col-
lapsed tubes, curled to look like Cs, Us, and Os. Longitudinal views of
cotton look like tiny twisted ribbons. Mercerized cotton fibers (see Figure
6.6b) are swollen to the size of cylinders or thick-walled tubes.<5>
Figure 6.7 shows silk in cross sections, which are irregularly shaped
three- four-sided polygons. Longitudinal views of silk fibers are striated
(see Figure 6.7).<5>
Figures 6.8-6.13 show man-made fibers. Their morphologies reflect
not only chemical composition, but also the manufacturers' design of the
spinnerette and treatment of the filament(s). Viscose fibers are character-
istically serrated in the cross section and grooved longitudinally (see Fig-
ure 6.8).<5>Terylene® polyester fibers (see Figure 6.9) vary in cross section
from approximately polygonal to circular. Nylon cross sections are more
116 Chapter 6

FIGURE 6.4. Scanning electron micrographs of flax. Courtesy of Shirley Institute.<5l

FIGURE 6.5. Scanning electron micrographs of cotton. Courtesy of Shirley Institute.<5>

Microscopical Properties of Fibers 117

FIGURE 6.6. Scanning electron micrographs of mercerized cotton. Courtesy of Shirley In-
stitute.<5l

FIGURE 6.7. Scanning electron micrographs of silk. Courtesy of Shirley lnstitute.<5J

118 Chapter 6

FIGURE 6.8. Scanning electron micrographs of viscose rayon fibers. Courtesy of Shirley
Institute. (S)

FIGURE 6.9. Scanning electron micrographs of Terylene® polyester fibers. Courtesy of Shir-
ley Institute.<5l
Microscopical Properties of Fibers 119

FIGURE 6.10. Scanning electron micrographs of nylon polyamide fibers. Courtesy of Shirley
lnstitute.<5l

nearly round (see Figure 6.10).<5l Orion® 42 acrylic fibers (see Figure 6.11)
resemble the figure eight in cross section but are slightly rough longit-
udinally.<5l Acrilan® acrylic fibers are somewhat kidney-shaped in cross
section and outwardly quite smooth longitudinally (see Figure 6.12).<5l
Dice}® acetate fibers* (see Figure 6.13) are quite nondescript [whether
viewed by SEM, TEM, or light microscope (the old standby)]. Thus we see
that the man-made fibers are not especially distinguished by morphology
alone. However, many of them are very distinguishable by means of their
optical properties. ·
For a light-microscopical examination of longitudinal views of fibers,
the general procedure is to mount the fibers on a microscopical glass slide
in mineral oil of refractive index 1.48 and cover with a standard glass

"Terylene®, Orion®, Acrilan®, and Dice!® are examples of trademarks registered with the U.S.
patent office. Each trademark belongs exclusively to a single owner (usually a corporation),
.for use with a specific product or products. Trademarks are not nouns, so they should be
followed by descriptive terms; for example there are Polaroid® cameras, films, or polars.
Trade name (two words) is used for a type of product, such as nylon for a polyamide fiber,
yam, or fabric, without regard to the producer, see A Guide to the Care of Trademarks, U.S.
Trademark Association, 6 East 45th St., New York, NY 10017.<6)
120 Chapter 6

FIGURE 6.11. Scanning electron micrographs of Orion® acrylic fibers. Courtesy of Shirley
Institute. (S)

FIGURE 6.12. Scanning electron micrographs of Acrilan® acrylic fibers. Courtesy of Shirley
lnstitute.(S)
Microscopical Properties of Fibers 121

FIGURE 6.13. Scanning electron micrographs of Dicel<l!> cellulose acetate fibers. Courtesy of
Shirley Institute. <5>

cover. Procedures for making fiber cross sections are covered in Chapter
20.
Morphology is usually sufficient for either describing or identifying
natural fibers; for man-made fibers however, the morphology may depend
more on spinning and drawing processes and on how the spun fiber was
physically treated than on composition of the material being spun. Never-
theless, every kind and variety of fiber has as many as eight different
optical properties and these properties can be described and measured by
means of polarized light and a polarizer and an analyzer. However the kind
and degree of each property is not so constant as with crystals. With
man-made fibers optical properties vary in kind and degree with variation
in treatment during and after spinning; but there is sufficient constancy to
be useful in both describing and identifying fiber species.
Figure 6.14 shows a simple fiber whose light waves vibrate in two
privileged directions: one vibration direction parallel (II) to the fiber's axis
and the other vibration direction perpendicular (.i) to the axis. To consider
the two directions simultaneously, a few fibers are cut to lengths of 1 or 2
<;m and mounted in a liquid to reduce light scattering by the fiber in air
(refractive index n = 1.00). Water (n = 1.33) is handy for mounting such
hydrophilic fibers as cotton, and mineral oil (n = 1.48) is commonly used
for such hydrophobic fibers as nylon. Fibers should be examined im-
 122 Chapter6

vibt'arlon
erouwise

FIGURE 6.14. Fiber in position of

brightness between crossed polars.
Drawn by C. Miller.

mediately on being mounted in liquid, because they may change. Cotton

for example swells in water with corresponding changes in optical prop-
erties, and nylon swells in some organic liquids. Such changes should be
noted as behavioral information, but initial characteristics are to be com-
pared with determinative literature.<2> At the same time subsequent be-
havior in liquids may indicate that more experiments are worthwhile.
With fibers properly mounted, the eight determinative optical prop-
erties can be observed in succession with the aid of a polarizing micro-
scope. The fibers are first brought into focus with the analyzer out, and
then observed between crossed polars while the stage is slowly rotated.
The eight properties to be noted follow.
1. Brightness or grayness (instead of darkness) in some or all positions
of rotation between crossed polars. Most fiber types show brightness in only
certain rotation positions (see Figure 6.15), but cotton is bright in all po-
sitions because its structure is spiral instead of axial. Inorganic glass fibers
show no change when rotated between crossed polars. Glass fibers are too
thin to show detectable retardation ordinarily, although thicker glass rods
and bottles do. The fact that glass fibers appear to be isotropic, even though
they are not strictly so, should not detract from the analytical importance
of their being the only kind of fiber that comes so close to showing no
retardation under the microscopical conditions just set forth. That alone is
determinative.
Microscopical Properties of Fibers 123

FIGURE 6.15. Positions between crossed polars of brightness and extinction (darkness); (
left) parallel extinction; (right) oblique extinction.

2. Extinction (darkness): complete or incomplete, every 90° of rotation

between crossed polars (see Figure 6.15). Cotton, with its spiral structure,
shows no substantial extinction. Linen (flax), hemp, certain other bast
fibers, and some mechanically treated man-made fibers show extinction in
certain segments at a time.
3. The extinction may be parallel or oblique to the fiber axis, and the
angle is determinative. Some fibers display segmented extinction, as in
bast fibers, silk filaments, and some crimped man-made fibers (see Figure
6.15).
4. Retardation: the distance in nanometers that one wave train falls
behind another (see Figure 6.14) is manifested as grays, whites, and colors
(see Figure 5.8), some of which (reds and pinks) are visible between
crossed polars to the eight order of classification of the interference be-
tween slower and faster ray bundles. Some polyester fibers show pinks of
such high order.
5. Sign of the birefringence depends on which of the two ray bundles is
the slower. Most fibers are positive, signifying that the rays vibrating
lengthwise are slower (i.e., have the higher refractive index, n), but an
important minority are negative. The sign is determined between crossed
polars using a retardation plate.
6. Quantitative birefringence, the numerical difference between the
high and low refractive indices, is independent of path length (thickness,
denier). It can be determined from the retardation per unit path length by
means of compensators<68> (see Chapter 5). Birefringence can also be deter-
mined at the fiber surface by measuring the two separate refractive indices
using immersion techniques.
7. n111 the refractive index for light (from the polarizer) vibrating parallel
to the length of the fiber, can be measured by comparing n11 with the
refractive index of a liquid standard. The resulting datum is determinative.
124 Chapter6

8. n.u the refractive index for light vibrating perpendicularly to the

length of the fiber, is measured in the same way as n11 but with the fiber
oriented at right angles to the position used for measuring nJ.. This datum
is determinative and can also be used to determine the quantitative bi-
refringence as in Property 6.

Brightness results when an anisotropic fiber on the rotatable stage is

oriented between crossed polars so that there is a vector of each vibration
direction parallel to the direction of vibrating light rays in the analyzer (see
Figure 6.14). The vector varies from nothing (darkness) at positions of
extinction to maximum brightness at a position of 45° between extinctions.
When there is no extinction, as in the case of a spiral structure such as that
of cotton, there is always brightness between crossed polars. In most kinds
of fibers, the brightness is strong enough for the microscopist to recognize
with confidence that the fiber is anisotropic. In a few cases, such as with
some cellulose acetates and some acrylics, the brightness may be so weak
as to leave doubt of anisotropy. In fact some cases of isotropy have been
reported in the literature for some trademarked products of these generic
types. When in doubt, bring the sensitive tint of a first-order red plate into
play. Also put a daylight blue filter into the path of yellowish artificial
light, so that the field will be bright red of the first order. If the fiber is at
all anisotropic in one diagonal set of quadrants, the fiber will be blue or at
least magenta. In the opposite set, the fiber will be yellow or at least orange
(see Figure 6.16). No further proof of anisotropy is needed.
Complete extinction (darkness) means that the whole fiber extinguishes
between crossed polars every 90° of rotation. Occasionally natural silk
fibers show some short portions that do not extinguish in the same posi-
tions of rotation as the major portions. Characteristically these positions
occur where the still-soft (recently spun) filament was crossed by another
part of the same filament as the silkworm was crisscrossing its double
filament to prepare its cocoon. Flax and other bast fibers are really bundles
of fibrils. Like a bundle of straws when bent, some of the fibrils will be
dislocated. Each dislocation results in a local relocation of extinction,
which shows up as a short, cross-hatched section. The number of disloca-
tions indicates the number of bendings. Thus old linen can be distin-
guished from new linen, and linen-rag paper can also be recognized.
Parallel extinction means that the axis of the fiber is parallel (or very
closely parallel) to one of the two cross lines in the eyepiece when the fiber
becomes dark during rotation of the circular stage between crossed polars.
It is assumed that the cross lines have been tested with a standard spec-
imen of parallel extinction so that one cross line in the ey~iece represents
the vibration direction of light from the polarizer (e.g., ~) and the other
Microscopical Properties of Fibers 125

cross line represents the direction in the analyzer (e.g., w~E) (see Figure
6.15).
Parallel extinction is shown by practically all man-made fibers, as
spun, because the structural units are arranged in the direction of flow
through the spinnerette. Treatments, such as crimping or texturizing, lo-
cally alter parallel extinction if the parallel arrangement is disturbed.
Retardation of one of two wave trains behind the other is the total effect
on the particular fiber by polarized light. In Figure 6.14, the polarizer is set
so that the vibration direction of emerging light waves is north-south,
applying terms of a magnetic compass to the cross lines of the eyepiece.
The fiber is either oriented NW\SE or SW /NE because (having parallel
extinction) it has maximum brightness in these two positions when the
analyzer is crossed (w~E) with the polarizer X· Referring to Figure 6.14
the birefringent fiber splits the polarized beam mto two polarized rays, one
vibrating lengthwise (II) to the fiber and the other crosswise {.l). They
travel through the fiber at different velocities without interfering with each
other because (as shown in Figure 6.14) they are in different planes. Yet
they must squeeze through the analyzer, which is set for vibration in a
single direction (w~E). Two rays, one from each wave train, each with the
same wavelength, may travel through a fiber of just the right thickness
(denier) to enter the analyzer in such a phase that they completely cancel
each other; that is, the color corresponding to that wavelength is lost in the
analyzer. But at some other wavelength, the rays will be in such a phase
that in going through the analyzer they reinforce each other. If, for ex-
ample, the velocity difference between the rays vibrating lengthwise and
those vibrating crosswise is such and the thickness of the fiber is such that
the slower ray is in phase but about 525 nm behind the faster ray, the color
of the light emerging from the analyzer will be red of the first order, as
shown in the Michel-Levy chart (see Figure 5.8). If retardation is about
1050 nm, the color is red of the second order; at about 1600 nm, third-order
red; at about 2100 nm, fourth order, and so on. Intermediate retardations
are manifested in other colors (at least through four orders) in Figure 5.8.
Second-order colors are the brightest in this (Newton's) series of inter-
ference colors. In the fourth order blues and yellows have faded out. In
higher orders than those shown in Figure 5.8, all colors but red have faded,
and even red has lightened to pink.
The order of retardation colors may be difficult to recognize. In such
cases a uniform quartz wedge of three or more regularly increasing orders
is useful. The wedge is placed in the slot of the body tube. If this slot is in
the NW\SE diagonal, start the test with the fiber oriented in the same
direction. If as the wedge is gradually inserted, colors in the fiber increase
in order, rotate the fiber 90° to the other direction. Now as colors decrease
126 Chapter 6

in order, count the orders to compensate (to darkness) the color band(s) in
question.
Drawn polyester fibers ordinarily exhibit orders higher than the three
or four orders of modestly priced quartz wedges. However higher orders
may not be needed in this particular test if the third or fourth red band can
be recognized so that the higher order reds (pinks) can be counted in the
thickest part of the fiber.
In addition to bands of different retardation colors due to differences
in fiber thickness, some are due to differences in birefringences, for ex-
ample in the skin-and-core structure of some fibers. These and other radial
heterogeneities may be detected in cross sections if they are not recognized
in the longitudinal views.
Retardation (polarization) colors and their order of extent (see Figure
5.8) are very determinative once recognized. Granted that thickness varies
widely among natural fibers, and purposely in different deniers of man-
made fibers, the variation is not too great to spoil the general value of
retardation colors and their orders. Low-order grays and colors are espe-
cially significant because thickness variations (denier) make so little dif-
ference in the order of retardation. Acrylics and cellulose acetates, from
fine deniers to carpeting sizes, always show first-order grays or colors,
whereas practically all drawn polyesters manifest high-polarization colors.
The difference in color order between undrawn and drawn nylon and
polypropylene is so great that drawing degrees can be recognized. Imma-
ture cotton fibers show yellow or blue on the red background of a first-
order red retardation plate. On the other hand mature cotton fibers are
distinguished by slightly higher order colors. Recognizing distinctive re-
tardation colors in a mixture of two or more kinds of fibers is not only
useful for forming quick qualitative opinions but also for estimating pro-
portions of those kinds of fibers.
The term sign of birefringence (or retardation) is preferred over the
term sign of elongation, since the sign of birefringence pertains to all
anisotropic fibers whether or not they have been elongated (stretched).
Most fibers are positive (+), i.e., they are length-slow: n11 > n.L" But some are
negative (-), i.e., length-fast: n11 < n1.· Those with negative sign at room
temperature(7J are acrylics, most cellulose triacetates, and at least some
sarans and vinyons. The fact that cellulose triacetate has a negative sign,
while cellulose diacetate has a positive one, indicates the ability of the
acetyl side group to slow down light. This suggests the best way of dif-
ferentiating between the two degrees of acetylation.
To determine the sign of fibers showing first-order gray, a first-order
red plate is inserted while the fiber is in a position of maximum brightness.
Microscopical Properties of Fibers 127

When the slot for the retardation plate is oriented NW\SE, the fiber is also
oriented NW\SE (see Figure 6.16); otherwise orientation is SW /NE for
both. If the sign of birefringence is positive (e.g., cellulose diacetate), the
fiber will be yellow or orange; if the sign is negative (e.g., cellulose tria-
cetate), the fiber will be blue or magenta. Gray on the Michel-Levy chart
(see Figure 5.8) amounts to a retardation of, say, 100 nm. Retardation of the
first-order red plate is about 500 nm, and its direction of slow vibration is
ordinarily oriented crosswise toward the long plate holder (see Figure
6.16). Thus 500 - 100 =400 (yellow); 500 + 100 =600 (blue).
In a fiber of positive birefringence, the slower ray vibrates parallel to
the fiber's axis. According to one convention, the vibration direction of the
slower ray is represented by an arrow shorter than the one representing the
vibration direction of the faster ray. As shown in Figure 6.16a, in the
diagram of a positive fiber, the shorter of the two perpendicular arrows is
drawn parallel to the fiber's axis and crossed with the (short) arrow of the
first-order red plate. The situation can be remembered if we think of the
two short arrows making a plus sign, signifying a positive sign of bi-
refringence for the fiber. All this is deduced from observing that the fiber
alone was gray between crossed polars and became yellow when the 1o red
plate was inserted. Since yellow represents a lower retardation than the 1o
red of the plate, there is a subtraction (reduction) in retardation of the 1o red
plate by the fiber when in the NW\SE position. Hence the vibration direc-
tion of the slower fiber rays must have been crossed with that of the
retardation plate; the slower rays must be lengthwise, and the fiber must be
positive.

(a) (b)

FIGURE 6.16. Determining the sign of birefringence of a fiber manifesting a first-order gray.
128 Chapter 6

In the other case (see Figure 6.16b), the fiber also happens to be gray
in position of brightness between crossed polars, but it turns blue in the
NW\SE position. Blue represents a higher retardation than the red of the
plate; that is, the two slow-ray directions of vibration are parallel, indicating
a negative sign of birefringence.
In some cases of skin-and-core construction,(7-9l such as in some acryl-
ics, the core has a different sign (+) than the skin (-).
With any retardation plate (or compensator),(10l be sure of the vibra-
tion direction of the slower ray. If there is only one arrow engraved on the
plate mount, it represents the vibration direction of the slower component.
But some manufacturers may choose to have that direction oriented
lengthwise to the plate's metal mounting; or if the markings are t x' -z', the
direction z' is that of the slower ray. Since the retardation disk itself may
become turned in its mount, check with a known material, such as cellulose
diacetate (+) or any acrylic (-) except Orlan® (which may be too weakly
negative to use as a strong standard). In cleaning any optical disk mounted
in a cylindrical well, be careful not to use sufficient circular pressure to
turn the disk. If this should happen, you or a repairman will have to loosen
the pressure cap and turn the disk back. Always make sure the pressure
cap is tight.
The first-order red retardation (sensitive tint) plate is good for deter-
mining the sign of birefringence of any material that shows a retardation
(polarization) gray or color (yellow, orange, red) of the first order. The
same plate will also indicate certain colors of the second order, such as blue
or green, by providing a dark or light gray during subtraction (crossing the
slow directions) and a blue or green of third order during addition (paral-
lel slow directions). But for yellow, orange, or red of the second order,
subtracting or adding first-order red will merely give the corresponding
color of the first or third order. These may be somewhat difficult to tell
apart.
The quarter-wave (0.251..) plate (light ray) is also good for determining
the sign of birefringence of a material showing a first-order gray. Sub-
tracting the two retardations will give a darker gray, and adding will give
a brighter gray (or a yellow). With a low-order color, the 0.251.. plate gives
a different color for subtraction than for addition.
For fibers showing retardation colors (see Figure 6.14), the quartz
wedge can be used as a retardation plate by moving it between crossed
polars over the fibers in a position of brightness. Subtracting results in a
succession of colors downward in order; adding results in an upward succes-
sion of colors. With knowledge of the direction of the wedge's slower
component, the sign of the fiber's birefringence is determined as indicated
by the arrows (not the colors) in Figure 6.16.
Microscopical Properties of Fibers 129

Dichroism is the preferential absorption of all* or part of the spectrum

in one preferred direction relative to the other preferred direction. The
phenomenon is especially visible with deeply dyed fibers. Since dye in the
fibers obscures the complementary polarization color, dichroism can sub-
stitute its own evidence of anisotropy. Absorption is usually greater in the
direction in which the slower ray vibrates. In positive fibers this direction
is parallel to the axis of the fiber; in negative fibers it is perpendicular.
Therefore dichroism can indicate the sign of retardation (birefringence).
The indication can of course be confirmed by using retardation plates with
the fiber in a diagonal position (instead of parallel) to the cross lines of the
eyepiece.
The arithmetic degree of birefringence is the difference between the two
principal refractive indices, e.g., n11 - n}11 l Indeed, both the birefringence
and its sign can be determined by measuring n11 and n.L and subtracting
them. However, the measurements may be long and tedious. Moreover,
they are determined only at the surface, which may be different in re-
fractive index from the interior, as in the case of skin-and-core construc-
tion,<3l Such radial heterogeneities are actually recognized by variations in
birefringence; therefore the degree of birefringence is a separate and in-
dependent optical property and a very important one.
Birefringence alone allows a broad classification of fibrous types into
those of weak birefringence, 0.001-0.01 (e.g., natural and regenerated pro-
teins, acrylics, modacrylics, acetates, saran, vinyon); those of moderate to
strong birefringence, 0.01-0.1 (e.g., natural and regenerated cellulose, silk,
nylons, polyolefins, vinyl); and those of intense birefringence, above 0.1, as
in polyesters.
Birefringence can also be defined as the ratio between retardation and
thickness of the specimen at a given point. Indeed, the quickest and sim-
plest way estimating birefringence is from the retardation color and the
thickness of the fiber, employing the Michel-Levy scale of birefringence
(see Figure 5.8). If the fiber is practically a cylinder, as in the case of some
nylons, polyolefins, polyesters, rayons, acrylics, and modacrylics, the
thickness is the same as the width of the longitudinal view and can there-
fore be measured directly with a calibrated ocular scale, as explained in
Chapter 5. Fibers of weak birefringence, such as acrylics and modacrylics,
usually display first-order gray between crossed polars. Note particularly
the shade of gray along the axis of the fiber and match this gray with the
likeness on the Michel-Levy scale. Go up the ordinate until the fiber

*It is interesting to note that dichroism is the fundamental mechanism in polarizing films,
such as Polaroid®. In the colorless variety practically all of the visible spectrum is absorbed
in one preferred direction but transmitted in the other preferred direction.
 130 Chapter 6

thickness is reached, then follow the diagonal line to the birefringence and
read the result.
Fibers with moderate to intense birefringence and the usual parallel
extinction display color bands parallel to the axis. The number of bands
(degree of retardation) depends on the fiber's birefringence and path
length. With a circular cross section, as shown in Figure 6.17, there is a
single central band of colors, and in this particular case, it is flanked by two
pairs of color bands of descending order. The colored line congruent with
the fiber axis represents the highest retardation of all, and it is the value,
together with the diameter of the fiber, used with cylindrical fibers in
translating into birefringence by means of the Michel-Levy scale (see
Figure 5.8).
If the cross section is not circular, or even roughly so, it should be
studied to anticipate where color band(s) of highest order will appear in
the longitudinal view of the fiber. For example in Figure 6.18 the cross
section is dumbbell shaped. Such a fiber will most probably lie on its side,
which is then seen as the width (W) in longitudinal view and measured on
the particular fiber in view. The thickness of the particular fiber is esti-
mated from the ratio of the thickest (1) part or the center (c) to the width
(W) of a typical cross section of the sample. Then the retardation colors of
either the central band or the twin bands are compared with the respective
thicknesses to estimate the birefringence from the Michel-Levy chart (see
Figure 5.8).
Likewise cross sections of fibers of some other consistent shapes, such
as dogbone, bean, cogwheel, and triangle (trilobal), can be used to estimate
what proportion of the width (measured in longitudinal view) is the thick-
ness with respect to a particular retardation color when determining bi-
refringence from the chart. Fibers with tubes are often regular in cross
section so thickness in longitudinal view can be estimated from the mea-
sured width. Even irregular cross sections, such as those commonly found

FIGURE 6.17. Diagram of a cylindrical fiber, showing a portion of the longitudinal view
between crossed polars. Hypothetically the birefringence and thickness (width w) exhibit
three orders of interference colors (1", 2•, and 3°).
Microscopical Properties of Fibers 131

l
w
twin bond of

centrol bond of
color

color

l twin bond of

longitudina l view
color

cross
section

FIGURE 6.18. Diagram of a hypothetical fiber with a cross section like a dumbbell and
hypothetically with one set of twin color bands. Considering the shape of the cross section,
the twin color bands must be of higher order than the single central band.

in cotton, some viscose and acetate fibers, and some dual-component

fibers, may give enough information about the ratio of thickness to width.
Fiber thickness can also be measured directly if it can be turned or
twisted to measure it on its edge. Or possibly a microscope is available
with a graduated, calibrated fine-adjustment focusing wheel for measur-
ing vertical distance from bottom to top, as explained in Chapter 5. Be sure
to multiply the apparent distance by the average refractive index of the
fiber. A very rough estimate of the average thickness may be made from
the following relation

denier =m:P I 4 x 9000 x density

Thickness, d, is in millimeters in the preceding formula and in the Michel-
Levy chart (see Figure 5.8). A still rougher estimate can be made by
remembering that most fabric-making fibers (except those for carpeting)
are between 0.01-0.03 mm in average diameter.
The Michel-Levy scale is inadequate for polyesters that manifest re-
tardation colors of a higher order than six. For such fibers a high-order
compensator is required(l 0>. This brings up the general subject of measur-
ing birefringence by means of a variable retardation device, the compen-
sator.111> A quartz wedge can be used directly if it is of a sufficient number
of orders and graduated. The Babinet compensator is more elaborate; it
consists of two opposing quartz wedges. One of them is moved over the
other by means of a graduated micrometer screw.l12> The Berek type of
compensator employs a different principle: It measures the degree of tilt in
an anisotropic plate to the point of compensation.<11 > Berek compensators
are available in various ranges. For highly drawn polyester fibers a range
of 10 orders is needed, and for experimental fibers, 15 or more orders may
be required. 18>
132 Chapter 6

Another important point is that exact compensation in white light can

be achieved only if the dispersion of birefringence of the fiber and that of
the compensator are the same.<8>For path differences under four orders, no
difficulty is found, but with higher orders the band of compensation is no
longer black but rather unsymmetrically colored and broad. One solution
is to establish the color of the compensation band. Another solution is to
cut a wedge out of the end of a fiber and examine it in a subtractive
position between crossed polars with the Berek compensator in place. A
count of the resulting fringes plus any additional fraction gives the retarda-
tion.<8> Thickness in millimeters still has to be determined or estimated as
previously described.
Determining birefringence has many applications in production and
quality control as well as in research. More and more birefringence de-
termination is being used to control the degree of elongation for optimum
strength and other desirable properties.
While birefringence alone may be a key to understanding the relation-
ship between elongation and strength, it can also be important to know the
actual individual values of the two principal refractive indices of a fiber.
The index for light vibrating parallel to the fiber's axis, n11, varies from a low
of about 1.47 in cellulose acetate to as high as 1.73 in some polyesters. The
span for n.L is not so great, but it is large enough to give a good spread
among types; it varies from about 1.47 in acetates to about 1.54 in polyes-
ters and about 1.61 in saran.<13>
The refractive index is generally determined by trial and error, and is
usually tedious. But experience, technique, cleanliness, and care can make
the results very satisfying. To separate n11 from n.L, polarized light is sup-
plied by the polarizer only. The other polar is removed from action. Figure 6.19
illustrates fiber orientations for determining n11 and n.11 respectively, with
the polarizer having its direction of vibration !, as represented by the
respective cross line of the eyepiece. In Figure 6.19a the fiber is aligned
parallel to the ~ cross line, so that n11 is being measured. In Figure 6.19b

WEffijE WWE FIGURE 6.19. The alignment of a fiber

to determine (a) n.L and (b) n.L for a fiber
with parallel extinction and positive bi-
s s refringence. The vibration direction of
(a) (b)
the polarized light is ~.
5
Microscopical Properties of Fibers 133

the fiber is aligned parallel to the w-E cross line, so that n1. is being mea-
sured. Incidentally the shorter arrow is shown lengthwise; that is, n11 is in
this instance greater than n1., and the fiber has positive birefringence.
There are two ordinary methods (among special procedures(14)) for
determining the refractive indices of a fiber. In both, the fiber is immersed
successively in liquids of known refractive index, and a search is made for
a match between liquid and fiber for n11 and n1.· One method for determin-
ing whether the fiber index is higher or lower than that of the liquid uses
oblique illumination, and the criterion is the position of the resultant
shadow cast by the specimen. (6) The other method uses the Becke refraction
line and its critical movement during focusing. The Becke method is pre-
ferred when the refractive index of the liquid is close to the fiber's. In either
case, specifications for illumination are very important, and a series of
liquids of known refractive indices is required. (15•16)
The Becke test requires axial transmitted illumination. If the con-
denser is in place, remove the top lens and nearly close the iris diaphragm,
leaving the polarizer in place. Focus up and down and notice the move-
ment of the Becke halo around the fiber. As you focus upward, the halo
moves to the medium of higher refractive index (see Figure 6.20). When
you are far from a match between fiber and liquid, the over- or under
match is very evident because the halo is in bold contrast. As you approach
a match, the halo becomes fainter. Darken the room if it helps; concentrate,
be patient. The Becke method is very satisfactory to the experienced ob-
server, and, of course, experience always helps. From the first six optical
properties and other information, you should have a good idea of what
your specimen is and what refractive indices are expected. For your first
liquid choose one with a refractive index in the middle of the probabilities.
Learn to estimate how much you are over or under the correct index; then
take a bold step, trying to bracket the index as soon as possible. Your
previous data, especially regarding the sign of birefringence (which is
greater, n11 or nJ.?) and the amount of birefringence (difference between n11
and nJ.), will help considerably in your choice of trial liquids. Use short
lengths of each of a few fibers to avoid entanglements and bending. Be
careful not to contaminate specimens with any other kind of fibers. Use

(OUTSIE)

FIGURE 6.20. The Becke line. Simulated fiber is in a liquid of higher index; (left) on focusing
upward slightly; (right) on focusing downward slightly.
134 Chapter 6

small cover glasses to save standard liquids and be able to fill the cover
glass and slide. When no Becke halo is observable and nothing seems to
move as you focus up and down, the fiber and liquid have the same
refractive index. Usually, however, the refractive index is an intermediate
between the values of those of two successive liquids in the series. Repeat
with the fiber in the other orientation, and you will determine both n.~. and

The oblique illumination test is advantageous when the specific re-

fractive index of the fiber is quite different from the test liquid's. This
situation can arise with an unknown fiber. The question is whether the
fiber has a much higher or lower index than the liquid. Sometimes it is
difficult to tell whether the bold, dark Becke line is moving in or out during
focusing, whereas it is easy to tell whether a dark shadow is on one side
or the other of the bright fiber illuminated obliquely across it. To accomplish
this, the full condenser is adjusted in focus with its diaphragm wide open.
1
The fiber is oriented or w-E, depending on which of the two indices
is to be determined. Tfien a long piece of black cardboard is placed to one
1
side underneath the condenser with its long edge or w-E, depend-
ing again on which index is being determined. This gives oblique illumina-
tion with half a cone of light. If the fiber is of higher refractive index than
the standard liquid, its dark shadow lies on the same side as the dark side
of the field (see Figure 6.21). If the fiber has a lower index than the liquid,

I
I
a ['

I I

'

FIGURE 6.21. Oblique trans-

mitted illumination. Glass tube
and rod in a liquid of lower re-
fractive index, which has partially
filled the tube.
Microscopical Properties of Fibers 135

the shadow lies on the side of the fiber that is opposite the dark side of the
field.
Sometimes it is important in research to determine n11 and n.L for a
series of samples to compare variations in index resulting from some
variation in a physical condition, such as percent elongation. Here the
birefringence may not vary much, but the precise values of the refractive
index may change in some characteristic manner. For example, some acryl-
ic fibers show an increase in both n11 and n.L when stretched, and even
though the difference between the two indices (the degree of birefrin-
gence) does not noticeably change, the magnitudes of the indices indicate
at once that the fibers have been stretched.
The microscopy of fibers is not yet a highly advanced science, but in
the relatively short time it has been applied to the technology of textile
fibers it has proven to be of great analytical value.<17> It allows the micro-
scopist to distinguish easily between natural and man-made fibers.<18l By
comparing results with published values for common fiber classes, some
very useful relationships become apparent.(19-21l Properties are found to
stem from (1) the chemical (molecular) composition of the fiber, as ex-
pected and (2) changes in orientation and spacing of constituent molecules
or other structural units during spinning, stretching, or other treatment.
This book is not a treatise on fiber chemistry or technology, but some
generalizations about structure and composition versus properties will
help the microscopist understand how anisotropy comes about, and what
governs it.

6.3. ANISOTROPY IN FIBERS

Fibers are usually birefringent due to molecular anisotropy, which

originates with electric polarity between adjacent atoms. Figure 6.22 illus-
trates the three simplest examples:
Figure 6.22a shows a pair of adjacent atoms aligned with the direction
of vibration of the polarized light.
Figure 6.22b shows a pair of adjacent atoms aligned at right angles to
the vibration direction.
Figure 6.22c shows two atoms relatively far apart.<16l
In all three cases the polarized light waves tend to orient electrons and
nuclei of the constituent atoms in the direction of the vibration of the
polarized light (e.g., ~), producing electric dipoles. The three kinds of
dipole alignments are of different strengths. Each of the adjacent dipoles
 136 Chapter 6

8
1-

G)G)
z
":J
a
! a.

(a) (b) (c)

s
FIGURE 6.22. Effect of polarized light waves on atoms and atom pairs.<16l

aligned parallel to the vibration direction is rendered stronger, and each of

the adjacent dipoles aligned perpendicularly is rendered weaker than each
of the separated dipoles shown in Figure 6.22c. In long molecules, such as
those of cellulose, nylons, polyolefins, and polyesters, atoms are largely
chained along the length of the molecule, so that dipoles are strongest
when light is vibrating in the lengthwise direction of the molecule.
Molecular birefringence is imparted characteristically to fibers, as sug-
gested in Figure 6.23. The degree with which long molecules grow or come

FIBER AXIS

I
11111111111111111111111111111111

111111111111111111 II 111111111111

11111111111111111111111111111111

II II
11111111111 111111 1111111 II II
111111111111111111111111111 II Ill
I FIGURE 6.23. Idealized alignment of long-chain mol-
ecules parallel to the axis of a fiber. (l6)
Microscopical Properties of Fibers 137

parallel to the axis depends on their formation and subsequent treat-

ment.<17l
With straight-chain molecules substantially like those indicated in
Figure 6.23, the refractive index (which is a positive function of the
strength of atomic dipoles) is greater for light vibrating parallel to the fiber
axis than for light vibrating perpendicular to it; that is, n.L > n.il and the fiber
is optically positive. For example polyesters are intensely positive; cel-
lulose, nylons, and polyolefins are strongly to moderately positive; but
acrylics and cellulose diacetate are only weakly positive<2> because of the
opposing polarity of the side groups on each molecule. In fact the third
acetyl group of cellulose triacetate has sufficient counterpolarity to turn the
positive diacetate into the negative triacetate at room temperatures. As
cellulose triacetate fibers are heated, the temperature for zero birefringence
(Tzn) is reached, above which fibers become optically positive. This phe-
nomenon is explained on the basis of the increasing diameter of the ra-
dially polarizing acetyl group as temperature increases. Thus, the dipole
movement of the acetyl group decreases to the point where axial polar-
izability takes over, and the fiber becomes optically positive.<17l
Polymeric fibers are generally assumed to be uniaxial. If so only the
lengthwise fiber axis is unique; that is, viewed endwise, i.e., in the cross
section, such fibers are isotropic. We have just discussed the two charac-
teristic refractive indices n11 and nv however certain processes (texturizing)
involving transverse pressures and heat can lead to a departure from such
symmetry and give rise to what may be called a biaxial fiber. Such a fiber,
traversely anisotropic, is characterized by three preferred refractive in-
dices-n.u nw and n.u. Transverse anisotropy has been observed in tex-
tured yarns where filaments have been given crimps, loops, coils, or crin-
kles (to give the yarns properties more like those of wool, for example).
Among the texturizing processes, the false-twist process is probably the
fastest growing. Its fundamental operations are twisting, heat setting, and
untwisting. The result, so delightful to the textile technologist, may appear
to the microscopist as a mixed-up mess (see Figure 6.24).<20> The problem,
would be relatively simple if we were dealing with one filament at a time
(see Figures 6.25 and 6.26); instead filaments are purposely linked. During
texturizing the shape of the cross section has been transformed from a
circle (see Figure 6.27) to a polygon (see Figure 6.27b).<21,22l

6.4. MOLECULAR ORIENTATION AND ORGANIZATION

Anisotropy due to the orientation and organization of molecules into

fibers occurs in many steps of growth or manufacture. Here we confine
138 Chapter 6

FIGURE 6.24. A typical, false-twist, textured yarn.<21 l

ourselves to man-made fibers, since the steps are designed to vary mech-
anical properties and much can be learned about the process from the
various optical properties.
1. Spinning. The organization, orientation, and distribution of mole-
cules begin with spinning, either spinning from the melt or spinning from
an aqueous (wet) solution or an organic (dry) solvent. Spinnerettes are

FIGURE 6.25. Textured nylon filament in white light, with crossed polars and universal
stage axis A4 at 45•.(21)
Microscopical Properties of Fibers 139

FIGURE 6.26. Same as Figure 6.25, but the universal stage axis A4 is at 315°.<21 >

designed to vary the external shape of the fiber, its internal solidarity or
hollowness (number of canals) and by dual spinning, the texture of the
fiber. The kind of liquid and design of the spinnerette can affect the
orientation of molecules radially (skin-and-core structure) as well as ax-
ially (flow birefringence).<9•17l
2. The method and rate of coagulation or jelling can affect the optical
and mechanical properties of the spun product.<23>The process can be
simulated to some extent under the microscope.(24> Postmortems can be
performed on commercial lots of dry-spun versus wet-spun fibers.< 9>Dry-
spun fibers can be coagulated quickly, and wet-spun fibers can be co-
agulated slowly; fast spinning can favor skin formation over slow spin-
ning.
Scott showed with experimental polyethylene terephthalate ribbons
that drawing conditions and treatment can produce a variety of structures

T
b

F E

l
FIGURE 6.27. (a) Diagram of a circular cross-section of an untextured filament.< 21 > (b) Dia-
gram of a polygonal cross-section of a textured filament.<21 >
140 Chapter 6

and corresponding properties.<17l The variation originates with the type of

neck produced in the beginning of the stretch-reduction process (necking
down). Figure 6.28 gives the shapes of three types of necks. Figure 6.28a
shows the usual shape with most plastics; it has sloping shoulders and a
concave throat. A core stretches before its skin. Polyester, as spun, draws
this way17 but if the spun polyester is first aged and then drawn, the skin
draws before the core, producing the second type (see Figure 6.28b), the
lustrous kind. Figure 6.28c shows the third type, produced by drawing the
aged as-spun polyester over a knife edge, so that the side away from the
edge draws first, producing a clear product of elongation.
Figure 6.29 shows slip planes and lines, the slip front, and filmy luster
voids; it also shows the spindle voids typical of polyester aged before
drawing, whether or not it also has filmy, luster-producing voids. Both
types of voids probably contribute to form-birefringence. The degree of
contribution can be checked by measuring birefringence immediately after
mounting in a penetrating liquid and again after penetration by the liquid,
or as Scott did,<17l by measuring birefringence in thick and thin sections.
Aging allows sufficient time (4 days) for crystallization to take place to
the extent of about 40%. At the end of 4 days, Scott observed spherulitic
nuclei. These nuclei tie molecules together, stiffening the as-spun structure
to the extent that slip planes form during deformation, as in metals.

(a)

CLEAR DRAWN

(b)

LUSTER DRAWN

KNIFE EDGE FIGURE 6.28. Scott's three types of reduction in an

AGED THEN CLEAR DRAWN experimental polyethylene terephthalate ribbon.<17)
Microscopical Properties of Fibers 141

SLIP PLANES

DRAW FRONT

FIGURE 6.29. Another view of Fig- "SPINDLE" VOIDS

ure 6.26b (Scott's diagram), polyeth-
ylene ribbon, showing slip planes,
draw front, and voids.<17J

Thus we see that birefringence in fibers is inherent in molecular struc-

ture, developed by crystallization, produced by the form orientation of two
or more phases, or developed by stress.

6.5. TRANSPARENT SHEETS, FOILS, AND FILMS

Strain as a result of stress in inorganic or organic glass is visible

between crossed polars. Indeed organic sheets, such as those of poly(me-
thyl methacrylate), are used in models of structures such as bridges and
buildings when studying the location and extent of strain under stress due
to the photoelastic effect between crossed polars. Some plastic sheets, such
as those used as windows, are prestressed uniaxially or biaxially. Trans-
parent foils and ribbons are often used to study dynamically the effects of
tension or compression.
Planar molecules are especially interesting if the molecular planes lie
in the plane of sheets or foils, as shown in Figure 6.30, for then a uniaxial
(or biaxial) interference figure is visible.<16>

6.6. FIBER IDENTIFICATION

A light-microscopical examination of a longitudinal section mounted

in mineral oil of refractive index 1.48 usually identifies wool easily (see
Figure 6.1). Should this approach fail, a cross section can be made with a
Hardy or another microtome to examine the fiber interior.<1•2> Natural fibers
can generally be identified by differentiating among their morphologies.
142 Chapter 6

OPTIC AXIS

--~---~------- /

- -
,.,'' \ 1-nmm .......... '
,"' \ J ....,,
I' -----:'\- , - - - -- '
1/ .,...-.;,;;-~ ~ \ I ._, • •;,.,.· ........... ' \

""' ',
o'r-----==--=-..--
\
\,,' ',.......-_...._. ____ / "-"ma•-
~ - _,~,,
- -J!____________ .,,', ~

',,,
-
... .............. ______ ------"" ,,
~' FIGURE 6.30. Planar molecules in
sheets or foils. (16)

Man-made fibers, however, vary in morphology within limits only of the

shape of the spinnerette' s orifice, almost regardless of the constituent
polymer's chemical composition. However man-made fibers differ widely
in their optical properties, particularly in the refractive index parallel to the
fiber's axis (E or n11) and perpendicular to the fiber's axis (oo or n.L), thus the
difference between the two (birefringence: E- oo or n11 - n.L). Some typical
values are shown in Table 6.U1l
While classifying fibers according to refractive indices appears
straightforward (as in Table 6.1), the quantitative determination can be
tedious and unnecessary because fibers have a total of eight determinating
optical properties, which are quite readily obtainable with a microscope
made for polarized light. The value of the birefringence is often sufficient
to identify most fibers.

TABLE 6.1.
Typical Values of Physical Properties for Identifying Fibers•
Refractive Indexc

Parallel to Perpendicular to Birefringencec,d

Fiber Mpb CC) Fiber Axis (E) Fiber Axis (ro) (E - ro)

Acetate 260 1.479 1.477 0.002

Acrylic dnmb 1.520 1.524 -0.004e
Anidex s 190 f f f
Aramid
Nomex® 371 1.790 1.662 0.128
Kevlar® 425 2.322 1.637 0.685
Asbestos 1.5-1.57 1.49 0.01-{).08
Cellulosic
Flax dnm 1.596 1.528 0.068
Cotton dnm 1.580 1.533 0.047
Microscopical Properties of Fibers 143

Refractive Indexc

Parallel to Perpendicular to Birefringencec,d

Fiber Mpb (OC) Fiber Axis (E) Fiber Axis (w) (E- w)

Fluorocarbon 288 1.37

Glass (inorganic) s 570 1.547 1.547 0.000
Modacrylic dnm 1.536 1.531 0.005
Novoloid dnm 1.650 1.648 0.002
Nylon
Nylon-6 219 1.568 1.515 0.053
Nylon-6,6 254 1.582 1.519 0.063
Qiana® 275 1.554 1.510 0.044
Nylon-4 265 1.550
Nylon-11 185 1.55 1.51 0.04
Nytril 176 1.484 1.476 0.008
Olefin
Polyethylene 135 1.556 1.512 0.044
Polypropylene 170 1.530 1.496 0.034
Polycarbonate 294 1.626 1.566 0.060
Polyester
2GTg 256 1.710 1.535 0.175h
4GTi 227 1.690 1.524 0.166
CHDM-Tj 283 1.632 1.534 0.098
Oxybenzoatek 225 1.662 1.568 0.094
Rayon
Cuprammonium dnm 1.548 1.527 0.021
Viscose dnm 1.547 1.521 0.026
Saran 170 1.603 1.611 -0.008
Silk dnm 1.591 1.538 0.053
Spandex 230 1.5
Triacetate 288 1.472 1.471 0.001
Vinal dnm 1.543 1.513 0.030
Vinyon (PVC) dnm 1.541 1.536 o.oo5•
Wool dnm 1.556 1.547 0.009
•courtesy of the American Society for Testing and Materials.
bdnm indicates the fiber does not melt; s indicates softening point.
cThe listed values are for specific fibers that warrant the highly precise values given. For identification
lurposes, these values should be regarded as indicating only the relative values of the properties.
Approximate values.
•varies; always weak; sometimes negative.
frhe fiber is opaque.
gethylene glycol type.
hstaple and fully oriented filament yarns (FOY), partially oriented yarns (POY), and undrawn yarns may
. have much lower values of birefringence and refractive index.
11,4-butanediol type.

jl,4-cyclohexanedimethanol type.
kp-ethylene oxybenzoate type.
144 Chapter6

6.7. SUMMARY

Microscopy is necessary for studying the morphology and structure of

fibers. Microscopic sizes of fibers and their parts are resolved in terms of
the metric system unit, the micrometer (J.tm), formerly called micron (J.lffi),
for one-millionth of a meter. The angstrom (A) (0.1 nm) remains in the
literature, although it is not an official unit. The theoretical and practical
limits of fiber morphology were first determined by means of light micro-
scopy. Transmission electron microscopy added its advantages of resolv-
ing power and resolution about the same time that various properties of
man-made fibers were being modified by varying manufacturing condi-
tions. Scanning electron microscopy added its own advantages, especially
the contrast and realistic, three-dimensional aspects; accordingly some 13
scanning electron micrographs introduce this chapter to illustrate varia-
tions in morphology. Such variations serve especially well in natural fibers
to describe and therefore identify them. Among man-made fibers, however,
variations in optical properties are more important for identification, since
they vary more than morphology. Such fibers are made of the melt or
solution of high-polymer extruded through a spinnerette of a certain size
and shape. During and/ or after spinning, filaments are subjected to a
certain kind and degree of stress. Such treatment results in a distinct
structure, composed of macromolecular domains, spherulites, and/ or
stress patterns. These are specifically manifested in reaction to polarized
light; indeed about eight different optical properties are determinable by
means of a polarizer, analyzer, Michel-Levy interference color chart, and
rotatable stage. Furthermore polarized light microscopy is both descriptive
and determinative with polymeric sheets, foils, moldments, castings, and
any other sufficiently transparent anisotropic material. Thus polarized
light microscopy is unique among all other kinds of microscopy.
Natural fibers.are named in terms of their origin: animal, vegetable, or
mineral, such as wool, cotton, or asbestos. Man-made fibers are named for
regenerated material or its treatment. Examples of a trade name (two
words) are nylon and acrylic (not capitalized). Orion• and Acrilan• are
trademarks (one word) that are registered in the U.S. Patent Office for
exclusive use by the sole owners.
 7

Microscopical Properties
of Crystals

7.1. STRUCTURAL CLASSIFICATIONS

A solid crystal is composed of atoms, ions, or molecules arranged in

a pattern that is periodic in three dimensions. !Il There are six structural!2>
classifications: isometric, tetragonal, hexagonal,!1> orthorhombic, monoclinic,
and triclinic (see Figure 7.1a).!2>
Bravais lattices for the 14 possible unit cells are shown in Figure
7.1b.(2al In a crystal unit cells are the basic space-filling units. They can be
repeated in the three dimensions and in this way generate the total crystal.
The uniqueness of the basic six crystal systems depends on their axial
dimensions and internal structural angles. Geometries of the crystal sys-
tems are given in Table 7.1.<2•>
The rhombohedral class (of which the calcite phase of CaC03 is a typ-
ical example) can be considered in the hemihedral class of the hexagonal
system, since it is made up of alternate faces of a hexagonal bipyramid.!2al
Structure determines the inherent properties of a crystalline compound; for
example the three crystalline phases of CaC031 calcite, aragonite, and vater-
ite, all have different crystalline structures; different solubilities; and differ-
ent melting points, hardnesses, and optical properties.

7.2. MORPHOLOGY

Crystals are also characterized by their morphology,!1l that is, their sizes
and shapes, as grown from solution, melt, vapor, or by altering other
crystals. The morphology of a crystal determines its rate of change, that is,
its rate of dissolution, melting, or other phase transformation, because

145
 146 Chapter 7

a Lamellar Tabular Equant Columnar Acicular

~ ~i
/ ' • fJtJJ/
; 1;..,
- ·'-·;:;"- ·
"'"l tiJIJI oto
)-· o·i···· ··-...
.• ot:Jr ,..

~ ~J
rn ~ n :::l
b:::l
a:
u Iii
~ ~
_g Iii
>-
0 a:
.s::.
t: (.)
0

CRYSTAL MORPHOLOGY (habitl

FIGURE 7.1. (a) Crystal structure and morphologyP> The trigonal (rhombohedral) system
is not represented here, but see Figure la. (b) The 14 Bravais lattices, representing the
fundamental, three-dimensional, space-filling pattems.<2aJ
Microscopical Properties of Crystals 147

Simple Body-centered End-centered Face-centered

§§§@@
orthorhombic orthorhombic orthorhombic orthorhombic

Simple Body-centered Face-centered s·1D1pIe B d te d

0 y-cen re
cubic cubic cubic
tetragonal tetragonal

FIGURE 7.1. (Continued J

morphology controls its specific surface. A crystal grown slowly from

solution, manifests characteristic faces related to the structure. Thus one
kind of face, called a form,(!) has constant angles, whether the crystal is
equally developed (equant), long (columnar or acicular), or flat (tabular or
lamellar).<2> Therefore each of the six or seven kinds of structure has five
kinds of naturally polygonal morphology, called habit, as illustrated in
Figure 7. 1.<2>
Crystals grown from solution<3> or by precipitation<4,5> on a micro-
scopical slide are seldom equant because the drop of mother liquor is

TABLE 7.1.
Axes and Axial Angles of Crystal Systems.:za
System Axes Axial angles

Cubic a1 = a2 = a3 All angles = 90°

Tetragonal a1 = a2 " c All angles = 90°
Orthorhombic a"b"c All angles = 90°
Monoclinic a .. b,.c Two angles = 90°; 1 angle ,. 90°
Triclinic a"b"c All angles are different; none equal 90°
Hexagonal a1 = a2 ( =a3) ,. c Angles = 90° and 120° (or 60°)
Rhombohedral a1 = a2 = a3 All angles are equal, but not 90°
148 Chapter7

shallow and difficult to stir while under the objective. Common salt (NaCl)
for example recrystallizes from a drop of solution on a slide not as equant
cubes but as long, flat rectangular shapes (see Figure 7.1a), sometimes
tipped to make interpretation all the more challenging. The terms for habit
vary in the literature,<~> but the structure remains constant. For this reason
face angles and optical properties are also constant, no matter what the
habit.

7.3. MILLER INDICES

Figure 7.1a shows the Miller indices for typical faces of equant habits.
These indices are reciprocals of the fractional intercepts that a face makes
on crystallographic axes. The axes are graphic (typical spacing directions
of the structural units, whether atoms, ions, or molecules). The form rep-
resenting the isometric system in Figure 7.1a is the cube, a special hexa-
hedron whose face angles are all right angles. It is a closed form; that is,
all of the faces are structurally alike (regardless of the dimensions of the
edges in the long flat habits). As the axes are set up in the equant habit in
Figure 7.1a, the front face cuts one axis by one unit (1) and the other two
axes at infinity (oc), so the Miller indices are 1/1:1/ oc:l/ oc =100. The back
face is the same, but to show that it is cut in back by one unit, its specific
Miller index is {iOO}. The right side is written {010} and the left {OiO}, the
top {001}, and the bottom {001}. The typical index for the cube is simply
written {100}. If we set up the axes diagonally, then the typical index would
be {110}, but in this instance nothing is to be gained by doing so. The Miller
indicesof the lamellar, tabular, columnar, and acicular habits are exactly
the same as those of the equant habit (cube). It does not matter that the
edges are of various lengths except that there are more unit cells (all
isometric) attached along the longer edges. How do we prove isometry
microscopically? If the crystals are isotropic (black) in every orientation as
observed between crossed polars, they are isometric.
It is easy to see why a cubic crystal is isotropic. Since unit spacing is
the same along the three axes, the velocity of light (and refractive index)
for a particular wavelength is the same in any direction at any one tem-
perature. For sodium chloride, NaCl, the refractive index, n0 'JJJ'c, is 1.544;
for potassium chloride, KCl, n0 'JJJ'c, is 1.490. While NaCl and KCl are both
isometric and isotropic, they are not isomorphous<1>; that is, they do not
crystallize together as mixed crystals in a solid solution. Sodium and po-
tassium ions are not interchangeable in the crystal structure (see Figure
7.2), but ammonium ions are interchangeable with potassium ions in crys-
tals of their chlorides to make a continuous isomorphous<1> series with
Microscopical Properties of Crystals 149

'
I

Ol •
I
: 0
FIGURE 7.2. Isometric ionic lattice. e represents pos- !&------·--
itive ion, such as K+ or NH4 +, interchangeably. 0 rep- /
0
resents negative ion, such as CI·, Br-, or I- (inter-
changeably).

refractive indices varying from n0 20·c = 1.49 for pure KCl to 1.64 for pure
NH4Cl, in direct proportion to the potassium and ammonium content.<6>
Hence the microscopical refractive index of a mixed crystal coupled with
the isotropy is very descriptive and determinative once the presence of
NH/ and K+ is established.<4>Rubidium and cesium ions are also inter-
changeable with K+ and NH4 + and thus could influence the optical prop-
erties, but their absence or presence can be detected by other tests.<4>
If NaCl in solution contains a small amount of OH- ion, such as from
urea, octahedral faces appear on its crystals with or without cubic faces.<7l
Miller indices of the octahedral faces of NaCl are all {111}; that is, all three
axes are intercepted equally. The structure of the NaCl remains the same:
isometric; therefore octahedral NaCl remains isotropic, with the same
refractive index, n0 2o·c = 1.544, as cubic NaCI.

7.4. ISOMORPHISM

The alums are a remarkable set of isometric-isotropic crystals with

interchangeable ions, all patterned after their namesake, potassium alu-
minum sulfate dodecahydrate, K2S04 • Al2(S04) 3 • 24Hp. The water of hydra-
tion of an alum, too, is in the space lattice<8>because it is in the ions'
coordination sphere. The K+ may be replaced by NH/, Na+, Cs+, Rb+, or
Ag+; the AP+ may be replaced by Cr3+, Fe3+, Mn3+, ln3+, Ga3+, or TP+; the
5042- may be replaced by Se042·.(3> Moreover a crystal of one pure alum
(e.g., a colored one) acts as a foundation for an overgrowth of another (e.g.,
a colorless one). The substitution of various ions for one another and the
overgrowth of one alum on another are both manifestations of isomorph-
ism.<3> All interchangeable ions fit into specific places in a single crystalline
pattern that is isometric in structure and octahedral in habit. True the re-
 150 Chapter 7

fractive index varies with composition, but it varies directly with the
proportional content of substituted ion. This is an analytical advantage, for
it allows rapid determination of composition once constituent ions are
known. Some other microscopical tests often identify ions, too: Cesium
alum for example is much less soluble than other alums, and it can be
detected as it precipitates at low concentration from a drop of solution on
a slide.(4l

7.5. SKELETAL MORPHOLOGY

The morphology of mature euhedraF~>-9l crystals is illustrated in Figure

7.la; no matter how small, they are filled out to the full extent shown.
Immature crystals can look different and be misinterpreted. If for example
NaCl crystals are grown from a very shallow drop (one spread over too
much of the microscopical slide), hopper-shaped crystals may develop. Be-
cause the drop is so shallow, the top face has run out of solute while the
meniscus of mother liquor attached to each vertical wall has supplied
solute to build the walls higher than the top surface. As such a crystal
grows, it becomes dished like a rectangular hopper. Unless we focus up
and down with an objective of short depth of focus (high NA) or use
oblique illumination, we may misinterpret the depression for an eleva-
tion-indeed for a set of faces.
Dendrites are even less filled-out crystals. They are fernlike, treelike,
branchlike skeletal crystals that have been grown so fast that they have not
had time to form faces and are therefore anhedral. (9>
Crystals grown from the meW 3•10•11l usually meet their neighbors in
more or less straight polygonal boundaries of crystalline grains, as in
igneous rocks, cast metals, frozen water, and other solidified liquids. This
action may be observed by watching water or other liquids freeze on a cold
stage.(4l Many organic and some inorganic solids can also be observed
during melting and freezing on a hot stage.(3•10•11 l Thus dynamic processes
can include changes in temperature gradients, annealing and quenching,
concentration and distribution of components and impurities, changes in
morphology (sizes and shapes of grains), and variations in structure (poly-
morphic transformations). Such knowledge can lead to a better under-
standing of materials as they undergo changes during manufacture, bulk-
testing, use, storage, weathering, or failure. The experience is also very
helpful in forensic investigations.( 12l Usually metals, ceramics, and other
inorganic systems melt at too high a temperature to be studied directly on
the hot stage of a microscope. In such cases experiments are conducted
elsewhere, and samples are taken and studied under the microscope. Here
Microscopical Properties of Crystals 151

experience with low-melting-point materials as they undergo dynamic

changes aids in interpreting appearances of high-melting-point or opaque
materials. (to)
The polyhedral boundaries of crystalline grains, such as those grown
from the melt in igneous rocks and certain ceramics, are the result of close
packing, like crowded soap bubbles. A crystalline grain can be a single
crystal, a twinned crystal, or a spherulite. Each has its characteristic optical
properties. Transparent single crystals grown from the melt can be studied
microscopically like euhedral<9l ones except that optical properties cannot
usually be related to grain boundaries because they do not consistently
represent crystallographic faces. Yet certain optical properties of anhe-
dral<9l crystalline grains can be interpreted in terms of the crystallographic
axes, and these are worth noting. For example (see Figure 7.1), in the
tetragonal and hexagonal (including trigonal) systems, there is only one
unique axis. Such crystals are uniaxial because the unique axis represents
the only direction down which the perpendicular field of view appears to
be isotropic. That is, viewed along that axis the field is dark throughout
360° of rotation between crossed polars. This occurs because the unique
axis (the c-axis) is at right angles to the plane of the other two (tetragonal,
a, a) or three (hexagonal, a, a, a) along which the spacing is the same.
Therefore looking down the c-axis, the isometric plane likewise is per-
pendicular to the axis of the microscope, and we see the one plane of isotropy.
In the orthorhombic, monoclinic, and triclinic systems (see Figure 7.1a),
spacings are different in all three crystallographic directions, so there is no
plane of equal spacing. However there are two directions in each whose
field of view is isotropic (dark throughout 360° of rotation of the specimen
between crossed polars). These, the two optic axes, are the directions in
which light travels with no apparent birefringence.<3l A bisectorial view
includes both optical axes if the angular apertures of the condenser and the
objective are wide enough. It follows that there is an acute bisectrix<1l and an
obtuse bisectrix,<1l and each is recognized by its characteristic interference
figure (see Chapter 5).
These and some other crystallographic and optical properties may be
determined on unicrystalline grains in thin sections (-30 ~-tm) of rocks,
minerals, and man-made products<6•13l within a wide range of grain sizes.
Searching for grains of adequate orientation<14-16l may be time consuming.
If so, a spindle<15l or a universal stage<17J to orient the specimen conveniently
may be desirable.
If it can be determined whether a crystal (euhedral or anhedral) is
isometric, uniaxial, or biaxial, a classification of sorts can be made on this
basis alone. Thus Table 7.2 shows the distribution of 935 frequently en-
countered crystalline phases, data compiled by Winchell<6•13l and classified
 152 Chapter 7

TABLE 7.2.
Distribution of 935 Crystalline Phases among Isotropic, Uniaxial,
and Biaxial Crystals<6•181
Number of phases Percentage
Total Inorganic
System Inorganic" Plus Organic~' Organic
Isotropic 86 (17.0, 9.2) 7 (1.6, 0.8) 10.0
Uniaxial 101 (19.9, 10.8) 49 (11.5, 5.2) 16.0
Biaxial 315 (62.1, 33.7) 359 (83.9, 38.4) 72.1
Intermediate 5 (1.0, 0.5) 13 (3.0, 1.4) 1.9

Totals 507 (100.0, 54.2) 428 (100.0, 45.8) 100.0

Grand total,l31 number of phases = 935

"Values in parentheses represent, respectively, the percentage of the total number of inorganic phases and
the percentage of the total number of inorganic plus organic phases.
hvalues in parentheses represent, respectively, the percentage of the total number of organic phases and
the percentage of the total number of inorganic plus organic phases.

by Kirkpatrick.<18> Only 10% of the 935 are isotropic, and most of these are
inorganic (probably because the coordination spheres of the constituent
ions require such a high degree of symmetry). Sixteen percent of the 935
phases crystallize in the uniaxial systems, one-third of them are organic
compounds. And 72.1% of the 935 phases are biaxial, more than half of
them organic compounds, showing how relatively large molecular units of
organic compounds pattern themselves.

7.6. ISOTROPIC SYSTEMS

For a given wavelength and a given temperature, there is only one

value for the refractive index of an isometric crystal, since it is isotropic.
Figure 7.3<181shows the distribution of the approximate refractive indices
(with white light at room temperatures) for inorganic isotropic substances.
The peak value is about 1.45 (25% of the total) for values falling between
1.33-2.0.(31 For each species of isometric crystal, there is some variation in
refractive index with respect to the wavelength, called optical dispersion. <1>
For example the refractive index of 2Na3P04 ·NaF·19H20 for the sodium
D line is 1.452, and the refractive index for the sodium C line is 1.451.
Optical dispersion is
nc25oc - no2soc = 1.451 - 1.452 = -0.001.(19)
Dispersion is just as much a determinative optical property as any other.
The degree of dispersion is also important in dispersion staining,<20•21> in
Microscopical Properties of Crystals 153

ORGANIC 1.4-1 1.45- 4 1. 5-1 1.55- 1

S m7

INORGANIC
30
s- 86

*- 20
(;
c
"'g
:I
10
...
u..

~
(n
-
(n
U1
in in
U1
Refractive index

FIGURE 7.3. Distribution of the approximate refractive indices (with white light at room
temperature) for inorganic isotropic substances.

which differences in refractive index for some wavelengths (but not others)
between crystals and mounting liquid lead to colored outlines (see Chap-
ter 9).
It is relatively easy to recognize octahedra {111} when they are equant;
see Figure 7.4a. The octahedron is lying on one of its equilateral triangles
(dotted). The opposite one is shown in Figure 7.4a (solid outline), pointed
the opposite way. The other six sides are alternately pointed up or down,
connected by edges that alternately go from top to bottom or vice versa.
These six zigzag edges, in perspective, make an equilateral hexagon, which
is not to be confused with a truncated bipyramid in the hexagonal system

--'Q' 0
I

-·--
I ',
I
(a) Equant ( b) Flat and equant (c ) Flat ond long

FIGURE 7.4. Drawings<3>of octahedra, each lying on an octahedral face and all three show-
ing the same angular symmetry.
154 Chapter 7

(or anything else), since there are the doubly shadowed up and down por-
tions that look like smaller, darker triangles in perspective. These small
dark triangles are also shown in the flat octahedra (see Figures 7.4b and
7.4c), but the equant and flat octahedron (see Figure 7.4b) shows in per-
spective 12 side lines instead of six, as shown in Figure 7.4a. The long, flat
octahedron (see Figure 7.4c) is most difficult to interpret because the side
lines are not of equal length. However the angular symmetry is still the
same, and that is what indicates the isometry; the isotropy clinches the case.
Incidentally the shadows (shading) in all three drawings (see Figure 7.4)
represent the partial reflection of incident light away from the micro-
scopical objective. The degree of shading represents the obliquity of the
side faces. 13>
In addition to the cube and octahedron, the isometric system is rep-
resented (less frequently) by the rhombic dodecahedron (as of hexameth-
ylene tetramine) and pentagonal dodecahedron (as of cerium formate).
There is also the hemihedral class of the octahedron, i.e., the tetrahedron
(as of sodium uranyl acetate).<4>

7.7. UNIAXIAL CRYSTALS

In the tetragonal and hexagonal systems (see Figure 7.1), there is one
unique axis, namely, the c-axis, along which spacing of the structural units
is shorter or longer than along the a-axes, which lie in a plane perpendicu-
lar to the c-axis. In the tetragonal system there are two a-axes at 90° to each
other; in the hexagonal system there are three a-axes at 120° to each other.
In either case in any plane, light vibrates in one direction at a constant
velocity, and therefore the ordinary refractive index ro is always" seen." On
the other hand the extraordinary refractive index £ is seen only if the
crystal is lying on a prism face, such as {100} or {110}. On an inclined face
(e.g., pyramidal or rhombohedral), the refractive index for light vibrating
at right angles to the always-seen ro is somewhere between the values for
ro and e; it is represented by the symbol e'. For crystals lying on a definite
face inclined to the c-axis (such as a pyramidal or rhombohedral face), the
value fore' is constant for that particular form (kind of face). Values fore' are
not generally given in compilations,<2.6•13l so the reader should start a collec-
tion of e' values in some retrievable manner. We emphasize that the values
ro, £, and e' are for crystallographically oriented vibrations. If there is any
doubt about to the orientation of the axes, the more abstract and general
symbols,<9> such as n1 and n21 should be used.
The plane of the a-axes is the basal pinacoid. Crystals resting on this
face appear dark in every position of retardation between crossed polars.
Microscopical Properties of Crystals 155

Most uniaxial crystals recrystallized on a microscopical slide do not

rest on a basal pinacoid, but some species do (for example iodoform, lead
iodide, cadmium iodide, and Na2SiF6}. Uniaxial crystals with low-enough
melting points, such as sodium nitrate, may be melted on a slide, covered
with a cover glass, and recrystallized from the melt. The resultant crys-
talline grains can be examined for the isotropic view. If either of these two
methods fails to produce an isotropic view and if a universal stage<m is
available, it can be employed to rotate the crystal180° in three perpendicu-
lar directions. Such a stage can be used with almost any small single
crystal, whether it is a polygon, fragment, sand grain, sawed section, or
product of fusion.
Once an isotropic view is sighted, the top lens of the condenser is put
in place and the diaphragm is opened all the way to give conoscopic illu-
mination and an interference figure.<3> (An objective of NA = 0.85 is pre-
ferred if its presence is assumed in tables of computation.)<14> Next the
Bertrand lens (see Chapter 5) is put into place in the microscope's tube
below the ocular to provide a compound microscope focused on the back
aperture of the first objective. If no Bertrand lens is provided, the ocular is
removed and the back aperture of the objective is viewed directly, i.e.,
without magnification. If the crystal is uniaxial and the microscope is
properly aligned, the isotropic view provides a centered uniaxial interference
figure or pattern. As shown in Figure 7.5, there is a dark Maltese cross
ringed with interference colors of numerous orders, depending on the
degree of birefringence. The rings of interference colors of Newton's series
and the Michel-Levy's chart represent the increased retardation derived
from increased angularity from the axis to the periphery of the cone of

FIGURE 7.5. Diagram of uniaxial interference figures, positive (left) and negative (right).
Vibration direction of slower components are indicated by arrows, c, c represent quadrants
in which compensation occurs. o represents the optic axisP>
156 Chapter7

illuminating rays. In other words rings of isochromes are summations of

retardations corresponding to variations in incident ray orientation, with
reference to the crystal's c-axis. The black arms of the Maltese cross are
called isogyres of the interference figure.<3> The cross is similar to the one
shown by spherulites, such as starch grains, and for the same reason. They
1
represent positions of extinction and w-E. Instead of a sphere of crys-
tallites, we have a sphere of directions. Indeed we can obtain the same
effect by illuminating a sphere made of the uniaxial crystal with axial light,
employing crossed polars.
What good is an interference figure? If it indicates that the crystal is
uniaxial, we immediately know that we are dealing with a species that
represents only 16% of a total of 935 crystalline species (see Table 7.1). If
the substance is organic, it is among only 5%; if it is inorganic, the prob-
ability is 11%. Thus uniaxiality alone is distinctive and determinative.
Moreover, the optic sign is easily obtained from an interference
figure.l3·15> If a first-order (fO) red retardation plate is inserted into the
NW\SE diagonal with its slower component vibrating SW /NE, the color
near the center of the (uniaxial) black cross is yellow (subtraction) in the
NW and SE quadrants, and the crystal is optically positive. If instead the
color is blue (addition), the crystal is negative. Or the quarter-wave (0.251..)
plate can be used. Then if a black dot is seen NW and another SE of the
center of the black cross, the crystal is positive; if NE and SW, it is negative.
Otherwise a quartz wedge can be moved in place with increasing order of
interference color. If the crystal's colors move outward, it is positive; if
inward, it is negative.<15> From Figures 7.6 and 7.7 we obtain a distribution
(percentage of probability) along the ordinates for uniaxial positive and
negative crystals among inorganic and organic species with regard to ro
(abscissa). This is the sole value seen down the unique axis (c-axis). Thus
the combination of uniaxiality and quantitative value for e' is very de-
scriptive. The combined data placed on punched cards or fed into a com-
puter become very selective, especially at the extremes of the distributions.
Off-centered figures can be used not only to obtain the optic sign but
also to show the vibration direction for e'.(3> For a given path length, the
more rings in the off-centered figure, the closer e' approaches e in value.
Therefore measuring the tilt angle of the optic axis (c-axis) and adding the
value for ro provide an estimate of e.<15>Determining values for ro and e for
different wavelengths of light establishes the degree of dispersion; in very
distinctive rare cases, the optic sign may even change.
A euhedral crystal resting on a prismatic face {100}, {110}, {1010}, or
{0100} presents ro and pure e. These may be measured directly by any of
the methods discussed in Chapter 6, or elsewhere.<3•15> The difference gives
 Microscopical Properties of Crystals 157

POSITIVE NEGATIVE

.,.

l..llll.. ::lll .I.... •

E

> s- 32 s- 67
u E
z
w
::l I·· I 1o I •
a
w
a:
u.
w

1.1111 .. ••••••
s- 32

... ... ... .... ... ,_. ,_. N

w :,. i.n ill " iD io 0
REFRACTIVE INDEX

FIGURE 7.6. Refractive index distribution for uniaxial positive and negative crystals among
norganic species with regard to ro and e.

POSITIVE NEGATIVE

50 5

4 40

3 30
E
20
e
>
u
s- 18 s- 29
zw 1
::l
a
w
a: 4
u.

w w
20
s- 18 S=30
10

... ...:,. ...i.n ...ill !"' !"' N !"' !"' ...i.n

w CD Ul 0w ~

REFRACTIVE INDEX

FIGURE 7.7. Refractive index distribution for uniaxial positive and negative crystals among
organic species with regard to ro and e.
158 Chapter 7

the birefringence and optic sign. If E>w, the crystal has a positive bi-
refringence; if E<W, a negative birefringence. Alternatively the birefringence
and optic sign can be determined directly, as mentioned earlier. Moreover
a prismatic view best presents the phenomena of dispersion and pleo-
chroism.*
A pyramidal, tetrahedral, or rhombohedral face presents w of course
but also a special and constant value for E', which is therefore descriptive
and determinative. Furthermore if the face angle is known, data depend-
ing on true E can be determined; traces of the uniaxial interference figure
may also be visible.
It is now obvious that attempts should be made to obtain crystal faces
for observation. While crystals as commercially received are not always
good enough for crystallographic and optical purposes, recrystallization
from a drop of solvent on a microscopical slide<Ml is generally fruitful.

7.8. BIAXIAL CRYSTALS

Table 7.1 shows that about 62% of inorganic crystals are biaxial, and
even more organic crystals, 84%, are in-this category. Figures 7.8 and 7.9
show distributions according to optic sign and the three principal refractive
indices n:u ny, and n)18l Since there are two optic axes, the angle between
them (2V) is also descriptive and determinative (see Figure 7.10). This
value is measurable or estimatable directly from the adequately centered
biaxial figures (see Figures 7.11a and 7.11b) whether the bisectrix is acute
or obtuse. Measurement is made easier by standardizing on a numerical
aperture of 0.85.<14l Then use a standard chart for 2V versus fl, the angle of
rotation of the stage necessary to cause the isogyres (curves around the
optic axes) to move from the center to the edge of the microscopical field,
to convert from 2E (the optic axial angle in air).<15l
It is probably already apparent that biaxial crystals are optically and
crystallographically complicated (see Figure 7.12).<22l This means that iden-
tifying the three principal refractive indices qualitatively and quantita-
tively may be difficult. But some values are always visible and capable of
measurement however vague their specific identity may be at the time of
observation. The important thing is to record them and to recognize them
on every repeated occasion, so that a pattern appears.<23l

*Pleochroism is the general term for absorption of different colors as light passes in different
directions through a crystal. If crystal exhibits two distinctive colors when viewed in two
unique directions in white light, it is said to be dichroic (see Chapter 6). If there are three
directions and three colors, it is trichroic.
Microscopical Properties of Crystals 159

POSITIVE NEGATIVE
401
30

:t Ill h•.:~':'.
40
I
·····-
snz= 134
.
lle3o
>-
~2
alo
w

w
ny
s= 144
a:
IL

40

nx
s = 136
t-1 ~""' ...,. .... ._. ...,. .... N
w~u,c,:...a.ioo

REFRACTIVE INDEX

FIGURE 7.8. Refractive index distribution for biaxial positive and negative crystals among
inorganic species.

7.9. OPTICAL PROPERTIES OF THE LIQUID CRYSTALLINE OR

MESOMORPHIC STATE

Some solid organic compounds assume the mesomorphic state, which

is intermediate between that of a solid crystal and a true liquid. Meso-
morphism that sometimes occurs when a solid crystal is heated is termed
thermotropism; what occurs when a solvent is added is called lyotropism. C24>
Most compounds that manifest mesotropism have long molecules,
sometimes also flat, such as those containing para-substituted benzene
rings. Mesomorphic substances also contain one or more polar groups. In
the solid crystalline state, the long and/ or flat molecules are arranged in
parallel positions, holding together by means of the polar groups and van
der Waals forces. When such crystals are heated or loosened with solvent,
if the polar forces are not too strong, the molecules find some freedom of
movement while retaining some degree of order.C24>
Mesophases are birefringent. In lyotropic systems form birefringence
160 Chapter 7

POSITIVE NEGATIVE

:: II
301
;z= 125

. ..&..-.JI•L-.·
_l.a.a..-1
_ . .. . . . . . .
L - . . .. . . . . .

40
30
20
nv
s= 180

10

40 40
30 30
nx nx
2 s= 132
20 s= 161

10 10

FIGURE 7.9. Refractive index distribution for biaxial positive and negative crystals among
organic species.

INORGANIC ORGANIC

~~t.ullll
2V 2V
s= 281 s= 318

z
w

I I
::>
0
w

~ :ot ::l 2E
5=281 10
2E
5==318

1''''''''''''''~···'''''''''·~
0 ~
0
~
0
~
0
~
0
~
0
0
~
~
0
~~
~~
0+
0

ANGLE, DEGREES
~
0
~
0
~
0
~ ~
0
~
0
~~
co+

FIGURE 7.10. Optic axial angle distribution for biaxial positive and negative crystals among
inorganic and organic species.
Microscopical Properties of Crystals 161

FIGURE 7.11. Vibration directions of slower components is indicated by arrows; c, c, quad-

rants in which compensation occurs; o, o, optic axes; Bxa, acute bisectrix.<ll (b) Estimation of
2V from curvature of isogyre, in optic axis interference figure. After F. E. WrightPl

(see Chapter 6), pertaining to the difference in refractive index between

liquid solvent and the long or flat solute molecules, may account for some
of the birefringence, but not all of it.(24l The fundamental cause of bi-
refringence in mesophases lies in the activity of polarized light waves as
they displace electrons in atoms in the vibration direction of polarized
light, producing electric dipoles oriented toward polarization. When the
162 Chapter 7

FIGURE 7.12. Crystallographic properties of melamine.<22)

System monoclinic Refractive indexes nx = 1.487
Class prismatic ny = 1.846
Axial elements a :b:c = 1.4121:1 :0.97288 nz = 1.879
= 112°2' Optic sign (-)
Birefringence 0.392 Optic axial angles 2E =53°30'
2V = 28°38'

vibration direction is parallel(-) to the lineup of the dipoles(+-)(+-)

(+-), the total electric field is greater than if the dipoles were widely
separated. When the light is vibrating at right angles to dipoles that are
side by side (+-), the total electric field is less than for widely scattered
atoms. When light is vibrating parallel to the long axis, the average
strength of the dipoles of long molecules, is greater (the refractive index
higher) than when light is vibrating crosswise.<24 )
Microscopical Properties of Crystals 163

OPTIC AXIS
I

lllllllllll
rrrrrrr1rrrr
lllllllllll
FIGURE 7.13. Smectic structures (schematic): Double
layers in so~. • represents terminal polar groups, e.g.,
rrrrrr1r1rr
-COONa.< J Courtesy of Micrpscope Publications.

7.10. THERMOTROPIC, MESOMORPHIC, SINGLE COMPOUNDS

Generally thermotropic mesophases of single compounds having long

molecules belong to one of the following three structure types:
1. Smectic mesophases is the type to which soaps belong (the Greek
word for soap is smega). Soaps have a polar group, such as-COONa at the
end of a long hydrocarbon chain. The polar group of one long molecule

OPTIC AXIS

I I I I 1 1 II I I I I
1
1 I I I
I I I
I II I I 11 II 1 II II II 1 I II 11
I I II II I I I I I I I I
II I 1 II 111 I I 11
1I 1 I 1 II
1
I I 11 I 11 I 1

FIGURE 7.14. Nematic structure (schematic).l24l Courtesy of Microscope Publications.

164 Chapter7

attracts that of another molecule head-to-head, forming a double layer in

the smectic phase, as illustrated in Figure 7.13. The layers are flexible and
glide over one another. Most smectic phases are optically positive and
uniaxial. <24>
2. Nematic mesophases are named for the Greek word for thread,
nema, because some members of this type manifest thready lines. All
members of this type are composed of single layers of more or less parallel
lengthy molecules, as shown in Figure 7.14. Nematic phases are optically
positive and uniaxial. (24)
3. Cholesteric mesophases are named for cholesterol and its derivatives
because they are in the majority of members of this type. Theirs is a spiral
arrangement of nematic layers, as shown in Figure 7.15. The pitch of the
spiral is about half that of a wavelength of visible light, giving the choles-
teric structure very special optical properties, including strong rotation of
the plane of polarized light around the axis of the spiral (which is also the
single optic axis). Cholesteric mesophases are optically negative because
long strands of such molecules are oriented in various directions around
the axis. Since these molecules are also flat and spaced close together, their
optical properties resemble those of planar molecules. When the layers are
parallel to the slide, they may show an off-center biaxial interference figure
with an optic axial angle of about 20° .<24>

FIGURE 7.15. Cholesteric structure

(schematic), with right-hand twist. The
planes mark levels in the structure be-
tween which a 45° rotation of the molec-
ular axes occurs. <24> Courtesy of Micro-
scope Publications.
Microscopical Properties of Crystals 165

Some smectic types show polymorphism, believed to be due primarily

to different lateral arrangement of the molecules. A smectic phase or
phases can also manifest either a nematic or a cholesteric phase, but not
both. The smectic phase always occurs at lower temperature than the
nematic or cholesteric phase, since the smectic phase is more ordered.
Because the crystalline phase of the same compound is still more ordered
than its smectic phase, the crystalline phase is stable at a lower temper-
ature.<24)

7.11. MORPHOLOGY TYPES

The morphology of mesophases is called texture by Hartshorne.<24)There

are several kinds of texture:

7.11.1. Homeotropic Textures

Homeotropic textures (named by Lehman and Friedel) refer to smectic

and nematic mesophases with the optical axis perpendicular to the glass
surfaces confining them. Hartshorne extends the definition to include also
cholesteric phases.<24) If for example a little (smectic) ammonium oleate is
pressed into a thick layer between a glass slide and cover glass, a confused
texture is seen between crossed polars because the specimen is too viscous
for the layers to flatten out. If the glass surfaces are cleaned with hot soapy
water without an etching agent, then rinsed with hot water, dried with a
clean cloth, and not handled thereafter and if the cover glass is gently
moved round and round by means of a rubber-tipped pencil, inspection
between crossed polars reveals a gradual disappearance of the polariza-
tion interference colors. When the field becomes quite dark and the illu-
mination is conoscopic, a uniaxial interference figure appears, as shown in
Figure 7.16. By means of a first-order red plate, the optical sign is shown
to be positive in the case of ammonium oleate because it is typically
smectic. Typical nematic mesophases are also optically positive, but chol-
esteric mesophases are generally negative.<24)

7.11.2. Focal Conic Textures

Focal conic textures arise when a mesophase is confined to surfaces

with which it forms strong local attachments, for example to a glass slide
and cover glass etched with hydrofluoric acid. Long molecules around
166 Chapter 7

ANALYZER ANALYZER
I I
I I

_P.QLARIZE !!__

I
8
(0) (b)

FIGURE 7.16. Determining the optical sire on an optic axial interference figure by using Red
1 plate. (B =blue; Y =yellow; R = red.)< >Courtesy of Microscope Publications.

each center of attachment are required to adopt a radiating arrangement

resembling a tipped hollow ring, known as a Dupin cyclide, as shown in
Figure 7.17. The Dupin cyclide is truncated by the glass slide and cover
glass. If the preparation is fairly thick and not too viscous and heated
between a slide and cover glass (both slightly etched) until the liquid phase
begins to appear, on lowering the temperature and agitating the cover
glass, a number of polygonal areas appear. In each polygonal area there is
a family of ellipses consisting of large ellipses with smaller ones in the

(a) (b)

FIGURE 7.17. Dupin cyclide. (a) Half-cyclide showing principal sections, (b) plan view.<24>
After Hartshorne and Stuart, Crystals and the Polarising Microscope, Arnold, 1970. Courtesy of
Microscope Publications.
Microscopical Properties of Crystals 167

interstices. The major principal axes of the ellipses in any one polygon meet
at one point within the polygon, as shown in Figure 7.18. If the upper
surface of the preparation is in focus and the analyzer is in place (without
the polarizer), each ellipse is crossed by a straight isogyre, whose nar-
rowest point coincides with one of the foci of the ellipse. If the polarizer is
employed alone, a similar pattern is in focus on the lower surface of the
preparation. If the focus is gradually lowered from the upper surface to the
lower surface of the preparation, each ellipse is joined by one branch of a
hyperbola that starts at the isogyre. All of the hyperbolas belonging to the
ellipses of one polygon meet at the lower surface at one point below the
intersection of the major axes of the ellipses. This point is a common corner
in a system of polygonal areas in the lower surface. Hartshorne explains
these phenomena. 24

7.11.3. Other Smectic Textures

Other textures of smectic substances are

Batonnets: separate little images of focal conic textures, often highly
ornamented; they may form on cooling the melt.
Fanlike textures: revealed by crossing the polars, successive radial
bands extinguishing at slightly different positions of orientation of the
stage.
Oily streaks: observed between crossed polars when a focal conic
texture is destroyed by shifting the cover glass on a preparation; the streaks
are birefringent bands having transverse striations. At sufficient magnifica-

(a) (b)

FIGURE 7.18. Polygonal texture. (a) Upper surface focused (analyzer, no polarizer), (b)
lower surface focused.'24> After Hartshorne and Stuart, Crystals and the Polarising Microscope,
Arnold, 1970. Courtesy of Microscope Publications.
 168 Chapter 7

tion the streaks' focal conic groups can be seen with the hyperbolas parallel
to the striae. The bands are length fast (n11 < n.L).

7.11.4. Nematic Textures

The texture of nematic mesophases is typically threadlike, formed by

rapid cooling the isotropic melt to nematic temperatures. These wormlike
textures mark structural discontinuities, analogous to ellipses and hyper-
bolas in smectic phases. However the nematic discontinuities do not con-
form to any definite geometrical plan because there is no stratification.
Approximately centered optic axis figures (see Figure 7.16) or optic normal
figures (see Figure 7.19) are usually found with which to determine the
optical sign (+). Incidentally the initial morphology of the nematic phase
from the melt is spherical droplets, not batonnets as from a smectic
phase.<241

7.11.5. Cholesteric Textures

The texture of a cholesteric phase, produced by cooling an isotropic

melt, resembles a cloud of very fine particles. When the cloud is disturbed
by moving the cover glass, a homeotropic texture is produced, with the
optic axis normal to the plane of the slide. The vivid colors in the specimen
held between crossed polars are very different from those of homeotropic
smectic and nematic phases. The specific color (or occasionally invisible

(a) (b)

FIGURE 7.19. Optic normal figure(" flash" figure). (a) 45° position showing example of color
distribution. In a positive mesophase, light vibrating along 0-0' (direction of the optic axis)
is slow. In a negative mesophase it is fast. (b) Extinction position.<241Courtesy of Microscope
Publications.
Microscopical Properties of Crystals 169

wavelengths) depends on the temperature and angle of observation. The

changes in color with temperature have been developed commercially to
register temperatures and temperature gradients.(24l
The second characteristic of cholesteric phases is the rotation of the
plane of polarized light. This phenomenon is manifested qualitatively
under the microscope by a lack of extinction between crossed polars.
Instead of darkness there is a color that changes as the analyzer is rotated,
due to the dispersion of optical rotation. The optical activity of most
cholestive substances is so great that several turns of the rotating stage are
necessary to restore extinction. Therefore a circularly wedged preparation
is used (see Figure 7.20). The number of rings indicates the orders of 180°
rotation by the specimen. A slight rotation of the analyzer to the right,
noticing whether rings go in or out, indicates whether the specimen is levo-
or dextrorotatory. A very low-power objective (•48 nm) and eyepiece
should be used. Since most cholesteric phases of single compounds are
stable above room temperature, a hot stage should be used, with the
temperature of the test recorded as a factor in the degree of rotation by the
specimen. Of course a plane wedge can be made from a large cover glass
propped up at one edge by another piece of cover glass. The specimen is
then run in between the sloping cover and a slide.(24l

7.12. LYOTROPIC PHASES

Solutions of mesomorphic phases behave like cholesteric thermotrop-

ic phasesJ24l Aerosol411 OT, a substance with peglike molecules of sodium-
di-2-ethylhexyl sulfosuccinate (a waxy smectic phase, difficult to handle)
is commercially liquified to a pourable lyotropic phase by adding about

FIGURE 7.20. Principle of determining optical rotation

of a cholesteric phase. (24) Courtesy of Microscope Pub-
lications.
170 Chapter 7

25% of an alcohol-water solution. Aerosol® OTis a typical amphiphile; that

is, the COONa group is strongly polar and hydrophilic (lypophobic), while
the double peglike chain is strongly lipophilic (hydrophobic), as shown in
Figure 7.2lb).<24>
The basic unit of amphiphile is the micelle, a cluster of polar molecules
with single or double chains with their polar groups oriented in the water.
Figure 7.22a shows a micelle in the water of a dilute, isotropic, colloidal
solution. Figures 7.22b-7.22d illustrate the chief kinds of birefringent lyo-
tropic mesophases. In Figure 7.22b the term neat comes from soap technol-
ogy referring to smectic molecules structured in a lamellar phase as a
double layer tail-to-tail with the polar groups facing the layers of water.
The resulting birefringence is positive, but the strength may be weakened
by some negative form of birefringence.<24>
In the soap industry middle phase (M1) pertains to single-chain mol-
ecules grouped into rodlike micelles of indefinite length arranged side by
side in a hexagonal pattern, as shown in Figure 7.22c. In each rod the
average orientation is radial, with the polar groups outside toward the
water between the rods. The optical sign is negative (in accordance with a
planar structure), but the crystal birefringence is probably lessened by
some degree of form birefringence.<24>
The inverse (M2) phase is in response to a higher concentration of the
long molecules, which then turn around so that their polar groups face
water now contained in hollow rods, as shown in Figure 7.22d. This phase
is also optically negative. <24>

GROUPS

t---LIPOPHILIC-----
CHAINS

(I) (2)

FIGURE 7.21. Types of amphiphillic molecules (schematic).<24> After Hartshorne and Stuart,
Crystals and the Polarising Microscope, Arnold, 1970. Courtesy of Microscope Publications.
Microscopical Properties of Crystals 171

WATER WATER

~\~
~~
WATER
\'mN}lmn WATER
(a) (b)

WATER

\~/.---
*1~ t\~1\'
-"'v'"\~------/
I ~1/'---.
...............
"-. -. /
-;i:;:>~r\
(c) (d)

FIGURE 7.22. Main types of micelles in amphiphile-water systems. Only molecules with
single lipophilic chains are shown, but peg-shaped molecules would be arranged similarly,
though such molecules are not known to form M 1 phases: (a) diametral section of spherical
micelle, (b) smectic layer in neat phase G, (c) middle phase M1 showing cross section of
micellar rods (according to Luzatti), (d) inverse middle phase M2 showing cross section of
micellar rods (according to Luzatti). <24>After Hartshorne and Stuart, Crystals and the Polarising
Microscope, Arnold, 1970. Courtesy of Microscope Publications.

Lyotropic phases can display spherulites and fan shapes. They are
usually positive; that is, the slow component is radial. However in neat
phases isogyres are merged as in Figure 7.23a, four of them producing a
larger, negative (radially fast) spherulite. A pinwheel effect (see Figure
7.23b) is apparent when the stage is rotated. These effects were not noticed
by Rosevear (1954) in spherulites of the middle phases.<25> He noted that the
neat phase had a greater birefringence than the middle phases. In applying
Rosevear's observations as a test of amphilic types, retardation must be
reduced to birefringence by taking thickness and orientation into con-
sideration. The Rosevear test does not apply to peg-shaped (double-chain)
molecules. Incidentally the sign of a spherulite is not necessarily the same
as the intrinsic optical sign of the mesomorphic structure; the sign of a
172 Chapter 7

ANALYZER ANALYZER

STAGE ROTATION

FIGURE 7.23. Merging of positive spherulites in neat phases of soaps and detergents. (a) the
four positive spherulites combine to give an apparently negative spherulitic region in the
center (radial direction "fast"). (b) On rotating the stage from the position in (a), a pinwheel
effect is obtained at the center.<24> Courtesy of Microscope Publications.

spherulite is merely an indication of the arrangement of units in the sphere

or disk. <24>
There are apparently isotropic phases in some lyotropic systems. For
example Winsor and Rogers<26> evaporated a drop of a 30% solution of
Aerosol"' OT in water and observed an isotropic phase between an M2 (-)
and a G (+) phase. While such a phase may not have an ordered structure,
it may have exactly compensating positive and negative structures, pos-
sibly including some form birefringence.<24,25>
When emulsions of water, soap, and a hydrophobic phase are frozen,
they sometimes break,<27l depending on whether or not the wall of third
phase<1> around each droplet is broken by the growing crystals of either
major phase. On the basis of current knowledge of mesophases of soap and
other emulsifying agents, breaking such emulsion on freezing should be
studied for their mesomorphic structure and habit at prevailing temper-
atures and gradients.

7.13. LIQUID CRYSTALLINE POLYMERS

Development of polymers capable of producing liquid crystalline

structures has greatly expanded in recent years; they are of both thermo-
Microscopical Properties of Crystals 173

tropic and lyotropic types. The three most important groups are (1) aro-
matic polyamides, (2) rigid-rod polymers, and (3) aromatic copolyesters.
An atlas of photomicrographs preceded by a survey introduction has
been presented by Woodward. <28> Liquid crystalline phases are normally
birefringent and often show one or more of the textures discussed in
Chapter 7.
Sawyer and Grubb state that essentially all microscopical techniques
have been used in characterizing these structures, including etching, son-
ication (for fibrillation), ultramicrotomy, fractography, and peeling to ex-
pose the internal structures. !29> They note that many thermotropics can be
transformed from one polymorphic configuration to another if the tempe-
rature is changed.

Crystalline - Smectic - Nematic - Isotropic

Solid

Increasing Temperature

It is important to note that surfaces, as with glass slides, often have a

strong influence on the preparation's observed order and the initially
observed structure ultimately relaxes to another structure with time: it
may take several days or longer for this relaxation to occur.
As discussed in Chapter 13, Young considers measurements of mo-
lecular orientation on aramid polymers and shows these have superior
sensitivity to those of X-ray diffraction.<30> He also shows the molecular
structures of Kevlar® and Nomex® polymers.

7.14. SUMMARY

Microscopists often use crystallography because crystals are distinc-

tive and have many precise characteristics, which are of enormous help in
identifying and studying substances. Crystallography is an extensive sub-
ject with a formidable vocabulary, but students can take comfort from the
fact that only six crystal systems are needed to embrace the (literally)
millions of known pure solid phases and their descriptions are logical and
orderly, although an ability to visualize solid geometric shapes helps.
Crystal axes are imaginary lines employed to describe the structure
and symmetry of crystals. The simplest crystal system, the isometric, is
represented by three mutually perpendicular axes, exactly like the familiar
x-y-z Cartesian coordinates. The structural units of such a crystal, whether
174 Chapter 7

ions, atoms, or molecules, are arranged in equidistant fashion along these

three equivalent axes. Hence the optical (and other physical) properties are
the same in any direction within the crystal, which is isotropic as well as
isometric. Such a crystal appears dark in all orientations when viewed
between the crossed polars of a polarizing microscope. In Winchell's very
useful lists of 935 commonly encountered crystalline solids, only 1.6% of
the organic and 17% of the inorganic substances are isometric, represent-
ing 94 of the 935 solids. Hence the fact that a crystalline solid is isometric
immediately limits the possibilities. If the index of refraction is then de-
termined, this further limits the choices within Winchell's list. Some judi-
cious chemical tests under the microscope then lead to a tentative
identification of the phase, and the choice can be verified by preparing a
few characteristic derivatives under the microscope and checking their
optical properties.
The second crystal system is the tetragonal, which also has three axes
at right angles, but one of them has different spacings along itself, and it
is considered to be of different unit length. Viewed along the unique axis
(the c-axis), a tetragonal crystal appears to be isometric because its other
two axes (the a-axes) are equivalent, but viewed along an a-axis, such a
crystal is birefringent because there are different velocities of light along
the two monequivalent axes. The third system, the hexagonal, has three
equivalent axes at 60° to each other, all perpendicular to the longer (or
shorter) c-axis. Hence a hexagonal crystal is also isometric when viewed
along the c-axis and birefringent in any other position. Tetragonal and
hexagonal crystals together are called uniaxial because they have only one
unique axis. These crystals account for 19.9% of the inorganic substances
in Winchell's lists and 11.5% of the organic substances. In appropriate
orientations and with conoscopic polarized light, uniaxial crystals produce
a characteristic interference figure (a Maltese cross centered on a series of
colored diffraction rings) that can be observed at the back of the objective.
The remaining crystal systems have more than one unique axis. The
orthorhombic system has three axes (a, b, c) all at right angles but all of
different length, so that its crystals are always birefringent in all orienta-
tions. The monoclinic system has two axes (a, b) at right angles, but the
third (C) is at some other angle to the plane of the first two. The last system,
the triclinic, has three unequal axes all at angles other than 90° to each
other. These three systems together make up 62.1% of the inorganic and
83.9% of the organic substances in Winchell's lists. Their identification is
more difficult than identifying isotropic and uniaxial crystals, but deter-
mining the angle between any two axes helps, as do the characteristic
interference figures. The refractive indices are determinative, likewise for
the sign of the birefringence. Even without quantitatively determining
Microscopical Properties of Crystals 175

refractive indices, the sign can be determined by using a retardation plate

or compensator.
In addition to six kinds of geometric structures, crystals have charac-
teristic morphologies by which they are recognized under the microscope.
Within each system crystals may be in the shape of flat plates (lamellar),
thicker tablets (tabular), more regular solids with edges more or less equal
(equant), elongated prisms (columnar), or tiny needles (acicular). No mat-
ter what the morphological type, a given pure phase always has the same
refractive index (or indices) and the same optical sign because its internal
structure remains constant. In addition to the five general morphological
types within each system, there are characteristic shapes and faces (forms)
by which the microscopist recognizes crystals. Thus isometric crystals
often show up as cubes, octahedra, or tetrahedra if they have had an
opportunity to grow regular faces and sides. (If crowded together, as when
crystallized from a melt or crushed or powdered, pieces of any crystalline
type are grains rather than regularly shaped units, but they have the same
optical properties as the beautiful equant shapes.) Well-developed tetra-
gonal crystals are rectangular prisms and tablets, sometimes with sloping
end faces. Hexagonal crystals may be the familiar hexagonal prisms, as in
quartz, or the equally-familiar rhombohedra (hemihedral hexagonal bipy-
ramids), as in calcite. Some crystallographers prefer to call the rhombo-
hedral class of the hexagonal system a separate (seventh) system. Ortho-
rhombic crystals are often tabular, as in the familiar form of sulfur.
Triclinic crystals have the "leaning prism" look, as in the familiar bright
blue pentahydrate of copper sulfate.
The faces (forms) of any crystal habit are best defined in terms of the
Miller indices, which are reciprocals of intercepts of a particular face with
the crystallographic axes. Thus the faces of a cube are represented by the
indices 100, 010, and 001, while any face of an octahedron is designated
111. Miller indices of the equant habits of all seven crystal systems are
given in Table 7.1, and the indices of lamellar, tabular, columnar, and
acicular crystals of the same system are equal to those shown there.
Most of the usefulness of crystallography to the microscopist comes
from long experience in recognizing particular systems from the way
their· crystals of characteristic habit appear as they lie on this face or that
or as they develop from solution on a slide or freeze from a sample held
on a hot stage or cold stage. Dozens of tips on how to do this are
illustrated with many examples, but the microscopist must be familiar
with all of the techniques and endure all the pitfalls before gaining the
facility that comes with that experience. The systematic study of pure
compounds, and especially faithfully recording all optical properties of
every substance that comes to the microscopist's attention (under some
176 Chapter 7

system that allows rapid retrieval of the information), will do wonders for
reaching that state.
Liquid crystals, which are manifestations of residual order held over
as a solid begins to melt or to dissolve in a solvent are discussed. The order
usually arises from the persistence of oriented electric dipoles, which is
why liquid crystals can be manipulated by electric fields to display digital
readouts in calculators and watches. The narrowly limited temperature
range of thermotropic liquid crystals is a reason why they are used also in
digital thermometers.
 8

Confocal Scanning
Light Microscopy

8.1. INTRODUCTION

Confocal scanning light microscopy, a new type of microscopy, has

generated considerable excitement. It gives higher resolution and thinner
noninvasive optical sections, or planar views, than those obtained by
classical bright-field or dark-field microscopy, and increased contrast is
another major advantage. Fluorescence microscopyC1> is also greatly im-
proved by using confocal scanning light microscopy, since three-dimen-
sional views can be generated, which lend themselves well to digital image
processing and possibly holography. In an image-processing system, a
hundred or more very thin optical sections can be stored and combined
into a composite, three-dimensional image. Then a display of the total
three-dimensional view, or selected parts, can be generated. The composite
three-dimensional views can resemble those from scanning electron micro-
scopy, but the specimen does not have to be in a vacuum, as with normal
scanning electron microscopy. Currently the technique is used only for
reflected light, bright-field and fluorescent microscopies, but additional
techniques are under development. Transmission and polarized light mi-
croscopes should be available in the future. The confocal microscopical
concept was patented in 1957, but the technique has only recently become
more common. Delay in its development is probably due to the only recent
common application of lasers and scanning techniques. Currently there are
some complications with all varieties of these instruments, and efforts are
continuing to decrease these.
Confocal means having the same foci. In general two lenses are ar-
ranged to focus on the same point. Because of pinholes, or apertures, on
each side of the specimen, as shown in Figures 8.1 and 8.2, only a very

177
178 Chapter 8

FOCUS
LENS
'
COLLECTED
UGifr

ILLUMINATION
&DETECTION
APERTURES

ABOVE BELON

FIGURE 8.1. Diagram illustrating the confocal principle. An important point is the aperture
labeled illumination and detection apertures. Lines emanating from the plane of focus pass
through this aperture or the confocal pinhole, which is at the center. Rays from above or
below the plane of focus are intercepted by the solid material surrounding the aperture. (2)

small volume is focused at any time. Light from only the actual plane of
focus enters the eyepiece, which eliminates practically all of glare and
scattered light. Fruition of the technique depends on recent developments
in many fields, mainly light microscopy, confocal imaging, scanning and
video microscopy, laser illumination, and computerized image analysisY>
The small volume of light entering at an instant does however create
problems in real-time imaging, as it does with photomicrography in scan-
ning electron microscopy.
The main difference between confocal and conventional microscopy is
that at any instant, the confocal microscope images points of light rather
than large volumes of lighU1- 3>As shown in Figures 8.1 and 8.2, a second
pinhole at the back focal plane of the objective acts as a spacial filter. This
second pinhole combined with the one in the front focal plane of the
objective permits the special features of confocal imaging.(2)
In theory the technique works in both transmission and reflection
modes, but back reflection is currently the only practical technique because
of alignment difficulties in matching conjugate components and the lack of
precision in the transmission mode. Most of the instruments are the epi-
Confocal Scanning Light Microscopy 179

Laser

Confocal
Pinholes

Objective

Objoct
e In focal Plano
0 not In Focal P1ono

FIGURE 8.2. Schematic illustrating the normal arrangement of the confocal pinholes in a
microscope. Courtesy of Leica.<23 >

illumination type, where epi-illumination means that a single objective

serves as both condenser and objective. Practical techniques have not yet
been developed to generate results of the type generated by polarized light
microscopy.
The principal difference between confocal- and conventional scanning
light microscopy is that the confocal uses a detector for points of light
rather than the traditional detector, which collects a much larger three-
dimensional volume of light. All of the unique features of confocal micro-
scopy stem from this difference_<l-3> Some confocal microscopes use con-
ventional illumination sources, while others use laser illumination. Each
technique has certain advantages over the other one which are described
in recently published surveys.<t-to)

8.2. SCANNING MODES

Scanning is required because only a small volume is illuminated at

any instant, but a signal from a larger area or volume must be collected for
a generally meaningful image. Scanning is accomplished by either beam
180 Chapter 8

scanning or stage scanning. Other techniques exist, but the three most
common scanning techniques are beam scanning by Nipkow disk scan-
ning,<1> illumination or mirror scanning, and stage scanning.<2> Relative
advantages and disadvantages of the different scanning techniques are
listed in Table 8.1.<10>

8.2.1. Nipkow Disk Scanning

Paul Nipkow (1884) discovered how to dissect an image by scanning

it in a raster pattern by using a rotating opaque disk perforated by a series
of rectangular holes arranged in a spiral. This discovery inspired early
attempts at television for the next 40 years, culminating in Jenkins's mech-
anical receiver in 1925.<1.4>
At any instant apertures on one side of the disk are illuminated and

TABLE 8.1.
Features of the Three Types of Confocal Microscopes<10>
(Nipkow)
Disk Scanning (Mirror)
Feature Stage Scanning (Tandem scanning) Beam Scanning
Light source Laser Hg Arc white light Laser
Beams One 100-500 One
Scan speed Slow (specimen mass) Very fast (Disk Medium
(limited by) rotational speed) (galvanometer
response)
Optimal Photo Detector:
Present Photomultiplier tube Cooled charge coupled Photomultiplier
device tube
Limitation Low quantum Leakage and electrical Low quantum
efficiency noise efficiency
Future Cooled or avalanche Same Same
photodiodes
Limitation Max. countrate = Same Same
106-107 I sec
Advantages On-axis optics Real-time image Best for
transmission mode White light quantitative
High detector analysis
quantum efficiency Suitable data for
digitizing
Disadvantages Slow Lacks sufficient High saturation
Vibrates specimen illumination and bleaching
Difficult to locate High detector noise Slow scanning
correct field of view Non variable pinhole speed
diameter
Confocal Scanning Light Microscopy 181

act as point light sources, while conjugate apertures on the disk's other side
serve as point detectors. Spiraling receiving perforations exclude light
emanating from points in the specimen not illuminated in the first set of
pinholes; this results in confocal illumination. This technique tends to be
limited by the relatively small amount of the light that is collected through
the pinholes, and precise mechanical alignment is necessary for relative
juxtaposition of all components and pinholes.
Advantages of disk scanning, as well as beam scanning, over stage
scanning involve being able to generate the total image from the small,
discrete volumes of light without physically moving the specimen. In-
stead, the stationary specimen is scanned by flying spots of light, so that
scanning speed is not limited by the movement of the specimen. Hence the
specimen can be observed in real time through an eyepiece,<1>which is
difficult with beam and stage scannings.

8.2.2. Beam Scanning

The stage scanning system has certain advantages over beam scan-
ning where the beam is scanned or deflected, since it is perhaps simpler to
translate the stage and all optical measurements are symmetrical about the
optic axis. <2A> If however the field of view is illuminated by beam deflection
rather than stage movement, faster scan rates can occur and specimen
preparation can be stationary. Such stability may be necessary to facilitate
specimen manipulation.

8.2.3. Stage Scanning

A major advantage of stage scanning where the specimen, and not the
light moves (as in beam scanning), is that total light intensity is much
greater and the necessary mechanical precision is not so great as with
Nipkow disk scanning.(l> Stage scanning is generally used with laser illu-
mination, but theoretically this is not necessary. However the image is not
actually generated in true real time, since the stage and specimen are
physically translated, and too much time is required for this movement to
permit real-time imaging.

8.3. ILLUMINATION

In theory any type of illumination source can be used. A commercial

microscope with illumination for both normal and confocal viewing is
182 Chapter 8

illustrated in Figure 8.3. Conventional sources provide both color and

contrast as the viewer is traditionally accustomed to them, but with a
conventional illumination source, it may be difficult to obtain sufficient
illumination without switching to a monochromatic laser source. If a laser
is used, artificial color contrast can be generated, as can be done for
scanning electron microscopy photomicrographs. Fluorescent microscopy
generally requires the use of higher intensity laser illumination than is
possible with white light because of illumination loss in exciting the
fluorescence. Confocal scanning fluorescence microscopy (CSFM) has pro-
duced a renaissance in fluorescence studies, since it essentially eliminates
out-of-focus blur.<5l
In normal microscopy scattered light is collected from both above and
below the plane of focus. This generates a fog or halo of nonfocused rays
around the true image. Such a halo does not occur with confocal micro-
scopy because of the two small apertures, and the resulting clarity of the
images is dramatic in comparison with conventional microscopy. In-

FIGURE 8.3. Illustration of a commercial confocal microscope. This model from Tracor
Northern permits the viewer to combine both confocal reflected light viewing and conven-
tional transmitted light viewing. Courtesy of Tracor Northem.<12)
 Confocal Scanning Light Microscopy 183

creased contrast and resolving power, and decrease in glare, are especially
useful in high-resolution, low-light intensity fluorescent microscopy.
These advantages are illustrated in Figures 8.4 and 8.5.
With UV or short wavelength light, epi-illumination is termed
epifluorescence. In conventional epifluorescent microscopy, the imaged
volume includes both considerable out-of-focus image as well as the in-
focus image. However with CSFM out-of-focus signals are nearly com-
pletely removed, as described earlier. This results in an image with very
high contrast, and such a possibility has generated a renewal of interest in
fluorescent microscopy, especially for biological studies. Optimized
reflection imaging in laser confocal microscopy has recently been re-
viewed.(7) Here we discussed how to decrease image degradation due to
internal microscopical reflections by using critically rotated polarizing
components in the microscope to eliminate or decrease internal, non-
image-forming reflections within the microscope. Custom selecting differ-
ent wavelengths of laser light, i.e., red, green, or blue, for different stains
is shown to have a significant influence on image quality.

8.4. COMPARING THREE TYPES OF CONFOCAL MICROSCOPY

No one type of the three practical varieties of confocal microscopes

has all of the desired features. Table 8.1 presents most of the individual

fiGURE 8.4. Mitotic in a moss protonema cell, microtubuli image. (a) Conventional
.epifluorescence and (b) confocal laser-scanning image. In conventional epifluorescence mi-
croscopy, stray light causes considerable distortion of fine details (especially with thick
samples) as shown in (a). As shown in (b) stray light is almost completely eliminated by the
confocal microscope, so that fine details become visible. Courtesy of Leica.(2a)
184 Chapter 8

FIGURE 8.5. Photomicrograph of drosophila embryo obtained by both confocal imaging

(bottom) and nonconfocal imaging Ctop). The photomicrograph shows dramatically the
power of confocal laser scanning to reject out-of-focus light. The specimen is a whole droso-
phila embryo. This is a rather thick object, roughly shaped like a football-1.5 mm by 1 mm.
The upper-half of the image is scanned nonconfocally, whereas the lower-half is in confocal
mode. It would be very difficult to quantitate the degree of improvement, since there is
almost no detail visible in the nonconfocal image. Courtesy of Leica.<28>

features. Because of low final-light collection efficiency and relative speed

of operation, most practical fluorescent microscopy is done by mirror
beam scanning. Confocal microscopy is very useful, but feasible tech-
niques are fairly new and will remain in a state of rapid evolution for a
period of time.

8.5. CONVENTIONAL, CONFOCAL, AND AXIAL RESOLUTIONS

Both lateral or horizontal and axial or vertical resolutions can be

increased with correctly used confocal microscopy, as shown in Table 8.2.
It is well known that lateral resolution increases as a function of the first
power of the NA. What is not generally known however is that resolution
differs when perpendicular to the plane of focus rather than in the plane
Confocal Scanning Light Microscopy 185

TABLE 8.2.
Practical Resolution Limits191
Conventional Confocal
Microscopical Microscopical
Numerical Horizontal Horizontal Axial Resolution
Aperture Resolution (,.un) Resolution (,.un) (,.un)
0.98 0.39 0.30 0.92
0.64 0.60 0.45 2.16
0.45 0.90 0.70 4.40
0.24 1.60 1.20 15.10
0.10 3.90 2.90 89.5

of focus. Along the axis of the microscope, above and below the focal plane
of a point source, the image resolution with a large NA is only one-half as
great as the lateral resolution.l111 In other words at high resolution theo-
retically, the axial resolution is only one-half the lateral resolution. This is
an important concept to remember when collecting vertically sequential
high-resolution images that may have to be as thin as possible.
Field depth in the specimen is also an important consideration, espe-
cially for confocal microscopy. Field depth is influenced by the light beam
spreading above and below the plane of focus, the microscopist's eye
accommodation, and magnification of the image.<111 In conventional
fluorescent and dark-field microscopy, areas above and below the sharp
plane or focus contribute light to the image. Fluorescent and light-scatter-
ing points also contribute light to a generally collected image. The ability
to reject much of this undesired light is a possible advantage of confocal
microscopy, which collects only a very thin planar image at any instant.l111
In the 1950s it was shown from information theory that lateral resolu-
tion is improved by V2 over the classical value when the field of view
becomes very small.l111 All of the preceding reasons contribute to why the
resolution of confocal microscopy is greater than that of conventional
microscopy. In addition to potential resolution, increased contrast,· spec-
imen transparency, and ease of three-dimensional optical sectioning are
also potential advantages. Total theoretical understanding however of
confocal microscopy does not yet exist.C111

8.6. DATA ACQUISITION AND PROCESSING

Powerful microcomputers, modem photomultiplier tubes, and im-

age-digitizing systems are useful combinations with the unprecedented
serial or optical sections provided by the confocal light microscope.l1.61
186 Chapter 8

Digitized image manipulation has expanded tremendously in recent years,

so that now a series of digitized planar images can be collected with a
video camera and a frame grabber (see Figure 8.6). In Figure 8.6 a set of
two-dimensional non invasive slices enters a digitized volume set called a
voxel.<6l The set requires considerable storage space by either magnetic
tape or high-capacity hard disk, and it is generally possible to exceed
storage capability with voxel data (a three-dimensional array).

8.7. SUMMARY

The confocal concept can be thought of as two microscopes with a

common optical axis, where both microscopes are focused on a common
focal plane.<8J The focused field of view is imaged in the optical viewing
tube at a second pinhole at the back focal plane of the objective. This
pinhole acts as a spatial filter. To have practical value and provide a
meaningful field of view, confocal light microscopes are of the scanning
type; that is, either the specimen is scanned or the illuminating beam is
scanned across the specimen. To generate a total image of the specimen,
the very small volume of light, termed voxel, is scanned across the spec-
imen by either deflecting the spot of light or scanning the specimen
through the illuminated volume.
Optical sectioning is now commonly used in confocal microscopes to
read compact disks.<9l It is not generally recognized that this operation is
essentially the same as that of a confocal microscope; however the stylus
on such monitors is a confocal microscope that collects information at only
a single axial point inside the plastic sheet and ignores covering surface

FIGURE 8.6. Series of confocal scans of a cell. Reprinted by permission from American
Laboratory 22, no. 11 (1990), p. 19. Copyright 1990 by International Scientific Communica-
tions.<6l
Confocal Scanning Light Microscopy 187

scratches and dust because of very thin planar images that are collected.<9>
In this manner a scanned image can be constructed that is an optical
section with very little noise outside the focused plane. Resolution is
controlled by the illumination wavelength and the NA of the objective and
the areas of the confocal pinholes.<9•10>
The scanning technique and confocal principles will theoretically
function for all standard light microscopical techniques, but they have not
yet been generally applied to more than bright-field and fluorescent mi-
croscopies.<13> Series of two-dimensional images can be collected into a
three-dimensional array, or voxel, which can be electronically acquired
and processed for three-dimensional viewing and manipulation.
 9

Micrography

9.1. MICROGRAPH: IMAGE PRODUCED BY LIGHT, ELECTRONS,

OR X RAYS

A micrograph is a reproduction of an image of an object formed by a

particular kind of microscope<1>; thus a photomicrograph is an image taken
by a light microscope and a light-sensitive material.<1•2>An electron micro-
graph is a photographic reproduction of an image formed by the action of
an electron beamY> Likewise an X-ray micrograph involves any kind of
X-ray microscopy.(3> Alternatively a microphotograph is a small, micro-
scopic photograph, requiring a lens system to view its detailsY>
There are at least two approaches to photomicrography: the artistic
approach and the scientific one. The artistic (subjective) mode is imaginative,
whereas the scientific (objective) mode is based on thought and memory.
Sometimes the purpose of a photomicrograph changes from scientific to
artistic; for instance photomicrographs produced by research and develop-
ment scientists and technologists can be exhibited and judged competi-
tively on their artistic appeal.<4>Furthermore scientific micrographs can be
used as advertisements,<S-7) greeting cards,<8>or strictly art,<8•> such as "mi-
croscapes" (imaginative landscapes or seascapes).<9>Much if not all of the
artistic appeal involves color; accordingly color is sometimes added to
scientific micrographs simply for artistic effects.<9>
However in science and technology the approach must be objective.
Photographs vividly describe objects and observations (see Figure 9.1) and
are relied on implicitly by the observer. Generally graphic descriptions of
these are interpretive illustrations of what has been repeatedly or charac-
teristically observed.
Another purpose of photography is to record a series of changes in a
specimen, such as those that occur during cooling, heating, extruding,

189
 190 Chapter 9

100J.Lm
FIGURE 9.1. Surface of wallpaper stained so that chemical pulp appears white and ground
wood appears dark. The photomicrograph tells a story.

stretching, relaxing, or immersing.<10>The action can be moderate, allowing

for snapshots<11l; fast,< 12l requiring high-speed techniques; or slow, using
time-lapse techniques. <13>
In electron microscopy, especially transmitting (rather than reflecting)
electrons, the specimen is heated by the electron beam and may change in
appearance. To show this or obtain the most accurate change as many
photomicrographs as possible are usually taken in a short time and studied
rather than studying the specimen visually (see Chapters 13-15). In any
event the story in a photomicrograph is told by the photomicrographer-
not by an armchair microscopist.
 Micrography 191

9.2. EXPERIENCE: RECORDS OF NEGATIVES

Experience is the only way of achieving satisfactory results in photo-

micrography. Keep a good record of each photographic exposure on a data
sheet, such as the one in Table 9.V6) It is especially important to record
readings of the exposure meter and the time of exposure. By correlating
these with results (to be entered in the Remarks column in Table 9.1), you
will gain enough experience to avoid having to take more than one frame
per specimen to ensure success.

9.3. IMAGINATION

Imagination helps in selecting the best combination of attributes con-

tributing to visibility: the best conditions, equipment, techniques, and
photosensitive materials to record the image both artistically and
scientifically. Above all, imagination helps combine the best of the old and
the most promising of the new.

9.4. RESOLVING POWER

Resolving power in photomicrography, as in visual work, is expressed

by NA. In both photomacrography and photography, resolving power is
expressed by f (focal length divided by the actual opening in the lens). By
neglecting the difference between the tangent, i.e., f and the sine of half the
angular aperture, the practical conversion is f = l/2NA. This estimate is
adequate for a compound microscope whose tube length is fixed. But
when photographic single-lens systems (simple microscopes) are used
with a bellows (see Figure 9.2), f varies with the ratio of image distance to
the Qbject distance (magnification), as shown in Figure 9.3. As the object
distance (Do) becomes smaller in focusing on a larger image (I), the angular
aperture (a) becomes greater; therefore NA becomes larger.

9.5. RESOLUTION WITH PHOTOMACROGRAPHIC LENSES

Resolution by means of photomacrographic lenses<14•) is as good or

better than with compound microscopical objectives because the macro-
lenses<14·16) are highly corrected for a wide field. They are particularly good
at bellows extensions of 250 mm or more, provided they have been
 TABLE 9.1.
Example of a Record of Photomicrography on Roll Film(6)
Pwpo~ Film Date
-~------
1\penure
Photo Iris Exposure
No. Objective Eyepiece Specimen Filter Voltage Diaphragm Adjustment Remarks
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
Micrography 193

FIGURE 9.2. The Polaroid MP-4 multipurpose camera system. Courtesy of Polaroid.<15)

mounted for close-up objects (the reverse of the situation where cameras
are focused on relatively distant objects). For close-up work the more
convex of the two outer glass surfaces is mounted toward the object; for
distant objects the more convex glass surface is mounted toward the pho-
tographic plane.<14l Macroobjectives of focal lengths 32 mm or shorter are
usually mounted with the standard microscopical thread.<17l If not, an
adapter is usually available for the lens board shown in Figure 9.2.
Because macrolenses are so well corrected to their extremities and
their characteristics are not modified by an eyepiece or projection piece,
they can be used with a very long bellows or even a dark room. Indeed
pictures taken this way represent a kind of photographic art.
Photomacrography with a bellows camera (see Figure 9.2) continues
to be a simple and inexpensive way of picturing macrosamples, such as
those shown in Figure 9.4. However shadows and other lighting problems
may be present; equipment and photographic films manufacturers<15l (see
Figure 3.10 for a fiber-optic cable illuminator).
194 Chapter 9

FIGURE 9.3. The NA of a macrolens system increases with magnification.<15) Courtesy of

Microscope Publications.

Photomicrography is a broad and detailed subject.<18l In the 1970s photo-

micrographs were still being taken by somehow assembling a visual mi-
croscope, camera or camera back, light source, and various accessories.
The literature reveals the high degree of microscopical success in various
scientific and technological areas; the degree and extent of failures are just
as well forgotten. Charge them to makeshift assemblies that invited vibra-
tions in the optical system, lengthy photographic exposures to slow photo-
graphic emulsions, breakable glass plates, complicated darkroom proced-
ures, etc. In the meantime various improvements were made along these
and other avenues. So-called students' microscopes, stable camera backs,
fast, roll, photographic film, or Polaroid® self-processing photographic film
are currently available.< 19•20l However professional microscopes now being
manufactured,<21 - 25l are or can be converted into photomicroscopes.<5l Figure
9.5 shows a photomicroscope fitted with a camera for 35-mm roll film<6l;
Figure 9.6 illustrates the addition of a camera back for 4 x 5 cut film.< 22l
Photomicroscopes fitted for Polaroid® self-developing, positive photomi-
crographs are available.<6l
Micrography 195

FIGURE 9.4. Photomicrograph taken simultaneously of a piece of plastic and a centimeter

scale recording the magnification.

9.6. SUMMARY

Photomicrography is the art of photographing images produced by a

microscope to provide a record for display, measurement, publication, or
future reference. Photomicrography combines aspects of photography
with the principles of microscopy with regard to both equipment and
techniques employed. The goal is a clear, bright picture of details chosen
by the microscopist that is large enough to serve its intended purpose but
has sufficient resolution to show the desired detail and in focus out to the
edges. If the photomicrograph is in black and white, proper contrast is
necessary to reveal that detail; if it is in color, there must be due regard for
correct color balance and density. Since a camera does not have the human
 196 Chapter 9

FIGURE 9.5. Binocular, monobjective microscope, with camera for 35-mm roll of film and
cabinet of controls. Courtesy of M. Abramowitz and Olympus Corporation.16l

eye's depth of focus and cannot accommodate itself, extreme measures

must be taken to obtain a flat field in the plane of the film. All this makes
photomicrography more exacting than visual microscopy, but results are
worth the additional effort; a good picture can convey more information
than thousands of words and can provide a compact way of storing results.
Photomicrographs can also be beautiful in their own right, quite apart
from their scientific content, and many have won prizes in art shows.
In the past several decades of photomicrography, camera and micro-
scope have become one: the photomicroscope. Among the advantages are
instantly switching from visual observation to photographic recording,
precise if not permanent design, mechanical stability and relief from vibra-
tions, standardized operation, and reduced training time for a technician.
Among the disadvantages are the cost of the purchase (and perhaps main-
tenance); reduced experimentation with technique by the technologist;
restricted availability for college graduate and undergraduate students
Micrography 197

FIGURE 9.6. Binocular monobjective photomicroscope, with two different camera backs:
one for "4 x 5" cut film and one for a 35-mm roll of film. Courtesy of Nikon Inc. Instrument
Group.<22>
198 Chapter 9

and certainly high school students, since the photomicroscope is used

more for industrial and subsidized research than academic research.
The photomicroscope has brought corresponding changes in photo-
graphic areas. It is designed to take 35-mm roll film and/ or Polaroid®
self-processing film. Some photomicroscopes accommodate 4 x 5 inch
sheet film and perhaps larger. Such sheet film as well as rolls can be
processed in commercial or specialized dark rooms rather than within the
microscopical realm.
 10

Contrast: Phase, Amplitude,

and Color

10.1. CONTRAST: COLORLESS AND COLOR

Contrast, as pointed out in Chapter 2, contributes to visibility, which

is next to resolution in importance. Indeed two parts of an object that are
resolved separately are not seen separately unless their images are con-
trasted against what is between them. In light microscopy we are con-
cerned with two kinds of contrast: colorless and color. In both kinds we are
also concerned with intensity, the amplitude of light waves. Intensity of
colorless contrast is in terms of black, white, and intermediate grays. This
kind of contrast comes from interference and reinforcement of light waves
originating at each point in the object but traveling different paths and
lengths through the optical system to form the final image.

10.2. INTERFERENCE: DESTRUCTIVE AND CONSTRUCTIVE

Destructive interference occurs when two waves are out of phase.<1> In

Figure 10.1 waves band c have the same length A., but they are exactly
one-half wavelength out of phase. Since they happen to have the same
amplitude, the net result is complete interference (zero intensity, i.e., dark-
ness). Constructive interference occurs when two waves are in phase. In
Figure 10.2, waves b and c still have the same length A., but they are exactly
in phase. Since they also happen to have the same amplitude, the net result
is constructive interference<2> to the extent of twice the original amplitude.
Intensity is proportional to the square of the amplitude.
A diffraction image is produced by interference and reinforcement
from various light waves from all points in the object (of specified thick-

199
200 Chapter 10

FIGURE 10.1. Two light waves band c of

the same length ). being propagated in
the same direction p and having started
exactly one-half wavelength out of phase.
At any point along the path p the net
result is complete cancellation (zero am-
plitude, Oa).

ness) along different paths within the angular aperture of the objective.
Thus by controlling angular apertures in the condenser and objective, the
microscopist can go a long way toward controlling the kind and extent of
contrast obtained in the image of a given object. In bright-field illumina-
tion<1l (see Figure 2.4) by transmitted light, control of the angular aperture
of the condenser is by means of a diaphragm, such as the well-known iris
diaphragm in Figure 10.3. It provides a variable annular stop, as shown in
Figure 10.4a.(3l With the iris diaphragm, nonscattering and poorly scatter-
ing parts or particles tend to appear brighter than highly scattering ones.
The reverse is true with a central stop, which eliminates some but not all
direct rays from the condenser into the objective, as indicated in Figure
10.4b. With this differential stop more highly scattering parts tend to
appear brighter than lesser scattering ones. This result is accomplished
most simply by inserting into the filter rack of the condenser a diaphragm
with an opaque central disk (Figure 10.5) just large enough to screen out
all direct rays to the objective when the condenser iris diaphragm is wide
open.<4l With this arrangement the field appears black, but those compo-

___
, ).

FIGURE 10.2. Two light waves b and c of the same length ). being propagated in the same
direction, but having started exactly in phase. At any point along the path p, the net result
is constructive interference (double amplitude, 2a).
Contrast 201

FIGURE 10.3. Iris diaphragm (left) (variable annular stop). Traviss type of expandable
central stop (right).

- -CONDENSER-
~~~~ -- ~ -~~~~~
ANNULAR STOP- IRIS {o) CENTRAL STOP ( b )

FIGURE 10.4. Reciprocal cones of bright-field illumination, illustrating the use of annular
(iris) stop (left) and complementary central stop (right). The shaded areas indicate portions
cut off by the particular variable stops.
202 Chapter 10

nents of the object that scatter light appear as bright spots against the dark
background.(5l As a consequence dark-field illumination is extremely con-
trasty and often very helpful in improving visibility.
If however the central disk of the diaphragm shown in Figure 10.5 is
transparent and colored, say, red and the annulus is blue, the background
appears red instead of black, and parts or particles that scatter light appear
blue instead of white. Such an arrangement is called a Rheinberg filter.(6•6•l
It produces a kind of optical staining that improves the visibility of many
transparent objects.
These are some of the many ways of obtaining contrast; they should
all be tried on any unfamiliar specimen rather than going directly to the
phase-amplitude-contrast method, which has its own problems of inter-
pretation. There are however times and circumstances when contrast by
earlier methods is inadequate, and staining or treating the specimen is
impossible or undesirable.

10.3. PHASE-AMPLITUDE CONTRAST

The phase-amplitude method of obtaining contrast builds on the use

of diaphragms for separating and recombining direct versus diffracted
rays.(s,7) Figure 10.6(5) illustrates a typical phase-amplitude system for trans-
mitted light. Kohler illumination is incident on the lower focal plane of the
condenser, where there is an annular diaphragm with an opaque central
stop. Rays through this diaphragm are focused as a hollow cone onto the
specimen.
In the back focal plane of the objective, there is a conjugate annular
diaphragm called a diffraction plate, because diffracted and undiffracted
rays strike different parts of it (see Figure 10.6). It is also called a phase plate

FIGURE 10.5. Fixed central stop, which fits under

the substage condenser.(4) The diameter of the per-
manent stop is selected according to the numerical
aperture of the objective and whether bright-field
or dark-field effects are desired.
Contrast 203

-r----.- IMAGE

r
I
I
I
I
I
I
I
I
I
J Amplltude~alterino film
; /on-annulus
11 1
deposit
phase ~ oller l ng

on annulus ~IZ · ~~2fi11ZI

Bock focal plane
of objective

PHASE PLATE
OBJECTIVE (DIFFRACTION PLATE)

CONDENSER

Lower focal
plane of
condenser
opaque
ANNULAR
DIAPHRAGM

From light source

FIGURE 10.6. Typical phase-amplitude microscopical system by transmitted light. In this

case the phase-altering material has been deposited in the annulus.(S)

because the phase relationship is altered here. If for example undeviated

rays are retarded by a transparent film of proper thickness, bright contrast
results (see Figures 10.7 and 10.8a).(7) If the phase~delay film is on the center
instead of on the annulus, dark contrast results (see Figures 10.7b, 10.8, and
10.9).(7) The actual degree of darkening or lightening depends on there-
tardation of the light waves from the object. In Figure 10.7c retardation is
204 Chapter 10

(0) (c)

-T
FIGURE 10.7. Principle of phase-contrast illumination.<6l (a) Bright-phase contrast. (b) Dark-
phase contrast. D, = direct (zero-order) wave, D, = resultant wave. (c) Phase object (with
refractive index slightly greater than that of surroundings). Courtesy of Microscope Pub-
lications.

assumed to be 0.25A. and the refractive index of the object is assumed to be

slightly greater than that of the medium,<7> If instead the refractive index of
the object is slightly lower than that of the medium, the results are re-
versed. With either a bright- or a dark-contrast phase plate, the annulus is
usually coated with a partially absorbing (very thin) film, such as silver
(Zernike) or carbon soot (Wilska),<5·7l to reduce the amplitude (intensity) of
the undiffracted direct rays, so they are commensurate with the low in-
tensity of the diffracted rays.
From the foregoing we see that a great variety of accessories and their
combinations is possible, and many are commercially available. The trend
of manufacturers has been to develop a wide variety of combinations with
interchangeable annuli on one condenser and phase- and amplitude-
modification plates built into the many objectives of various contrasts and
numerical apertures. Annuli are interchanged in the condenser by rotating
them in a turret fitted with labeled click stops. The turret must be "cen-
terable," so that a particular annulus can be made conjugate with the
corresponding objective's ringed phase plate. Coincidence may be ob-
served by means of a special compound microscope (telescope) in place of
the eyepiece. It your microscope is already fitted with a Bertrand lens (see
Chapter 7), use it instead.

10.4. PHASE-AMPLITUDE CONTRAST IN DETERMINING

REFRACTIVE INDEX

Figures 10.8a-c indicate that phase-amplitude contrast may be helpful

in determining refractive index by immersion methods, especially as
matching refractive indices between specimen and immersion medium be-
comes close, as in Figure 10.8c. Reportedly precision and accuracy in deter-
mining refractive index by the Becke test have advanced an additional dec-
imal point by means of phase-amplitude contrast.<5l The commercial
 Contrast 205

'

-11-
lpm

FIGURE 10.8. Phase contrast in practice. (a) Glass particles, bright-contrast phase.(7l (b)
Glass particles, dark-contrast phase.(7) (c) Glass particles, bright-field. Same field, same
mounting medium (Clarite) as Figures 7a and 7b.(7) Poor contrast.
206 Chapter 10

FIGURE 10.9. Head of unstained nematode worm; dark-contrast phase. Electronic flash.(7)

equipment just described is useful only with isotropic or weakly anisotrop-

ic specimens however, since the illumination is conoscopic. As discussed in
Chapter 7, the Becke test depends on strictly unidirectional rays passing
through the crystal,<7al and using conoscopic illumination violates this prin-
ciple. Figure 10.9 indicates one way out of the difficulty: (a) a clear slot for
the condenser and (b) a groove for dark contrast or a ridge (for bright con-
trast) inserted in the objective. Both the slot and the groove or ridge are at
right angles to the vibration direction of the polarized light.
The slot provides the plane that contains the vibration direction and
the {perpendicular) propagation direction of the incident light. The vibra-
tion plane is parallel to only one axis of the crystal at a time. In the one view
of the crystal mounted on the microscopical slide, we are interested in only
two axes of the crystal; they correspond to the two perpendicular positions
of extinction when the crystal is rotated between crossed polars. Therefore
the procedure is to rotate the mounted crystal between crossed polars until
one of the two perpendicular positions of extinction is reached. Remove
the analyzer and match the refractive index for light vibrating from the
Contrast

polarizer (see Figure 10.10a) with a standard liquid (i.e., of known re-
fractive index). After this is done, tum the mounted crystal to the other
(perpendicular) position of extinction, then determine the other principal
refractive index for this particular view of the crystal. This method is more
accurate than the ordinary Becke method because the accessories shown in
Figure 10.10 put more contrast into the Becke band.(7aJ
Unfortunately, such accessories may not be commercially available
for the particular microscope being used. Saylor<"> describes how to make
a negative phase strip in the laboratory; Hartshorne and Stuart<7•> show
how to make a corresponding substage slot. An alternative method in-
volves using regular phase-amplitude equipment (see Figure 10.6) but
places a slot diaphragm perpendicular to the vibration direction of the
polarizer over the substage annulus. The result is the same as before but
with much reduced intensity.!7•>
Phase-amplitude contrast is especially applicable when examining
parts or particles that differ little in refractive index from their natural or
designed mounting medium; it is also useful when examining very thin
sections. One application is sampling ultrathin sections for transmission
electron microscopy (see Chapter 14). Another application is deliberate
thinning sections to bring them within the prevailing depth of focus and
thereby improve the sharpness of the image.<9>
To examine dry ultrathin sections and other uncovered specimens in
air, objectives corrected for use without a cover glass are used, with trans-
mitted phase-contrast illumination provided by a properly diaphragmed
condenser. <9>

10.5. VARIABLE PHASE-AMPLITUDE MICROSCOPY

Microscopists employing fixed phase-amplitude plates sometimes

need bright contrast on a darker background or dark contrast on a brighter

8 8 Objectivn
Condenser._ Phase
Slat Groove Plate
orRidQI--

(a)
!bl

I Vibralian Direction
In Pdarizer

FIGURE 10.10. Special phase-amplitude plates. Substage slot (a) and directional phase plate
(b) for determining a specific refractive index of an anisotropic specimen by the Becke test. (7a)
208 Chapter 10

background, with increased or decreased contrast within a specimen. Such

specimens include unstained cells and tissues, biopsy tissues, ultrathin
sections, and any other unalterable system of poor refractive contrast. The
Polanret'M microscope provides continuously variable phase and ampli-
tude alterations. Polanret is coined from polarizing, analyzing, and re-
TM

tarding. As Figure 10.11 indicates there is a polarizer and analyzer and a

quarter-wave retardation plate. The diagram also shows a turret of four
Polazone•M plates, one for each objective: NA =0.25, NA =0.50, NA =0.66,
and NA =1.25 (oil). Each Polazone•M plate has the conjugate area and the
complementary area polarized at right angles to each other. The upper
right insert in Figure 10.11 indicates that there are four types of phase
plates (A, B, C, and D): solid area, absorbing film, stippled area, and
dielectric film. Figure 10.12 shows a pair of phase-amplitude photomicro-
graphs taken at optimum selective settings on the Polanret micro- TM

scope.<10·11>
Figure 10.13 shows another pair of phase-amplitude photomicro-
graphs: protoplasmic bridges, Diospyros discolor, taken at optimum selec-
tive settings on the Polanret'M microscope.<10>

A B C D
~~ ~ ~-~~ [_~

~ Poo..onoTM ....a eyepiece

-
relay 1
6
t==:::J
:'~/::'(' ~)!~
' 4J I ' "'-

~(ill'~- \
I'\\
·~' (\_analyzer
mi~ro~cope { [1 \ % wave plate
obJective - Q \._ polarizer
0

FIGURE 10.11. Diagram of the Polanret'" type system. Upper right insert: Cross-section
diagrams of the four types of diffraction plates. Solid area, absorbing film; cross hatched area,
dielectric film. <10>
Contrast 209

lO,..m

FIGURE 10.12. Diatom, Pinnularia sp., photomicrographed with Polanret'" variable phase-
amplitude microscope.<10>(a) Bright-contrast plate A, dial setting 0.3; retardation setting 9.25
A.. (b) Dark-contrast plate B, dial setting 0.2; retardation setting 0.25 >..<10>

FIGURE 10.13. Protoplasmic bridges, Diospyros discolor, photomicrographed with Polanret '"
variable phase-amplitude microscope.<10>(a) Bright-contrast plate A, dial setting 0.1; retarda-
tion setting 0.5 1... (b) Dark-contrast plate B, dial setting 0.55; retardation setting 0.5 1...<10>
210 Chapter 10

10.6. MODULATION-CONTRAST MICROSCOPY

Chapter 2 discusses the use of oblique illumination to increase resolu-

tion and unidirectional oblique illumination to increase contrast. In our
present discussion of the PolanrefM phase-amplitude microscope, variably
crossing polars<1> is mentioned as a means of modulating the intensity of a
light beam.
The novelty of Hoffman's system<12•13> for modulating contrast lies in
the variety of effects obtainable by using a sliding, rotatable, slit dia-
phragm of variable width, all fitted under the condenser. The variable slit
is imaged in the back focal plane of the objective, where the modulator is
located with its permanently dark, gray, and bright segments. Positioning
and adjusting these components must be carefully controlled if optimum
visibility is to be achieved.<12> Indeed the components must be adapted and
especially fitted to the particular manufacturer's model.<13>
Figure 10.14<13> illustrates the general principles of modulation con-
trast: P1 represents a rotatable polarizer of incident light; behind the po-

PORTION OF THE SLIT

CONTROLLED BY pi and p2

G 8

D
MODULATOR
•*E·~!CI ===::J
OBJECTIVE
c:::::::::==:::
SPECIMEN

CONDENSER
c:::::::::==:::
SLIT
p l11:m •
2

UlllllllijlQ?Zld

FIGURE 10.14. A diagram showing components for converting a bright-field microscope to

a modulation-contrast microscope. The left side shows the modulator regions; dark (D), gray
(G), and bright (B). The right side shows the slit image correctly registered and superimposed
on the modulator. P1 and P2 are polars.!13l
 Contrast 211

larizer P1 and in front of the condenser is a sliding slit aperture with a

second polar P2 covering a variable part of the slit. The combination of the
slit and P1 is a rotatable slide. The modulator with its dark region (D), gray
region (G), and bright region (B) is placed in the back focal plane of the
objective.
Centered Kohler's illumination (see Chapter 2) is used in the system.
The modulator (see Figure 10.14) is viewed through a telescope or eyepiece
and a Bertrand lens (see Chapter 5). The slit plus polarizer P2 are slid into
place and oriented so that their image is superimposed on the modulator,
as shown in Figure 10.14. The width of the P2 area (cross-hatched) is
controlled by the degree to which the operator slides P2 • The darkness of
the P2 area is controlled by rotating the polarizer P2• Focusing the slit and
P2 is performed with the condenser. Finally, the telescope (or Bertrand
lens) is removed, and the eyepiece is replaced (or left in).<13>
The following figures show what can be done with modulated con-
trast. Figure 10.15 is a fresh human cheek cell, showing the three-dimen-

FIGURE 10.15. Modulation-contrast image of the surface of a fresh human cheek cell show-
ing the three-dimensional appearance of bacteria (B), membrane folds (M), cell folds (C), and
small particles (P). Taken with a 100x Neofluar objective.<13l Courtesy of Robert Hoffman
(1977).
212 Chapter 10

sional appearance manifested by the shadows alongside the bacteria and

the cell's folds.<13> Figure 10.16 shows carcinoma cells from a mouse, com-
paring (a) modulation contrast with (b) phase contrast. Figure 10.17 shows
that modulated contrast can be used on a stained section to give more
detail than that manifested by a bright field. Figure 10.18 compares photo-
micrographs taken by modulation contrast (a), phase contrast (b), and
bright field (c). The indefinite halo (H) around the phase-contrast image (b)
presents difficulty in locating the edge when measuring the diameter, area,
or volume. The bright-field image (c) shows a sharp circumference but
with little contrast for micrometry. Micrograph (a), taken by modulated
contrast, gives a good sharp edge and minute detai1.<13>

10.7. DISPERSION STAINING

Optical dispersion is the variation of refractive index n with wavelength

1...<1> One way of expressing the dispersion of a particular substance is by
the nu value. v.<14>

8J,Im
FIGURE 10.16. Comparison photomicrographs using modulation contrast (a) and phase
contrast (b) to view mouse peritoneal exudate containing Ehrlich carcinoma cells (E) and a
relatively flat macrophage (M). Red blood cell (RBC), vacuole (V), and granules (G) are more
apparent in the modulation contrast view. Cell R shows multidirectional resolution of the
granules. The bright halo (BH) in phase contrast corresponds to position of optical gradients
revealed by modulation contrast. The faint halo (FH) in phase contrast is an artifactP>
Courtesy of M. Padnos and R. Hoffman.
Contrast 213

141J.m

FIGURE 10.17. Comparison of photomicrographs using modulation, contrast (a) and bright
field (b) to view a cross section of a mouse skin stained with hematoxalin and eosin.<13>The
granular nature of the secretory cells (s), hair shaft (h), and stroma (st) are clearly revealed
by modulation contrast. The nucleus (n) is clearly revealed by both systems. Courtesy of
Robert Hoffman (1977).
214 Chapter 10

111lm

FIGURE 10.18. Photomicrographs comparing the image of a rounded phase object by

modulation contrast (a), phase contrast (b), and bright field (c).l131 In (a) note the three-
dimensional appearance, image dimension, and edge detail. In (b) the edge detail is only
partially visible. The image dimension is indefinite if the bright halo (H) is considered to be
an artifact. The halo however represents structure and corresponds to optical gradients. The
bright-field image (c) is barely visible except for its extent and some edge detail. Courtesy of
M. Padnos.

v=

wherein 0, C, and F, are particular wavelengths A. in the visible spectrum,

usually chosen as follows:
D = 589 nm C=656nm F=486nm
If a specimen is immersed in a fluid of the same refractive index as its own
for a single wavelength A. and the dispersion is quite different for the two
media, either the specimen or the mounting medium will show color
contrast, depending on the position of focus. The phenomenon is called the
Christiansen effect,<5> and it is often noticed when determining the re-
fractive index by the Becke method. The Christiansen color may be natural
or fortuitous; it can also be produced purposely, and then the process
becomes a kind of optical staining. Crossman, who advocated dispersion
staining in 1949,<15>used mixtures of cinnamic aldehyde with butyl carbi-
tol<16> to vary the refractive indices to suit the specimen being optically
stained. Various other mixtures are suggested by Hartshorne and Stuart,<7a>
including a commercially available<171 series of miscible liquids. Each bottle
of liquid is labeled with the refractive index for the D line, its temperature
coefficient, and its dispersion.
McCrone and his colleagues have published data for the dispersion
staining of hundreds of minerals, chemicals, and other materials.<18•191 In
each case at a given temperature (25° C), values are given for nF (486 nm),
Contrast 215

n0 (589 nm), and nc (656 nm). NOTE: These refractive indices are for three
different liquids measured for the D line at 25° C.<7•·20> The liquids are those
whose refractive indices match that of the specimen at the specific wave-
length (color). While McCrone's tables are primarily for determinative
purposes, they also give information about optical staining to improve
contrast.
Generally speaking, liquids have greater optical dispersion than sol-
ids, as shown in Figure 10.19.<14> Therefore if a particle of a typical solid is
microscopically mounted in a typical liquid whose refractive index of the
solid in red light (nc) is greater than the (nc) for the liquid and whose
refractive index of the solid in blue light (np) is less than the np of the liquid,
as the objective is focused upward, the particle will appear red and the
liquid appears blue, as indicated in Figure 10.20.<14> The light (f..o) of match-
ing refractive index (say, n25°) will not be refracted but will be transmitted
parallel to the optical axis of the microscope. The corresponding analytical
dispersion staining "curve" (A) is shown in Figure 10.20. the abscissa is
1/'A.2 instead of 'A. (the wavelength for the matching refractive index of
liquid and solid) to straighten out the curves. In curve B in Figure 10.20,
the difference in dispersion between liquid and solid is much less than in
A. In C there is no difference. In D the solid has greater dispersion than the
liquid. The corresponding spectra seen in the back aperture of the objective
are indicated in Figure 10.21.<14>

10.8. SPECIAL ACCESSORIES

For situations A and B in Figure 10.21, a large annular stop transmits

the central rays Y, whereas a large central stop shuts them off. In situation C
in Figure 10.21 (rare), no stop can help the color contrast significantly. In

400 500 600 700

X. (nm)
FIGURE 10.19. Dispersion curves.<14l
216 Chapter 10

"0
·:;
.!:!
t:========~~~~===~
0
o::: D
Axial c:

White
Light

400 500 600 700

1/"A~ (nml

FIGURE 10.20. Refraction occuring at particle liquid interface (left) corresponding to dis-
persion staining curve A (right); other possible dispersion staining curves are also shown
(right). <14>

situation Din Figure 10.21 (unusual), the coloration is the reverse of situa-
tions A and BY4>In 1962 Malies designed a turret with two stops that is
fitted with the standard objective thread.<21 >The housing screws into a stan-
dard<22> microscope and carries an objective of NA = 0.25 (see Figure 10.22).
The turret in the housing has three openings: plain, annular stop (0.2-nm
hole), and central stop.<21 >These produce the three effects just noted.

10.9. SCHLIEREN MICROSCOPE

Schlieren are regions of varying refraction in a transparent medium,

often caused by differences in temperature or pressure, and detectable

-Eit-[f}-{ IJ
B R BR YMITE R B

A B c D
FIGURE 10.21. Objective back focal planes corresponding to dispersion staining curves in
Figure 10.20 (right). The specimen is assumed to be the one shown in Figure 10.20 (left).<14l
Contrast 217

FIGURE 10.22. Turret carrying three openings (plain,

annular stop, and central stop) over the objective, NA
= 0.25.(21> Courtesy of Microscope Publications.

especially by photographing the passage of a beam of IightY> The schlieren

microscope(23l uses stops only 10-12 11m in diameter, which makes it the
most sensitive dispersion-staining system.(14>Such small stops cannot be
used with objectives of high NA, wherein they would do the most good,
because the back focal plane of such objectives is inside the objectives.
Instead the stop is placed at the eye point of an ordinary eyepiece with an
auxiliary eyepiece placed above, as shown in Figure 10.23. The original
eyepiece now becomes a transfer lens; the new eyepiece should be a 1x
telescope. A well-corrected, high-aperture (f =1.5), short-focal-length (50-
mm) camera lens is adequate. Its mount should be 'centerable' with the
condenser's iris diaphragm. The overall tube length is 200 mm (instead of
160-170 mm). Kohler's illumination is used. After the proper schlieren stop
is placed at the eye point, the condenser's iris diaphragm is opened as far
as possible while maintaining a dark field. A high-NA objective may be
used, even an oil-immersion objective.(24l
Using dispersion staining to determine specific refractive indices of an
anisotropic substance involves no monochromator, special illuminators, or
filters, no index match with a specific liquid, and no problem with ambig-
uous movement of the Becke line. (23>It does require determining the dis-
persion-staining curve by noting the matching wavelength (Ao) for several
different liquids in the matching range. The intersection of the best line
through 'A. =589.3 nm is n0. The index nF is given on the bottle for Ao at 486.1
nm, and nc is given on the bottle for '-a at 656.3 nm.(14>
Alternatively the three '-a can be determined by mounting the spec-
imen in a Cargille liquid showing 1-o near 700 nm at room temperature.
Using the temperature coefficient of the refractive index for that liquid
enables us to calculate the index for each Ao as the temperature is raised on
a hot stage (see Chapter 12). A quantitative schlieren method is described
by Kafi and Glatt, who give a theoretical explanation of the schlieren
method.(25>
218 Chapter 10

New eyepoint

Auxilory
ocular

Schlieren stop Original eyepoint

Microscope
ocular

Objective back
focal plane Fourier plane
Objective
Object plane Substage
condenser
Schlieren aperture___..:~~:::::..- Iris

Lamp FIGURE 10.23. Light path through

condenser a microscope with a schlieren eye-
LiQht source piece.<23>

10.10. SUMMARY

If two adjacent particles of a microscopical specimen are not resolved

by the physical quality of the optical system (as explained in Chapter 2),
then they cannot be distinguished, no matter what the magnification. And
if they are resolved but do not stand out against the background with
sufficient contrast, they still cannot be seen. This second situation arises fre-
quently when examining biological tissue if the watery material differs
very little in refractive index from the watery medium in which it is sus-
pended. In such instances the microscopist can gain the necessary contrast
by resorting to phase contrast, phase-amplitude modulation, color contrast,
continuously variable contrast modulation, or dispersion staining.
Since the diffraction image of an object is produced by various degrees
of destructive interference and reinforcement of the light waves from all
Contrast 219

parts of the object included in the angular aperture of the objective, one
way of varying the contrast is to vary the paths of light within that cone.
Closing down the iris diaphragm of the condenser reduces the prominence
of highly scattering parts of the object and thus emphasizes nonscattering
transparent parts. Conversely inserting a diaphragm with a small opaque
disk into the condenser (with a wide-open iris diaphragm) cuts off some
direct rays and emphasizes wide-angle rays from highly scattering parts.
The extreme of this technique involves cutting off all direct rays and
producing dark-field illumination, so that the opaque or highly scattering
particles in the object appear bright against a black background.
A variation of this technique uses multicolored diaphragms (Rhein-
berg filters) instead of opaque stops. For example a condenser diaphragm
with a red transparent center and a blue annulus around it produces a red
background against which any highly scattering particles appear to be
blue dots. Phase-amplitude contrast is achieved by a more elaborate ar-
rangement in which a condenser diaphragm with an opaque central stop
is complemented by a phase plate, a small diaphragm inserted in the back
focal plane of the objective. The phase plate has a film of material depos-
ited on it that delays light rays passing through it, e.g., by one-quarter of
a cycle, so that the delayed rays interfere with the direct rays and enhance
the contrast. If the phase-delay film is deposited on the annulus of the
phase plate (and the refractive index of the object is greater than that of the
medium), the object appears brighter than its background (the mounting
medium). If the phase-delay film is deposited on the central portion of the
phase plate instead, then the object appears darker than its background. In
either case interference caused by phase delay reduces intensity, so a
compensating reduction of intensity in the oblique rays is achieved by
depositing a very thin film of silver or carbon on the annulus film. The
combined effect is then phase-amplitude contrast.
There are many devices and accessories designed to achieve phase
contrast conveniently, all of which involve modifying the condenser and
the objective to include the necessary diaphragms. An arrangement that
allows quick change from bright-field to dark-field to phase-contrast illu-
mination with parfocal objectives is preferred. All devices require provi-
sion for centering the complementary diaphragms. Once the chosen equip-
ment is installed, phase-amplitude contrast can be used to improve the
accuracy and precision of determining refractive indices.
There is call for continuously variable alteration of phase and ampli-
tude. The Polanret'M-type microscope obtains the desired variability by a
system of regular polars, a turret of Polazone rM phase-amplitude polar
plates, a turret of objectives, and dial settings on a specialized microscope
for varying either bright or dark contrast.
220 Chapter 10

The Hoffman system for modulating contrast custom fits a set of only
three added units: a polarizer for the incident light; a rotatable holder for
a sliding slit with a partial polar to fit under the condenser; and a mod-
ulator disk with dark, gray, and bright areas to fit in the back focal plane
of the objective. The result is directional oblique illumination with mod-
ulated contrast between image and background. Such an arrangement
provides modulation shadows and sharp outlines without halos, a very
three-dimensional effect. Comparing the modulated contrast image with
that of a bright field or phase amplitude is easy.
Dispersion staining takes advantage of the Christiansen color effect that
is sometimes noticed when determining the refractive index for white light
by the Becke method (see Chapter 6). The Christiansen color is manifested
when the variation of the refractive index with the wavelength in white
light is different (usually greater) for the immersion liquid than for the
immersed solid. If for a typical example the refractive index in red light is
greater for the solid than for the liquid and if the index in blue light is less
for the solid than for the liquid and the microscopical objective is focused
upward, the solid will appear red and the liquid blue. In recent years dis-
persion staining has gained usage for determinative purposes by virtue of
the commercial appearance of the required microscopical accessories and
standardized immersion liquids of high dispersive power.
 11

Interference Microscopy

11.1. INTERFERENCE OF TWO WHOLE BEAMS

In Chapter 11 we are essentially concerned with two whole micro-

scopical beams (rather than individual rays) that are deliberately caused to
interfere with each other. The graphic result is a pattern of interference
fringes analogous to Newton's rings.(l) With incident white light, the
fringes are from Newton's series of color bands more or less superimposed
on the pictorial image. Figure 11.1 for example shows three different
micrographs of the same surface area of crystalline grains.<Z> All three
micrographs were taken on the same simple microscopical interferometer,
shown schematically in Figure 2. Micrograph (a) in Figure 11.1 was taken
with practically no tilting angle a to the reference surface [see reference
reflector (4) in Figure 11.2]; hence there was practically no interference.
Incidentally the reference beam was sufficiently out of phase with the
specimen's beam to produce interference contrast. Thus interference micro-
scopy is related to phase-amplitude contrast (see Chapter 10).
Photomicrographs (b) and (c) in Figure 11.1 show definite interference
bands. They are contour bands, parallel within grain boundaries but dis-
jointed at the boundaries because the grain surfaces are at uneven levels
due to etching rates corresponding to crystallographic orientations of the
three-dimensional grains. Differences in detectable surface level among
grains are slight, varying from a fraction of a wavelength to 10 J.tm (1958)<1>
to 0.5 J.tm (1970).<3>While interferometry is a delicate kind of vertical mi-
crometry, for purposes of explaining the significance of interference
fringes as contour lines or bands, our model (see Figure 11.3) is crudely
made for the sake of simplicity. Nevertheless the stepwise elevation in-
dicated in the side view (a) of Figure 11.3 is suggested by the microsteps
on the faces of some crystals, which show a plan view similar to that of the
model (b).<4>

221
222 Chapter 11

FIGURE 11.1. Three micrographs of the same surface area of crystalline grains, (a) inter-
ference contrast, (b) broad bands, and (c) narrow bands, taken on the same interference
microscope (see Figure 11.2) but with the interfering reference beam at a tilt angle a varying
from zero for (a) to a larger angle in (b) and largest in (c), so that band width varies from
infinity in (a) to narrowest in (c).<2>Taken through Zeiss optics. Courtesy of Carl Zeiss.

11.2. TYPES OF INTERFERENCE MICROSCOPES

11.2.1. Single Microscopes

In Figure 11.2,<2>light from the source (1) is split into two beams by the
semitransparent mirror (2). One beam is reflected from a reference reflector
(4) and a semitransparent reflector (2) into the simple or compound micro-
scope (5). Along the way to the screen (6) (or eye), the reference beam

FIGURE 11.2. Schematic diagram of a typical beam-splitting interference system.<Z>

Interference Microscopy 223

(O) (b)

FIGURE 11.3. Model of a stepwise pyramidal elevation: (a) side view showing elevation and
(b) plan view showing contour lines.<4l

interferes with the beam from the specimen (3), producing interference
images, such as are shown in Figure 11.1.<2>
While the single-lens system in Figure 11.2 belongs to a simple micro-
scope with the semitransparent reflector (2) in front of the lens system, a
prototype interference microscope is based on a microscope fitted with a
vertical illuminator (see Chapter 4) and an optically flat glass plate in close
contact with the specimen surface. The surface of a metallic specimen is too
reflecting when compared with the surface of the glass plate, so the glass
plate must be given a semireflecting coating of commensurate reflectance
with silver or other metal. With monochromatic light, adjacent fringes
correspond to differences in depth of half a wavelength, so that even
shallower depths can be measured. This principle has been modified by
Mirau, who converted the glass plate into a beam splitter, so that one beam
is directed to a separate metal-coated reference surface while the second
beam is reflected from the surface under examination, as shown in Figure
11.4<5> The Watson manufacturers have converted the system into a unit
objective by bringing the illumination into the side of the objective instead
of the side of the microscope's tube. Therefore the semireflecting surface in
Figure 11.4, or the equivalent, must be removed if the microscope is fitted
with a vertical illuminator. As shown in Figure 11.5, the Watson inter-
ference objective fits on almost any microscope equipped with a standard
thread. <6> The light from a sodium lamp (no filter) or mercury lamp (plus
D filter) enters the collimating condenser Con the side of the objective. The
beam splitter E sends some light down to the specimen (and then through
the objective system 0). The beam splitter E sends its second beam to the
reference mirror L. The reference beam from L is aimed back to the beam
splitter E by adjusting screws G, H, and K, so that the reference beam is
reflected into the objective system 0. There the reference beam interferes
224 Chapter 11

From i<¥1f
sol6Ce

FIGURE 11.4. Mirau interference objective principle.<5l Courtesy of Microscope Publications.

B (f~sRMS)

A ( I<!U'ted lodlinQ 0 (objective

nng) system )

C (~ knl system)

E (beam sp6tter)

FIGURE 11.5. Watson interference objective. (Drawing adapted from illustration courtesy
the Watson Microscopy Division of M. E. L. Equipment Company.)<5l Courtesy of Microscope
Publications.
 Interference Microscopy 225

with the beam from the object to produce tell-tale fringes depicting the
specimen's surface. Shutter F shuts off the interfering beam when the
specimen is to be observed without the interference fringes.<5>
These single beam-splitting interference microscopes (see Figures
11.2, 11.4, and 11.5) have been used in examining metallic surfaces and
edges (such as razor blades) and thin films (such as lacquers and electron-
microscopical substrata). Thin biological specimens can be studied in simi-
lar fashion by mounting them between half-metallized optical flats. In this
case internal structural features of the specimen determine local resistance
to cutting, and so features are reflected in the surface topography of the
thin section.<1>
In the interference microscopes illustrated in Figures 11.2 and 11.4, the
specimen surface and reference surface are separated by a few centimeters
so the two wavefronts can be separated by as much. However another way
of separating the two wavefronts employs a doubly refracting substance,
such as calcite (see Chapter 5). Then separating the two wavefronts is
reduced to as little as the resolving power of the light microscope (...0.2 JAm;
see Chapter 2). Such fine beam splitting is called differential interference
contrast or differential separation of the wavefronts.<2> Figure 11.6 shows a

Analyzer

Wollaston
prism 2

Ob jecl ive

Specimen

Condenser

Wollaston
prism 1

FIGURE 11.6. Schematic diagram of a two-beam in- Polor\zer

t~rference microscope for differential interference con-
trast in transmitted light. <2> (Separation between the
Lamp f ield stop
two wavefronts is exaggerated.)
226 Chapter 11

schematic diagram of differential beam splitting by transmitted light, and

Figure 11.7 shows beam splitting by reflected light.<2>
Both microscopes (see Figures 11.6 and 11.7) feature the Wollaston
prism as modified by Nomarski.<2>This prism receives polarized light, then
splits it into two wavefronts, separated by only 1 ~m (exaggerated in
Figures 11.6 and 11.7). The Zeiss-Nomarski equipment is designed to
complement the Zeiss phase-amplitude contrast equipment. This inter-
changeability is in itself an advantage; at the same time it emphasizes that
the chief purpose of this type of interference equipment is to gain contrast.
Such contrast is gained without loss in resolution or optical sectionability;
at the same time there are no troublesome halos and no loss in path length.
The brilliance of colors is especially striking by reflected light.<2> The Nom-
arski method is also recommended for examining polymer crystals, which
are otherwise low in contrast<4> because they differ so little from their
surroundings in refractive index.
The Baker interference microscope incorporates a continuously vari-
able-phase difference between images in and out of focus, as produced by
doubly refractive rotatable plates. The image is in color contrast, which
shows very small differences in thickness and refractive index. Like the
Nomarski microscope, the American Optical Baker interference micro-
scope is recommended for the contrast it provides to specimens low in
contrast<4> (see Figure 11.8).

WoHoston
prism Bean splitter

~Objective FIGURE 11.7. Schematic diagram of

a reflected light system for differen-
Reflecting/
object f : I tial interference contrast micro-
scopy.<2> (Separation between the two
wavefronts is exaggerated.)
Interference Microscopy 227

FIGURE 11.8. Taken with the A-0 Baker interference microscope. Contrast can be changed
to show desired detail, as in these photomicrographs of unstained protozoa-part of a series
used in the Scientific American and on educational television. Courtesy of American Optical
Co. and R. W. Richards.

11.2.2. Mach-Zehnder Systems

The double interference-contrast microscope, devised by Linnik, is

built like Michelson's interferometer.(!) The illuminating beam is split and
sent into two microscopes: one for the specimen and the duplicate for the
reference beam. In the Leitz interference microscope designed by Mach
and Zehnder, the two beams are separated by 62 mmP> The isolation of the
two beams removes all concern about the identity of the specimen area and
the reference area. An anisotropic specimen produces a fringe shift of a
particular magnitude and direction. If the same specimen is placed under
the other microscope, the fringe shaft is equal but opposite. (7)
An important use of this type of interference microscope is in deter-
mining refractive indices.(7•8> Since most specimens are anisotropic and
therefore have two or more specific indices, the instrument uses polarized
incident light. Part of the polarized light passes through the specimen,
which is mounted in a liquid of known refractive index and oriented at one
of the two positions of extinction. The other part of the polarized light goes
through only the standard mountant. The two beams are then combined,
and the resultant retardations appear as amplitude differences in the im-
age. These are measured by means of a built-in compensator, such as the
Senarmont type. If the thickness at one given point in the specimen is
known, the refractive index can be calculated. Or the specimen can be
228 Chapter 11

mounted in a second standard liquid and the second retardation can be

observed as before. Then it becomes possible to calculate the refractive
index without knowing the thickness. This method gives a more accurate
value for the refractive index than the Becke method. Besides interferom-
etry gives the average value through the cross section of the specimen,
while the Becke method gives the refractive index of the superficial layer.
Therefore if the specimen is a coated (e.g., weathered) crystal or a fiber
with a characteristic skin, values from the two different methods are dif-
ferent and thereby determinative.
Another brand of interference microscope that has had wide accep-
tance in recent years is manufactured by aus Jena. Its split beams do not
go through two essentially structurally independent microscopes as does
the Leitz interference microscope previously described. This means that
the aus Jena model cannot be used for examining large samples as can the
Leitz, but the aus Jus model is simpler, easier to maintain, and less ex-
pensive. It is effectively used for fields of view of 200 J.tm or less. The a us
Jena model does use a Mach-Zehnder interferometer, as does the Leitz
model, which has not been manufactured for a considerable number of
years.
The aus Jena model uses a shearing method to separate the two
mutually polarized beams to generate two images of the same object, and
a small Mach-Zehnder interferometer is located on the image side of the
optical path. Like most true interference microscopes, this microscope
determines refractive indices to an accuracy of 2 x 10-4, path lengths of a
few nm up to 5 J.tm, as well as precise measurement of contact angles. <9>The
initial beam into the specimen is generally polarized by diffraction from a
slit in the front focal plane of the microscope.<9•10l This instrument functions
through a Mach-Zehnder interferometer composed of a pair of Wollaston
prisms on the image side of the objective, and here the beam is converted
into two beams that are mutually polarized by the first of these two prisms.
In the rear focal plane, after the first Wollaston prism, one of the two beams
goes through a shearing prism to generate two images and then through
a wedge compensator to set the phase difference between two wavefronts;
the other beam goes through adjustable compensator plates. A diagram of
the light raypath for the aus Jena interference microscope is shown in
Figure 11.9.(9> Similar microscopes for reflected light also are produced.<9l
Basic concepts of the aus Jena microscope can be somewhat com-
plicated, but its use is straightforward after a procedure is established. In
addition to the basic reference of I<rug et al.,<10l additional references can
provide further background material. <11•12>
Interference Microscopy 229

22 21 27

FIGURE 11.9. Optical diagram of JENAVAL-interphako. 0-object; 0' ... 0"'-intermediate

images of the object; AP'-exit pupil; AP-image of exit pupil; 1-light source; 2-collector; 3-field
iris; 4-collimator; 5-slit diaphragm; 6-condenser; 7-specimen; 8-objective; 9-first tube lens;
10-relay system; 11-angled mirror; 12-interferometer prism; 13-shearing wedges; 14-com-
pensation wedges; 15,16-adjusting wedges; 17-compensating wedge; 18-measuring wedge;
19-interferometer prism; 20-second tube lens; 21-Bertrand lens; 22-deflectin~ prism; 23-field
lens; 24-imaging system; 25-eyepiece; 26-relay system; 27-photomultiplier.< l
230 Chapter 11

11.3. APPLICATIONS TO HIGHLY BIREFRINGENT SPECIMENS

If the birefringence of a man-made fiber is low, the fringe shifts for n11
and n.L are easily determined. Then if the thickness of the fiber is known,
the actual values for n11 and n.L can also be determined (see Chapter 6).
However when the fringe shift is more than three orders, the thickness of
the sample changes so rapidly at the edge of the fiber that it becomes
difficult to assign an exact order number to a fringe. To solve such a
problem, Scott<7> mounted highly birefringent fibers in a liquid of refractive
index near n11 or n.L. Using unpolarized light he obtained the Moire effect
of superimposing the interference pattern for n11 in the fiber on the inter-
ference pattern for n.L in the fiber. The resulting dark regions correspond
to places where X orders of interference at n11 overlap Y orders of inter-
ference at nv X and Y being whole numbers.
Considerable advances have been made in computer processing these
data in recent years. 13·14 In addition to the more common need to measure
radial birefringent gradients in optical fibers and textile fibers, a difficult
problem has recently occurred with high-speed production of fibers in
excess of 6000 m/ min and possible void formation under some conditions.
Voids generate form (two-phase) birefringence, which, if confounded with
general (one-phase) birefringence, can make the calculated molecular bi-
refringence invalid. This possible evaluation problem is somewhat ne-
gated by calculating the independent Lorentz density or optical den-
sity.l4,15
The refractive index of a material through the Lorenz-Lorentz rela-
tionship depends on the polarizability of all molecules in the field. 15 Op-
tical density is also an independent way of calculating the material's
internal order or density if the polymer contains voids. 16

11.4. SUMMARY

Interference microscopy provides a way of gaining contrast by super-

imposing the image of a specimen and interference rings or bands of
controllable width and density. This is accomplished by splitting the illu-
minating radiation into two separate beams, putting one beam through the
sample, introducing a controlled delay or phase difference into the refer-
ence beam, and then combining the two beams so that they interfere with
each other and produce the rings. If white light is used, the rings are like
the familiar Newton's rings in color. When monochromatic light from a
sodium lamp or a filtered mercury arc is used, the image is sharper and
more easily interpreted. Interference microscopy also provides clear per-
Interference Microscopy 231

ception of very small changes in depth or elevation in the sample, and such
changes can be measured by counting the successive interference rings.
Still further refinement of the method, using polarized light, allows precise
calculation of the refractive indices of birefringent fibers and crystals.
An interference microscope can be quite simple or extremely complex,
depending on what is required of it. The simplest arrangement splits the
illuminating beam by means of a lightly silvered semireflecting glass plate
placed at an angle of 45°, then reflects one beam from the sample while the
other beam is reflected from a reference surface that may be tilted through
small angles to introduce a phase difference. The two beams are then
recombined within the optical system of a simple or compound micro-
scope to produce the interference rings or bands in the image. Alterna-
tively the semireflector and its associated illuminating system can be con-
verted into an objective designed for vertically illuminating opaque
objects. Thin transparent objects, such as biological specimens, can be
mounted between half-silvered optical flats.
Another way of splitting the illuminating beam involves using a
prism made of a doubly refracting crystal, such as calcite (the Wollaston or
Nomarski prism). The Baker system uses doubly refractive rotatable plates
to achieve a continuously variable phase difference. It is especially useful
for examining thin crystals or thin sections of minerals.
The greatest flexibility and capability in an interference microscope is
achieved by using two separate compound microscopes, one for the object
beam and the other for the reference beam. The Leitz interference micro-
scope is an advanced example: It splits the illuminating beam into two
parallel beams by means of a prism, sends one beam through the sample
via a fully equipped compound microscope, sends the other beam through
a duplicate optical system fitted with tiltable-plate phase-delay devices
and a wedge compensator, and then combines the two beams in a dupli-
cate prism before sending them through the eyepiece. Both optical systems
are fitted with polarizers, analyzers, and all appropriate diaphragms and
stops. This arrangement allows the specimen to be moved about at will
and keeps the specimen area separate from the reference area. Further-
more the specimen can be shifted from one stage to the other, so any fringe
shift of a particular magnitude or direction caused by the sample is re-
versed when the sample is shifted to the other stage. Thus all confusion is
removed from the interpretation. A simpler microscope that provides
similar information for small samples is the aus Jena, which also uses a
Mach-Zehnder interferometer.
 12

Microscopical Stages

12.1. INTRODUCTION

Practically all of microscopy is staged in some way and to some

degree. In its simplest state a microscopical stage is a device used to hold
the specimen in a desired position in the optical path.<1>On stereoscopic,
biological, and metallographic microscopes, the fundamental stage is usu-
ally rectangular, flat, and drilled to take clamps to hold the microscopical
slide or the specimen itself. Some rectangular stages are made with built-
on mechanical stages (see Figure 12.1).<2>Otherwise there are usually tapped
holes to take the fastening screws of a mechanical stage<2•3>to move the
specimen in directions X and Y. Graduations plus their vernier scale on
rack-and-pinion scales usually allow measurements in tenths of a milli-
meterP> Some specialized mechanical stages<~> operate with micrometer
screws with vernier measurements to within hundredths of a millimeter
(see Figure 11.2). Micrometer screws can be integrated into a single mech-
anical stage so that in a single traverse of a composite specimen, paths over
separate constituents can be measured separately (see Figure 12.3),<2> This
mechanical method of areal analysis has been automated by means of
motors and counters.
All stages should be waterproof and oilproof; they should also be
reasonably resistant to solvent, chemicals, and heat. The plane of the main
stage must be exactly perpendicular to the axis of the microscope, other-
wise the optical quality is set back hundreds of years. If the microscope is
ever dropped or the stage is struck in any way, the microscope should be
carefully examined, and if damaged, it must be repaired. The central
opening should be at least 25 mm in diameter so that the condenser can be
raised level with the upper surface of the stage.(7)
Metallographic and other microscopes for thick specimens should
provide movement in the third (Z) direction (see Figures 12.3 and 12.4).

233
234

FIGURE 12.1. This biological microscope

has a built-on, graduated mechanical
stage.<2>

Polarizing microscopes should be provided with a rotatable stage,

graduated in degrees (and a vernier in tenths) at the circumference (see
Figure 12.4).<2>Centering screws should be provided unless the objectives
and condenser are "centerable." Ball bearings ensure against wear and
keep the stage from wobbling when turned. A rotatable stage should be
large enough so that the central 40 mm of a 75-mm slide can be explored
and rotated without having the slide hang over the edge of the stage. If the
rotatable shape is easily removed, a hot stage or other accessory can be
inserted in its place. A removable rotatable stage is also easily lubricated
and otherwise serviced.(7) A locking screw or other device will prevent
rotation whenever a small attached mechanical stage is used (see Figure
12.4).m
The glide stage is relatively new (see Figure 12.5).(3> It is designed to
speed up scanning or searching an entire large specimen, such as a biolog-

FIGURE 12.2. Film stretcher

equipped with graduated mi-
crometer screws and Kofler hot
stage.<4>
Microscopical Stages 235

FIGURE 12.3. Integrating stage (Leitz). Its

six micrometer screws permit the quantita-
tive approximation of six constituents or
groups of constituents in a single traverse of
the specimen. (2l

ical or crystalline culture in a Petri dish. Glide stages should provide ease
of motion on the glide plane and rotational bearings and yet be resistant
to slight unintentional pressures.
There are many special stages and holders(3,7J; some are designed to
level a polished section to be perpendicular to vertical illumination. Other
stages and holders are intended for special objects, such as a chuck for

FIGURE 12.4. Rectangular briquet fitted in a mechanical stage (Leitz) on a rotatabk stage
(Leitz).(2)
236 Chapter 12

Slots

frame

_;...-..c-;--Fosrens lo micro-
scope slond

Cenlerimo~
screws

FIGURE 12.5. Glide stage princi-

ples. (After photographs courtesy
Top View of Carl Zeiss.)(3) Courtesy of Mi-
Cenlerino Piece
croscope Publications.

bullets. (3> Chucks can be used for a variety of purposes; Figure 12.6, for
example, shows a chuck used to hold a dental drill for removing a mi-
crosample from an ore fraction.(2l The chuck in this case is on a separate
stand (coarse micromanipulator) because the rotating cable and its sheath
are too stiff to be mounted directly on the microscopical stage.
Another important use for the mechanical stage is in locating and
recording a certain position on a specimen by means of numerical co-
ordinates x and y. To return to a position, the specimen must be put back
into the mechanical stage in its original orientation of front versus back
and left versus right. A rectangular specimen, such as a microscopical slide
or a rectangular briquet (see Figure 12.3), properly labeled, presents no
orientation problem. Circular specimens need an index such as a notch
engaged by a lug. The return to a location is initiated with a low-power
objective.(2•3>
Microscopical Stages 237

FIGURE 12.6. Dental drill and micromanip-

ulator. The chuck of the dental drill is held in
a special clamp attached to a Leitz-type mi-
cromanipulator.<2>

Another way of returning to a location in a specimen is to mark the

spot with a qualitative marker (see Figure 12.7)<3>or a quantitative hardness
indenter (see Figure 12.8)<2> that fits into the standard thread.<8> The Bier-
baum scratch-hardness tester<9> can be used to mark a microscopic place.
As shown in Figure 12.9,<2> the Bierbaum tester fits a square stage, such as
that of the stereomicroscope. The test scratch can be made long enough to
be easily located and followed.

12.2. MICROMANIPULATORS

Various operations require mechanical aid in controlling the X, Y, and

Z directions of moving a microtool. Coarse movements can be provided by
ordinary rack-and-pinion assemblies, as shown in Figure 12.6. The vertical
mechanism is much like that of a simple microscopical stand on which the
X-Y movements could be added with an ordinary mechanical stage.
Somewhat finer adjustments are provided by micrometer threads of or-
dinary screw-micrometers. <>-5>

~~ Morllet Stylus Sol Scrow

Sly1ut)

FIGURE 12.7. Sheaff microobject 8 ~ A I En9rovi>9 Slylut I

marker. (After illustration courtesy of
Arthur H. Thomas Company.) Cour-
tesy of Microscope Publications.
 238 Chapter 1Z

FIGURE 12.8. Vickers-type impression

hardness tester (Eberbach). The Vickers-
shaped diamond point is attached to a
spring in a holder fitted with Society objec-
tive thread. A standardized compression of
the spring, using the microscope's fine-fo-
cusing adjustment, is indicated by a signal
light on the panel of the box.<21

Useful magnifications in the range of 300-600x require finer move-

ments in the X and Y directions. The Chambers system is based on one to
four units, two of which are shown in Figure 12.10.(31 The X andY move-
ments in each unit are provided by modified micrometer screws with
control knobs attached to flexible shafts. The shafts also absorb vibrations
from the operator's hands. Clamps for holding microtools are mounted on

FIGURE 12.9. Bierbaum scratch-hardness tester. The tester fits a square-type microscope
stage or the stand shown. The specimen is moved under the diamond point by means of a
micrometer screw. The degree of hardness is indicated by the width of a scratch as measured
microscopically.<21
Microscopical Stages 239

Coarse vertical/ [ ]
controls "" I

FIGURE 12.10. Typical operational layout, Micrometer

Chambers micromanipulators. (After photo- syrilge
graphic illustration courtesy Brinkman Instru-
ments.)(3l Courtesy of Microscope Publica-
tions. y

vertical rods for leeway in adjusting to the Z direction. Up to four units can
be clustered around a microscope. Figure 12.10 shows two mounted in a
moist chamber for biological purposes.
The Emerson rnicrornanipulator<5>works on a different principle: A
single vertical lever Goystick) controls an omnidirectional movement in the
plane of the microscopical field. The measurement of the joystick is in the
same direction as the apparent direction of motion. The extent of travel.can
be varied from about 0.025-3.2 rnrn for about 90 rnrn of travel by the hand.
A rnicroworrn gear controls movement in a horizontal arc of zoo. The
mechanical stage can be tilted about 10°. The tool holder is a tubular chuck
that allows attaching tubing for gas or electrical connections through a
hollow needle.P>

12.3. HEATABLE STAGES

Heatable (hot) stages are a necessity in direct microscopical observa-

tion of thermal transitions above room temperatures. The maximum tern-
perature to be reached is set by the materials of construction in ordinary
light microscopes: about 350° C. Stages heatable to such temperatures are
240 Chapter 12

adequate for studying most organic crystalline phases,<10•11 > fibers,< 12l many
other polymeric materials, and many inorganic crystalline transitions.(7)
Although most metals require higher temperatures for significant thermal
studies, there are parallel physical-chemical transformations among or-
ganic systems that are very instructive in the fields of metallography<13l and
ceramography. Heatable stages within the range up to 350° C can be
divided into two categories: those with long working distances and those
with short ones.<11>Working distance usually refers to the distance between
the specimen in the stage and the outside lens surface of the objective with
high-enough NAto obtain an interference figure.(7) In Chapter 7, we de-
termined that the ideal NA for interference figures is 0.85 because tables of
reference between measurements on interference figures and other optical
properties are based on anNA of 0.85.<141This value of 0.85 is not absolute,
since there are special objectives for NA .. 0.85 with longer than ordinary
working distance, such as those objectives intended for use with universal
stages (see Chapter 7).

12.3.1. Hot Stages with Long Working Distances

The Kofler hot stage is a model<10•11•15l and originated in Austria with

Reichert. The U.S. version was designed by H. Alber for the A. H. Thomas
Company. It is very useful on stereomicroscopes with long working dis-
tances,<4.Sl on chemical microscopes,<7•10•15> and wherever interference
figures are either not needed or cannot be observed under the circum-
stances. As shown in Figure 12.11,<161 the Thomas-Kofler hot stage is
housed in a metal cylinder A, fitted with metal rim B and glass cover C. The
heating coil HE (see Figure 12.12) is electrically supplied and regulated by
its variable transformer. The stage takes a half-slide (25 x 38 mm). The
half-slide can be placed within fork Fin Figures 12.11 and 12.12, so that the
specimen may be moved from outside the stage. A glass baffle D (see
Figure 12.11) can be used to ensure more uniform heating. Two thermom-
eters are supplied (30--230° C and 60--350° C), and each is precalibrated
while in its protective metal sheath M. There are many accessories, each
with its own heat capacity, so the thermometer used with a particular
assemblage of accessories should be checked with the appropriate refer-
ence melting point standards, CR (see Figure 12.11).<161 More precise mea-
surement of the temperature of the specimen can be performed by placing
a fine-wire thermocouple on the specimen just beyond the microscopical
field of view. The other end of the thermocouple is kept in ice and water,
bubbled with air to keep its temperature constant, in a Dewar flask. <51
In Figure 12.12, the Thomas-Kofler stage has its own condenser R. As
Microscopical Stages 241

....... 4 0\~I I I P

FIGURE 12.11. Accessories for the Kofler hot stage.! 16l

with a stereoscopic microscope,<4•5l this lens should be removed by taking

out the screws RS. The lens is also removed when dark-field illumination
is intended with a polarizing microscope. For dark field a Leitz objective
(UM-4, NA = 0.20) of long working distance is used.<17l The top lens of the
regular substage condenser is removed, and an adequate central stop (see
Figure 10.5) is placed in the slot under the condenser. The UM-4 objective

IP

FIGURE 12.12. Cross section of Kofler microscopical hot stage.0 6l

242 Chapter 12

has an iris diaphragm that is closed until a dark field is obtained. With this
setup of the Kofler hot stage and dark-field illumination, the growth or
decline of crystalline units, such as spherulites with variation of tempe-
rature, has been reported.<l7)
The Leitz Company<18> has produced a hot/ cold stage for temper-
atures from 360 to -20° C, which allows the use of long working objectives
(UM), originally designed for the universal stage. This provision for NA up
to 0.6 permits observation of useful interference figures.< 11>
The Mettler<19> hot stages FP2 and FP52 feature two heating coils, one
above the specimen and one underneath, thus practically eliminating the
vertical thermal gradient of a single coil. A second advantage of this model
is the use of a small fan (Pin Figure 12.13)(1 1) to circulate air inside the stage.
In the older stage, FP2, air is cooled by introducing a cold gas through a
fitting underneath the fan. In the newer model, FP52, cold gas is in-
troduced directly through a tube in the housing of the stage; otherwise the
two stages are alike.
In Figure 12.13, the specimen is moved by control knobs K. The
specimen is thermally protected by a mirrored housing 0 and a current of
air from fan P. The window Q is also a heat filter, which together with 0
and P, protects the microscope as well as the specimen. The temperature,
20-300a C, is measured by a platinum resistance thermometer in fixed
position. The automatic alternative rates of heating, 0.2° C, 2o C, or 10° C
per minute, can be preselected by push buttons overridden by another
push button. The temperature is continuously recorded digitally to within
0.1 o C at the top of the panel, while significant events (melting or other
transition points) can be recorded by pressing the appropriate button. A
special button arrests the temperature and maintains it as long as the
button remains depressed.<11> Instructions and correction factors have been
published. <20>

•
FIGURE 12.13. Mettler FP2 stage: side section (Mettler Instrument AG).<11 >
Microscopical Stages 243

There was an addition to the Mettler FP52 heatable stage: the photo-
electric sensor employed in thermal depolarization analysis (TDA) (not differ-
ential thermal analysis, DTA).<11> The photoelectric sensor records changes
in light transmission that accompany phase transitions of a specimen
heated or cooled between crossed polars<11> (preferably synchronously ro-
tating crossed polars). The most striking transition is melting to the point
where the whole field becomes dark.<21> One eyepiece of the binocular
microscope is replaced by the photoelectric sensor that signals changes in
light transmission.<11> The technique has been applied to the study of ther-
mal changes in polymers and mesomorphic systems.<21>
The mutual problem with all heatable stages having long working
distances is that ordinary objectives with sufficiently high NA for observ-
ing interference figures cannot be focused close enough to the specimen.
The use of long working objectives, such as those designed for universal
stages,<18> has been mentioned as a solution to the problem. Another more
general solution that includes using ordinary objectives employs a stage
with a short working distance.

12.3.2. Hot Stages with Short Working Distances

Hot stages heated up to 150° C can be tolerated by ordinary objectives

of nominal4-mm focal length (NA = 0.85).<11> The toleration criterion re-
quires the objectives' ability to portray interference figures not to be im-
paired by continual use with the hot stage. Thus the idea is to have the
working distance of the stage as short as that of a 4-mm objective. Hart-
shorne designed two thin heatable stages: a circular one to fit onto a
polarizing microscope with the conventional circular stage and a rectangu-
lar stage to fasten directly onto a microscope with the stationary rectangu-
lar stage.
The circular thin hot stage consists of a single brass disk about 10 em
in diameter and about 4 mm thick. The disk is recessed about 2 mm,
leaving a rim for peripheral screws and a central ring containing the axial
hole. The recess in the disk holds the nichrome heating ribbon threaded
through holes in a sheet of mica. This heating unit is insulated from the
disk by a whole sheet of mica. The disk's axial hole is tapered to the shape
of the cone of light from the condenser (about 4 mm in upper diameter
and 8 mm in lower diameter). The hot junction of the thermocouple is
made by securing copper and constantan wires separately to the central
boss with very small countersunk screws. A third mica sheet covers the
stage, and it is fastened with small screws. There are four knurled hand
nuts that attach the whole stage to the main rotatable stage of the polariz-
244 Chapter 12

ing microscope. Two knurled nuts are drilled to accommodate stage clips.
Heating is controlled by a variable transformer. The resulting voltage is
read on a millivoltmeter with a scale calibrated with the known melting
temperatures of pure crystalline standards observed to be in equilibrium
with their melts at corresponding voltages.<ll) The cold junction of the
thermocouple is immersed in ice water bubbled with air in a vacuum
flask.< 5>
The circular Hartshorne hot stage has two major limitations, both
arising from the thin design of its heating element in the metallic shell on
the microscope's stage. One limitation is that the metallic shell raises the
specimen above the substage condenser that has to furnish conoscopic
illumination to obtain an interference figure. Perhaps the stop on the
condenser's substage can be adjusted to raise the condenser sufficiently; if
not, perhaps an auxiliary lens or different condenser can be inserted. The
second limitation to the circular design is that even if the round stage is not
heated over 150° C, it may be hot enough to damage the microscope's stage
or polarizer or some other part. A solution may be to substitute a thermal
insulator, such as an asbestos composite, for the metallic shell. (If a thermal
insulator is merely stuffed under the metallic stage, the specimen is raised
even farther above the condenser).<11> In any case the polarizer should be
of a polarizing plastic, such as Polaroid®, rather than calcite, because the
former is much less expensive to replace. If the plastic sheet must be
replaced, the replacement should be a heat-resistant variety such as Po-
laroid Corporation makes.
If the microscopist wishes to go slightly higher than 150° C, select
Hartshorne's rectangular design, because the heating element hangs under
(rather than sits on) the metallic shell. Of course the rectangular model is
not rotatable. The stationary aspect means that the electrical leads are also
stationary and do not twist back and forth, as with the rotating stage. A
point to be considered is that polarizing microscopes with synchronously
rotatable polars may not be available. If such a microscope is available, the
rectangular heatable stage shown in Figure 12.14 is appropriate.(11> The
metallic shell of the rectangular stage is made of thin copper sheet (not
more than 0.8 mm), rolled to a slight concavity upward (see Figure 12.14a,
[i]), so that mounted on the microscope's stage, it will straighten out when
pulled down at the comers. A thin sheet of mica (see Figure 12.14a, [ii]) has
a central hole in which a circular cover glass rests on a thicker sheet of mica
(see Figure 12.14a, [iii]), with an extension carrying the binding posts for
the leads from the heating coil and for the thermocouple. This sheet (see
Figure 12.14a, [iii]) has a central hole about 6 mm in diameter for illumina-
tion. The heating coil is made of nichrome wire or ribbon that conducts 1
or 2 A at about 36 V. The sheet also safely and carefully carries a copper-
Microscopical Stages 245

Leads for
Cover slip
Lc;_"•mooooplo
heolinQ
(i)........ ;,,t;~ aL:nt
(ij)-:rf::~~
.:.~.- ~Pn
"\ Thermo-
(iii)-~== :} couple
leads
(y)
I
I
(ivl_/:-• "·~~w·---~--
I
(a) Section showinQ compo-
1 ne11ts separated.

Leads for
heatinQ
curre111

CID
(b) Pla11.

FIGURE 12.14. Simple thin hot stage. (After Hartshorne and Stuart.)<11l Courtesy of Micro·
scope Publications.

constantan thermocouple junction as near to the center of the stage as

possible. Another thin sheet of mica (see Figure 12.14a, [iv]) and one of
asbestos paper (see Figure 12.14a, [v]) serve as thermal insulators. They
carry holes about 6 mm in diameter for low powers and 3 mm for high
powers.<11l

12.3.3. Hot-Wire Stages

A hot wire can be used to heat a microscopical specimen directly

between the cover glass and slide. The resulting temperature gradient can
produce two or more phases of a system in a single microscopical field. For
example if we stretch a wire in a melt that freezes at a temperature within
the ordinary hot-stage range (between-300° C and room temperature) and
if we remelt the solid by flowing an electric current through the wire, we
obtain two or more phases on both sides of the wire, as shown in Figure
12.15.<11l Jones<22l uses no. 24-gauge platinum wire about 25 mm long, held
between two clips. To keep the heating wire straight, each clip is soldered
246 Chapter 12

...

FIGURE 12.15. Digrammatic representation of a field represented by a compound having

two mesophases under a hot wire. L, liquid; II, mesophase stable at the higher temperatures;
I, mesophase stable at the lower temperatures; C, crystalline phase.<11 > Courtesy of Micro-
scope Publications.

to a stiff copper wire held firmly in place. The other ends of the two copper
wires are soldered to flexible wires leading to a transformer. The slide and
cover glass containing the specimen rest on an aluminum ring to protect
the condenser from the hot slide.<11•22>
Hartshorne has elaborated on the hot-wire principle.<23> His wire stage
fits onto his polarizing microscope with its rectangular stage. The polars
rotate together instead of having a rotatable stage. As shown in Figure
12.16, the nichrome heating wire is stretched on a rectangular block A (6
mm thick) of a hard asbestos composite carrying a central hole 20 mm in

G
c'
o·

FIGURE 12.16. Hot-wire stage in plan.<23l Courtesy of Microscope Publications.

 Microscopical Stages 247

diameter. Two brass platelets C,C' are bolted to A and contain binding
posts and finger nuts D,D' and G. The nichrome wire E, even when hot, is
kept taut by a strong spring hook F. The posts Hand H' are of such a height
that the wire E just misses touching the cover glass over the specimen on
slide/. Cover glasses and slides are selected within narrow limits of thick-
ness, so that the level of wire E does not have to be readjusted. Clip L holds
the slide J tightly against cardboard strips K and K', which thermally
insulate the slide from asbestos composite base A. The temperature gra-
dient can be increased by means of a brass or copper cooling plate M,
shown in Figure 12.17. Soldered underneath the plate M is a brass shim N
the same thickness as the cover slip. The wire E is heated by about 6 V from
a variable transformer.
Jones<22> determined melting points by means of a tiny thermocouple.
Hartshome<23> calibrated a linear eyepiece scale oriented at right angles to
the heated wire. He used a compound having at least three known tran-
sition points<7•11•13> and measured the distance on the eyepiece scale be-
tween phase boundaries. (The wire's thermal gradient is not linear, and so
at least three phases are needed to calibrate the eyepiece.) The standard
and unknown specimen are mounted under separate strips of cover glass,
as indicated in Figure 12.18. The strips are melted separately under their
separated cover glasses, and any excess solidified substance is cleaned off
with a razor blade. After the strips are mounted at right angles to the hot
wire, they are heated and observed.<23>

FIGURE 12.17. Cooling plate used to increase temperature gradient. Courtesy of Microscope
Publications.
248 Chapter 12

STANDARD UNKNOWN

FIGURE 12.18. Comparison preparation

for determining transition temperatures.
Courtesy of Microscope Publications.

12.4. VERY HOT STAGES

Temperatures up to 1000° C can be obtained on a hot stage, which is

a miniature water-cooled furnace.<24> Such a furnace has been used to
observe heated samples of pyrotechnic systems.<25>The furnace is heated by
means of nichrome wire, and temperatures are determined with a plate-
type thermocouple (platinum versus 13% rhodium-platinum alloy) con-
nected to a potentiometric recorder. The sample is placed in a 6-mm
flat-bottom Inconel or platinum pan, such as is used in DT A. The pan is
placed on the thermocouple plate in the furnace, whose lid has a quartz
window. The lid can be rotated 360° while maintaining a gas-tight seal so
that condensed reaction products can be removed from view. <25>
The sample is viewed by reflected light through a stereomicroscope
fitted with a trinocular head for photomicrography. The illumination is
from a 150-W quartz-iodine light through a fiber-optics cable at an effec-
tive angle. An event marker is used to record every exposure on the
temperature record. Agfachrome® SOL professional color reversal film
(50AS) is usually employed.<25>

12.5. COLD STAGES

Cold stages are essential for studying frozen biological tissues, chem-
ical or physical fluids, emulsions<26> or suspensions,and for transitions. All
of these can be studied by light, electron, and/or X-ray microscopy.<27l
Microscopical Stages 249

FIGURE 12.19. Thermal stage on a light microscope. Courtesy of Sensortek.<28)

12.6. THERMAL STAGES: HOT AND COLD

Dual purpose, hot-cold stages are now available. Figure 12.19 shows a
modem thermal stage on a light microscope.<28) Figure 12.20 shows a close-
up of the thermal stage, and Figure 12.21 illustrates details of the thermal
stage's size and construction.<28) Figure 12.22 shows the automatic thermal

FIGURE 12.20. Close-up of thermal stage.

Courtesy of Sensortek.(2B)
250 Chapter 12

Countersinking
Check turret clearance A
Check mechanical stage clearance B and C
Maximum microscope objective diameter
for full 1/•• travel • P/1rt

NOT DRAWN TO sc:ALE

FIGURE 12.21. Detail of cold stage (not to scale). Courtesy of Sensortek.<28>

Microscopical Stages 251

FIGURE 12.22. Automatically controlled cooling and freezing unit fitted to a microtome
capable of producing precise serial sections of a frozen specimen, such as for electron
microscopy. Courtesy of Sensortek.<28>

unit fitted to a microtome capable of producing single or serial sections pre-

cisely for electron microscopy, light microscopy, or any other kind.<28>Such
a thermal unit usually fits or can be made to fit manual microtomes. The
Sensortek units need AC electricity and a trickle of water.<28>

12.7. OTHER SPECIAL CELLS AND CUVETIES

There are many qualitative and quantitative kinds of special micro-

scopical cells. Among the quantitative kinds are those of specific volume,
such as hemacytometer cells, 0.10 mm deep with coordinate squares of
0.0025 mm2 engraved on the bottom.(7)
The Nichols stage refractometer is a set of two cylindrical cells ce-
mented to a metal slide.(3> Each cell is equipped with two glass prisms (see
Figure 12.23). One set of prisms has a refractive index of 1.52, and it is for
liquids having refractive indices between 1.40-1.65. The used second set
has prisms of 1.72, and it is used for liquids with refractive index 1.85 and
higher. In calibrating a cell it is filled with liquid of a known index, and the
252 Chapter 12

Distance between
these lines is
measured

t
Light
A. CELL CONSTRUCTION B.~

FIGURE 12.23. Nichols stage refractometerPl Courtesy of Microscope Publications.

distance between two lines as seen through the microscope is measured

with an eyepiece micrometer. Ocular distances for other standards are all
plotted against the refractive index at a constant temperature to obtain the
calibration curve at that temperature.
Colorimetry can be performed to some extent under the microscope
using special cells and a photoelectrometer.!7J Polarimetry on liquids can be
performed in a polarizing microscope if it has a clear straight barrel that
will take a long-enough tube filled to a known length to contain the liquid
specimen. In this case a cap analyzer, rotatable in a ring, is used to measure
the degrees of rotation of polarized light to the left or right by the fluid in
the microscopical tube.!7l
There are also many kinds of qualitative cells, most of them supple-
mentary to the usual stage of the microscope. Illumination by radiation in
the near UV requires UV-transmitting slides and perhaps cover glasses.
Far UV or infrared radiation requires quartz or fused silica slides and cover
glasses.(7) Qualitative microscopical analysis for any elements in both the
specimen and the ordinary glass slide requires a special slide, such as a
plastic one.<29l
Orthogonal unilateral illumination, as in slit ultramicroscopy, requires a
special cell for holding the fluid to be illuminated from the side.<7l A similar
cell fitted with electrodes can be used in microelectrophoresis. !7l
Pressure cells are required in the microscopical study of some tech-
nological problems, such as those dealing with aerosol cans or polymeriza-
tion under pressure. The primary problem lies in specifying and testing the
glass windows. !30J
In biology and allied technology there are many types of microscopical
cells, chambers, and cuvettes for specimens in vivo or in vitro-stationary,
moving, or changing with time. Some fundamental designs are shown in
Figure 12.24.131> Some of these cells are to be used vertically, and therefore
the microscope is used horizontally.!31l
Microscopical Stages 253

a DD
d

. r.;·,•·····;·····.. ······
1-
.' :
-,
:·
.··
/
·._-.l ............,.,. .....,.... e
c

FIGURE 12.24. Typical cuvettes for microscopical specimens in vivo or in vitro. (Jl) (a) Support
for cover glass provided by droplets of paraffin wax (after Kuhl, 1949). (b) Wedge-shaped cell
viewed from above and from the side, designed to prevent damage to very tender specimens
by weight of the cover glass. At the left a wall of paraffin holds the edge of the cover glass;
at the right paraffin legs and pads support and hold the opposite corners of the cover glass
(after Kuhl, 1949). (c) Three-sided enclosure of special advantage for living material. The
reserve supply of water (drop extending at right) can be replaced as needed to prevent drying
the sample and unnecessary motion of the liquid (after Kuhl, 1949). (d) Staining bridge
shown in top and side views. The glass blocks are cemented to the slide, the cover glass d is
fastened to them by clumps of wax w, and p is a pipette used to transfer staining and washing
liquids (after von Wasielewski and Kiin, 1914). (e) Ring-shaped cell for larger living spec-
imens shown in top and side views. A mantel of water surrounds the ring, preventing
evaporation of the experimental liquid (after Kuhl, 1949). (f) Adjustable cell for larger spec-
imens made of glass blocks cemented to and covered by photographic film shown in top and
side views (after Kuhl, 1949). (g) Combination of moist chamber and ring cell for larger living
specimens or monitored sampling; made of base plate, cemented glass blocks, and large
cover glass fastened at the corners with paraffin (after Kuhl, 1949). (h) Above: Simple cuvette.
Below: Arrangement for constant renewal of culture medium in the cuvette. By siphon action
the liquid streams from the supply vessel V into the cuvette K and is then sucked into the
flask F (after Bode, 1954). (i) Microaquarium after Schaudinn: The cutout in the glass block
is enclosed on both sides by cover glasses D1 and D2. The blocks s used for protection (after
BeJar, 1928).
254 Chapter 12

:_:·
~ ..
.. .
. .

I I I I
f h

@-11--- - -·- - -·- - - -<}

(
IKI>~ .)

FIGURE 12.24. (Continued)

12.8. SUMMARY

Every microscope has a stage on which to examine the specimen. As

in a theater the stage can be stationary and rectangular in shape or circular
and rotatable. At the present time polarizing microscopes are usually
equipped with circular stages for rotating the specimen between the pol-
ars. However there is some advantage to the old-fashioned polarizing
microscope with a stationary rectangular stage and synchronously rota-
table polars. The rectangular stage is relatively easy to provide with X and
Y coordinate mechanical movements either at the factory or by an auxiliary
attachment. The rectangular shape is also relatively easy to fit with other
auxiliary stages, such as those for heating or cooling, micromanipulating,
testing, or otherwise treating the specimen.
Heatable stages facilitate the study of physical-chemical changes,
such as melting, crystallization, and polymorphic transformations. Com-
mercial heatable stages are appreciably thick, presumably to provide ade-
quate insulation, heat distribution, and chamber space, and therefore the
microscopical objective must have an appropriately long working dis-
Microscopical Stages 255

tance. Hot stages on the market that are limited to use with ordinary
objectives of NA =0.25 (lOx) are adequate for most work requiring heat.
However to obtain interference figures, dark-field illumination, or certain
other facilities, a higher NA is required. For these purposes special ob-
jectives can be used with both long working distance and adequate NA
(0.85) for obtaining interference figures. Such objectives are provided by
manufacturers of universal stages, and these require placing a glass hemi-
sphere about 1 em in radius above the specimen.
An alternative to thick hot stage with high NA objective of long
working distance is a hot stage thin enough to be used with ordinary
objectives of NA up to 0.85. A thin hot stage may have to be especially
built.<11>With a high-powered objective, the thin stage is not recommended
for temperatures much above 150° C without further insulation and con-
sequent compromise in purpose.
Another alternative to the thick hot stage is the hot-wire stage.<23>This
is especially designed to study phase transformations, including meso-
phases.<11> The wire can be heated to 300° C without apparent damage to
the polarizing microscope. Temperatures are measured directly with a tiny
thermocouple<5> or indirectly by observing the behavior of an adjacent
standard system of phases.<23>
Very hot stages for temperatures of 1000° C and above are really
miniature water-cooled furnaces. They require the long working distance
of an ordinary stereoscopic microscope, a simple microscope, or a tele-
scope. Some conventional hot stages for use below 300° C can be cooled by
liquid<18> or gas.<19> Cold stages specifically designed for cooling below
room temperatures are commercially available.<27l
There are many kinds of special slides, chambers, and cells used in
microscopy. Plastic slides are substituted when ordinary inorganic glass or
special silica slides are inappropriate. Hollow slides are used to hold more
liquid than will stay in place by viscosity or capillary action. To quantita-
tively determine particles in a known volume of liquid, such as blood,
hollow slides are manufactured with precise cavities, sometimes with
micrometric engravings on the bottom. The Nichols stage refractometer is
a cylindric cell containing two crisscross glass wedges of known refractive
index. When the cell is filled with a liquid of unknown refractive index, the
measured distance between two bright refraction bands indicates its re-
fractive index.
In biology there are many other types of microscopical cells, cham-
bers, and cuvettes for specimens in vivo or in vitro. Specimens can be
stationary, moving, or changing with time. Some of these cells are filled
with liquid that will run out unless the cell is kept vertical and the micro-
scope is used horizontally.
 13

Fourier Transform
Infrared Microscopy

13.1. INTRODUCTION

Infrared spectrometry and microscopy were combined in the late

1940s when all-reflective objectives became available<1.1•l; these also served
as condensers. When the entire microscopical system was made to trans-
mit the infrared spectrum, spectroscopists developed FT-IR microscopy,<2l
which allowed them to analyze samples in the milligram range. Accord-
ingly, FT-IR microscopy became highly developed in the 1980s and
1990s.<:Hil However microscopists, who routinely work with subnanogram
samples,<3l lately have found applications for what they may term IR
microscopy. <2>

13.2. EQUIPMENT

The FT-IR microscopes have two sources of illumination, one for the
IR and the other for the visible; these are used sequentially. TheIR source
is normally an incandescent ceramic rod or tube. Other special items for IR
microscopes are mirror lenses, called Cassegranian lenses, for focusing,
and beam splitters for generating interferograms over the IR spectra range
being used. A schematic for an FT-IR microscope is shown in Figure 13.1,<4>
and details of a Cassegranian objective is shown in Figure 13.2.<2>
Images with reflecting Cassegranian lens cannot equal the quality of
those from a proper transmitting lens, because reflecting objectives cannot
be corrected for chromatic aberration. A refracting lens cannot be used,
since it absorbs too much IR radiation.<2l The wavelength range generally

257
258 Chapter 13

EYEPECES
REFLECTED I.JGHT
LU.U~T~ SOURCE
<D

FROMIR
SOURCE

LOWER VARIABI.£ APERT\JRE

FIGURE 13.1. General schematic of an FT-IR microscope. Courtesy of Marcel Dekker.(4)

Fourier 'Iransform Infrared Microscopy 259

FIGURE 13.2. Ray paths through

a mirror-surfaced Cassegranian
objective. Courtesy of Royal Mi-
croscopical Society.<2>

used is 2.5--25 ...,m for the infrared to approximately 0.5 ...,m for the visible.
As with other sophisticated tests, Ff-IR microscopy can provide mislead-
ing results and therefore requires skill to be properly used.
The reader is referred to the numerous Ff-IR references for informa-
tion on general Ff-IR principles (e.g., References 9,10). Suffice it to state
here that an interferometer generates a final interference wave pattern of
suitable wavelength IR illumination. A series of mirror lenses then trans-
mits the resulting IR beam to the specimen on the stage. Specific IR fre-
quencies are absorbed, and the emitted interferogram is modified from its
original shape. Final signal modifications are functions of the absorb-
ing chemical groups. The result is the FT-IR trace, which is normally
characteristic of the viewed material.(2> A laser beam follows the same
optical path as the IR beam and serves as an internal reference for the
wavelength.
260 Chapter 13

FIGURE 13.3. An FT-IR microscope (left), shown together with a FT-IR system (right).
Courtesy of Spectra-Tech, division of Nicolet Instrument Corporation.<11 >

13.3. FT-IR MICROSCOPES

Figure 13.3 shows an IR microscope and a FT-IR system. The FT-IR

microscope can be used in either transmission or reflection modes. In
reflection, if the angle of incidence from vertical is about 80°, a surface
thickness in the range of a nanometer is analyzed, but if the angle of
incidence is on the order of 30°, then micrometer thicknesses are as-
sessed.<4·5> The amount of reflected radiation depends on the angle of
incidence, surface roughness, refraction index, and the sample's absorp-
tion properties.<4·5>
With a computer controlled X-Y specimen stage, it is possible to scan
and generate a map showing the distribution of measured components
over the observed specimen. The specimen must be flat, and the scanning
and general procedure are similar to that used for X-ray microanalysis
with electron microscopy.
Actually the two procedures of FT-IR microscopy and X-ray micro-
analysis (EDX) are quite complementary. Elemental composition can be
obtained from EDX, and the specific compound identification can be ob-
Fourier Transform Infrared Microscopy 261

tained from the FT-IR microscope. Absorption information from the part
of the spectrum below 1500 nm is especially useful. This region is often
termed the fingerprint" region because it contains the unique compo-
II

nents of the spectrum,!2>

13.4. SPECIMEN ANALYSIS

Specimen preparation is generally simple: For observation by trans-

mission the specimen should be 10 !J.m or less in thickness; if a specimen
is too thick, some of its bands can be totally absorbed. Specimens are often
squeezed to correct thickness and then sandwiched between I<Br win-
dows. Any reasonable thickness however can be used for the reflection
mode. Individual particles can be used if they are mounted on a fine point
or microtomed.
One of the more frequent complications is shown in Figure 13.4.<4>
Spectra from an acrylic fiber sample are shown where different apertures
were used for each spectrum.<4>First the specimen was embedded in poly-
styrene and microtomed from its original 20-J.tm diameter to 10-J.tm diam-
eter thickness. The spectra were obtained under identical conditions ex-
cept for changes in the aperture size to delineate better fiber from the
embedding resin. Different chemical groups are contained in the fiber and
the resin. The first spectrum with a large aperture primarily shows the
spectrum for the embedding resin, and the bottom spectrum, which con-
tains more noise, shows primarily the spectrum for the fiber. The result can
be greatly influenced by experimental conditions. Figure 13.4 illustrates
some of the insight needed to use the FT-IR microscope properly.
Infrared microscopy is increasingly useful with modern high-perfor-
mance fibers. A specific example is using FT-IR microscopy to establish
relationships between dichroic ratios, from IR microscopy, and filament
modulus for different types of Kevlar fibers.< 8>It is interesting that X-ray
orientation measurements did not discriminate among different types of
Kevlars with quite different moduli, whereas IR microscopy does. Polar-
ized IR is needed for such an application, and a schematic of the micro-
scope used is shown in Fig. 13.5.(7)
The FT-IR microscopy has been useful in a wide range of applications
for both routine and research applications. For example, pharmaceutical,
petrological, electronic, medical, biological, polymer, and forensic indus-
tries have all benefitted considerably from this new technology. Much of
the work, to date, has been conducted by spectroscopists, but micro-
scopists have recently become deeply involved, especially in forensic cases
with very small samples. The FT-IR microscopy has been used to deter-
262 Chapter 13

NON APT ACIM.IC FIBER

3250 3050 2850 2650 2450 2250 2050 1850

WAVE NUMBER

3250 3050 2850 2650 2450 2250 2050 1850

WAVE NUMBER

RED N'r ACAVliC FIER

3250 3050 2850 2650 2450 2250 2050 1850

WAVENUMBER
FIGURE 13.4. Signal modification from stray or spurious energy in an FT-IR microscope. All
three spectra are for an acrylic fiber embedded in polystyrene. Top: Fiber has wide apertures.
Middle: the fiber is defined by a single aperture. Bottom: Redundant or dual conjugate
apertures define the fiber area. Courtesy of Marcel Dekker.<4>

mine contaminants and polymers in the plastics and pharmaceutical in-

dustries<12>; it can even be used to analyze surface lubricants and surface
contaminants.
Different perspectives and backgrounds give these two groups some-
what different views of what to expect from FT-IR microscopy. The mi-
croscopists are more concerned with corroborating data with microscopi-
cal observations, whereas IR spectroscopists are more concerned with
corroborating the image with theIR spectrum.<13·14>
 Fourier Transform Infrared Microscopy 263

EYEPIECE

Aperture

lliuminator beamsplitter

1 I
I !
L. _ .............. ,

FIGURE 13.5. Paths of light in the Digilab microsampling accessory with the polarizer.
Courtesy of Marcel Dekker.(7)

13.5. SUMMARY

Applications for FT-IR microscopy are as broad as those for light

microscopy. The three sciences of light microscopy, analysis by EDX, and
FT-IR microscopy all complement each other. Each can provide additional
information for analysis. The FT-IR microscopy not only identifies ele-
264 Chapter 13

ments but also the specific molecular compounds.<5l Low atomic numbers
can easily be detected with FT-IR microscopy, whereas it is difficult with
EDX. Skill and experience are needed with light microscopy for the proper
use of FT-IR microscopy because erroneous results can be generated,<4.6l
but these can be decreased with the combination of proper light micro-
scopical applications. Resolution with FT-IR microscopy is always less
than with light microscopy because of the use of uncorrected and reflecting
lenses rather than corrected, transmitting lenses. This is due to the very
high absorption of IR radiation in a transmitting lens and the fact that a
reflecting lens cannot be corrected for chromatic aberrations over the wide
range of wavelengths that are used.
 14

Transmission Electron
Microscopy and
Electron Diffraction

14.1. ELECTRON MICROSCOPES

An electron microscope is an optical device for producing high resolu-

tion of detail in the object by a beam of electrons.*(I,Ia) There are three
principal kinds of electron microscopes, classified according to how detail
in the specimen is revealed by electrons: transmission, scanning, and emis-
sion. In the first two types, free electrons are discharged from an electron
gun to act on the atomic nuclei of the specimen, whereas in the field-
emission type, the specimen itself is the source of radiation.(!) The field-
emission microscope (discussed in Chapter 16), employs no lenses, where-
as the TEM and the SEM (see Chapter 15) employ focusable lenses.
The focusability and extremely short wavelength of electron beams
are responsible for the theoretically high resolving power of the TEM and
SEM. The rapid technological development of practical resolution, con-
trast, and other attributes contributing to visibility account for the useful-
ness of these electron microscopes. Although the NA of electronic lenses
is still relatively low, a compensating factor is their great field depth.
Electrons react with the various atomic nuclei in the object rather than with
the much larger domains in light microscopical specimens, so that electron
microscopical images give unique information. Since preparing electron
microscopical specimens is often different than preparing light micro-
scopical specimens, different changes are introduced into a specimen. This
must be taken into account when interpreting images.
*An electron is a subatomic particle having a negative charge of 4.8025 x w-IO esu and a
charge-to-mass ratio or specific charge of 5.2737 ± 0.0015 = 1017 esu/ g. (I)

265
 266 Chapter 14

In the TEM the image is formed by electrons passing through the

specimen. The resultant beam contains some of the original free electrons
that have not been changed in velocity or direction and some that have
been changed either way or both.<2>
The TEM is both a projection microscope and a photomicroscope.
Since the electron image cannot be viewed directly by the eye, the image
is projected onto a fluorescent screen, and when ready it is transmitted to
a photographic plate or film. As such the electron microscope can be
compared optically with the light projection microscope or photomicro-
graphic camera, as shown in Figure 14.1.<3>
A beam of free electrons emitted in a vacuum by a pointed filament
Fin Figure 14.1 can be condensed by an electromagnetic (or electrostatic<4>)
lens C to an even smaller spot (2 or 3 ~-tm) on the specimen S. Another
electron lens, the objective 0, focuses the transmitted beam to an inter-

Lamp filomenl _v_F

con- c
denser
Slide spectmen s
objective

_ _inter- _ _ 1
mediate
image

projector P

fluorescent
screen or M FIGURE 14.1. (left) Light-projecting microscope and
photomicro(J'aph
(right) electron microscope compared schematically.P>
Transmission Electron Microscopy and Electron Diffraction 267

mediate image I, which is enlarged by the projector lens(es) P to form the

image on the fluorescent screen or photosensitive material M.
The beam of electrons is generated and accelerated in a typical elec-
.tron gun,(l) shown in Figure 14.2,<2> The Wehnelt gun cylinder acts as a
cathode shield, and the base has a circular ap.!rture (1-3 mm in diameter),
centered with the filament's apex. Most modem electron microscopical
guns have a bias resistor applied between the cathode shield and the
filament. Such guns are actually self-biased because the negative potential
between shield and filament is produced by the flow of beam current
through the bias resistor (rather than by a battery). As in an unbiased gun,
the distance between the tip of the filament and the aperture of the gun is
adjusted to yield the most intense spot as seen on an intermediate screen
or the final screen at low magnification. With a biased gun the adjustment
is much easier. Moreover the filament tip can be focused on a tiny single
spot, resulting in much higher intensity for a given beam current. Another
distinct advantage is the relative insensitivity to fluctuations in beam
current. <2>

FILA~T

WEHELT
CYLINDER BIAS
RESISTOR

HIGH
VOLTAGE
SUPPLY
+

FIGURE 14.2. Self-biased electron gun showing lens-type constriction of the beam to a focal
spot S (a =0.50 angular aperture) (from Practical Scanning Electron Microscopy, J. I. Goldstein
and H. Yakowitz, [eds.]). New York: Plenum, 1975. Adapted from Hall.<2>
 268 Chapter 14

14.2. ELECTRON LENSES

The chief lens in transmission microscopes is the objective. It deter-

mines the degree of resolution in the image.<2l A simplified scheme of a
magnetic objective lens is illustrated in Figure 14.3.<5l Although the iron
yoke Y and electrical windings W are relatively large and externally im-
pressive, the important pole pieces P, and Ph have very precise bores B, an
aperture A, and specimen S. The specimen can be placed between the two
pole pieces, it can be slightly above the top pole piece P,. As long as the
distance above P1 is greater than a certain minimum, the distance is not
precise. So the specimen may be tilted through large angles. While there
is less angular tolerance if the specimen is placed between pole pieces,
there is great tolerance of lateral motion, a simple mechanism for inserting
the specimen, and shorter attainable focal lengths; however longer focal
lengths give better contrast. An aperture A is usually inserted to increase
contrast by removing scattered electrons; of course the smaller the aper-
ture, the lower the resolving power and the more difficulty in keeping the
microscope clean. Stigmators 51, devices to correct for spherical aberration
(usually six or eight) are placed around the space Sp and between the two
pole pieces P, and Ph to adjust electron rays along the respective azi-
muths.<6l

• • • • • • • • • •
• • • • • • • • • •
• • • • • • • • • •
• • •
w • • • • • • •
• • • • • • • • • •
• • • • • • • • • •
• • • • • • • • • •
• • • • • • • • • •
• • • • • • • • ••

FIGURE 14.3. Simplified scheme of 'lectromagnetic objective lens with iron yoke Y,
electrical windings W, pole pieces P1 ami Pb, aperture A, specimen S, space between pole
pieces Sp, stigmators s,.<5l
Transmission Electron Microscopy and Electron Diffraction 269

The projector lens is required to receive the small stream of electron

rays and produce a relatively large image. To gain more variation in
magnification, the projector lens system is usually a doublet(2l (see Figure
14.4}.(5)
The condenser forms an image of the crossover point of between the
annular rays from the aperture of the electron gun. Again a double lens
adds flexibility to the illuminating system in terms of intensity versus area
of the illuminated specimen(2l (see Figure 14.4).(5>
The Zeiss EM910 electron microscope, shown as Figure 14.5, features
automated controls designed to produce Kohler illumination.(t,taJ This
innovative system automatically selects the appropriate aperture and lens
conditions to illuminate homogeneously only the imaged area of the
specimen. Whereas in light microscopy Kohler illumination eliminates
glare from extraneous light, in electron microscopy, Kohler illumination's
main advantage has been in protecting the specimen from damage by
heat.(7)

FIGURE 14.4. Transmission electron

L FWORESCENT
microscope with double condenser
SCREEN
and double projector.(5J
PLATE
270 Chapter 14

FIGURE 14.5. Zeiss electron microscope EM910 features automated controls designed to
produce Kohler illumination. Courtesy of Carl Zeiss.(7)

14.3. RESOLVING POWER

As in light microscopes resolving power is fundamentally what you

pay for: the potential ability to distinguish between two points, expressed
as a minimum distance. Hall pointed out<2> that this reasoning is not strictly
logical, since we say that the resolving power is higher the smaller the
II

distance." He preferred the definition used in spectroscopy: Resolving

power is inversely proportional to the minimum wavelength difference
between spectrum lines.
For the approximate minimum resolving power as distance d, we bor-
row from light microscopy: d =0.51../ sin a, where A represents the wave-
length and a equals one-half the angular aperture.< 1>
Ruska<8> has estimated attainable" resolving powers for some beam
II

voltages (kV) and consequent wavelengths (A.). For the common voltages
50 and 100 kV and the especially high voltage of 1000 kV, his values are
given in Table 14.1.
High-voltage TEMs, such as the one shown in Figure 14.6, are built to
attain the limiting resolving power and test their practicability<S-10> (see
Figure 14.7).<8 >
Abbe had postulated that (in the light microscope) only one of the two
diffracted rays of the first order is necessary (in combination with the zero
order ray) to form an image of a periodic structure. Under this condition,
Transmission Electron Microscopy and Electron Diffraction 271

TABLE 14.118l

Beam Voltage (kV) Wavelength, [A. (A)] Resolving Power, [d (A)]

50 0.054 2.1
100 0.037 1.7
1000 0.009 0.7

a diffraction grating could be twice as fine as the limit resolved by axial

rays. The required double angle of aperture 2a can be achieved by tilting
the illuminating beam. Gold crystals have a periodic structure of atoms
separated by 2.06 A (or 0.21 nm), and they have been resolved by tilting
the electron beam to 2a.l2l

14.4. RESOLUTION

Resolution is what you achieve with your microscope from day to day.
Practical resolution depends on the value and constancy of the voltage as
it controls the monochromatic wavelength "A., and it is inversely propor-
tional to the angular aperture a. The objective's aperture depends not only
on the size of the diaphragm as delivered, but on the size before or after
cleaning, as the case may be. Resolution is reduced from 0.3 nm when the
objective has a focal length of 2 mm to 3.5 nm with a local length of 14 mm.

FIGURE 14.6. 750-kV electron microscope of

the Electron Microscope Section in the Caven-
dish Laboratory, Cambridge University, Cam-
bridge, England.
272 Chapter 14

FIGURE 14.7. Commercial

(drawn) polyethylene terephthal-
ate filaments etched with 42%
aqueous solution of n-propyl-
amine at 30° C, showing stress
corrosion and extremely fine
etching. Taken at 1000 kV on a
high-voltage electron micro-
scope. The filament in the lower
left is quite in focus; the central
filament manifests the halo of un-
derfocus.<10l

14.5. CONTRAST

Contrast in the electron microscopical image is a function of the nature

of the specimen and the properties and adjustment of the electron optical
system. Hall<2> takes up six factors: contour fringes, amorphous scattering,
diffraction contrast, refraction, deflecting fields, and instrumental factors.
Contour fringes are most prominent at edges bounded by free space.
They also occur at the edge of dense particles embedded completely in a
matrix of lower scattering power. At true focus contour fringes are in-
visible, but deviation from true focus affects resolution adversely.
Amorphous scattering is a function of spherical aberration resulting in
the loss of ·part of the incident intensity from the imaging beam. Such
scattering increases contrast as if a smaller diaphragm were used on the
objective. For the same reason resolution is lost as contrast is increased by
amorphous scattering from spherical aberration.l2>
Diffraction contrast occurs in crystals as an interaction among dif-
fracted and undiffracted waves, as well as multiple scattered beams. It is
a complicated phenomenon manifested as fringes at the edge, extinction
contours at grain boundaries, and curious patterns of dislocations.l2l
'li'ansmission Electron Microscopy and Electron Diffraction 273

Refraction contrast occurs at boundaries between refractive material

and a vacuum as a result of interference (phase delay) between the respec-
tive waves. The resulting phase contrast is manifested by over- or under-
focusing. 12>
Deflecting beam electrons by a magnetic or electrostatic field is an
especially important source of contrast. Boundaries between ferromagnet-
ic domains are manifested in this manner. Phenomena in electrostatic
fields in highly resistant materials are more complicated and may be
erratic.12>
The chief instrumental factors affecting contrast are the beam voltage
and using an objective aperture. Decreasing the voltage increases the
contrast, but the thickness of the specimen must be decreased accordingly;
thus there is a very practical lower limit to the voltage. Contrast can also
be increased by reducing the aperture of the objective. The limit to this
procedure is set by the degree of diffraction error to be tolerated. Another
limitation is that the smaller the aperture, the sooner it will be plugged by
contaminant<2> (chiefly a carbonaceous product of charred organic matter).
Additional contrast is generated for most biological and polymeric spec-
imens by "shadowing"-spraying at an angle with a heavy metal.
Staining with heavy metal compounds is a major technique for cre-
ating contrast within specimens that inherently have little contrast due to
their composition from mainly lighter elements.

14.6. ABERRATIONS

Aberrations in electron microscopical images can be caused by either

mechanical or optical problems. Mechanical problems arise from difficul-
ties in machining pole pieces precisely and from inhomogeneities in the
iron pieces. Either or both difficulties are manifested in astigmatism, i.e.,
appreciable lack of symmetry around the optic axis. In modem instru-
ments correction is effected by stigmators, whereby the azimuth and mag-
nitude of correcting asymmetry can be applied while the microscope is
operating. A mechanical method uses two compensating iron pieces. Their
distance from and along the optic axis can be varied until correction is
accomplished. Another method uses electrostatic electrodes equally ar-
ranged around the optic axis. The azimuth and direction of the correcting
asymmetrical field are controlled by separate potentiometers. The electro-
static method has no moving parts or vacuum seals, and the appropriate
compensating circuit can be switched in simultaneously with a change
in the beam potential. Stigmators are used on objective and condenser
lenses.<2l
274 Chapter 14

Aberrations resulting from the projector lens system do not affect

resolving power, but they can produce distortion in the final image, espe-
cially near the outer zones of the lens. The obvious safeguard is to avoid
going to the magnification limit set by the bore of a particular pole piece.
The temptation to overextend the limitations of the projection lens is not
so great in the modem two-lens projector system as in the single lens. The
two-lens projector permits a greater range of magnification without ex-
changing pole pieces.l2>
Hall lists seven optical aberrations from electron (as well as light)
lensesl2> of which axial-spherical aberration is the most important. This is
the only theoretical aberration that occurs for a point on the optical axis as
well as for all other (extra axial) points in the specimen. The explanation
is that from any point in the object, the power of the lens is greater the
longer the distance at which rays pass through the lens. Electron lenses are
always convergent; the power of outer zones of the lens is always too great
compared with that of the inner zones. Since at present there are no
divergent electron lenses to combine with convergent ones, correcting for
axial-spherical aberration cannot be accomplished with electrons as it is
with light. The tendency, therefore is to keep the angular aperture small to
reduce the aberration, and this limits the resolving power.<2>Ruska<8>and
Spetier<11>have attempted to overcome spherical aberration and attain the
theoretical limit of the TEM.
Some varieties of spherical aberration peculiar to electron projector
lenses can be corrected. They are manifested as either pincushion or barrel
distortion. In either case the image lies on a curved surface, so that dis-
tortion occurs when projected on a flat surface. Correction is by means of
a second lens that tends to neutralize the spherical aberration of the first.
The pair can be either a doublet or a two-lens system. <2>

14.7. CLEANLINESS

Cleanliness in electron microscopes involves taking precautions

against contamination and removing whatever contamination has never-
theless accumulated. A contaminant can be any product of bombardment
by the intense beam of electrons in a high vacuum. The chief kind of
contaminant is a carbonaceous product of decomposed organic matter:
commonly grease used as vacuum seals or oil leaking in as vapor from the
forepump. Grease is avoided by using greaseless seals such as dry, non-
volatile, elastomeric gaskets, metallic bellows, a grease-free specimen, and
photographicallocks. Oil is reduced by having a remote forepump with a
main valve that is kept closed until the diffusion pump has warmed up.<2>
Hydrocarbon vapors from oil, etc., decompose under electronic born-
Transmission Electron Microscopy and Electron Diffraction 275

bardment and are deposited as such or as polymers. Silicone oils may also
deposit silica. If either kind of oil vapor is trapped away from the spec-
imen, water vapor may be the chief offender by becoming ionized and
etching any organic specimen. The apparent source of water is photo-
graphic negative material,<2>which therefore should be thoroughly desic-
cated before use. In some instances contaminants are controlled by using
a trap cooled by liquid nitrogen in the microscope's column.
Of prime importance is protecting the specimen itself from contamina-
tion. Precautions should also be taken to clean scrupulously containers,
instruments, reagents, substrata, replicating materials, and room atmo-
sphere. Reagent and wash water should be freshly distilled, not stored in
a metallic tank and run through pipes.
Cleanliness also means having clean lenses, open apertures, etc., just
as in light microscopy.

14.8. FOCUS DEPTH

Focus depth means in practice how far the viewing screen or photo-
graphic plane can be moved along the microscope axis and still receive
satisfactory focus of the image. This distance is relatively long. Hall esti-
mates that the viewing screen and photographic plate or film can be
separated successfully by several inches.<2>

14.9. FOCUS

Focus is the point at which rays originating from a point in the object
converge or from which they diverge under the influence of a lens.<t> As in
light microscopy, an outline of a particle is imaged most sharply when
focus is exact, though contrast may then be poorest. Sometimes an over- or
underfocus halo is preferred or inadvertently obtained (see Figure 14.7).

14.10. ILLUMINATION

Illumination in the TEM takes place by transmitting electrons through

the specimen. The electron source is usually an incandescent filament in
the shape of a hairpin. While its relatively large size provides mechanical
stability and long life, its hairpin shape is surrounded by an asymmetrical
electrostatic and electromagnetic field. A pointed filament is much better
electron optically, but it is of course more fragile, and it has a shorter life.<2>
The condenser in the TEM serves principally to control the size and
 276 Chapter 14

therefore the intensity of the illuminated specimen field. Even higher

intensities can be obtained electronically by means of a double condenser.
The second condenser also allows a greater reduction in the area illumin-
ated, thus protecting the specimen outside the field of view from injury by
the illuminating beam.<2l
Illumination intensity is expressed in amperes per square centimeter,
i.e., in the illuminated area of the specimen. Intensity is inversely propor-
tional to the square of the magnification. Brightness is defined as the flux
per unit area per unit solid angle, usually measured in amperes per square
centimeter per steradian. <2l
Tilted illumination, as mentioned before, is a way of increasing resolv-
ing power. With the TEM it is also a method of obtaining oblique illumina-
tion or a dark-field view. Another method involves placing an eccentric
diaphragm in front of the specimen. Either way a bright image is sought
against a dark background, especially with crystalline substances, which
manifest diffraction images. <2l
Radiation illuminating the electron microscopical specimen has a con-
stant wavelength to the degree that the accelerating potential on the tung-
sten filament is kept constant.

14.11. ANISOTROPY

Anisotropy is considered in electron microscopy because the motion of

electrons is somewhat analogous to the behavior of light in anisotropic
materials. <2l Moire patterns can render the structure of crystalline and other
anisotropic specimens visible. For example, Holland(l 2l has demonstrated
symmetry in a crystal of polyethylene lying on a substrate of crystalline
polyethylene. The Moire pattern is the same in two opposite quadrants but
different from the pattern in the other pair of quadrants.

14.12. USEFUL MAGNIFICATION

Useful magnification in electron (as well as in light) microscopy refers

to the minimum magnification required to show the desired detail in a
specimen. Of course the finest detail may be beyond the practical limit of
resolution by means of electrons. In such cases it may be better to define
empty magnification, beyond which no new information is revealed.*<1l

•some empty magnification may however be advantageous in making measurementsPl

Transmission Electron Microscopy and Electron Diffraction 277

The required resolution must be in the electron micrograph before en-

largement, and the enlarger must be in sharp focus on the original micro-
graph. Moreover photographical copying procedures generally lose reso-
lution.

14.13. FIELD OF VIEW

Field, the portion of the object in view, is very small in transmission

electron microscopy-on the order of a few square micrometers at 20,000x
within the microscope. (3>At any given magnification, the field is at present
limited by a low NA (...0.002); greater apertures carry unacceptable aberra-
tions and distortions. Other limiting factors are faintness of image for
viewing or photographing, specimen susceptibility to damage from more
intense illumination, practical selection, and the number of fields to be
viewed.
Experience in field selection may come directly through repetitive
work in a specialized field, such as virology, but in broad scientific, tech-
nological, or industrialized areas, mapping out significant fields comes
from experience with macroscopy and light microscopyP>
Relevant fields selection lies within the general science and art of
sampling. The ability to discriminate a representative from a nonrepre-
sentative field takes a great deal of experience, thought, and judgment.

14.14. ARTIFACTS

An artifact is a spurious image that does not correspond directly with

the true microstructure of the original specimen.(1>Obviously artifacts can
result in serious misinterpretations. Avoiding or reducing artifacts de-
pends on understanding how and why they become visible. Some electron
microscopical artifacts represent damaging changes in the specimen dur-
ing exposure to the beam of electrons. For example films representing the
specimen, substrate, or replica may shrink, tear, or curl, if they absorb
energy from the electron beam. Textile or paper fibers or biological tissues
may swell or evolve gas, while microorganisms may change in size or
shape. (J) Observing variations in such changes with time of exposure or
intensity of the electron beam should lead to preventing artifacts. Re-
member that no electron diffraction is observed if the crystallinity has been
destroyed by impinging electrons during the preliminary viewing.
Those artifacts discussed in connection with the electron microscope
 278 Chapter 14

itself are introduced by contaminations. For instance specimens of carbon

black particles may be observed to increase,<3>resulting from the deposition
of such carbonaceous products as pump oil, gaskets, substrates, or spec-
imens themselves.!2> Another result of contamination is due to the image
drifting. If after cleaning the microscope and taking precautions against
contamination from pump oil and gaskets, such artifacts recur, changes in
the specimen preparation should be considered. If the specimen itself is
volatile, encasing it in an evaporated film of metal or another stable ele-
ment may help. At any rate changes in the specimen caused by the high
vacuum in the electron microscope should always be considered and
constantly studied. Some specimens may require thorough precondition-
ing in a vacuum comparable to the electron microscope.

14.15. DEYfH CUES

Cues to structure depth in an electron microscopical specimen include

the built-in cue of the relatively great depth of field in a reasonable focus,
about 10 J.tm, which is about 10 times the depth of high-aperture light
microscopical objectives. The result is a more realistic three-dimensional
appearance of the electron microscopical image than in high-powered light
microscopy. <3>
Shadows are very important specimen cues in a depth variation, but
they are obtained in a much different way than in light microscopy be-
cause electron microscopical lenses do not have a high aperture. Con-
sequently oblique illumination cannot be obtained in the electron micro-
scope by blocking off one side of the aperture of the condenser lens (as in
light microscopy). Instead the specimen is shadowed before it enters the
electron microscope; that is the specimen is prepared by obliquely deposit-
ing a material, usually a heavy metal, at an angle. The metal is evaporated
from an electrically heated filament in a vacuum at great enough distance
from the specimen for the metallizing atoms to approach as a beam. A
vertical projection of height h casts a shadow of length l =h/tan e, where
e is the horizontal angle of obliquity. Thus the height h can be estimated
by measuring the length of the shadow cast at a known angle e. In the
positive-electron micrograph, such shadows appear light, making depth
interpretations difficult for the observer accustomed to viewing dark shad-
ows. Therefore a negative print is usually made from a transparent pos-
itive printa> (see Figure 14.8). The print should be oriented so that the
shadows fall below the object (rather than above it), since we are ac-
customed to illumination from above (rather than below) an object.
A third cue to depth is stereoscopy, made possible by the great field
Transmission Electron Microscopy and Electron Diffraction 279

FIGURE 14.8. Polystyrene emulsion stabilized and shadowed by coating lightly with gold
(at an angle of 10°). A negative print was used to make dark shadows. Electron micrography
by Mrs. E. Gagnon Davis and E. J. Thomas, American Cyanamid Co.

depth in focus in the electron microscope. Two electron micrographs are

taken with the specimen tilted at two different angles to the axis of the
microscope. The specimens are best viewed in a stereoscope. Most com-
mercial electron microscopes are built so that the specimen can be tilted to
the appropriate angles for stereoscopy.<2>
At the present time Fourier transforms have been applied with the
TEM to image periodic structures of molecules in three dimensions with a
resolution of 7 A, nearly three times the best resolution previously attain-
able. Fourier microscopy combines data from low-contrast electron micro-
graphs with data from electron diffraction. Various views of the specimen
 280 Chapter 14

are obtained by having the specimen on a tiltable stage. From such projec-
tions an image of high resolution is constructed in three dimensions. <131

14.16. SPECIMEN THICKNESS

Specimen thickness in transmission microscopy is very important. For

optimum visibility the specimen must be thin enough to allow electrons to
penetrate some parts significantly and yet thick enough for other parts to
absorb electrons preferentially. Even atoms with a low atomic number
scatter electrons appreciably. Therefore sections of biological tissue and
other organic materials must be much thinner than the thinnest specimens
(1 f.A.m) used in light microscopy. Inorganic materials must be even thinner
if they are to transmit any electrons in contrast to a silhouette. The thinner
or smaller a specimen, the more likely it will be unable to support itself on
the grid of the stage. Supporting films are usually no thicker than 200-500
A (20-50 nm) to be sufficiently transparent to electrons.<14l
Particulate specimens, such as dusts, precipitates, pigments, and
strengthening fillers, must be sufficiently dispersed to differentiate units
from aggregates. However during preparation the size of the effective
particle should not be affected if the electron micrograph is to be inter-
preted in terms of a problem, such as lung disease, air or water pollution,
pigmentation, or particulate strengtheningP>

14.17. FIELD DEPTH

Field depth in the TEM is sufficient to ensure that the whole field in
view of an adequately thin specimen is in focus. To this extent field depth
in electron microscopy is not a problem but an advantage in visibility. Of
course field depth varies with the angular aperture 2a. While 2a is always
small in electron microscopes as compared to light microscopes, angular
aperture and therefore field depth can be varied between limits. For a
resolution d of 10 A, a is about 2.5 x 10-3 rad for beam potentials of 50-100
kV; field depth Dis 4000 A, and D/2 on the underfocused side is 2000 A
or 0.2 !!m. For a resolution of 50 A, D/2 is 1 !!m.<2l

14.18. SPECIMEN STRUCTURE

Specimen structure also influences visibility. For example a periodic

structure such as that of a single crystal of gold is resolved (d =2.04 A) when
Transmission Electron Microscopy and Electron Diffraction 281

illuminated across the periodic structure by a tilted beam at an angle equal

to the angle a of the objective. This resolution is five times the usual limit of
10 A for a nonperiodic structure. The example is simply Abbe's diffraction
principled= 'A./2 sin a when the undiffracted ray and one of the first-order
diffracted rays are both included within the angular aperture of the ob-
jective. The resulting image is really a diffraction pattern of the (gold)
crystal. It is a special example of how specimen structure affects visibility.
Nevertheless it is a practical case, since all metals and their alloys represent
periodic (crystalline) structures that can be resolved if they are illuminated
by a properly tilted and directed beam of electrons.<2> Structures of other
crystalline specimens also play an important role in visibility.<15>

14.19. SPECIMEN MORPHOLOGY

Morphology is the shape and size of particles, lines, areas, or volumes

in a structure.(l) Seeing morphology is what microscopy is all about. In-
terpreting shape is relatively easy when the size is large and other attri-
butes contributing to visibility are favorable. However some microscopists
wish to see smaller and smaller particles or parts regardless of larger
neighbors with the same composition and shape. Other microscopists are
not particularly interested in cracking the barriers of resolution, but they
have no larger relevant particles to look at. These microscopists would do
well to seek or grow larger specimens. The chemists's method of digesting
the finest particles and precipitating the resultant solute (in time) on the
larger particles may be useful, or sublimation or annealing can be tried.
Otherwise it may be worthwhile to study analogous or isomorphous spe-
cies of crystals. Likewise biologists may learn relevant morphology by
studying closely related but larger species. The point is that morphology,
true to its Greek derivation, pertains more to shape than to size.

14.20. SPECIMEN INFORMATION

Information is a very important attribute in interpreting TEM images.

If the required information does not come with the sample, the electron
microscopist should ask such questions as, What is the problem? Is there
macroscopical and light microscopical information relevant to the prob-
lem? How were the samples taken? Are they stable in a vacuum? Are they
heat sensitive? In what are they soluble and insoluble? Are there good and
bad examples present or obtainable? Old and new? If there have been
changes in environment, treatment, etc., are there examples of each?
282 Chapter 14

Similarly the microscopist should furnish adequate information about

the electron microscopical examination with every micrograph submitted
in a report. Magnifications must be accurately given and usually are, but
other essential information is sometimes omitted. Which is the top side of
the electron micrograph? Remember to orient the picture so that the illu-
mination appears to be from above if you wish the viewer to interpret
shadows of elevations and depressions as they would be seen macro-
scopically. Also remember to indicate a positive or negative print and how
you made the shadows dark. You may be educating and informing your
sponsor, and in the long run, the benefit rebounds to you. This means that
exact details of sample preparation should be given with advantages and
limitations of the method.
Procedures labeled standard should have been standardized by an
official society like the American Society for Testing and Materials
(ASTM).(16l The ASTM standards are the result of democratic deliberation
and voting by peers representing the producer on one hand and the
consumer on the other. The ASTM standards are published annually and
reconsidered at least every 5 years. The ASTM procedures are reproduc-
ible by consultants and referees, and the results constitute admissible
evidence in court. Microscopists should keep bound notebooks of informa-
tion about procedures, interpretations, illustrations, and experiments for a
filing system and maintain electron micrographs.

14.21. EXPERIMENTATION

Experimentation should be devised to test the interpretation of all

images not obtained by standard or repetitive procedures. The micro-
scopist's reputation for accurate interpretation is always at stake, and only
the microscopist knows when to take time to vary procedures in sampling,
preparing specimens, and checking for specimen contamination and ag-
ing. Other experiments involve gradient heating or cooling and chemical
or physical reactions. Admittedly electron microscopical procedures re-
quire a lot more time than procedures using light microscopy.

14.22. SPECIMEN PREPARATION

Specimen preparation is too large a topic to be discussed in procedural

detail. The reader who wishes laboratory directions is referred to mono-
graphs on the subject(tf'>-2tl and to ASTM recommended practices.(t6,22l It
must be emphasized here that any preparation of a microscopical spec-
Transmission Electron Microscopy and Electron Diffraction 283

imen changes it in some way and to some degree. In transmission electron

microscopy the primary goal is to prepare a tiny, thin specimen that will
fit into the microscope holder. In most instruments the holder is intended
for a screen or grid with about 200 openings per inch. One popular screen
shape is a disk about 3 mm in diameter.(Z.I4.17J In most cases the TEM
specimen represents a very small portion of the sample (see Chapter 20).
The first problem is whether to select a representative or nonrepre-
sentative specimen. If we are interested in contamination, impurities,
forensic traces, spots, specks, or other isolated portions, a nonrepresenta-
tive specimen is in order. In the process the original sample has been
changed drastically, so that an accurate record of the selective process
should be made. One good way of doing this is to photograph at a low
resolution an area that pinpoints the field taken at high resolution (see
Figure 14.9a and b).(21 l
If sampling for TEM is representative, there are other considerations.
Even a dry powder composed of only one phase but small and large
particles is difficult if not impossible to represent in a specimen of TEM
size.(3J If the original sample is large enough, it may be advisable to make
a quantitative size analysis macroscopically and then take random spec-
imens of the resultant size fractions for electron microscopical illustrations.
Size fractionation can be by rate of settling in a gas or liquid,(16•24l and
settling in a liquid can be by gravity or centrifugation. Liquids and some
gases can cause aggregation of particles, so unless such aggregates are

1pm

FIGURE 14.9. Transmission electron micrograph of a thin section of a cotton fiber; (a) cross
section of whole dry fiber of cotton; (b) higher resolution of an area in (a). USDA micrograph
by Southern Regional Center, New Orleans.
284 Chapter 14

penetrated by the electron beam, they will appear in silhouette and can be
mistaken for single, much larger particles. Without sufficient information
it should not be assumed that dispersion in any one preparatory liquid
duplicates commercial dispersion in a different medium. Many a false
interpretation of practical particle size or shape has been made by assuming
the morphology to be that of the ultimate<2> (finest) size of the particles
before introduction into the commercial product.
Particles often deposited directly onto a filmy substrate, such as
Formvar® polyvinyl formal resin, by settling from a dispersion in air or by
dusting or rolling from a cotton swab dipped into a powdery sample.
Other powders may need more shear force, such as that provided by a
Tesla coil operating on a small portion of powder resting on a Formvar®
film. An alternative involves an ultrasonic vibrator operating on the pow-
der suspended in acetone so as to spray the suspension onto a carbon film.
Magnetic particles, or any other dry materials that would contaminate the
TEM, must be embedded in an adhesive, such as a 1% solution of collodion
(cellulose nitrates) in amyl acetate. Dispersion is by means of a spatula-or_
muller on a glass plate<l7) or a tiny mortar and pestle. Enough solvent is
added to leave a sufficiently thin film after evaporation. If aggregation
occurs during this treatment, it may be better to use a vehicle and a thinner
that represent natural or industrial conditions. Particles are usually sha-
dowed by evaporating a heavy metal at a known angle<17l (see Figure 14.8).
Surfaces are generally prepared for the TEM by replication. Carbon
replicas are preferred because they are thermally and electrostatically
conducting, and they can be made thinner than plastic replicas. A typical
method of making a carbon-positive replica of a solid surface is shown in
Figure 14.10. First a 4% solution of collodion in amyl acetate is applied,
dried, stripped off, and discarded to clean the surface. Then several layers
are stripped off and shadowed by evaporating a heavy metal on the
replicating side. Next carbon is applied as a positive replica from a carbon
arc in a vacuum.U7l

,--.,yr---,
Metal &rloce with Scrotch -~.--• Corbon Arc
v ~-Collodion ,/,? '\~\\
' ' \ . Carbon
Fa-motion of Collodion Replica
I. I
/ / t
"~ .
\ \ . I
I Film
v.-Metol Voporized in
Carbon Replication
"':.;--, Tunosten Basket

c. ·. . .,a
•• •• ·., ,
«1;:
ShodowinQ
Collodion
-~:co
Stripping
~~¥P!!!!!!!!!!!!!I

Corban Replica

FIGURE 14.10. Steps in making a carbon-positive replica.

Transmission Electron Microscopy and Electron Diffraction 285

Surfaces sensitive to a plastic solvent can be replicated dry by using

polystyrene disks made by flattening pellets at 165° C. Powders, such as
dry yeast, are especially amenable specimens; they are simply sprinkled on
the disks, covered with a weighted glass slide, and placed in the oven at
165° C for about 5 minutes. After cooling the specimen is removed, and
carbon is projected from a carbon arc in a vacuum as usual. Since carbon
replicas are very delicate, special techniques have been developed to han-
dle them. Ct7l
Microtomy is discussed in Chapter 20.
Freeze fracturing biological and other aqueous specimens is accom-
plished by freezing the specimen in Freon® 12 refrigerant and producing
a fracture surface with a precooled razor blade. The specimen is trans-
ferred to a special accessory for the Ladd vacuum evaporator, where at the
precise moment the specimen is shadowed with platinum and carbon,
then replicated with carbon. The replica is protected with a drop of 1%
collodion in amyl acetate, and a slice of the specimen and replica is cut off
with the razor blade. The specimen is dissolved in Chlorox® sodium hypo-
chlorite solution, and the collodion is dissolved by fumes of amyl acetate,
leaving the carbon replica for electron microscopical examination.CI7)

14.23. ELECTRON MICROGRAPHY

Accelerating voltages of 50-100 kV produce electrons, each one of

which is capable of passing through as many as 100 photosensitive grains
and losing enough energy to make at least some of the grains developable.
The number of grains developed depends on the developer and develop-
ment time. The information in a ray of electrons is the signal. There is also
noise (without information); However the signal-to-noise ratio for electrons
is 100-1000 times greater than that for light! The photographic record also
has a signal-to-noise ratio; the signal is the optical density, and the noise
is the developed granularity.C26> If there is no granularityC1> developed, the
signal-to-noise ratio in the electron micrograph is the same as in the elec-
tron beam. Such a film is on Kodak® high-resolution plates,C25> which are
practically without graininessC1> but far too slow for any except the most
stable instruments and specimens.C26>
Among glass photographic plates, the projector slide grades are the
most practical. !25> As far as electrons are concerned, there is little difference
between the two grades medium and contrast. One great advantage to
both grades is that useful exposure time to electrons (speed) can be
changed by adjusting conditions during development. Normally Kodak®
developer D-19® solution is recommended for 3 minutes at 68° F (20° C).
286 Chapter 14

However development time can be varied from as little as 1 minute to as

much as 8 minutes.<26> Photographic plates have the greatest dimensional
stability, and they are readily prepumped before use.<27l But the disadvan-
tages of heavy, bulky, and breakable glass plates are obvious when it
comes to storage, both before exposure and after processing.
Fortunately Kodalith® LR film, Estar® polyester base available in cut
sheets overcomes the problems of glass plates. Moreover the polyester
base pumps down almost as fast as glass and much faster than acetate
bases. Polyester also has less curl and more dimensional stability than
acetate. Kodalith® LR film, when developed in D-19® for 2 minutes at 68°
F (20° C), has the same electron reaction speed as medium projector slide
plates with 50-kV electron acceleration and two-thirds the electron reac-
tion speed with 100 kV. Because of gradual fogging however, increased
development is impractical, and a Kodak® safelight filter 1A (instead of 1)
is required during all handling prior to fixation. Kodalith® LR Estar® film
is available in all sheet sizes.
For TEMs equipped for 35- or 70-mm roll film, the fine-grain variety
has been the most popular. A drawback for some users is the fact that
acetate base requires four times as long to pump down as glass plates. The
electron reaction speed of this film is slower than that of projector slide
plates, but this is acceptable because the bellows length is shorter and
therefore the electron beam intensity is higher. The signal-to-noise ratio is
low enough to be just barely detectable by the eye; this characteristic may
or may not be important. Kodalith® LR Estar® film was available in rolls 35
or 70 mm wide.<26>
Besides potential granularity in a photographic emulsion, the spread
function limits the acceptable extent of enlargement in TE micrographs.
The spread function measures the area over which an electron's energy is
expended in its deviated path through the gelatin (not the photosensitive
grains) of the photographic emulsion. Two narrow electron beams sep-
arated by less than the radius of spread (scatter) will not record separate
information. For accelerating voltages of 50-100 kV, the spread function is
about 5-10 !J.m, giving a maximum enlargement of 20x. Less than 20x,
developed granularity limits the photographic resolution of information
with all materials except Kodak® high-resolution plates, which have prac-
tically no granularity.<26>
After exposure to electrons and specific development of the negative,
stopping, fixing, washing, drying, and printing are similar to processes
used after exposure to light;(27l the reader is referred to current literature. <28>
There is however a drawback in the photographical systems of TEMS
that do not accommodate the fast self-developing, direct-positive camera
backs, such as Polaroid®. These cameras must be used outside the vacuum
Transmission Electron Microscopy and Electron Diffraction 287

system of the TEM. Focusing on the outside of the fluorescent screen is

unsatisfactory because of the granular nature of the screen. Instead a
grainless single crystal of europium-activated calcium fluoride is em-
ployed. The activated crystal, assembled with a mirror, is placed in one of
the two portholes provided for accessories in most TEMs. As indicated in
Figure 14.11, a special light microscope M, consisting of an objective
camera lens (f = 1.4) and an ocular, is placed in the other porthole. Con-
nected to the microscope M is a Polaroid® camera<30l C, supported on a
stand S. The fluorescent crystal's assembly F includes a vacuum-tight
sleeve that permits focusing on the fluorescent image by viewing through
a hand magnifier held in the plane of the camera back. The sleeve F also
allows the whole assembly to be drawn out of the way when not in use.
Figure 14.12 is an electron micrograph of a diffraction grating taken as a
test for resolution and calibration.<29l
With the TEM it is important to select an area to be micrographed
before the specimen changes, bums up, or contaminates the microscope.
This is especially difficult at first if the image is weak or low in contrast.
While some TEMs are equipped with image intensifiers, others are not. For
the unequipped microscope there is an accessory that intensifies both
brightness and contrast of the image, and it fits on the microscope in place
of the binocular viewer. The accessory focuses the original image on a
photocathode and reproduces it on a phosphor associated with an anode.
The net gain in luminance is about 4 x 1Q4.<29l
The accessory image intensifier can be connected to a cathode ray
(video) tube. Such a system has been used to examine electronmicroscopic
particles from celestial space. Some of these particles decompose readily in

FIGURE 14.11. Polaroid® camera system attached

to a TEM. A fluorescent crystal mirror assembly (F)
is on table in foreground.<29)
288 Chapter 14

FIGURE 14.12. Micrograph of 1134line/ mm grating replica at 5000x made with a Polaroid®
camera system of a TEM.<29 l

the electron beam and therefore are examined at extremely low-beam

intensities with the image intensifier. The video aspect of the system is an
apt introduction to the STEM (see Chapter 15).
Additional useful magnification can be obtained with photomicrog-
raphy and subsequent enlarging. Figure 14.13 shows a lattice image with
a stacking fault. Most of the useful magnification occurred in the micro-
scope, but additional useful magnification was achieved in photographic
printingP1>

14.24. ELECTRON DIFFRACI'ION

Electron diffraction is routinely conducted with conventional trans-

mission electron microscopy to obtain additional information, such as
crystalline structure, crystalline habit, and molecular orientation. Related
insight is provided by X-ray diffraction (XRD), but the required sample
sizes are quite different. Electron diffraction (ED) from an area 111m x 111m
is relatively easy to obtain with a suitable sample for transmission electron
Transmission Electron Microscopy and Electron Diffraction 289

FIGURE 14.13. A high-resolution TEM photomicrograph of Cd Te, a semiconductor com-

pound with a face-centered cubic structure, observed in the (110) direction. The lattice image
clearly shows a stacking fault on the (Ill) plane. Shifts in the lattice plane at the fault are
indicated by the arrows. Courtesy of JEOL, USA, Peabody, MA.(31)
290 Chapter 14

microscopy, whereas a sample size of more than twenty times this is

necessary to provide XRD.<32l Thus with small apertures or masks in the
electron beam, many subareas can be analyzed even though they are too
small to provide diffraction information by XRD. In addition considerably
more information is provided in the ED pattern than in the XRD pattern
because of differences in the utilized wavelengths, ca. 0.002 A for ED
versus ca. 2 A for XRD. This difference is due to differences in wave-
lengths, a topic beyond the scope of this book but involving a larger Ewald
sphere for ED and the sphere's intersection with the crystal's reciprocal
lattice points. This concept is often explained in texts on ED<32l and XRD.<33l
While crystalline lattices are discussed in Chapter 7, it is pertinent to
illustrate the crystallographical advantage of the high resolution of the
TEM. Figure 14.13 is a TEM micrograph of CdTe, a semiconducting com-
pound with a face-centered cubic structure, observed in a certain direction
(specified as 110). The lattice image shows a stacking fault on the 111 plane,
indicated by the arrows.<31l
A simple cubic unit cell is represented in Figure 14.14. Spacings be-
tween such planes are given by Bragg's law from X-ray diffraction, i.e., A
=2d sin e, where A =wavelength of electrons, dnkl =spacing between the
periodically spaced planes in the unit cell and crystals that diffract the
waves, and e =angle between the incident electrons and the specific set of
atomic planes. Constructive interference occurs only when many parallel
planes are at an angle such that Bragg's law is satisfied; otherwise scattered
waves intersect destructively and are not seen.
Small differences do exist between ED and XRD. Because the wave-
lengths of electrons are typically one thousand or more less than those of
X-rays, Bragg's law is fulfilled by crystallographic planes almost parallel to
the original, undeviated electron beam. Also because thin crystals are used

FIGURE 14.14. Cubic-unit cell showing

" - - - -- x the d100, d01 0> d001 , and d111 lattice plane
spacings. After Beeston, Home, and
MarkamP2l
Transmission Electron Microscopy and Electron Diffraction 291

with ED, typically 10-50 nm thick, limited constructive interference, or

incomplete destructive interference, does occur when crystal alignment
deviates slightly from the Bragg condition.<34>
As indicated in Figure 14.15, when an image is formed in the image
plane of the objective lens, a diffraction pattern (VW) is formed in the
objective's back focal plane. It also indicates that all rays from the same
point in the specimen are focused at the same corresponding point in the
image plane. Rays from point A are focused at B, and rays from Care
focused at D. Figure 14.15 also shows that all rays parallel from the spec-
imen are focused at the same points in the back focal plane where con-
structive and destructive interferences occur.<32> It is also understood in
Figure 14.15 that rays scattered or diffracted in the same direction are
patterned in the back focal plane of the objective. Rays from a point in the
specimen are collectively focused at the same point in the image plane, and
rays scattered in the same directions from the specimen are collected at the
same points in the back focal plane.<32>On being combined in the back focal
plane, the rays undergo constructive (additive) or destructive (subtractive)
interferences, and small electron diffraction patterns can be formed. When
an image is focused in the image plane, interference of diffracted waves
can occur in the back focal plane. This concept is similar to the concept of
light interference patterns discussed in Chapter 5.
Figure 14.16 illustrates electron scattering from stacked crystalline
lattice planes. For scattering angles other than those indicated by the Bragg
law, waves associated with the scattered electrons interact destructively,

R
Objective lens

FIGURE 14.15. Schematic diagram of

electron rays passing through a periodic
Back focal plane
structure, such as a crystal, to fonn a
characteristic electron diffraction pattern
in the back focal plane of the electron
lens. After Beeston, Home, and Mark-
am. (32) B«oe-------' First image plane
292 Chapter14

'
FIGURE 14.16. Schematic diagram illus-
trating the origin of an electron diffrac-
tion paHem from parallel crystal laHice
planes of spacing dhld, lying at an angle 8
Optic axis to the incident electron beam. After Bees-
ton, Home, and MarkamP2l

and only a small amount of diffuse scattering is observed if the specimen

is noncrystalline. If the specimen is crystalline, the diffraction pattern is
composed of discrete spots if the crystals have a preferred orientation;
"preferred" means that the crystals are not randomly oriented. In contrast
if the crystals are randomly oriented, they diffract at all angles, and the
observed diffraction pattern is composed of complete rings with inter-ring
distances determined by inter-lattice planar spacing; this is due to an
inverse relationship.
A very high magnification photomicrograph of a crystalline lattice
with atomic resolution is shown in Figure 14.17. Superimposed on this
lattice image is its ED pattern. Such an ED pattern could be made at much
lower bright-field magnification, where the atomic structure could not be
resolved. However through the ED pattern considerable information could
be generated on such parameters as interatomic spacings, type of unit cell,
approximate crystal size, and atomic orientation in the matrix.
Generally two projector lenses function together to magnify the initial
image formed by the objective lens. For viewing the ED pattern however,
the first projector lens is weakened because less current flows through its
lens coil to decrease its focal length; therefore it becomes focused on the
back focal plane of the objective rather than on the first image plane. The
image is given additional magnification by a succeeding lens, or several
lenses, and finally the image is focused on the viewing screen.<32> Of course
when the general image is viewed, the strength of the lenses after the
objective lens must be changed. Thus the resulting image is of the first
Transmission Electron Microscopy and Electron Diffraction 293

FIGURE 14.17. Transmission electron micrograph with crystalline lattice resolution of a gold
single crystal. Magnification on the original print was 4,000,000. Superimposed on the lattice
image is an electron diffraction pattern from the same microcrystaJ.<31 > Courtesy of JEOL,
USA, Peabody, MA.

image plane rather than the back focal plane as it was in the diffraction
case.
Specimens normally interact destructively with electron radiation,
and such interaction of electrons is generally destructive to the specimen,
especially many crystalline, organic specimens. The radiation dose is so
concentrated that it is often compared to what occurs in nuclear explo-
sions.<35> Specimen heating is usually not a severe problem because of the
specimen's large surface-to-volume ratio, although it can create artifacts
with temperature-sensitive specimens. This point emphasizes why it is so
important to minimize as much as possible the specimen's exposure to
beam current by using low-intensity currents. The potential increase in the
specimen's lifetime is also a major advantage of the scanning transmission
294 Chapter 14

electron microscope (STEM) over the conventional transmission electron

microscope (CTEM). Often images, especially those of organic materials,
degrade detectably while being viewed or even before first viewing if the
microscopist does not work quickly. This is often a main reason for using
cold stages to decrease the rate of specimen degradation.
In summary considerable information about a specimen suitable for
transmission electron microscopy, can often be fairly easily obtained with
ED.

14.25. SUMMARY

Electron microscopy began with the realization that a beam of elec-

trons behaves, like visible light, both as a train of waves and a stream of
particles. Since the de Broglie wavelength of electrons decreases with their
kinetic energies, fast-moving electrons have very short wavelengths asso-
ciated with them and so are capable of very high resolution if that wave-
length can be used in an appropriately designed instrument.
The TEM is one such instrument. It is arranged much like an ordinary
microscope designed for examining translucent specimens by the trans-
mission of visible light except that magnets are used instead of light-
bending lenses to deflect and focus the beam of electrons. The electrons
originate in an electron gun that usually has a hot filament (sometimes a
cold-cathode emitter) as the source and an arrangement for defining and
accelerating a narrow beam of electrons. The accelerating voltage can be
anywhere from 30,000-1,000,000 V or more, but it is usually in the range
of 50,000-100,000 V. The accelerated beam is then focused on a tiny area
of the specimen by a pair of concentric toroidal electromagnets that act as
condensing coils. The sample is held on a screen of extremely fine wires,
so the beam passes through one of the holes in the screen and on through
the objective and projection electromagnets. Since different parts of the
specimen absorb electrons differentially, as the projected beam of electrons
falls on a fluorescent screen, it shows bright areas where the sample has
absorbed least and darker areas where the sample has absorbed more of
the electrons. By removing the fluorescent screen and allowing the pro-
jected beam to fall on a photosensitive plate or film, electron micrograph-
ical negatives can be taken. Direct positive quick-developing systems, such
as Polaroid require an external adapter.
Since the electron beam would be scattered by collision with air mol-
ecules, the interior of the electron microscope has to be pumped down to
an extremely low pressure by efficient diffusion pumps and forepump. It
follows that any specimen that dries out or loses volatile components in a
Transmission Electron Microscopy and Electron Diffraction 295

vacuum is altered, and this limits the applicability of the method and
tempers the interpretation of the image. Furthermore volatile carbon-
aceous material is decomposed by the hot cathode and the stream of
energetic electrons, which deposits decomposition products in the fine
bores of the coils and fouls the microscope. Sample decomposition by the
energetic beam produces the same undesirable result. Volatile components
of lubricants, sealing compounds, and pump oil must be eliminated. Some
contamination of the microscope is unavoidable though, and periodic,
thorough cleaning of critical parts is necessary.
The resolving power of a TEM is theoretically proportional to the
wavelength of the electrons and inversely proportional to the angular
aperture. The theoretical limit is never attained in practice, but a resolution
of about 4 A at 50 kV and 3 A at 100 kV is attainable. Tilting the sample
improves the resolution. In general greater resolution is obtained on an
electron photomicrograph than on the visual fluorescent screen in the
microscope, because the photographic emulsion has much finer grains.
Contrast in the TEM image depends on many factors, but operation-
ally contrast can be increased by reducing the beam voltage. This allows
greater differential absorption by the sample, but of course it reduces
resolution. Similarly reducing the aperture of the objective (by means of a
diaphragm) increases contrast at the expense of resolution. Greater con-
trast in the photomicrograph can be obtained by using high-contrast plates
and developer and/ or stains.
Some aberrations of the magnetic lenses can be corrected by adjust-
able electrostatic deflectors within the TEM. Some progress is being made
to reduce spherical aberration to approach theoretical resolution.
Focus depth presents no problem in the TEM, which is adequate for
all purposes. Deliberate under- or overfocus increases contrast but reduces
sharpness.
The useful magnification of the TEM depends on its resolving power.
At a resolution of 10 A, a magnification of 50,000 is routine. The field of
view under such conditions is very small, only a few square millimicrons.
Hence the operator must usually choose between several kinds of field,
based on experience, and justify the choice. Recognizing spurious images
as the result of damage to the specimen by the electron beam or fouling of
the microscope is also a matter of experience.
Preparing the specimen is an especially important part of operating
the TEM. The sample must be thin enough to transmit electrons yet thick
enough to show differential absorption. Variations in depth can be en-
hanced greatly by shadowing the sample-depositing a heavy metal at an
acute angle by evaporating the metal from a hot filament off to one side in
a vacuum chamber. Further details of structure can be revealed by tilting
296 Chapter 14

the sample in two or more directions. Experiments can be conducted on

the sample material to select a range of particle size, to determine the effect
of drying or heating, or to gain greater dispersion of solids, but of course
any alteration of the sample material deemed necessary as a result of such
experiments must be justified in the report. Many suggestions for sampling
material, preparing a specimen, and interpreting the image are given, but
for details see references.
Electron diffraction conducted can easily be on a specimen suitable for
transmission electron microscopy. Properly obtained results from ED can
tell whether the specimen is crystalline or not; and if it is, the ED can tell
the crystalline habit and whether or not the crystals have a preferred
orientation.
 15

Scanning Electron Microscopy

and Compositional Analysis

15.1. INTRODUCTION

In an SEM the image is formed in a cathode ray tube synchronized

with an electron probe as it scans the surface of an object.( 1l The resulting
signals are secondary electrons, backscattered electrons, characteristic X
rays, Auger electrons, and photons of various energies.
In a typical SEM an electron gun and multiple condenser lenses pro-
duce an electron beam whose rays are deflected at various angles off the
optic axis by the first set of electromagnetic scan coils. The second set of
scan coils deflects the beam back across the optic axis. Both sets of scan
coils are in the bore of the final lens. Figures 15.1 and 15.2(2l help show the
components; Figure 15.1 also indicates that all the rays pass through the
final aperture of the final lens. From this final crossover, the rays, one at a
time, strike the specimen at various points. The scan coils and the cathode
ray tubes are powered by the same scan generator, so that each scanned
point on the specimen is unique as reproduced on the displaying or the
recording cathode ray tube and video amplifiers. To these amplifiers, one
or more of the resultant signals are fed: high-energy backscattered elec-
trons; low-energy secondary and/ or backscattered electrons, X rays, and
cathode-luminescent radiation in the UV, visible, and infrared regions. All
results can be monitored separately or simultaneously by means of the
appropriate detectorsP-5> Many types of signals exist, as illustrated in
Figure 15.2. Consequently SEMs are complicated and expensive instru-
ments; see Figure 15.3.
Interpreting scanning electron micrographs is different from interpret-
ing images formed directly by bending light or electron rays from object
to image. The SEM indirectly constructs a pattern or map that can be

297
298 Chapter 15

Wehnelt Aperture -+--E<:7&

~ ~~~~

Electron Beam --4----:111

Vacuum

Deflection Yoke

Electromagnetic
Lens

Image on
Flourescent Screen

SEM T.V. TUBE

FIGURE 15.1. Schematic of an SEM and a television. Many of the general principles are the
same for both instruments. Courtesy of Philips Electronic Instruments Company.

interpreted as an image of the object. Interpretation of the SEM image is

facilitated by the many other attributes contributing to visibility, particu-
larly resolution, contrast, focus depth, morphology (topography), appar-
ent illumination, and its three-dimensional aspect.
One of the most striking aspects of the SEM image by secondary and
backscattered signals is its resemblance to images of depressions and
elevations illuminated from above by an oblique beam of light. For ex-
ample the ant and chip shown in Figure 15.4 look as though they were
illuminated by an oblique beam of light from above. We have been familiar
with this type of illumination in macroscopy since childhood. It is also the
type of illumination most frequently used with simple magnifiers and
Scanning Electron Microscopy and Compositional Analysis 299

WCIOENT
ELECTRON
BEAM
CHARACTERISTIC
X.fiAY PHOTONS
BREMSSTRAHWNG
(X-ray oontinwm) SECONDARY
ELECTRONS
VISIBLE LIGHT
(cathodolumll"oHc.nc:e)

ABSOR9ED
ELECTRONS
(lj>eeimen currenl)
El.ASTlCAI..LY TRANSMmED AND
SCATTERED tNEL.ASTICAl.LY SCATTERED
ELECTRONS ELECTRONS

FIGURE 15.2. Signal types generated by electron-specimen interactions.(2l

FIGURE 15.3. Typical SEM; the left-hand display screen shows the specimen, and the
right-hand display screen shows elemental analysis. Courtesy of Philips Electronic Instru-
ments Company.
 300 Chapter 15

FIGURE 15.4. Scanning electron micrograph, not a photo montage, of a chip in the man-
dibles of an ant. Original magnification was 30x, and the chip is approximately 2 x 2 mm.
This award-winning micrograph was made in the Application Laboratory for EM, Philips
Einhoven, and is reproduced by courtesy of Philips Einhoven, The Netherlands.

stereoscopic compound microscopes; consequently structure and mor-

phology are easily inferred.
Even when magnification is boosted to 2000x so that resolution and
focus depth are beyond that of the light microscope, a scanning electron
micrograph is easy to interpret because it looks as though it were illumin-
ated by a unidirectional beam of light from above the specimen<6> (see
Figure 15.5). Another aid to interpretation is the perspective these scanning
electron micrographs manifest.

15.2. MAGNIFICATION AND RESOLVING POWER

Magnifications from around ' 10-200,000x or more are possible with

SEMs. Normally lower magnifications solve the problem and the micro-
scopist should always ascertain that resolution is adequate to support the
magnification. Magnification calibrations are often established by observ-
ing diffraction gratings of known spacing. Since no actual image is formed
Scanning Electron Microscopy and Compositional Analysis 301

FIGURE 15.5. Cotton fiber coated with a thin layer of gold and palladium by evaporation in
a vacuum and photomicrographed by SEM. <6> The photo vividly shows thickening of flat
fiber, fibrils, and two spiral structures. USDA micrograph by Southern Regional Center, New
Orleans.

in the microscope's column, magnification is the ratio of the area scanned

to the display area. Since the size of the display is generally fixed at
between 10-20 em, magnification is increased by decreasing the area
scanned, as depicted in Figure 15.6. Magnification is generally calculated
as the ratio of the length of the display divided by the corresponding
length of the scanned area.
The resolving power of the SEM depends primarily on the effective
beam diameter of the probe. The secondary collection efficiency is as-
sumed to be unity and the specimen capable of producing the 25% contrast
necessary to satis~ Raleigh's criterion (see Chapter 2); a theoretical limit
of resolution at 90 A (9 nm) was calculated earlier for the ideal specimen.<3>

15.3. RESOLUTION

The practical resolution of the SEMis limited to 200 A(20 nm) for most
specimens in optimum position relative to the detector for secondary
302 Chapter 15

DISPLAY

- - - - - L -----~

FIGURE 15.6. Image formation and magnification in the SEM. Courtesy of The Mineralogical
Record.<2>

electrons. Some pairs of points on the surface of the specimen may not be
in the optimum position for detecting secondary electrons, and therefore
they are not resolved even though the limiting pairs in optimum position
are resolved. <3>
Nonconducting specimens are coated with a metal to reduce damage
by the thermal effects of the electron beam and to eliminate electric charges
on the surfaceP> Thin as such coatings are (100-300 A[10-30 nm]), they do
cover up the finest topographical features, thereby reducing the practical
resolution of the very finest detail.

15.4. CONTRAST

Contrast in scanning electron microscopy is defined as the ratio of the

change in signal between any two points on the specimen and the average
signal. Differences in atomic number, i.e., nuclear charge, of the atoms
composing the specimen, are important in the SEM just as in the TEM. In
the SEM however, we are concerned chiefly with backscattered electrons
rather than transmitted electrons. The higher the atomic number, the greater
the backscattering and the less the transmission of electrons. For a given
phase, such as that of an alloy, ore, rock, ceramic, or cement, the effective
backscatter coefficient is the weighted average of the backscatter
coefficients of the pure elements. In smooth plane surfaces we are dealing
entirely with atomic contrast<3>(see Figure 15.7).
In the case of rough surfaces, we are dealing with topographic contrast,
Scanning Electron Microscopy and Compositional Analysis 303

FIGURE 15.7. Pure gold wire after annealing to a smooth surface (plus voids, black) at 950°
C (1742° F) for 2 hrJ13l Courtesy of A. C. Reimschuessel.

whether backscattered or secondary electrons are detected. When a plane

specimen is tilted away from the normal incidence of the bee "11, electron
backscattering increases, gradually approaching unity at grazing incidence.
Therefore a faceted surface (see Figure 15.8) with its facets at various
angles to the beam emits signals of varying strengths. The peak in back-
scattering by each facet lies in a plane containing the normal to the surface
and the direction of the incident beam(3> (a sort of oriented luster). The
Everhart-Thornley (E-T) detector is highly directional and picks up only
trajectory electrons traveling toward the detector. Thus the resultant image
contains a high proportion of black (shadowed) facets. If the specimen's
current signal (SCS) is employed to form the image, trajectory effects are
lost, and topographic contrast is due solely to the relative number of
electrons from each facet. (3>
Secondary electrons can escape from the surface of most materials at
depths of less than about 10 nm (100 A). At such shallow depths there is
minimal elastic scattering; electrons travel in a beam parallel to the direc-
304 Chapter 15

FIGURE 15.8. Lead sulfide, galena (isometric), taken by F. G. Rowe with field-emission
electron gun (see Figure 15.11).

tion of incidence. A greater number of electrons escape from surfaces at a

higher tilt.(ll
Contrast in the SEM can be varied in other ways(3l:

Electron channeling contrast, related to the crystallographic nature of

such a specimen.
Magnetic contrast related to crystalline structure.
Magnetic contrast related to noncrystalline specimens.
Voltage contrast from beam-induced current contrast.
Electron-beam-induced current contrast, a phenomenon of electron
hole pairs in semiconductor devices.
Cathode luminescences from the emission of long-wavelength elec-
tromagnetic radiation of light. A very important advantage of ca-
thodoluminescence is that it introduces natural color contrast into
the SEM, so that photomacrographs or photomicrographs can then
be taken in colorPl
Scanning Electron Microscopy and Compositional Analysis 305

Major influences on contrast are the incidence angle of impinging

electrons onto the specimen and the collecting angle from the specimen to
the positively charged collector. Synthetic color can be introduced into a
series of correlated black and white (e.g., X-ray) images of the very same
area (e.g., an alloy) by arbitrarily assigning a different color to a different
image.<3>

15.5. ABERRATIONS

Spherical aberration is caused in the SEM when electrons move more

strongly in trajectories away from the optical axis being focused than those
near the axis (see Figure 15.9 top). Consequently the image of point Pis a
disk or circle of confusion. Aberration can be reduced by diaphragming
with a lower intensity of the probe, or the object can be placed nearer the
lens; however a decrease in working distance and a loss of freedom from
the condenser's magnetic influence result. <7l
Chromatic aberration is caused by a variation in the energy from E0 to
E0 + AE (see Figure 15.9 center). Of course a variation in the magnetic field
also produces a disk of confusion in the scanning probe, but sources of
trouble can be reduced by stabilizing the respective power supplies. Nev-
ertheless there remains a fundamental variation AE in the energy rep-
resenting the spread of energies leaving the cathode (2-3 eV for a tungsten
or lanthanum boride source; 0.2-0.5 for field-emission cathodes).<3,7)
Since there is wave motion in the electron beam and a diaphragm of
radius R, diffraction causes its own disk of confusion dr in the scanning
probe of electrons (see Figure 15.9 bottom). The radius of the first diffrac-
tion minimum dr subtends an angle 8 at the lens.<7l
Astigmatism results when a circular object does not yield a circular
image. Astigmatism enters the electron probe lens through machining
deviations, inhomogeneities within the iron pieces, asymmetry in the
windings, and an uneven accumulation of contaminants. Astigmatism is
usually corrected by a stigmator with two controls: one for the magnitude
of asymmetry and the other for the direction of asymmetry. The two con-
trols are alternately adjusted and refocused at medium magnification,
5,000-10,000x.<J>

15.6. CLEANLINESS

Cleanliness in the SEM is as important as in the TEM and for the same
reasons: to keep the apertures open and avoid accumulating carbon and
 306 Chapter 15

I LENS

SPHERICAL
ABERRATION

CHROMATIC
ABERRATION

LENS

DIFFRACTION

FIGURE 15.9. Schematic drawing showing spherical and chromatic aberrations as well as
diffraction at a lens aperture.<2) Adapted from Ha11(7) and Oatley.<8l

any other matter that can deviate the electron beams. Again the chief
offender is carbon or carbonaceous matter from sealing grease, pump oil,
or the specimen itself. Precautions, too, are the same: greaseless seals, cold
oil traps, and prompt removal of carbonaceous specimens. In Auger elec-
tron analysis,(8•9l an oil-free, ion pump is required.
In X-ray analysis contamination by hydrocarbons may lead to poly-
merization rather than decomposition. X rays of long wavelength, such as
from beryllium, boron, or carbon, may be highly absorbed, leading to a
high degree of polymer contamination and an increasing carbon Ka count
Scanning Electron Microscopy and Compositional Analysis 307

as a function of impingement time. One preventive method is to direct a

stream of gas at low pressure onto the specimen. Air is then introduced to
bum the carbon deposit. Another method is to provide a nearby cold
"finger" on which the organic molecules preferentially collect. A combina-
tion of the two devices may be worthwhile<3>(see Section 15.8).

15.7. FOCUS DEPTH

Focus depth of the SEM is the greatest among microscopes, because the
final condenser lens has such a very small aperture to demagnify the image
of the electron source sufficiently to make it an adequate probe. Focus
depth is a great advantage for keeping in focus all parts of a rough top-
ography, but a compromise must be made between field depth and resolu-
tion (see Figure 15.5). Since the specimen is tilted toward the electron
collector, there is a substantial difference in the path length from one side
of the specimen to the other (see Figure 15.1). Most manufacturers have
introduced a second lens to correct the focus as the beam moves across the
specimen (dynamic focusing).<3,9,JO)

15.8. FOCUSING

Focusing the SEM is much like focusing a television set. The operator
should first set the contrast and brightness controls to suit personal pref-
erence. It is generally easier to focus on a screen that is not very bright. If
the contrast is not suitable, consider using either the black level or the
gamma control. Increasing the black level makes a flat surface appear less
flat. Increasing the gamma control is useful in looking at the bottom of
holes or in obtaining a picture when the specimen is changing. Both the
black level and gamma controls produce artificial pictures, so their use
should always be recorded.<10>
For magnifications over 2000x, the image should be tested for astig-
matism with the fine-focusing control by focusing. If the picture seems to
be smeared at an angle to the scanning axes on one side of focus and at the
opposite angle on the other side of focus, there is astigmatism. Use the
stigmator, going back and forth through focus until correction is opti-
mum.<10>
Focusing and astigmatic adjustments should be checked at a higher
magnification than the one to be recorded. If the beam voltage, aperture,
or lens currents are changed, the image should be refocused and correc-
tions for astigmatism should be made again.
308 Chapter IS

15.9. ILLUMINATION

Illumination in the SEM picture should appear to go toward the top of

the specimen, because the observer is accustomed to such illumination and
therefore finds it the easiest to interpret in microscopy. Therefore shadows
are below elevations and in the upper part of depressions. For single
pictures illumination appears to come from the top, but with stereopairs
the illumination usually appears to come from the side of the integrated
image. Effective illumination from the top of the stereopair can be obtained
by tilting the specimen around an axis parallel to the line between spec-
imen and detector. Since this kind of tilt is not usually provided in SEM
stages, a goniometer stage can be used to rotate the specimen on an axis
normal to the specimen-detector line.<3>
The source of illumination in the SEM must be an electron gun of
great intensity because the specimen's responsive signals are relatively
weak. The thermionic gun made of tungsten filament (the same as for
TEM; see Figure 14.2) is reasonably satisfactory at moderate magnifica-
tions, but for higher magnifications or shorter scanning times, more in-
tensity is needed. The lanthanum hexaboride gun(3> (see Figure 15.10) of
Broers is 10 times as bright as the tungsten filament and has a longer life

Evaporation
shield

lcm

FIGURE 15.10. Electron gun with LaB6 cathode<3> (from Broers).

Scanning Electron Microscopy and Compositional Analysis 309

at a cost of additional complication and expense.<8l The field-emission

gun<3l (see Figure 15.11) is capable of very high intensity and small spot
(less than 0.2 J.tm).<8l Its major problems are the high vacuum required and
relative instability.<3l

15.10. RADIATION

The radiation used in the SEM is one or more of eight different types
of signal, as indicated in Table 15.1.<9) So far we have discussed emission
and reflection from a surface of secondary and backscattered electrons as
information signals in the SEM. However some electrons are transmitted to
give information about the thickness and composition of foils and films.
The degree of electron absorption can thereby be determined to give com-
plementary information about the specimen.
In certain specimens high-energy electrons create pairs of electron
holes. If the pairs combine, energy is manifested as radiation of long
wavelengths. Such cathodoluminescence can include visible light of various
wavelengths (colors). Cathodoluminescence is usually weak and requires
an efficient light-collecting system, including elliptical mirrors and fiber
optics. <3) Results are both qualitative (pictorial) and quantitative (photo-
metric), and these are feasible from both industrial and biological spec-
imens. Figure 15.12 illustrates the more common signals and the location
of the specimen volume from which they originate.

FIGURE 15.11. Electron gun with field emission.<3) Adapted from Crewe et al.
310 Chapter 15

TABLE 15.1
Types of Radiation Signaling Information in SEM(91
Radiation Signal Location Information
Emission Secondary electrons Within 5 11m of Topography of surface
surface
Reflection Backscattered electrons Within 1 or 2 11m of Nature of specimen
surface
Transmission Transmitted electrons Thin foils and films Thickness and compo-
sition
Absorption Specimen current Through specimen Complemen'tary to pre-
ceding information
Beam induction Current in external Within specimen Semiconductors
circuit
Cathodolumin- Photons of selected Light Various phases in a
escence wavelength specimen
X rays Selected wavelengths Kinds of atoms Spectrochemical
of X rays analysis
Auger Auger electrons, Auger electrons Chemical elements
selected wavelengths present

X rays are photons of electromagnetic radiation. Therefore they have

a wavelength A related to their energy E through the equation i.. = he/ eE,
where his Plank's constant, cis the velocity of light, and e is the electron's
charge.(3l In microanalysis X-ray spectrometry can employ either a wave-
length-dispersive spectrometer (WDS) or an energy-dispersive spectrom-
eter (EDS). Since the SEM of the EDS is easier to use, it is more common
than the WDS type, and the current discussion is limited to the EDS.
When a electron beam of sufficient energy impinges on a solid, X ray

PRIMARY
ELECTR)N
BEAM

SPECIMEN SECOODARY
SURFACE ELECTRONS

CHARACTERISTIC
~~-=-=--- X-RAYS FIGURE 15.12. The volume within a
,_~,.-- X-RAY cornNUUM specimen from which various types
of signals originate. Courtesy of The
Mineralogical Record. (Z}
Scanning Electron Microscopy and Compositional Analysis 311

photons may be emitted by core scattering, manifested as a continuous

background (bremsstrahlung) of X rays and inner-shell ionization, which
yields the characteristic spectrum of X rays. (3l
The intensity of the continuum is a function of atomic number and
accelerating voltage. As the voltage increases the continuum manifests
shorter wavelengths and increased intensity. The extent of continuum
radiation also increases with increasing atomic number, because heavier
elements have more nuclear scattering and less energy loss from interac-
tions among electrons. Continuum radiation forms the background x-
radiation(tJ in the electron microprobe SEM. In most instances it is desirable
to keep the continuum (background x-radiation) to a minimumYl
Characteristic x-radiation results from the interaction of incident elec-
trons with electrons belonging to the inner shells of atoms in the specimen.
If an incident electron has sufficient energy, it may dislodge an electron
from an inner shell (e.g., K, L, and M(t) [see Figure 15.13],(3) leaving the atom
in an excited (ionized) state. The atom returns to its original state by
transitioning an outer electron into the vacancy in the inner shell. The atom
loses specific energy by emitting a photon of x-radiation (see Figure 15.14).
The discrete energy level is described by a principal quantum number of
the atom: one energy level for n = 1 (K shell), three energy levels for n =
2 (L shell), five energy levels for n = 3 (M shell), etc. The emitted X-ray
photon has a discrete energy level equal to the difference in energy be-
tween the initial and final states of the atom. That is wavelengths of the
characteristic X-ray photons from a specific chemical element are critically

FIGURE 15.13. Electronic transitions in

an atom.(3) Courtesy of J. I. Goldstein et al.
312 Chapter 15

related to a discrete energy level E in keV.* Determinative tables are

available. <3>
Figure 15.14 indicates that following ejection of an orbital electron
(top), electron relaxation (center) may cause an Auger electron to be ejected
(bottom left) instead of an X-ray photon (bottom right). In either case the
energy is characteristic of the emitting element. The Auger yield is very
high for light elements, while the X-ray yield is low, so Auger analysis may
prove to be more useful than X-ray analysis for light elementsP>
The qualitative and quantitative use of X rays excited by a microprobe
of electrons has been offered separately by manufacturers of the EPMA.
However the EPMA and SEM can be one and the same instrument,(3> and
they are treated as such in this chapter.
If a solid-stage X-ray detector (SD in Figure 15.15) is placed close to the
electron probe of an SEM or EPMA, X rays pass through a thin beryllium
window into an evacuated chamber containing a cooled reverse-bias lith-
ium-drifted silicon crystal. X ray absorption produces a charge pulse that
is converted into a voltage pulse by a sensitive preamplifier. After more
amplification a multichannel analyzer sorts the voltage pulses and dis-
plays them on a cathode ray tube, x-y recorder, or computerP>
A schematic depicting principal components of an EDS is shown in
Figure 15.15. A primary electron beam is scanned across the specimen in
synchronism with the cathode ray tube's scanning electron beam. As with
normal image scanning, the area of the specimen being scanned can be
controlled, and hence the resulting magnification is variable in a controlled
manner. Beam intensity in the display cathode ray tube is modulated by
detecting a characteristic X ray, and this causes a single dot of light to
appear on the cathode ray tube.<M> In this manner as the electron beam is
scanned across the specimen, a set of characteristic dots can be generated.
This dot pattern forms a map of the elemental distribution superimposed
and displayed on the more common image of the specimen. Thus we can
visually associate the elemental distribution with the specimen morphol-
ogy.
"Electronic transition involves a change from one discrete energy level to another, and the
change in energy &E (by Planck's law) is equal to hv, where v is the frequency associated
with the transition. In terms of wavelength i.., since
i..v =c (the velocity of light)
then
v =eli..
and
&E = hc/2
where hand care constants and &E can be expressed in kiloelectron volts (keY) instead of
ergs (1 keY = 1.602 x Io- 9 erg).
 @)- -•
,/ l ',•
EJECTED ORBITAL
ELECTRON
''
jl '
SCATTERED PRIMARY
INCIDENT e ELECTRON
ELECTRON

@ ELECTRON RELAXATION
AND PHOTON
GENERATION

'
I. \.• PHOTON
INTERNALLY
~
r> X- RAY PHOTON
EMITTED

CONVERTED
AND AUGER
ELECTRON
EMITTED

FIGURE 15.14. Electron excitation manifesting characteristic X rays or Auger electrons<3l

Courtesy J. I. Goldstein et a/.

OISPI..AY (CAl)

X-RAY SIGNAL

FIGURE 15.15. Principal components of an EDS.(3)

 314 Chapter 15

Detecting light elements (Be, B, C, N, 0, and F) is difficult because of

the low-energy X rays they generate. Because of their low energy, these
signals are absorbed by the beryllium window generally used as the de-
tector cover shown in Figure 15.15. It is possible to detect the low-energy
signals, but this is beyond our discussion, so the reader is referred to
specialized texts.<3•4>
Superposing the detected distribution of identified elements on the
image of the specimen provides considerable structural information. Not
only can an elemental content be obtained, but the microdistribution of
elements, as shown in Figure 15.16, is also provided. Considerable caution
must be taken in interpreting relative dot concentrations as ratios of ele-
mental composition because the relative intensity of dots depends on such
factors as specimen surface smoothness and different excitation energies
(which are related to atomic numbers).<4>

FIGURE 15.16. Elemental analysis obtained at the cross in the upper part of a microscopical
reference photomicrograph obtained at 2500x. The elemental distribution displays peaks for
SiKa and RuKa, which with other information proves that ruthenium osmium silicide
composed this region. This analysis was performed by an EDAX elemental detector and
analyzer. Courtesy of EDAX International, Inc.
Scanning Electron Microscopy and Compositional Analysis 315

Conventional maps with discrete dots representing detected charac-

teristic X rays (called continuous rastering) have been produced for many
years. In this case the emitted X-ray beam signal is displayed in synchro-
nism with continuous scanning of the electron beam in the display cathode
ray tube. Even though the scanning is termed continuous, dot images are
not continuously recorded, since the detector system produces a pulse up
to tens of microseconds long for each processed X-ray signal.C4> Because of
this required time per signal, the detector system can be overloaded, so
that generated signals may not be collected nor displayed.
Discrete rastering is now more common than continuous rastering.
Actual initial generation of the detected X-ray signals is similar in both
cases however. With the discrete technique, each point at which the scan-
ning beam stops and information is generated is called a pixel.C4> Generally
128-512 points are detected along the scanned line, and the same number
is often used in the display. The point-by-point scanned area is stored in
the computer memory as a two-dimensional array of pixels.
A typical analysis of the components in an alloy is shown in Figure
15.16. By computer processing the collected signal, much more can be done
with the collected signals. For example different signals can be displayed
with different colors to enhance visibility, or component signals can be
artificially added or removed to assist in understanding the problem.
An excellent survey of compositional imaging and X-ray spectrometry
has been published.(5) Activities during the past 35 years are surveyed. The
first 25 years concentrated on operating with dot maps for compositional
imaging. Each dot represented a detected X ray from the specimen. How-
ever in recent years there has been a dramatic contribution from the
computer-controlled electron beam and computer-controlled manipulated
compositional images. This has permitted routine mathematical smooth-
ing across the discrete dots and the display of a continuous colored region
or area to represent each detected element. Recent inexpensive digital
storage components with multimegabit capacity have greatly expanded
existing procedures.(5)

15.11 USEFUL MAGNIFICATION

One aspect of the SEM' s useful magnification is different from the TEM:
Micrographs taken on the SEM cannot be enlarged very much because of
raster lines.(12> The limit of directly useful magnification is about 20,000x,
since higher magnifications tend to be fuzzy.( 11 >
Useful magnification can be determined on manufactured linear
scales of a variety of spacings. A composite photomicrograph should be
316 Chapter 15

made of a particular scale in two positions of rotation 90° apart; a better set
of standards uses cross-ruled diffraction gratings. All standards should be
photomicrographed at the working distance used to record the specimen
image.

15.12. FIELD OF VIEW

The field of view of the SEM is comparable to that of the light micro-
scope, and it is large compared to that of the TEM0°> (see Figures 15.4,<60>
15.17, and 15.18<13>).

15.13. NOISE

Noise should not be greater than one-fifth the signal change caused by
the specimen if the eye is to distinguish the true signal change in the

9tJm

FIGURE 15.17. Eyes and back of head of sweat bee. Taken by F. G. Rowe with a Coates and
Welter SEM, Modell06A with field-emission electron gun.
Scanning Electron Microscopy and Compositional Analysis 317

FIGURE 15.18. Vascular bundle in Scirpus lacustris, a reedJ13) Courtesy of A. C. Reim-

schuessel.

scanning-electron image. Noise can be introduced at all stages in the signal

path from the specimen to the cathode ray tube (see Figure 15.1). In the
transmission electron scintillator-photomultiplier system, the number of
informative electrons or photons is smallest at the intersection between
beam and specimen, where secondary electrons are collected. In an
efficient scintillator the signal is increased preferentially but without fur-
ther information in the image. Noise manifests itself as graininess(" snow")
in the image. Noise results from too little current for the scanning beam,
which should be at least twice the threshold (minimum acceptable) cur-
rent.(3l

15.14. DEPTH CUES

The depth cues in the SEM image are very impressive and effective.
With single SEMs the main cues are the distinct shadow and the apparent
 318 Chapter 15

perspective. In the SEM image the microscopic specimen appears as

though you were looking at macroscopic elevations and depressions of
wet sand (see Figure 1.4). In both cases depressions have their shadows at
the lower half of the visual image. The illustrator must assume responsi-
bility for correctly orienting photocopies of photographs in reports and
publications. Shadows should fall in the natural azimuth, and perspective
should also be natural. Parallel lines should appear to converge; larger
parts should be in front of smaller ones. Note these aspects in Figures 15.5,
15.8, and 15.17.

15.15. WORKING DISTANCE

The long working distance of the SEM is the distance between the
surface of the specimen and the front surface of the objective lens,<1>which
is of special advantage in arranging movement of the specimen. Working
distance from the bottom pole piece of the objective lens to the specimen's
surface is around 5-25 mm,(3> so that the specimen and its low-energy
secondary electrons are outside the magnetic field of the lens. The working
distance also makes it possible to have a large space to hold the specimen
on a special stage for heating, cooling, bending, ion etching, or electromi-
gration. <12>

15.16. FIELD DEPTH

The great field depth<U> is one of the most important advantages of the
SEM (see Figure 15.4). It is hundreds of times greater than in the light
microscope of the same magnification,<8>and it is capable of showing an
inclination with its highest and lowest points in simultaneous focus.

15.17. STRUCTURE

Structures are composed of units of increasing complexity: atoms,

molecules, domains, crystalline grains, and biological cells. <1> Periodic
structures are particularly interesting at this point. Figures 15.19 and 15.20
for example show the three-dimensional periodicity of certain diatoms.<13>
Figures 15.21-15.24 show the linear periodicity, respectively, of yellow,
pink, orange, and black scales taken from colored butterfly wings.<13>The
same figures also show interlinear structure that may or may not con-
tribute to the structural color.< 14>
FIGURE 15.19. Filter aid: diatomaceous earth. Taken by F. G. Rowe with a Coates and Welter
SEM, Model 106A with field-emission electron gun (see Figure 15.11).

FIGURE 15.20. A single fragment of shell in diatomaceous earth.<13) Courtesy of A. C.

Reimschuessel.
FIGURE 15.21. Yellow scale of the wing of the butterfly Colias eurythyme. Periodicity is~
~tm.< 1 3) Courtesy of A. C. Reimschuessel.

FIGURE 15.22. Pink scale of wing of the butterfly Colias eurythyme. Periodicity is 2-4~tm.<13>
Courtesy of A. C. Reimschuessel.
Scanning Electron Microscopy and Compositional Analysis 321

FIGURE 15.23. Orange scale of wing of the butterfly Colias eurythyme. Periodicity between
ribs is 1.5 ~tm; between cross ribs, 0.5--D.7 Jtm.(13l Courtesy of A. C. Reimschuessel.

15.18. MORPHOLOGY

Morphology, as has been stated,(!> involves the size and shape of a

structure. In Figures 15.21-15.24, the distance between lines should be
compared to the wavelength (color) of light from butterfly wings.

15.19. INFORMATION

To continue our search for information about the color of scales from
butterfly wings, we must know macroscopically whether the color is yel-
low, pink, orange, or black. Is a pigment present to contribute to the total
color by reflected light? What is the color by transmitted light with the
specimen mounted in a permeating liquid of similar refractive index, as
compared to the color in a liquid of very different refractive index?(l4l With
such information, scanning electron micrographs, such as those in Figures
15.21-15.24 have much more meaning than would otherwise be possible.
322 Chapter 15

FIGURE 15.24. Black scale of wing of the butterfly Colias eurythyme. Periou'u'y ,~ 3--6 J.lm.<13l
Courtesy of A. C. Reimschuessel.

15.20. DYNAMIC EXPERIMENTATION

Dynamic experimentation is a specialty of the SEM, which has great field

depth in focus and a large working space. Substantial specimens can be
moved with considerable freedom with or without accessories.
SEM stages strong enough to stretch metals, alloys, and semicon-
ductors have been devised,<Jl and micromanipulators have been built to
operate on specimens in the SEM. Closely related mechanisms are made
for observing and measuring scratch and indentation hardnesses.<12>
Special stages have also been developed to change the temperature,
magnetic field, electric field, liquid medium, etc.(3J Heating, cooling, mech-
anical manipulation, etching, and electromigration can all be performed on
the specimen within the SEM. For example explosive chemicals have been
studied as they slowly decompose at carefully controlled temperatures
below 200° C. Nickel particles have been thermally etched at 1000° C or
1300° C. Change in topography of metal surfaces has been studied at
various temperatures within the SEM.<12l
Ion etching has been performed by an argon ion beam at 5 keV
Scanning Electron Microscopy and Compositional Analysis 323

striking the specimen at right angles. Some of the materials studied in situ
by ion etching are dental tissues, iron oxide films, aluminum on oxidized
silicon,(l 2> and a wide range of inorganic and organic substances. <10> Elec-
tromigration is a process of moving metal atoms along a conductor in
which a current is flowing. Aluminum has been studied in this way.<12>
With beam diameter greater than about 1000 A, the current can be
made strong enough to employ television rates for scanning. Then dy-
namic events can be recorded on either cinefilm or video tape. With nar-
rower beams the SEM is limited to imaging repetitive events that can be
examined stroboscopically.<12>

15.21. SPECIMEN BEHAVIOR

Specimen behavior can be observed postmortem by exposing the spec-

imen to conditions outside the TEM' s chamber, stopping the reaction, and
putting the spent specimen into the TEM. Such procedures include weath-
ering, wear, fracture (see Figure 15.25), corrosion, erosion, annealing (see
Figures 15.20 and 15.26), and certain manufacturing processes.

FIGURE 15.25. Intragranular fracture in alumina.<13l Courtesy of A. C. Reimschuessel.

324 Chapter 15

FIGURE 15.26. Pure gold wire before annealing. Compare with Figure 15.7 sample after
annealing.(13l Courtesy of A. C. Reimschuessel.

15.22. SPECIMEN PREPARATION

Preparing a specimen for examination in the SEM by secondary elec-

trons can range from almost nothing to do to a complex procedure. No
preparation is needed if the surface is of interest and is conductive as
received. If the interesting surface is an insulator, it can become charged
and ruin the image. If the insulating layer is thin enough for some electrons
to penetrate an underlying conductor, a satisfactory image can be obtained
from the secondary electrons. If not, the usual procedure is to coat the
surface by vaporizing with a thin layer (-100 A) of metal such as gold or
gold plus palladium. A different approach involves viewing the insulating
surface through a hole in a foil of aluminum.
Surfaces of wet specimens are preserved by freeze-drying. For ex-
ample, fibers of wet paper fresh from the machine are not collapsed during
freeze-drying, whereas they are by ordinary drying. Of course, in finished
paper, fibers are not only flattened by commercial drying but even more
so by the calendering (glazing) process. The freeze-dried surface does not
Scanning Electron Microscopy and Compositional Analysis 325

represent the surface of paper, but it helps us understand changes in fibers

during paper making. <3>
In many instances a suitable surface of the material has to be made for
the SEM. Biological material can be cut once with a razor blade or mi-
crotome knife (see Figures 15.18 and 15.27), and watery tissue can be
frozen and fractured. Ice crystal size can be controlled by adding glycerine
or gelatin or both, or the water can be displaced by alcohol or acetone, the
sample quenched in liquid nitrogen, and then fractured.< 12> Treatment with
hydrophobic liquids, such as chloroform, ether, amyl acetate, or epoxy
resin (unpolymerized), may change the appearance of the material so
much that the image may not be interpretable. More complicated proce-
dures, such as chemical digestion, may make interpretation easier.

15.23. PHOTOMICROGRAPHY

Photomicrography is simpler with the SEM than with the TEM because
the image is exposed to the photosensitive material outside rather than

FIGURE 15.27. Section of Scripus lacustris, a reed, to show distribution of stomata.<13>Cour-

tesy of A. C. Reimschuessel.
 326 Chapter 15

inside a vacuum. With the SEM, Polaroid® or other fast-developing photo-

sensitive material may be used directly in the commercial camera back, so
that photomicrographs can be made very quickly.
On the other hand photomicrographic exposure is longer with the
SEM than with the TEM, because SEM signals are integrated slowly into
the image [less noise (fewer dots) appears during slow integration than
during fast exposure]. An exposure of 40 seconds is usual; 100 or 200
seconds may be required to clear up the noise. Yet a mere 20-seconds
exposure records a transient image and prevents damage to the specimen
by the electron beam. The number of lines per frame should be adjusted
to 1000 or up to 2000 with higher resolution. More lines per frame may
overlap and blur the picture, while fewer lines may be so far apart that they
show in the final photomicrograph.<10> In any event a canning electron
micrograph, with its raster structure cannot be enlarged to the same extent
as a transmission electron micrograph, with its very fine grain structure.
Many SEMs have cameras that automatically select exposure times
according to overall brightness and contrast in the image. If the operator
is interested in optimum brightness and contrast for a small part of the
image, it is possible to override the automatic mechanism with a manual
control. With instruments not having automatic cameras, the operator has
to make a decision by studying a line across an interesting part of the
image in darkness. The scan line should not be left very bright on the
record screen in darkness because the screen is easily burned, and it is
expensive to replace. Using Polaroid® positive film matched to the ASA
rating of the negative film speeds up the trials with fewer errors.<10> Scan
rotation units make it easier to arrange the image in a position that lends
itself more readily to interpretation.

15.24. SUMMARY

The SEM differs from the TEM in several important respects:

1. The SEM deals with various signals scattered from the surface of the
specimen.
2. These signals are received by cathode ray tubes which are synchro-
nized with the scanner probing the surface of the specimen.
3. The picture (pattern) on a cathode ray tube, like that on a television
tube, is composed of scanner (raster) lines that limit the degree of useful
total magnification but permit photomicrography with a camera placed
outside any vacuum. The Polaroid® type of quick photography is the most
popular.
Scanning Electron Microscopy and Compositional Analysis 327

4. The photomultiplier receives the secondary and/ or backscattered

electrons at an average angle to the surface of the specimen and at various
angles to various facets of grains. The resultant image manifests mod-
ulated shadows and a perspective, which add three-dimensional aspects
and render the image easily interpreted.
5. A faceted or rough surface manifests topographic contrast because
the detector is highly directional. Only certain facets are so oriented that
most of their backscattered electrons are aimed exactly at the detector,
while the remaining facets contribute fewer electrons thereby providing
more contrast between facets.
6. Crystalline specimens also contribute contrast by electron channel-
ing and magnetic or semiconducting effects.
7. In addition there are noncrystalline specimens contributing con-
trast by other effects of the initial electron beam: magnetic, voltage (from
beam-induced current), and luminescence.
Like the TEM, the SEM has great depth of field in focus because the
wavelength is so short and the angular aperture is so small. Resolution by
means of the SEMis somewhat less than that of the TEM, but it is of course
greater than the resolution of the light microscope.
The most important comparison however emphasizes the differences in
the kinds and extent of information provided by the three different types of
microscopes.
8. The qualitative and quantitative use of X-rays excited by a micro-
probe of electrons is offered separately by some manufacturers as EDS,
WDS, or EPMA. The EDS is the most common of these instruments.
 16

Emission Microscopies

16.1. INTRODUCTION

Emission microscopes without lenses can achieve high resolution by

using a very small source or detector in combination with scanning of
either the source, detector, or spedmen,<I> Chapter 16 discusses field-emis-
sion or point-projection microscopes, scanning tunneling and atomic force
microscopes, and scanning near-field light microscopes.

16.2. FIELD-EMISSION MICROSCOPES

Field-emission (point-projection) microscopes are usually operated

with the projection point cold. In the electron-emission microscope the
specimen can be heated (see Figure 16.1).<2.2al In other hot-emission micro-
scopes, the specimen can be broad and glow with visible or infrared
radiation (becoming the source of illumination). If however the surface of
a broad hot specimen is irradiated with UV light, photoelectrons can be
extracted at high potential and imaged on a fluorescent screen. <3•4>Two
kinds of field-emission microscopes<2>were invented by E. W. Muller (see
Figure 16.2). The older device, a specialized cathode ray tube (see Figure
16.1),<2>uses field-emitted electrons from the negatively charged tip of a very
sharp needle into a vacuum by point projecting the image onto a positively
charged, fluorescent screen.<2>The newer device (1950) emits ions from an
anode.<2a>
The electron field-emission microscope* is unique in its ability to

"The field electron microscope is sometimes loosely called a shadow microscope. It is not to
be confused with another, very different shadow microscope, much like the TEM except that
it produces an image by direct (point) projection instead of by a projection lens. <3>

329
330 Chapter 16

FIGURE 16.1. A schematic drawing of

the electron field-emission microscope,
showing the remarkable simplicity of
the apparatus. The coil coated with an
impurity can be heated to evaporate its
atoms onto the point for the purpose of
making emission studies.(2) Courtesy of
E. W. MUller.

detect if not resolve single atoms or small groups of atoms on the surface
of the needle tip. Thus the purity of the metal is established. If the coil
shown in Figure 16.1 is coated with a potential additive, such as barium,
and heated, such atoms migrate to the metallic tip of the cathode where
they are visible as bright spots in motion. The device is useful in studying
electron emission as a function of surface structure. Organic compounds
can be substituted for the inorganic additive and evaporated onto a clean
metal point. Phthalocyanine shows a pattern curiously like the structural
formula of its molecule.(2l Caution must be taken however when interpret-

FIGURE 16.2. Principle of both kinds of

point-projection or field-emission micro-
scopes.(2)
Emission Microscopies 331

ing such patterns, since a large number of molecules, regardless of their

shape or symmetry, produce similar patterns.<5-6l
Muller's newer device (1950) is the field-ion microscope (FIM).<2·7l It
operates with the metallic needle tip positively charged in the high vacuum.
A gas, such as helium, is introduced and ionized at the tip of the cooled
specimen. The tip is needled down from fine metal wire (e.g., tungsten) by
etching to give it a radius of only 5-100 nm. The etching roughens the tip
of the metal so that its atoms stand out in a terrace of ledges (see Figure
16.3).<4> A potential of several kilovolts is applied to the tip; these are
positive with respect to the negatively charged fluorescing screen. Figure
16.4 explains the formation of an image (or pattern) on the fluorescent
screen.<2l Incident atoms of the gas (e.g., helium) are attracted to the rough,
sharp tip of the specimen. Some of them hop along the tip, losing electrons
to the specimen cooled by a cryogenic bath. The resulting gas ions are
projected onto the fluorescent screen, where they release light in a typical
pattern (micrograph).
Figure 16.5<4> shows a diagram of the typical apparatus. Field-ion
microscopy is concerned with the study of the atom itself, not only in
metallurgy, but also in fundamental physics and chemistry.<S-ll)
Professor Muller added the atom probe to the FIM to identify atoms
chemically. Selected atoms are fed into an ion detector. Data are collected
slowly by a skilled operator and require careful interpretation.<4•10>
Figure 1.6 shows a field-ion micrograph of a platinum crystal that was
taken in the 1960s<2> at the Pennsylvania State University by the late Pro-
fessor E. W. Muller. The micrograph shows the distribution of Pt atoms in
a Pt crystal. The micrograph was provided by his successor, Professor T.
T. Tsong.< 10l In 1933 Professor Tsong published an up-to-date account of
field-ion microcopy.<2aJ This technique "now permits the study of mecha-
nisms and energetics of atomic processes on solid surfaces and the single-
atom and atomic-layer chemical analyses of surfaces."<2aJ

FIGURE 16.3. Illustration of a ball mod-

el that approximates the end of a field-
ion emitter. The surface is not smooth;
protruding atoms are imaged in the
fiM.(4)
332 Chapter 16

POLARIZ£0 He ATOM

FIGURE 16.4. A polarized helium atom is attracted to the metal tip, slowed down in a
number of hops, and ionized in the ionization zone (0.2-A thick) over a protruding atom. The
helium ion is accelerated toward the screen.<2>

FIGURE 16.5. Schematic drawing of a FIM.'2l

Emission Microscopies 333

16.3 ATTRIBUTES CONTRIBUTING TO VISIBILITY BY

FIELD-EMISSION MICROSCOPY

16.3.1. Thought, Memory, and Imagination

The mental processes of thought, memory, and imagination are espe-

cially important when interpreting images by field-emission microscopy.
In the absence of lenses, illuminating devices, and automatic controls, a
field-emission microscope is less of an apparatus and more of a direct aid
to the eye and brain. Together the mental processes furnish, Muller wrote,
the skill, experience, patience, and ingenuity which the microscopist
can muster for the preparation of the specimen, the making of the
observation, and, finally, the interpretation of the image. It seems as
if the evasive atoms still hide from the curious eye of the casual sight-
seer, and reveal themselves rewardingly only to the serious re-
searcher. Ill

16.3.2. Resolving Power

The FIM is capable of visually separating individual atoms.<2> Each

atom is depicted as a bright dot on a black background, like stars in the
night sky. The theoretical resolution is about 1.5 A, since the wavelength
of ions (protons) is so much less than that of electrons and the temperature
of liquid hydrogen or nitrogen is used to reduce thermal motion. <3>

16.3.3. Resolution

The practical resolution of the modem FIM is as small as a triangular

spacing of 2.3 A. Resolution amounts to separating bright spots or halos
with certain elements-helium ions and liquid nitrogen. Neon, nitrogen,
oxygen, and argon can be used instead of helium, but the images are
inferior. Elemental specimens that give best resolution and those that give
satisfactory images are discussed in Section 16.2.15.<2•10>
It should be emphasized that resolution with the FIM involves sep-
arating atoms as halos (bright spots or tiny stars). There is no resolution
within the atom. With the field-emission electron microscope, while res-
olution of phthalocyanine (see Figure 16.3) is 12 Aon a side,<3> each square
is divided into two or four parts, giving a resolution of at least 6 A.
334 Chapter 16

16.3.4. Contrast

There is usually plenty of black and white contrast between bright

spots. In 1960 Muller used color contrast to detect quick changes in two
successive images by printing one on red film and the other on green.
When the two are superimposed, deletions from the first pattern are red,
additions are green, and unchanged portions are yellow.(s,JO)
The frequent problem of low intensity in field-ion images is due to
contrast. Muller found some success by post accelerating ion beams
through a fine, high-transmission wire mesh, but disturbing double im-
ages may occur. Converting the ion image into an electron image by using
secondary-electron emission from a fine grid is somewhat more promising.
External electronic image intensifiers based on photoelectric emission have
proved to be more successfulPl

16.3.5. Aberrations

Aberrations in field-emission microscopy originate at either the tip

emitter or the fluorescing screen (since there are no lenses). The tiny tip is
the chief source of aberrations. Most of them arise from the mechanical
stress exerted by the electric field on the conductive surface, about 1
ton/nm,(3l which may exceed the yield strength of the particular metal by
a factor of 50. The shear component of the stress, due to nonsphericity of
the tip, may cause dislocations to move until the tip fractures. One correc-
tion involves reducing the tip's radius to less than 200 A, so that there is
insufficient room for defects to grow.(2)
Another kind of correction involves reducing the field strength to
shape the tip without introducing severe lattice imperfections. Hydrogen
promotes field evaporation and therefore shaping of the tip; it can reduce
the required field strength by 5-20% for most metals used as tips.

16.3.6. Cleanliness

Cleanliness is of supreme importance in field-ion microscopy because

some of its main applications involve the purity of metals-detecting and
locating impurities and studying solids, liquids, or gases adsorbed on the
surface. Therefore the metal specimen used for the emitting tip should be
as pure as possible, as should be gases for ionization or adsorption and any
other substance introduced into the microscope. All these ingredients must
also be scrupulously free of dust, dirt, oxidation, and any other contamina-
Emission Microscopies 335

tion. Metal for the specimen tip should be kept free of mechanical changes
that would alter crystallographic conditions, such as dislocations, slip-
page, twinning, orientation, etc.

16.3.7. Focus Depth

Focus depth does not apply to the microscope itself, since it is without
lenses, but it does apply to the photographic lenses in the cameras used to
record either snapshots or moving pictures. Especially with the FIM im-
ages are of such low intensity that the lens should have a high aperture
(low !-number). This means short focus depth, which in tum means that
the fluorescent screen should be as flat as possible.<6> Another reason for a
flat screen in either kind of emission microscope is that images are often
changing or migrant, and this means short exposures, wide-open lens, and
consequently short focus depth.
Atoms are generally in focus, but occasionally an atom jumps out of
focus and appears brighter on the fluorescent screen.<10>

16.3.8. Illumination

Illumination in emission microscopy is due to the point projection of

electrons or ions from the specimen-emitter onto the fluorescent screen.
Intensity of image in the FIM depends on the specimen, its tip, the ioniz-
able gas, etc. When these factors cannot be improved upon sufficiently for
visual examination or for photography, Muller has used an external image
intensifier (see Figure 16.6).<2>

16.3.9. Radiation

Radiation in the field-emission electron microscope forms a cone of

electrons, as in an ordinary cathode ray tube. In the FIM, a stream of
polarized atoms of a gas, such as helium, is attracted to the cold specimen
at a high positive potential. Here gas atoms are slowed down in a number
of hops and ionized and accelerated toward the screen.<2•14>
Most of the metals examined to date are isometric and thereby iso-
tropic. Some however such as rhenium, are in the hexagonal system and
therefore anisotropic.
Orientation of the metal grains does, however, contribute to the field-
emission image, so that grain boundaries, twins, dislocations, etc., are
recognized.(2l In the field-electron microscope, the orientation of some
 336 Chapter 16

4:1 ,F:Q87

WATER COOLED COIL INTENSIFER TUBE

FIGURE 16.6. Schematic drawing of a FIM with external image intensifier.!2>

molecules, such as phthalocyanine, apparently makes a difference in the

image, but the explanation is not clear.(5l

16.3.10. Magnification

Magnification is quite incidental in field-emission microscopes. It de-

pends primarily on the radius of curvature of the tip. But the variation in
local magnification due to bumps on the tip are not directly related to the
radius of the bump because of a strong compression factor. Magnification
of course also depends on the projection distance.

16.3.11. Field of View

The field of view in emission microscopes is small because the tip has
a volume of only about 10-21 m3• Larger fields require higher potentials
than the usual30 kV. Larger volumes also result in higher stresses on the
specimen with greater potential damage.

16.3.12. Artifacts

The chief artifacts in field-ion microscopy come from disturbances to

the specimen by extremely high mechanical stresses induced by very high
Emission Microscopies 337

electrical fields. Such stresses are great enough to cause fracture. Stress
both nucleates and rearranges dislocations, and it can induce deformation
twinning; it usually leads to relatively high artifactual vacancies. As long
as such artifacts are understood, they can be turned into an advantage in
performing experiments in situ.
In the FIM the third dimension is limited to one or two layers of atoms,
a true surface. Yet in a homogeneous specimen, we can infer much about
the third dimension from the surficial arrangement of atoms and from
previous knowledge of the space lattice of the respective metal (see Figure
16.3). Field-ion microscopy is already accepted in studying three-dimen-
sional metallography.<t3)

16.3.13. Working Distance

The working distance in both kinds of field-emission microscopes is

fixed by dimensions within the sealed chamber. Nevertheless the working
distance is ample for any practicable experiment, since no lenses are in-
volved.

16.3.14. Field Depth

The field depth in emission microscopes is completely adequate be-

cause there are no lenses (and no focusing), and the object space consists
of only one or two rows of atoms.

16.3.15. Structure

The atomic and crystalline structures of the metal are very important
attributes contributing to visibility in the FIM. The chemical nature of the
ionizable gas is also important. By the ionization of helium, for example,
the following metals at the temperature of liquid hydrogen yield sat-
isfactory fj.eld-ion images: tungsten, rhenium, iridium, platinum, molyb-
denum, tantalum, niobium, and rhodium; marginal are zirconium, vana-
dium, palladium, titanium, nickel, and iron. With neon gas gold, iron,
nickel, copper, and zinc can also be imaged at or below their evaporation
rate, as can metals in the first of the two preceding lists. Various changes
in crystalline structures can be seen at the atomic level, such as disloca-
tions, vacancies, impurity atoms, twinning, slip bands, and fatigue cracks.
 338 Chapter 16

16.3.16. Morphology

Morphology of the field-emission microscopical specimen at present

refers chiefly to sizes and shapes of particles of a second phase distributed
in an alloy. Quantitative particle-size distribution is difficult to obtain due
to the complex shape of the specimen. Calculations are best performed on
a computer.(4l

16.3.17. Information

Crystallographic information about the specimen amenable to field-ion

microscopy is published and generally available.(9- 11 l

16.3.18. Experimentation

Various experiments can be performed inside the FIM. By preferred

field evaporation of weakly bound, substitutional impurity atoms or by
corrosion through field-induced chemical reactions with strongly ad-
sorbed oxygen, carbon monoxide, or nitrogen, surface vacancies can be
made to appear. Slip bands can be produced in situ by pulling at the tip by
applying a reduced electric field at elevated temperature. The growth of
fatigue cracks has been seen by superimposing an alternating voltage
component onto the tip voltage to cycle the stress. The difference between
annealed and field-evaporated specimen tips can be observed. The re-
versed action-rearranging atoms when annealing a field-roughened tip-
can be witnessed, and at the same time activation energies can be mea-
sured in situ. (Z) These are a few examples of experimentation in the FIM.
Experiments can also be performed by field-emission electron micro-
scopy. Emission is critically influenced by monatomic layers, and single
atoms or small groups of atoms can be detected as scintillating bright spots
on the fluorescing screen. Migration of the atoms is readily observed as the
surface structure is varied. Organic molecules can be introduced by evap-
oration from the heater coil within the microscope. The scintillating spots
have structure, but interpretation awaits further experimentation.(3,5l

16.3.19. Behavior

Behavior can be observed in field-emission microscopes, since the en-

vironment is changed relatively easily in such a small, sealed space. Oxy-
Emission Microscopies 339

gen, carbon monoxide, nitrogen, and other atmospheric gases can be in-
troduced at will; likewise various other molecules can be introduced if
they can be volatilized satisfactorily within the microscopical chamber by
means of the high vacuum and localized heat. In this way corrosion and
stress corrosion can be studied in situ. Specimen behavior in organic atmo-
spheres may also be studied,<3>

16.3.20. Specimen Preparation

Specimen preparation is an important factor imaging by radial projec-

tion. The first crude steps are chemical etching and electropolishing a fine
wire of the specimen metal, such as platinum. The etched specimen tip is
spot welded to the tungsten leads sealed in glass (see Figure 16.5). The
microscope is evacuated, and evaporation occurs at the tip when thermal
activation permits atoms to leave the surface. However when a sufficiently
high electric field is applied, some surface atoms may leave as ions by
shedding an electron or two apiece. Field evaporation takes place pref-
erentially at the sharp tip, which had been roughened by the etching. The
tip becomes an oblate microhemispheroid, characterized by a uniform
field strength. <2>
The problem of preparing photographs of field-emission images lies in
recording a fleeting or moving subject at a low level of light intensity. A
fast photosensitive emulsion is required. Fortunately there is plenty of
contrast: white spots on a black background. Visual monitoring can be
done in a darkened room. The external image intensifier shown in Figure
16.6 helps in severe cases, especially in taking moving pictures.<2. 10l
Even with fast film, fast lenses are required in field-ion microscopy.
Hydrogen-ion still pictures can be taken at f = 1.4, but with helium ions f
=1.0 is required. For 16-mm motion pictures f =1.0 is also required.<5•10>
Muller reports the technique of taking two successive exposures and
superimposing them to make a positive picture of very fast changes; print-
ing the first picture on red film and the second on green is another tech-
nique.<2·10>

16.4. SCANNING-TUNNELING AND ATOMIC FORCE

MICROSCOPIES

The scanning-tunneling microscope is among the latest inventions,

having been developed in the 1980s. It has subatomic resolution; un-
fortunately the STM does not operate with nonconductive specimens,
340 Chapter 16

whereas the subsequently developed AFM can be properly operated with

insulating specimens.
Although developed almost independently of the earlier FIM, the two
instruments are related, but the STM has many advantages over the FIM.
The STM has greater resolution than the FIM and generally scans over the
surface in a raster pattern to assess a much larger field of view than is
obtained with an FIM. Scanning is similar to that with a SEM, but here
scanning is a physical movement of the probe, whereas it is electronic in
an SEM.
The AFM is similar to the STM but its probe tip or objective actually
contacts the specimen's surface, whereas that of the STM does not. Resolu-
tion is greater on the STM than on the AFM.
For both types of microscopes, no free particles are collected. The
tunneling microscope detects voltage changes that vary exponentially with
the probe's distance from the surface. With the quantum tunneling con-
cept, there is no sharp discontinuity between surfaces, just an exponen-
tially decreasing probability of the two sets of electron clouds overlapping.
The AFM detects surface contours through either the tunneling principle,
capacitive techniques, or interferometry.
Tunneling current provides a very sensitive atomic-level sensor or
probe of the surface profile-in other words it is a microscope. Since
vertical position of the objective must quickly respond to changes in atom-
ic elevation, computer controls are necessary.

16.4.1. Scanning-Tunneling Microscopy

The STM produces an image by monitoring electrons that tunnel

between the two electron clouds bridging the narrow gap separating the
conductive specimen surface from the probe. The probe is approximately
triangular in shape with the sensing end ideally terminating in a single
atom<12•17•18> (see Figures 16.7 and 16.8). Wave functions in both the micro-
scope's probe and that of the specimen overlap, so that with a small
applied voltage between the probe tip and the specimen, electrons tunnel
between the two surfaces. Since the tunneling current is extremely sensi-
tive to distance, a rapid-response, computer-controlled feedback mecha-
nism maintains a constant distance between surface and probe. In this
manner the probe measures the surface contours while scanning, and its
motion is interpreted by a computer and displayed on a monitor.<15•16>
Scanning over the field of view yields a three-dimensional image of
the surface. For proper operation the two surfaces are less than 10 A
Emission Microscopies 341

ELECTRONICS

COMPUTER SCREEN

SCANNING NEEDLE TIP

FIGURE 16.7. Electron tunneling, (bottom); scanning an electron tunneling tip and a screen
display of the surface morphology are shown (top). Electrons tunnel across very small
separations, and because the tunneling current depends exponentially on the degree of the
separation, the tunneling current provides a very sensitive microscope of the surface's
morphology. From Gerg Binnig and Heinrich Rohrer, "The Scanning Tunneling Micro-
scope," copyright© Scientific American (Aug. 1985); all rights reserved.<17l

apart,<17•18l and the applied voltage is in the range of O.l-1V.02•17J The in-
teraction strength of the two overlapping sets of electron clouds decreases
exponentially with distance, which makes the instrument extremely sen-
sitive to minute surface irregularities. A change in the separation distance
of approximately one atomic diameter changes transmitted voltage by as
much as 1000 times.<17J
Natural atomic and subatomic scale irregularities isolate the tunneling
current, and these are the source of the extreme resolution.<12l Vertical and
 342 Chapter 16

zPIEZO

xPIEZO

~
SAMPLE

FIGURE 16.8. Schematic diagram of vacuum tunneling showing the tip and piezoelectric
controls for the x, y, and z directions.<12l Tips are generally less than 10 Ain diameter. The
piezoelectric control marked z controls the working distance between the specimen and tip.
X and y piezoelectric controls scan the tip in a raster pattern over the field of view. During
scanning the tunneling current is monitored and creates the displayed image.

horizontal resolutions are approximately 1 A (0.1 nm).<19•20> With the very

tip of the probe generally one atom in size, the sensitivity or resolution is
subatomic. In fact a new tip is checked by simply measuring the resolution:
If resolution is not atomic or subatomic the tip is normally replaced with
another fairly inexpensive one. The tip is often of tungsten or platinum-
iridium.<20l Since sensitivities yield magnifications of 10-100 million; how-
ever isolation from any vibrational noise and precise translational drives
are critical. (IZ,t7)
Both the STM and the AFM can scan a field of view as large as 130 nm.
Because scanning is a contact technique, it does not resolve steep walls
well.<20> The AFM has provided high-resolution images on nonconducting
materials, such as polymers, and biological materials, such as amino acids
and blood cells. Both air and liquids have been used as the media around
the specimen.<27l Excellent recent contributions on this subject were re-
Emission Microscopies 343

viewed in Ultramicroscopy, which has a series of relevant symposium arti-

cles. (2£>-28>
Figure 16.9 shows the most critical parts, a larger view of the instru-
ment AFM's appears in Figure 16.10. A nanoscope AFM is shown in Figure
16.11. The upper part of the instrument is a reflected light microscope, and
the "head" of the AFM is in the protective housing at the front focal plane
of the light microscope. With the light microscope initial alignment to
within 1 f.!m can be accomplished. Figure 16.12 shows the atomic resolu-
tion of NaCI atoms.

SOUDSTATE
LASER

SPUT-DIODE
PHOTO DETECTOR

XYZ
SINGLE TUBE
PIEZO
SCANNER

FIGURE 16.9. Schematic of an atomic force microscope showing the optical lever for detect-
ing cantilever deflection. Reprinted from American lAboratory 23, 4(1991), 40. Copyright 1991
by International Scientific Communications.(25>
344 Chapter 16

lUNNEUNG SIGNAL

3 AXIS
PIEZOELEClRIC
SCANNER

SVSlEM
CONTROLLER/
FEEDBACK
COMPUTER &
DISPLAY

BIAS VOLlAGE
SAMPLE

FIGURE 16.10. Diagram of a scanning-tunneling microscope. Reprinted from American Lab-

oratory 23, 4(1991 ), 36. Copyright 1991 by International Scientific Communications. (2S)

16.4.2. Scanning Near-Field Light (Optical) Microscopy

The SNLM is still in the experimental stage, but it has already ex-
tended the resolving power of light microscopy by more than an order of
magnitude over the resolution limit developed from classical theories (i.e.,
from 20 nm to 0.2 nm)(29> (see Figure 2.1). Although sometimes called an
optical microscope, light is the preferred term, since the medium is light
and optical also pertains to other media.(S,Sa) Development of the SNLM
was stimulated by the earlier appearance of the STM, and they both use the
same close proximity between the probe objective and the specimen's
surface.
Scanning near-field light microscopes, invented at Cornell University
by Professor Michael Isaacson and his associates, do not have lenses. The
resolving power of lenses is limited, not only by the wavelength(s) of the
emanation, but also by aberrations imposed on an image formed relatively
far from the object, that is, in the far field. Professor Isaacson uses a simple
analogy: a garden hose with a nozzle of a certain diameter supplied with
water at a certain pressure. If we observe the aperture of the nozzle at close
hand, we will be sprayed by water in a spot whose diameter is close to the
diameter of the hole in the nozzle. But if we stand, say, 3 feet from the
FIGURE 16.11 Nanoscope AFM and Nikon light microscope with video display. As shown
on the video screen, one can position the probe tip while simultaneously viewing the sample
magnified by light from above. The tip can be initially positioned before commencing the
AFM scanning to within 1 urn. Courtesy of Nanoscope, Digital Instruments, Santa Barbara,
CA.
346 Chapter 16

FIGURE 16.12. Resolution of atoms on the surface of NaCI to show the ability of the AFM
to resolve atoms on the surface of nonconductors. Courtesy of Nanoscope, Digital Instru-
ments, Santa Barbara, CA.

nozzle, we will be sprayed all over with water.<33l Therefore instead of

focusing light with a lens, Isaacson passes light through a very small hole
and a sample so close to the hole that the light beam has no chance to
spread out. Using yellow green light (A. = 550 nm), Isaacson's team obtains
a resolution of about 40 nm. Thus they are not resolving atoms (as with an
STM), but they are using light. The limit is not so much the wavelength of
the light as the fact that the amount is small enough to be undetectable.<33l
Aaron Lewis at Hebrew University in Jerusalem and Raoul Koppel-
man at the University of Michigan plug the 50-nm hole with a tiny crystal
of anthracene because it fluoresces in UV light, yielding excitons that pass
easily through the tip and turn into visible light (fluoresce). The result is
a much brighter image, say, of a biological cell, from a near-field light
microscope.133l
Emission Microscopies 347

Since the resolution of SNLM is similar to that of a SEM, what are its
advantages? Potential advantages include its different type of illuminat-
ing, lack of need for a vacuum environment, potentially considerably
lower price, and use of well-developed light contrast mechanisms over
electron microscopy.<30>In the future this technique maybe used to develop
a near-field infrared microscope for chemical analysis of submicron spec-
imens. <31- 34>
At first the SNLM seems to contradict the basic laws of optics; how-
ever these laws exist for the far field, while the SNLM functions only in the
near field. Figure 16.13 shows a schematic of the SNLM.
The far field is well explained by the classical work of Abbe and
others, but theoretical explanations for the behavior of light in the near
field have not yet been completely developed, although the general un-
derstanding does exist. Near-field behavior depends on evanescent or
transient waves. These waves cannot propagate in free space, but they
provide the mechanism for light to transmit through an aperture smaller
than the wavelength.(31> Light emanating from an aperture of submicron
diameter forms an illuminated spot of subwavelength diameter and this
spot of light is scanned over the specimen to generate the image.<31 >During
light transmission through the submicron-sized aperture the light's wave-
length is collimated to the aperture size, and the transient light through the
aperture is mainly independent of the initial wavelength-this evanescent
light extends only a distance approximately equal to the aperture's ra-

\...---Optical Fiber
} /ZPiezo
Piezo Voltage ' / /""Pipette

••
I
Illumination
I

FIGURE 16.13. Schematic of the near-field scanning light microscope developed at Cornell
University.<30l
 348 Chapter 16

dius,(32> at greater distances its behavior follows classical laws. For a 500-A
diameter aperture, the near field extends only about 200 A.<32> Tunneling
current from protrusions on the aperture has been used in conjunction
with the light probe to control the objective's or probe's working distance
from the sample.<30>
At first forming the submicron-sized apertures seemed a major ob-
stacle, but this was overcome by drawing hollow glass capillary tubes to
the breaking point.<32> Metal is then evaporated onto the drawn capillary,
making it opaque, and the hole is left in the end of the capillary.
Work has been done with the aperture functioning as the illumination
source, and some recent developments use it as the collecting objective.<30>
The illumination system is scanned across the specimen similar to the
scanning in an STM or SEM. A major problem with continually decreasing
the diameter of the aperture is the amount of light emitted through the
minute aperture. The emitted light can be as little as one ten-thousandth
or less of the illuminating light, and techniques are being developed for
increasing the illumination.<33> Betzig and Trautman provide a compre-
hensive review of the SNLM. <34>

16.5. SUMMARY

Emission microscopy is one of the most rapidly evolving develop-

ments, and it potentially has major importance. It permits atomic resolu-
tion and detection of the chemical composition at the atomic scale. The
latest development in this class of microscopes uses light and the in-
vestigators have achieved a resolution which is a small fraction of the
light's wavelength.
In a field-emission microscope, the specimen is the source of radiation
by virtue of a strong electrical field and a very high vacuum. The specimen
source is cold (at or below room temperature) unlike thermal emission.
Lacking lenses, cold field-emission microscopes achieve magnification by
straight projection of electrons or ions rays; therefore the point of the
thin-wire specimen is made extremely sharp (5-100 nm radius). With a
projection distance of 10 em, magnification (over one million times) is
useful to the extent of resolving atoms or small groups of atoms, depend-
ing on whether ions or electrons are emitted.
The electron field-emission microscope is much like an ordinary cath-
ode ray tube except that the specimen, which is the cathode, is pointed at
a resolving power of 2 nm. In this state the field-electron microscope is
useful for studying the emission of electrons as a function of surface
structure.
Emission Microscopies 349

The ion field-emission microscope employs the extremely pointed

specimen as the anode, which is kept very cold in a liquified gas. Helium
atoms are introduced and find their way to the cold tip, where they are
slowed down in a series of lower and shorter hops on the roughly etched
tip .of the anode. Some of the slow helium atoms lose an electron to a
protruding metal atom. The resultant helium ions are shot onto the lu-
minescent screen, producing a dim pattern that can be photographed on
fast film during long exposure or intensified photoelectronically. There-
sultant pattern (image) is of refractory metal atoms organized or dis-
organized on a surface or succession of surfaces. Grain boundaries, slip
planes, dislocations interstices, and vacancies are also made visible. Inter-
pretations lead to surficial binding energies and work functions; surface
chemical reactions of interest include corrosion and catalysis.
The STMs, AFMs, and SNLMs are among the latest and potentially
most important technological developments. No free particles are collected
with either microscope. The detector is either within tunneling distance of
the surface or actually contacting the surface.
Scanning-tunneling microscopy is related to earlier field-ion micro-
scopy, but it has many advantages: Scanning-tunneling microscopy has
greater resolving power than field-ion microscopy and generally scans
over a larger field of view. The AFM is similar to the STM, but its probe
tip actually contacts the specimen's surface, whereas the STM collects
information by sensing electronic tunneling signals from the specimen's
surface. The AFM' s resolution is less than that of the STM, but it can
examine the surface of an insulator, whereas the STM cannot.
The SNLM is the latest in this evolving series. Its resolution is sub-
wavelength through use of a condenser objective of much smaller inside
diameter than the initial wavelength. Like the STM and AFM, the scanning
near-field light microscope is a near-field instrument and therefore its
fundamental resolution and capabilities do not follow the classical law
developed for the far field. Understanding is still at an early and evolving
stage. In a strict sense an SEM is an emission microscope, but since it is so
well established independently of the others, it is discussed in Chapter 15.
The scanning probe microscopes discussed in Chapter 16 and others
are reviewed in considerable detail in an article, "The Scanning Probe
Microscope,"<35> which should be studied for additional detail. Unique
capabilities of these microscopes make them an exciting topic, and they
hold considerable potential for the future.
 17

X-Ray Microscopy

17.1. X RAYS

X rays are electromagnetic radiation of the same nature as visible light

but with only one-thousandth of the wavelength. (I) X rays behave like light
toward photosensitive materials. For example when a dentist takes a ra-
diograph she/he places an unexposed photosensitive film next to the teeth
and turns on the X rays. Cavities show up dark on the negative film
because they transmit X rays so well. Sound teeth show up less dark
because they are denser than air or flesh. Ceramic and plastic fillings are
still darker if they are denser than teeth and bone. Silver and gold show up
very dark when exposed to X rays because they are very dense (such have
high atomic numbers).
The dentist's radiograph is approximately life-size (lx). Dioptric
magnification by transmitting X rays is not feasible because lenses have not
been developed for it. X rays at glancing incidence have been reflected
from concave mirrors or curved surfaces of crystals, but so far focusing is
not sharp enough to construct an X-ray microscope this way.
The dentist's radiographs and other radiographs may be usefully
enlarged to lOx, but not much more. An X-ray microscope based on the
principle shown in Figure 17.1 has no more magnification than the simple
light microscope or equivalent that enlarges the contact negative. The
enlarger cannot remove the contact negative's blur, which is caused by the
crossover of peripheral X rays from such a large source.<2,3> Still the or-
dinary source of X rays may be useful in low-power microradiographic
studies of the distribution of heavy elements among lighter ones in an
essentially inorganic specimen. In an organic system contrast in the pref-
erential absorption of X rays is increased if a selective stain can be found
bearing a heavy element in its composition. In any case it helps in simple

351
352 Chapter 17

--Negative film

r-1
Source FIGURE 17.1. Ordinary source of X rays
for microradiography blurs the contact
image PI

microradiography to have the specimen as thin as commensurate with

selective absorption among the specimen's constituents.
-The boom to moderately high-power (100 or 200x) microradiography
came with the development of a point source for X rays. As shown in Figure
17.2, the point source provides a very narrow bundle of almost parallel X
rays to impinge on the specimen. As the source point becomes smaller and
smaller, the crossover angle of the peripheral X rays through the specimen
is reduced accordingly. Therefore the contact image is made sharper, and
the permissible thickness of the specimen may be a little greater.

17.2. CONDENSER LENSES

One way of producing a concentrated point of X rays is to use the

objective lens of an electron microscope to focus electrons on a target

·~========+--H--===

\ SOURCE
FIGURE 17.2. Point source of x rays for
microradiography improves the con-
tact image.<J>
X-Ray Microscopy 353

situated near the specimen and photosensitive film. Figure 17.3C4>shows a

homemade adapter for the point projection of X rays from a target through
the specimen to the filmC4>Subsequently electromagnetic lenses were built
into commercial point-projection X-ray microscopes.'2•3•5>Figure 17.4 shows.
a diagram of a double electromagnetic X-ray microscopeP> Such a micro-
scope is ideal for studying inorganic chemical constituents, especially
those with high atomic number. Nevertheless the specimen should be thin.
If it is thick, the catalytic pellets for absorbing automotive leaded gasoline
vapors, the pellets are abraded until they are only 25 t-tm thick. Figure
17.5C6>shows a negative microradiograph of thinned unused experimental
pellets, taken on a Philips instrument.C5>The photosensitive film was Ko-
dak fine grain, processed through a fine-grain developer. The finished
negative was enlarged 20x. Other pellets that had been exposed to fumes
from leaded gasoline were treated and x-radiographed in the same way.
The commensurate negative enlargement is shown in Figure 17.6. While
peripheral rings show the distribution of lead atoms in the exposed pel-
lets.C6>
Point-projection radiography is also very useful in studying organic
systemsP·7- 9>Botty and his colleagues studied plastic foams by employing
soft {low-voltage) X rays of low-penetrating power, in which low-mass
materials {e.g., air and polymers) are differentiated. In Figure 17.7, an
enlarged negative microradiograph of an organic artificial foam, air bub-
bles photograph black, and the polymeric walls photograph white and
grey.C7) The point of X rays is so sharp and the specimen is so thin that a
useful magnification of 100x was achieved.

ELECTRON BEAM focused

by Objective Lens to gwe
a Pomt Source of X-rays
at Target
Pole Piece
Target

Adapter Insert Obi Lens I

I
Specimen I
Photographic Film ----11:-:""""

FIGURE 17.3. Schematic drawing of adapter showing target, specimen, pole piece, and
paths of electron and X-ray beams. C4>
354 Chapter 17

FWORESCENT SCREEN
OR PHOTOGRAPHIC PLATE

X-RAYS

OBJECTIVE

B..ECTRON SOURCE

FIGURE 17.4. Diagram of the point projection X-ray microscopeP>

17.3. RECENT DEVELOPMENTS

X-ray microscopy, with either synchrotron radiation(10l or laser-pro-

duced plasmas as x-ray sources,(11 l has recently begun to realize its promise
in biology, engineering, radiobiology, geology, and materials science.
Both illumination sources and detectors have been improved in recent
years.(12> Brightness for example has been increased millions of times over
that of conventional X-ray tubes by means of synchrotron radiation, X-ray
lasers, and X-ray-emitting plasmas. Detectors have been improved
through both new electronic developments and such recording techniques
as microscopic resist materials.(12>
Synchrotron radiation is being used as the source of soft X-ray pho-
tons of 0.1-1 keV energy with 1-10 nm wavelength. There are three major
potential advantages of soft X-ray microscopy over light and electron
microscopies( 13>:
FIGURE 17.5. Microradiograph of experimental auto exhaust catalyst pellets before ex-
posure to leaded gasoline vapors. Thin ground sections (approximately 25 J.lm thick).<6>
Philips CMR Instrument.<5> Pellet diameter: 1.5 mm.

FIGURE 17.6. Microradiograph of experimental auto exhaust catalyst pellets after exposure
to leaded gasoline vapors. Ground thin sections. The white peripheral rings show the dis-
tribution of Pb condensate.<6> Philips CMR Instrument.<5J Pellet diameter: 1.5 mm.
356 Chapter 17

100~tm

FIGURE 17.7. Contact microradiograph of a thin section of thermosetting polymeric foam

showing holes, or thin-walled air bubbles (black spots), at the periphery of the thin cell
windows.m Exposure conditions: 2 kV, 3.5 mm. Philips(S) CMR Unit: standard specimen
holder; Kodak film .

X-ray microscopy has a potential resolution 25-50 times greater than

light since their wavelengths are much shorter than those of light.
In the X-ray spectrum from 2.3-4.4 nm wavelengths, there exists a
large difference, an edge, in the absorption of carbon containing
molecules and water.
Due to their high penetrating power relative to electrons, the internal
structure of relatively thick (10-20 1-1m) specimens can be examined
in transmission.

Scanning microscopes with such sources have been constructed at centers

throughout the world. An example is the National Synchrotron Light
Source at Brookhaven National Laboratory in Upton, New York.( 12l Images
can be recorded digitally, so that quantitative information on specimen
absorptivity as a function of position can be quickly obtained.
With synchrotron X-ray sources it is possible to choose different wave-
lengths of radiation to use the atomic absorption coefficients of structural
materials selectively.(14l From such information elemental maps can be
X-Ray Microscopy 357

drawn.<15> Resolution is currently better than 600 A, and current microfab-

rication techniques are increasing the resolution.<12,16> In addition to syn-
chrotron radiation sources, considerable activity in laser development is
occurring in applications of X-ray lithography, and X-ray microscopy is
also benefiting.<11,17l A schematic of this technique is shown in Figure
17.8.!l7)
The basic technique, which is similar to contact printing and in fact
termed contact microradiography,<17l involves placing a specimen near the
recording medium, which is then exposed to the X-ray source. High res-
olution is possible; X-ray photoresists are used<1>to record the image. Much
of the necessary technology evolved from fabricating integrated circuits
using microlithography. While earlier techniques to miniaturize circuits
involved electron beams, X rays have been used more recently. For this
type of microscopy, a specimen is placed in intimate contact with the resist
surface. A typical high-resolution resist is poly(methyl methacrylate)
(PMMA). After exposure to X rays and subsequent development, there-
sulting relative heights in the contact image are functions of the specimen's
X-ray absorption; Figure 17.9 illustrates the contact-imaging process.!17l
After preparing the contact image, it is magnified in either an SEM or a
TEM. Damage can occur to the photoresist image in the electron beam used
for viewing so techniques are being developed to form metal replicas of the
resist image.<11>
LASER
PULSE
I t
I I

••
I t

WINDOW
I I
~ I ~APERTURE
I I I

i=t=i--
I I
LENS

,•r~
I I I

11 : : • SUBST.AATE
'1. ~·RESIST I
1II1 11 / , ',1 SPECIMEN
~J~ J'/1/x-RAYS
.•!/~
PLAS~TARGET
FIGURE 17.8. Schematic of a
plasma generated by a laser pulse.
Drawing shows a possible config-
uration of the source and specimen
(from p. 288 in Examining the Sub-
micron World, Plenum, 1986).<17l
358 Chapter 17

FIGURE 17.9. Schematic of contact

X-RAYS imaging. (a) Contact imaging of spec-
imen into resist where incident X-ray
beam is modified by the absorption of
the specimen. (b) The resist after de-
X-RAYRESISTA
velopment and metallization. The re-
sist profile is then magnified and read
SUPPORTING out by electron microscopy (from p.
WAFER
282 in Examining the Submicron World,
a b Plenum, 1986).<17)

Imaging X-ray microscopy and scanning X-ray microscopy are more

direct techniques than soft X-ray contact microscopy.<12l These microscopes
depend on one or more Fresnel zone plates. A circular Fresnel lens is
defined by ASTM as "a sheet of transparent material into which concentric
groves have been formed in such a pattern that light will be focused as
with a lens."<1l A Fresnel zone plate is similar to a Fresnel lens, and it
consists of alternating transparent and opaque rings whose spacing dimin-
ishes with distance from the center.<12l Waves are diffracted as they pass
through the transparent rings. By controlling the spacing between the
rings, or the wavelength, we can control the distance from the plate to the
common focal point. Recently zone plates have been constructed with
grating spacings as fine as 30 nm. Fresnel zone plates can serve as con-
denser and objective X-ray lenses, and these function much as lenses in a
transmitted light microscope.<12l
Scanning X-ray microscopes are becoming more common. A zone
plate is used to sequentially focus an X-ray beam onto a small, localized
area of the specimen. As the beam is scanned, picture elements (pixels) are
generated much as in an SEM. The size of the illuminated spot determines
the resolution. Scanning minimizes radiation damage, and since a
sufficiently coherent soft X-ray beam is needed, scanning X-ray micro-
scopy can be accomplished with only a major synchrotron source.<12l

17.4. SUMMARY

X rays hold great microscopical promise for several reasons:

X rays for X-ray microscopy are generated by conventional X-ray
sources and now also by synchrotrons, X-ray lasers, and X-ray
emitting plasmas.
Common wavelengths are short enough (0.1-4 nm) to give. high-
resolving power.
X-Ray Microscopy 359

X rays absorption spreads among the chemical elements as the cube

of the atomic number.
Since X rays travel well in air, making a vacuum unnecessary, wet or
living specimens can be examined directly without desiccation.
Specimens are so transparent and X rays have such penetrating power
that thicknesses of 20-50 11m are permissible for shorter wave-
lengths.
If parts of a specimen differ (or are treated to differ) in atomic number,
there is plenty of contrast. Also with soft X rays of 1-10 nm wave-
length, even materials with lower atomic number provide sufficient
contrast without any treatment. However with these longer X rays,
specimens must be sufficiently thin.
With synchrotron radiation, different wavelengths can be selected to
use absorption coefficients. In this way elemental maps are ob-
tained. Resolution better than 600 Ahas been achieved, and higher
resolution is anticipated. This resolution is possible by recording on
photoresists with subsequent magnification by scanning or trans-
mission electron microscopy.
Since X-ray micrographs are pictures of absorption, they may give
better contrast than light micrographs of inorganic parts or particles
of metals, ores, minerals, rocks, ceramics, silicones, pigments, and
fillers. Moreover elements with a high atomic number can be added
as selective stains to parts of organic systems<8•9l for automatic anal-
ysis.
The full potential of X-ray holographic microscopy has not been real-
ized. Submicron resolution has been achieved but considerable
difficulties exist because of the low sensitivity of the recording
media and difficulties in recording contrast readout reconstruction.
Research is currently underway to develop X-ray microscopical
holography as a practical technique.<18l
 18

Acoustic Microscopy

18.1. ULTRASOUND WAVES FROM MICROSPECIMENS

Acoustic microscopes employ electromagnetic signals and transform

them into acoustic waves by means of a piezoelectric transducer. The
resultant acoustic waves are the same propagating elastic type as water
waves in the ocean and sound waves in air.(l-11> Being of very high or
ultrahigh frequency, 100-2000 megacycles/ sec or megahertz (MHz), mi-
croscopical acoustic waves are ultrasonic (they cannot be heard). As in
macroscopical use of ultrasound,<7l the primary significance is that such
acoustic waves are reflected or deflected by the specimen's variations in
density or stiffness rather than by differential refraction, absorption, or
reflection (as with light or electrons). Consequently a whole new micro-
scopical area of structural information is open for correlation with prop-
erties or behavior and composition or treatment. Much of the contrast with
acoustic microscopes is due to variation in the material's elastic constants.
With light microscopy however much of the contrast is due to variation in
dielectric constants. Therefore acoustic microscopy complements light mi-
croscopy.
In acoustic microscopy the specimen is contained in a cell filled with
a liquid, often water. Incident acoustic waves are deviated by the specimen
and are collected and converted into electronic signals that are translated
into an image on a cathode ray tube, in video fashion. Figure 18.1<9>shows
what can be resolved in acoustic microscopy.

18.2. ACOUSTIC MICROSCOPES: SLAMs VERSUS SAMs

There are two versions of the scanning acoustic microscope, which are
classified according to how scanning is performed. In the Sonomicro-
scope® scanning laser acoustic microscope (SLAM), scanning is done with

361
362 Chapter 18

.. .. o.modulator end
PhotocMtectot

..
••
lrMglng Optlca-
•
•

•
~·

~~~, .. f~ . . ~~
•
•
Lilt--*~ •

FIGURE 18.1. Simplified block diagram of the Sonomicroscope® SLAM.<9)

a laser. As Figure 18.1 shows, the specimen is mounted in shallow liquid

and held on a stationary stage that is activated acoustically at 100 or 500
MHz. The specimen is covered with a mirrored semitransparent cover slip.
The resultant differential disturbances of the mirrored slip are detected by
the scanning laser (jlying spot) and converted into electrical signals by a
photodetector. The (weak) signals are amplified and decoded to produce
an acoustic image display (lower right, Figure 18.1). An optical image can
simultaneously be produced with the small amount of laser light trans-
mitted differentially and displayed (lower left, Figure 18.1) or differ-
entially reflected from the specimen and displayed (not specified in Figure
18.1). Hence a special feature of SLAM is that the laser light image sim-
ultaneously be compared with the acoustic image.<9)
An optional feature of the Sonomicroscope® involves feeding acoustic
and optical signals into separate channels of a color television monitor.
Thus the separate identities of the acoustic and optical images are main-
tained in contrasting colors. This feature is important in interpreting the
novel acoustic image in terms of the conventional optical image. Inter-
ference patterns can be produced by means of an electronic phase refer-
ence. The result is a graphic display of changes in density and elasticity
within the specimen.
Acoustic Microscopy 363

FIGURE 18.2. Photograph of the SLAM by Sonomicroscope®, Inc., for producing acoustic
micrographs of specimens.(9l

In the SAM, scanning is performed mechanically by moving the spec-

imen, as shown in Figure 18.3.
Figure 18.4 (left) shows an electromagnetic input signal being trans-
formed into an acoustic beam by a piezoelectric film of zinc oxide on the
input sapphire crystal. The acoustic beam travels through the liquid (water)
in the cell that also contains the object, supported by Mylar® polyester film.
The surfaces of the input sapphire crystal and the output sapphire, which
are in contact with the spherical part of the specimen's cell, are concave.
Thus the input acoustic beam is focused (condensed) on the specimen,
which scatters the output acoustic beam into the output (objective) lens
(liquid+ sapphire) system. The output acoustic beam is converted into the
output electromagnetic signal by the respective piezoelectric film transduc-
er (see Figure 18.4, right). The acoustic cell in the middle constricts the
acoustic beam to less than one wavelength (approximately 2 J.tm) in diam-
eter. The object is scanned in a raster pattern. The acoustic output mod-
ulates the intensity of the output electrical signal and displays it on a ca-
thode ray tube (see Figure 18.3). The resultant image corresponds either to
amplitude modulation or phase interference. In Figure 18.5a the amplitude
(intensity) mode is shown in an acoustic micrograph of a fixed preparation
364 Chapter 18

RF P/~1
CRT IMAGE
DISPlAY

OUTPUT

FIGURE 18.3. View of the SAM showing scanning drives and associated electronic equip-
ment. (lO,ll)

UQL() IN CEU.. TO
PERt.'IT SCANNING OB.£CT A!i._ATTACHED
MOTION OF OB..ECT TO MY~ PUYESTER FILM

INPUT SAPPHIRE CRYSTAL

MYLAFfll> POL'YESTER FILM
SlPPORT OB..ECT

FIGURE 18.4. Schematic representation of the acoustic cell as used in the SAM.(l.lO)
Acoustic Microscopy 365

of red blood cells. The change in amplitude of the acoustic beam gives in-
formation about acoustic absorption and impedance. Figure 18.5b (the
phase model) shows a point-by-point display of changes in the velocity of
sound when the section is uniformly thick.(l.t~ 14>
Difficulties exist in routinely aligning separate transducers for trans-
mitting and collecting signals in acoustic microscopes. With reflection
rather than transmission, only one transducer is needed. Techniques have
also been developed for generating V(z) curves to assess changes in in-
tensity of the acoustic reflection as a function of sample depth. Such plots,
which provide information about acoustic velocities and elastic properties,
are unique for each material.<6>
Results of a study of composites show that the internal angles of
individual fibers can be determined in an opaque-to-light matrix by ob-
serving the interference fringes that appear on the specimen's surface.<6>
Another useful result is that interference of surface acoustic waves,
i.e., Raleigh waves, detect the existence of microcracks near the surface,
that are far smaller than the image resolution of the microscope. These
cracks may be a nanometer in diameter, and their interference results are
easily seen.<6•13> A potential limitation of the microscope is that at a resolu-
tion of ca. 1 ~m with a frequency of ca. 1 GHz (1000 MHz), signal attenua-
tion limits microscopical information to a depth of only about 1-100 ~m.<6>

FIGURE 18.5. Scanning acoustic micrographs (a) amplitude modulation and (b) phase in-
terference of red blood cells fixed with methanol. 1000 MHz. May 28, 1976. Courtesy of C. F.
Quale.
366 Chapter 18

The SAM is being applied to such medical problems as malignance in

blood cells, mammillary glands, lymph glands, and lung tissue.<4>Prob-
lems involving even greater constituent differences in density and stiffness
could include teeth, bones, and various types of kidney stones. Foreign
microscopic particles involved in lung diseases include siliceous minerals,
asbestoses, other fibers, soil minerals, and air pollutants. The physiolog-
ically detrimental compounds of lead and mercury also seem to be acous-
tically determinative.
In nonbiological fields acoustical studies are being done on solid-state
materials, such as a thin aluminized pattern deposited on silicon. Another
example is a circuit of silicon on sapphire, in which microscopic flaws were
discovered solely by acoustic microscopy. In cold-worked aluminum
acoustic micrographs revealed not only the surfacial slip lines but also
structural changes below the surface. Other variations in stiffness and
density may be revealed among constituents in alloys, welds, ores, slags,
ceramics, cements, pigmented materials, and reinforced plastics, among
others. Thus the SAM provides considerable quantitative information that
is otherwise unobtainable on composites.
Conceivably the acoustic frequency in the SLAM could be changed to
improve the resolving power. Even at 500 MHz, resolving power of the
SLAM depends on the distance between the object plane of interest and the
cover slip, since the SLAM operates like a general x-microradiography.

18.2.1. Theoretical Resolving Power of Acoustic Microscopes

The theoretical resolving power of the acoustic microscope of course

depends on the acoustical wavelength in a particular medium. Table 18.1
shows how the wavelength at 100 MHz varies with the medium according
to its density and stiffness. The resolving power of the Sonomicroscope®
100 (MHz) type of SLAM is stated to be between 0.7-1.3 wavelengths. In
the Sonomicroscope«~ 500, the input frequency is 500 MHz, with a cor-
respondingly higher theoretical resolving power.<9>
At present a still higher frequency in the SAM (2000 MHz) ensures a
correspondingly higher theoretical resolving power. In water the wave-
length is 1.5 f..lm, and the theoretical resolving power is possibly half of
that. The net optical resolvability is less than the audio but comparable.
Both audio and optical resolvabilities are less than the resolving power of
the conventional light microscope, but again they are comparable.<1>Liquid
helium has been employed instead of water to reduce acoustic velocity to
as little as one-eighth and yield a resolution near 100 nm (1000 A).<U.s>
Acoustic Microscopy 367

TABLE 18.1
Typical Wavelengths at 100 MHz in J.tmm19>
Material Compressional Waves Shear Waves
Air 4
Water 15
Soft tissue 14-17
Hard tissue 30-60
Plastic-acrylic 27 11
Metals
Aluminum 63 31
Brass 43 20
Iron 59 32
Titanium 60 31
Glass 56 34

18.2.2 Practical Resolution of Acoustic Microscopes

In addition to the wavelength, practical resolution from an acoustic

microscope depends primarily on the effective diameter of the detector. In
the lensless SLAM, the laser beam diameter can be adjusted to the limit of
the optical wavelength. In the laserless SAM the diameter of the acoustic
beam at the waist of the cell (see Figure 18.4) is slated to be less than one
wavelength in water, i.e., <1.5 J.lm.l1> The resulting resolution limit is about
2 J.lm. Water is used as a mounting liquid not only because it is compatible
with biological and other specimen types, but also because it supports a
sufficiently low acoustic velocity to effect good practical resolution at a
given input frequency. At the same time water has high enough absorption
to prevent stray multiple acoustic reflections from degrading the image
(provided that input and output electrical connections are well shielded
from one another).l10>
For maximum practical resolution, all relevant adjustments must be at
their best. When used as a transmission microscope, the two lenses (con-
denser and objective) in the SAM, must be carefully aligned in all three
directions, since the beam's diameter is so small (• 1 J.Uil). This difficulty
is removed by using the instrument in the back reflection mode.
In both types of acoustic microscopes, practical resolution also de-
pends on maintaining the frequency of incident ultrasound. In the SLAM
the laser beam scanner must be sharply controlled, and in the laserless
SAM the mechanical scanning mechanism must function within certain
specifications.
368 Chapter 18

18.2.3. Contrast in Acoustic Images

Contrast is generally adequate in acoustic images because the varia-

tions in elastic parameters are greater than those in optical parameters.
Furthermore, in either the SLAM or the SAM, electronic amplification of
contrast is readily available within the bounds of the signal-to-noise ra-
tio. <1l Both types of acoustic microscopes provide an interference mode of
operation by comparing either the transmitted or the reflected signal with
a constant portion of the initial electrical oscillation. The resulting phase-
interference micrograph can then be compared with the amplitude (in-
tensity) micrograph to a study contrasts. Using color contrast between
acoustic and laser micrographs has already been mentioned as an optional
feature of the SLAM. <9l

18.2.4. Aplanatic Lenses in SAMs

Aberrations in the spherical lens of the cell (see Figure 18.4) are neg-
ligible at this stage in development of the mechanical SAM. In fact the lens
is inherently aplanatic, because the concave spherical face of the sapphire
contacts water instead of air. The acoustic velocities of water and sapphire
are in the ratio of 7.45:1, so that acoustic rays are sharply refracted at an
angle closely parallel to the radii. The focal length of the lens is only 15%
greater than the radius<12l (a very low f number and a very high NA).

18.2.5. Cleanliness in Acoustic Microscopes

Cleanliness is a factor in both types of acoustic microscopes because the

specimen is immersed in the liquid medium (e.g., water). Solutes bleeding
from the specimen into the liquid medium could change the velocity of
sound significantly, and local concentrations of solute would introduce
aberrations. Such considerations pertain largely to biological specimens
that may contain a natural serum, an isotonic solution, or a fixing reagent
unless it were cleaned of such matter. Of course when light is introduced
into either type of acoustic microscope, any turbidity becomes an optical
problem.

18.2.6. Focus Depth in SAMs

The field in focus depth (penetration depth) in the SAM is probably on

the order of 1-100 J..lm. However with a thick section (6-8 J..tm) section,
Acoustic Microscopy 369

although more information is obtained about differential densities and

stiffness. But with opaque or relatively dense materials, such as metals,
inorganic glass, kidney stones, or bone, the image comes chiefly from the
surface and is therefore in sharp focus.C12> The SLAM accommodates sam-
ples of up to several millimeters thick. The acoustic penetration is gener-
ally inversely proportional to the acoustic frequency of the specimen (see
Table 18.1 ). Evidently the field depth in focus is greater in the SLAM (at 100
or 500 MHz) than in the SAM (at 1000 MHz).

18.2.7. Focusing SAMs

The SAM is focused with a micrometer screw that holds the specimen
in the cell (see Figure 18.3). When the object plane coincides with the nar-
rowest cross section of the acoustic beam, the sharpest focus is achieved. (lOJ

18.2.8. Acoustic Radiation

The acoustic radiation in the Sonomicroscope1111 100 is at a frequency of

100 MHz (or, as an option, 500 MHz).(4l As shown in Table 18.1, compres-
sional (and in some materials, shear) waves of characteristic wavelength
are set up in the mounting medium and the specimen. Coincidentally a
flying spot of laser-coherent light scans the microfield, as shown in Figure
18.1. The flying spot detects perturbations of the mirrored slip covering the
vibrating specimen. A photo detector converts the signaling laser beam
into electric signals that are amplified and decoded into the amplitude-
(intensity-) modulated acoustic micrograph. At the same time some of the
incident light of the laser beam is reflected and/ or transmitted by the
specimen. Either the reflected or transmitted light beam can be photo-
detected and displayed by a separate video system. The resultant optical
micrograph can be compared with or superimposed on the SLAM acoustic
micrograph. (9>
The radiation currently in the SAM is up to 2000 MHz with water
immersion. The angle of incidence for the acoustic lens can have a total
aperture of 120° or more.(5l The acoustic cell (see Figure 18.4) can be tilted
90° to place the acoustic axis in a vertical position. Thus the entire cell can
be immersed in a medium suitable for maintaining the cell's environment
in a viable condition. Then living cultures can be viewed over an extended
period of time.(7) The 90° tilt places the specimen in a horizontal position.
With the condenser (input lens) on top, the acoustic microscope resembles
an inverted light microscope, with some of the same advantages.
370 Chapter 18

The incident angle in the Sonomicroscope® is normally 10° for aqueous

media. Stages with other incident (insonification) angles are optional.<9l In
the SAM the incidence is axial (0°).

18.2.9. Magnification

The magnification quoted< 15> for the Sonomicroscope® 100 is lOOx; for
the 500 it is quoted as 5000x. At a practical resolution of 20 f..lm, a mag-
nification of 10 or 20x would be sufficient to meet the minimum require-
ment (200 llm of the eye). At a resolution of 4 llm for the 500, 50 or lOOx
would be useful. How much more magnification is useful rather than
empty (see Chapter 2) depends on use (such as in micrometry). The
quoted<15> magnification of the SAM is 500-lOOOx. At a practical resolution
of 2 f..lm, 100 or 200x is sufficient for the eye. How much more magnifica-
tion is useful depends on other uses besides simple qualitative visual
examination. Obviously if the theoretical resolution of 0.5 f..lm is achieved,
500-lOOOx is useful magnification.

18.2.10. Field of View

The field of view with the Sonomicroscope® is 1-12 mm2,<1.1 5l and 30

images are produced each second on the television monitor, making it
possible to observe dynamic activity. The field of view with the SAM is
much smaller, 0.25 mm on a side. This area amounts to 5 x 1()4 elements
of information and takes about 1 sec to scan.<10•15l Larger areas are com-
posited (see Figure 18.6).<12!

18.2.11. Stray Acoustic Radiation

Stray acoustic radiation produced in the SAM by multiple reflections is

attenuated enough to avoid degradation of the image (glare) if care is taken
to shield the electrical in and out connections from each other.

18.2.12. Three-Dimensional Aspect of SLAMs

The three-dimensional aspect of the SLAM is heightened by superimpos-

ing acoustical and optical micrographs.<16•17l The Sonomicroscope® accom-
modates any sample, transparent or reflecting, as thick as several milli-
meters.
Acoustic Microscopy 371

FIGURE 18.6. Human liver tissue as invaded by hepatitis. Acoustic frequency of 900 MHz
in the SAM.(l 2) (Sample prepared and analyzed in the laboratory of Dr. J. Edward Berk.)

18.2.13. Specimen Thickness

The depth of thick biological sections for use in transmission with the
SAM is 6-8 f.!m and often cut from frozen fresh samples. The depth of thin
sections, generally cut from mounted fixed specimens, is about 1 f.lm.< 6)
Reflective materials, such as metals, their compounds, coal, cements, and
ceramics, are studied at their surfaces. Specimen thickness is therefore a
dimension of convenience. An example of limited field depth is shown in
Figure 18.7. This can be a very useful feature of acoustic microscopy.

18.2.14. Working Distance

Working distance above a specimen in the SLAM is apparently ample

for experimenting with the specimen in situ. Specimens can be placed
directly on the stage or a glass slide; however they are covered with a
semireflective cover slip. Interchangeable stages are available for the
Sonomicroscope® for polarized shear-wave insonification, alternate
FIGURE 18.7. Two views of a woven composite. (a) Graphite fibers are in the focal plane; (b)
the image is focused 10 11m below the field in view (a). (b) A void is clearly distinguished by
good contrast with the resin. Original composite specimen courtesy of Mansour Mohamed,
NCSU. Photomicrograph courtesy of D. Downs, X. Cui, A. El-Shiekh, P. Tucker and J. Russ,
NCSU.<6>
Acoustic Microscopy 373

insonification angles, and other special work.C3> In the SAM the specimen
is mounted on a Mylar® polyester membrane stretched across a thin metal
ring (see Figure 18.3). The ring is connected to the movement of a loud-
speaker cone. The ring assembly is enclosed in the cell, which is filled with
a liquid (see Figure 18.4). Consequently there is no practical working
distance beyond the specimen. Working distance with the SAM in the back
reflection mode is similar to that in a light microscope.

18.2.15. Specimen Structure

Structure contributes fundamentally to acoustic microscopy, since dif-

ferentiating features of the image represent microvariations in density,
stiffness, viscosity, or viscoelastic properties. Table 18.1 shows certain
types of matter that differ greatly in acoustic properties. Even in micro-
scopic amounts such different materials as air, water, soft tissue, hard
tissue, plastic, elastomer, metal, or inorganic glass are sharply contrasted
against one another in their composite acoustical image. Indeed brass is a
composite in itself, and various possible constituents, such as a and ~ solid
solutions of copper and zinc, are probably distinguishable, since the form-
er are based on the crystallographic space lattice of copper and the latter
on that of zinc. Iron can be present as a, y, or other crystallographic
structure. Acoustic absorption probably differs not only with the iron's
structure but also with the amount of carbon in solid solution in steels.
Moreover alloy steels vary in crystal structure and physical properties with
the kind and amount of alloying element, such as vanadium, chromium,
or nickel. There is perhaps no more dramatic example of the effect of
structure on elastic properties than that of carbon crystallized as either
diamond or graphite. Other industrial examples of crystalline allotropy are
the white pigment Ti02 as rutile and/ or anatase, and CaC03 as calcite,
aragonite, or vaterite.

18.2.16. Anisotropy

Anisotropy is a related structural phenomenon that can be acoustically

important. Variations in elasticity according to propagation direction of
ultrasound waves may be due to anisotropy in particular molecules-
crystals, glasses, fibers, films, or other solids or semisolids.
Structure change has been investigated in stretched and unstretched
elastin or collagen fibers.<t> The same kind of experiments would be inter-
esting with elastomeric fibers and strained polymers.
374 Chapter 18

18.2.17. Specimen Morphology

The study of morphology in both the SAM and the SLAM is limited by
their respective resolving powers. Hence in pioneering research at this
stage of development, it may be well to choose systems or problems
involving relatively large microscopic morphology.

18.2.18. Information about Acoustical Images

Relevant information is inherent in the SLAM, since laser light images

are readily available and superimposable on acoustical images.<9•18l With
the SAM ordinary light images are independently formed.<l.1°· 11> In either
case information accumulated during the long history of light microscopy
(see Chapter 1) is available for interpreting acoustical images. Likewise
macroscopical information about elastic properties and material strength
should be considered when interpreting acoustical images.

18.2.19. Experimentation

Among all the dynamic experiments that can be performed with acous-
tic microscopes, the most relative are studies of the hearing mechanism in
humans, land and aquatic animals, insects, and other creatures. Aquatic
studies are especially relevant because of the use of water as the mounting
medium. Indeed biological material in general can conveniently be studied
in vivo or in vitro. Inanimate aqueous or hydrophilic material is likewise
amenable to acoustical experimentation; examples are wet-strengthened
paper, wet fibers, tow, yam, cloth, wood, or sponge. It is conceivable that
such specimens can be dynamically stressed in situ. Acoustic studies of the
static and dynamic stiffening of fibers (dyed as well as undyed), at various
temperatures seem to be logical experiments. Similar studies with other
plastic materials, especially those containing pigments, carbon black, pul-
verized minerals, or man-made fibers, are needed by industry. Hydro-
phobic materials can be studied in a nonaqueous liquid suitable in the
SAM cell. Metallic fibers, powders, foils, wires,<19l and fabrics can be stud-
ied acoustically with regard to cold working, annealing, plating, welding,
alloying, and compositing. Copper-plated paper laminates, widely used as
microcircuits in the electronic industries, present many problems that
might be solved uniquely with the SAM. Solid-state materials and devices
have already been examined. <11.20> Acoustic microscopy is providing
unique information about a wide variety of subjects, including biological,
electrical microcircuits, and composites.
Acoustic Microscopy 375

18.2.20. Specimen Behavior

Behavior of disease on tissue has been studied in the SAM by post

mortem examination and from samples of live objects. Comparing the
elasticity and density of diseased tissue with that of healthy tissue is a
unique capability of acoustic microscopes. Also unique is the opportunity
to compare changes in elasticity and density (by audio scanning) with
changes in optical parameters (by coherent or noncoherentlUO,IIJ light). In
the SLAM the two kinds of images can be compared by separate displays
or a single superimposition with differentiating colors, as when locating
the advancing front of a disease.l9l
While the study of elasticity and density in most biomedical spec-
imens is new,l21 l the study of such properties in industrial materials, even
in microscopical specimens, is an old part of physically testing stress-
strain relationships. In the acoustic microscope however, we have a new
and distinctive feature that allows us to view directly the changes in
elasticity. For example it is well known that natural rubber ages poorly,
particularly in oxygen or ozone; with an acoustic microscope actual aging
can be viewed. Changes in elasticity can be studied microscopically in
other materials, such as plastics, metals, alloys, ceramics, and inorganic
glasses. l20l

18.2.21. Specimen Preparation

Preparing a polished thick section of a specimen, such as metal, ore,

rock, plastic, bone, or teeth, for SLAM or SAM is similar to the preparation
for examination by reflected light (see Chapter 4). The only special require-
ment is that the so-called thick section be only a few millimeters thick.
For thin (1-J.tm) microtomed sections, the less preliminary prepara-
tion, such as dehydration and fixation, the better until it is learned what
effect the treatment has on the specimen's elastic properties. Microtomy of
a frozen specimen was, however, found to be more difficult than fixing,
mounting in, and releasing from poly(methyl methacrylate).(l 2l

18.2.22. Photomicrography

Photomicrography is provided by the full-range of camera backs and

automatic fast-developing photographic film. With the advantage of
speed, simplicity, and convenience of the Polaroid® system come the re-
strictions on choice of photosensitive materials, variations in photopro-
cessing, multiple photoprinting, and transparencies for projection.
376 Chapter 18

Photomicrography by means of the video image(9-t2>brings limitations

to resolution caused by raster lines. A video tape recorder(9l can relieve
some of that limitation, at least during information storage, and at the same
time permit dynamic events to be recorded, such as cardiovascular con-
traction and the fracture process in steel.

18.3. SUMMARY

There are two kinds of acoustic (ultrasound) microscopes. The SLAM

employs a scanning laser beam microphone.<9> The other acoustic micro-
scope, the SAM, employs mechanical movements.(10•11>
The SLAM is lensless; it is an acoustic shadowgraph projection micro-
scope whose resolving power is limited by the wavelength of the ultra-
sound and diameter of the laser flying spot. With an input frequency of 100
MHz, the limiting resolution of the SLAM is about 20 J.tm; with an input
of 500 MHz, the limiting resolution is about 4 J.tm.(9l In transmission, the
SAM possesses two small, identical planoconcave lenses: One acts as a
condenser of the acoustic beam, and the other acts as the objective. Both
lenses are sapphire crystal, immersed in a liquid usually water (see Figure
18.1). Although each lens is a single component, it is practically aplanatic,
because the ratio of acoustic velocity in sapphire to that in water is so high
(7.45:1). As a result the acoustic rays are very sharply focused. Since the
focal length of each water-immersion lens is only 15% longer than the
radius, the NA is very high. The velocity of sound in water is so low that
the wavelength (A.) at a frequency of 1000 MHz is only 1.5 J.tm. This
corresponds to a limiting resolving power in the SAM of 0.5 J.tm. (12>Because
of the difficulty in aligning both the transmitting and receiving lenses at
higher resolution, most recent SAMs have used back reflection with a
pulsed signal. In addition to alignment problems, this is also because
acoustic waves need a solid or liquid for conduction, and they have an
attenuation proportional to the square of the frequency in most fluids.< 5>
After characteristic interaction with the specimen in the SAM, acoustic
energy is converted by a piezoelectric transducer into an electromagnetic
signal that is imaged on a television screen. In the SLAM the output
acoustical beam modulates the laser beam as it scans the field. The mod-
ulated laser beam is received by a photodetector that yields two signals:
One depends on the optical features of the specimen; the other is propor-
tional to the acoustic amplitude within the specimen. Each type of signal is
television monitored separately, but the two images may be superim-
posed, even in contrasting color. With the laserless SAM, ordinary light is
used separately to make an optical image for comparison with the acous-
 TABLE 18.2
Guide to Various Applications for the Three Most Common Techniques of Acoustic Imaging19l
C-scan SLAM SAM
Frequency 1 MHz . . . 10 MHz ... 30 MHz . . . 100 MHz ... 200MHz ... 500MHz ... 1GHz
Wavelength 1.5 mm 15mm 1.5mm
Medical ultrasound
Conventional ultrasonic nondestructive testing
Coarse-grain metals: defect detection
Cracks in plastic encapsulated integrated circuit (IC) devices
Composite materials
Cracks in ceramic IC packages
Spot welds
Heat sealing food pouches
Ceramic chip capacitors: delaminations and cracks
Hermetic seal reliability
Polymer-foil package lamination
IC die attach
Thick-film adhesion and porosity
Laser spot welds
Fine ceramics: defect detection
Fine-grain metals: defect detection
Seam welds on tin cans
Lead bonds on hybrid circuits
Ceramic substrate porosity and cracks
Cracks in silicon wafers
Thin-film adhesion
Grain structure determination
Fine-line inspection on silicon
Source: Courtesy Sonoscan<9>
378 Chapter 18

tical image of the same field. In both acoustic microscopes the optical
image is helpful in interpreting the acoustical image, especially during
experimental applications.
Acoustic interference microscopy can be incorporated into either in-
strument by combining signals from attenuated acoustic rays with a ref-
erence signal. The resultant interferogram consists of light and dark bands.
By analyzing the position of the bands, quantitative data on density and
elastic properties are derived on a microscale.
Applications of acoustic microscopy in the micron range stem from
experience in the macroscopical (millimeter) range with the familiar pulse-
echo devices used in medical diagnoses and in material flaw detection. The
micron range of resolution is also that of light microscopy. Thus the ex-
ploration of elastic or dense microstructure by ultrasound waves is where
light microscopy was four centuries ago. It is now hoped that acoustic
microscopy will progress to areas where electron microscopy began.
Table 18.2 lists various applications for the three most common tech-
niques of acoustic imaging (also see the frontispiece).
 19

Image Collection, Analysis,

and Reconstruction
by Computer

19.1. INTRODUCTION

Automated image processing has facilitated new insight into complex

data; for example it is no longer necessary to view a three-dimensional
specimen as a series of two-dimensional slices. A three-dimensional image
can be reconstructed, and this new image can be electronically rotated or
subsections of it studied at high resolution, although a more common
application is automated statistical characterization of a complex two-
dimensional image. Parameters can be measured and aspect statistics calc-
ulated on a large enough population to make such numerical characteriza-
tion impossible by manual means. We must recall the concept of empty
magnification versus resolution when we hear that it is possible to magnify
up to one thousand or more by video microscopy with a low-resolution
objective. Classical principles still apply to these new techniques.( 1>
As with most tools hardware and software can be fairly simple or
extremely complex. A typical system includes a microscope, video system,
display monitor and (or) a recorder, and for a few systems integration into
a multisite or even multicountry network. The specific microscope selected
determines requirements for other components by determining or greatly
influencing available light levels, resolution, contrast, image size, and type
of necessary information storage.
The human eye and brain plus conventional photomicrography do an
excellent job of acquiring and processing two-dimensional information,
but these are deficient when processing and reassembling three-dimen-
sional information or four-dimensional information that has time as a

379
380 Chapter 19

variable. Such three-dimensional and four-dimensional processing was

much more difficult and expensive until the advent of relatively inexpen-
sive, automated image acquisition equipment, such as frame grabbers, and
image-processing equipment, such as personal computers (PCs). This
hardware has been accompanied by numerous packaged software pro-
grams from many suppliers. Of course increasing the complexity of image
manipulation may exceed the abilities of a PC and thus require an in-
tegrated image analysis system from one of the larger microscopical deal-
ers or even connection to a massive computer analysis network. However
simpler, less-expensive systems satisfy most needs.
All involved subtopics are undergoing such rapid changes that it is
difficult to keep current. Inoue<2> lists many journals with useful informa-
tion, including Journal of Microscopy, IEEE, Transactions, and Microscopia
Acta; others that should be included are Computer-Assisted Microscopy and
Bioimaging, and a complete list would include major journals for almost
every discipline. Also recommended are relevant trade journals. Inoue also
provides a useful glossary of technical terms to assist the user with com-
plex terminology acronyms.<2>
Numerous small companies now provide packaged systems, and
such companies often have considerable expertise or occasionally very
limited expertise. If expert advice is needed with the purchased items, it is
wise to check on the professional reputation and support ability of the
potential supplier, since software development is a complicated, evolving
field currently lacking clearly documented guidelines.
Proper use of automated image processing always depends on cor-
rectly using the microscope, which has been the main thrust of this text.

19.2. MICROSCOPES

Automated image collection can be done through any type of macro-

scope or microscope. Automated stages are special features that translate
in three directions, X, Y, and Z, in synchronization with data collection.
Such automated translation is normally done discretely rather than con-
tinuously, and sufficient time must be allowed at each increment or step
to allow accumulation of desired data from the field of view. Image equip-
ment can also be purchased to refocus automatically the microscope as the
stage is translated. Such systems can free the microscopists of many com-
plex or tedious procedures, and the tasks can be automated; however
automated stage systems are expensive, and it may be necessary to employ
many people to approximate their operations manually.
Image Collection, Analysis, and Reconstruction by Computer 381

19.3. VIDEO SYSTEMS

The standard digitizing board or frame grabber provides a 525-line

image, with each line composed of 525 pixels (discrete picture elements)
and 30 displays or frames per second. Many of the newer systems provide
640 pixels per scan line and 480 scan lines per display.<3•4>The standard-
format camera for generating the pixels in black and white is known as the
RS-170; other systems exist outside the United States, and even within the
United States, for color.<1> Cameras can achieve higher resolution, even
beyond 2000 lines; a tube camera, such as the vidicon camera, is the
standard, earlier model. Similar, newer cameras are the nuvicon and plum-
bicon, which have greater light sensitivity;<M> however vidicon cameras do
have spectral sensitivity closer to that of the human eye.
More recent solid-state devices that function as cameras are the
charge-coupled device (CCD) and the charge injection device (CID); the
CCD is the more common of the two. The basic concept for both devices
involves using separate devices or elements for each picture point or pixel
to be measured and measuring all points simultaneously. For low-light
intensities, such as that used with fluorescent microscopy, solid-state cam-
eras can be obtained to integrate the intensity for as long as several mi-
nutes. Such cameras are often cooled to control the amount of noise.<3>
The CCD and CID cameras are simpler and more rugged than tube-
type cameras;<3> however these former lack the resolution of tube-type
cameras because of the need for separate sensing cells for each picture
point to be shown. The CCD cameras are recently being marketed with up
to 2048 x 2048 pixels with 12 bits per pixel of resolution, so they are rapidly
evolving. <3a)
Digitization by digitizing boards or frame grabbers converts the cam-
era's analogue (A) signal to a digital (D) signal for the computer; the
digitizing boards and frame grabbers are known as ND converters.
Resolution with frame grabbers can be improved by collecting a series
of scans of the same image and averaging across this series. <4>A special
C-mount adapter is normally required to connect a video camera to the
microscope, just as a similar adapter is needed to connect a light-photo-
graphic camera.
Since the components are in series, there is no advantage in using a
microscopical resolution higher than that imposed by the camera. Actually
there may be disadvantages, such as unnecessary loss of size and field
depth.<5>As a general rule 300 dots/inch appear continuous at a normal
viewing distance, and Webb and Dorey note that a person with 20/20
vision can distinguish dots 1 arc-second apart, which is equivalent to a
 382 Chapter 19

200-t-tm separation at a 1-m viewing distance.(5l Lack of an adequate true

three-dimensional display of images is a limitation with videoimaging,
especially when micromanipulating specimens as with a stereomicro-
scope.

19.4. COMPUTERS AND SOFTWARE

All current larger PCs can be used for image processing. As with other
microscopical equipment, it pays to first decide what tasks are to be done
and learn the general concepts before purchasing equipment, since soft-
ware selection is a complicated task.
It is generally advisable to see prospective software demonstrated on
your specific tasks before making a purchase. A vendor's ability to provide
continuing technical support should also be considered.*
Software exists for both image-acquisition software and image-pro-
cessing software, and it can be obtained to conduct almost any desired
measurement or data manipulation. Major light and electron microscopi-
cal companies also have software in depth, and it should be considered if
such a wide range of programs is necessary for your tasks.
Digitized images occupy considerable storage space. An image of 512
x 512 pixels and 1 byte per pixel requires one-fourth of a megabyte (Mb)
of storage.<7J Storage media can either be archival as with magnetic tape,
which has a lower cost but longer access time; more expensive Winchester
hard disk with fast access time; or more reasonably priced optical disks,
which can store up to 650 Mb of data.(7) Programs and systems have
recently been developed to operate with workstations that would pre-
viously have required main frame computers because of the complexity of
the operations.(8l

19.5. DISPLAY AND NETWORKS

If you use a single unit without windowing capabilities, you can

alternate between the video image displayed on the computer screen and
the computer menus. With a double-monitoring system, you can dedicate
one monitor to display results from the frame grabber and use the other for
computer menus.(7) Multiple monitors can be used to compare results from
different processes.

•More than 40 suppliers of image analysis products have recently been listed. (6)
Image Collection, Analysis, and Reconstruction by Computer 383

Most monitors have a resolution of 512 pixels per line and 480 lines
per display,<6> but special monitors have higher resolution. Polaroid for
example has a higher resolution system where the original image is re-
corded photomicrographically onto film and then scanned into a computer
using a digital scanner with approximately four times the resolution of
standard ceo cameras. (9)
Palatini et al. review their use of a PC to capture images and then a
Digital Equipment Corporation VAX computer to store, retrieve, and dis-
tribute images on site and intemationally.<10> Such an image system is
illustrated in Figure 19.1. It is not necessary to illustrate the video camera's
image in the final report; the electronic camera and computer results can
be processed for the desired data and a conventional photomicrograph
recorded as an illustration, since high-resolution video printers are at the
stage "where instant film was 15 years ago."<1>
A review of terminology for computer networking has been pub-
lished;<10al critical concepts, such as modem (an acronym for MOdula-
tor /DEModulator) and baud rate, are defined and recommendations are
made for purchasing total systems. A modem converts the digital com-
puter signal into an analog signal that can be sent by telephone transmis-
sion; baud rate refers to the data transmission speed of the modem.<10•>

COMPUTER VIDEO
MONITOR MONITOR

COMPUTER

DATA
RECORDING

FIGURE 19.1. A typical image analysis system.

384 Chapter 19

19.6. CONTRAST, LOOK-UP TABLES, AND COLOR

Contrast and color, i.e., gain and offset, of the video image can be
artificially influenced if desired; of course a fairly representative color of
the original can also be produced.
For artificial color each pixel is associated with a specific gray that is
coded from zero for the darkest gray (or black) to 255 for the brightest
white.( 12l A similar manipulation exists for color images with a much wider
possible range of values. We are able to distinguish only about 30 different
gray levels, but we can distinguish hundreds of thousands of colors.
Shades of gray can be converted into artificial colors, and such pseudo
color can improve contrast. On the other hand if improperly used such
artificial color contrast may lead a viewer to concentrate on image details
and neglect the information.(4l
Gray or brightness values of discrete pixels are converted into color by
using look-up tables (LUTs) to find the gray value of a specific pixel, then
converted it into a number from 0-255. Numbers in this range are divided
into three groups: blue, green, and red, and the shade of each is related to
the grayness of the original signal.(12l In this manner artificial colors are
assigned to different shades of gray from a monochromatic camera. True
color images are considerably more expensive to collect(4l because three
different collection sensors must be used: one each for blue, green, and red.
After the color image is collected it is also considerably more expensive to
store these three images, and the software is also much more complicated
for analyzing the three color combinations.

19.7. SUMMARY

Automated image analysis is practical and beneficial. While the hu-

man eye and brain do an excellent job of acquiring and processing simpler
two-dimensional images with conventional photomicrography, reassem-
bling three-dimensional information or four-dimensional information,
which varies as a function of time, proves to be more difficult. Resources
are generally not available to collect and process large amounts of routine
microscopical data, which is relatively simple with an image analysis
system.
With most microscopical systems a video camera is needed with the
microscope to supply the analog signal to a digitizing board or frame
grabber, which converts the analog signal to a digital signal for the com-
puter. With SEMs for example, separate initial digitizing boards are not
Image Collection, Analysis, and Reconstruction by Computer 385

needed, since an image is not initially generated since there is a direct feed
of the electrical signal to the computer.
Numerous packaged programs exist to process collected data. Either
two-dimensional or three-dimensional images can be reconstructed. Two-
dimensional digitized information units are called pixels, and three-di-
mensional digitized information units are called voxels. Series of images as
a function of time can also be obtained if needed. Most monitors display
512 pixels per line and 480 lines per display, but higher resolution can be
obtained at considerably increased cost. Since all components are in series,
the final resolution is no better than the poorest link, and this means that
resolution of the total system must be increased to increase the final resolu-
tion. Collected information can be processed on the computer and then
stored or distributed on a network.
Details on a total system involving many programs and different
features are published in Microscopy: The Key Research Tool,<11>which should
be consulted for information on three-dimensional reconstruction and
computer graphics.<11 > These software programs are written in generic C
language and run on a variety of machines using the UNIX operating
system.
The MSA Bulletin, published semiannually, contains a section, "In the
Computer Comer." The MSA has a growing list of computer programs,
reference spectra, and tables of physical constants that can be obtained
through an electronic network, BITNET. Communication can also be made
by electronic mail. These services are free or available at nominal cost.*

*Bulletin of the Microscopy Society of America, MSA, P.O. Box EM, Woods Hole, MA 02543;
e-mail: EMSA/MAS BBS.
 20

Specimen Preparation

20.1. INTRODUCTION

Specimen preparation depends to a great extent on what information

is already known and what is sought about composition, treatment, prop-
erties, behavior, structure, and morphology (see Figure 20.1).11> We usually
know the composition and condition but seek the structure and morphol-
ogy to predict properties or behavior; of course we could know the compo-
sition and condition and test for properties and behavior to determine
structure and morphology-and so on.
Often while testing for properties, microscopical information is ob-
tained simultaneously. For example Figure 20.2(2> shows the fracture sur-
face of a test bar of thermoreacted resin that exhibited a certain statistical
strength according to ASTM standards.(3l Coincidentally the close-up view
discloses the granular texture of the fracture surface imparted by the wood
flour filler.<2•4>
Figure 20.3 shows typical areas in two sheets of rubber, one (left)
varnished and the other (right) unvarnished. Both surfaces were weath-
ered simultaneously by a standard procedure.(3l The unvarnished surface
(right) became cracked in a typical granular pattern while the varnished
rubber surface seemed to be unchanged (by comparison at the time with
a piece of original sample).(2l
Figure 20.4 shows a cross section of a sheet of artificial rubber after
testing in acid and simply cutting across it with a wet razor blade. (2l
Sometimes a macrotest can be turned into a microtest to allow for
some microscopy. For example the paper industry's most important resin
(the one that makes writing paper, not blotting paper) is rosin, a natural
mixture of complex unimers derived from the black liquor by-product of
pulping coniferous wood. However recovered rosin is so pure that it
sometimes crystallizes out of solution before it can be applied to the paper

387
 388 Chapter 20

..=8.
c
0

•:r
Ill

Science
E ~
lJ f FIGURE 20.1. Facets of the scien-
tific area: composition in physical-
chemical terms; condition as used
in mechanical tests; properties for
practical utility; behavior in use,
weather, or test; morphology in
terms of size and shape; structure
in terms of construction units.

FIGURE 20.2. A fractured test bar of thermoreacted resin by oblique reflected light; left:
Fracture surface; right: Milled slot that directed the fracture during an impact test.(2l Courtesy
of McCrone Research Institute.
Specimen Preparation 389

FIGURE 20.3. Sheets of rubber after weathering; by reflected light; left: varnished; right:
unvamishedPl Courtesy of McCrone Research Institute.

pulp. Inhibitors are added, so there are standard macrotests<4> for crystal-
lizability. The Kirk flask< 5> (see Figure 20.5) was devised to be large enough
to give reproducible test results yet small enough and with side sufficiently
flat to reveal crystals between crossed polars with a stereoscopic micro-
scope (see Figure 20.6).<2>
By looking into a stereoscopic microscope with one eye and putting a
camera over the other eyepiece, an automated microdynamometer was
developed<6> (see Figure 20.7) and improved<7l (see Figure 20.8). Figure 20.9
shows the dimensions of a standard microspecimen adopted for the orig-

~ ~-
. .
I •.. . •
J. • .... ' • ·: "":,.. •

r:'llllll!ll .... .. . ·. 1" ·:

~ ........ ,),.

~..... . ~ - _'~ . . .

FIGURE 20.4. Cross section of an artificial rubber sheet; oblique, reflected light. Specimen
shows cracks and voids after testing in acid. Preparation: As cut with wet razor blade.<2>
Courtesy of McCrone Research Institute.
 390 Chapter 20

FIGURE 20.5. The Kirk Flask

#1000 for incubating rosin or rosin
size and subsequent microscopi-

::-::J
cal examination; devised by A. F.
Kirkpatrick and furnished by R. C.
Ewald.<5l This cell is thick enough

c_____
to permit reproducible growth of
crystals from the viscous medium.
It is easily filled yet easily sealed
by drawing the neck closed in a
flame. The sides are flat enough
for viewing under a stereomicro-
I' II I' II I' I' I 'I'I I' Ill' Jllll '111111)
IIICHal 2
1)11 'IIJ 'I' IIIII q I) II
3
scope. Courtesy of McCrone Re-
search Institute.

inal automated microdynamometer.<2•6l Figure 20.10 shows a typical mi-

crospecimen stretched to breaking in the microdynamometer.<2•6l Figure
20.11 shows a load time curve for Lexan® polycarbonate, plotted by a
microdynamometer, with frames from a cinamatographic record corre-
sponding to the times shown by selected points on the graph.<6l In this
particular case the testing device was brought to the microscope instead of
vice versa.

FIGURE 20.6. A specimen of rosin size after a crystallizability test in a Kirk flask (see Figure
20.5). The average crystal length is 19 mm, yet the crystals are well within the focus depth
of the stereomicroscope. The crystals, shown here between crossed polars, are highly aniso-
tropic, as indicated by those that happen to be in positions of brightnessPl Courtesy of
McCrone Research Institute.
FIGURE 20.7. Prototype of an automated microdynamometer developed at the Research
Laboratories of American Cyanamid Company in Stamford, CT.(6) Courtesy of McCrone
Research Institute.

TENSILI! HOLOER•NO. 70000

FIGURE 20.8. The Ladd Tensile Holder. Courtesy of Ladd Research Industries.(7J
392 Chapter 20

~19 =~·:..:.~ ~~ I
~ ~ 0.976 11 ~
F; 0
FIGURE 20.9. Diagram . and dimen-
sions of a standard(3) microspecimen
of transparent plastic foil (film) for
the microdynamometer.(6l Courtesy
0 of McCrone Research Institute.

20.2. PREPARING FIBER, FUR, AND HAIR

Natural and man-made fibers are important units in textiles, paper,

cordage, forensics, etc. Such fibers are fit for the polarizing light microscope
because of their relevant microscopic size, characteristic anisotropy to-
ward light (see Chapter 6), and ease of preparation.(2•4>Indeed for longi-
tudinal views, fibers need no more preparation than immersion in a color-
less, medicinal grade of mineral oil (n = 1.48).(4•>
Cross-sectional views can be prepared with a "microbread" slicer,(8>
an assemblage of two single-edge razor blades firmly clamped togetherP>
Perhaps even shorter lengths can be obtained with a dual-edge razor.
Another quick method of cross-sectioning fibers is to stuff them into one
of the 36 ready-made holes in a piece of black vinyl board the size of a

FIGURE 20.10. Microspecimen of Lexan® polycarbonate stretched and broken between

crossed polars in the microdynamometer.(6) Courtesy of McCrone Research Institute.
Specimen Preparation 393

FIGURE 20.11. Automated resinography of a plastic film. Load-time curve for Lexan® poly-
carbonate plotted automatically on the microdynamometer with frames from a cinemato-
graphic record corresponding to times shown by selected points on the graph.(6) Courtesy of
McCrone Research Institute.

microscopical slide.(9l Since only one hole is used per sample, a slide can
hold as many as 36 different specimens, easily filed for future reference.
Stuffing involves the following steps:

Push a flexible needle threader through a hole until the wire loop
opens up. The open loop is filled with specimen fibers plus a strong
black thread. Pull the black thread into a loop. When the loop be-
comes small, it is filled with specimen fibers.
Pull the black thread so that the bundle of specimen fibers bends,
doubles, and stuffs the hole firmly.
Cut away excess fibers with scissors.
Shave with a sharp (fresh) single-edge razor blade.
 394 Chapter 20

In the old-fashioned method of cross-sectioning fibers, (1) a loop of

thread is inserted into a hole in a cork stopper with a sewing needle, (2)
fibers are inserted into the loop and pulled into the cork, and (3) the fibers
are sliced. A newer method involves stuffing fibers or yam into a piece of
plastic tubing and making thin slices of the composite0 2al; this is an espe-
cially good technique when using an SEM.
For light microscopical examination, the perforated slide is placed on
an ordinary glass slide on the microscope's stage and illuminated from
below. Some textured yams and all dull (pigmented) yams require a
mounting liquid (such as mineral oil) to decrease the scattering of light.
Compared with cork the perforated plate produces a more uniform and
reproducible section, which is simultaneously mounted and preserved.
Both methods are suitable for classroom use (see Figure 20.12). A draw-
back to purchasing perforated plate is the minimum number per order of
1000.(9) Perhaps one or more of our microscopical supply houses would be
willing to accept smaller orders.(IO,ll,lla,I2J

FIGURE 20.12. Cross sections of Antron® II nylon fibers mounted in a hole in a black vinyl
card, then cut on both sides with a razor blade and photomicrographed in class.(2l Courtesy
of McCrone Research Institute.
 Specimen Preparation 395

20.3 MICROTOMY

Microtomy is the technique of cutting sections suitable for examination

in the light microscope by transmitted illumination.<13) A microtome con-
sists of a holder for the specimen, a feeding mechanism for regulating the
thickness of the cut, and a guide for the knife.<14•) Microtomes range in size
from the small Hardy type<15) for fibers to heavy bench types for three-
dimensional specimens, including those extensively prepared by fixing,
polymerizing, impregnating, etc. Figure 20.13 illustrates the use of a mi-
crotome<tf>-ts) for fibers, including the use of collodion<12) to attach the sec-
tion of a fiber to a microscopical slide. Figure 20.14, which is related to
Figure 20.1, shows the correlation of a composition and condition with
structure and morphology to learn about properties and behavior through
cross-sectioning (see Figure 20.13) and employing light microscopy.
If the material to be sectioned is porous, friable, or has poor tenacity,

FIGURE 20.13. Sketches showing the use of a small microtome for fibers.<2) (a) The slotted
top chromium-plated for protection against scratching by razor blade, forceps, etc.; (b) filling
the slot; (c) plunger pushing fibers together; (d) trimming bundle with razor blade; (e)
appearance after trimming; (f) further compaction with plunger; (g) embedding ends (or
edges) prior to sectioning with a lacquer, such as MICO-LAC solution; (h) preliminary
cutting with unused blade; (i) coating with collodion or other lacquer; OJ slicing; (k) removing
slice; (I) lubricating with water; (m) removing slice; (n) firming slice. Courtesy of McCrone
Research Institute.
 396 Chapter 20

a:: 4
w
z
~ A
'
(/)
::E
<t
a::
(.!)

z
H
(/)
(/)
w
...a::
(/)

--~•o,um

0 5 25 30 35 40
STRAIN AS % ELONGATION
FIGURE 20.14. Resinographic study of experimental acrylic fibers cut successively from the
same wet tow and given various treatments before drying. Corresponding structures and
stress-strain properties are depicted. (I)

some sort of embedding must be used. Dry, porous materials can be

simply and quickly infiltrated by molten paraffin( 12) or harder vegetable
waxes, provided they are not affected by the medium at a temperature of
60-70 oc (140-158 °F). Paper, leather, and other porous materials can be
supported this way during microtomy. Wax however is not strong or
tough enough to hold textile fibers, and these must generally be embedded
in plastic resin, as described for ultramicrotomy. Moist or wet materials,
such as plant and animal tissues,( 19) shrink or are otherwise distorted
during heating. Such materials generally behave well during quick freez-
ing, and evaporating ether or carbon dioxide in an attachment to the
microtome keeps the specimen frozen while it is being sectioned.

20.4. ULTRAMICROTOMY

Ultramicrotomy is the technique of cutting sections thin enough for

examination in electron microscopes. (13•14) In the 1940s and 1950s, specimens
Specimen Preparation 397

had to be ultrathin, less than 100 nm (0.1 J.tm) thick at the available electron
accelerating voltages. With the development of higher accelerating vol-
tages, and especially the construction of STEMs, thicker specimens (0.1-2.5
~-tm and even thicker) are acceptable (see Figure 14.9); nevertheless ultra-
thin specimens cut on an ultramicrotome continue to be required. Because
now-a-days the specimen is moved past the cutting edge (sharp glass or
diamond), the bulk material must be firm or it becomes distorted while
being cut. From the 1970s to the present, biological and aqueous non-
biological specimens are dehydrated and embedded in a resin. The results
are not always consistent, apparently because of variations in the embed-
ding medium, process, ageing, temperature, humidity, etc. Because most
biological specimens and many nonbiological materials contain water or
can be wetted, they can be frozen and ultramicrotomed at low tempe-
ratures; elastomers and other soft materials can be stiffened and then
ultramicrotomed at low temperatures. C13l
Reid and Beesley<13l describe ultramicrotomes on the market as of
1991, and list sources of ultramicrotomes, microtomes, cryoattachments,
steel, glass, diamond, or sapphire knives, equipment for embedding, block
trimming, and other items. HayatC20l gives directions in transmission elec-
tron microscopy as of 1986, for preparing fixatives, tissue blocks, section-
ing, staining, support films, and specific biological preparations.C20•21 •l

20.5. REPLICATION

To answer the question, "why one side of a specimen of commercial

nylon film slides easily when folded against itself but encounters great
resistance when folded the other way," each of the two sides was posi-
tively replicated by evaporating silica and then electron micrographed.(!)
Figure 20.15 shows that the rough side is due to projecting crystalline
grains of nylon, whereas the other side is smooth. Figure 20.15 also shows
that the molten nylon was cast against a smooth solid to form a smooth
surface, but on the other side of the film, spherulites were free to grow in
air, creating a relatively rough surface.
Another type of replica employs the extraction technique. There are
three kinds: (1) direct carbon replica, (2) carbon replica shadowed with
platinum, and (3) two-stage plastic-<:arbon replica.C21 l The purpose is not
only to extract particles from their matrix but also to maintain them in their
relative positions. Accordingly particles must be small enough to be sup-
ported by a carbon film; instructions are provided by Goodhew,C21 l who
also gives advice on mounting and storing specimens and a list of mater-
ials and suppliers as of 1984.
398 Chapter 20

FIGURE 20.15. Commercial film of nylon showing differences in texture between the two
sides. Electron micrographs of positive silica replica; no shadowing metal. Right: smoother
side cast against a smooth solid; left: rougher side cast in air.< 1l

20.6. FRACTURE SURFACES

Many materials are too hard to be satisfactorily cut with a microtome

or ultramicrotome. Such materials may be brittle, or they can be embrittled
sufficiently to be prepared for microscopical examination by fractur-
ing.<1.2·14> For revealing macromolecular domains, freeze-fracturing has
been successful.<22 >The Botty<23 > fracture technique involves wrapping a
specimen, such as test bar or sheet, in aluminum foil, then cooling it to
about -60 oc (-76 °F) in contact with solid C02 for 2 hours. When the
aluminum foil is removed, the test bar or sheet is fractured in a cooled
tensile tester. Aluminum foil is used as a wrapper to contain fragments and
prevent contamination. The fracture surface is replicated (negatively) with
a 5% solution of gelatin in water at a temperature of about 55 oc (131 °F).
This layer is strengthened with a layer of 10% gelatin solution to facilitate
stripping off the negative replica from the fracture surface. A positive
replica is then made of silica by vacuum deposition to a thickness of about
150 A<23>(see Figure 20.16).<22>
Specimen Preparation 399

FIGURE 20.16. Unshadowed Si02 replica of fractured melamine-formaldehyde molded res-

in.(22) Macromolecular domains 70-80 nm in diameter are evidently composed of smaller
domains 15-20 nm in diameter ..

Another way of producing a fracture surface in a test bar of con-

solidated polymeric plastic involves bending the bar, which is held by
screw clamps at each end, over a central fulcrum as indicated in Figtlre
20.17. The bar flies apart, usually with a loud noise, if a drop of ethyl
alcohol is placed on the part of the bar over the fulcrum.( 24> The fracture
surface is best observed by an SEM.

FIGURE 20.17. Sample apparatus for wet stress crazing of test bars. After turning the
thumbscrews to the desired extent of stress, a drop of wetting liquid (e.g., ethyl alcohol) is
placed on the specimen (e.g., PMMA) over the fulcrum.(2)
400 Chapter 20

20.7. THIN SECTIONS OF HARD MATERIALS

Such materials as bone, hom, sea shells, and some minerals are too
hard to be microtomed, but they are soft enough to be hack-sawed by
hand.!25> Moreover hacksaw dust from a clean blade, is adequate for a
preliminary microscopical examination by transmitted light. <25> However
for a microscopical examination of rocks, minerals, and the like, a petro-
graphic thin section is required. <25> This involves cutting off a specimen
with an abrasive wheel, such as a metallic disk rimmed with diamond
dust.
The usual sequence of operations for preparing the petrographic type
of specimen is illustrated in Figure 20.18. The standard thickness of pet-
rographic sections is 0.03 mm. Since the most common mineral in a rock
is quartz, which has a very recognizable first-order polarization color at a
thickness of 0.03 mm, the professional technician grinds rock sections to
the color for quartz, as indicated on the Michel-Levy scale of birefringence
(see Figure 5.8). Of course with other types of specimens, a thickness
micrometer may (or may not) be used. Since preparing petrographic (thin)
sections is a specialized technique, the general microscopist may wish to
send occasional requirements to a professional technician. <26>

20.8. TRANSPARENT PARTICULATE SPECIMENS

Transparent powders, granules, foils, papers, yams, and fabrics must

be consolidated before they can be thin-sectioned by either microtomy or
grinding. Most of these specimens are either thermosensitive, porous, or
otherwise not amenable to briquetting by means of a pressure-sensitive
resin, such as phenol-formaldehyde. If the specimen is not thermoplastic,
it can be embedded in paraffin<12> or a mixture of camphor (62%) and
(napthalene), which freezes at 32 oc (89.6 °F),<14> for microtomy; a pre-
formed capsule is convenient for this purpose.!2•12>If the specimen is porous
like some yams, fabric, paper, or sponge, warming at an allowable tem-
perature with or without a vacuum is desirable.<2>

20.9. MONOMERIC EMBEDDING MEDIA

Butyl methyl methacrylate embedding medium has regained favor

with some electron microscopists because it can be cured with UV radia-
tion at room temperature or even refrigerated.<28> Another reason for its
return to favor is the availability of azo-1-cyanocyclohexane as a catalyst
 1. SKTlONING Pe-tlonned wuh a dtamond cul-ofl wheel lo reduce the
parent sample to a workable ond represent011ve PleC'ft

~ 2. lOUGH GIINDING Performed on 0 rotalln9 lap woth a h•ed or loose abra.ovo

~ to Pfoduee a Hot smoolh surface I~ from delormallon
I
~
~"­.) 3. CEMENTING THE " CHIP" TO THE GLASS SliDE
~ Pedormed on a No 30-8010 AB Sl1de Wormer 'WI.ttth o lhenno-plasnc
cemctnl. or with a ccld mor.,.~nlmg a: men I to hold lheo ch•p to the sbde
~
/01
~~COlD ~
0 _ • . RNISN GIINDING
MOUNfiNG ~ 4o. t•Mdiolti,. fOpfioffoiJ
Performed wuh a dtomond cut.off.wheel and 11-3100
~ AB ReuociLOntng Vtse to remove e,.;ees.s molena] and
~ .subs1an11clly redur:e omounl of Coarse Fmish Gru1dm9

41>. Coa ... ' l• i""l••

Performed on a ro1ahn9lap w1th 320 and 600
Gnt SoC and a No 30-8000 AB Th1n Secloon
Shae Holder or w11h a medJum d1am.ond dJ5C
~ ~ and a No 30-8005 AB Petr09rophic Slide
Hotder. unhl the S«11on 15 reduced. to op-
/ prolamaleiy .50 m1c:rons

~
~\
........
' /01 ~
~~
~/2EJ . '"'•
""· n.. ' '"'""'
Performed by ..hand on o No
39-1430 AB PetrQ9rophoc Hand
~ ~ Gnnder with 5- 10 Mocron AI,O, or
r:s::..~~ ~ on o slowly rolohng lap untd the
1 ~tOn ~s reduced to 30 micron$

5. COM~LITING THE SECTION Ground surface may be covered woth a ccver gi<Hs (5o ). or poliShed (5bl

FIGURE 20.18. Scheme for preparing thin sections of

rock and other hard, relatively transparent spec- ~~
imens.(27) Courtesy of Buehler.(27) ~~ ~ ~
.5cr. Movftll~to rite C~•r GIGu Jb.. ,olialdn• tM Secflo11
Porformed woth a No 30-8010 AB
Shde Wormer end o suttable
~' Petlormed on a rotating lap in
two steps. 6 M.cron Metadi• and Alummum
comen1 to proled and "wet" 1he O•ide (Q J.Q05 MocronJ unlu a high poll5h
s.uc·tJon surface is oblamed
 402 Chapter 20

to replace the potentially explosive benzoyl peroxide. Poly(butyl methyl

methacrylate) may also appeal to the light microscopist, who should fol-
low directions for keeping quantities of the catalyzed monomer down to
capsule size (or thereabouts). Methacrylate polymerization is a strongly
exothermic reaction that is catalyzed by heat, light, and active oxygen.
Since the monomer must not come in contact with the skin, it is necessary
to wear gloves and goggles and to use an active exhaust hood.(2l
Epoxy-anhydride embedding media have certain advantages over the
methacrylate media in resinography: Since resultant (cured) epoxy resins
are thermoset, they can be sawed, abraded, polished, or heat-treated with-
out getting sticky. At the same time they can be formulated to be soft
enough to be microtomed into thin sections(29> or to be hard enough to be
abrasively sawed and polished into thin sections.(30l A favorite mixture is
Dow Epoxy Resin (DER) 332 (harder) and DER 732 (softer) with diethylene
triamine as a curing agent; a good combination to start with is 45% by
weight of DER 332, 45% by weight of DER 732, and 10% of diethylene
triamine.(31 >Specimens should be cured at 60° C (140° F) for 35 minutes and
they must be dry. The mixed resin should be stored in a desiccator or on
top of the curing oven; the amine, which is more hygroscopic than the
mixed resin, must be stored in a desiccator. The separate mixed resin has
a long shelf life. Use a disposable paper cup and a wooden stick(2l to
combine the mixed resin with the amine hardener.
Flexible capsules of silicone potting agent<32l are cast in a negative
mold, as shown in Figure 20.19. This negative mold is fashioned after
Sanders' s(33>; but none of the pieces has to be turned on a lathe; instead they
can all be sawed from a 0.25-in. sheet of poly(methyl methacrylate). The
sawed edges must be straight and smooth.
To cement the pieces together, clamp them, then use a medicine
dropper to lubricate the joints with a good, volatile solvent, such as methyl
ethyl ketone. If air spaces return, fill them again with solvent. Keep the
pieces clamped together overnight, then inspect them again. If there are no
air spaces at the joints, you can remove the clamp. If there are air spaces,
fill them again, this time using a syrup of PMMA and solvent. When the
negative mold is ready, fill it to the top with the silicone potting agent,
which has been mixed according to directions. After curing, remove the
flexible mold, then cut shallow slots with a razor blade at the apex and the
center of each capsule to hold a sample of yarn. Note: Do not make the slots
until you need them because they can easily be cracked as you flex the
mold to remove the capsules.
Composite yarn made on the integrated spinning system(29> is a typical
example of the preceding method of preparation by cutting a sledge mi-
crotome. This type of yarn combines the strength of a bundle of continuous
Specimen Preparation 403

TOP
VIEW

(a) • : SOLD

sr~ ~~~~SS·SECTIONAL
(b) IZ'J : souo
FIGURE 20.19. Flexible silicone negative
mode (a and b) to produce (c) positive
casting(s) of specimens for either micro-

l~ ~
tomy or grinding/polishing.<2) (a) Flex- E
ible, negative silicone mold, top view; (b) E ,·,
side view of flexible, negative silicone ~1 ~
mold; (c) positive, hollow, unit cell to re- 1----t
ceive specimen. (c) 9mm

filaments with the appearance and thermal insulation of cotton, wool, or

man-made staple yarn. The staple is stuck onto the continuous filaments
of polyester or nylon or continuous ribbon of polyolefin. The assembly is
then twisted to integrate the staple fibers. To perform resinography with-
out disturbing the yarn's cross section, a short length of a composite yarn
is encapsulated in 50:50 DER 332:732 catalyzed with 10% diethyl amine.
The yarn is cured at 60 oc (140 °F) for 35 minutes, then thin sections are cut
on the sledge microtome.<29)
In an earlier investigation,<30) two-component Bobtex integrated com-
posite-spun yarn was embedded in epoxy resin that became hard enough
to slice on a cut-off wheel and to prepare as though it were a piece of rock.
The resin was Epon 828 (88%) cured with metaphenylene diamine (12% ).
First specimens were placed in a small cardboard box and heated to 60
oc for a half hour to expel moisture. Then the specimen was transferred
to the bell jar shown in Figure 20.20. The vacuum pump was used for a
half hour to expel air from the specimens. Then Epon 828 metaphenylene
diamine mixture was allowed slowly to fill the specimen box almost
completely. Evacuation of air and volatiles was continued for 8 hours at
25 oc to jell the resin; curing was at 53 oc (127.4 °F) for 2 hours and at
149 oc (300.2 °F) for 2 hours. The block of hard, dark resin was trimmed
404 Chapter 20

I ' - - -- - - FUNNEL
IIJL--- - - ---RESIN

l'=h.:t----..TO VAaJUM
PUMP

BASE PLATE

FIGURE 20.20. The vacuum system by which entrapped air is removed from the sample.<2l

with a diamond-studded cut-off wheel<33l (see Figure 20.20), and enough

of the block was cut away to expose the specimen. Then one side of a thin
slice of the specimen was fastened with water-soluble adhesive (gum
arabic solution) to a solid, nonporous block as a handle. The opposite
(exposed) side of the specimen was smoothed with successively finer
abrasive papers. The abraded surface was washed with water and dried
with a current of air. This surface was polished on successively finer
diamond-impregnated laps.<34l When free of microscopic scratches and
pits, the handle was dissolved in water. The polished surface was washed
and dried, then permanently cemented to a microscopical glass slide with
Epon 828 catalyzed with metaphenylene diamine (see Figure 20.21). After
curing the adhesive resin, the other side of the thin section was abraded

FIGURE 20.21. Diagram of spec-

imen assemblage with the rough
surface still to be polished. The
polished surface is permanently
fastened to a microscopical glass
WATER slide with hard epoxy resin. The
SOWBLE GWE
other side of the glass slide is held
HARD EPOXY RESIN on the holding block with water-
soluble gluePl
Specimen Preparation 405

and polished like the first. Care was taken not to go right through the
wafer when polishing.
Epoxy resins make good permanent specimen mounts and coverslip
seals. Since they are fairly permanent however, this may create difficulties
in later studying the sample in various refractive index liquids. Small,
convenient, dual packages of epoxy fluid and hardener are marketed as
patching and mending materials. They all probably make good seals; as
mounting media, they should be tested to assure that they are sufficiently
transparent when cured.
All epoxy materials but particularly amine hardeners are harmful to
the skin, especially the eyes, so it is important not to rub the eyes while
working with catalyzed uncured epoxy resin. If you get epoxy material
on your hands, wash it off immediately and wear thin gloves during
preparation. Some of the amines, especially crystalline metaphenylene
tetramine as melted for action, are dark-colored and can stain clothing so
it is important to wear an apron and keep hands clean.

20.10. PREPARING FOR REFLECTED ILLUMINATION

Thermoset resins, ores, coal, and other opaque materials are prepared
for examination by reflected light, electrons, X rays, etc. If transparent
constituents are closely associated, they, too, are prepared to be examined
by reflected emanation. If the specimen is solid and it can be cut to a handy
size, it is already fit to be abraded, polished, etched, stained, etc.; otherwise
the specimen must be consolidated into a convenient size and shape. If the
specir:nen is in parts, these are compressed, impregnated, composited, or
otherwise consolidated into a convenient size and shape.<1•2•14.2S,:ZSJ

20.11. PREPARING METALS AND OTHER OPAQUE MATERIALS

Metallographic specimens preparation is well documented.<3·3a·14•25•3S-37J

Similar methods have been adapted for other opaque materials, such as
ores, coal, filled thermoset resins (see Chapter 4), tires,(l) composites,(l)
ceramics, plasma coatings on metals, and cemented tungsten carbide.<38>
Ceramics, plasma coatings, cemented tungsten carbide, and other hard
materials require diamond dust for cutting, abrading, and polishing.<39l
Figure 20.22 shows a diamond loaded cut-off wheel on a low-speed saw
fitted with a sample-holding arm fitted with an electronic load-sensing
circuit to prevent injury to the operator (or the machine).<39> For softer
406 Chapter 20

FIGURE 20.22. Buehler Isomet® low-speed diamond saw.<26a) Courtesy of Buehler.<26•>

specimens silicon carbide and/ or aluminum oxide abrasive and polishing

materials are recommended.<33>
Figure 20.23 is of a polished cross section of a commutator brush for
an electric generator. The brush is a composite of particulate copper and
particulate graphite consolidated in thermoreacted melamine-formalde-
hyde resin. The specimen had been prepared by mounting the article in
thermoreactive black bakelite BM120 and using the compression mold
with rectangular cavities shown in Figure 20.24. Compared to round bri-
quets, rectangular briquets are easier to hold, orient, and return to their
previous locations. Once the front side has been designated (by inscrip-
tion) and a particular location has been recorded, it can be relocated by
means of graduated coordinates on a rectangular mechanical stage, such
as the one shown in Figure 20.25. Moreover this briquet fits in the mech-
 Specimen Preparation 407

FIGURE 20.23. Polished cross

section of a composite commuta-
tor brush for an electric generator.
White =copper (for conductivity),
light gray = graphite (for lubrica-
tion}, dark gray = melamine-for-
maldehyde resin (consolidation),
and black = air (pores).P>

anical holder of the particular automatic polishing machine (see Figure

20.26}.(25>
Briquetting with a thermosetting resin permits surfacing and polish-
ing a wide variety of coexisting phases, for example soft and hard con-
stituents, nonreflecting and reflecting ones, and particulate as well as solid
materials.
Figure 20.27 shows the cross section of an adhesive bond between two
sheets of aluminum alloy as polished for examination. Before coating each
sheet with phenolic varnish (two dark layers), each aluminum sheet had
been anodized (dark dots) to make the phenolic resin adhere better. The
central layer (medium gray) is an elastomer to give some more resilience
to the joint. The results of tensile tests on this composite were good, but
costs of so complicated a sandwich structure were much higher than the
cost of using a composite, one-step "spread" (see Figure 20.28). The con-
tinuous phase is a phenolic varnish, and the particulate phase is an elas-
 FIGURE 20.24. Four-cavity spedmen bri-
quet mold (Eagle Tool Co.): (a) bottom plate;
(b) chase; (c) top plate; (d) stripping plate.

FIGURE 20.25. Rectangular briquet fitted in a mechanical stage (Leitz) on a rotating stage
(Leitz).( 25)
Specimen Preparation 409

FIGURE 20.26. Mechanical holder (Mico Instrument Co.) for a rectangular briquet.l25>

FIGURE 20.27. Resinous bond for aluminum alloy. The white areas, top and bottom, are
aluminum alloy; the central, medium gray layer is elastomer; the two dark gray layers are
varnish primer.(25>
410 Chapter 20

FIGURE 20.28. Elastic mixture or spread for one-step bonding of aluminum sheets. Con-
tinuous phase is phenolic varnish; particulate phase is an elastomer.(25l

tomer. An advantage of the particulate system is its three-dimensional

application and properties.(25l

20.12. SUMMARY

The preparation of a material for microscopical examination depends

upon what is to be examined and by what kind of microscope. In most
scientific, technical, and practical endeavors, light microscopy and electron
microscopy are the most important kinds. Since methods of preparation
are generally related, the two types are discussed together. A list of topics
follows:
Microscopical examination of the specific specimen surface that has
failed in service or a specific test
Microscopical examination of fibers, fur, or hairs by methods that
require little preparation
Specimen Preparation 411

Microtomy for light microscopy

Ultramicrotomy for electron microscopy
Surface replication as is or as prepared
Fracture surface as is or as prepared
Thin sections of hard materials, such as materials, rocks, and ores, in
the manner of petrography
Transparent particulate specimens, such as powders, granules, fibers,
foils, papers, yarns, and fabrics embedded in paraffin or a mixture
of camphor and naphthalene
Monomeric embedding media that polymerizes to a solid
Thermoset resins, ores, coal, etc., prepared for examination by re-
flected light
Metal preparation (metallography).
References

Chapter 1

1. C. Jacker, Window on the Unknown: A History of the Microscope (New York: Scribner's,
1966).
2. S. H. Gage, "Brief History of Lenses and Microscopes," in The Microscope, 17th ed.
(Ithaca, NY: Comstock, 1947).
3. Milestones in Optical History (Rochester, NY: Bausch and Lomb, 1954).
4. I. Asimov, Biographical Encyclopedia of Science and Technology (Garden City, NY: Double-
day, 1972).
5. D. J. Hamlin, "What a Spectacle! Eyeglasses and How They Evolved," Smithsonian 13,
1983, 100-111.
6. "Origin and Development of the Microscope," in Catalogue of the Royal Microscopical
Society, (Oxford, England: The Royal Microscopical Society, 1928).
6a. S. Schulman, "Micromagic," Technology Review Nov.-Dec. 1989, 11-12.
7. M. Rooseboom, "The History of the Microscope," in Proceedings of the Royal Microscopical
Society 2 1967, 266--93.
7a. G. L.'E. Turner, The Great Age of the Microscope, Bristol and New York: Adam Hilger, 1989.
8. J. Kenseth, ed., The Age of the Marvelous (Hanover, NH: Hood Museum of Art, Dart-
mouth College, 1991).
9. R. Hooke, Micrographia (Royal Society of England, 1665; reprinted by Dover: New York,
1961).
9a. J. Hogg, The Microscope, Its History, Construction, and Applications (London: Herbert
Ingram, 1856), 3-6.
10. J. van Zuylen, "The Microscopes of Antoni van Leeuwenhoek,"]. of Microscopy 121, part
3 (Mar. 1981), 309-28.
11. B. J. Ford, "Reappraising the History of Microscopy: Revelation and the Simple Micro-
scope," Microscopy and Analysis (Sept. 1989), 7-10.
12. B. J, Ford, "Bacteria and Cells of Human Origin on van Leeuwenhoek's Sections of
1674," Transactions of the American Microscopical Society 101 (Jan. 1982), 1-9.
13. B. J. Ford, "The van Leeuwenhoek Specimens," Notes and Records of the Royal Society
36,(1) (Aug. 1981), 37-59.
14. B. J. Ford, "Robert Brown, Brownian Movement and Teeth Marks on the Hatbrim," The
Microscope 39 (3d-4th quarter 1991); "Brownian Movement in Clarkia Pollen: A Reprise
of the First Observations," The Microscope 40 (3d-4th quarter 1992).

413
414 References

15. 0. W. Richards, "Microscopy: Yesterday and Tomorrow," Transactions of the American

Microscopical Society 91 (1972) 529-32.
16. S. Bradbury, "The Quality of the Image Produced by the Compound Microscope:
1700-1840," in Proceedings of the Royal Microscopical Society 2 (1967) 151-74.
17. S. Bradbury, "Now It Can Be Proved," The Vickers Magazine (Autumn 1966), 13-14.
18. C. W. Mason, Handbook of Chemical Microscopy, Vol. I, 4th ed. (New York: Wiley, 1983).
19. D. B. Weiner, Raspail, Scientist and Reformer (New York: Columbia University Press,
1968).
20. D. L. Padgitt, A Short History of the Early American Microscopes (Chicago: Microscope
Publications, 1975).
20a. J. G. Delly, Photography through the Microscope (Rochester, NY: Eastman-Kodak, 1988).
21. "100 Years of the Carl Zeiss Foundation," Microscopy and Analysis (Sept. 1989), 48.
22. ASTM designation E-211, "Specifications and Methods of Test for cover Glasses and
Glass Slides for Use in Microscopy" Index to Annual Book of ASTM Standards (Philadel-
phia: American Society for Testing and Materials, 1990).
23. Components of Microscopes, Part 2: Dimensions of Microscopes (London: British Standards
Institution, 1963).
24. Index to Annual Book of ASTM Standards (Philadelphia: American Society for Testing and
Materials, 1993).
25. R. Vallery-Radot, The Life of Pasteur, trans. R. L. Devonshire (Garden City, NY: Double-
day, Page, and Co., 1926).
26. T. G. Rochow, "Some Incidental History of Metallography and ASTM Committee E-4,"
in ASTM Symposium on "Metallography 75 Years Later," May 8-10, 1991, Atlantic City,
NJ, 83-87. Published as ASTM STP1165, (Philadelphia: ASTM, 1993).
27. D. W. Humphries, "The Contributions of Henry Clifton Sorby to Microscopy," The
Microscope and Crystal Front 15 1967, 351-62.
27a. J. Clark, "Milestone in Polarizing Microscopy," Australian E. M. Newsletter 171988,7-8.
28. A. H. Bennett, et al., Phase Microscopy, Principles and Applications (New York: Wiley,
1951), chap. 1, 1-3.
29. W. Krug, et al., Contributions to Interference Microscopy, trans. J. H. Dickson (London:
Hilger and Watts, 1964).
30. "ASTM Symposium on Interference Microscopy (abstracts)," P. H. Bartels, ed., Materials
Research and Standards 2 1962, 672.
31. J. H. Richardson, Handbook for the Light Microscope (Park Ridge, NJ: Noyes, 1991).
32. M. Locquin and M. Langeron, Handbook of Microscopy (Butterworth, 1983).
33. "In Memoriam: Dr. Edwin Herbert Land, May 1, 1909-March 1, 1991," Optics & Pho-
tonics News (May 1991).
34. E. H. Land eta/., "Color Translating Ultraviolet Microscope," Science 109 (1949), 371-74.
35. R. E. Gore, "Infrared Spectrometry of Small Samples with the Reflecting Microscope,"
Science 110 (1949), 710-11.
36. J. A. Reffner and W. T. Wihborg, "Microanalysis by Reflectance FT-IR Microscopy,"
American Laboratory (April1990) 26-34.
36a. J. A. Reffner, "The Commercial Development of FT-IR Microspectrometry," Microscopy
Today 93-3, (April 1993).
37. A. Fisher, "Superscopes," Popular Science Ouly 1990), 66-69.
38. Carl Zeiss, Inc., 1 Zeiss Drive, Thornwood, NY 10594.
39. Leica, Inc., 24 Unk Drive, Rockleigh, NJ 07647.
40. Nikon, Inc., 1300 Walt Whitman Road, Melville, NY 11747-0299.
41. Olympus Corp., Precision Instrument Div., 4 Nevada Drive, Lake Success, NY 11042-
1179.
References 415

42. C. E. Hall, Introduction to Electron Microscopy 2d ed. (Hightstown, NJ: McGraw-Hill,

1966).
43. T. Mulvey, "The History of the Electron Microscope," in Proceedings of the Royal Micro-
scopical Society 2 1967, 201-27.
44. E. F. Burton and W. H. Kohl, The Electron Microscope, an Introduction to Its Fundamental
Principles and Applications, 2d ed. (New York: Reinhold, 1946).
45. T. G. Rochow, "Significant Early Days in Industrial Electron Microscopy," EMSA Bul-
letin 13, 1 (spring 1983).
46. M. W. Ladd, "Electron Microscopy," in Encyclopedia of Industrial Chemical Analysis, vol.
1 (New York: Wiley, 1966), 649~85.
47. M. W. Ladd, The Electron Microscope Handbook (Burlington, VT: Ladd Research In-
dustries, 1973).
48. V. E. Cosslett, "Beyond Optical Limits," The Vickers Magazine (autumn 1966), 15-16.
49. Electron Microscope Society of America, Yearbook (1943); now the Microscopy Society of
America, Box MSA, Woods Hole, MA 02453.
50. R. B. Barnes et al., "Electron Microscopical Replica Techniques for the Study of Organic
Surfaces, Journal of Applied Physics 16 (1945), 730--39.
51. J. l. Goldstein and H. Yakowitz, eds., Practical Scanning Electron Microscopy (New York:
Plenum, 1975).
52. J. W. S. Hearle et al., The Use of the Scanning Electron Microscope (Elmsford, NY: Per-
gamon, 1972).
53. T. T. Tsong, "Atom-Probe Field Ion Microscopy," Physics Today (May 1993), 24-31.
54. E. W. Muller, "Field-Ion Microscopy," Science 149, 3684 (Aug. 1965) 591~01.
55. Sales literature from Sonoscan®, Inc., 752 Foster Ave., Bensenville, IL 60106 (1975).
56. R. A. Lemons and C. F. Quate, "Acoustic Microscopy: Biomedical Applications," Science
188 (1975) 905-911.
57. A. Fisher, "Seeing," Popular Science (April1989) 102, 104-107.
58. M. C. Botty and F. G. Rowe, U.S. Patent 2,843,751, July 15, 1958.
59. M. C. Botty and E. J. Thomas, "Applications of Microradiography with the EMU-I
Electron Microscope," paper presented to the Electron Microscope Society of America,
Columbus, Ohio, Sept. 9, 1959 (unpublished).
60. For example, Philips Electronic Instruments, Mount Vernon, NY 10550; Canal Industrial
Corp., Bethesda, MD 20014.
61. T. W. Ford et al., "Improved Imaging of Biological Specimens by Soft X-Ray Contact
Microscopy Using Laser-Produced Plasmas as X-Ray Sources," in EMAG-MICRO 89,
vol. 1: Physical P. J. Goodhew and H. Y Elder, eds. (Bristol, London and New York:
Institute of Physics, 1990), 527-30.
62. W. C. McCrone, "INTERMICR0-92," The Microscope 40, 3 (1992), 175.

Chapter 2

1. Glossary of Microscopical Terms and Definitions, 2d ed. (New York: NY Microscopical

Society, 1989).
1a. S. Zeki, "The Visual Image in Mind and Brain," Scientific American (Sept. 1992), 68-76.
2. Compilation of ASTM Standard Definitions, 6th ed. (Philadelphia: American Society for
Testing and Materials, 1986).
2a. A. J. Olson and D. S. Goodsell, "Visualizing Biological Molecules," Scientific American
(Nov. 1992), 76-83.
416 References

3. M. W. Davidson, "Fabrication of Unusual Art Forms with Multiple-Exposure Color

Photomicrography," The Microscope 38 (1990), 357--65.
3a. D. M. Schwartz, "Revealing the Hidden Beauty of Commonplace Crystals," Smithsonian
(May 1993), 112-14.
4. L. Wilson, "Photomicrographs of Rods and Cones of the Human Eye," Life Magazine
(Oct. 1971), 58-59.
5. J. G. Hollyfield, "Photomicrography of the Retina in Eye Research," American Laboratory
(Oct. 1991), 31-32.
Sa. J. H. Richardson, Handbook for the Light Microscope (Park Ridge, NJ: Noyes, 1991).
6. T. Mulvey and C. J. R. Sheppard, eds., Advances in Optical and Electron Microscopy, vol.
12, (Academic Press, 1991), 245.
6a. "Edwin Land's Retinex Theory: An Important Contribution to Understanding Color
Vision," In Focus, Fall-Winter, (1992), 8, 9, 15 .
7. T. G. Rochow and E. G. Rochow, Resinography (New York: Plenum, 1976), 233.
8. E. Baer et al., "Biological and Synthetic Hierarchical Composites," Physics Today (Oct.
1992), 60-67.
9. J. A. Reffner and W. T. Wihborg, "Microanalysis by Reflectance FT-IR Microscopy,"
American Laboratory (Apr. 1990), 26-34.
10. C. W. Mason, Handbook of Chemical Microscopy, vol. 1, 4th ed. (New York: Wiley, 1983).
11. 0. W. Richards, "Instrument Myopia-Microscopy," Amer. f. of Optometry and Phys-
iological Optics 53, (10) (1976), 658--63.
12. T. G. Rochow and R. L. Gilbert, "Resinography," in Protective and Decorative Coatings, J.
J. Mattiello, ed. (New York: Wiley, 1946).
13. John G. Delly, Photography through the Microscope, 9th ed. 2d (Rochester, NY: Eastman
Kodak, 1988).
14. "Excellence in Transmission Electron Microscopy" (Thornwood, NY: Carl Zeiss, 1990).
15. H. W. Zieler, The Optical Performance of the Light Microscope, part 1 (Chicago: Microscope
Publications, 1972).
16. E. H. Land et al. "A Color-Translating, Ultraviolet Microscope," Science 109 (1949),
371-74.
17. R. E. Gore, "Infrared Spectrometry of Small Samples with the Reflecting Microscope,"
Science 110 (1949), 710-11.
18. N. H. Hartshorne, The Microscopy of Liquid Crystals (Chicago: Microscope Publications,
1974).
19. N.H. Hartshorne and A. Stuart, Crystals and the Polarising Microscope, 4th ed. (London:
Edward Arnold, 1970).
20. G. E. Schleuter and W. E. Gumpertz, "The Stereomicroscope: Instrumentation and
Techniques," American Laboratory (Apr. 1976), 61--64, 66.
21. V. Betz, "High-Magnification Mineral Stereophotomicrography," The Mineralogical Re-
cord 21 (Sept.-Oct. 1990), 475-480.
22. A. N. Winchell, The Microscopic Characteristics of Artificial Inorganic Solid Substances or
Artificial Minerals (New York: Wiley, 1938).
23. N. H. Hartshorne and A. Stuart, Crystals and the Polarising Microscope, 4th ed. (New York:
American Elsier, 1970).
24. T. G. Rochow et al., "Light and Electron Microscopical Studies of Pleurosigma Angu-
latum for Resolution of Detail and Quality of Image," The Microscope and Crystal Front
15 (1966), 177-201.
25. T. G. Rochow, "Significant Early Days in Industrial Electron Microscopy," EMSA Bul-
letin 13 (spring 1983) 1.
References 417

26. F. T. Jones, "Two Apparent Exceptiors to Abbe's Theory of Resolution," The Microscope
16 Gan. 1968), 4-11.
27. T. G. Rochow eta/., "Scanning Electron Microscopy of Pleurosigma Angulatum (Que-
kett) for Resolution of Detail and Quality of Image," The Microscope 32 (1984), 151-
62.
28. R. P. Loveland, Photomicrography, vols. 1 and 2, 2d printing (Malibar, FL: R. E. Krieger,
1985).

Chapter 3

1. H. W. Zieler, The Optical Performance of the Light Microscope, part 1 (Chicago: Microscope
Publications, 1972).
2. McCrone Research Institute, 2820 S. Michigan Ave., Chicago, IL 60616-3292.
2a. Bausch & Lomb Scientific Optical Products Div., Rochester, NY 14602.
3. V. E. Cosslett, Modern Microscopy (Ithaca, NY: Cornell University Press, 1968).
4. Edmund Scientific Co., 101 E. Gloucester Pike, Barrington, NJ 08007-1380.
5. C. W. Mason, Handbook of Chemical Microscopy, 4th ed., vol. 1 (New York: Wiley, 1983),
180-81.
6. Swift FM-31 field microscope, Swift Instruments, Inc., 1190 N. 4th Street, P. 0. Box 562,
San Jose, CA 95106.
7. G. E. Schleuter and W. E. Gumpertz, "The Stereomicroscope: Instrumentation and
Techniques," American Laboratory (Apr. 1976), 61-64, 66, 68-71.
8. "Leica's New Flex-Arm System Provides Greater Flexibility for Microscope Work Sta-
tion," The Microscope 39 (3d/4th quarter 1991), v-vi.
8a. Ring-Light, Hacker Instruments, Inc., Box 657, Fairfield, NJ 07006.
9. Leica, Inc., 24 Link Dr., Rockleigh, NJ 07647.
10. R. G. Scott, "The Structure of Synthetic Fibers," ASTM Symposium on Microscopy,
ASTM Special Technical Publication 257 (Philadelphia: American Society for Testing
and Materials, 1959), 121-31.
11. Olympus Corp., Precision Instrument Div., Lake Success, NY 11042.
12. M. Abramowitz, Microscope Basics and Beyond, vol. 1 (Lake Success, NY: Olympus Corp.,
1985).
13. M. Abramowitz, Contrast Methods in Microscopy, vol. 2 (Lake Success, NY: Olympus
Corp., 1987).
14. J. G. Deily, Photography through the Microscope, 9th ed. (Rochester, NY: Eastman Kodak,
1988).
15. Kodak techbits® (Rochester, NY: Eastman-Kodak); published three times a year (spring,
summer, and fall).
16. In Focus (Cambridge, MA: Polaroid Corporation).

Chapter 4

1. T. G. Rochow and R. L. Gilbert, "Resinography," in Protective and Decorative Coatings,

vol. 5, J. J. Mattiello, ed.(New York: Wiley, 1946).
2. "Metallography-A Practical Tool for Correlating the Structure and Property of Materials,"
 418 References

ASTM Special Technical Publication 557 (Philadelphia: American Society for Testing
and Materials, 1975).
2a. ASTM Symposium, "Metallography: 75 Years Later," May s-10, 1991, Atlantic City, NJ,
published as ASTM STP 1165 (Philadelphia: ASTM, 1993).
3. J. H. Richardson, Optical Microscopy for the Mnterials Sciences (New York: Marcel Dekker,
1971).
4. T. G. Rochow, "Resinography," in Encyclopedia of Polymer Science and Engineering, vol. 14,
2d ed. (New York: Wiley, 1988), 421-37.
5. J. B. Nelson," Apparatus for the Microscopical Determination of Ore Minerals," Amer-
ican lAboratory (Apr. 1975), 81-82, 84, 86, 86-91.
6. W. C. McCrone, "Surface Characterization by light Microscopy," preprints of papers
presented at the 171st meeting, ACS, Div. Organic Coatings and Plastics Chemistry 36,
no. 1 (Apr. 1976), 2.
7. T. G. Rochow and E. G. Rochow, Resinography (New York: Plenum, 1976).
8. D. W. Skalla and S. R. Mather, "A Nondestructive Method of Observing and Recording
Wear Characteristics of Phonograph Records," The Microscope 23 (1975), 55-60.
9. E. M. Slayter, Optical Methods in Biology (New York: Wiley-Interscience, 1970).
10. V. P. Miniutti, "Reflected-light and Scanning Electron Microscopy of Ultraviolet Ir-
radiated Redwood Surfaces," The Microscope 18 Oan. 1970), 61-72.
11. H. W. Zieler, The Optical Performance of the Light Microscope, part 2 (Chicago: Microscope
Publications, 1973).
12. F. G. Rowe and H. F. Nicolaysen, "Reflected light Microscopy of Waxes on Paper," in
International Microscopy Symposium, W. C. McCrone, ed. (Chicago: McCrone Associates,
1960).
13. C. W. Mason, Handbook of Chemical Microscopy, Vol. 1: Physical Methods (New York:
Wiley, 1st ed., 1930; 4th ed., 1983).
14. R. B. McLaughlin, Accessories for the Light Microscope (Chicago: Microscope Publications,
1975).
14a. M. Abramowitz, "Dark-Field lllumination," American lAboratory (Nov. 1991), 60.
15. M. Abramowitz, "Reflected light Microscopy: An Overview" (Lake Success, NY: Olym-
pus Corp., 1990).
16. J. G. Deily, Photography through the Microscope, 9th ed., 2d printing (Rochester, NY:
Eastman Kodak, 1988).
17. Simply Revolutionary (Lake Bluff, IL: Buehler, 1988). Catalog of equipment for preparing
metals, etc., for microscopical examination by reflected light.
18. E. N. Cameron, Ore Microscopy (New York: Wiley, 1961).
19. ASTM designation El12, "Estimating Average Grain Size of Metals," Annual Index to
ASTM Standards (Philadelphia: American Society for Testing and Materials, 1970, re-
viewed 1989).
20. ASTM designation E384, "Test for Microhardness of Materials," Annual Index to ASTM
Standards (Philadelphia: American Society for Testing and Materials, reviewed 1989).
21. ASTM National Committee E-4 on "Metallography, Scope and Organization," annual
ASTM Yearbook; standards, Annual Index to ASTM Standards (Philadelphia: American
Society for Testing and Materials).
21a. G. F. Vander Voort, Metallography, Principles and Practice (New York: McGraw-Hill,
1984).
22. L. D. Nichols et al., "Some Structural and Physical Properties of Yam Made on the
Integrated Composite Spinning System," part 1, Textile Research ]ourna/42 Gune 1972),
336-44.
References 419

23. T. G. Rochow, Light-Microscopical Resinography (Chicago: Microscope Publications,

1983).
24. ASTM designation E407, "Microetchants for Metals and Alloys," Annual Index to ASTM
Standards (Philadelphia: American Society for Testing and Materials, 1970, reviewed
1989).

Chapter 5

1. Glossary of Microscopical Terms and Definitions, 2d ed. (New York: NY Microscopical

Society, 1989).
2. Compilation of ASTM Definitions (Philadelphia: 6th ed., American Society for Testing and
Materials, 1986).
3. T. G. Rochow, Light Microscopical Resinography, vo!. 47 (Chicago: Microscope Publica-
tions [Division of McCrone Research Institute] 1983).
4. C. W. Mason, Handbook of Chemical Microscopy, 4th ed., vol. 1 (New York: Wiley, 1983).
5. N.H. Hartshorne and A. Stuart, Crystals and the Polarising Microscope, 4th ed. (New York:
American Elsevier, 1970).
6. F. D. Bloss, An Introduction to the Methods of Optical Crystallography (New York: Holt,
Rinehart, and Winston, 1961).
7. J. G. Deily, "Microscopy's Color Key," Industrial Research (Oct. 1973), 44-50.
8. E. M. Slayter, Optical Methods in Biology (New York: Wiley, 1970).
9. N. H. Hartshorne, The Microscopy of Liquid Crystals (Chicago: Microscope Publications,
1974).
10. T. G. Rochow and E. G. Rochow, Resinography (New York: Plenum, 1976).
11. F. Ruch, "Physical Techniques in Biological Research," in Cells and Tissues, G. Oster and
A. W. Pallister, eds. (New York: Academic Press, 1955), Vol. 3, 149-74.
12. H. S. Bennett, "The Microscopical Investigation of Biological Materials with Polarized
Light," in Handbook of Microscopical Techniques, 3d ed., R. M. Jones, ed. (New York:
Hafner Press, 1950), 591-677.
13. Carl Zeiss, Inc., 1 Zeiss Dr., Thornwood, NY 10594.
13a. Leica, Inc., 24 Link Dr., Rockleigh, NJ 07647.
14. "MICROPHOT-FXA," 1989, Nikon Instrument Group, 623 Stewart Ave., Garden City,
NY 11530.
15. M. Abramowitz, "The Polarizing Microscope," American Laboratory Oune 1990), 72.
16. Olympus Corp., New Hyde Park, NY 11042.
16a. "Edwin Land's Retinex Theory: An Important Contribution to Understanding Color
Vision," In Focus 4 (fall/winter 1992), 8, 9, 15.
17. "Method of Test for Determination of Birefringence in Fibers of Circular Cross Section
by a Variable Compensator Technique," Index to Annual Book to ASTM Standards (Phil-
adelphia: American Society for Testing and Materials).
18. R. B. McLaughlin, "Accessories for the Light Microscope" (Chicago: Microscope Publica-
tions, 1975).
19. N. A. Crites et al., "Photoelastic Techniques," Product Engineering 33, part 3 (Sept. 1962),
57-69.
20. R. C. Emmons in A. N. Winchell, Microscopic Characters of Inorganic Solid Substances (New
York: Wiley, 1931); R. C. Emmons, "The Universal Stage" (Boulder, CO: Geological
Society of America, 1943).
420 References

21. ASTM designation E-211, "Specifications and Methods of Test for Cover Glasses and
Glass Slides for Use in Microscopy," Annual Index to ASTM Standards (Philadelphia:
American Society for Testing and Materials, 1982, reviewed 1990).
22. W. C. McCrone, Fusion Methods in Chemical Microscopy (New York: Wiley, 1957).
23. T. G. Rochow and R. J. Bates, "A Microscopical Automated Microdynamometer Mi-
crotension Tester," ASTM Materials Research and Standards 12 (1972), 27-30.
24. T. G. Rochow and R. L. Gilbert, "Resinography," in Protective and Decorative Coatings, J.
J. Mattiello, ed., vol. 5 (New York: Wiley, 1946).
25. J. G. Deily, "Photography through the Microscope" 9th ed., 2d printing (Rochester, NY:
Eastman Kodak, 1988).
26. Kodak techbits11 (Rochester, NY: Eastman Kodak); published three times a year (spring,
summer, and fall).
27. M. Abramowitz, How to Improve Photography through the Microscope (New Hyde Park,
NY: Olympus Corporation of America, 1988).
28. In Focus 3 (1991), Polaroid, Cambridge, MA, 02139.
29. R. P. Loveland, Photomicrography, vols. 1 and 2, 2d printing (Malibar, FL: R. E. Kriger,
1985).

Chapter 6

1. ASTM designation 0276, "Identification of Textile Fibers," in Annual Book of ASTM

Standards (Philadelphia: American Society for Testing and Materials, 1991).
2. "AATCC Test Method, Fibers in Textiles: Identification," AATCC Technical Manual
(Research Triangle Park, NC: American Association of Textile Chemists and Colorists,
1993 and annually).
3. P. A. Tucker, "Fibers," in Encyclopedia of Polymer Science and Engineering, 2d ed. (New
York: Wiley, 1987).
4. A. C. Reimschuessel, "Scanning Electron Microscopy," /. Chemical Education 49 (1974),
A413-A449.
5. Scanning electron micrographs by the Shirley Institute via the British Technology
Group, Manche!iter, England.
6. Guide to the Care of Trademarks, U.S. Trademark Association, 6 East 45th St., New York,
NY 10017.
6a. C. W. Mason, Handbook of Chemical Microscopy, Vol. 1: Physical Methods, 4th ed. (New
York: Wiley, 1983).
7. M. A. Sieminski, "The Temperature for Zero Birefringence of Arnel® and Other Fibers,"
Textile Research /ournal34 (1964), 918-24.
8. M. A. Sieminski, "A Note on the Measurement of Birefringence in Fibers," The Micro-
scope 23 (1975), 35-36.
9. R. W. Singleton et al., "The Effect of Radial Heterogeneity on Fiber Properties," Textile
Research Journal 31 (1961), 917-25.
10. E. Leitz, Instructions for Tilting Compensator K (Rockleigh, NJ: E. Leitz, 1971).
11. Index to Annual ASTM Standards (Philadelphia: American Society for Testing and Ma-
terials, 1993).
12. N. H. Hartshorne and A. Stuart, Crystals and the Polarising Microscope, 4th ed. (London:
Edward Arnold, 1970).
13. T. G. Rochow and R. L. Gilbert, "Resinography," in vol. 5 of Protective and Decorative
Coatings, J. J. Mattiello, ed. (New York: Wiley, 1946).
References 421

14. C. P. Saylor, "Heterodoxy in Refractive Index Measurement," NYMS Dialogues, May

17-19, 1977, New York Microscopical Society, American Museum of Natural History,
New York, NY 10024.
15. Cargille Scientific, Inc., 55 Commerce Rd., Cedar Grove, NJ 07009, for example.
16. N. H. Hartshorne, The Microscopy of Liquid Crystals (Chicago: Microscope Publications,
1974).
17. R. G. Scott, "A Few Observations Concerning the Structure of Synthetic Fibers," ASTM
Symposium on Microscopy, F. F. Morehead and R. Loveland, eds., ASTM Special Tech-
nical Publication 257 (Philadelphia: American Society for Testing and Materials, 1959).
18. Federal Trade Commission, "Rules and Regulations under the Textile Fiber Products
Identification Act," March 3, 1969, Federal Trade Commission, Washington, DC 20580.
19. A. 0. Mogensen, "Microscopical Apparatus and Techniques for Observing the Fiber-
Forming Process," in Resinographic Methods, ASTM Special Technical Publication 348
(Philadelphia: American Society for Testing and Materials, 1964), 31-35.
20. T. G. Rochow, Light Microscopical Resinography (Chicago: McCrone Research Institute,
1983), 48.
21. T. G. Rochow et al., "Transverse Anisotropy in False Twist Textured Nylon 66 and Its
Characterization with the Universal Stage," The Microscope 28 (1980), 129-140.
22. F. D. Bloss, The Spindle Stage (Cambridge, UK: Cambridge University Press, 1981).
23. T. G. Rochow and E. G. Rochow, Resinography (New York: Plenum, 1976).

Chapter 7

1. Compilation of ASTM Standard Definitions, 6th ed. (Philadelphia: American Society for
Testing and Materials, 1986 and following).
2. A. N. Winchell, The Microscopic Characters of Artificial Inorganic Solid Substances or
Artificial Minerals (New York: Wiley, 1938).
2a. L. H. VanVlack, Materials Science for Engineers (Reading, PA: Addison-Wesley, 1970).
3. C. W. Mason, Handbook of Chemical Microscopy. Vol. 1: Physical Methods, 4th ed. (New
York: Wiley, 1983).
4. E. M. Chamot and C. W. Mason, Handbook of Chemical Microscopy, Vol. 2: Chemical
Methods, 2d ed. (New York: Wiley, 1940).
5. H. Behrens and P. D. C. Kley, Organische Microchemische Analyse, Voss, Leipzig (1922);
translated by R. E. Stevens, Microscopical Identification of Organic Compounds, (Chicago:
Microscope Publications, 1969).
6. A. N. Winchell and H. Winchell, The Microscopical Characters of Artificial Inorganic Solid
Substances: Optical Properties of Artificial Minerals, 3rd ed. (New York: Academic, 1964).
7. C. P. Saylor, J. Physical Chemistry 32 (1928), 1441-460.
8. R. E. Stevens, "Microscopical Identification of Water in Crystals," Analytical Chimica
Acta 60 (1972), 325-34.
9. C. W. Mason, Report of Chairman of American Chemical Society Committee on Re-
commended Practice for Microscopical Reports on Crystalline Materials in A. C. S.
Publications, Industrial and Engineering Chemistry 17 (1945), 603-04 and Analytical Chem-
isty 20 (1948), 274.
10. E. M. Chamot and C. W. Mason, "Chemical Microscopy, Part 1: Crystallization Experi-
ments as an Introduction to Metallography," Journal of Chemical Education 5 (1928),
10-24.
11. W. C. McCrone, Fusion Methods in Chemical Microscopy (New York: Wiley, 1957).
422 References

12. P. L. Kirk, Crime Investigation (New York: Wiley, 1953).

13. A. N. Winchell, Optical Properties of Organic Compounds (Madison, WI: University of
Wisconsin Press, 1943).
14. F. D. Bloss, An Introduction to the Methods of Optical Crystallography (New York: Holt,
Rinehart and Winston, 1961).
15. N.H. Hartshorne and A. Stuart, Crystals and the Polarising Microscope, 4th ed. (New York:
American Elsevier, 1970).
16. E. E. Wahlstrom, Optical Crystallography, 4th ed. (New York: Wiley, 1969).
17. R. C. Emmons, "The Universal Stage," Geological Society of America Mem. 8 (1943), 205;
see also Reference 2a, Chapter 8.
18. A. F. Kirkpatrick, unpublished recapitulation of data collected by A. N. Winchell.<6•13l
19. W. C. McCrone et al., The Particle Analyst (Ann Arbor, MI: Ann Arbor SCience Pub-
lishers), a journal for one year (1968); reprinted as a monograph (1969).
20. W. C. McCrone, "A New Dispersion Staining Objective," The Microscope 23 (1975),
221-226.
21. T. G. Rochow, in the Encyclopedia of Chemical Technology, vol.13 (New York: Wiley, 1967),
499.
22. Cyanamid Melamine, Booklet 1C 9055, American Cyanamid Co., Wayne, NJ 97470 (1959).
23. M. L. Willard, Chemical Microscopy (State College, PA: W. B. Keeler Bookstore, 1952).
24. N.H. Hartshorne, The Microscopy of Liquid Crystals (Chicago: Microscope Publications,
1974).
25. F. G. Rosevear, "The Microscopy of the Liquid Crystalline Neat and Middle Phases of
Soaps and Synthetic Detergents,"]. American Oil Chemists Society 31 (1954), 628-39.
26. J. Rogers and P. A. Winsor, "Changes in the Optical Sign of the Lamellar Phase (G) in
the Aerosol OT/Water System with Composition and Temperature,"]. Colloid and
Interface Science 30 (1969), 247-57.
27. T. G. Rochow and C. W. Mason, "Breaking Emulsions by Freezing," Industrial and
Engineering Chemistry 28 (1936), 1296--300.
28. A. E. Woodward, Atlas of Polymer Morphology (New York: Hanser Publishers, 1988).
29. L. C. Sawyer and D. T. Grubb, Polymer Microscopy (Chapman and Hall, 1987).
30. P. L. Young, "The Characterization of High-Performance Fibers Using Infrared Micro-
scopy," Spectroscopy 3 (1988), 9.
(NOTE: Publications listed in references 4, 5, 6, 11, 13, 20, and 24 are available from McCrone
Research Institute, 2820 S. Michigan Avenue, Chicago, IL 60616-3292.)

Chapter 8

1. J. B. Pawley, ed., Handbook of Biological Confocal Microscopy (New York: Plenum, 1990).
2. A. Boyde, "Confocal Optical Microscopy," in P. J. Duke and A. G. Michette, Modern
Microscopies, Techniques and Applications (New York: Plenum, 1990), 185-203.
2a. Leica, Inc., 24 Link Drive, Rockleigh, NJ 07647.
3. T. Wilson, ed., Confocal Microscopy (New York: Academic Press, 1990).
4. R. Keeler, "Confocal Microscopy," R & D Magazine (Apr. 1991, 40-42.
5. D. Shotton, "The Current Renaissance in Light Microscopy II. Blur-Free Optical Section-
ing of Biological Specimens by Confocal Scanning Fluorescence Microscopy," Royal
Microscopical Society, Proceedings 23/5 (1988).
6. M. Richardson, "Confocal Microscopy and 3-D Visualization," American Laboratory
(Nov. 1990), 19-24.
References 423

7. D. H. Szarowski eta/., "Optimized Reflection Imaging in Laser Confocal Microscopy

and Its Application to Neurobiology: Modifications to the Biorad MRC-500," Scanning
14 (1992), 104-11.
8. A. M. Hallet a/., "Confocal Microscopy-The Basics Explained," Royal Microscopical
Society, Proceedings 26/2 (1991), 6~8.
9. R. H. Webb, "Confocal Microscopes," Optics and Photonics News Ouly, 1991), 8-13.
10. J. B. Pawley, "Fundamental and Practical Limits in Confocal Microscopy," Scanning 13
(1991), 184-98.
11. S. Inoue, "Foundations in Confocal Scanned Imaging in Light Microscopy," in J. B.
Pawley, ed. Handbook of Biological Confocal Microscopy (New York: Plenum, 1990).
12. Tracor Northern, Inc., 2551 W. Beltline Hwy, Middleton, WI 53562-2697.
13. Cover of Science, June 4, 1993, instrumentation issue, by R. W. Wagner eta/., using
fluorescence confocal microscopy, Gilead Sciences, Inc., 353 Lakeside Dr., Foster City,
CA 94404. "Antisense Gene Inhibition by Oligonucleotides Containing C-5 Propyne
Pyrimidines," Science 260 (1993), 1510-13.

Chapter 9

1. Glossary of Microscopical Terms and Definitions (New York: NY Microscopical Society,

1989).
2. Compilation of ASTM Standard Definitions, 7th ed. (Philadelphia: American Society for
Testing and Materials, 1986).
3. M. R. Howells eta/., "X-Ray Microscopes," Scientific American (Feb. 1991), 88-94.
4. Nikon annual Small World photomicrographical competition, Instrument Division,
Dept. SW, 623 Stewart Ave., Garden City, NY 11530.
5. ]. G. Deily, Photography through the Microscope, 9th ed, 2d printing (Rochester, NY:
Eastman Kodak, 1988).
Sa. M. Peres and C. Murphy, "Precise Black-and-White Tone Reproduction in Photomicrog-
raphy," Kodak techbits, no. 1 (1991), 2-9.
6. M. Abramowitz, How to Improve Photography through the Microscope (New Hyde Park,
NY: Olympus Corporation of America, 1988).
7. MICROPHOT-FXA, NIKON, INC. Instrument Group, 623 Stewart Ave., Garden City,
NY 11530 (1989).
8. Lucy and Walter McCrone, annual holiday greeting cards, McCrone Research Institute,
2820 S. Michigan Ave., Chicago, IL 60616-3292.
Sa. D. M. Schwartz, "Revealing the Hidden Beauty of Commonplace Crystals, Smithsonian
(May 1993), 112-114.
9. J. Gerakaris, "New Photographic Possibilities through the Use of Scanning Photo-
macrography," Kodak Techbits (summer 1988), 7-11.
9a. N. L. Kedersha, "Photomicrography of Immunofluorescently Labeled Cells," American
Laboratory (Apr. 1991), 28.
10. T. G. Rochow and E. G. Rochow, Resinography (New York: Plenum, 1976).
11. T. G. Rochow and C. W. Mason, "Breaking Emulsions by Freezing," Industrial and
Engineering Chemistry 28 (1936), 1296-300.
12. S. A. Reigel and R. P. Bundy, "High-Speed Cinematography," Research/Development
(Dec. 1975), 24-28.
13. J. Schwartz, "Some Uses of Time-Lapse Cinemicrography in Contemporary Research,"
American Laboratory (Apr. 1970), 37, 39, 40.
424 References

14. T. G. Rochow and R. L. Gilbert, "Resinography," in Protective and Decorative Coatings;

vol. 5, J. J. Mattiello, ed. (New York: Wiley, 1946).
14a. Of the former B&L Tessar type. Try the Cambridge Instruments Co., P. 0. Box 123,
Buffalo, NY 14240.
15. In Focus 2 (1991), 2.
16. H. W. Zieler, The Optical Performance of the Light Microscope, part 2, (Chicago: Microscope
Publications, 1973).
17. ASTM designation E-210, "Microscope Objective Thread," Annual Index to ASTM Stan-
dards (Philadelphia: American Society for Testing and Materials, 1990).
18. R. P. Loveland, Photomicrography, vols. 1 and 2, 2d printing (Malibar, FL: R. E. Kriger,
1985).
19. Biology Catalog (Rochester, NY: Ward's Natural Science Establishment, 1991).
20. Catalog (Barrington, NJ: Edmund Scientific Co., 1992).
21. Leica, Inc., 24 Link Drive, Rockleigh, NJ 07647.
22. Nikon, Inc., 1300 Walt Whitman Road, Melville, NY 11747-0299.
23. Olympus Corp., Precision Instrument Div., 4 Nevada Drive, Lake Success, NY 11042-
1179.
24. Carl Zeiss, Inc., 1 Zeiss Drive, Thornwood, NY 10594.
25. Kodak techbits (Rochester, NY: Eastman Kodak); published triannually (spring, summer,
and fall).

Chapter 10

1. Compilation of ASTM Standard Definitions, 7th ed. (Philadelphia: American Society for
Testing and Materials, 1990).
2. Glossary of Microscopical Terms and Definitions, 2d ed. (New York: N.Y. Microscopical
Society, 1989).
3. T. G. Rochow et al., "Light and Electron Microscopical Studies of Pleurosigma Angulatum
for Resolution of Detail and Quality of Image," The Microscope and Crystal Front 15
(1966), 177-201.
4. M. J. Abramowitz, "Darkfield Illumination," American Laboratory (Nov. 1991), 60, 61.
5. C. W. Mason, Handbook of Chemical Microscopy, volt, 4th ed. (New York: Wiley, 1983).
6. R. P. Loveland, Photomicrography, vols. 1 and 2, 2d printing (Malibur, FL: R. E. Krieger,
1985).
6a. J. G. Deily, Photography through the Microscope (Rochester, NY: Eastman Kodak, 1988).
7. A. H. Bennett et al., Phase Microscopy (New York: Wiley, 1951).
7a. N.H. Hartshorne and A. Stuart, Crystals and the Polarising Microscope, 4th ed. (London:
Edward Arnold, 1970).
8. C. P. Saylor, in Advances in Optical and Electron Microscopy, R. Barer and V. E. Cosslett,
eds. (New York: Academic Press, 1966).
9. S. G. Ellis and W. Hunn, "High-Resolution-Transmitted Light Phase Microscopy of
Unmounted Specimens," The Microscope 23 3 (1975), 127-131.
10. 0. W. Richards, "The Polanret •• Variable Densiphase Microscope," Journal of Microscopy
98, 1 (May 1973), 67-77.
11. Information on the Polanret'" microscope, American Optical Corp., Scientific Instru-
ment Div., Buffalo, New York 14215.
12. R. Hoffman and L. Gross, "Modulation Contrast Microscope," Applied Optics 14 (1975),
1169-1176.
References 425

13. R. Hoffman, "The Modulation-Microscope-Principles and Performance," journal of

Microscopy 110, 3 (Aug. 1977), 209-219.
14. W. C. McCrone, "A New Dispersion Staining Objective," The Microscope 23 (1975),
221-226.
15. G. C. Crossman, Stain Technology 24 (1949), 61.
16. G. C. Crossman, American Industrial Hygiene Quarterly 18 (1957), 341.
17. Special set of liquids for dispersion staining, R. P. Cargille, Inc., Cedar Grove, NJ 07009.
18. W. C. McCrone, "Why Use the Polarized Microscope," American Laboratory (Apr. 1992),
17-21.
19. W. C. McCrone and J. G. Deily, Particle Atlas, 6 vols. (Ann Arbor, MI: Ann Arbor Science
Publishers, 1973-1978).
20. W. C. McCrone, reply to letter to the editor by D. L. Faulkner and T. G. Rochow, The
Microscope 20 (1972), 228-230.
21. W. C. McCrone and R. I. Johnson, Techniques, Instruments and Accessories for Microan-
alysts, A User's Manual (Chicago: Walter C. McCrone Associates, 1974).
22. ASTM designation E-210, "Microscope Objective Thread" (Philadelphia: American So-
ciety for Testing and Materials, 1990).
23. W. C. McCrone, "Determination of n0 , np and nc by Dispersion Staining," The Micro-
scope 23 (1975), 213-20.
24. J. Dodd and W. C. McCrone, "A Schlieren Eyepiece," The Microscope 23 (1975), 89-
92.
25. 0. Kafri and I. Glatt, The Physics of Moire Metrology (New York: Wiley-Interscience, 1990).

Chapter 11

1. C. W. Mason, Handbook of Chemical Microscopy, vol. 1, 4th ed. (New York: Wiley, 1983).
2. W. Lang, "Nomarski Differential Interference Contrast," American Laboratory (Apr. 1970),
45-46, 48, 50, 52.
3. S. Tolansky, Multiple-Beam Interference Microscopy of Metals (New York: Academic Press,
1970).
4. P. Sullivan and B. Wunderlich, Interference Microscopy of High Polymers, Office of Naval
Research, Technical Report No.4, Contract Nonr-401 (44), Task No. NR 051-428 (Ithaca,
NY: Cornell University, Department of Chemistry, 1963).
5. R. B. McLaughlin, Accessories for the Light Microscope (Chicago: Microscope Publications,
Ltd., 1975).
6. ASTM designation E-210, Microscope objective thread, Annual Index to ASTM Standards
(Philadelphia: American Society for Testing and Materials).
7. R. G. Scott, "A Few Applications of the Interference Microscope to the Study of Fibrous
Materials" (in German), Leitz Mitteilungen fiir Wisseschaft und Technik 5, (5) (Mar. 1971),
132-140.
8. N. H. Hartshorne and A. Stuart, Crystals and the Polarising Microscope, 4th ed (London:
Edward Arnold, Ltd., 1970).
9. ]ena Review, 1987/1, Jenoptik, Carl Zeiss Str., Jena, Germany.
10. W. Krug, J. Rienitz, and G. Schulz, trans. J. H. Dickson, Contributions to Interference
Microscopy (London: Hilger & Watts, 1964).
11. S. Tolansky, An Introduction to Interferometry (London: Longmans, Green, and Co., 1966).
12. M. Francon and S. Mallick, Polarization Interferometers, Applications to Microscopy and
Macroscopy (New York: Wiley, 1971).
426 References

13. E. J. Roche, B. Rubin, and R. F. van Kavelar, "Automatic Analysis of Structural gradients
in Fibers, 3A-2~3A-15," Proceedings of Fiber Producer Conference, Oct. 14-16, 1986,
Clemson University, Clemson, SC.
14. C.-Y. Un, "Poly(ethylene terephthalate) Melt Spinning via Controlled Threadline Dy-
namics," Ph.D. diss. NCSU, Raleigh, 1990.
15. H. G. Elias, Macromolecules, vol. 1 (New York: Plenum, 1977).
16. A. Ziabicki and L. Jarecki, in High-Speed Fiber Spinning, A. Ziabicki and H. Kawai, eds.
(New York: Interscience, 1985).

Chapter 12

1. Glossary of Microscopical Terms and Definitions, 2d ed. (New York: Mirroscopical Society,
1989).
2. T. G. Rochow and R. L. Gilbert, "Resinography," in Protective and Decorative Coatings, J.
J. Mattiello, ed., vol. 5 (New York: Wiley, 1946).
3. R. B. McLaughlin, Accessories for the Light Microscope (Chicago: Microscope Publications,
Ltd., 1975).
4. T. G. Rochow, "Some Microscopical Aspects of Resinography," ]. Royal Microscopical
Society 87 (1967), 39-45.
5. T. G. Rochow and R. J. Bates, "A Microscopical Automated Microdynamometer Mi-
crotension Tester," ASTM Materials Research and Standards 12, 4 (Apr. 1972), 27-30).
6. J. I. Goldstein and H. Yakowitz, eds., Practical Scanning Electron Microscopy (New York:
Plenum, 1975).
7. C. W. Mason, Handbook of Chemical Microscopy, vol. 1, 4th ed. (New York: Wiley, 1983).
8. ASTM designation E-210, Microscope objective thread, Annual Index to ASTM Standards
(Philadelphia: American Society for Testing and Materials, 1962, confirmed 1990).
9. ASTM designation E-384, Standard method of test for microhardness of materials, An-
nual Index to ASTM Standards (Philadelphia: American Society for Testing and Materials,
1989).
10. W. C. McCrone, Fusion Methods (New York: Wiley, 1957; reprinted by McCrone Research
Institute, Inc., 2820 S. Michigan Ave., Chicago, IL 60616-3292).
11. N.H. Hartshorne, The Microscopy of Liquid Crystals (Chicago: Microscope Publications,
1974).
12. D. G. Grabar and R. Haessly, "Identification of Synthetic Fibers by Micro Fusion Meth-
ods," Analytical Chemistry 28 (1956), 1586-589.
13. E. M. Chamot and C. W. Mason, "Chemical Microscopy. I. Crystallization Experiments
as an Introduction to Metallography," ]. Chemical Education 5 (1928), 9-24.
14. F. D. Bloss, An Introduction to the Methods of Optical Crystallography (New York: Holt,
Rinehart, and Winston, 1961).
15. L. Kofler and A. Kofler, Thermomikromethoden (Innsbruck, Austria: Wagner, 1954).
16. Kofler micro hot stage, Directions for Use (Philadelphia: Technological Service, Arthur H.
Thomas Co., 1958 to date).
17. C. D. Felton, "Dark-Field Microscopy," Analytical Chemistry 34 (1962), 880.
18. Leica, Inc., 24 link Drive, Rockleigh, NJ 07647.
19. W. C. McCrone, Applications of Thermal Microscopy (Princeton, NJ: Mettler Instrument
Corp.). 22 Pp.
20. Y. Julian and W. C. McCrone, "Accurate Use of Hot Stages," The Microscope 19 (1971),
22.>-41).
References 427

21. E. M. Barrall and M. A. Sweeney, "Depolarized Light Intensity and Optical Microscopy
of Some Mesophase-Forming Materials," Molecular Crystals 5 (1969), 257-71.
22. F. T. Jones, "Fusion Techniques in Chemical Microscopy," The Microscope 16 (1968),
37-43.
23. N. H. Hartshorne, "A Hot-Wire Stage and Its Application," The Microscope 23 (1975),
177-90.
24. Stanton Redcroft, Cooper Mill Lane, London SW170BN, England.
25. E. L. Charsley and D. E. Tolhurst, "The Application of Hot-Stage Microscopy to the Study
of Pyrotechnic Systems," The Microscope 23 (1975), 227-237.
26. T. G. Rochow and C. W. Mason, "Breaking Emulsions by Freezing," Industrial and En-
gineering Chemistry 28 (1936), 1296-1300.
27. P. Echlin, Low-Temperature Microscopy and Analysis (New York: Plenum, 1992).
28. Sensortek, Inc. (formerly Bailey Instruments), 154 Huron Ave., Clifton, NJ 07013.
29. E. M. Chamot and C. W. Mason, Handbook of Chemical Microscopy, vol. 2: Chemical Methods,
2d ed. (New York: Wiley, 1940).
30. J. A. Davidson, "Pressure Cells in Optical Microscopy," The Microscope 23 (1975), 61-71.
31. H. Reumuth and T. Loske, "Kuvette-Mikroskopie im Biologie und Tecknik," Mikroskopie
17 (1962), 149-178.

Chapter 13

1. R. C. Gore, "Infrared Spectrometry of Small Samples with the Reflecting Microscope,"

Science 110 (1949), 710-11.
1a. J. A. Reffner, "The Commercial Development of FT-IR Microspectrometry," Microscopy
Today (Apr. 1993), 3.
2. G. Nichols, "FT-IR Microscopy: Microanalysis Using Infrared Spectroscopy," Proceed-
ings, Royal Microscopical Society 25 (May 1990), 205-11.
3. W. C. McCrone, "Microscopy in the 1990s," American Laboratory (Apr. 1991), 17-26.
4. R. G. Messerschmidt, "Minimizing Optical Nonlinearities in Infrared Microscopy," in
Infrared Microspectroscopy, ed. R. G. Messerschmidt and M.A. Harthcock (New York:
Marcel Dekker, 1988), 11-19.
5. J. A. Reffner et al., "Chemical Microscopy of Surfaces by Grazing Angle and Internal
Reflection FT-IR Microscopy," American Laboratory 46 (Apr. 1991), 48-50.
6. D. L. Grieble and S. A. Gardner, "Applications of Infrared Microscopy to Textile Sam-
ples," in Book of Papers (Greenville, SC: AATCC Warp Sizing Symposium, 1989), 40-42.
7. S. L. Hill and K. Krishnan, "Some Applications of the Polarized FT-IR Microsampling
Technique," in Infrared Microspectroscopy, ed R. G. Messerschmidt and M. A. Harthcock
(New York: Marcel Dekker, 1988), 115-27.
8. P. H. Young, "The Characterization of High-Performance Fibers Using Infrared Micro-
scopy," Spectroscopy 3, 9 (1988), 24, 26-30.
9. W. D. Perkins, "Fourier Transform-Infrared Spectroscopy-Part I. Instrumentation," ].
Chemical Education 63, 1 (1986), A5-Al0.
10. W. D. Perkins, "Fourier Transform-Infrared Spectroscopy-Part II. Advantages of FT-
IR," ]. Chemical Education 64, 11 (1987), A269-A271.
11. Spectra-Tech, 652 Glenbrook Rd., Stamford, CT 06906.
12. H. Goldner, "Two New Attachments Advance FT-IR Microscopy," R & D Magazine 35
(Apr. 1993), 65.
13. J. Ryan, et al., International Laboratory auly/ Aug. 1989), 26-31.
428 References

14. J. Sellers, "FT-IR Spectroscopy for the Analytical and Research Laboratory," American
Laboratory (Apr. 1993), 22-30.

Chapter 14

1. Compilation of ASTM Definitions, 7th ed. (Philadelphia: American Society for Testing and
Materials, 1990).
1a. Glossary of Microscopical Terms and Definitions, 2d ed. (New York: NY Microscopical
Society, 1989).
2. C. E. Hall, Introduction to Electron Microscopy, 2d ed. (Hightstown, NJ: McGraw-Hill,
1966).
3. C. W. Mason, Handbook of Chemical Microscopy, vol. 1, 4th ed. (New York: Wiley, 1983).
4. G. Rempfer, et al., "An Electrostatic Transmission Microscope," American Laboratory
(Apr. 1972), 39-44, 46.
5. B. M. Siegel, Modern Developments in Electron Microscopy (New York: Academic Press,
1964).
6. E. L. Thomas, "Electron Microscopy," in Encyclopedia of Polymer Science and Engineering,
2d ed., vol. 5 (New York: Wiley, 1988).
7. "Excellence in Transmission Electron Microscopy" (Thornwood, NY: Carl Zeiss, 1990).
8. E. Ruska, "Past and Present Attempts to Attain the Resolution Limit of the Transmission
Microscope," in Advances in Optical and Electron Microscopy, vol. 1, R. Barer and V. E.
Cosslett, eds. (New York: Academic Press, 1966), 116-79.
9. P.R. Swann, C. J. Humphreys, and M. J. Goringe, eds., "High-Voltage Electron Micro-
scopy," in Proceedings of the Third International Conference (New York: Academic Press,
1973).
10. P. Tucker and R. Murray, "A Study of Amine-Etched Poly(ethylene terephthalate)
Filaments by HVEM and SEM," in Thirty-third Annual Proceedings of the Electron Micro-
scopy Society of America, G. W. Bailey, ed. (Baton Rouge, LA: Claiter, 1975), 82-83.
11. A. Septier, "The Struggle to Overcome Spherical Aberration in Electron Optics," in
Advances in Optical and Electron Microscopy, vol. 1, R. Barer and V. E. Cosslett, eds. (New
York: Academic Press, 1966), 204-274.
12. V. F. Holland, private communication toT. G. Rochow, Monsanto Triangle Park De-
velopment Center, Inc., Research Triangle Park, NC, 1973.
13. A. L. Robinson, "Electron Microscopy: Imaging Molecules in Three Dimensions," Sci-
ence 192 (1976), 360-362, 400.
14. E. M. Slayter, Optical Methods in Biology (New York: Wiley, 1970).
15. J, M. Cowley and S. lijima, "Electron Microscopy of Atoms in Crystals," Physics Today
(Mar. 1977), 32-40.
16. Annual Book of ASTM Standards and Index (Philadelphia: American Society for Testing
and Materials, 1992).
17. M.A. Hayat, ed., Principles and Techniques of Electron Microscopy Techniques, 5 vols. (New
York: Van Nostrand Reinhold, 1970-1975).
18. J. I. Gedney, M. C. Botty, and E. J. Thomas, "The Preparation of Rigid Foams for
Microscopical Examination," Special Technical Publication No. 414 on Resinography of
Cellular Plastics (Philadelphia: American Society for Testing and Materials, 1963).
19. T. G. Rochow, "Microscopic Domains in Some Synthetic Polymers," J. Applied Polymer
Science 9 (1965), 569-81.
20. Freezing Stages: Physitemp Instruments, 154 Huron Ave., Clifton, NJ 07013.
References 429

21. W. R. Goynes and J. A. Harris, "Structural Characterization of Grafted Cotton Fibers by

Microscopy," Journal of Polymer Science, part C, no. 37 (1972), 277-89.
22. R. F. Bils, Electron Microscopy, Laboratory Manual and Handbook (Los Angeles: Western
Publishing Co., 1974).
23. N. Reid and J. E. Beesley, Sectioning and Cryosectioning for Electron Microscopy, ed. A. M.
Glavert (New York: Elsevier, 1993).
24. W. C. McCrone, J. G. Deily, and McCrone Associates, The Particle Atlas, 2d ed., vol. 3,
Electron Microscopy Atlas (Ann Arbor, MI: Ann Arbor Science Publishers, 1973).
25. R. P. Loveland, Photomicrography vols. 1 and 2 (New York: Wiley, 1970).
26. "Some Things Every Electron Microscopist Ought to Know," Tech Bits, no. 65-2 (1965),
3-10.
27. "Photographic Techniques in Electron Microscopy," Kodak Pamphlet no. P-109 (1970),
10-70.
28. Techbits (Rochester, NY: Eastman Kodak Co.).
29. T. R. Turnbull, et al., "An Image Intensifier and a Polaroid® Camera for the TEM," in the
Thirty-fourth Annual Proceedings, Electron Microscopy Society of America, G. W. Bailey, ed.
(Woods Hole, MA: Electron Microscopy Society of America, 1976). See also sales lit-
erature, E. F. Fullam, Inc., P. 0. Box 444, Schenectady, NY 12301.
30. In Focus, Polaroid magazine.
31. JEOL, U.S.A., Inc., 11 Dearborn Rd., Peabody, MA 01960.
32. B. E. P. Beeston et al., "Electron Diffraction and Optical Diffraction Techniques," in
Practical Methods in Electron Microscopy, A. M. Glauert, ed. (London: North-Holland
Publishing Co., 1972).
33. L. H. Schwartz and J. B. Cohen, Diffraction from Materials (New York: Academic Press,
1977).
34. D. Campbell and J. R. White, Polymer Characterization (London, New York: Chapman
and Hall, 1989).
35. L. C. Sawyer and D. T. Grubb, Polymer Microscopy (London, New York: Chapman and
Hall, 1987).

Chapter 15

1. Glossary of Microscopical Terms and Definitions, 2d ed. (New York: NY Microscopical

Society, 1989).
2. B. M. England, "Scanning Electron Microscopy," The Mineralogical Record 22 (Mar.-Apr.
1991), 123-132.
3. ]. I. Goldstein and H. Yakowitz, eds., Practical Scanning Electron Microscopy (New York:
Plenum, 1975).
4. J. I. Goldstein et al., Scanning Electron Microscopy and X-Ray Microanalysis, 2d ed. (New
York: Plenum, 1992).
5. C. E. Lyman, "Compositional Imaging in the Electron Microscope: An Overview," Micro-
scopy: The Key Research Taal22, 1 (Mar. 1992), 1-9.
6. W. R. Goynes and J. A. Harris, "Structural Characterization of Grafted Cotton Fibers by
Microscopy," journal of Polymer Science, part C, 37(1972), 277-89.
7. C. E. Hall, Introduction to Electron Microscopy, 2d ed. (Hightstown, NJ: McGraw-Hill,
1966).
8. C. W. Oatley, The Scanning Electron Microscope (London: Cambridge University Press,
1972).
430 References

9. T. Mulvey and R. K Webster, eds., Modern Physical Techniques in Materials Technology

(London: Oxford University Press, 1974).
10. J. W. S. Hearle, et al., The Use of the Scanning Electron Microscope (Elmsford, NY: Pergamon,
1972).
11. T. E. Everhart and T. L. Hayes, "The Scanning Electron Microscope," Scientific American
226 Oan. 1972), 55-69.
12. 0. C. Wells, Scanning Electron Microscopy (New York: McGraw-Hill, 1974).
13. A. C. Reimschuessel, "Scanning Electron Microscopy," ]. Chemical Education 49 (1972),
A413-A449.
14. C. W. Mason, Handbook of Chemical Microscopy, vol. 1, 4th ed. (New York: Wiley, 1983).

Chapter 16

1. A. Howie, "Microscopy without Lenses," Proceedings, Royal Microscopical Society, 26, 3

Ouly 1991), 144-148.
2. E. W. Muller, "Field-Ion Microscopy," Science 149 3684 (Aug. 1965), 591-601).
2a. T. T. Tsong, "Atom-Probe Field-Ion Microscopy," Physics Today (May 1993), 24-31.
3. C. E. Hall, Introduction to Electron Microscopy, 2d ed. (Hightstown, NJ: McGraw-Hill,
1966).
4. B. Ralph, "Field-Ion Microscopy," in Modern Physical Techniques in Materials Technology,
T. Mulvey and R. K Webster, eds. (London: Oxford University Press, 1974).
5. Compilation of ASTM Standard Definitions, 3d ed. (Philadelphia: American Society for
Testing and Materials, 1976).
Sa. Glossary of Microscopical Terms, 2d ed. (New York: NY Microscopical Society, 1989).
6. R. Gomer, Field Emission and Field Ionization (Cambridge, MA: Harvard University Press,
1961).
7. "Field-Ion Microscope," private communication, Materials Research Corp., Orange-
burg, NY, December 15, 1965.
8. S. J. Fonash, "Advances in the Understanding of Image Formation in FIM," Micro-
structures (Aug./Sept. 1972), 17-21.
9. J. J. Hren and S. Ranganathan, eds., Field-Ion Microscopy (New York: Plenum, 1968).
10. E. W. Milller and T. T. Tsong, Field-Ion Microscopy (New York: Elsevier, 1969).
11. R. F. Hockman, et al., Applications of Field-Ion Microscopy (Atlanta: Georgia Technical
Press, 1969).
12. Calvin F. Quate, "Vacuum Tunneling: A New Technique for Microscopy," Physics Today
(Aug. 1986), 26-33.
13. A. Cerezo et al., "The Position Sensitive Atom Probe: Three-Dimensional Reconstruction
of Atomic Chemistry," EMSA Bulletin 20, 2 (1990), 77--83.
14. T. T. Tsong, Atom-Probe Field-lon Microscopy (Cambridge, Great Britain: Cambridge
University Press, 1990).
15. G. D. W. Smith, Atom Probe Microanalysis: Principles and Applications to Materials Problems
(Pittsburgh: Materials Research Society, 1989).
16. ASTM National Committee E-4 on Metallography, organizational information in the
annual Yearbook (Philadelphia: American Society for testing and Materials, 1993).
17. G. Bennig and H. Rohrer, "The Scanning Tunneling Microscope," Scientific American
(Aug. 1985), 50-56.
18. Rudy M. Baum, "Scanning Tunneling Microscope Achieves Atomic Scale Resolution,"
Chemical and Engineering News (Apr. 1986), 22-25.
References 431

19. M. E. Weiland and M. E. Taylor, "Scanning Tunneling Microscopy," in Modern Micro-

scopies, P. ]. Duke and A. G. Mitchette, eds. (New York: Plenum, 1990), 231-54.
20. D. R. Denley, "Practical Applications of Scanning Tunneling Microscopy," Ultramicro-
scopy 33 (1990), 83-92.
21. P. E. Russel and I. H. Musselman, "Scanning Tunneling Microscopy of Polymers: A
Status Report," Proceedings of the Forty-Seventh Annual Meeting of the Electron Microscopy
Society of America, G. W. Bailey, ed. (San Francisco: San Francisco Press, 1989), 330-
31.
22. A. Stemmer and A. Engel, "Imaging Biological Macromolecules by STM: Quantitative
Interpretation of Topographs," Ultramicroscopy 34 (1990), 129-40.
23. Microscopy, Microanalysis, Microstructures 1, 5/6 (1990), 299-518. Complete journal issue
devoted to atomic level microstructure and concentrating on field emission microscopy,
scanning tunnel microscopy, and scanning force microscopy.
24. ]. D. Baldeschwieler et al., "The Scanning Probe Microscope: A Powerful Tool for
Visualizing the Micro World," American lAboratory (Feb. 1991), 34-39.
25. M. Thompson and V. Elings, "Scanning Tunneling and Atomic Force Microscopy:
Applications in Life Sciences," American lAboratory (Apr. 1991), 36-42.
26. Arthur Fisher, "Seeing Atoms," Popular Science (Apr. 1989), 102-107.
27. P. Dietz and P. K. Hansma, "Atomic-Force Microscopy of Synthetic Ultrafiltration
Membranes in Air and under Water," Ultramicroscopy 35 (1991), 155-59.
28. "Scanning Tunneling Microscopy and Related Techniques," selected contributions from
a symposium, ed. M. Isaacson, Ultramicroscopy 33, 2 (Aug. 1990).
29. D. W. Pohl, "Scanning Near-Field Optical Microscopy (SNOM)," in Advances in Optical
and Electron Microscopy, vol. 12, T. Mulvey and C. ]. R. Sheppard, eds. (London: Aca-
demic Press, 1991).
30. E. Betzig et al., "Collection Mode Near-Field Scanning Optical Microscopy," Appl. Phys.
Lett. 51, 25 (Dec. 1987), 2088-90.
31. U. Diirig et al., "Near-Field Optical-Scanning Microscopy," ]. Appl. Phys. 59, 10 (May
1986), 3318-27.
32. E. Betzig et al., "Progress in Near-Field Scanning Optical Microscopy (NSOM)," Pro-
ceedings of the Forty-Sixth Annual Meeting of the Electron Microscopy Society of America, G.
W. Bailey, ed. (San Francisco: San Francisco Press, 1988), 436-37).
33. Arthur Fishen, "Super Scopes," Popular Science (July 1990), 66-69.
34. E. Betzig and J. Trautman, "Near-Field Optics: Microscopy, Spectroscopy, and Surface
Modification beyond the Diffraction Limit," Science 257 (July 1992), 189-95.
35. J. Jahanmir et al., "The Scanning Probe Microscope," Scanning Microscopy 6, 3 (1992),
625-60.

Chapter 17

1. Compilation of ASTM Standard Definitions, 7th ed. (Philadelphia: American Society for
Testing and Materials, 1990).
2. CANALCO leaflet. The Micro Source of X Rays" (Bethesda, MD: Canal Industrial Corp.,
1959).
3. S. B. Newman and D. Fletcher, "Soft X-Ray Microscopy of Paper," Technical Association
of the Pulp and Paper Industry 47 (1964), 177-80.
4. M. C. Botty and E. ]. Thomas, "Applications of Microradiography with the EMU-1
Electron Microscope," paper presented to the Electron Microscope Society of America,
432 References

Columbus, Ohio, September 9, 1959 (unpublished). M. C. Botty and F. G. Rowe, US

Patent 2,843,751 (July 15, 1958).
5. Philips Electronic Instruments, Mount Vernon, NY 10550.
6. M. C. Botty, unpublished work, American Cyanamid Co., Stamford, CT; private com-
munications, May 21 and June 2, 1976 toT. G. Rochow.
7. J. I. Gedney et al., "The Preparation of Rigid Foams for Microscopical Examination,"
paper presented to the American Society for Testing and Materials, Atlantic City, NJ,
June 29, 1966 (unpublished).
8. W. C. Nixon in Modern Methods of Microscopy, A. E. Vickers, ed. (London: Butterworth
Scientific Publications, 1956).
9. E. P. Bertin and R. J. Longobucco, "Practical X-Ray Contact Microradiography," Part 1,
RCA Scientific Instrument News 5, 4 (1960); and part 2, RCA Scientific Instrument News 6,
1 (1961).
10. D. Sayre et al., eds., "Introduction," in X-Ray Microscopy II, vol. 56, T. Tamir, ed. (New
York: Springer-Verlag, 1988).
11. T. W. Ford et al., "Improved Imaging of Biological Specimens by Soft X-Ray Contact
Microscopy Using Laser-Produced Plasmas As X-Ray Sources," in EMAG-MICRO 89, vol.
1: Physical, (P. J. Goodhew and H. Y. Elder, eds. (Bristol, England and New York: Institute
of Physics, 1990), 527-30.
12. Malcolm R. Howells et al., "X-Ray Microscopes," Scientific American 264, 2 (1991), 88-94.
13. Robin Cotton and Julian Fletcher, "Bringing Soft X-Ray Contact Microscopy to the Bi-
ologist," Proceedings, Royal Microscopical Society 27, 2 (Apr. 1992), 77-79.
14. E. Spiller, "The Scanning X-Ray Microscope-Potential Realisations and Applications,"
in SCilnned Image Microscopy, E. A. Ash, ed. (New York: Academic Press, 1980).
15. C. Jacobsen et al., "Scanning Microscopy with Soft X Rays," in Proceedings of the Forty-
Third Annual Meeting of the Electron Microscopy Society of America, G. W. Bailey, ed. (San
Francisco: San Francisco Press, 1985), 594.
16. D. Sayre, "Imaging Properties of the Soft X-Ray Photon," in Proceedings of the Forty-Third
Annual Meeting of the Electron Microscopy Society of America, G. W. Bailey, ed. (San Fran-
cisco: San Francisco Press, 1985), 592.
17. R. Feder and V. Mayne-Banton, "X-Ray Contact Microscopy," in Examining the Submicron
World, NATO ASI Series B: Physics, vol. 137, R. Feder et al., eds. (New York: Plenum,
1986), 277-98.
18. C. Jacobsen et al., "Progress in High-Resolution X-Ray Holographic Microscopy," in
Proceedings of the Forty-Third Annual Meeting of the Electron Microscopy Society of AmeriCil,
G. W. Bailey, ed. (San Francisco: San Francisco Press, 1985), 253-261.

Chapter 18

1. C. F. Quate, "Acoustic Microscopy," Trends in Biochemical Sciences (June 1977), N127-

N129. See section on emerging techniques.
2. A. D. Briggs, "An Introduction to Scanning Acoustic Microscopy," (Oxford, England:
Oxford University Press, 1985).
3. C. F. Quate, "The Acoustic Microscope," Scientific AmeriCiln 241, 4 (Oct. 1979), 58.
4. C. F. Quate, "Acoustic Microscopy Recollections," IEEE Transactions on Sanies and Ultra-
sonics, SV-32, 2 (Mar. 1985), 132-35.
5. A. Briggs, "An Introduction to Scanning Acoustic Microscopy," in Microscopy Handbook
12 (Oxford, England: Oxford University Press, 1985).
References 433

6. D. A. Downs et al., "Interference Patterns in Scanning Acoustic Microscope Images of

Composites,/. Computer-Assisted Microscopy 1, 2 (1989), 19.>-215.
7. D. White, ed., vol. 1; D. White and R. Barnes, eds., vol. 2; D. White and R. Brown, eds.,
vols. 3A and 3B, Ultrasound in Medicine (New York: Plenum, 197.>-1977).
8. C. F. Lewis, "Ultrasonics Reveal the Inside Picture," Materials Engineering (Mar. 1988),
39-43.
9. Sales literature of Sonoscan®, Inc., 1981, 720 Foster Avenue, Bensenville, IL 60106.
10. R. A. Lemons and C. F. Quate, "Acoustic Microscopy: Biomedical Applications," Science
188 (1975), 90.>-911.
11. R. Kompfner and C. F. Quate, "Acoustic Radiation As Used for Microscopy," Physics in
Technology (Nov. 1977).
12. C. F. Quate, "Scanning Acoustic Microscope," material prepared for the Symposium
Workshop, Indianapolis Center for Advanced Research, Inc., Feb. 4-18, 1977.
13. W. Rohrbeck and E. Chilla, "Detection of Surface Acoustic Waves by Scanning Force
Microscopy," Physica Status Solidi. a: Applied Research 131, 1 (May 1992).
14. C. F. Quate, Edward L. Ginzton Laboratory, Stanford University, Stanford, CA.
15. Information distributed at the First Acoustic Microscopy Symposium Workshop at the
Indianapolis Center for Advanced Research, Feb. 14-18, 1977.
16. L. W. Kessler, "High-Resolution Visualization of Tissue with Acoustic Microscopy," in
Proceedings of the Second World Congress on Ultrasonics in Medicine, M. deVlieger, ed.
(Excerpta Medica Foundation, 1974).
17. L. W. Kessler, "Acoustic Microscopy: Progress and Applications 1977," ASA Ninety-
fourth meeting, Miami Beach, FL, Dec. 1977.
18. L. W. Kessler et al., "Simultaneous Acoustic and Optical Microscopy of Biological Spec-
imens," Nature 239, (Sept. 1972), 111-12.
19. A. Madeyski and L. W. Kessler, "Initial Experiments in the Application of Acoustic
Microscopy to the Characterization of Steel and to the Study of Fracture Phenomena,"
IEEE Transactions on Sanies and Ultrasonics, SU-23, 5 (Sept. 1976).
20. L. W. Kessler and D. E. Yuhas, "Acoustical Microscopy-A New Tool for Materials
Analysis and NOT," Thirty-seventh National ASNT Fall Conference Proceedings, Oct.
3-6,1977.
21. R. C. Eggleton and F. S. Vinson, "Heart Model Supported in Organ Culture and Analyzed
by Acoustic Microscopy," Acoustical Holography Vol. 7-Recent Advances in Ultrasonic
Visualization, L. W. Kessler ed. (New York: Plenum, 1977).
22. EMAG-MICRO 89, vol. 1: Physical, Institute of Physics Conference Series, no. 98, P. J.
Goodhew and H. Y. Elder, eds. (Bristol and New York: Institute of Physics, 1990), 13.>-
56.

Chapter 19

1. Tim Studt," Add Video to Your Microscope for Even Better Imaging," R&D Magazine
Uuly 1992), 32--36.
2. Shinya Inoue, Video Microscopy (New York: Plenum, 1986).
3. John C. Russ, The Image Processing Handbook (AJln Arbor, MI: CRC Press, 1992).
3a. Photometries, Ltd., 3440 East Britannia Dr., Tucson, AZ 85708.
4. John C. Russ, Computer-Assisted Microscopy (New York: Plenum, 1990).
5. R. H. Webb and C. K. Dorey, "The Pixelated Image" in Handbook of Biological Confocal
Microscopy, J. B. Pawley, ed. (New York: Plenum, 1990), 41-51.
434 References

6. Robert Keeler, "Shop Like a Pro for Your Image Analysis System," R&D Magazine
(March 1991), 48-52.
7. M. Richardson, "Confocal Microscopy and 3-D Visualization," American Laboratory
(Nov. 1990), 19-24.
8. The Imagist 1, 2 (1991).
9. In Focus, The Magazine for Instant Scientific Imaging 3 (Spring/Summer 1991).
10. D. J. Palatini, "Distributing Images to Remote Sites Using a Computer Network," EMSA
Bulletin 21, 1 (spring 1991).
lOa. "The Computer Comer," EMSA Bulletin 22, 3 (Nov. 1992), 78-81.
11. David Hessler et al., "SYNU: Software for Visualization of 3-Dimensional Biological
Structures," Microscopy: The Key Research Tool, (Mar. 1992), 73-82.
12. D.-P. Hader, ed., Image Analysis in Biology (Boca Raton, FL: CRC Press, 1992).

Chapter 20

1. T. G. Rochow and E. G. Rochow, Resinography (New York: Plenum, 1976).

2. T. G. Rochow, Light Microscopical Resinography (Chicago: Microscope Publications, 1983).
3. Annual Index to ASTM Standards (Philadelphia: American Society for Testing and Ma-
terials, 1993).
3a. G. F. VanderVoort, Metallography, Principles and Practice (New York: McGraw-Hill,
1984).
4. Technical Association of the Pulp and Paper Industry (TAPPI), 1 Dunwoody Park,
Atlanta, GA 30338.
4a. R. R. Cargille Laboratories, Inc., 55 Commerce Rd., Cedar Grove, NJ 07009; refractive
index standards, microscopical slides, reference materials, etc.
5. R. C. Ewald, 67-79th St., Middle Village, NY 11379.
6. T. G. Rochow and R. J. Bates, "A Microscopical Automated Microdynamometer Mi-
crotension Tester," ASTM Materials Research and Standards 12 (1972), 27-31; ASTM Tem-
plin award.
7. The Ladd Tensile Holder, information request from Mr. Ted Willmarth, 1209 Dogwood
Drive, Kinston, TN 37763.
8. FRL fiber cutter, obtained from Fabric Research Laboratories, Inc., 1000 Providence
Highway, Dedham, MA 02026.
9. lnsulfab Plastics, Inc., 834 Hayne St., P.O. Box 4277, Spartanburg, SC 29303; the mini-
mum direct order of perforated black vinyl slides is 1000.
10. Earnest F. Fullam, Inc., 900 Albany Shaker Rd., Latham, NY 12110.
11. Ward's, P.O. Box 92912, Rochester, NY 14692-9012.
lla. SPI® Source Book, Supplies Division of STRUCTURE PROBE, Inc., 569 E. Gay St., West
Chester, PA 19381-0656.
12. Microscopy and Histology Catalog (Warrington, PA: Polysciences, 1993-1994).
12a. D. Stadden, "Cross-Sectioning Method for Yarns," EMSA Bulletin 22 (May 1992), 25.
13. N. Reid and J. E. Beesley, Sectioning and Cryosectioning for Electron Microscopy, vol. 13 of
Practical Methods in Electron Microscopy, ed. A.M. Glavert (New York: Elsevier, 1991).
13a. L. C. Sawyer and David T. Grubb, Polymer Microscopy (New York: Chapman and Hall,
1987).
14. T. W. Robards and A. J. Robards, principal eds., Procedures in Electron Microscopy New
York: Wiley-Liss, 1993); looseleaf format with 6-monthly update service.
References 435

14a. M. V. Locquin and M. Langeron, Handbook of Microscopy (Boston: Butter Worth, 1983).
15. Hardy, U.S. Dept. Agric. Cir. 378 (1935); Textile Research 6 (1936), 270.
16. G. L. Royer and C. Maresh, "Application of Microscopy to the Textile Industry," J. Soc.
Dyers and Colorists (Sept. 1947), 287-293.
17. Mico Instrument Division of KFK Machine, 1944 Main St., Marshfield Hills, MA, P.O.
Box 451.
18. M. C. Botty, et al., "Application of Microscopical Techniques to the Evaluation of Ex-
perimental Fibers," Textile Research Journal 30 (1960), 959-965.
19. W. A. Burns and A. Bretschneider, Thin Is In: Plastic Embedding of Tissue for Light
Microscopy (Chicago: American Society of Clinical Pathologists, 1981).
20. M. A. Hayat, Basic Techniques for Transmission Electron Microscopy (Orlando, FL: Aca-
demic Press, 1986).
20a. Ted Pella, Inc., P.O. Box 492477, Redding, CA 96049-2477, Catalog 8, Dec. 1990; catalog
supplements and further issues to date.
21. P. J. Goodhew, Specimen Preparation for Transmission Electron Microscopy of Materials,
RMS Microscopy Handbook 03 (Oxford, UK: Oxford University Press, 1984).
21a. Polysciences, Inc., 400 Valley Rd., Warrington, PA 18976, 1990--91 Polymer & Manomer
Catalog, supplements and subsequent issues.
22. T. G. Rochow and F. G. Rowe, "Resinography of Some Consolidated Separate Resins,"
Analytical Chemistry 21 (1949), 461-66.
23. M. C. Botty, "Morphology of Polyphase Solids," Journal of Polymer Science, part C, no.
3 (1%3), 151~2.
24. R. E. Coulehan, "Stress Crazing Failure-a Mechanism of Surface Formation through
Wetting," American Society for Testing and Materials Special Technical Publication, 348
(Philadelphia: 1964), 168-86.
25. T. G. Rochow and R. L. Gilbert, "Resinography," in Protective and Decorative Coatings, ].
]. Mattiello, ed. (New York: Wiley, 1946).
26. Such as American Petrographics, Inc., 20 Appletree Lane, Roslyn Heights, NY 11577. See
also Geotimes 6.9 aan. 1993), 36.
27. "Petrographic Sample Preparation for Microstructural Analysis," Buehler Digest 24, 1
(1987).
28. T. G. Rochow, chairman, "Symposium on Resinographic Methods," ASTM Special
Technical Publication 348 (Philadelphia: American Society for Testing and Materials,
1964).
29. M. H. Mohamed et al., "Some Structural and Physical Properties of Yarn Made on the
Integrated Composite Spinning System, Part II: Three-Component Yarns," Textile Re-
search Journal 44 3 (1974).
30. L. D. Nichols, et al., "Some Structural and Physical Properties of Yarn Made on the
Integrated Composite Spinning System, Part 1: Two-Component Yarns," Textile Research
Journal 42, 6 (1972).
31. J. H. Sanders, "An Epoxy Resin System for Embedding Fiber Samples for Microtomy,"
unpublished (Williamsburg, VA: Dow Badische Co., 1970).
32. Such as General Electric RTV 630 mold-making silicone elastomer. Use the 20:1 ratio of
silicone to catalyst for high tensile strength, elongation, and tear strength.
33. Buehler Consumables Buyer's Guide (Lake Bluff, IL: Buehler, 1993).
34. Metal Digest 1.7/13, 1 (1973), 16-17.
35. Metallography-A Practical Tool for Correlating the Structure and Property of Materials,
ASTM Special Technical Publication 557 (Philadelphia: American Society for Testing
and Materials, 1975).
436 References

36. ASTM, "Symposium on Metallography: 75 Years Later," May ~10, 1991, Atlantic City,
NJ, published as ASTMSTP1165 (Philadelphia: ASTM, 1993).
37. G. VanderVoort, Metallography: Principles and Practice (New York: McGraw-Hill, 1984).
38. Buehler® Dialog'•, Microstructural Analysis Reference Manual for preparing metals, cer-
amics, composites, rocks and minerals, as of 1992 (Lake Bluff, IL: Buehler, 1992).
39. Simply Revolutionary (Lake Bluff, IL: Buehler, 1985). Catalog of equipment and supplies
for preparing hard materials for microscopical examination.
Author Index

Amer. Assoc. of Textile ASTM, 6, 10, 24, 30, 58, 80, Bausch & Lomb Co., 1, 12,
Chemists and Color- 81, 143, 151, 282, 387, 39, 413, 417
ists, 113, 122, 141, 414, 415, 418 Becke, 133, 134, 206, 207,
420 ASTM Annual Index, 387, 220,228
Abbe, E., 8, 10, 24, 41, 51, 405, 414, 419--421, Beesley, J. E., 395, 397, 429,
270, 281 424--426, 429, 434 434
Abramowitz, M., 50-59, 69, ASTM, Committee E-4 on Beeston, B. E. P., 290, 291,
71-73, 75, 78, 94, 95, Metallography, 340, 292, 429
103, 106, 192, 196, 431 Behrens, H., 148, 421
200, 202, 417, 418, ASTM Special Tech. Pub., BeJar, 251
420, 423, 424 405, 414, 417, 418, Bennett, A. H., 10, 94, 202--
Airy, G. B., 8 435,436 206, 414, 425
Alber, H., 240 ASTM Standard Bennett, H.S., 91, 94, 419
American Cyanamid Co., Definitions, 189, 199, Bennig, G., 340-342, 431
13, 62, 76, 86, 92, 210, 265, 267, 269, Berek, 100, 131, 132
158, 171, 279, 391, 270, 275, 276, 281, Berger, 18
422 285, 331, 334, 335, Berk, J. E., 371
American Laboratory, 343, 338, 339, 351, 358, Berry, Mrs. G., 131
344 419, 421, 424, 428, Bertin, E. P., 359, 432
American Microscopical 430, 431 Betz, V., 416
Society, 8 ASTM Symposium on Met- Betzig, E., 347, 348, 431
American Optical Co., 12, allography (1991), Bils, R. F., 282, 429
208, 226, 227, 425 405 Binnig, 13, 17, 18, 341
American Petrographics, Bioimaging, 380
400,435 Baer, E., 25, 416 Bloss, F. D., 91, 94, 137, 151,
American Society for Test- Baez, A. V., 19 155, 158, 240, 419,
ing and Materials, 8, Baker, 226 421, 422, 427
12, 33, 61, 81, 87-89, Baldeschwieler, J. D., 431 Bode, 254
91, 100, 106, 113, 131, Barnes, R. B., 14, 415, 433 Botty, M. C., 19, 282, 353,
141-143, 145, 147, Barrall, E. M., 243, 427 355, 395, 398, 415,
148, 193, 203, 237, Bartholin, E., 9 432,435
282, 283, 414, 419 Bates, R. ]., 107, 233, 237, Boyde, A., 178, 181, 423
Amici, G. B., 6, 8 239,240,244,389- Boyle, R., 2
Ardenne, M. von, 14 392, 420, 426, 434 Bradbury, S., 6, 414
Asimov, 1., 1, 413 Baum, R. M., 431 Bretschneider, A., 435

437
438 Author Index

Briggs, A. D., 432, 433 Davis, Mrs. E. Gagnon, 279 Federal Trade Commission,
Broers, A. N., 15, 308 de Broglie, L., 12 135, 158, 421
Brookhaven National Lab., Dekker, M., 263 Felton, C. D., 242, 427
356 Deily, J. G., 6, 28, 59, 74, 76, Fisher, A., 11, 17, 18, 343,
Brown, R., 4, 5, 20, 433 87, 91, 98, 108, 189, 346-348,414,415,431
Buehler®, 401, 402, 404-406, 194, 202, 214, 283, Fletcher, D., 353, 354
418, 435, 436 414, 416-420, 423, Fletcher, J., 354, 432
Bundy, R. P., 190, 424 424 Focus, 418, 420, 424, 429,
Burns, W. A., 396, 435 Denley, D. R., 342, 431 434
Burton, E. F., 13, 415 Dietz, P., 342, 343, 431 Fonash, S. L 331, 430
Busch, 12 Dodd, J., 217, 425 Ford, B. J., 3-5, 9, 413
Dolland, G., 5 Ford, T. W., 354, 357, 415,
Cambridge Scientific In- Dorey, 381, 382, 434 432
struments, 15, 191 Downs, D. A., 361, 365, 371, Francon, M., 228, 426
Cambridge University, 271 372, 433 Fresnel, A. L 19, 20, 358
Cameron, E. N., 419 Drebbel, C., 2 Friedel, 165
Campbell, D., 291, 429 Dupin, 166 Fullam, E. F., 287, 288, 394,
CANALCO, 352, 353, 432 Diirig, U., 347, 431 434
Cargille, 133, 214, 277, 392,
421, 425, 434 Echlin, P., 248, 427 Gage, S. H., I, 9, 413
Cassegranian lenses, 257 Edmund Scientific Co., 40, Galileo, Galilei, 2, 3, 4
Cavendish Lab., 271 43, 194, 417, 424 Gardner, S. A., 257, 427
Cerezo, A., 337, 338, 430 EDAK, International, Inc., Gedney, J. 1., 282, 353, 355,
Chamot, E. M., 145, 148- 314 432
151, 154, 157, 158, Eagle Tool Co., 408 General Electric Co., 402
240, 247, 252, 421, Eggleton, R. C., 433 Gerarkaris, T., 189, 424
422,426 Electron Microscopy Soci- Geotimes, 435
Charsley, E. L., 248, 427 ety of America, 14, German Standardization
Chilla, E., 433 19, 415 Institute, 43
Clark, J., 9, 414 Elder, H. Y., 415, 432, 433 Gilbert, R. L., 27, 61, 63-65,
Coates, D. G., 15, 316, 319 EMSA Bulletin, 383, 385, 71, 81, 107, 132, 191,
Cock, C., 2 434 233-238, 400, 405,
Cohen, J.B., 290, 429 Elias, H. G., 230, 426 407-410, 416, 420,
Computer-Assisted Micro- Elings, V., 343, 344, 431 426, 435
scopy, 380 Ellis, S. G., 207, 425, 426 Glatt, 1., 217, 425
Cook, Troughton, & Sims El-Shiek, A., 372 Goldner, H., 262, 428
Co., 12 Emmons, R. C., 107, 157, Goldstein, J. 1., 14-16, 233,
Cornell Univ., 397 420,422 237, 267, 311-315,
Cosslett, V. E., 13, 37, 38, Engel, A., 431 317, 318, 322, 325,
415, 417 England, B. N., 297, 299, 415, 426, 429, 430
Cotton, R., 354, 432 429 Gomer, R., 331-335, 430
Coulehan, R. E., 399, 435 Everhart, T. E., 15, 315, Goodhew, P. J., 397, 415,
Cowley, J. M., 281, 428 430 432, 433, 435
Crewe, A. V., 15, 309 Everhart-Thornley, 303 Goodsell, D. S., 415
Crites, N. A., 97, 101, 420 Ewald, R. C., 381, 390, 434 Gore, R. C., 11, 30, 257, 414,
Crossman, G. C., 214, 425 416, 427
Cui, X., 372 Faber, G., 2 Goringe, M. J., 270
Fabric Research Lab., 392, Goynes, W. R., 282, 283,
Davidson, J. A., 252, 427 434 300, 429, 430
Davidson, M. W., 23, 416 Feder, R., 357, 358, 432 Grabar, D. G., 426
 Author Index 439

Gregory, D., 6 Howells, M. R., 189, 354, Kohl, W. H., 415, 427
Grieble, D. L., 257, 427 356-358, 423, 432 Kohler, A., 9, 27-29, 36, 70,
Gross, L., 210, 425 Howie, A., 329, 430 71, 211, 217, 269, 270
Grubb, D. T., 173, 422, 429, Hren,]. J., 331, 338, 430 Kompfner, R., 364, 365,
434 Humphreys, C. J., 270 374-376, 433
Gumpertz, W. E., 32, 44-47, Humphries, D. W., 9, 414 Koppelman, R., 11, 346
416, 417 Hunn, W., 207, 425 Krishman, K., 257, 261
Huygens, C., 4, 6, 51 Krug, W., 10, 228, 421, 426
Hacker Instruments, Inc., Kuhl, 253, 254
417 IBM Research Lab, 18 Kiin, 254
Hader, D. P., 384, 434 IBM Zurich Research Lab,
Hall, A. M., 179, 423 18 Ladd, W. A., 13, 391, 415,
Hall, C. E., 13, 15, 267, 269- I.E.E.E., 380 434
273, 274, 305, 306, Iijima, S., 428 Land, E. H., 10, 11, 30, 99,
329, 334, 338, 339, Inoue, 5., 380, 423, 433 414, 416, 420
415, 428, 430 Insulfab Plastics, Inc., 393, Lang, W., 221-223, 226, 425
Hamlin, D. J., 1, 413 394, 434 Langeron, M., 395, 414, 435
Hansma, P. K., 342, 343, Isaacson, M., 11, 343, 344, Leeuwenhoek, A. van, 2--5,
431 431 9, 20,40
Hardy, 435 Lehman, 165
Harris, J. A., 282--284, 300, Jacker, C., 1, 9, 413 Leica, 11, 47, 48, 94, 179,
430 Jacobsen, C., 357, 359, 432 183, 184, 194, 242,
Hartshorne, N.H., 30, 32, Jahanmir, J., 349, 431 243, 414, 419, 423,
91, 93, 95, 96, 102, Jarecki, I., 230, 426 424
131, 133, 135, 136, lena Review, 426 Leitz Co., 11, 12, 128, 131,
141, 142, 151, 155, JEOL, 288-290, 293, 429 231, 408, 421
158, 159, 162--172, Johnson, R. I., 217, 425 Lemons, R. A., 17, 18, 364,
227, 240, 242--247, Jones, F. T., 33, 245--247, 365, 367, 369, 370,
416, 419, 421, 422, 417, 427 374-376, 415, 417,
425--427 Josephson, B., 16 433
Harvard-Smithsonian As- journal of Microscopy, 380 Lewis, A., 11, 346
trophysical Ob- Julian, Y., 242, 427 Lewis, C. F., 261, 433
servatory, 19 Jung Co., 12 Lin C.-Y., 230, 426
Hayat, M. A., 282, 283, 397, Lister, J. J., 6, 9
429, 435 Kafri, 217, 425 Locquin, M., 10, 395, 414,
Hayes, T. L., 315, 430 Keeler, R., 179-181, 382, 435
Hearle, J. W. 5., 307, 326, 383, 423, 434 Longobucco, R. J., 359, 432
335, 415, 430 Kedersha, N. L., 424 Lorenz-Lorentz, 230
Hessler, D., 385, 434 Kenseth, J., 2, 413 Loske, T., 253, 427
Hill, S. L., 257, 261, 427 Kepler, J., 1, 2 Loveland, R. P., 35, 108,
Hillier, J., 13, 15 Kessler, L. W., 18, 370, 374, 194, 202, 285, 417,
Hockman, R. F., 331, 333, 375,433 420, 424, 429
430 Kirk, P. L., 150, 422 Lyman, C. E., 297, 315, 430
Hoffman, R., 210-212, 220, Kirkpatrick, A. F., 152, 158,
425 389, 390, 422 Mach, E., 10, 227
Hogg, J., 2, 413 Kley, P. D. C., 148, 421 Mach-Zender, 228, 231
Holland, V. F., 276, 428 Kodak®, 59, 106, 194, 285, Madeyski, A., 374, 433
Hollyfield, J. G., 23, 416 352,418,420,424,429 Malies, 216
Hooke, R., 2, 3, 4, 20, 413 Kofler, A., 240 Mallick, 5., 228, 426
Home, 291 Kofler, L., 240, 427 Malus, E. L., 9
440 Author Index

Marcel Dekker, 258, 262, Miller indices, 148 Nipkow, 180, 181
263 Mineralogical Record, 202, Nixon, W. C., 15, 359, 432
Maresh, C., 395, 435 310 Nomarski, 226, 231
Markam, 291, 292 Miniutti, V. P., 62, 65, 81,
Marton, L. L., 13 86,418 Oatley, C. W., 15, 306, 309,
Mason, C. W., 6, 10, 26, 28, Mogensen, A. 0., 421 430
32, 34, 41, 65, 69-71, Mohamed, M. H., 372, 402, Olson, A. J., 415
78, 87, 90-95, 97, 98, 403,435 Olympus Corp., 12, 50, 52,
104, 119, 123, 147- Moire, 276 54-57, 71-73, 94, 103,
151, 153-155, 158, MSA Bulletin, 19, 20, 385 105, 192, 194, 196,
190, 202-204, 214, Muller, E. W., 16, 17, 329- 414, 417, 420, 424
221, 225, 227, 233, 336, 338, 339, 415,
240, 247, 248, 251, 430
252,257,266,276- Mulvey, T., 23-25, 307, 309, Padgitt, D. L., 6, 414
278, 280, 283, 318, 310, 415, 416, 430, Padnos, M., 212, 214
321, 414, 416-422, 431 Palatini, D. J., 434
424--430 Murphy, C., 189 Pasteur, L., 9, 20, 414
Mather, S. R., 61 Murray, R., 428 Pawley, J.B., 177, 179, 180,
Mayne-Banton, V., 358, 432 Musselman, I. H., 431 181, 185, 186, 423
McCrone, W. C., 21, 37, 61, Pease, R. F. W., 15
150, 152, 189, 212, Pella, Ted, Inc., 435
214-218,240,242, Nanoscope Digital Instru- Peres, M., 189, 423
283, 415, 417-422, ments, 345, 346 Perkin-Elmer Co., 11
424-429,430 National Bureau of Stan- Perkins, W. D., 428
McLaughlin, R. B., 69, 73, dards, 77 Philips Co., 13, 19, 298-300,
75, 87, 101, 223-225, National Synchrotron Light 353, 355, 356
233, 234, 236, 237, Source, 356 Philips Electronic Instru-
239, 251, 252, 418, Nelson, E. M., 9, 27, 28, 36 ments, 415, 432
420, 425, 426 Nelson, J. B., 61, 68, 81, 418 Photometries, 381, 434
McMullen, D., 15 Newman, S. B., 353, 354, Physitemp Instruments,
Messerschmidt, R. G., 257, 432 Inc., 282, 428
258,260-262,427 Newton, 1., 102, 221 Planck, 312
Metal Digest, 404, 435 N.Y. Microscopical Soc., 23, Podnos, 212-214
Metropolitan-Vickers, 13 24, 30, 33, 89, 90, Polysciences, 394-397, 400,
Michel-Levy interference 189, 199, 233, 265, 434,435
chart, 97, 100, 106, 268, 297, 311, 318, Prebus, A., 13
129, 131, 144, 155, 331, 338, 415, 419,
400 423,424,426,428- Quate, C. F., 17, 18, 340-
Michelson, A., 10, 227 430 342, 361, 364-370,
Mico Instrument Co., 409, Nichols, G., 257, 258, 261, 373-376, 383, 415,
435 427 430, 432, 433
MICO-LAC, 395 Nichols, L. D., 87, 402, 403,
Microscopia Acta, 380 419,435 Ralph, B., 329, 331, 338, 430
Microscopy, Microanalysis, Nicol, W., 9 Ramsden,]., 6, 51
and Microstructures, Nicolaysen, H. F., 62, 67, Ranganathan, S., 331, 338,
431 418 430
Microscopy Society of Nikon Co., 12, 74, 76, 94, Redcroft, S., 248, 427
America (MSA), 19, 109, 110, 189, 194, Reffner, ]. A., 11, 25, 30,
20,385 197, 414, 420, 423, 257, 260, 264, 414,
Miller, C., 122 424 416,427
Author Index 441

Reichert Co., 12 Ruch, F., 91, 94, 419 Smith, K. C. A., 15

Reid, N., 395, 397, 429, 434 Ruska, E., 12, 13, 270, 271, Snyder, R. L., 15
Reigel, S. A., 190, 424 274,428 Sokolov, S., 18
Reimschuessel, A. C., 113, Russ, J., 372, 381, 384, 433, Sorby, H. C., 9, 20
303, 316, 317, 319, 434 Spectra-Tech, 260, 428
320-324, 420, 430 Russel, P. E., 431 Spiller, E., 350, 432
Rempfer, G., 428 Ryan, J., 262,428 Stadden, D., 394, 434
Reumuth, H., 252, 427 Stanford Univ., 18
Richards, 0. W., 6, 26, 208, Sanders, J. H., 435 Stemmer, A., 431
209, 227, 414, 416, Sawyer, L. C., 179, 422, 429, Stevens, R. E., 149, 422
418,425 434 Structure Probe, Inc., 394,
Richardson, J. H., 10, 61, Saylor, C. P., 133, 149, 207, 434
414, 416 421, 422, 425 Stuart, A., 30, 32, 91, 102,
Richardson, M., 179, 186, Sayre, D., 354, 357, 432 131, 151, 155, 158,
382,423 Schaffer, C., 287, 288 166, 167, 171, 206,
Robards, A. J., 398, 405, Schaudinn, 254 207, 214, 215, 227,
435 Schleuter, G. E., 32, 44--47, 416, 419, 421, 422,
Robards, T. W., 398,405, 416, 417 426
435 Schott, 0., 8, 10 Studt, T., 379, 381, 383, 433
Robinson, A. L., 280, 428 Schulman, S., 1, 18, 413 Sullivan, P., 221, 223, 226,
Roche, E. J., 230, 426 Schwartz, D. M., 23, 189, 425
Rochow, E. G., 25, 61, 81, 416,424 Swann, P. R., 270, 428
89, 91, 93, 139, 190, Schwartz, J., 190 Sweeney, M. A., 243, 427
387, 396-398, 405, Schwartz, L. H., 290, 429 Swift Co., 12, 40, 42, 417
407, 419, 434 Science, 423 Szarowski, D. A., 179, 181,
Rochow, T. G., 10, 13, 25, Scientific American, 341 423
27, 33, 61, 6~5, 71, Scott, R. G., 48, 49, 135-137,
81, 87, 89, 91, 93, 139-141, 417, 421, Taylor, M. E., 341, 342, 431
107, 132, 137-139, 426 Tech. Assoc. of Pulp & Pa-
152, 190, 191, 193, Sellers, J., 262, 428 per Industry (TAP-
200, 233--240, 244, Senarmont, 100, 227 PI), 387, 434
248, 282, 387-400, Seneca, 1 The Imagist, 382, 434
402-410, 414--422, Sensortek, Inc., 249-251, Thomas, A. H., Co., 240,
424, 426, 427, 429, 427 241,427
434,435 Septier, A., 274, 428 Thomas, E. J., 279, 282, 353,
Rogers, J., 172, 422 Shephard, C. J. R., 23-25, 415, 432
Rohrbeck, W., 365, 433 416, 431 Thomas, E. L., 268, 428
Rohrer, H., 17, 340-342, 431 Shirley Institute, 113--121, Thompson, M., 343, 344,
Rontgen, W., 18 420 431
Rooseboom, M., 2, 6-8, 413 Shotton, D., 179, 182, 423 Thomley, R. F. M., 15
Rosevear, F. G., 171, 172, Siegel, B. M., 268, 269, 428 Tolanski, S., 221, 225, 228,
422 Siemens & Halske Co., 13 425, 426
Ross, A., 8 Sieminski, M. A., 126, 128, Tolhurst, D. E., 248, 427
Royal Microscopical Soc., 1, 131, 132, 421 Tracor Northern, 182, 423
259, 413, 414, 415, Singleton, R. W., 128, 139, Transactions, 380
427 421 Trautman, J., 347, 348, 431
Royer, G. L., 395, 435 Skalla, D. W., 61, 418 Traviss, 201
Rowe, F. G., 19, 62, 67, 304, Slayter, E. M., 61, 91, 280, Tsong, T. T., 16, 17, 329,
316, 319, 398, 415, 283, 418, 419, 428 331, 333-335, 338,
418,435 Smith, G. D. W., 340, 431 339, 415, 430
442 Author Index

Tucker, P. A., 113, 270, 272, Ward's Natural Science Es- Winchell, H., 151, 152, 422
372, 420, 428 tablishment, 424 Winsor, P. A., 172, 422
Turnbull, T. P., 287, 288, Wasielewski, 254 Wollaston, 228, 231
429 Watson Interference Div. of Wood, R. W., 16
Turner, G. L. E., 2, 413 M.E.L. Equipment Woodward, A. E., 173, 422
Co., 224 Wright, F. E., 161
Unitron Instruments, 80 Webb, R. H., 179, 181, 185- Wunderlich, B., 221, 223,
U.S. Department of Agri- 187, 382, 423, 434 226
culture, 283 Webster, R. K., 307, 309,
U.S.D.A. Southern Regional 310,430
Center, 283, 301 Wehnelt, 267 Yakowitz, H., 14-16, 233,
U.S. Steel Research Lab., 85 Weiner, D. B., 6, 414 297, 303-305, 307-
U.S. Trademark Associa- Weiland, M. E., 341, 342, 314, 317, 318, 322,
tion, 119, 420 431 325, 415, 426, 429
Wells, 0. C., 318, 322, 323, Young, P. H., 257, 261, 428
Vallery-Radot, R., 9, 414 430 Young, P. L., 173, 422
Vance, 13 Wenham, F. H., 9 Yuhas, D. E., 374, 375, 433
VanderVoort, G. F., 81, White, A., 78
381, 405, 419, 434, White, D., 361, 369, 433
436 White, J. R., 291, 429 Zeiss Co., 6, 8, 10, 11, 24,
VanVlack, L. H., 145, 147, Wihborg, W. T., 11, 25, 30, 28, 29, 74, 94, 194,
421 414, 416 222, 226, 267, 269,
Van Zuylen, J., 3, 413 Wild Co., 11, 46 270, 414, 419, 424
Vinson, F. S., 433 Willard, M. L., 158, 422 Zeki, 5, 23, 415
von Ardenne, M., 14 Willmarth, T., 389, 434 Zender, 10, 227
von Borries, 13 Wilson, L., 23, 416 Zernike, F., 10
Wilson, T., 179, 423 Ziabicki, A., 230, 426
Wagner, R. W., 187 Winchell, A. N., 32, 33, Zieler, H. W., 28, 29, 37, 40,
Wahlstrom, E. E., 151, 422 145-147, 151, 152, 62, 66, 67, 80, 191,
Ward's, Rochester, N.Y., 154, 174, 416, 420- 214, 416-418, 424
194, 394, 434 422 Zworykin, V. K., 13, 15, 24
Subject Index

Abbe condenser, 55 acoustic microscopes (Cont.)

Abbe's law, 8, 10 SAMS, 363-366
aberrations, 6 scanning systems in, 362-364
correction, 25, 67 schematic diagrams of, 362-364
achromat, 51, 54 SLAMS, 361-366
achromatic lens, 6, 54 Sonomicroscope® scanning laser acoustic
achromatic objectives, 50, 54 microscope (SLAM), 361-362
acicular crystals, 147 study of structure with, 373
acoustic microscopes (SLAMS, SAMS), 18, structure of specimen, 373
361 summary of, 376-378
acoustic optical images combined, 362 third dimension, 370
acoustic beam for, 362 useful magnification of, 370
anisotropy, 373 working distance in, 371-372
aplanatic lenses in, 368 acoustic radiation for SAM and SLAM,
applications of, 366 369-370
biological specimens, 365, 371 acoustic shadowgraph microscope, 376
cleanliness, 368 acoustic waves, 326-327
color display, 362, 376 acute bisectrix, 154-155, 161-162
contrast of, 368 Aerosol® OT, wetting agent, 169-176
depth of field in focus, 368-369 aging of fibers after spinning, 140
experimentation with, 375 alums, 149-150
field of view in, 370 amphiphilic molecules, 170-171
focusing, 369 amplitude-contrast microscope, 199-202
interferometry in, 368 analysis of crystals, 145-162,
morphology, 374 analyzer, 10, 90, 97
nonbiological applications of, 366, 373- angular aperture, 94
375 angular symmetry of crystals, 154-159
photomicrography with, 375-376 anhedral crystals, 30
preparation of specimens for, 375 anisometric crystals, 91
principles of, 361-366 anisotropy, 30, 58, 91
radiation for, 369 birefringent, 30
resolution of, practical, 367 molecular, 9, 30
resolution versus wavelength, 366-367 polarization, 9, 30
resolving power of, theoretical, 368 strain, 9, 30
sample specimens for, 373 streaming, 30

443
 444 Subject Index

anisotropy of fibers, 30 biological microscopes, 49-60

annular stops, 215--216 anisotropy in specimens for, 58
anthracite coal, 66 attitudes in use, 49-50
antiglare coatings, 78-79 condensers for, 51-53
antiglare devices, 16 corrections for aberrations in, 50, 54-55
apochromat, 54 cues to depth in, 32, 43
apochromatic lens, 6, 11, 54 depth of field of, 41
apochromatic objectives, 11, 50, 54 experimentation with, 34
aragonite, 145 eyepieces for, 51
arc lamps, 88 focusing of, 26--27
arsenopyrite, 76 illumination of, 56---58
asbestos, 65 magnification obtained with, 58-59
astigmatism, 25 objectives for, 50-51
atom probe, 331 photomicrography with, 59
atomic force microscopes, 18, 340, 342, 345, preparation of specimens for, 49
346--348 resolution obtained with, 41
atomic number, 280 birefringence, 97-99
attributes contributing to visibility, 25--35 negative, 126-129
correcting for aberrations, 25 positive, 126-129
definitions, 35, 36 Bobtex yam, 403
experimentation, 34 bright-field illumination, 27, 73, 201
focus depth, 26 Brownian movement, 5
illumination, 27 butterfly wings, color of, 318-321
importance of relaxed eye, 26
magnification, 31
morphology, 35 calcite, 145
photomicrography, 35 camera, 193, 196-197
preparation of specimens, 34 charge coupled device (CCD), 381
quantity and quality of sample, 25 charge injection device (CID), 381
radiation, 26 carbon arc, 8
stereoscopy, 32 carbon replicas, 284
structure of specimen, 32 carbon steel, 70, 77-73
video, 35 case-hardened steel, 69-70
Auger effect, 297, 306, 312---313 cathode luminescence (cathodolumin-
auto exhaust catalyst, 353-355 escence), 304
cathode-ray display, 297-300
cells, 4
Babinet compensator, 131 cells and cuvettes, special, 252---253
backscattered electrons (in SEM), 297, 302--- central stop, 200-202, 241
303 cesium alum, 150
bagasse, 63 Chambers micromanipulator, 238-239
basal pinacoid, 154 chemical microscope, 94
batonnets, 167 cholesteric phases and textures, 164
Becke line, 133, 207 Christiansen effect, 214
bee, head of (electron micrograph), 316 chrysotile asbestos, 65
behavior of specimen, 34--35 cleanliness and orderliness, 68, 102
Berek compensator, 100, 207 coal, 66
Bertrand lens, 96, 103, 105, 106, 155, 204 cold-cathode electron emitters, 331
biaxial crystals, 152, 158-159 cold stages, 248-251
biaxial interference figures, 151, 154 color, structural, 318, 320-321
Subject Index 445

color-contrast microscopy, 199, 202 condensers (Cont.)

colors, retardation, 98, 118--119 oil-immersion, 52, 55--56
columnar crystals, 146 with reflected light, 68--71
coma, 20 with transmitted light, 8
compensation for retardation, 100 substage, 51
compensator, 100 for x-ray microscopy, 352-354
compound microscopes, 40 confocal microscopy, 177
compound microscopes using reflected scanning modes, 179-181
light, 61--88 conoscopic illumination, 103-104
behavior of specimen, 63, 86 contrast, 24, 199-221
contrast obtained with, 66-67 by reflected light, 66
corrections for aberrations in, 67 in electron microscopes, 272-273
cues to depth in, 79 interference, destructive and con-
dark-field illumination for, 71, 73 structive, 199-202
depth of field in, 79--80 perception of, 24-25
depth of focus in, 68 correction for aberrations, 101
experimentation in, 83 astigmatism, 25
field of view of, 77-78 chromatic, 54
focus, 68 curvature of field, 25
glare, 78--79 distortion, 25
illumination for, 68--75 phase-amplitude, 202-209
information about specimen, 83 reflected light, 67
magnification of, 75-77 refractive, 36
morphology, 82-83 spherical, 25
photomicrographic techniques with, 87 cotton, 66
polarized light in, 66, 76 cover glass, 51
preparation of specimens for, 86--87 critical illumination, 28
radiation for, 71 crossed polars, 89, 103
resolving power of, 62, 65-66 crystal axes, 173-174
structure, 81-82 crystal habit, 146-153
study of morphology with, 82-83 crystal structure, 81-82, 145-159
study of surfaces with, 68-88 crystal structure and morphology (chart),
summary of, 87 146
thick sections for, 61-66 crystals, microscopical properties of, 145-
transparent reflectors for, 70 176
vertical illumination with, 70--71 biaxial crystals, 158--162
working distance of, 80--81 birefringence, + and -, 149-150
computer aided image, 379, 380, 382 Bravais lattices, 147
analysis, 382-384 distribution of refractive indices, 152,
collection, 380 157, 159
processing, 382 interference figures, 155, 156, 158
reconstruction, 379, 385 isogyres, 156
condensers, 8, 51 isomorphism, 148--150
for acoustic microscopy, 88 isotropic crystals, 152-154
aplanatic-achromatic, 54, 55 liquid crystals, 159, 172, 176
dark-field, 200--202 morphology, 145-147, 150
for electron microscopy, 269, 275-276, Miller indices, 148
297,307 optical dispersion, 152
focusable, 51-53, 55, 57 polygonal morphology, 150
history, 8--9 refractive index, 148
446 Subject Index

crystals, microscopical properties of (Cont.) electromigration, 323

rhombohedron, 14~ 175 electron diffraction, 288--294
crystals, microscopical properties of (Cont.) electron excitation, 313
trigonal classification of rhombohedra, electron field-emission microscope (FEM),
151 329-338
skeletal morphology, 150 electron lenses, 268--269
spherulites, 171-172 electron micrography, 189, 285-288
structure, 145-162 electron microscopes, 265-270
summary, 173-176 history, 12-14
twins, 92 electron microscopy, 270
uniaxial crystals, 154-158 aberrations, 273
uniaxial interference figures, 155-156 anisotropy, 276
cubic crystals, 147-149 artifacts, 277
cues to depth, 14, 32, 79-80 contrast, 272-273
curvature of field, 196 cleanliness, 274-275
electron microscopy without lenses, 329-
349; see also field emission micro-
dark-field illumination, 9, 200-202
scopes
by reflected light, 66, 71, 73
electron-probe microanalyzer (EPMA), 16
deBroglie theory, 12
electron sources, 267-269, 275-276
definitions, 23-25, 35-36
electron transitions, 311
contrast, 24
Emerson micromanipulator, 239
microscope, 23
emission microscopies, see field emission
microscopy, 23
microscopes
resolution, 24
empty magnification, 59, 104
resolving power, 23
enlargement of electron micrographs, 288
dendrites, 150
equant crystals, 146
dendritic crystals, 150
erecting prisms, 44, 45
depth of field, 79
etchants, 66, 70, 82, 87, 88
depth of focus, 26
etching, 66, 70, 82, 87, 88
depth of specimen, 106
euhedral crystals, 151, 156, 158 ·
denier, 131
experimentation, 36, 83, 107, 282
developers, photographic, 286
extinction, 90--91
diatomaceous earth, 319
extra-long working-distance objectives, 107
dichroism, 129, 158
extraordinary image, 89
digestion, 281
eyeball, structure of, 23-24
direction images, see interference figures
eyepiece, 4
dispersion, optical, 152
development of, 6, 8, 40, 44, 51
dispersion staining, 152-153
Huygens, 4, 6, 51, 55
dissecting (stereo) microscopes, 43-49
Ramsden, 6, 51, 55
distribution of optic axial angles, 160
distribution of refractive indices, 153, 157,
fibers, 113-114
159, 160
identification of, 141-143
Drebbel microscope, 2
microscopical properties of, 113-144
Dupin cyclides, 166
Acrilan®, 119, 120, 142, 144
Dutch microscopes, 1
acrylic fibers, 120, 144
alignment of molecules, 135-141
elasticity and compression, microstudy of, analytical value of, 135
361-362 anisotropy, 135-137
electromagnetic lens, 12-13, 268--269 Becke refraction line, 133-134
Subject Index 447

fibers (Cont.) fibers (Cont.)

microscopical properties of (Cont.) microscopical properties of (Cont.)
birefringence versus strength, 132 sources of anisotropy, 135
brightness or grayness between spinning, wet or dry, 138--141
crossed polars, 122, 124 structure and composition versus
cellulose acetate fibers, 121 properties, 135
coagulation of, 139 summary, 144
color bands in, 126, 130 between crossed polars, 122--124
colors, retardation or polarization, use of compensators, 126-128
125-132 use of first-order red plate, 128
comparison with crystals, 121 use of quarter-wave plate, 128
cotton, 116, 117 use of quartz wedge, 128
degree of birefringence, 123-124, 129- variations in birefringence, 129-130
130 viscose, 118
dichroism, 129 wool, 114
eight optical properties, 122--124 field-emission microscopes, 16, 329-339
extinction between crossed polars, 124 artifacts, 336-337
flax, 116 behavior of specimen, 338--339
flow birefringence, 139 cleanliness, 334--335
form birefringence, 140 contract obtained with, 334
goat, 115 correction for aberrations in, 334
identification, 141-143 experimentation with, 338
mercerized cotton, 117 field depth, 337
Michel-Levy scale (Figure 5.8), 97-100 field of view, 336
molecular orientation and organiza- focus and depth of focus of, 68, 335
tion, 137-141 illumination, 335
morphology, 113-121 image intensifiers for, 334
mounting liquids, 121-122 magnification obtained with, 336
nomenclature, 144 morphology, 338
nylon, 119 photomicrography with, 339
oblique extinction, 123 polyamide fibers, 119
oblique illumination test, 134--135 preparation of specimens for, 339
optic axes, 151 principles of, 329-331
optical principles, 121-141 resolution, limit of, 333
order of retardation, 125-126 radiation, 335-336
Orion®, 119, 120, 142, 144 resolving power of, 333
parallel extinction, 124 results, reference to, 338, 430
with polarized light, 122--137 schematic diagrams for, 330, 332
polyamide, 119 source of electrons for, 329
polyester, 118 sources of contamination in, 334, 335
positive and negative birefringence, structure of metal sample, 337
126 summary of, 348--349
quantitative birefringence, 123, 125, types of, 329
128, 129, 132 field-ion, 17, 331
rayon, 118 working distance in, 337
refractive index, parallel, 132--135 field-ion microscope, 16
refractive index, perpendicular, 132-- field microscope, 42
135 field of view, 77
retardation, 123, 125-130 fillers for resins, 63-65
silk, 117 films, 141
448 Subject Index

first-order red plate, 100 history (Cont.)

flow birefringence, 30 scanning tunneling microscopes, 16-17
focal conic textures, 165 summary, 20-21
focus, 26, 68, 102 transmission electron microscopy, 12-14
foils, 141 x-ray microscopy, 18-19
forensic investigations, 150 x-ray laser microscopy, 19
form birefringence, 93, 140 homeotropic textures, 165
Formvar® film substrate, 284 hot stages, 239-248
Fourier transform-infrared (FT-IR) micro- Huygens eyepiece, 4, 51, 55
scopy, 11, 257-264 hyperopia, 37
frame grabbers, 381, 383
freeze-drying of specimens, 324 illuminants, 48, 56
Fresnel zone plate to focus x-rays, 358 illumination, 27, 58
furnace, water-cooled, 248 bright-field, 27, 73, 201
conoscopic, 103
galena, 304 critical, 56
Galileo microscope, 2 dark-field, 27, 73, 201
telescope, 2 infrared, 58
glare, 88 Kohler's, 9, 29, 56, 57, 269-270
by reflected light, 78-79 Nelsonian, 9, 28, 56
glass, 1 oblique, 27
gold wire, surface of, 303, 324 ultraviolet, 58
granite, 92 vertical, 70, 73
graphic arts, 189 image
Greenough binocular microscope, 44, 45, 60 real, 4
Gregory correction for abberations, 6 virtual, 38
image analysis, 380, 382, 384
habit, crystalline, 146-148 collection, 380
hand microscope, compound, 40 processing, 382
hardness testers, 238 reconstruction, 379, 385
Hartshorne hot stage, 243, 246 image intensifiers, 387, 334
hemacytometer cell, 251 immersion objectives, 8, 41, 54--56
hexagonal crystals, 145-147, 151, 154 immersion oils, 8, 41, 54-56
history, 1-21 information about specimen, 83
atomic force microscopy, 18 infrared microscope, 257
condensers, S-9 infrared microscopy, 257-261
correction for aberrations, 6-8 Fourier transform microscopy (FT-IR),
dark-field microscopy, 8 257
early, 1-6 illumination, 257
electron probe microanalyzers, 16 lenses, Cassegranian, mirror, 257
field-emission microscopes, 16, 17 limitations, 264
manufacturers of light microscopes, 11- microscope, FT-IR, 260
12 objective, Cassegranian, 257
Microscopy Society of America (MSA), specimen, analysis of, 261
19-20 x-ray micro-analysis, 260
near-field scanning light microscopes, interference, light wave, 199-200
11-22 interference figures, 73, 76, 94, 95, 97, 105,
polarized light, 9-11 106
scanning electron microscopes, 15 interference films, 102
scanning acoustic microscopy, 18 interference microscopes, 221-231
Subject Index 449

interference microscopy, 221-231 light microscope manufacturers, 11-12

advantages of, 230-231 light, polarized, 95-97
aus Jena model, 228--229 light sources, 28--29
Baker interference microscope, 226-227 limiting resolution, 37
beam-splitting system for, 222 liquid crystals, 159-172
differential interference contrast in, 226 lyotropic phases, 169-172
double interference microscopes, 225-226 lyotropism, 159
highly birefringent specimens for, 230
interference fringes obtained by, 221, magnification, 31, 38, 58, 59
230 designation, 58--59
interference, 221 empty, 59
interference objectives for, 214-215 reflected light, 75
Leitz system, 227 total, 104
Mach-Zehnder system, 227-229 useful, 41
Mirau objective for, 223-224 magnifying glass, 39
Nomarski microscope, 226 Malies turret, 216-217
refractive index determination by, 227- Maltese cross, 155
229 measurement of thickness, 98--99
single microscopes, 222-225 mechanical stages, 233-235
summary of, 230-231 melamine, crystallographic properties of,
Watson objective for, 221 158, 162
Zeiss-Nomarski equipment for, 226 Melmac®, melamine-formaldehyde resin,
inverted microscopes, 72, 76 65
ion detector, 331 mesomorphic stage, 159, 163-172
ion-etching, 322-323 metallography, 84, 85
ion field-emission microscope (field-ion mi- metals, study of structure in field-emission
croscope, FIM), 331-332 microscope, 337
isogyres, 158, 165, 166 Mettler hot I cold stage, 242-243
isometric cyrstals, 145-149, 152-154 micelles, 170-171
isomorphism, 149-150 Michel-Levy chart, 97-100
isomorphs, 149-150 Michelson interferometer, 227
isotropic system, 152-154 microcline, 92
microdynamometer, 107, 389-392
Kepler eyepiece, 1 microelectrophoresis, 252
Kirk flask, 389, 390 micrograph, 189
Kodalith®, 286, 356 definition of, 189
Kofler hot stage, 240-241 electron micrograph, 189
Kohler's illumination, 9, 56, 57, 202, 269- photography, purposes of, 189-190
270 photomicrography, 189, 193, 194
by reflected light, 70-71 x-ray micrography, 189
records of negatives, 191
Ladd vacuum evaporator, 264 resolving power, 191-192
lamellar crystals, 146 resolution, 191
Land's inventions, 10 summary, 195-198
lanthanum boride electron gun, 308--309 micrography, 189-198
lanthanum boride emitter, 308--309 micromanipulators, 81, 237-239
laser-produced plasmas, 354 micrometry, 41, 45
leaded gasoline, 353, 355 microphotograph, 189
lens coatings, 78--79 microscope
Lieberkiihn illuminator, 69 acoustic, 361-378
450 Subject Index

microscope (Cont.) Newton's series of interference colors (Fig-

binocular, 42-49 ure 5.8), 97-100
biological, 49-59 Nichols stage refractometer, 251-252
compound, 40 Nicol polarizer, 94
definition of, 2 nu value (of optical dispersion), 212, 214,
Dutch, 1 215
electron, 265-296, 297-327 numerical aperture (NA)
field, 41 definition, 8
field-emission, 16, 329-339 of biological microscope, 50, 67
infrared, 11, 257-264 of eye, 26, 31
interference, 199 of simple microscope, 59
origin of, 1-4 of stereomicroscope, 45, 46
phase-contrast, 202-209 practical limit, 41
polarizing, 89-111 relation to magnification, 31, 32
simple, 2, 4, 38 with reflected light, 73, 88, 94, 101-103,
stereo, 43-49 106
x-ray, 351-359 nylon, 119, 126, 143, 144
microscopic, 23
microscopical, 23 objective, 6, 8, 50, 54
microscopy reflected light, 65
dark-field, 9 oblique bright-field illumination, 71
definition of, 23 oblique extinction, 91, 123
history of, 1-21 oblique illumination, 210-213
Microscopy Society of America (MSA), 19- obtuse bisectrix, 151
20 octahedral crystals, 149, 153
microtome sections, 325, 392, 394-397 oculars
microtomes, 395-397 Huygens, 4, 51, 55
Miller indices, 148-149, 175 Ramsden, 6, 51, 55
Mirau interference objective, 223-224 oil-immersion objectives and condensers,
mirror, 4 41, 56, 67, 88
modulation-contrast microscopy, 210- oligoclase, 92
212 opaque materials, study of, 61, 87
Moire effect, 230 optic axes of crystals, 161, 165, 166
Moire patterns, 276 optic sign of crystals, 166
molecular anisotropy, 137-141 optical activity, 9
molecular birefringence, 137-141 optical dispersion, 152, 212
monoclinic crystals, 145, 146, 147, 151, optical lever atomic force microscope, 18
162 optical principles, 23-32
morphology optical sign, 155-156
of crystals, 145, 146, 150 optical staining, 214-215
of fibers, 113-121 ore, 62
of specimen, 33-34 organic versus inorganic crystals, 152
by reflected light, 82 Orlon®, 144
mounting liquids, 133 orthoclase, 92
mounting media, 63-65, 70, 87 orthorhombic crystals, 146, 147, 174
Mylar® polyester film, 363 orthoscopic illumination, 99, 102
overhead projection, 89
near-field scanning light microscope
(SNLM), 12 parallel extinction, 91
nematic phases and textures, 164 peanut shells, 64
Subject Index 451

periodic structure, 280-281, 318-321 photomicrography (Cont.)

perspective and interpretation of SEM, attitudes employed in, 189
297-298 of biological specimens, 59
petrographical microscope, 94 color in, 189
phase, amplitude, and color contrast, experience involved in, 191-192
methods for, 199-220 imagination in, 191
analytical dispersion staining, 215-216 limitations of modernization, 196
annular stops, 201 macrolenses for, 191-193
bright-field contrast, 201 magnification, useful in, 193
bright-field illumination, 201 micrograph, 189
central stops, 201-202 photomacrography, 193
Christiansen color, 214 photosensitive materials for, 194
constructive interference, 199-201 photomicroscopes, 194, 196, 197
dark contrast, 204 polarimeter, 9
dark-field illumination, 200-202 polarized light and crossed polars in, 108
destructive interference, 199-201 Polaroid® self-processing film, 194
diffraction image, 100 purposes of, 189-190
dispersion staining, 212-217 records kept in, 191-192
Hartshorne and Stuart, substage slot, 207 resolution obtained in, 191
illumination, 203 resolution by photomacrographic lenses,
liquids for dispersion staining, 215 191, 193
modulation contrast, 210-214 resolving power, 191
optical dispersion of solids versus summary of 195, 196, 198
liquids, 212 35-mm cameras for, 194
optical staining, 214-215 photomicroscope, 193, 194, 196, 198
phase-amplitude contrast, 202-209 piezoelectric transducer, 361
phase plate, 204 pixels, 382-383, 385
phase relation (diagrams), 204 planar molecules, 93
Polanret'" microscope, 208, 209 Planck's law, 312
polarized light, 206 pleochroic crystals, 158
refractive index determination, 204 pleochroism, 158
Rheinberg filters, 202 point source of x rays, 352
Saylor negative phase strip, 207 Polanret'" microscope, 208-210, 219
schlieren eyepiece, 218 polarization (interference) figures, 73, 76,
schlieren microscope, 216-218 94, 95, 97, 105, 106
special accessories, 204, 215-216 polarization by reflection, 99
summary,218-220 polarized light, 9
variable phase amplitude, 218 polarizer, 10, 97
phase-contrast microscope, 219 polarizing microscopes, 9, 96, 97, 103, 105,
phenolic resin for mounting, 63-64 109, 110
photoelastic effect, 93-94 Bertrand lens for, 96
photographic materials for electron micro- compensators for, 100
scopy, 285-288 condensers for, 96
photography with microscopes, 189-198 contrast obtained with, 97
photomacrography, 191-193 correction for aberrations in, 101, 102
photomicrographs versus microphoto- cues to depth in, 106
graphs, 189 depth of field in, 106
photomicrography, 35, 189-198 depth of focus in, 102
artistic approach, 189 direction of vibration of polarized light,
scientific approach, 189 104
452 Subject Index

polarizing microscopes (Cont.) polyethylene terephthalate ribbon, 139-140

eyepieces, 96 polyhedral grains, 150
experimentation with, 108, 112 polymeric foam, 356
fiber identification with, 121-137 polymorphism, 165
field of view in, 105 positive and negative uniaxial crystals, 157
first-order red plate, 97 positive replicas for TEM, 284, 285
focusing, 102 potassium chloride, 148, 149
function of polarizer and analyzer in, 96, precipitation hardening, 82, 83, 84-85
97 preparation of samples, 49
glare in, 101, 102, 105, 106 preparation of specimen for electron micro-
illumination for scopy, 387-411
conoscopic, 102 embedding media, 400-405
orthoscopic, 99, 102 embedding transparent particles, 400-405
information about specimens for, 108 fibers, fur, and hair, 392-396
interaction of specimen and polarized fracture surfaces, 398-399
light in, 92, 94 microtomy, 395-396
interference phenomena in, 94, 95 preparing metals and other opaque spec-
interpretation of Michel-Levy chart, 99 imens, 405-410
magnification obtained with, 104 preparing for reflected illumination, 405
Michel-Levy chart, use with, 92-99 replication of surfaces, 397-398
micrometer, linear, 96 thin sections, 400
morphology of specimens employed, ultramicrotomy, 396-397
107 pyrite, 76
objectives, 96
overhead projector, comparison with, quarter-wave plate, 156
89-91 quartz, 94
photographic methods for, 108 quartz-iodine lamp, 248
polar, 88, 96 quartz wedge, 100
polarizer, 96
preparation of specimens for, 108 radiation, 29, 30, 71
principles, 95-97 color, 30
quartz wedge, 97 sources, 104
radiation for, 104 wavelength, 29, 30
recrystallization of samples, 108 Ramsden eyepiece, 6, 51, 55
resolution obtained with, 95-96 real image, 4
retardation plates for, 97, 100 receptors of eye, 23
sensitivity of eye, 99 records, 192
slot for retardation plates, 97 reflected light, study of surfaces by, 61, 88
structure of specimens investigated by, reflective microscopes, 61, 65, 66
107 refractive index, 30, 93, 98
summary of, 109-111 determination, 132-135
thickness of specimens for, 101 of crystals, 148, 149, 152, 153, 159, 160,
working distance of, 106-107 162
Polaroid® analyzer; polarizer, 89, 97 of fibers, 121, 123, 124, 126, 142-143
Polaroid® camera, 10, 93, 193, 287, 288 refrigerated stages, 248-251
Polaroid® photographic materials, 10, 59, replication of surfaces, 284-285
194, 326 resolution, 24, 25
Polaroid® polars, 89, 95, 97, 129 of electron microscope, 271
polars (polarizing devices), 89, 97 principles, 270
PolazoneT" plate, 208, 219 theoretical limit, 271
Subject Index 453

resolving power, 6-8 scanning electron microscope (Cont.)

limit of, 24-25, 41 specimen behavior and preparation, 323-
principles, 25 324
of transmission electron microscope, structure, 318-323
270-271 summary of, 326-327
retardation, 98 synthetic color, 305
colors, 98, 99 topographical contrast in, 303
plates, 100 types of radiation involved in, 303
reticle (reticule), 75, 96 working distance of, 318
retina, eye, 23, 24 x-ray detection in, 314
rock, 92 x-ray emission and absorption in, 310,
311-313
sapphire lenses, 363, 376 scanning near-field microscope, 344, 346
scan coils (of SEM), 297 scanning transmission electron microscope
scanning electron microscope (SEM), 14, (STEM), 13
297-327 scanning tunneling microscope, 16, 339-344
aberrations, 305 schlieren microscope, 218
automatic cameras for, 326 schlieren microscopy, 216-218
cleanliness, importance of, 305-307 Schott glass, 8, 10
color contrast in, 305 scratch-hardness tester, Bierbaum, 238
contrast obtained with, 302-305 secondary and backscattered electrons in
correction of aberrations in SEM, 299
astigmatism, 305 SEM, scanning electron microscope/micro-
chromatic aberration, 305 scopy, 297-310
diffraction disk, 305 Senarmont compensator, 100
spherical aberration, 305 Seneca's magnifier, 1
depth of field of, 317, 318 sensitive-tint plate, 100
depth of focus of, 307 serpentine, 65
detectors employed in, 273 shadow microscopes, 329
display of image in, 300 shadowing specimens, 278, 279, 285, 295
dynamic focusing of, 322 sign of birefringence, 123, 126-128, 129
experimentation with, 322, 323 signal-to-noise ratio, 285, 316-317
field of view of, 316 simple microscope, 38-40
focusing procedure for, 307 smectic phases, 163, 167
history, 12, 15 soap, structure of, 163
illumination, 308-309 "Society" threads, 8
interpretation of image obtained with, sodium chloride, 148, 149
297-300, 321 Sonomicroscope®, scanning laser acoustic
kinds of signal employed in, 297 microscope (SLAM), 361, 362, 363,
magnification, 300, 315-316 366, 369, 370
manipulation of sample in, 324, 325 special cells and cuvettes, 251-253
morphology, 321 spectacles, 1
noise, 316 spherical aberration, 25
polyamide fibers, 119 spherulites, 91, 171
photomicrography with, 325 spread function, 286
principles of, 297-300 stage micrometer, 75, 77
radiation, 309-315 stages, microscopical, 233-255
resolution, limit of, 301-302 cold, 248-251
resolving power of, 300-301 cooling plate, 247
schematic diagram of, 298 glide, 234-235
 454 Subject Index

stages, microscopical (Cont.) substage condensor, 51, 52, 53, 55

hardness, 238 sugar cane, 63
heatable, 239-248 synchrotron radiation, 354
hot/ cold, 249
hot-wire, 245
hot with long working distances, 240- tabular crystals, 140
243 Tech Bits, E-K C., 285, 286, 429
hot with short working distances, 243- temperature measurement, hot and cold
245 stages, 239-251
Koffler hot stage, 240-242 tetragonal crystals, 146, 147, 154, 158
Leitz integrating, 233, 235 textures, 167-169
mechanical, 233-237 thermal depolarization analysis, 243
Mettler hot, 242--243 thermionic electron gun, 308
microhardness, 238 thermocouple, 240, 243, 244, 245
micromanipulators for, 237-239 thermotropism, 163
polarizers for hot, 244 threadlike (smectic), 163
pressure cells for, 252 trademark, 119, 144
refrigeration system for, 250, 251 trade name, 119, 144
rotatable, 234 transmission electron microscope (TEM),
special, 235--237, 251-253 12, 265--296
spindle, 151 aberrations in, 273-274
stage refractometer, 251, 252 accelerating voltage for, 270, 271, 294
summary,254-255 amorphous scattering in, 272
temperature measurement, 240 angular aperture of, 273
thermoelectric cold plate, 249 anisotropy in, 276
universal, 151 artifacts in, 277
very hot, 248 astigmatism and its correction in, 273
stainless steel, structure of, 82 axial-spherical aberration in, 274
stereoimages, 43, 44 cleanliness, importance of, 274-275
stereomicroscopes, 43 comparison with light microscope, 266
advantages of, 44, 45 condensers for, 275--276
binobjective, 43, 44 contour fringes in, 272
binocular, 43, 44 contrast obtained with, 272--273
common objective, 44, 45 contrast versus voltage in, 273
depth of field, 45, 46 cues to depth in, 278--280
Greenough, 44 depth of field in, 280
limitations of, 44, 45 depth of focus in, 275
resolving power of, 46 diffraction contrast obtained with, 272
stands for, 47 dispersion of particles required in, 283,
stereoscopy, 32 284
stigmator, 268, 273, 305 electron diffraction, 288--294
strain-free objectives and oculars, 95, 96, 111 electron lenses for, 268--270
structural classifications of crystals, 145 electron micrography, 285
structural color, 320, 321, 322 experimentation in, 282
structure of specimen, 32, 33, 146 field of view of, 277
anisometri~ 33, 319 flux of electrons in, 276
anisotropic, 152, 154-162 focusing of, 275
crystalline, 145--149 freeze-fracturing specimens for, 285
isometric, 146, 148 illumination in, 275
isotropic, 148 image intensifier, 287
Subject Index 455

transmission electron microscope (Cont.) vaterite, 145

information about the specimen, 281-282 vertical illumination, 69-74
magnetic objective lens for, 268 vertical micrometry, 75-77
micrography with, 285 video image in SAM, 376
Moire pattern anisotropy in, 276 virtual image, 53
morphology of specimen for, 281 visual acuity, 37
photographic materials employed with, voxels, 385
285-288
Polaroid® camera attachment, 287 Watson interference objective, 223-225
preparative procedures for, 282-285 wetting agents, 169-172
principles of, 265-269, 294-296 Wollaston prism, 225, 226
projector lens for, 269 wool, 113
records kept for, 282 working distance, 80, 106
refraction contrast obtained with, 273 Wright eyepiece, 105
replication of surfaces for, 284
resolution obtained with, 271 X-ray analysis, 306, 310--315, 327
resolution of micrographs from, 271 X-ray holography, 359
resolving power versus voltage em- X-ray laser microscopy, 19
ployed in, 270 X-ray micrograph, 189
sampling for, 283 X-ray microscopy, 18, 351-359
scanning transmission electron micro- advantages of, 354
scope (STEM), 15, 288 condensers for, 352-354
spherical aberration and its correction in, contact radiographs from, 357-358
274 electromagnetic lenses for, 352
structure of specimens, study of, 280-- history, 18
281 holography with, 359
summary of, 294-296 micrography with, 352
surfaces, preparation of, 284-285 microradiographs from, 351-359
thickness of specimens for, 280 point-projection radiography in, 352-354
types of, 265 principles of, 351-359
ultramicrotome sections for, 396, 397 radiographs from, 355-356
use of Polaroid® camera in, 287 scanning x-ray microscopes, 358
useful magnification, 276 schematic diagrams of, 383
transparent reflectors, 69-71 sources of x rays for, 354
transparent sheets, foils, and films, 141 summary of, 358-359
Traviss expandable stop, 201 synchrotron radiation, 354
triclinic crystals, 146, 147 X rays in electron microscopy
tube length, 53 absorption, 306, 359
twinned crystals, 91, 92 analysis by, 306
characteristic, 310
ultraviolet light, 10, 11, 30, 58 continuum, 310
uniaxial crystals, 154-158 detectors, 315
uniaxial interference figures, 155-156 spectra, 315
universal stage, 151
useful magnification, 276 zoom objectives, 45