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 Confocal Microscopy and
Multiphoton Excitation Microscopy
The Genesis of Live Cell Imaging
 Confocal Microscopy and
Multiphoton Excitation Microscopy
The Genesis of Live Cell Imaging

Barry R. Masters

Bellingham, Washington USA

Library of Congress Cataloging-in-Publication Data

Masters, Barry R.
Confocal microscopy and multiphoton excitation microscopy : the genesis of live cell
imaging / Barry R. Masters.
p. cm.
“Press monographs v. PM161”—Provided by publisher.
Includes bibliographical references and index.
ISBN 0-8194-6118-0 (alk. paper)
1. Confocal microscopy. 2. Multiphoton excitation microscopy. I. Title.

QH244.M37 2005
502'.82—dc22
2005026105

Published by

SPIE—The International Society for Optical Engineering

P.O. Box 10
Bellingham, Washington 98227-0010 USA
Phone: +1 360 676 3290
Fax: +1 360 647 1445
Email: [email protected]
Web: http://spie.org

Copyright © 2006 The Society of Photo-Optical Instrumentation Engineers

All rights reserved. No part of this publication may be reproduced or distributed in any form or by
any means without written permission of the publisher.

The content of this book reflects the work and thought of the author(s). Every effort has been made
to publish reliable and accurate information herein, but the publisher is not responsible for the
validity of the information or for any outcomes resulting from reliance thereon.

Printed in the United States of America.

Cover image: Confocal microscopy of a fixed, stained, vertical section of human skin. This is a
biopsy specimen from the upper arm. The horizontal field width is 1400 µm.
To our teachers who taught us,
so that we can teach others
On looking back to this event, I am impressed by the great limitations of the
human mind. How quick are we to learn, that is, to imitate what others have
done or thought before. And how slow to understand, that is, to see the deeper
connections. Slowest of all, however, are we in inventing new connections or
even in applying old ideas in a new field.

Frits Zernike, Nobel Lecture, December 11, 1953

Contents

List of Abbreviations xiii

Preface xv

Part I. Optical Microscopy 1

Chapter 1 A Brief History of the Microscope and its Significance

in the Advancement of Biology and Medicine 3
1.1 Timeline of Optical Microscope Development 3
1.2 Key Developments of Fluorescence Microscopy and its
Limitations, Genesis, and Some Applications 9
1.3 Key Advances in Biology and Medicine Made Possible
with the Microscope 14
1.4 Summary 16

Chapter 2 The Optical Microscope: Its Principles, Components,

and Limitations 19
2.1 What is an Optical Microscope? 19
2.2 Image Fidelity: Mapping the Object into the Image 19
2.3 Optical Aberrations 21
2.4 The Compound Microscope 22
2.5 Chief Components of an Optical Microscope 23
2.6 Microscope Objectives 28
2.7 Sets of Conjugate Planes in the Optical Microscope 33
2.8 Epi-Illumination Fluorescence Microscope 34
2.9 Summary 36

Chapter 3 Abbe Theory of Image Formation and Diffraction

of Light in Transmitted Light Microscopes 37
3.1 The Contributions of Abbe 37
3.2 Abbe Diffraction Theory of Image Formation and Optical
Resolution in the Light Microscope 40
3.3 Summary 46

Chapter 4 Optical Resolution and Resolving Power: What It Is,

How to Measure It, and What Limits It 49
4.1 Criteria for Two-Point Resolution 49
4.2 The Role of Depth Discrimination 51

ix
x Contents

4.3 Point Spread Functions Characterize Microscope Performance 52

4.4 Summary 54

Chapter 5 Techniques That Provide Contrast 55

5.1 Nonoptical Techniques 55
5.2 Optical Techniques 57
5.2.1 Phase contrast microscopy 57
5.2.2 Differential interference contrast (DIC) microscopy 60
5.2.3 Video-enhanced contrast microscopy 63
5.3 Summary 64

Part II. Confocal Microscopy 67

Chapter 6 Early Antecedents of Confocal Microscopy 69

6.1 The Problem with Thick Specimens in Light Microscopy 69
6.2 Some Early Attempts to Solve These Problems 69
6.3 Scanning Optical Microscopes: How Scanning the
Illumination Reduces Light Scatter and Increases Contrast 71
6.4 Some Early Developments of Scanning Optical Microscopy 73
6.5 Summary 80

Chapter 7 Optical Sectioning (Depth Discrimination) with

Different Scanning Techniques: The Beginnings
of Confocal Microscopy 83
7.1 The Confocal Microscope: The Problem and Its Solution 83
7.2 Stage-Scanning Confocal Microscope Invented by
Marvin Minsky 85
7.3 Mojmir Petràn, Milan Hadravsky, and Coworkers Invent the
Tandem-Scanning Light Microscope 89
7.4 Guoqing Xiao and Gordon Kino Invent the One-Sided
Confocal Scanning Light Microscope 94
7.5 Effect of Pinhole Size and Spacing on the Performance
of Nipkow Disk Confocal Microscopes 96
7.6 Akira Ichihara and Coworkers at Yokogawa Institute
Corporation Invent a Microlens Nipkow Disk
Confocal Microscope 98
7.7 Svishchev Invents an Oscillating Mirror Scanning-Slit
Confocal Microscope 100
7.8 Laser-Scanning Confocal Microscope Designs 102
7.9 Analytical Expression of Resolution in a Confocal Microscope 107
7.10 Comparison of Different Confocal Microscope Designs:
Which One Should You Purchase? 109
7.11 Limitations of the Confocal Microscope 111
7.12 Summary 115
Contents xi

Chapter 8 The Development of Scanning-Slit Confocal Systems for

Imaging Live Cells, Tissues, and Organs 117
8.1 Scanning-Slit Confocal Microscope 118
8.2 Statement of the Problem: Slit Width Versus Field of View 120
8.3 Goldmann’s Wide-Field Microscope 120
8.4 Maurice Invents Several Types of Specular Microscopes 120
8.5 Svishchev’s Invention of a Scanning-Slit Confocal Microscope 124
8.6 Baer Invents a Tandem-Scanning-Slit Confocal Microscope
with an Oscillating Moving Mirror-Slit Assembly 124
8.7 Maurice Invents a Scanning-Slit Wide-Field
Specular Microscope 125
8.8 Koester Invents a Wide-Field Confocal (Specular)
Microscope for In Vivo Imaging 127
8.9 Masters Develops a Confocal Microscope based on the
Maurice Design with an Axial Scanning Microscope
Objective 128
8.10 Thaer Real-Time Scanning-Slit Clinical Confocal
Microscope 130
8.11 Summary 133

Chapter 9 The Components of a Confocal Microscope 135

9.1 Light Sources 135
9.2 Scanning Systems 139
9.3 Dichroic Mirrors and Filters 141
9.4 Pinholes 142
9.5 Detectors 144
9.6 Microscope Objectives 147
9.7 Summary 149

Part III. Nonlinear Microscopy 151

Chapter 10 The Development of Nonlinear Spectroscopy and

Microscopy 153
10.1 Nonlinear Optical Processes in Spectroscopy and Microscopy 154
10.2 The Nonlinear, Scanning, Harmonic Optical Microscope
is Invented at Oxford University 156
10.3 The Role of Lasers in the Development of Nonlinear
Microscopy 158
10.4 Summary 160

Chapter 11 Multiphoton Excitation Microscopy 161

11.1 Göppert-Mayer’s Theory of Two-Photon Absorption 161
11.2 The Denk, Strickler, and Webb 1990 Science Publication
and 1991 Patent 162
xii Contents

11.3 Comparison of Multiphoton Excitation Microscopy

and Confocal Microscopy 165
11.4 Summary 168

Chapter 12 Theory and Instrumentation of Multiphoton

Excitation Microscopy 169
12.1 Theory 169
12.2 Instrumentation 171
12.2.1 Laser sources 172
12.2.2 Laser beam diagnostic instrumentation 173
12.2.3 Laser pulse spreading due to dispersion 174
12.2.4 Microscope objectives 175
12.2.5 Scanners 175
12.2.6 Detectors 176
12.3 Summary 177

Part IV. The Path to Imaging Live Cells, Tissues, and Organs 179

Chapter 13 Remaining Problems, Limitations,

and Their Partial Solutions 181

Chapter 14 Speculation on Future Directions for Confocal

and Multiphoton Excitation Microscopy 185
14.1 Correlative Microscopy 185
14.2 Multimodal Microscopes 186
14.3 In-Vivo Microscopy or Live Cell and Tissue Imaging 186
14.4 Instrument Development 187
14.5 Summary 188

Chapter 15 Safety and Cleanliness Considerations 189

15.1 Laser Safety 189
15.2 How to Clean Optics 189

Epilogue 191

Appendix: Reference Materials and Resources 193

Index 205
List of Abbreviations

AOTF acousto-optical tunable filter

APD avalanche photodiode
CCD charge-coupled device
CRT cathode-ray tube
CT computed tomography
CSLM confocal scanning laser microscope
DIC differential interference contrast
DOF depth of focus
DPH diphenylhexatriene
FISH fluorescence in situ hybridization
FLIM fluorescence lifetime imaging
FRAP fluorescence recovery after photobleaching
FRET fluorescence resonance energy transfer
fs femtosecond
GFP green fluorescent protein
LED light-emitting diode
LSCM laser scanning confocal microscope
MIAM multiple imaging axis microscopy
NA numerical aperture
OCT optical low-coherence tomography
PDT photodynamic therapy
PMT photomultiplier tube
ps picosecond
PSF point spread function
RMS Royal Microscopical Society
SHG second-harmonic generation
SNR signal-to-noise ratio
SPAD single-photon avalanche photodiode
STED stimulated emission depletion

xiii
Preface

This text explains the fundamentals of confocal microscopy and multiphoton exci-
tation microscopy. It presents the big picture of technological development in opti-
cal microscopy and provides insight into the origins, development, modification,
and application of confocal and multiphoton excitation microscopes and their use
in biology and medicine. This insight is presented in light of the key problems that
each new invention attempted to solve, the various paths to the solution, the myriad
interactions of various inventors and their associated technologies, and the practi-
cal limitations of each step of discovery and technological development. The hu-
man side of these technological developments is also revealed by describing the in-
dividual motivations that drove different scientists to their inventions, as well as the
parallel developments that preceded each stage of technological development.
The repeated convergence of disparate techniques, instruments, theoretical stud-
ies, inventions, and reinventions from a wide variety of disciplines partially solved a
series of problems in the field of microscopy and produced the current renaissance in
modern optical microscopy. Innovative ideas and technical developments came from
many individuals living and working in several countries around the world. Innova-
tion evolves from a broad knowledge base, an awareness of advances in disparate
fields of science, the courage to radically depart from mainstream thinking, and a
clear understanding and statement of the problem to be solved. In many cases, inno-
vations arise from technology transfer and not true invention.
Only recently have technical developments in many separate fieldsfor exam-
ple, medical imaging and cell biologyspread across disciplines. There are many
more examples of advances in different fields finding applications in optical mi-
croscopy. The field of digital image processing was first developed for air and
space imaging applications. The field of adaptive optics, now being developed into
optical microscopes and medical laser imaging devices, was first developed in the
fields of astronomy and military laser weapons. Finally, the emerging medical im-
aging field of optical low-coherence reflectometry and tomography was first devel-
oped for the telecommunications industry as devices for checking fiber optics and
integrated optical devices.
The biomedical applications of optical microscopy constitute an emerging
field driven by spectacular advances in the field of in vivo microscopy. Advances in
confocal microscopy are providing new and important technical solutions in the
fields of endoscopy, minimally invasive surgery, dermatology, and ophthalmol-
ogy. New technical advances in the fields of neurobiology and developmental biol-
ogy build on the instruments described in this book. In many cases, the solutions to
these problems required the optimization of one or more other solutions; typically,
designs compromised one or more parameters (resolution, contrast, time for image
acquisition) to serve a specific purpose. Optical microscopy began with the obser-

xv
xvi Preface

vation of living specimens, and recently there has been a revolution to return to the
observation of in vivo specimens. The combination of spectroscopic techniques and
optical microscopy has resulted in important advances in the field of “optical
biopsy.” Furthermore, these promising new diagnostic techniques are transitioning
from the laboratory to the clinic.
There is an advantage to staying aware of the theoretical and technical ad-
vances of disparate fields of science. Such awareness may prove to be useful in the
development of techniques that seem far from the problem at hand. Being aware of
current and interesting problems in the biomedical area as well as advances in mod-
ern techniques of imaging, signal processing, nanotechnology, and integrated op-
tics creates the conditions for success in interdisciplinary research. I hope the
reader will find these themes useful for stimulating developments in new instru-
mentation as well as innovative, clinically useful applications in the exciting field
of optical microscopy.
Since many excellent books, courses, and Internet resources are available that
describe many aspects of modern microscopy, is there anything new to write on this
subject? I delayed the completion of this book over a period of years while I at-
tempted to answer this question myself. I believe the answer is a definite yes. In this
book, I present several new ways of approaching these two topics. First, I present the
recent developments as partial solutions to existing long-term problems. Second, I
show that many developments are advances on previous instruments and techniques;
there was an intellectual lineage in the development of modern microscopes. Third, I
connect the developments of unique types of microscopes in disparate fields of sci-
ence and medicine, and demonstrate their similarities. Fourth, I indicate the prob-
lems, limitations, artifacts, and experimental difficulties with modern microscopes.
And fifth, I describe the techniques that use optical microscopes for studies on liv-
ing tissue and organs and explain why the new types of microscopes are emerging
as important clinical tools for medical diagnostics. In vivo microscopy and optical
biopsy are active fields of research. This is evident from the exciting research in de-
velopmental biology, ophthalmology, dermatology, oncology, and brain imaging.
Another unique feature of this text is the discussion of the historical develop-
ments of optical microscopy and the technology’s critical impact on the fields of bi-
ology and medicine. The reader may ask why this is necessary for an understanding
of the modern instruments and their applications. There are several reasons. First,
an appreciation of the chain of invention may serve to correct some incorrect attri-
butions of priority and rediscovery of previous inventions. Second, an understand-
ing of the historical development of both instruments and techniques has an impor-
tant educational value in demonstrating serial and parallel approaches to problem
solving in optical microscopy. Third, the study of the antecedents to various technical
developments can put each invention and advance in its proper perspective and per-
haps stimulate innovation. So many excellent books focus on applications that I de-
cided it would be redundant to present extensive reviews of applications. Applica-
tion notes are available online from the companies that manufacture confocal and
multiphoton microscopes.
Preface xvii

The projected audience for this text includes those who wish to gain insight
into confocal microscopy and multiphoton excitation microscopy, and who intend
to apply these techniques to biology and medicine. Therefore, it cannot be stated
that this book was written for a single group of individuals. The projected audience
includes undergraduate students who seek a global insight into the field of modern
optical microscopy, graduate and postdoctoral students who will work with these in-
struments, and physicians who work with engineers and scientists to design and de-
velop new, noninvasive, diagnostic instruments based on confocal or multiphoton
excitation microscopy.
Optical microscopy is a nexus of theory, techniques, and devices from a wide
variety of sources and disciplines, and the organization of this book reflects this
fact. The text is divided into four parts. The largest part is devoted to confocal mi-
croscopy, with an introduction and a part devoted to multiphoton excitation mi-
croscopy. The emphasis is not on the main types of optical microscopes, but on
how various technical developments served to solve the common problems of opti-
cal microscopy. However, throughout the book there are common themes, connec-
tions, and technical solutions to the problems of light microscopy that necessitated
the deliberate repetition of some central concepts and ideas.
Each chapter of this text begins by introducing the materials to be covered and
explaining their role in the book. A summary of key points at the end of each chap-
ter reinforces those critical points. Hopefully the text contains everything that is es-
sential and excludes those topics and details that are not critical for an understand-
ing of the principles and their applications in microscopy. Further insight into the
theory and practice of optical microscopy may be garnered by perusal of the printed
and electronic resources that I have recommended in the appendix.
Part I covers the background, significance, and principles of the optical micro-
scope. Chapter 1 presents a history of the microscope and the development of fluo-
rescent microscopy, and describes the role of microscopy in the advancement of bi-
ology and medicine. Chapter 2 introduces the reader to the optical microscope by
describing its chief components and limitations. Chapter 3 describes the contribu-
tions of Abbe, including the Abbe theory of image formation in an optical micro-
scope. Chapter 4 discusses optical resolution in a microscope. When the major
problems of optical resolution and optical aberrations were solved, the new primary
problems concerned the development of techniques to provide contrast. These
techniques resulted in the emergence of live cell imaging in optical microscopy.
Chapter 5 explains both the nonoptical and optical techniques (phase contrast and
differential interference contrast microscopy) that provide contrast.
Part II describes the partial solutions to the following problem: how to image
thick, highly scattering specimens with an optical microscope. The invention of the
confocal microscope, with its many technical variants, provided one partial solu-
tion. Confocal microscopy improves the resolution, contrast, and optical sectioning
capability of the light microscope. The connecting theme in Part II is that a variety
of techniques were invented and reinvented to solve the same problem: how to con-
struct an optical microscope that has depth discrimination, and thus provide a mi-
xviii Preface

croscope with the capability to “optically section” thick, scattering specimens.

Chapter 6 formulates this problem and then describes several early antecedents to
the development of confocal microscopy. Chapter 7 analyzes the myriad solutions
to the problem of depth discrimination: the various types of confocal microscopes
and their limitations.
Chapter 8 describes the development of scanning-slit confocal microscopes,
which were developed in disparate fields: ophthalmology, neurobiology, and cell
biology. Chapter 8 also plays a special pedagogical role in this book. While superfi-
cially it may seem that the theme is of interest only to ophthalmologists because the
applications are predominantly imaging of the in vivo eye, there is a much deeper
motivation to include these topics. This chapter demonstrates the linkages, connec-
tions, and technology transfers from numerous sources in the progression of tech-
nological development of the confocal microscope. For example, the inventions of
Baer were motivated by the desire to develop a confocal microscope for cell biol-
ogy, and the inventions of Svishchev were motivated by the desire to develop a
confocal microscope to study neurobiology.
The primary message contained in Chapter 8 is that technical problems are
solved by building on the previous and parallel work of others. The insights exposed
in this chapter were derived not only from reading the published papers and patent lit-
erature, but also from personal conversations with Maurice, Svishchev, Petràn,
Hadravsky, Baer, Koester, Kino, and Thaer. I also gained insight from working in
the laboratory with Kino, Maurice, and Thaer. This chapter also provides an impor-
tant lesson: teachers should teach not only those techniques that are popular; they
must have a larger objective to teach how to solve problems by devising creative
solutions. Many of the technical advances developed in Chapter 8 have found their
applications in modern biomedical confocal instruments: scanning-slit confocal
microscopes to investigate the cochlea, study in vivo human skin, and study the
normal and pathological eye. Confocal microscopes based on slits are also being
developed to image large embryos and study their development.
Chapter 9 describes the components of a confocal microscope. Even with the
invention and development of the many types of confocal microscopes, problems
remain. First, the ultraviolet excitation light used to excite many fluorescent dyes in
molecular biology, ion indicator dyes, and endogenous molecules such as
NAD(P)H and neurotransmitters, with absorption bands in the ultraviolet, is toxic
to live cells, tissues, and organisms. Second, the depth of penetration of thick, scat-
tering specimens is a few hundred microns and therefore precludes the imaging of
thicker specimens. Third, the highly intense visible and short wavelength light
causes photobleaching of the specimens during observation. The partial solution to
these problems came with the invention and development of nonlinear microscopy.
Part III describes nonlinear optical microscopy with an emphasis on multi-
photon excitation microscopy. Chapter 10 presents the development of nonlinear
spectroscopy and microscopyin particular, the seminal role played by the inven-
tion of the laser. Chapter 11 presents a detailed description of multiphoton excitation
microscopy, from the Göppert-Mayer theory (Maria Göppert, 1929) to the Denk,
Preface xix

Strickler, and Webb 1990 Science publication. Chapter 12 summarizes the theory
behind and describes the instrumentation of multiphoton excitation microscopy.
Part IV discusses the path to imaging live cells, tissues, and organs. Chapter 13
sets out the remaining problems and describes the limitations of nonlinear micros-
copy. Chapter 14 presents future directions for confocal and multiphoton excitation
microscopy. Chapter 15 addresses the important topic of laser safety and includes a
section on how to clean optics. An epilogue discusses humans as tool makers and
tool users.
The book concludes with an appendix containing an annotated listing of care-
fully selected reference materials and resources. They present applications in great
detail as well as experimental protocols. The appendix also contains a partial listing
of the author’s publications in ophthalmology and dermatology that illustrate the
benefits of confocal and multiphoton microscopy in clinical medicine.
This book tells the story of the development of solutions to formidable prob-
lems in optical microscopy. It also tells the story of the limitations of optical mi-
croscopy: optical aberrations, optical artifacts, fundamental physical limitations of
signal and noise, the quantum nature of light, stray light, background fluorescence,
and light damage to the specimen. The information in this book will be an ongoing
storymicroscope development continues as an active field of progress toward the
partial solution of the following problems: resolution, contrast, and optical micros-
copy of live cells, tissues, and organisms with minimal toxic and destructive ef-
fects. There is much work to be done, as we have only partial solutions to these
problems. The state of the art is a moving target.
Finally, I gladly thank Margaret Thayer and Sharon Streams of SPIE for their
help with the manuscript.

Confocally yours,

Barry R. Masters
November 2005
 Part I
Optical Microscopy
Chapter 1

A Brief History of the Microscope and its

Significance in the Advancement of
Biology and Medicine

This chapter provides a historical foundation of the field of microscopy and out-
lines the significant discoveries in the fields of biology and medicine that are linked
to the microscope. Microscopes, which are devices to image those objects that are
invisible to the naked eye, were transformed from interesting instruments used by
hobbyists to serious scientific instruments used to explore and understand the mi-
croscopic world. Because the technique of fluorescence microscopy is a major, if not
the most widely used, application of both confocal microscopy and multiphoton ex-
citation microscopy, I present a series of key developments of fluorescence micros-
copy. Microscopy began with the observation of live specimens and continues its
growth with technical developments in the fields of intravital microscopy, endos-
copy, and in vivo microscopy. In this chapter, I cite and discuss many of the ad-
vances in both biology and medicine that critically depended on the development
of the optical microscope. These sections provide a framework for the book and
support the premise that technical advances in microscopy have led to both the gen-
eration of new knowledge and understanding as well as advances in diagnostic and
clinical medicine, which has ultimately resulted in an improvement of the human
condition.

1.1 Timeline of Optical Microscope Development

The invention of the microscope (ca.1600) and its improvements over a period of
400 years has resulted in great advances in our understanding of the microscopic
world as well as extremely important advances in biology and medicine. The opti-
cal microscope, a device that in many cases was used as an interesting toy, became
a key instrument in basic science and clinical research: it gives the observer a view
of inner space, that is, the world that cannot be observed with the naked eye because
of insufficient resolution, such as atoms, molecules, viruses, cells, tissues, and mi-
croorganisms.
The reader may ask, why were the numerous early advances made in the design
and manufacture of telescopes not rapidly transferred to the microscope? A partial
answer is that telescopes were the domain of physicists and mathematicians,
whereas the design, construction, and use of the early optical microscopes were left
to laypersons, those whom today we call hobbyists. As we shall see, there were bril-

3
4 Chapter 1

liant exceptions, and the application of mathematics and physics ultimately had
great impact on the development of optical microscopes.
The history of the microscope is intimately connected with advances in optics.
Advances in optics took place over hundreds of years, with contributions from
scholars in many lands. One outstanding example is the work of Abu Ali al-Hasan
ibn al-Hasan ibn al-Haytham, also known as Ibn al-Haytham or Alhazen. He was a
Persian mathematician and astronomer who worked in Cairo, Egypt. Ibn
al-Haytham wrote his treatise Kitab al-Manazir (“Optical Treasures”) in the second
quarter of the 11th century A.D. The first Latin translation, which reached Europe
at about 1200 A.D. was called Perspectiva or De aspectibus; in 1572 a Latin ver-
sion was printed in Basel with the title Opticae Thesauris. He described the laws of
rectilinear propagation of light and of reflection and refraction. In the late 16th and
17th centuries, the Opticae Thesauris was known to Willebrord Snellius (who by
experimentation rediscovered the law of refraction), René Descartes, Johannes
Kepler, and Christiaan Huygens.
The laws of reflection and refraction were used to design optical instruments
for many years, but after the 1690 publications of Huygens’ Traité de la Lumiére,
the Huygens construction was used to trace geometrical wavefronts. The seminal
work of Abbe applied the wave properties of light, specifically light diffraction, to
image formation and optical resolution in the light microscope.
In the 17th century, advances in optics such as the law of refraction, geometri-
cal optics, ray tracing, and Huygens’ theory of light contributed to advances in mi-
croscopy. In the 19th century, the theory of diffraction was exploited by Abbe to
explain optical resolution in a microscope. In the 20th century, the theories of inter-
ference and light polarization were developed into the interference microscope and
the phase contrast microscope. These technical advances resulted in optical micro-
scopes that provide contrast in living, unstained cells and tissues. The development
of the electron microscope built on advances on the understanding of wave optics, es-
pecially in the design and construction of magnetic lenses to focus the electron beam.
Many books in many languages are devoted to the historical development of
the optical and electron microscope. The World Wide Web also contains several
very interesting websites devoted to the history of the microscope, which can be
easily found by means of a search engine such as Google. The following is a brief
timeline of some of the microscope developments and findings that resulted from
advances in microscopy. What follows is neither comprehensive nor complete; it is
only a brief survey of some of the many points of interest. If you are stimulated to
further explore these fascinating topics, then please continue your learning with
some of the excellent books, papers, and websites devoted to them.
In 1590 the Dutch spectacle makers Johannes Jansen and his son Zacharias pro-
duced a microscope based on two lenses held within a tube. In 1667 Robert Hooke
published his book Micrographia, which included his many wonderful observa-
tions of the microscopic world. He correctly described the fruiting structures on liv-
ing molds. The drawings of these microscopic observations contained in his book
helped promote rising public interest in microscopy.
A Brief History of the Microscope 5

In 1675 Antony van Leeuwenhoek, a cloth merchant in Delft, constructed a

single-lens microscope (see Fig. 1.1). He used a small double-convex lens with a
maximum magnification of about 270×. The source of illumination was the sun,
and the eye was the light detector. Leeuwenhoek observed and reported on bacteria,
spermatozoa, red blood cells, simple plants, the structure of the cornea, the ocular
lens, the optic nerve, the cornea, and striated muscle. Thus began live cell imaging
with the microscope.
In 1830, Joseph Jackson Lister demonstrated how a combination of several
lenses could minimize the problem of spherical aberrations. He used one lens with a
small spherical aberration and then added a series of lenses to form a high magnifi-
cation from the entire set. The additional lenses do not add to the spherical aberra-
tion of the first lens, but they increase the total magnification. This important ad-
vance allowed the objective to be constructed with increased apertures, which
resulted in increased resolution.
Another difficult problem was chromatic aberrations. In 1813, the Italian bota-
nist Giovanni Battista Amici solved this difficult problem by inventing a horizontal
achromatic reflecting microscope based on mirrors. Later, in 1850, he used wa-
ter-immersion microscope objectives that had improved resolution. In 1816, Fraun-
hofer invented a single achromatic lens that consisted of two different glasses in
contact. Until the 1830s, with the development and wider availability of achromatic

Figure 1.1 A typical Leeuwenhoek single-lens light microscope.

6 Chapter 1

Figure 1.2 Ernst Abbe.

microscopes, the optical quality of microscopes did not surpass the quality of
images obtained with the simple, single-lens microscope!
Following the inventions of Lister and the subsequent solution offered by ach-
romatic microscope objectives, the next important problem was to increase the res-
olution of the optical microscope. In the 1870s Ernst Abbe (see Fig. 1.2) in Jena
worked out the diffraction theory for image formation and derived a formula (Abbe
formula) that related resolution to the wavelength of the illumination light and the
numerical aperture (NA) of the lens. Abbe showed that in order to maximize the
resolution of the microscope, it is necessary to collect as large a cone of light from
the specimen as possible. Chapter 3 further describes the development of the Abbe
resolution formula.
Although the principle of immersion microscope objectives was known for 200
years, Abbe began 10 years of work on the design of new immersion objectives in
1878. In the 1890s, he introduced several oil-immersion microscope objectives
with a NA of 1.4, which were incorporated into the Carl Zeiss microscopes (see
Fig. 1.3). These newly developed optical microscopes achieved their theoretical
resolution of 0.2 µm with visible light.
Another productive scientist at the Carl Zeiss Corporation was August Köhler
(see Fig. 1.4). In 1893, Köhler invented the subsequently named Köhler illumina-
tion system for microscopes. This important advance permitted uniform illumina-
tion of the specimen, as well as offering the highest obtainable resolution. Today,
all commercial light microscopes are designed for Köhler illumination. Köhler illu-
mination is described further and illustrated in Sec. 2.7.
A Brief History of the Microscope 7

Figure 1.3 Carl Zeiss. Figure 1.4 August Köhler.

Critical illumination uses a light source such as a filament lamp followed by a

field stop. The light passes through an aperture stop and then onto a condenser lens.
The aperture stop sets the NA of the condenser lens. The light from the condenser
lens is directly focused on the specimen.
With Köhler illumination, a different optical setup is used that provides uni-
form illumination of the specimen in the object plane. A lens and a field stop image
the light source onto the back focal plane of the condenser, which provides uniform
illumination in the object plane. Note that critical illumination is much brighter
than Köhler illumination; however, it is very uneven, especially with low-power
microscope objectives.
Another important technical advance derived from Abbe and incorporated by
Zeiss into its microscopes was the Abbe microscope condenser, which is a common
form of the bright-field condenser. It was constructed from two single-lens ele-
ments. The Abbe microscope condenser is designed to have a NA large enough to
match that of any achromatic microscope objective.
Abbe invented, designed, and constructed new optical instruments and compo-
nents for the light microscope; furthermore, he developed a theory of the light mi-
croscope and performed experiments to validate his theory. The Abbe diffraction
theory of image formation, several methods for forming contrast, and a discussion
of various definitions of resolution in the optical microscope will be discussed in
subsequent chapters. Between 1888 and 1895, Abbe published a series of articles
describing his complete theory of image formation in the optical microscope. With
8 Chapter 1

the use of apochromatic objectives and the homogeneous immersion technique, the
optical microscope achieved an Abbe resolution of about 0.2 µm. In fact, the Abbe
resolution limit depends on many physical parameters and will vary under different
circumstances: wavelength, NA, coherence of light. These advances permitted the
observation of many types of bacteria.
In 1903 Richard Zsigmondy (Nobel Prize recipient in 1926) and Wilhelm
Siebenkoph, while working at Carl Zeiss in Jena, invented the ultramicroscope. Ba-
sically, ultramicroscopy is a form of dark-field microscopy using a very bright
source of illumination that is perpendicular to the optical axis of the microscope.
The optical axis is defined as a straight line joining the centers of curvature of lens
surfaces. Ultramicroscopy can detect colloidal particles that are much smaller than
the calculated classical limit of resolution in an optical microscope. While these
particles can be detected by the ultramicroscope, they are not resolved!
In 1911 and afterwards, all the microscope objectives made by Zeiss were
parfocal. Parfocal objectives, which comes from a suggestion from Köhler, means
that the image remains in focus when the observer changes one microscope objec-
tive for another. This advance makes it easy to work with several different
microscope objectives.
After the technical solution to the problems of resolution in the optical micro-
scope arrived, the next set of major technical developments were solutions to the
problems of contrast; i.e., how to produce contrast in thin, transparent specimens
such as living, unstained cells and tissues, which have little inherent contrast. Mi-
croscopic observation of thin transparent living cells are phase objects and are diffi-
cult to observe under a standard light microscope; the main effect of the light in
transmitting through the cells is to change the phase of the light by different
amounts as it travels through various regions of the cells. Unfortunately the human
eye cannot detect differences in phase; but it can detect differences in light
intensity.
The solution to this problem was the work of Fritz Zernike. In 1932, Zernike
(Nobel Prize recipient in 1953) invented a phase contrast microscope, which con-
verts small differences in the phase of the light interacting with a specimen into cor-
responding differences in intensity that the human eye can detect. This important
invention resulted in the widespread application of the phase contrast microscope
to the field of cell biology; in particular, to the microscopic observation of living
cells in tissue culture.
In 1953, the French physicist Georges Nomarski invented the differential inter-
ference contrast (DIC) microscope, which can image transparent cells and tissues.
The DIC microscope converts gradients of phase of the light interacting with a
specimen into intensity differences. This technique is very useful for the observa-
tion of unstained biological specimens and permits the observation of internal
structures in transparent cells.
It should be clear that the invention of improved optical microscopes was a nec-
essary but insufficient condition to lead to many advances in biology and medicine.
In addition to the new microscopes, it was necessary to develop instruments and
A Brief History of the Microscope 9

techniques for sample preparation. The techniques of tissue fixation, embedding,

sectioning, and staining were critical to the success of the microscope. For exam-
ple, the invention of the microtome in 1856 by Welcker, used to produce very thin
sections, was crucial to many of the advances in anatomy and histology that are as-
sociated with the microscope. The microtome provided a technique to section soft
tissue after fixation, thus opening the door for the observation of bacteria in tissues
and the start of microbiology.
Other highlights in the area of sample preparation include the following. The
optical microscope is inherently two dimensional; therefore, a three-dimensional
specimen has to transform into two dimensions, i.e., a very thin specimen. This was
achieved by producing thin smears or by mechanically cutting or sectioning a
fixed, hardened specimen.
Paul Ehrlich wrote his dissertation in 1878 on the theory and practice of stain-
ing tissues with aniline dyes. In 1882, he developed his method for staining the tu-
bercle bacillus. Ehrlich showed that dyes could be classified as basic, acid, or neu-
tral. His work became the basis of future work in hematology and the staining of
cells and tissues.
In 1884 the Danish physician Christian Gram invented what became known as
the Gram stain. His method consisted of staining with gentian violet and potassium
iodide, which results in differential staining or the ability to separate bacteria into
two classes: gram-positive and gram-negative. The invention of what is known as
the Golgi silver stain by Camillo Golgi in 1873 permitted the observation of single
neurons within the complex nervous systems of animals. This technique was modi-
fied and exploited between 1877 and 1900 by Santiago Ramón y Cajal in his semi-
nal, extensive studies on the histology of the nervous system. In more modern
times, the development of the scanning electron microscope, together with
freeze-etching and freeze-fracture techniques, resulted in the observation of the in-
ternal fine structure of cells and membranes. For a wonderful study of the role of
staining in microscopy, the reader is referred to History of Staining (Clark and
Kasten, 1983). With the exception of a brief review of fluorescence microscopy,
these advances in tissue preparation are not discussed further in this book.
Once the problems of optical aberrations and optical resolution were suffi-
ciently solved to permit the manufacture of optical microscopes with sufficient res-
olution to resolve bacteria, the next stage was to develop techniques and methods to
provide improved contrast and specificity. One of the great advances in optical mi-
croscopy, used in both confocal and multiphoton excitation microscopy, is the
invention of fluorescence microscopy.

1.2 Key Developments of Fluorescence Microscopy and its

Limitations, Genesis, and Some Applications
This section integrates the genesis of the fluorescence technique with its physical
basis and points out some important applications. Fluorescence microscopy is a
means to achieve high specificity and contrast. For example, using fluorescent tech-
10 Chapter 1

niques it is possible to label single proteins, single-cell organelles, cytoskeleton

structures, cell membranes, parts of chromosomes, and single neurons; to monitor
intracellular ion concentrations, transmembrane potential differences in excitable
tissues, the expression of specific genes, and detect single molecules.
It is valuable to briefly review the origins of fluorescence spectroscopy, since
this is the foundation of fluorescence microscopy. I strongly recommend that the
reader who wishes to exploit the many aspects of fluorescent microscopy (single
photon or multiphoton) become familiar with Lakowicz’s excellent book Princi-
ples of Fluorescence Spectroscopy and also the catalog provided by Molecular
Probes, Inc.
In 1838, David Brewster observed the phenomenon that today we call fluores-
cence. The great utility and specificity of fluorescence techniques in microscopy is
related to two fundamental properties observed in Cambridge by George G. Stokes
in 1852. Stokes, a physicist and professor of mathematics, observed what he coined
“fluorescence” from a solution of quinine. The source of excitation was sunlight,
the excitation filter was the colored glass of the church window, the emission filter
was a colored glass of wine, and his eye was the detector. Stokes observed that the
fluorescence typically is observed at longer wavelengths than the excitation light;
consequently, today we label this effect the Stokes shift. It is because of the Stokes
shift that sets of fluorescent filters can be used to isolate the fluorescence light from
the excitation light. Stokes performed many experiments with the sun as the source of
excitation light and liquid excitation filters to isolate the ultraviolet light. He used a
yellow barrier filter made from a solution of potassium dichromate to separate the
fluorescence from the excitation light.
The second property of fluorescence that is extremely useful in microscopy is
that the absorption and emission of light from a fluorescent molecule is related to its
structure. The Stokes shift varies for different fluorescent molecules; therefore, dif-
ferent fluorescent molecules can be used in parallel with different fluorescence fil-
ter sets. Fluorescent probes can be designed to cover the spectrum of available light
sources. Modern confocal or multiphoton excitation microscopes can simulta-
neously image two or three different fluorescent channels, i.e., two or three differ-
ent types of fluorescent molecules can be imaged simultaneously in the specimen.
Stokes and others observed that many natural substances, such as chlorophyll,
show fluorescence. Autofluorescence was documented in 1911 by Hans Stübel,
who investigated the natural fluorescence of teeth, bacteria, protozoa, proteins, and
hemoglobin. Over the next several decades, the natural fluorescence of porphyrin
breakdown products, lipofuscin, elastin fibers, and, more recently, the natural fluo-
rescence of the cornea, ocular lens, and human skin were observed.
Fluorescent probes, stains, and intravital dyes also have a fascinating history.
The development of these stains and fluorescent probes is integrated with the ad-
vances of microscope instrumentation for fluorescence microscopy. Haitinger in
1931 coined the word fluorochrome for a fluorescent stain that induces secondary
fluorescence in tissues. One early example is the molecule fluorescein. It was first
synthesized by Baeyer in 1871. It is of interest to note that in 1882 Paul Ehrlich
A Brief History of the Microscope 11

used fluorescein to study the pathways of the aqueous humor in the animal eye.
This may be the first reported used of an intravital dye in physiology.
Perhaps a precursor of the fluorescence microscope was the ultraviolet micro-
scope developed at the Carl Zeiss factory by August Köhler at Jena, Germany.
Shortly before that development, Köhler and Moritz von Rohr developed quartz
monochromatic ultraviolet microscope objectives that transmit at 275 and 280 nm.
From the first fluorescence microscopes by Köhler and Siebenkoph, to more ad-
vanced models by Carl Zeiss in Germany and Carl Reichert in Vienna, fluorescence
microscopes gained performance and utility. Major technical advances included
the development of objectives for the ultraviolet, new powerful light sources, and
new types of excitation and emission filters. There were immediate successful ap-
plications in medicine. For example, Ehrlich used the fluorescence microscope and
fluorescence dyes to observe bacteria in tissues.
In 1911, Hans Stübel used an ultraviolet fluorescence microscope to demon-
strate cell damage caused by short-wavelength excitation light. He described the ul-
traviolet-light-induced death of paramecia. The problem of phototoxicity and
photodamage is still a major limitation of in vivo microscopy for both the confocal
and the multiphoton microscope.
In 1929, based on the work of Phillip Ellinger and August Hirt, Carl Zeiss pro-
duced a fluorescence microscope. Known as an intravital microscope, it used a wa-
ter-immersion microscope objective, an ultraviolet light source, filters, and a verti-
cal or epi-illumination system. The intravital microscope was used for studies of
kidney function, liver function, and the detection of vitamins and bacteria in living
tissues. Ellinger used the device to investigate the structure and alteration of the
microvasculature.
Following the development of the new Zeiss microscope, Leitz in Germany
produced what was called the Ultropak. This microscope was used for many studies
of the intravital fluorescence of living organisms. Other manufacturers, such as
Reichert in Germany and Bausch & Lomb, manufactured fluorescence micro-
scopes.
What were the key technical advances that led to the widespread use of fluo-
rescence microscopes in biology and medicine? In 1959, E. M. Brumberg pub-
lished a paper, “Fluorescence microscopy of biological objects using light from
above”(Brumberg, 1959). He described a special illuminator with interference di-
viding mirrors to separate the excitation light from the fluorescence emission.
Brumberg’s invention was further developed by J. S. Ploem to form the inter-
changeable dichroic mirrors that are used in fluorescence microscopes with inci-
dent light illumination.
In 1946, Larionov and Brumberg observed living mammalian cells with a re-
flected light microscope that used an ultraviolet light source. They observed that
the appearance of living mammalian cells differs from that of injured or dead cells.
This indicates the importance of live cell imaging with the light microscope.
Brumberg’s reflected light fluorescence microscopy is an example of a conven-
tional epi-illumination microscope. The entire field of view is simultaneously illu-
12 Chapter 1

minated (full-field or wide-field illumination), and fluorescence or reflections from

the complete depth of the specimen are imaged. Since the fluorescence comes from
all regions and not just the focal plane, the resulting image is degraded with blur and a
loss of contrast. This epi-illumination system has important advantages over the
transmission light fluorescence microscope: the full NA of the microscope objective
is utilized, and fluorescence microscopy can be combined with Nomarski differential
interference microscopy. The dichromatic beamsplitter or dichroic mirror reflects the
incident light at 90 deg. through the microscope objective to the specimen. The mi-
croscope objective functions as both the condenser and the image-forming lens.
Specificity is one requirement of the development of fluorescent probes. The
word fluorochrome was coined in 1934 by Max Haitinger to describe fluorescent
dyes used to induce fluorescence in tissues. The invention of immunofluorescent
probes by Albert Coons in 1941 was a major development in the field of fluores-
cence microscopy. Coons invented a method that could localize specific classes of
proteins in cells by chemically attaching fluorescein to an antibody. The very high
specificity of the antibody-antigen interaction is the molecular basis. The invention
and development of immunofluorescence was a great advance for clinical medi-
cine. Today a number of fluorescent probes exist, such as various types of green
fluorescent proteins, that can be expressed by cells and used as markers of gene
expression in the study of complex developmental processes.
Both single-photon fluorescence confocal microscopy and multiphoton excita-
tion microscopy depend on, and take advantage of, important previous develop-
ments in fluorescence microscopy. Therefore, it is important to present an over-
view of the historical development of fluorescence microscopy and fluorescent
probes and staining techniques in order to place this technique in its proper context.
See Kasten (1989) for a more detailed account.
Microscope components such as dark-field illumination, dichroic mirrors,
epi-fluorescence illumination systems, and intravital microscopy were all in use
prior to the invention of confocal and multiphoton excitation microscopes. In addi-
tion, autofluorescence, fluorescence probes, fluorescence-linked antibody probes,
and light damage of specimens during microscopic observations were well known.
Many types of fluorophores are used in biological imaging. They include molecules
that show autofluorescence (intrinsic or endogenous fluorescence) such as NAD(P)H
and flavins. Another class of fluorescent molecules, called fluorochromes, is intro-
duced into the specimen and results in extrinsic or exogenous fluorescence. Exam-
ples of the latter include molecular fluorescent probes, fluorescent antibodies, and
green fluorescent proteins.
The second requirement of fluorescent probes to be used for live cell and or-
ganism studies with fluorescence microscopy is that the incorporation of the probe
or its genetic expression in cells does not alter the normal structure and function of
the cells. Finally, the fluorescent probe must not kill or damage the cells in the pres-
ence or absence of excitation light.
The field of quantum dots fluorescent probes is a very active area of research
and development for application in both confocal microscopy and multiphoton ex-
A Brief History of the Microscope 13

citation microscopy. Quantum dots are semiconductor nanoparticles composed of

thousands of atoms with several unique properties that are exploited in their role as
fluorescent probes. Quantum dots can be thought of as nanoparticles in which an
electron is confined in a three-dimensional well. The use of quantum dots as fluores-
cent probes provides several advantages over organic fluorophores. First, quantum
dots can be produced in a wide spectrum of emission wavelengths; the emission spec-
trum is a function of the size of the nanoparticles. By selecting nanocrystals of a spe-
cific size, it is possible to tune the emission wavelength. In addition, by selecting the
materials (e.g., CdSe, CdS, InAs) as well as the particle size, a very wide spectrum of
emission wavelengths can be obtained, which is extremely useful for bioimaging. Sec-
ond, quantum dot fluorophores have emission bands that are narrower than those for
organic fluorophores. Third, the fluorescence lifetime of quantum dots is of the order
of hundreds of nanoseconds. This property is useful when time-gated detection is
used to separate the emission from the quantum dots from the much shorter lifetimes
of cell autofluorescence. Fourth, almost no photobleaching of the quantum dots oc-
curs. However, the quantum efficiency of the quantum dots is low, which results in a
low fluorescence intensity. Major developments include water-soluble quantum
dot fluorophores, quantum dots linked to specific biomolecules, and the develop-
ment of biocompatible quantum dot fluorophores for cells and tissues.
As with many microscopic techniques, at least two major limitations are asso-
ciated with fluorescence microscopy. The first is photodamage, which is associ-
ated with the fluorescent probe and living cells and tissues. It was noted many years
ago that living cells and organisms are more sensitive to ultraviolet light illumina-
tion in the microscope following the application of fluorescent probes. In recent
times, the photophysics of this process has been exploited in the therapeutic tech-
nique of photodynamic therapy (PDT) for cancer.
The second limitation is photobleaching, which is associated with the destruc-
tion of the fluorescence molecules. Experimentally this is observed as the loss of
fluorescence of a stained specimen following continuous illumination with ultravi-
olet light and also with visible light, which causes the fluorophore to fluorescence.
The basis for this phenomenon is the photochemical transformation of the fluores-
cent molecule into another molecule that is not fluorescent. It has been found that
oxygen plays an important role in this process; therefore, reducing the concentra-
tion of oxygen (not advisable for living cells and organisms) can mitigate, but not
eliminate, photobleaching.
Wide-field fluorescence microscopy is a highly useful technique that has ex-
tremely high specificity. For very thin mechanically cut sections, the image of the
specimen is sharp and shows high contrast. Its limitations become evident for thick,
highly scattering specimens, such as in vivo human skin, intravital microscopy of
tissues and organs, in vivo brain imaging, and whole, living embryos. For these
thick specimens, the image is blurred and the contrast degraded because of the fluo-
rescent and scattered light from above and below the focal plane that contributes to
the image. A wide-field fluorescence microscope has no depth resolution. The sig-
nal remains a constant value as the degree of defocus is increased.
14 Chapter 1

Parts II and III of this book explain how the development of confocal micros-
copy and multiphoton excitation microscopy have solved this limitation.

1.3 Key Advances in Biology and Medicine Made Possible with the
Microscope
In 1939 Kausch and Ruska in Germany made the first photomicrographs of the to-
bacco mosaic virus. For the first time, it was possible to observe a virus. After 1945,
the invention of the electron microscope provided the researcher with a resolution
that could not be obtained with optical microscopes. This development led to the
understanding of the fine structure of viruses, the cell and its organelles, the nu-
cleus, cell membranes, and neuronal synapses. It is important to state that these ob-
servations were made on nonliving cells and tissues and therefore could not capture
structural changes, e.g., cell division.
We now briefly review the role of the optical microscope in biology and medi-
cine. We will select some of the highlights in the history of microscopy to illustrate
the connection between the discoveries and the optical microscope.
One of the last advancements in our knowledge of anatomy made during the Re-
naissance was the 1628 discovery of blood circulation by William Harvey. He used a
magnifying glass, which he called a multiplying glass, to study the pulsations of
blood flow in small animals and in his studies of the structure of dissected hearts,
lungs, and blood vessels. Since he used only a magnifying glass and not a light micro-
scope, he could not resolve and therefore could not observe what we call capillaries.
But in 1660 Marcello Malphigi discovered capillaries with his microscopic observa-
tions of frog lungs. He also made many original observations in studies of chick em-
bryology and the structure of human organs, such as the liver and kidney. Malphigi
also used the microscope to discover taste buds and their associated nerves.
Robert Hooke in 1664 described the plant cells in wood, and details of the flea
and the louse. His book, Micrographia, awakened the interest of the general public.
During the same period, Jan Swammerdam observed erythrocytes and the two-cell
division of a frog’s egg. The mammalian ovarian follicle was discovered by Reinier
de Graaf in 1672. From 1650 onwards, the light microscope was an important tool
in the hands of anatomists.
The work of Leeuwenhoek stands out, not only because he built his own micro-
scope, but also because he made many important observations: protozoa, striated
muscle fibers, bacteria, spermatozoa, yeast cells, leukocytes, and the axon and my-
elin of nerve fibers.
With the development of the achromatization of the microscope and, hence, the
correction of chromatic aberrations, another important set of medical advances oc-
curred. In 1857, Pasteur discovered the lactic acid bacterium with an optical micro-
scope. Another milestone that depended on the microscope was Pasteur’s 1857 ex-
periments that refuted the theory of spontaneous generation.
In the 1800s, the optical microscope was used in many studies of anatomy and his-
tology. The concept of the cell is intimately linked with the optical microscope. The
A Brief History of the Microscope 15

publications of Schleiden (1838) and Schwann (1839) on cell theory were other impor-
tant milestones. About 100 years later in 1938, Rudolf Virchow published his book
Cellular Pathology, which became the basis of the new science of pathology.
After 1878, when microscopes were developed with oil-immersion objectives,
a series of important discoveries on the pathogenic nature of microorganisms oc-
curred. The use of oil-immersion objectives increased the NA to about 1.4 and pro-
vided the maximum theoretical resolution with visible light. In the field of microbi-
ology, Robert Koch used the microscope with the new Zeiss immersion objectives
to discover the pathogenic protozoa and bacteria that caused tuberculosis and chol-
era, and the achromatic microscope permitted him to describe the life cycle of the
anthrax bacillus.
In the 1880s, Eli Metchnikoff made important advances in understanding in-
flammation and the process of phagocytosis. The brilliant work of Ramón y Cajal
on the structure of the nervous system took place over several decades and was
made possible with the use of a Carl Zeiss microscope with a 1.4 NA oil-immersion
microscope objective.
Spectroscopy was first applied to chemical analysis in flames and later com-
bined with telescopes to analyze the light from stars. When spectroscopy was com-
bined with the optical microscope, the result was enhanced chemical specificity
and a long series of important advances in fluorescence microscopy.
The microscope was also used to study cells and tissues based on their absorp-
tion and emission spectra. The application of spectroscopy to medicine has a long,
innovation-filled history. The light microscope was an integral part of instruments
designed for both microabsorption studies and microfluorometric studies. The com-
bination of the microscope and the spectrometer or fluorometer permitted the local-
ization of the signal to specific regions of a cell. With the microscope, it became pos-
sible to measure the fluorescence from a specific organelle within a single cell—for
example, to characterize the fluorescence of nucleic acids and nucleoproteins that
were stained with acridine orange. Microfluorometric studies permitted the quanti-
tative measurement of the autofluorescence from the mitochondria under a variety
of physiological states.
In the late 1800s, Charles Alexander MacMunn investigated the spectra of
heme proteins in different states of oxygenation. He summarized his spectroscopic
findings in two important books: The Spectroscope in Medicine (1880) and Spec-
trum Analysis Applied to Biology and Medicine (1914). These early investigations
were extended by David Keilin from 1925 to 1960, during which he used optical
spectroscopy to study the respiratory chain and cytochromes common to plants,
yeasts, and higher animals.
Otto Warburg in the early 1930s observed the fluorescence of NADH in solu-
tion. He used near-ultraviolet excitation light and observed the fluorescence at 460
nm. Warburg’s work was seminal to later studies on the fluorometry of NADH in
mitochondria and muscle.
In 1950 Torbjoern O. Caspersson of the Karolinska Institute, Sweden, pub-
lished Cell Growth and Cell Function, a Cytochemical Study, which summarized
16 Chapter 1

his 20 years of research on microspectrophotometry of cell organelles, nucleotides,

and proteins during the cell cycle, growth, and differentiation. Later, Rudolf Rigler,
Jr., developed microscope-based instrumentation to study nucleic acids within
cells using the technique of microfluorometry. There is a direct link among the
works of Keilin on respiratory proteins, the prolific work of Caspersson on cellular
microspectrophotometry, the microspectroscopy studies of Bo Thorell, the cellular
fluorescence microscopy studies of Joseph Hirschberg, Elli Kohen, and Cahide
Kohen, the work of Rudolf Rigler on cell microfluorometry, and the innovative
studies of Britton Chance on the application of spectroscopic techniques to cellular
respiration. Analytical cytology made great gains in Stockholm from 1945 to 1950.
The light microscope was initially used to explore the microscopic living
world. Ancillary techniques such as fixing, mechanical sectioning, and staining
were necessary components for its contributions in the life sciences and medicine.
The development of the fluorescence microscope together with the continuing de-
velopment of new, more specific stains and dyes resulted in tremendous gains in
specificity and contrast. The invention of the phase contrast microscope and the
differential contrast microscope permitted the observation of live cells and tissues;
however, the long-term observations of thick, highly scattering tissues, embryos and
organisms were still extremely difficult, if not impossible. In vivo microscopy be-
gan with Leeuwenhoek and continues today as a robust microscopic tool in the
neurosciences, developmental biology, and as a clinical diagnostic tool in ophthal-
mology and dermatology.
In this chapter I have placed the development of the optical microscope in its
important place in the history of biology and medicine. In Chapter 2, I present the
principles and components of the optical microscope and discuss its limitations.

1.4 Summary
• The invention of the microscope (about 1600) and its improvements over a pe-
riod of 400 years resulted in great advances in our understanding of the micro-
scopic world and extremely important advances in biology and medicine.
• In 1816 Fraunhofer invented a single achromatic lens that consisted of two dif-
ferent glasses in contact.
• In 1830, Joseph Jackson Lister demonstrated how a combination of several
lenses could minimize the problem of spherical aberrations.
• In 1878 Ernst Abbe in Jena worked out the diffraction theory for lens image
formation and derived a formula (Abbe formula) for the maximum resolution
in optical microscopes.
• With the use of apochromatic objectives and the technique of homogeneous
immersion, the optical microscope achieved the Abbe resolution of about 0.2
µm, which permitted the observation of many types of bacteria.
• The brilliant work of Ramón y Cajal on the structure of the nervous system
took place over several decades and was made possible with the use of a Carl
Zeiss microscope with a 1.4 NA oil-immersion microscope objective.
A Brief History of the Microscope 17

• In 1929, based on the work of Ellinger and Hirt, Carl Zeiss produced a fluores-
cence microscope. This new fluorescence microscope used a water-immersion
microscope objective, an ultraviolet light source, filters, and a vertical or epi-il-
lumination system.
• The invention of immunofluorescent probes by Albert Coons in 1941 was a
major development in the field of fluorescence microscopy.
• In 1959 E. M. Brumberg described a special illuminator with an interference
dividing (dichroic) mirror to separate the excitation light from the fluorescence
emission. Similar dichroic systems are used in all modern fluorescence micro-
scopes.
• The invention of the electron microscope permitted the investigation of the fine
structure of cells, synapses, and viruses. Its resolution, which exceeded that of
light microscopy, is due to the shorter wavelength of the electrons in the elec-
tron microscope.
• The limitation of wide-field fluorescence microscopy becomes evident for the
case in which the specimen is a thick, highly scattering object. For these speci-
mens, the image is blurred and the contrast degraded because of fluorescent
and scattered light from above and below the focal plane that contributes to the
image. A wide-field fluorescence microscope has no depth resolution. The sig-
nal remains a constant value as the degree of defocus is increased.
Chapter 2

The Optical Microscope: Its Principles,

Components, and Limitations

2.1 What is an Optical Microscope?

How does a slide projector differ from a microscope? A slide projector magnifies
the image on the slide; hence, it projects a small image into a larger image on a
screen. A slide projector does not increase the resolution of the object.
A microscope also provides a magnified image for the observer, although its
most important function is to increase the resolution! With a microscope, we can
observe microscopic specimens that would not be visible and resolve details that
were unresolved to the naked eye. But unless there is sufficient contrast, no details
can be observed. So, optical microscopy depends on both sufficient resolution and
sufficient contrast.

2.2 Image Fidelity: Mapping the Object into the Image

As in all imaging systems, the optical microscope maps an object into an image. An
ideal system would make this mapping with the highest fidelity between the object
and the image. Even so, the finite aperture of the lens as well as many forms of opti-
cal aberrations place fundamental limits on the fidelity of this mapping. The aim of
microscope design, manufacture, and practice is to minimize the aberrations, maxi-
mize the resolution, and approach the highest fidelity possible.
What are the requirements for spatial and temporal resolution in optical mi-
croscopy? Spatial resolution denotes the ability of the microscope to resolve or sep-
arate adjacent points on the object. Microscopic observations may only involve the
detection or absence of a particle, or may require the full three-dimensional struc-
ture of a thick, highly scattering specimen such as the eye or skin. The microscope
should be capable of resolving the highest spatial frequencies that are required to
form an image that is appropriate to the questions posed by the observer.
In order to map the object into the image with high fidelity, it is necessary to
map the intensities and the spatial frequencies of the object. Spatial frequency is
the frequency in space for a recurring pattern, given in units of line pairs/mm. The
Nyquist theorem, which is valid for both spatial and temporal frequencies, defines
how to sample the object. The theorem states that the sampling must be performed
at a minimum of two times the highest spatial frequency in the object to accurately
reproduce the object in the image.
If the imaging system does not meet the Nyquist criterion, then there is aliasing
in the image. Aliasing is the phenomenon that occurs when periodic structures in

19
20 Chapter 2

the object are not correctly mapped into the image; hence, the image has the incor-
rect periodic structure. Specifically, aliasing causes spatial frequencies higher than
the Nyquist frequency to be displayed at lower frequencies. Aliasing is another
form of artifact in the image.
Note that there is a trade-off between field of view and resolution. If we require
a large field of view in the image, then the image will have a lower resolution com-
pared with a high-power microscope objective and a high NA. Recently, some mi-
croscope manufacturers (Leica and Nikon) have produced new, non-Royal Micro-
scopical Society (RMS) threaded microscope objectives that combine high NA
(higher resolution) with a lower magnification. The area of the aperture in the back
focal plane and the threaded diameter of the objective are increased over the stan-
dard RMS threaded diameter in order to manufacture these new microscope
objectives.
In addition to the spatial resolution, the transverse resolution (in the plane of
the specimen) and the axial resolution (along the optical axis of the microscope),
there is also temporal resolution. If the specimen is fixed, nonliving, and stationary,
then time is not a consideration. But if we are using the optical microscope to ob-
serve time-dependent events, e.g., changes in ion concentration, calcium waves in
excitable tissue, alterations of intensity in live brain optical imaging, or cell and tis-
sue changes in cell division, fertilization or embryo development, then temporal
resolution is important. In general, we are required to acquire separate images (time
sequence of images) that do not distort the temporal events observed. To do this
correctly, the microscope should acquire images at a rate at least twice that of the
most rapid process. This image acquisition speed will ensure that the time events
are not distorted.
Finally, what contributes to a loss of image fidelity? First, if the resolution of
the optical microscope is too low to image the fine details of the specimen, i.e.,
those parts with a high spatial frequency, then there will be a loss of fine details.
Second, if the kinetics of the process under observation is too rapid compared to the
image acquisition time, then the observed kinetics of the events will be distorted.
Third, optical aberrations in the microscope can degrade the resolution of the im-
age. Fourth, in order to achieve the maximum diffraction-limited performance of a
microscope, it is necessary to use the microscope objective at its full NA. If the illu-
mination source does not completely fill the back focal plane of the objective, the
resolution will be compromised. Fifth, resolution is one requirement for image
formation; appropriate contrast levels are also required.
The optical surfaces of all elements of the microscope, especially the objective,
must be free from dirt, oil, dust, fibers, and mechanical scratches. Dust and me-
chanical scratches degrade the image quality, reduce resolution, and contribute to
increased stray light with concomitant decrease in image contrast. Stray light in an
optical microscope must be minimized since it also degrades contrast. In subse-
quent chapters, we will discuss other factors that reduce image fidelity, including
photophysical bleaching of fluorescence of the specimen, illumination-induced
cell and tissue death and damage, signal-to-noise ratios (SNR) and their effects on
The Optical Microscope: Its Principles, Components, and Limitations 21

image detection, and the role of statistics and the quantum nature of light in light
detectors.
The next section introduces the various types of optical aberrations. Although
modern microscope objectives are available with high degrees of correction for
various types of aberrations, the history of our understanding, measurement, and cor-
rection of optical aberrations represents a major achievement in the advancement of
optical microscopy.

2.3 Optical Aberrations

Optical aberrations represent the failure of an optical system to produce a perfect
image. They are the deviation caused by the properties of the lens materials or the
geometric forms of the refracting and reflecting surfaces.
Modern microscope objectives are manufactured to minimize five categories
of optical aberrations: spherical aberrations, coma, astigmatism, field curvature,
and distortion. This set of monochromatic optical aberrations is called Seidel Aber-
rations in honor of Ludwig von Seidel, who classified them. The aberrations must
be corrected in the listed order; i.e., to correct for astigmatism it is first necessary to
eliminate spherical aberrations and coma.
The presence of spherical aberration results in the lack of a sharp focus point;
instead there is a zone of confusion or caustic. This aberration is caused by a lens with
spherical surfaces for which the peripheral regions refract light more than the central
regions. The optimal correction for spherical aberration of a microscope objective re-
quires a defined object and image distance. This explains why the results of high-NA,
oil-immersion objectives used with a coverslip to image thick specimens are severely
limited by the generation of spherical aberrations at increasing distances below the
coverslip. Other sources of spherical aberration are mismatch of tube length and ob-
jective, nonstandard thickness of coverslips, and poor-quality immersion oil.
Coma is a lens aberration that occurs when light is focused at points off the op-
tical axis. The optical axis is perpendicular to the plane of the lens and passes
through the center of a circular lens. The name, derived from the Latin term for
comet, is due to the fact that the aberrated image of a point looks like a comet.
Astigmatism must be corrected after spherical aberration and coma are cor-
rected. The Seidel aberration of astigmatism is not equivalent to the term astigma-
tism as applied to human vision. For the human eye, the nonspherical shape of the
lens results in different foci for different meridional planes. In contrast, Seidel
astigmatism can occur with perfectly spherical lens surfaces.
It is first necessary to define two planes in the optical system. The meridional
or tangential plane contains both optical axis and the object point. The sagittal
plane is perpendicular to the tangential plane and contains the object point. What is
observed is that points will be blurred only in a circular direction in the tangential
focal plane. In the sagittal focal plane, only the radial direction has blurring.
Field curvature is another aberration that persists after spherical aberration,
coma and astigmatism are corrected. In the presence of a lens with field curvature,
22 Chapter 2

object points that are in a plane will be imaged onto a paraboloidal surface. Field
curvature makes a flat field appear curved and various regions of the image to be
blurred. In the presence of field curvature, when imaging with a high-aperture mi-
croscope objective, one observes that either the center or the peripheral of the field
of view is sharply focused.
Distortion is a displacement of the entire image rather than a blurring of the in-
dividual points that form the image. Distortion occurs when the lens magnification
varies from the center to the periphery. Distortion can occur as either pincushion or
barrel distortion.
In addition to the previous Seidel aberrations, corrections must be made for ax-
ial and lateral chromatic aberration, which cause the focus position to depend on
the wavelength of the illumination light. Spherical and chromatic aberrations affect
the entire field; in contrast, the other types of aberrations are only important for
off-axis image points.
Axial chromatic aberration occurs when different light wavelengths are not
focused at a single point on the optical axis. Each color of light will focus at a dif-
ferent point on the optical axis. The image is surrounded by fringes of different col-
ors that change with varying focus. A concave lens of a glass of one refractive index
can be joined to a second convex lens of a different refractive index to form an ach-
romatic lens in which several wavelengths focus at the same point on the optical
axis. Note the definition of refractive index: the ratio of the speed of light (phase
velocity) in a vacuum to that in a given medium.
Lateral chromatic aberration occurs when different wavelengths are magni-
fied at different ratios. This effect is greatest at the outside of the visual field of the
object where the light rays are more oblique. Each object is surrounded by a colored
fringe. This effect can be compensated by eyepiece design and the microscope ob-
jective (in older microscopes) or in the objective alone (in modern microscopes).

2.4 The Compound Microscope

The compound optical microscope uses two lenses (microscope objective and
eyepiece lens) to project a magnified image of the specimen onto the image detec-
tor (solid state detector or the eye of the observer). Figures 2.1, 2.2, and 2.3 show
the layout of the compound microscope, its conjugate planes, the illuminating
light path, and the image-forming light path. The first lens is the microscope ob-
jective and the second lens is the ocular or eyepiece. Image formation interpreted
in terms of the Abbe diffraction theory, to be discussed in the next chapter, is criti-
cally dependent on two lenses: the microscope objective and the condenser lens.
The function of the microscope objective is to collect the light diffracted by the
specimen and to form a magnified real image at the intermediate image plane near
the ocular. The function of the condenser lens is to evenly illuminate the speci-
men.
It is necessary to define real and virtual images. A real image can be observed
on a screen or captured on photographic film or on a solid state detector. In contrast
The Optical Microscope: Its Principles, Components, and Limitations 23

Figure 2.1 Schematic diagram of a compound microscope.

to the real image, a virtual image can only be observed or detected with the use of
another lens or lens system.

2.5 Chief Components of an Optical Microscope

As shown in Fig. 2.1, the components of the microscope include the light source, il-
lumination system, condenser, various diaphragms, the stage, immersion fluid,
cover glass (or coverslip), microscope objective, tube, tube lens, eyepiece, and var-
ious filters, polarizers, and other optical elements. The detector (remember that in-
tensity is detected) is either the naked eye or a film or electronic imaging system.
24 Chapter 2

Figure 2.2 Schematic diagram showing the four conjugate field planes and the four
conjugate aperture planes, showing bright-field Köhler illumination.

The light source illuminates the object and is the ultimate source of light that
forms the detected image. The components located below the specimen include
those of the condenser system. In addition to the condenser lens, there is a field dia-
phragm and an aperture iris. These components provide uniform illumination.
Above the specimen in Fig. 2.1 are two important magnifying lenses. The mi-
croscope objective is the most important optical element to form a high-resolution
image; it collects the light from the various points in the specimen and redirects the
light to the corresponding points in the image. A real image of the object is pro-
jected into the upper part of the microscope (microscope body tube). The second
optical element, the eyepiece lens, forms a real image on the retina or the camera.
We begin by defining some useful terms. These terms and many others in English,
German, and French are defined in the RMS Dictionary of Light Microscopy (1989).
An aperture is the area of a lens that is available for the passage of light. A pu-
pil is defined as the apparent minimum common cross section of all light-ray bun-
The Optical Microscope: Its Principles, Components, and Limitations 25

Figure 2.3 Schematic diagram showing the illuminating and image- forming light
paths for an infinity-corrected light microscope with bright-field Köhler illumination.

dles both on the object side (the entrance pupil) and the image side (the exit pu-
pil). A diaphragm provides a mechanical limitation of an opening normal to the
optical axis that restricts the cross-sectional area of the light path at a defined place
in the optical system. An aperture diaphragm is a diaphragm in the plane of any
aperture of an optical system that limits its pupil (see Fig. 2.4). A field diaphragm
is one in the object plane or any plane conjugate to it (see Fig. 2.4). The word con-
jugate means linked together by the rules of geometrical optics. It is also the name
of the iris diaphragm in front of the collecting lens of the light source. With Köhler
illumination, the condenser focuses the image of the field diaphragm onto the
image plane (see Fig. 2.2).
26 Chapter 2

Figure 2.4 Schematic diagram of an epi-illumination fluorescence microscope.

A condenser iris diaphragm is located at the front focal plane of the con-
denser lens of a microscope. With Köhler illumination, the iris is located in a plane
conjugate to the back focal plane of the objective lens. This iris continuously alters
the numerical aperture of the condenser.
Dioptric describes optical elements, indicating that they operate by refraction,
i.e., using lenses. Catoptric describes an optical system that operates by reflection.
Catadioptric refers to an optical system with both reflecting and refracting sur-
faces that are used to form the image.
Two common terms used with lenses are back and front focal planes. The back
focal plane of a lens is the focal plane that lies behind the lens when viewed in the
direction of the passage of light. The front focal plane lies in front of the lens when
viewed in the direction of the passage of light.
The Optical Microscope: Its Principles, Components, and Limitations 27

The mechanical parts of a classical optical microscope include the tube that
contains both the microscope objective and the eyepiece, the stand that supports the
complete instrument, and the specimen stage. The tube is that part of the micro-
scope that connects the objective and the eyepiece.
The mechanical tube length is the distance from the top of the microscope ob-
jective housing to the top of the tube into which the eyepiece is set. For microscope
objectives with short focal lengths, the mechanical tube length is approximately
equal to the optical tube length that is defined as the distance from the back (rear)
focal plane of the microscope objective to the intermediate image plane. Modern
microscope manufacturers use different tube lengths: Nikon and Leica (200 mm),
Olympus (180 mm), and Zeiss (160 mm).
Once the light microscope is properly aligned and adjusted, the spatial posi-
tions of all the components are fixed. Note that temperature variations and mechan-
ical vibrations from the microscope system itself, or motors, controllers, shutters,
lamp and laser cooling fans, or cooling water flow are greatly magnified and ad-
versely affect operation. In addition, vibrations from sounds, elevators, motors, and
street traffic can be transmitted to the microscope. Therefore, microscopes are usu-
ally placed on anti-vibration optical tables, and great efforts are made to isolate the
microscope from all sources of vibration. The microscope stand is designed to
provide a stable mechanical system to hold the various components.
The focal plane of the microscope objective is displaced through the volume
of the specimen in two ways. First, the mechanical stage that contains the speci-
men can be manually or mechanically moved by a precise stepper motor driven
along the optical axis of the microscope. Second, for very small but highly precise
movements along the optical axis, a piezoelectric micropositioning device that is
attached to the microscope tube on one side and the microscope objective on the
other can be used to displace the objective relative to the stationary microscope
stage. In each case, the distance between the specimen and the front lens of the
microscope objective is changed. It is important to note that mechanical motion
of any kind can and will induce transient mechanical vibrations in the micro-
scope. Therefore, in an automated series of z-measurements or optical sections at
different depths within the specimen, for example with the confocal microscope,
it is important to allow mechanical vibration to be damped before each sequence
of image acquisition.
The eyepiece or ocular is a lens system that is responsible for the angular mag-
nifications of the final virtual image that it forms at infinity from the primary im-
age. This is converted into a real image by the observer’s eye or other converging
lens system (see Fig. 2.2).
Another important optical element is the tube lens (see Fig. 2.3), defined as an
intermediate lens designed to operate as an essential component of infinity-cor-
rected objectives and located in either the body tube or the viewing tube of the mi-
croscope. The tube lens is associated with the objective lens system and will influ-
ence the effective magnifying power and possibly the state of correction of the
system.
28 Chapter 2

Finally, it is necessary to define two confusing terms: depth of field and depth
of focus. Depth of field (depth of sharpness in object space) is the axial depth of the
space on both sides of the object plane within which the object can be moved with-
out detectable loss of sharpness in the image, and within which features of the ob-
ject appear acceptably sharp in the image while the position of the image plane is
maintained. Depth of focus (depth of sharpness in image space) is defined as the
axial depth of the space on both sides of the image plane within which the image ap-
pears acceptably sharp while the positions of the object plane and of the objective
are maintained.

2.6 Microscope Objectives

The microscope objective is a critical component of the optical microscope. Typi-
cally, a modern optical microscope contains several different objectives that are
contained in a rotatable turret connected to the microscope tube. The sets of micro-
scope objectives are parfocal; that is, they are mounted so that with the specimen
in a fixed position each of the objectives is at the same level of focus within the
specimen. This feature makes it easy to switch microscope objectives. A modern
microscope objective is designed to minimize optical aberrations, stray light, and
fluorescence from its components. Table 2.1 shows the various types of micro-
scope objectives and their corrections.
Microscope objectives are optimized for specific applications and classified
into broad groups with differences in the degree of correction from aberrations and
also in cost. Achromats are objectives that are corrected at 540 nm for spherical ab-
errations. They are also corrected for chromatic aberration at both red and blue
wavelengths (656 nm and 486 nm). They have excellent performance when used
with monochromatic light. When used for low-magnification work, i.e., below 40x,
they are a good selection based on performance and price.

Table 2.1 Microscope objectives and their corrections.

Type of Corrections for Aberrations Field Curvature

Microscope Spherical Chromatic Correction
Objective Correction Correction Needed?

achromat 1 wavelength 2 wavelengths no

plan achromat 1 wavelength 2 wavelengths yes
fluorite 3 wavelengths 3 wavelengths no
plan fluorite 3 wavelengths 3 wavelengths yes
apochromat 4 wavelengths 4 wavelengths no
plan apochromat 4 wavelengths 4 wavelengths yes
The Optical Microscope: Its Principles, Components, and Limitations 29

The word plan placed in front of the type of microscope objective indicates
that the objective is corrected for field curvature. Fluorite or semiapochromate
objectives are corrected for both spherical aberration and chromatic aberration at
three wavelengths. These objectives can be used to the near-ultraviolet and have
good contrast and high transparency. With these characteristics, they are useful for
differential contrast, polarization, and immunofluorescence microscopy.
Apochromats, originally designed by Abbe in 1886, are the most expensive
class of microscope objectives, but are highly corrected at four wavelengths for
both spherical and chromatic aberrations. Useful for color microscopy with white
light, they are available with large (1.4) NAs and are also transparent in the near-ul-
traviolet. Therefore, they are also very useful for low-light fluorescence micros-
copy and for fluorescence microscopy using dyes that have absorption bands in the
ultraviolet region.
An aplanatic lens is corrected for both spherical aberrations and coma. Achro-
matic microscope objectives or achromats are corrected for spherical aberrations at
one color (green) and for chromatic aberrations at two colors. Apochromats are
corrected for spherical aberrations at two and for chromatic aberrations at three
wavelengths.
A very useful technical advance is the advent of infinity optical systems. In a
microscope based on a finite optical system, the light from a specimen passes
through the objective and converges toward the primary image plane (see Fig. 2.5).
The focus in the primary image plane is also the eyepiece focus point. That is the
basic light path in a standard light microscope.
A microscope with an infinity optical system is very different. The micro-
scope objective focuses light from a point source (i.e., a small fluorescent bead) to
form a parallel beam of light. The infinity-corrected microscope does not form an
image; instead the parallel beam of light is focused by a tube lens to form a real im-
age in the primary intermediate image plane, which is conjugate with the object
plane and the retina. In the space between the microscope objective and the tube
lens (the infinity space), the light from the specimen is a set of parallel rays.
Many modern microscopes use infinity-corrected microscope objectives. An
example, manufactured by Nikon for fluorescence microscopy, is the Plan Apo 60×
oil-immersion, NA 1.40 with a free working distance of 0.21 mm. The free work-
ing distance is defined as the distance or depth of free space between the top or
front lens of the objective and the surface of the specimen or the cover glass.
This microscope objective is designed to work optimally with a microscope
that has a tube length of 200 mm, a thread diameter of 25 mm, and a parfocal dis-
tance of 60 mm. Parfocal distance of a microscope objective is the distance in air
between the object plane (the uncovered surface of the object) and the locating
flange of the microscope objective.
One advantage of infinity-corrected objectives is that it is simple to insert opti-
cal elements into the microscope tube (i.e., infinity space); for example,
waveplates, filters, or compensators. The only optical requirement is that they have
plane-parallel surfaces. In that case, their location is not critical and there is mini-
 Another Random Document on
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He obtained the good opinion of all his fellow-students; but José was
his friend, and to serve him he would have gone through fire and
water.
Solon has, I think, said: "No praise before death;" and he said
wisely, for one moment of forgetfulness might tarnish even the most
irreproachable life. Who can boast of being infallible, especially in
youth? José, the prudent José, learned this to his cost; for,
unhappily, these reflections apply to him. It was his first fault; but it
was a serious one, as we shall show.
Occasionally, during the summer, José's companions formed
themselves into little parties, and spent the day in the country in an
inexpensive manner; for they had both good legs and a good
appetite, and required only simple fare. They went into the environs
of Paris, and returned home in the evening, after spending a
pleasant day. But José, though keenly alive to the pleasure of these
parties, often refused to join them, as they occasioned a loss of time
which to him was very precious. However, the fête of Saint Cloud
was approaching, and Francisco proposed going to see the fountains
play. This proposition was eagerly acceded to, and José felt a strong
desire to accompany them. He had never seen the fountains play,
and this sight possesses powerful attractions to a Parisian, and
especially to a young man like José, who was ignorant of almost
everything foreign to his studies. It was, therefore, decided that they
should form a party of twelve, dine at Saint Cloud, and share the
expenses between them. José communicated his project to Dame
Robert, and this excellent woman loved him too tenderly to oppose
what appeared likely to afford him so much pleasure; nevertheless,
at the moment of his departure, she followed him to the door,
recommending him not to lose his purse in the crowd, and not get
into any quarrel with the boothkeepers at the fair. José smiled at her
fears, and hastened to rejoin his friends, who were to meet him at
the Tuileries.
The young people merrily pursued their way, already amused with
the procession of carriages, horses, carts, and pedestrians, like
themselves, all taking the same direction. On arriving at Saint Cloud,
they commenced with a simple breakfast, the greater part of their
little treasure being reserved for their evening meal. They then took
a survey of the booths, admired the cascades, listened to the bands,
marvelled at the conjurors, and even laughed at Punch's buffoonery,
as the numerous spectators of this fête are annually accustomed to
do at the same season of the year. They several times fell in with a
troop of young men, pupils of a different master, and their rivals in
glory and talent. These two studios were jealous and inimical, as
well from party spirit as from a sentiment of attachment to their
masters; and this animosity had been manifested in more than one
encounter of class against class, for there existed between them no
individual aversion. On this occasion, they looked at each other with
an expression of irony.
"Oh, oh!" cried José's companions, "here are the Princes of
Babocheux and Flou-flou."[6]
"Yes, gentlemen," replied the others, "ready to admire your Croûtes
aux épinards."[7]
Each made a grimace; but they separated without saying anything
more.
Returned to the inn, after having wandered about for a considerable
time, José and his companions were prepared to enjoy a repast,
dainty to them, from their simple habits; and they contemplated it
with a degree of satisfaction, which would have made many young
people, spoiled either by fortune or by their parents, shrug their
shoulders with contempt. Their table was laid in what was called the
garden, a small enclosure surrounded by walls, and covered with a
trellis work, ornamented with honey-suckle and vine. This spot was
capable of containing five or six tables, separated by partitions, also
of trellis work, and though very warm, still there was a little more air
there than in the house; besides the circumstances of the guests
permitted them no choice, and our young students were therefore
very well satisfied at being so comfortably located.
As may be imagined, there was no lack of conversation; this turned
at first upon their good cheer, which they had time enough to enjoy,
as the waiters were so much occupied, that they allowed full half an
hour to intervene between each course.
"Well! Angevin, my friend," said Francisco,—for José's protection had
caused him to be received into the party,—"what do you think of this
Marinade?[8] something better than your usual fare, hey!"
"I should think so," replied the Angevin, holding out his plate for the
third or fourth time. "Plague take the stew, I shan't touch it to-
morrow."
"What!" cried the young folks, laughing; "what do you mean by the
stew?"
"Oh! nothing, nothing," replied the Angevin, already regretting his
indiscretion; but his companions insisting, and José joining in their
request, he told them, laughingly, that, finding it impossible to live in
Paris in any other than the most economical manner, he had ended,
after trying various plans, by purchasing a large stew-pan and an
earthen stove. He filled it once a week with turnips, potatoes, and a
few slices of bacon, which he boiled altogether, and this ragout,
which was hot only for the first time, served him for dinner during
the whole week. He was so much accustomed to call it his stew, that
the word had inadvertently escaped him in the presence of his
companions.
"My poor fellow!" said José, holding out his hand to him. "Poor
Angevin!" repeated the others; and, so far from laughing, a
momentary silence pervaded the whole party.
"Gentlemen," said Francisco, who blushed at the remembrance of
the murmurs which often escaped him on account of what he called
his father's unnecessary economy; "I am going to propose a toast:
to the success of our worthy comrade! May he gain the prize, even
though I should myself have to be left behind him."
The young friends rose, and eagerly touched their glasses, while the
Angevin, deeply moved, repeated, in a tone of emotion, "Oh! Berr,
Berr, it is to you that I owe all this!"
Their conversation then turned upon painting, and upon the hopes
entertained by Francisco and José, who flattered themselves with
being this year permitted to compete for the prize, not, however,
with the presumptuous hope of obtaining it, for they were both very
young, especially José; but the mere fact of being admitted to the
competition counted for much, and they might perhaps deserve
honourable mention. Francisco had, moreover, an additional motive
for desiring, as soon as possible, to distinguish himself. Glory was
not the only passion which agitated his breast; for some time past
he had grieved at being without fortune or reputation, which
prevented him from aspiring to an alliance which would have
crowned his fondest wishes. But this prospect was so distant and so
uncertain that he had never spoken of it, even to José, except once,
and then very vaguely.
Whilst, then, they were conversing upon art, with an enthusiasm
worthy of the subject, they were interrupted by a loud noise, which
proceeded from a room on the first-floor, immediately above the
spot where they were dining. As the window was open, it was easy
to overhear what passed, and, by a natural feeling of curiosity, the
young guests checked their conversation, in order to listen to their
joyous neighbours.

"By the powers!" cried one, "here's a splendid charge[9] it ought to

be hung up in Barbe's shop; the veriest rapin[10] would recognise it!"
"Yes," said another, "it is his very self, with his vagabond air! Ah! ah!
my gentlemen of the green and yellow school! you fancy you are
going to carry off the next prizes from us, do you? We shall see, my
lads! we shall see!"
Our young friends looked at each other with indignation, and softly
approached the window, in order to hear more, for they recognised
their antagonists, who doubtless little imagined they were so near.
"For my part," said one of the rival students, "I fear neither Rivol nor
Enguehard, nor even the famous Berr, about whom they make such
a fuss; he is ready enough, and up to the tricks of the art, and that's
all. Enguehard is an idle dog, who does no good, while Rivol is too
well off ever to be anything more than an amateur and a dauber. So
down with the Purists, and long life to the Colourists!"
"Long life to the Colourists!" shouted his companions, and they
added many other jests so bitter and so personal, that José and his
friends, already animated by a few glasses of wine, to which they
were unaccustomed, could no longer restrain their indignation, and
commenced the attack by throwing into the room plates, knives, and
anything else which happened to come in their way. The enemy
hastened to the window, and recognising their adversaries, uttered
shouts of laughter, which completely exasperated the others. A
decanter, thrown by José, struck the forehead of one of the
Colourists, who in their turn became furious, and began to make a
descent, by means of the trellis-work placed beneath the window,
for the purpose of crushing their antagonists. A battle then ensued,
amidst bitter insults. Fragments of broken chairs flew about in all
directions, the women at the neighbouring tables screamed, the
children cried, and the men rushed forward to separate the
combatants, without being in the least able to understand the
invectives with which they overwhelmed each other, under the
names of Purists and Colourists. The landlord of the inn, attracted by
the noise, ran towards the scene of action, followed by his waiters,
and they succeeded, without much difficulty, in calming those who
were only soldiers—for they fought solely for the honour of their
corps. But the chiefs did not so readily listen to reason; Enguehard
was stretched upon the ground, his arms pinioned by the two stout
hands of a Colourist, and José, absolutely out of his senses, was
stifling, with the weight of his knee, the young man who had spoken
of him with so much contempt, and who had just been conquered
by his impetuosity.
These four madmen would listen to nothing, and were at length
obliged to be separated by main force; but José, while still
struggling, slipped over some pieces of the broken plates, and gave
himself so violent a twist that he was unable to rise, and was obliged
to remain seated on the ground, suffering excruciating pain.
It being proved by the testimony of eye-witnesses, that the young
people in the garden had commenced this memorable battle, by
throwing plates into the room, and that the Colourists had only
broken the trellis-work in descending, the landlord contented himself
with a slight sum as indemnification, and allowed them to depart;
but José and his friends had done considerable damage, and had
been the first to commence the disturbance; they had only sufficient
money to defray the expenses of their dinner, and the innkeeper
declared that he would be paid, and that he should send for the
police. Francisco increased the man's anger, by the rage into which
he put himself; the poor Angevin employed prayers and tears, to
soften the innkeeper; while José, ashamed, and in despair,
maintained a gloomy silence, abandoning himself to the most
melancholy reflections, when his name, pronounced by a severe and
well-known voice, made him utter a cry, and hide his face in his
hands.
The voice was that of the good and vigilant Gabri, who had been
induced by his active friendship for José to follow him to the fête,
and to watch over the inexperience which he very justly attributed to
him. He had watched the young men from a distance, and
determined not to make his appearance, except in case of accident;
finally, having been able to find accommodation only at the farther
end of the place occupied by them at the inn, he had been the last
to arrive at the scene of action.
"Sir," he said coldly to the innkeeper, "estimate the damage done,
and make out your account; I will discharge the debts of these
madcaps, who are of my acquaintance, and we will afterwards settle
matters together."
The host, who was no cheat, and who was, moreover, too happy to
be paid without any further trouble, made out a tolerably reasonable
account, which Gabri immediately discharged. Then telling Francisco
and the Angevin to support José, who was unable to walk, he placed
him in a carriage, and drove off with him, after having saluted the
troop of students, who were still too much bewildered by what had
taken place even to think of thanking him.
Gabri had placed José in the cabriolet in as convenient a position as
possible for his injured leg, while he went upon the box himself, and
during the whole of their way home never once addressed a single
word to the poor sufferer, nor even turned his head towards him,
notwithstanding the complaints which the constant jolting of the
rude vehicle drew from the culprit. The well-paid coachman took
them as far as Dame Robert's door. "There, there he is," said Gabri
to the terrified woman, "and now good evening; I will see him again
when he has recovered, and grown wiser;" and he turned away
without listening to Dame Robert's exclamations, who in her trouble
did not perceive that José had almost fainted. He was conveyed to
bed, his dislocated ankle set, and his numerous bruises attended to:
but the wine which he had taken, and the violent excitement which
had followed an excess altogether new to him, brought on a
somewhat severe illness, which lasted for several days; and even
when it was subdued he was obliged to remain six weeks with his
foot resting upon a chair, without being able to move. We may judge
of his grief and remorse, which many circumstances contributed to
augment. Gabri allowed his heart to be touched by his repentance,
and consented to see him; but he was sad, and Dame Robert
uneasy; and José was one day deeply grieved to see her, while
thinking herself unobserved, lock up a bottle of brandy which was
standing near him.
Soon afterwards he had to endure a far more bitter trial. The time
for competing for the prizes arrived; Francisco was admitted for the
sketches; while José, who was only just beginning to walk, and
whose studies had, moreover, been too much interrupted, was
obliged to give up all hope for that year, and endure the
mortification of finding himself left behind by companions
considerably less advanced than himself. Francisco, though sincerely
grieved at his friend's misfortune, felt his ardour increased from not
having to compete with so formidable a rival. He made astonishing
efforts to sustain the honour of the school, but he only obtained the
second prize, which did not send its possessor to Rome: the first was
carried off by that same chief of the Colourists who had spoken of
José with so much contempt: and thus the poor boy remained with
the bitter remembrance of two months passed in suffering, of a
triumph lost, and of a folly committed.
However, as it is not considered that a young man must necessarily
be dishonoured because he has once been intoxicated and beaten,
José, after having passed some time in a state of complete apathy,
at length took courage. He perceived, that instead of abandoning
himself to vain regrets he ought to endeavour to repair his fault,
while that intimate consciousness of power, in which even the most
modest cannot help believing, told him, that he could repair
everything. It usually happens after a first fault, that a young man
either turns from the evil path, or pursues it for the rest of his life.
José had too much superiority of nature not to profit by experience.
Redoubling, therefore, both his assiduity and zeal, he made such
marked progress during the course of the current year, that
Monsieur G. decided that he also might compete as well as Francisco
and Rivol.
The place in which the young people then worked at their prize
pictures, was situated at the top of that same Pavilion du Musée, of
which we have already spoken. It was divided into several little
compartments, or cells, called boxes, in each of which a student was
shut up, so as to allow him no communication with his companions,
and still less with his master or with strangers. The subject for the
picture was chosen by the professors of painting of the Institution;
the programme was distributed to the candidates, and when their
sketches were made, and received, they were all to commence their
pictures at the same time, according to those sketches, without
changing anything. Each morning, on arriving, they were rigidly
searched, in order to make sure that they brought with them no
drawings or engravings which could in any manner aid them. Thus
left to their own resources, they passed two months in this manner,
en loge, as it is termed; and these pictures, the figures in which
were one third the size of life, were publicly exhibited during three
days before the prizes were awarded. But although it was strictly
forbidden for the pupils to see their respective works, in order,
doubtless, to prevent the weak from being aided by the strong, or to
take care that a happy idea should remain the sole property of its
author—notwithstanding, I say, all these precautions, the students of
that time, less sensible perhaps than those of the present day, found
means of visiting each other without being perceived. The windows
of their cells all looked in the same direction, upon a small, dirty, and
almost unfrequented square, in which is now situated one of the
gates leading to the quay. These temporary abodes were, as we
have already said, situated in the roof, all the windows opening upon
wide leads, unprotected by railings. These madcaps, at the imminent
risk of breaking their necks by falling from an immense height,
glided by this way from one cell to another. The more scrupulous
closed their windows, so as to prevent intrusion; but two days
before the expiration of the time allowed for the pictures, each
student permitted, without difficulty, his work to be inspected by his
companions, and the little Areopagus, with remarkable sagacity and
impartiality, precisely anticipated the decrees of the greater one, and
awarded the first and second prize in such a manner, that there is
scarcely an example of their decisions having turned out erroneous.
José, who took the first rank in the sketches, now prepared to
submit to this trial, so severe, but, at the same time, so important to
him. Monsieur G. had recommended the reputation of his studio to
his pupils. Three times had they competed, without any of them
obtaining the first prize. It was necessary to repair this disgrace, and
be avenged for the late success of the Colourists. In addition to two
formidable rivals in the opposition school, José had to contend
against his two friends, Francisco and Rivol, who, besides having
already competed for the prize, had, also, the advantage of age—
José was then only fifteen years and a half old; but these
considerations by no means discouraged him; and fired by that
enthusiastic and true love of art which overcomes all difficulties, he
commenced, though not without emotion, the required picture, the
subject of which was the "Death of Hippolytus."
Dame Robert, as may be imagined, was greatly excited, and her
mind wholly absorbed by her darling boy's undertaking. Certainly,
had she been consulted, José would have had nothing to fear; but
neither the good woman's indulgence, nor Gabri's affection, could
avail poor José anything—they must wait. "If," said Dame Robert, "I
could only see what they are doing, I should soon find out whether
José had not left them behind; but they are cloistered up like so
many monks, and when the boy comes home at night, he does not
even so much as give us a hint as to how things are going on."
Gabri, equally anxious, but more discreet than Dame Robert, did not
seek to elicit anything from José; but he watched him carefully,
sighed when the poor boy appeared depressed, and rubbed his
hands with glee when he seemed happy.
The good-natured Angevin, who was not yet sufficiently advanced to
compete for the prize, was deeply interested in the success of his
friend; but he felt little uneasiness, for he knew that José was very
far superior to his rivals. He too would have liked to have seen his
work, but he was obliged to content himself with walking beneath
the windows of the young captives, and see their heads pop out and
in occasionally, like so many marionettes, with now and then a mahl-
stick accompanying them, and serving to complete the resemblance.
Six weeks had passed away, the pictures were advancing, and as,
with the exception of José and his companions, the competitors
were of different schools, he had seen only the work of his friends;
and his own was so far superior to theirs, that a hope which he
scarcely dared own, even to himself, made his heart beat high within
his breast. He had nothing to fear from the other students, as they
were all inferior to Francisco and Rivol. He was standing, therefore,
contemplating with a kind of secret pleasure the group of terrified
horses which he had just completed, when Francisco tapped at his
window, and immediately afterwards leaping into the room, told him,
with a countenance expressive of the utmost concern, that he was in
despair, and should never succeed with his figure of Aricia, which
was in the programme distributed for the picture. Subjects are
usually selected with but few female figures, these being more
difficult for the young artists, as they cannot have models; and the
unfortunate Aricia, which almost all of them had reserved till the
last, had completely wrecked both the courage and talent of
Francisco. He looked with admiration on José's Aricia, for he had
been entirely successful, at least in his sketch. José, anxious to
soothe the agitation of his friend, accompanied him across the leads
to his cell, in order to examine the figure which so much distressed
him: he found it awkward, ill-drawn, and in bad taste, and could not
conceal from his friend that he thought it detestable. This, of course,
served only to increase Francisco's despair. He dashed his palette to
the ground, stamped upon it, broke his brushes, and ended by
crying with rage. José embraced and tried to soothe him, and at
length, by dint of kindness and encouragement, succeeded in
persuading him that all was not yet lost, and that he could still
repaint the figure during the week that yet remained to them. He
pointed out to him what he had to avoid, and raised his courage by
dwelling on the merits of the rest of the picture. At last, after having
spent two hours in this manner, he left him, if not entirely consoled,
at least sufficiently recovered to resume his work.
The following days Francisco repainted his unfortunate figure, but
still without success; he effaced it, recommenced, again effaced it,
and at last succeeded in completing it; but in a manner so far
inferior to the other parts of the composition, that it formed a
blemish which destroyed the general effect. Such was the opinion of
his companions, when, according to the rule established among
them, they visited each other to judge of the respective merits of
their productions. They had still four days to remain at work, and the
pictures were not completely finished, but it was easy to judge
which would obtain the prize; and José was regarded as the
conqueror, provided he completed the figure of Aricia as he had
done the group of Hippolytus and his horses. Next to his, came
Francisco's picture, then Rivol's, the others were very far from the
mark, and need, therefore, cause them no anxiety.
Francisco, deprived of the last ray of hope by the decision of his
companions, as well as by that of his own judgment, shut himself up
in his cell, and would not allow José to enter, though he entreated
for admittance. He gave no reply to these friendly solicitations, and
the intensity of his annoyance had rendered him so unjust, that to
avoid seeing José, who lay crouched upon the narrow ledge of the
window, he took a large piece of linen, which served him for a blind,
and fastened it before the window. José listened to him for some
time pacing up and down and groaning with despair; but seeing that
his perseverance was useless and importunate he retired, deeply
grieved at his distress.
He passed a sleepless night, and the next morning no sooner had he
reached his own cell than he ran to Francisco's; but he was not
there, his picture still rested upon the easel, and for a moment José
thought of retouching the figure of Aricia. But this would have been
a palpable fraud, and his honour revolted from its commission.
Francisco, moreover, would never have consented to triumph by
such disgraceful means. José, therefore, laid down the brush which
he had taken up, and with a heavy heart returned to his own cell.
Whilst painting the figure which had proved so fatal to poor
Francisco, he vainly sought some method of serving him, and his
tender friendship made him almost desire that his Aricia might not
be better than his companion's. He worked with so little care, that,
had any one else been in the case, his wishes would have been
accomplished; but, as it often happens with artists, the very thing
that he took the least pains with turned out the best; and, to make
use of a familiar expression, this figure came so happily, that even
an experienced painter would not have been ashamed to own it.
With a mind absorbed in reflection, José painted on almost without
heeding what he did, and it was not until he rose up, when all was
completed, that he perceived that the last touches seemed to have
been given by the hand of a master, rather than by that of a pupil.
His first feeling was one of intense joy, but it was soon
overshadowed by the thought of Francisco. He felt that the prize was
his, but soon one of those noble inspirations which elevated minds
alone receive in their happiest moments, presented itself to his
imagination, and showed him that the safety of his friend depended
solely upon him.
By one of the old rules of the professors, the pupil who presented
his picture with a figure completely erased, or otherwise defaced,
was on this account excluded from the competition; his picture was
exhibited with the others, but was not taken into account in the
awarding of the prizes, even though it were a masterpiece in
comparison with the rest. This rule, which it was found rarely
necessary to apply, was unknown to most of the students. José had
become informed of it during his residence at M. Barbe's, but he was
quite sure that Francisco knew nothing about it. His friend's picture
was the best, after his own; and by having the courage to destroy
the figure of Aricia, which alone would have ensured the prize to a
work of less merit, Francisco would remain without a rival.
At first José seized upon this idea with all the warmth of generous
affection, but, on raising his eyes to his work, he began to think the
sacrifice beyond his strength. Pacing his cell with agitation, he
thought of the honour of being crowned at the age of sixteen, of the
pleasure of going to Italy, and of the advantage his studies would
derive from the journey.
"But," said he, turning his back upon his picture, "Francisco needs it
almost as much as myself; the means of his parents are almost
exhausted by the efforts they have made for his education; his
mother's health requires a warmer climate; if Francisco gains the
prize his family will follow him,..." and José again approached his
easel.
"Francisco is nearly twenty," he continued; "he has already obtained
a second prize, and thus cannot have it again; his age will soon
exclude him from the competition, while I have still two or three
years before me; moreover, he spoke to me of a vague hope which
he entertained of a happy marriage, to which his want of fortune
might one day be the only obstacle. If a brilliant success were to
overcome this obstacle? If the happiness of his future life depended
upon what I am about to do?..." José trembled, opened a box, took
out his palette knife, and approached the head of the charming
Aricia—but again he paused.
"If I were only to injure it a little," he thought, "alas! it would still be
better than my poor friend's!"... and he cast a look of approbation
upon the canvas. But soon a thought presented itself, which
dispelled his irresolution, and strengthened his wavering heroism. He
recalled that painful moment when, despised, falsely accused, on the
point of being driven from the house by Barbe, and without hope of
justification, Francisco did not fear to own the truth, and to re-
establish, at his own cost, the honour of the poor little Savoyard.
The honourable career which was now before him commenced from
that moment; all that he was, all that he hoped to be, sprang, in the
first instance, from Francisco's generous confession.... José no
longer hesitated, he resumed his knife, and with a firm hand so
erased the figure that nothing but the sketch remained—and thus
nobly repaid the debt of friendship formerly contracted to his young
companion.
 José erasing his Figure of Aricia, p. 301.
Satisfied with himself, and more calm after this trial of strength—an
act of high virtue in a young man of sixteen—José gave the last
touches to the other parts of his picture, and so cleverly managed
the erasure, that nothing more could be inferred from it, than one of
those movements of irritability by no means uncommon among
students. He kept his secret until the day previous to the one on
which the pictures were to be removed. He then called upon
Francisco at his father's, and told him that his figure of Aricia was
unfinished, and indeed in a great measure effaced, and that there
was not time to repaint it. Francisco, recovered from his unjust
displeasure, grieved for and blamed his friend; but, being ignorant of
the rule of exclusion, he assured him that the prize would still be his,
and José did not attempt to remove his impression.
But José had still severe trials to encounter: he foresaw the grief of
Dame Robert, Gabri's disappointment, and finally a whole year's
work before he could again reach the desired goal, which he had so
nearly attained; but the most painful moment was past, and he
awaited that in which Francisco should be triumphant, as the only
compensation worthy of him.
The exhibition of pictures was held, as usual, in a small room on the
basement floor, now appropriated to another use. The artistic crowd
arrived, and was constantly renewed during three entire days; and
the young students, mingling with it, heard alike the censure and
praise unreservedly bestowed, and often even with the knowledge
that the young authors of the works were present. The universal
opinion was in favour of the pictures of José and Francisco; but the
spectators were constantly heard to exclaim, "A figure erased! what
a pity! what madness!"
At length, on the fourth day, after a private conference, the
professors summoned before them the trembling candidates, and
José's sacrifice did not prove unavailing. He heard Francisco
Enguehard proclaimed for the first prize, Rivol for the second, and
he scarcely heard the honourable mention made of himself,
notwithstanding the fatal figure which had excluded him from the
competition.
Francisco, surprised and bewildered at such unexpected happiness,
scarcely knew what he was about; he did not hear the felicitations of
his companions, but allowed himself to be led away by José, who
made him run until he reached his father's house.
"He has gained the prize!" cried José, at the foot of the stairs,
"Francisco has gained the prize!" and seeing his friend in the arms of
his parents, who wept while they blessed him, this noble youth was
rewarded by a pleasure more intense and more elevated than any
which his own triumph could have afforded him.
Leaving Francisco in the arms of his happy mother, who was never
weary of looking at him, and who even thought him handsomer, now
that the laurel decked his brow, José bent his steps homeward, and
perceived in the distance Dame Robert and Gabri anxiously awaiting
his return.
"He walks rapidly," said Dame Robert; "so much the better, he bears
us good news."—"He looks happy," continued Gabri: "Oh, if he has
gained the prize! at sixteen, too!" and already a smile of joy shone
upon the countenance of this excellent man.
"Congratulate me, my friends," cried José, as he approached them;
"I am happy in my failure; Francisco has gained the prize!"
"Francisco!" exclaimed Dame Robert, letting fall her arms, already
extended to embrace him; "and you? Have you gained nothing? On
my word there must be some abominable trickery in the affair."
"No," replied José smiling, "but be comforted, my good mother, I am
neither depressed nor discouraged, and next year you shall see the
laurels on my brow."
"But," said Gabri, in a tone of vexation, "who obtained the second
prize?"
"Rivol," replied José; "and I might perhaps have had it if ..." and he
looked timidly at Gabri, "if I had not erased my figure of Aricia."
"Yes!" exclaimed Gabri, as if talking to himself, "I was sure of it, I
suspected as much at the exhibition.... José, José, embrace me, my
son. Gracious Heaven! this is the first day I have passed without
regretting the loss of my own noble boys."
Gabri was too familiar with artistic matters not to have divined the
sacrifice which José's friendship had induced him to make, and his
heart was capable of appreciating and rejoicing in it; but Dame
Robert, who understood nothing of the matter, save that her boy
was rejected, gave free vent to her dissatisfaction.
"Indeed, M. Gabri, it is very fine to pet him up after such a failure as
that. Who would have thought it? It was well worth while to be shut
up for two months without uttering a syllable, to let others walk off
with the prize; still your picture was very fine, my boy, though, to tell
you the truth, your female figure was too pale. I told you, however,
not to spare your colours, but young people will always have their
own way."
José smiled, and hastened to tranquillize the good woman. So far as
concerned himself he succeeded without much difficulty; but she
was for some time out of humour with Gabri, whose triumphant air
annoyed her, because she did not understand it. Nor did she gain
any information on the subject, for Gabri was discreet, and would
not divulge José's secret; he did not even seek an explanation from
the lad himself; but his marks of friendship were increased, and he
more frequently repeated, "My son José!"
At the annual meeting of the Academy, when the students publicly
receive the laurel crown, awarded for the merits of their works, José
appeared more pleased than Francisco. He was restless, busying
himself with his friend's toilet, &c.; and, placed in a corner of the
room during the ceremony, the spectators might have imagined,
from his excitement and his looks, when Francisco Enguehard was
proclaimed, that he was the happy father of the young laureate,
were it not that his almost childish features precluded the
supposition.
A month after this great epoch for the two friends, they were
separated; Francisco and his parents took the route to Italy; and
José having returned to his studies, pursued them with ardour and
contentment in thinking of the happiness which he had been the
means of securing to three persons.
The year passed, and when again about to compete for the prize,
José wrote to his friend, and told him to expect him in three months
from that date. He felt confidence in himself, and had acquired so
much power, that notwithstanding the merits of seven competitors,
all older than himself, his picture was unanimously declared the best.
It was even so superior to anything usually seen at these
competitions, that it was thought proper to allow the exhibition to
remain open several days longer than usual, in order to gratify the
crowd of amateurs who flocked to see it. Dame Robert fully enjoyed
José's triumph, and the almost equal pleasure of relating its history
to her neighbours. Gabri rubbed his hands, and bent his head while
listening to the praises of the young artist, and the honest Barbe
exultingly boasted of having supplied for this famous picture the
finest and the best canvas in his shop.
José, overwhelmed with honours, and full of joy, set out on his way
to Rome, where he found Francisco, who had still four years
remaining of the five granted by the government. Monsieur and
Madame Enguehard received José as a second son; he lived in the
same house with them, and enjoyed, in all its fulness, the delights of
a life devoted to friendship and the fine arts, in that beautiful land
where these arts so naturally flourish.
Many years have passed away since these events took place.
Monsieur and Madame Barbe, grown rich and old, have retired, and
given up their business to the excellent Gabri. A new generation of
artists and students frequents the shop, and pursues pretty nearly
the same habits as that which preceded it. But it is not in the same
spot; the theatre of José's first exploits no longer exists. The two
large posts may still, indeed, be seen; but Barbe's house has been
taken down, and in its place monkeys and learned birds, attract by
their various tricks, numerous spectators. Francisco Enguehard,
steady and talented, is married, as he wished, to the only daughter
of a rich antiquary, who desired to have for a son-in-law, a man of
genius. Dame Robert has given up her business to her eldest son,
and rests her fingers, if not her tongue, for she is never weary of
relating to any one who will listen to her, how that José was a poor
orphan, how she took him and put him to sleep on her counter, &c.,
&c. Philip, a worthy fellow, and a passable tailor, is married and
settled, as he says, in his wife's native province, that is to say in the
Marais. The poor Angevin, still a bad painter, notwithstanding all his
efforts and perseverance, has returned to Angers. There, at least, he
has talent, and directs in his turn the same school which sent him to
Paris. He who was called poor José is now one of our most
distinguished artists. He possesses a respectable fortune, acquired
by his talents, and, what is far more valuable to him, the universal
esteem granted to the most noble character and the most
irreproachable conduct. Faithful alike to delicacy and friendship,
Francisco never knew the sacrifice which obtained for him his crown.
José's laurels are suspended in his magnificent studio, beside his
first palette, and his shoeblack's knife. He watches over Gabri, as a
son over a father; listens to the long stories of the good old Dame
Robert, without the least sign of impatience; and, finally, though
young, handsome, and sought after, he always wears clothes made
by Philip, and boasting of little elegance, with shoes of the same
kind from Dame Robert's shop: and this is not the least remarkable
trait in his history.
 Caroline:
OR,

THE EFFECTS OF A MISFORTUNE.

"How delighted I am that Robert is gone!" exclaimed Caroline de
Manzay, as she entered her mother's room; "I never knew anybody
so disagreeable!"
"What!" said Madame de Manzay, "not even Denis?"
"Oh! that is quite different; Denis is teazing and troublesome,
meddles with everything, and is angry when prevented; he jeers and
laughs at one, and becomes passionate and insulting when
contradicted; but then he is a mere child, and one overlooks it."
"You did not seem very ready to do so: you were always quarrelling,
and could say very insulting things yourself sometimes."
"For all that I like him better than Robert."
"Yet Robert never teazed you; he is very reasonable."
"To be sure he is; he is twenty years old: and how proud he is!
Because he is five years older than I am he treats me like a little girl,
and to-day he told me I was a spoiled child."
"Robert is not the first person who has said that, my dear; but for
what reason did he pay you this compliment?"
"It was because Denis, who always takes delight in seeing me
vexed, came to tell me, with an air of triumph, that when we took
him and Robert to the village, we were to go by the road which I do
not like. I said we were not to go that way; he asserted that we
were, because he had heard my father give orders to his forester to
wait for him at the green-gate, that he might see on his way back
the fir-trees which are to be cut. Then I declared that I would not go
out at all, and Robert laughed at me, and insisted that if my father
chose it I should be obliged to go, and to take the road he wished.
All this made me angry, and when papa came up I teazed him so, till
he said we should go the way I liked best, and that he would look at
the fir-trees another time. 'Well,' said I to Robert, when my father
was at a little distance, 'it is my turn to laugh at you now;' 'I would
recommend you not,' he replied, very contemptuously, 'there is no
glory in being a spoiled child, and in abusing indulgence,' and then
he turned his back on me. Oh! I detest him! So when he got into the
carriage I would not say good-bye, and when he came up to kiss
me, I turned my back upon him in my turn."
"And did that appear to grieve him?"
"He did not care in the least; he began to laugh, and said, 'Adieu,
Caroline, try to become a little more reasonable, you need it
greatly.'"
"And how did you part with Denis?"
"Oh, very well, for I spoke to him."
"What did you say to him?"
"I told him I was delighted that he was going away, because he was
so rude; and he replied, that he was quite as glad, because I was so
wilful and captious. In fact, I am not at all fond of Denis, either, and
it is a great relief to be rid of him. It will be a long time, will it not,
before we see him again?"
"Much too long; his guardian thinks of going to America, and taking
Denis with him. God only knows when he will come back."
"Oh! I shall have quite enough of him; he is so insufferable! And
Robert?"
"He is going on his travels for four or five years."
"That is a great blessing."
"But, my dear child, you should reflect that Robert is your father's
nephew, and that Denis is my poor sister's son; they are both of
them your nearest relatives, and ought to be your best friends."
"Fine friends, indeed! the one teazes me, and the other despises
me."
"I allow that Denis is fond of teazing, and that Robert is scornful, but
they will out-grow that."
"No, that they won't."
"What! do you, then, really think that Denis, at twenty years old, will
spoil your drawing, or blow out your candle?"
"He will do something as tiresome; and even if he should improve,
Robert will always remain the same."
"I hope not; he will gain with years the gentleness in which he is
deficient. But, even supposing he should not change, you yourself
will alter, and when you are no longer a spoiled child, he will not call
you such."
"I don't know that; he is so unamiable. However, it is all the same to
me; I do not care for his opinion."
"So I perceive, my dear," said her mother, smiling, "you speak of it
so calmly."
At this moment, Caroline heard her father calling her, and ran out to
join him; she was always happy to be his companion, and responded
with all her heart to the passionate affection which he showed her.
Caroline was the only survivor of Monsieur and Madame de Manzay's
eight daughters, and during her infancy her health had been so
delicate, as to cause them the greatest anxiety. Continually agitated
by the fear of losing her, their only thought had been to preserve
their treasure: they trembled lest the slightest opposition should
endanger her fragile existence, or cast a cloud over a life which
might have so short a duration. For some years past, these terrible
apprehensions had ceased, but Caroline had been so long
accustomed to have her own way, that the effect survived the cause.
She was accustomed to no other rule than her caprice, or the
prompting of a disposition naturally upright and generous. When her
fancies or her self-love did not interfere, she was ready to do
everything to oblige, and diffused around her all the cheerfulness
natural to her age: but if it at all crossed in her wishes, nothing
could be obtained from her, and even her kindness of heart was
insufficient to conquer her temper. In such unhappy moments, which
were but too frequent, she would answer her mother with petulance,
refuse to walk with her father, or sing him the airs he loved, and
behave roughly to her little brother, whom she nevertheless loved
with all her heart, and considered almost as her own child. Being ten
years old, when Stephen was born, she had never thought of him as
a rival, but as a protégé. She was habitually kind and indulgent, and
would spend whole hours in building card-houses for him, or in
telling him stories. It is true she did not like him to amuse himself
with others: as she could not appropriate him to herself, like his
parents, she devoted herself to him; but she did appropriate him, in
fact, and one of the principal causes of her dissatisfaction with Denis
was, that Stephen preferred his stories to hers, and his noisy games
to the more tranquil pleasures procured him by his sister.
"What does it signify, if Stephen enjoys himself better with Denis
than with you?" said Robert to her one day.
"It displeases me."
"But why?"
"Because he is so whimsical; a week ago he was interrupting me
perpetually, to make me tell him over and over again the story of the
Wonderful Cat, and now, when I call him on purpose, he says it
wearies him."
"Naturally enough, when you propose telling it to him at the very
time that Denis is just in the finest part of a story about robbers or
battles."
"And twenty times have I begged Denis not to tell him any more
such stories: but he does not care for a word that is said to him."
"Stephen would be very sorry if he left off, I can assure you: look
how attentive he is."
"Yes, and what am I to do while Stephen is listening to Denis?"
"You might finish the drawing which your father asked for this
morning, and which, as you said, you had not time to complete."
"Indeed I shall not, it is too tiresome; and if anything more is said
about it, I will tear it to pieces."
"Surely not, you are not silly enough to do that."
"And why then should I be silly to tear this drawing? It is my own, I
hope."
"A fine reason truly! The château yonder is mine also. What would
you say if I were to burn it down?"
"There is no resemblance in the two cases."
"In fact, I should be a madman, and you merely a child."
"A child! Do you know that I am fifteen?"
"So they say, but I cannot believe it."
"Why not? I am taller than the gardener's daughter, who is sixteen."
"Yes; but you are not as reasonable as Stephen, who is only five."
"And, how not, pray?"
"Come, do not be angry; you are, perhaps, about as much so, but
that is all I can grant you. Now do not put yourself in a passion, that
will not frighten me; you cannot tear me to pieces like your
drawings. Adieu, make yourself happy: I am going to carry off Denis
to hunt, so you may tell Stephen the story of the Wonderful Cat as
many times as you please."
It was by conversations like these that Robert had drawn upon
himself the animadversions of Caroline. Unaccustomed to any
opposition to her wishes, she could not forgive the harsh manner in
which her cousin contradicted her, and, spoiled as she was by
continual marks of affection, she was astonished at the
contemptuous disapprobation which she had to encounter from one,
whose good opinion she was desirous of obtaining. Never had she
heard the name of Robert de Puivaux mentioned without eulogium.
He had completed his studies most successfully, and had particularly
distinguished himself at the Polytechnic School, which he had just
left, after spending two years there, simply for instruction. His
character was extolled, his judgment esteemed, and his
understanding and acquirements were considered by all as beyond
his years; but all these advantages were effaced, in Caroline's mind,
by his ungracious conduct towards herself—or, rather, they served to
render it the more vexatious to her. It must be allowed that Robert
had treated her in a manner far from pleasant. Naturally serious,
and disposed to regulate his conduct on principles of reason and
duty, he could not comprehend the inconsiderateness of Caroline,
and the importance which she attached to her own whims; he had
no patience in seeing everyone yield to her, and was as angry with
her for their weakness as for her own defects; he, therefore, never
lost any opportunity of showing his disapprobation and contempt:
and, wholly engrossed by the unfavourable impressions with which
she inspired him, he did not remark the good qualities which lay
hidden under this petulant exterior, and which the future would
develope.
Shortly after the departure of Robert and Denis, Madame de Manzay,
who had been an invalid ever since the birth of Stephen, was
suddenly snatched from her family, after a few days' illness. We will
not attempt to describe this sad event: there are sorrows which can
never be comprehended by those who have not felt them, and which
it is needless to relate to those who know them by experience. The
language of man cannot adequately express all that the soul of man
is capable of feeling, and such feelings are not learned but revealed;
a single moment—one of those moments which are equal to a whole
life—can explain more than years of reflection, and convey to the
heart, what all the knowledge of the mind would be unable to grasp.
A week had elapsed since the death of Madame de Manzay, and her
unhappy family were not yet roused from the first stupor of grief;
their hearts had not yet recovered composure; they had not
returned to their usual habits; no one obeyed, for no one
commanded; and each one, engrossed by his affliction, forgot his
duties. There was neither regularity nor labour; confusion alone
reigned in the desolate household. Poor little Stephen was left all
day long to himself; Monsieur de Manzay wandered about in the
park; his daughter shut herself up in her room; and no one
attempted to assist anyone else in supporting the weight of grief, by
which each was oppressed. Caroline, as usual, was weeping in her
own apartment, when an old servant, who had been in the family
from the birth of her father, and who had just seen his master,
seated, alone, in his wife's room, thinking he would like to see his
daughter, went to her, and said, "Pray go, Miss Caroline, to my
master. Poor gentleman! he has no one now but you."
"And Stephen, Peter; you do not reckon him."
"Oh! that is quite another thing, miss; master loves the dear little
fellow with all his heart, but he is not company for him; he cannot
talk with him, and divert his thoughts, as you could. Oh! Miss
Caroline, you are the very image of my good mistress; try then to
resemble her in everything. You cannot remember it, for you were
too young, but when my mistress lost four of her children in one
year, and you alone were left—well, miss, it was she who then
consoled master. He was like one distracted, and said he felt
tempted to throw himself in the water, and the poor lady was
obliged to appear perfectly calm, in order to tranquillize him. I have
sometimes seen her leave my master's room, to go and cry, and
then she would return, and urge him to submit to the will of God;
she would make him walk with her, or read aloud to divert his
thoughts; she would even amuse him with music: and how he loved
her in return! Oh! Miss Caroline! you had a treasure in your mother;
endeavour to be as good as she was."
Caroline's sobs prevented her from making any reply; but she held
out her hand to the aged Peter, and rose immediately to follow him
to her father. She was told that he was in the park, and repaired
thither; but, absorbed in her affliction, and in the reflections
suggested by Peter's artless observations, she mistook the path, and
did not perceive her error, for she went on without thinking whither
her steps were directed. For the first time in her life, perhaps, she
became aware that she had a duty to fulfil towards others, and that
she was not placed in this world merely to be loved and indulged.
She had just been told—"Your father has no one but you." It was the
truth; but of what use had she been to her father, during the past
week? Had she afforded him consolation or assistance, when, given
up to her own affliction, she had scarcely bestowed a thought on
his; when he had been obliged to try and comfort her, and had
sought to do so in vain; when her tears and cries had shaken the
resolution he found it so difficult to maintain; when she had kept out
of his presence, and abandoned him at the time he most needed
her? Was it thus that her mother had acted, when, struck by
misfortune, she had, for the sake of calming her husband's despair,
begun by controlling her own feelings? Yet who, more than her
father, possessed a claim to her active gratitude, to her affectionate
devotion? Her earliest recollections were associated with his
kindness and tenderness. He had consecrated his leisure to her
instruction, relinquished for this purpose studies in which he took
delight, and renounced all recreations but those which he could
share with her; he had made her the companion of his walks, and
allowed her to direct them as she chose. If she wished for an
excursion in the neighbourhood, M. de Manzay would leave all his
occupations to procure this pleasure for her; in a word, he never
refused her a request, and yet her demands had not been few. And
what had she done, on her part, to requite such great affection?
How had she repaid the extreme indulgence of her parents? She
loved them heartily, and they were convinced of this, but she had
done nothing more: whilst they thought only of her, she had never
considered them, and had found it perfectly natural to be continually
the recipient of benefits, without ever giving anything in return. "Oh,
how wicked I have been!" she exclaimed, clasping her hands; "can
God ever forgive me, or mamma?" She threw herself on her knees,
and, melting into tears, promised, as if still in the presence of her
whom she could never again behold in this world, to repair, by her
attention to the objects of affection she had left, the faults which
she had committed against her. She felt that her resolution was
accepted and blessed; that the relations of those who love each
other are eternal; and that her mother was pleased with her earnest
endeavours, as she would have been if still living. She felt that it was
her soul which responded to her own, and inspired her with the love
of virtue, the hope of perseverance, the joy of pardon. She arose,
and returned to the château, eager to find her father, and begin her
new part. "Hitherto, he has devoted his life to me," said she to
herself, "now, it shall be my care to live for him;" and immediately,
with the ardour so natural to youth, she depicted to herself all the
various ways in which she could be useful to him, and was
enchanted at the idea of being at last good for something in the
world; no obstacle or difficulty presented itself to her mind, so
natural did the performance of her duty appear to her at this
moment.
On approaching the château, she found Stephen sitting quite alone,
under a tree, crying. "What is the matter, Stephen?" she asked,
kissing him.
"I am hungry."
"Hungry! why what o'clock is it?"
"It is twelve o'clock."
"But you have already had your breakfast?"
"No; Mary forgot to make my soup. Nobody thinks about me now
that mamma is gone."
"I will think of you, my dear child. Come with me, I will get you
some breakfast, and tomorrow you shall not have to wait so long."
On entering the house, she inquired for her father. She was told that
he had come in, and had asked for her, and, after waiting some
time, had gone out again. "But he has had his breakfast, I
suppose?"
"No, miss, the cook has gone out."
"Things must not go on thus," thought Caroline; "I must have some
order in the household." She perceived at this moment her father
coming in, and hastened to meet him; she was eager to have some
conversation with him, and impart to him her good resolutions; but
the very first was, to attend to others rather than herself, and she
therefore sacrificed to Stephen's appetite her desire of
communicating to her father her new projects. After breakfast, M. de
Manzay was going towards his wife's sitting-room, where he passed
all the time which he spent in-doors. Caroline, who wished to follow
him, paused for an instant at this sight: she never yet had sufficient
resolution to enter her mother's apartment, and trembled at the idea
of revisiting a spot so filled with her image. "But how can I ever be
of service to my father, if I cannot go where it is his desire always to
remain? Come, I must go to him;" and, making an effort to
command her feelings, she went to her father. Surprised and pleased
to see her in this room, where his recollections became almost
realities, he embraced her with even more than his wonted
tenderness; and, comparing, with a pleasure mingled with grief, the
portrait of his wife with the features of his daughter,—"Oh, my
child!" he exclaimed at length, his voice checked by tears, "I have
only you now." She threw her arms round him, and for some time
neither father nor daughter could utter a word. At length,
overcoming her emotion, she said, "My dear papa, I have hitherto
done very wrong, but I will endeavour to repair my faults. I have
been a selfish, ungrateful child, and lived only for myself; henceforth
my life shall be devoted to you. Forgive me for having been so
useless to you; forget the past; you shall see that I am no longer the
same, and you shall be satisfied with my conduct. Kiss me, dear
papa; I will correct all my faults, and endeavour to be like mamma."
"God bless you, my child, for having formed such a project! but you
are very young to make even the attempt."
"Not too young, I hope. I shall hardly succeed at first, but the
recollection of mamma will come to my assistance. I know what she
used to do, and I will endeavour to imitate her. I will come and see
you in your study, and be always ready to give up my own
occupations to please you. I will give Stephen his lessons. I will keep
the accounts. You shall see how steady I will be; only try me, papa."
"Do what you like, my child; I am in no state to make any decision; I
can think of nothing. I leave you mistress of the house, of your
brother, of myself. If there are still any peaceful moments in store for
me on this earth, I shall enjoy them through you, and you alone."
"And Stephen, papa, you forget him."
"Poor child! no, I do not forget him; go and bring him here."
Caroline brought her little brother to her father, who took him in his
arms, saying, "Stephen, you loved dear mamma, did you not?"
"With all my heart," replied the child, sobbing.
"And you were also obedient to her. Well, now you must love your
sister, and obey her; she will be a mother to you henceforward."
"Would you like it, Stephen?" asked Caroline, "would you like me to
take care of you, and give you your lessons?"
"Yes, if you promise not to scold me."
"My dear child, I will not scold you; I will try to be kind like
mamma."
"Oh, you are very kind already, I am sure," said Stephen, caressing
his sister, "only sometimes you get out of patience, and that
frightens me."
"Make yourself easy, I intend to grow better; but you must also be
very good, to please papa, who has so much sorrow."
"Oh! yes; for that I will learn my lessons better than I used to do."
"My beloved children," said M. de Manzay, encircling them both in
his arms, "my dear children, this is the first moment of comfort I
have had for a week past. Go, my own Caroline, assume your new
functions; take possession of the keys; direct, command, re-establish
the regularity which formerly reigned in the house; take the same
care of your brother that he has been accustomed to; but first come
to me, that I may give you my blessing, before the portrait of your
mother."
After some moments devoted to these tender and afflicting
emotions, Caroline left the room with Stephen. Her first care was to
see if his apartment was in order: she found it completely stripped
of all the articles which he was in the habit of using.
"What has become of your little table, Stephen?" she enquired.
"Oh! I dare say it is in the garden; I took it there the day before
yesterday, and they have forgotten to bring it in."
"And your arm-chair?"
"I tied it to Turk's tail, for a carriage, and he broke it."
"You might have expected as much, my dear."
"What could I do? I was alone, and tired of doing nothing."
"You have recollected, I hope, to give water to your birds."
"Oh! gracious! I have never given them any but once. Poor
creatures! they must be very thirsty. But, Caroline, do not scold me,
it is not my fault. Every morning, mamma used to ask me if I had
taken water and seeds to my birds, leaves to my rabbits, and grass
to my fawn; and now, who is there to think of all these things?"
"I will. Let us go to your aviary, and I will talk to you by the way."
Caroline then explained to her brother all her plans concerning him.
She told him that he should work with her, and that she would
amuse him, and take care of all his things; in a word, that she
would, as far as possible, supply the place of a mother to him. She
had his books brought into her sitting-room, and such of his
playthings as he had been accustomed to keep in his mother's
apartment; she gave him a shelf in her library, and the lower part of
a closet, and established his little table by the window, as he wished.
At first, she intended to place it elsewhere, for this was her own
favourite place; but she recollected that last year, when she had
remarked that her mamma was happy in being able to enjoy, while
sitting at table, the prospect over the valley, her mother had yielded
to her the place she coveted. "I cannot be so good as mamma," she
thought, "unless I do as she did, so I will remove my table from the
window."
Such were the feelings and views with which Caroline undertook the
reformation of her character, and she begun the task with the blind
ardour so natural to youth: that happy privilege bestowed by
Providence, to remove all hesitation from their resolutions, and leave
nothing doubtful but the execution. But this first strong and happy
impulse does not always last; when the sentiment which gave it
birth ceases to be exclusive, things which had been forgotten
reappear, the realities of life and the peculiarities of character
resume their claims, and what we still desire above all things is,
nevertheless, not our sole object. This was precisely the case with
Caroline. For a considerable time her heart was so full of the idea of
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