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Principles of Fluorescence Spectroscopy

Book · January 2006

DOI: 10.1007/978-0-387-46312-4

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Principles of
Fluorescence Spectroscopy
Third Edition

Joseph R. Lakowicz
University of Maryland School of Medicine
Baltimore, Maryland, USA

Springer
 cps

Ontroduction to Fk • rescence 2.2. Light Sources 31

2.2.1. Arc Lamps and Incandescent
1.1. Phenomena of Fluorescence Xenon Lamps 31
1.2. Jablonski Diagram 3 2.2.2. Pulsed Xenon Lamps 32
1.3. Characteristics of Fluorescence Emission 6 2.2.3. High-Pressure Mercury (Hg) Lamps 33
1.3.1. The Stokes Shift 6 2.2.4. Xe—Hg Arc Lamps 33
1.3.2. Emission Spectra Are Typically Independent 2.2.5. Quartz—Tungsten Halogen (QTH) Lamps 33
of the Excitation Wavelength 7 2.2.6. Low-Pressure Hg and Hg—Ar Lamps 33
1.3.3. Exceptions to the Mirror-Image Rule 8 2.2.7. LED Light Sources 33
1.4. Fluorescence Lifetimes and Quantum Yields 9 2.2.8. Laser Diodes 34
1.4.1. Fluorescence Quenching 11 2.3. Monochromators 34
1.4.2. Timescale of Molecular Processes 2.3.1. Wavelength Resolution and Emission
in Solution 12 Spectra 35
1.5. Fluorescence Anisotropy 12 2.3.2. Polarization Characteristics of
1.6. Resonance Energy Transfer 13 Monochromators 36
1.7. Steady-State and Time-Resolved Fluorescence 14 2.3.3. Stray Light in Monochromators 36
1.7.1. Why Time-Resolved Measurements? 15 2.3.4. Second-Order Transmission in
1.8. Biochemical Fluorophores 15 Monochromators 37
1.8.1. Fluorescent Indicators 16 2.3.5. Calibration of Monochromators 38
1.9. Molecular Information from Fluorescence 17 2.4. Optical Filters 38
1.9.1. Emission Spectra and the Stokes Shift 17 2.4.1. Colored Filters 38
1.9.2. Quenching of Fluorescence 18 2.4.2. Thin-Film Filters 39
1.9.3. Fluorescence Polarization or Anisotropy 19 2.4.3. Filter Combinations 40
1.9.4. Resonance Energy Transfer 19 2.4.4. Neutral-Density Filters 40
1.10. Biochemical Examples of Basic Phenomena 20 2.4.5. Filters for Fluorescence Microscopy 41
1.11. New Fluorescence Technologies 21 2.5. Optical Filters and Signal Purity 41
1.11.1. Multiphoton Excitation 21 2.5,1. Emission Spectra Taken through Filters 43
1.11.2. Fluorescence Correlation Spectroscopy 22 2.6. Photomultiplier Tubes 44
1.11.3. Single-Molecule Detection 23 2.6.1. Spectral Response of PMTs 45
1.12. Overview of Fluorescence Spectroscopy 24 2.6.2. PMT Designs and Dynode Chains 46
References 25 2.6.3. Time Response of Photomultiplier Tubes 47
Problems 25 2.6.4. Photon Counting versus Analog Detection
of Fluorescence 48
2.6.5. Symptoms of PMT Failure 49
2. Onstrumentation for Fluorescence 2.6.6. CCD Detectors 49
Spectroscopy 2.7. Polarizers 49
2.8. Corrected Excitation Spectra 51
2.1. Spectrofluorometers 27 2.8.1. Corrected Excitation Spectra Using
2.1.1. Spectrofluorometers for Spectroscopy a Quantum Counter 51
Research 27 2.9. Corrected Emission Spectra 52
2.1.2. Spectrofluorometers for High Throughput 29 2.9.1. Comparison with Known Emission
2.1.3. An Ideal Spectrofluorometer 30 Spectra 52
2.1.4. Distortions in Excitation and Emission 2.9.2. Corrections Using a Standard Lamp 53
Spectra 30 2.9.3. Correction Factors Using a Quantum
Counter and Scatterer 53

xv
 CONTENTS
xvi

2.9.4. Conversion between Wavelength and 4.1.3. Examples of Time-Domain and

Wavenumber 53 Frequency-Domain Lifetimes 100
2.10. Quantum Yield Standards 54 4.2. Biopolymers Display Multi-Exponential or
2.11. Effects of Sample Geometry 55 Heterogeneous Decays 101
2.12. Common Errors in Sample Preparation 57 4.2.1. Resolution of Multi-Exponential
2.13. Absorption of Light and Deviation from the Decays Is Difficult 103
Beer-Lambert Law 58 4.3. Time-Correlated Single-Photon Counting 103
2.13.1. Deviations from Beer's Law 59 4.3.1. Principles of TCSPC 104
2.14. Conclusions 59 4.3.2. Example of TCSPC Data 105
References 59 4.3.3. Convolution Integral 106
Problems 60 4.4. Light Sources for TCSPC 107
4.4.1. Laser Diodes and Light-Emitting Diodes 107
4.4.2. Femtosecond Titanium Sapphire Lasers 108
3. Fluorophores 4.4.3. Picosecond Dye Lasers 110
4.4.4. Flashlamps 112
3.1. Intrinsic or Natural Fluorophores 63 4.4.5. Synchrotron Radiation 114
3.1.1. Fluorescence Enzyme Cofactors 63 4.5. Electronics for TCSPC 114
3.1.2. Binding of NADH to a Protein 65 4.5.1. Constant Fraction Discriminators 114
3.2. Extrinsic Fluorophores 67 115
4.5.2. Amplifiers
3.2.1. Protein-Labeling Reagents 67 4.5.3. Time-to-Amplitude Converter (TAC)
3.2.2. Role of the Stokes Shift in Protein and Analyte-to-Digital Converter (ADC) 115
Labeling 69 4.5.4. Multichannel Analyzer 116
3.2.3. Photostability of Fluorophores 70 116
4.5.5. Delay Lines
3.2.4. Non-Covalent Protein-Labeling 4.5.6. Pulse Pile-Up 116
Probes 71 117
4.6. Detectors for TCSPC
3.2.5. Membrane Probes 72 117
4.6.1. Microchannel Plate PMTs
3.2.6. Membrane Potential Probes 72
4.6.2. Dynode Chain PMTs 118
3.3. Red and Near-Infrared (NIR) Dyes 74
4.6.3. Compact PMTs 118
3.4. DNA Probes 75
4.6.4. Photodiodes as Detectors 118
3.4.1. DNA Base Analogues 75
4.6.5. Color Effects in Detectors 119
3.5. Chemical Sensing Probes 78
4.6.6. Timing Effects of Monochromators 121
3.6. Special Probes 79
4.7. Multi-Detector and Multidimensional TCSPC 121
3.6.1. Fluorogenic Probes 79
4.7.1. Multidimensional TCSPC and
3.6.2. Structural Analogues of Biomolecules 80
DNA Sequencing 123
3.6.3. Viscosity Probes 80
4.7.2. Dead Times, Repetition Rates, and
3.7. Green Fluorescent Proteins 81
Photon Counting Rates 124
3.8. Other Fluorescent Proteins 83
4.8. Alternative Methods for Time-Resolved
3.8.1. Phytofluors: A New Class of
Measurements 124
Fluorescent Probes 83
4.8.1. Transient Recording 124
3.8.2. Phycobiliproteins 84
4.8.2. Streak Cameras 125
3.8.3. Specific Labeling of Intracellular
4.8.3. Upconversion Methods 128
Proteins 86
3.9. Long-Lifetime Probes
4.8.4. Microsecond Luminescence Decays 129
86
3.9.1. Lanthanides 4.9. Data Analysis: Nonlinear Least Squares 129
87
3.9.2. Transition Metal—Ligand Complexes 4.9.1. Assumptions of Nonlinear Least Squares 130
88
3.10. Proteins as Sensors 4.9.2. Overview of Least-Squares Analysis 130
88
3.11. Conclusion 4.9.3. Meaning of the Goodness-of-Fit 131
89
References 4.9.4. Autocorrelation Function 132
90
Problems 4.10. Analysis of Multi-Exponential Decays 133
94
4.10.1, p-Terphenyl and Indole: Two Widely
Spaced Lifetimes 133
4, Time - Dornain Lifetime 'Heesurenlene 4.10.2. Comparison of x R2 Values: F Statistic 133
4.10.3. Parameter Uncertainty: Confidence
4.1. Overview of Time-Domain and Frequency- Intervals 134
Domain Measurements 98 4.10.4. Effect of the Number of Photon Counts 135
4.1.1. Meaning of the Lifetime or Decay Time 99 4.10.5. Anthranilic Acid and 2-Aminopurine:
4.1.2. Phase and Modulation Lifetimes 99 Two Closely Spaced Lifetimes 137
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY xvii

4.10.6. Global Analysis: Multi-Wavelength 5.5. Simple Frequency-Domain Instruments 173

Measurements 138 5.5.1. Laser Diode Excitation 174
4.10.7. Resolution of Three Closely Spaced 5.5.2. LED Excitation 174
Lifetimes 138 5.6. Gigahertz Frequency-Domain Fluorometry 175
4.11. Intensity Decay Laws 141 5.6.1. Gigahertz FD Measurements 177
4.11.1. Multi-Exponential Decays 141 5.6.2. Biochemical Examples of Gigahertz
4.11.2. Lifetime Distributions 143 FD Data 177
4.11.3. Stretched Exponentials 144 5.7. Analysis of Frequency-Domain Data 178
4.11.4. Transient Effects 144 5.7.1. Resolution of Two Widely Spaced
4.12. Global Analysis 144 Lifetimes 178
4.13. Applications of TCSPC 145 5.7.2. Resolution of Two Closely Spaced
4.13.1. Intensity Decay for a Single Tryptophan Lifetimes 180
Protein 145 5.7.3. Global Analysis of a Two-Component
4.13.2. Green Fluorescent Protein: Systematic Mixture 182
Errors in the Data 145 5.7.4. Analysis of a Three-Component Mixture:
4.13.3. Picosecond Decay Time 146 Limits of Resolution 183
4.13.4. Chlorophyll Aggregates in Hexane 146 5.7.5. Resolution of a Three-Component
4.13.5. Intensity Decay of Flavin Adenine Mixture with a Tenfold Range of
Dinucleotide (FAD) 147 Decay Times 185
4.14. Data Analysis: Maximum Entropy Method 148 5.7.6. Maximum Entropy Analysis of FD Data 185
References 149 5.8. Biochemical Examples of Frequency-Domain
Problems 154 Intensity Decays 186
5.8.1. DNA Labeled with DAPI 186
5.8.2. Mag-Quin-2: A Lifetime-Based Sensor
3. [Frequency-Domain UNtime for Magnesium 187
Gei easurements 5.8.3. Recovery of Lifetime Distributions from
Frequency-Domain Data 188
5.1. Theory of Frequency-Domain Fluorometry 158 5.8.4. Cross-Fitting of Models: Lifetime
5.1.1. Least-Squares Analysis of Frequency- Distributions of Melittin 188
Domain Intensity Decays 161 5.8.5. Frequency-Domain Fluorescence
5.1.2. Global Analysis of Frequency-Domain Microscopy with an LED Light Source 189
Data 162 5.9. Phase-Angle and Modulation Spectra 189
5.2. Frequency-Domain Instrumentation 163 5.10. Apparent Phase and Modulation Lifetimes 191
5.2.1. History of Phase-Modulation 5.11. Derivation of the Equations for Phase-
Fluorometers 163 Modulation Fluorescence 192
5.2.2. An MHz Frequency-Domain Fluorometer 164 5.11.1. Relationship of the Lifetime to the
5.2.3. Light Modulators 165 Phase Angle and Modulation 192
5.2.4. Cross-Correlation Detection 166 5.11.2. Cross-Correlation Detection 194
5.2.5. Frequency Synthesizers 167 5.12. Phase-Sensitive Emission Spectra 194
5.2.6. Radio Frequency Amplifiers 167 5.12.1. Theory of Phase-Sensitive Detection
5.2.7. Photomultiplier Tabes 167 of Fluorescence 195
5.2.8. Frequency-Domain Measurements 168 5.12.2. Examples of PSDF and Phase
5.3. Color Effects and Background Fluorescence 168 Suppression 196
5.3.1. Color Effects in Frequency-Domain 5.12.3. High-Frequency or Low-Frequency
Measurements 168 Phase-Sensitive Detection 197
5.3.2. Background Correction in Frequency- 5.13. Phase-Modulation Resolution of Emission
Domain Measurements 169 Spectra 197
5.4. Representative Frequency-Domain Intensity 5.13.1. Resolution Based on Phase or Modulation
Decays 170 Lifetimes 198
5.4.1. Exponential Decays 170 5.13.2. Resolution Based on Phase Angles
5.4.2. Multi-Exponential Decays of and Modulations 198
Staphylococcal Nuclease and Melittin 171 5.13.3. Resolution of Emission Spectra from
5.4.3. Green Fluorescent Protein: One- and Phase and Modulation Spectra 198
Two-Photon Excitation 171 References 199
5.4.4. SPQ: Collisional Quenching of a Problems 203
Chloride Sensor 171
5.4.5. Intensity Decay of NADH 172
5.4.6. Effect of Scattered Light 172
 CONTENTS

7.5. Picosecond Relaxation in Solvents 249

6. Solvent and Environmental Effects
7.5.1. Theory for Time-Dependent Solvent
6.1. Overview of Solvent Polarity Effects 205 250
205 Relaxation
6.1.1. Effects of Solvent Polarity 7.5.2. Multi-Exponential Relaxation in Water 251
6.1.2. Polarity Surrounding a Membrane-Bound 7.6. Measurement of Multi-Exponential Spectral
Fluorophore 206
Relaxation 252
6.1.3. Other Mechanisms for Spectral Shifts 207
7.7. Distinction between Solvent Relaxation
6.2. General Solvent Effects: The Lippert-Mataga 253
208 and Formation of Rotational Isomers
Equation
210 7.8. Comparison of TRES and Decay-Associated
6.2.1. Derivation of the Lippert Equation 255
212 Spectra
6.2.2. Application of the Lippert Equation 255
213 7.9. Lifetime-Resolved Emission Spectra
6.3. Specific Solvent Effects 257
215 7.10. Red-Edge Excitation Shifts
6.3.1. Specific Solvent Effects and Lippert Plots
216 7.10.1. Membranes and Red-Edge
6.4. Temperature Effects 258
217 Excitation Shifts
6.5. Phase Transitions in Membranes
219 7.10.2. Red-Edge Excitation Shifts and
6.6. Additional Factors that Affect Emission Spectra
Energy Transfer 259
6.6.1. Locally Excited and Internal
7.11. Excited-State Reactions 259
Charge-Transfer States 219
7.11.1. Excited-State Ionization of Naphthol 260
6.6.2. Excited-State Intramolecular Proton
7.12. Theory for a Reversible Two-State Reaction 262
Transfer (ESIPT) 221
7.12.1. Steady-State Fluorescence of a
6.6.3. Changes in the Non-Radiative
222 Two-State Reaction 262
Decay Rates
6.6.4. Changes in the Rate of Radiative Decay 223 7.12.2. Time-Resolved Decays for the
223 Two-State Model 263
6.7. Effects of Viscosity
7.12.3. Differential Wavelength Methods 264
6.7.1. Effect of Shear Stress on Membrane
Viscosity 225 7.13. Time-Domain Studies of Naphthol Dissociation 264
6.8. Probe—Probe Interactions 225 7.14. Analysis of Excited-State Reactions by
6.9. Biochemical Applications of Environment- Phase-Modulation Fluorometry 265
Sensitive Fluorophores 226 7.14.1. Effect of an Excited-State Reaction
6.9.1. Fatty-Acid-Binding Proteins 226 on the Apparent Phase and Modulation
6.9.2. Exposure of a Hydrophobic Surface Lifetimes 266
on Calmodulin 226 7.14.2. Wavelength-Dependent Phase and
6.9.3. Binding to Cyclodextrin Using a Modulation Values for an Excited-State
Dansyl Probe 227 Reaction 267
6.10. Advanced. Solvent-Sensitive Probes 228 7.14.3. Frequency-Domain Measurement of
6.11. Effects of Solvent Mixtures 229 Excimer Formation 269
6.12. Summary of Solvent Effects 231 7.15. Biochemical Examples of Excited-State
References 232 Reactions 270
Problems 235 7.15.1. Exposure of a Membrane-Bound
Cholesterol Analogue 270
7. Dynarnics of Solvent and Spectral nellaxation References 270
Problems 275
7.1. Overview of Excited-State Processes 237
7.1.1. Time-Resolved Emission Spectra 239
7.2. Measurement of Time-Resolved Emission
Spectra (TRES) 8 Quenching of Fluorescence
240
7.2.1. Direct Recording of TRES 240
7.2.2. TRES from Wavelength-Dependent 8.1. Quenchers of Fluorescence 278
Decays 8.2. Theory of Collisional Quenching 278
241
7.3. Spectral Relaxation in Proteins 242 8.2.1. Derivation of the Stern-Volmer Equation 280
7.3.1. Spectral Relaxation of Labeled 8.2.2. Interpretation of the Bimolecular
Apomyoglobin 243 Quenching Constant 281
7.3.2. Protein Spectral Relaxation around a 8.3. Theory of Static Quenching 282
Synthetic Fluorescent Amino Acid 244 8.4. Combined Dynamic and Static Quenching 282
7.4. Spectral Relaxation in Membranes 245 8.5. Examples of Static and Dynamic Quenching 283
7.4.1. Analysis of Time-Resolved Emission 8.6. Deviations from the Stern-Volmer Equation:
Spectra 246 Quenching Sphere of Action 284
7.4.2. Spectral Relaxation of Membrane-Bound 8.6.1. Derivation of the Quenching Sphere
Anthroyloxy Fatty Acids 248 of Action 285
PR1NCIPLES OF FLUORESCENCE SPECTR SCOF"( xix

8.7. Effects of Steric Shielding and Charge on 8.16.2. Molecular Beacons Based on Quenching
Quenching 286 by a Gold Surface 314
8.7.1. Accessibility of DNA-Bound Probes 8.17. Intramolecular Quenching 314
to Quenchers 286 8.17.1. DNA Dynamics by Intramolecular
8.7.2. Quenching of Ethenoadenine Derivatives 287 Quenching 314
8.8. Fractional Accessibility to Quenchers 288 8.17.2. Electron-Transfer Quenching in a
8.8.1. Modified Stern-Volmer Plots 288 Flavoprotein 315
8.8.2. Experimental Considerations 8.17.3. Sensors Based on Intramolecular
in Quenching 289 PET Quenching 316
8.9. Applications of Quenching to Proteins 290 8.18. Quenching of Phosphorescence 317
8.9.1. Fractional Accessibility of Tryptophan References 318
Residues in Endonuclease 290 Problems 327
8.9.2. Effect of Conformational Changes
on Tryptophan Accessibility 291
8.9.3. Quenching of the Multiple Decay 9. Mechanisnis and Dynarnics of
Times of Proteins 291 Fluorescence Quenching
8.9.4. Effects of Quenchers on Proteins 292
8.9.5. Correlation of Emission Wavelength 9.1. Comparison of Quenching and Resonance
and Accessibility: Protein Folding of Energy Transfer 331
Colicin El 292 9.1.1. Distance Dependence of RET
8.10. Application of Quenching to Membranes 293 and Quenching 332
8.10.1. Oxygen Diffusion in Membranes 293 9.1.2. Encounter Complexes and Quenching
8.10.2. Localization of Membrane-Bound Efficiency 333
Tryptophan Residues by Quenching 294 9.2. Mechanisms of Quenching 334
8.10.3. Quenching of Membrane Probes 9.2.1. Intersystem Crossing 334
Using Localized Quenchers 295 9.2.2. Electron-Exchange Quenching 335
8.10.4. Parallax and Depth-Dependent 9.2.3. Photoinduced Electron Transfer 335
Quenching in Membranes 296 9.3. Energetics of Photoinduced Electron Transfer 336
8.10.5. Boundary Lipid Quenching 298 9.3.1. Examples of PET Quenching 338
8.10.6. Effect of Lipid–Water Partitioning 9.3.2. PET in Linked Donor–Acceptor Pairs 340
on Quenching 298 9.4. PET Quenching in Biomolecules 341
8.10.7. Quenching in Micelles 300 9.4.1. Quenching of Indole by Imidazolium 341
8.11. Lateral Diffusion in Membranes 300 9.4.2. Quenching by DNA Bases and
8.12. Quenching-Resolved Emission Spectra 301 Nucleotides 341
8.12.1. Fluorophore Mixtures 301 9.5. Single-Molecule PET 342
8.12.2. Quenching-Resolved Emission Spectra 9.6. Transient Effects in Quenching 343
of the E. Coli Tet Repressor 302 9.6.1. Experimental Studies of Transient
8.13. Quenching and Association Reactions 304 Effects 346
8.13.1. Quenching Due to Specific Binding 9.6.2. Distance-Dependent Quenching
Interactions 304 in Proteins 348
8.14. Sensing Applications of Quenching 305 References 348
8.14.1. Chloride-Sensitive Fluorophores 306 Problems 351
8.14.2. Intracellular Chloride Imaging 306
8.14.3. Chloride-Sensitive GFP 307 0. Fluorescence Anisotropy
8.14.4. Amplified Quenching 309
10.1. Definition of Fluorescence Anisotropy 353
8.15. Applications of Quenching to Molecular
310 10.1.1. Origin of the Definitions of
Biology
Polarization and Anisotropy 355
8.15.1. Release of Quenching upon
310 10.2. Theory for Anisotropy 355
Hybridization
10.2.1. Excitation Photoselection of Fluorophores 357
8.15.2. Molecular Beacons in Quenching
311 10.3. Excitation Anisotropy Spectra 358
by Guanine
311 10.3.1. Resolution of Electronic States from
8.15.3. Binding of Substrates to Ribozymes
Polarization Spectra 360
8.15.4. Association Reactions and Accessibility
10.4. Measurement of Fluorescence Anisotropies 361
to Quenchers 312
10.4.1. L-Format or Single-Channel Method 361
8.16. Quenching on Gold Surfaces 313
10.4.2. T-Format or Two-Channel Anisotropies 363
8.16.1. Molecular Beacons Based on Quenching
10.4.3. Comparison ofT-Format and
by Gold Colloids 313
L-Format Measurements 363
 CONTENTS

10.4.4. Alignment of Polarizers 364 11.4.6. Example Anisotropy Decays of

10.4.5. Magic-Angle Polarizer Conditions 364 Rhodamine Green and Rhodamine
10.4.6. Why is the Total Intensity Green-Dextran 394
Equal to 4 + 364 11.5. Time-Domain Anisotropy Decays of Proteins 394
10.4.7. Effect of Resonance Energy Transfer 11.5.1. Intrinsic Tryptophan Anisotropy Decay
on the Anisotropy 364 of Liver Alcohol Dehydrogenase 395
10.4.8. Trivial Causes of Depolarization 365 11.5.2. Phospholipase A2 395
10.4.9. Factors Affecting the Anisotropy 366 11.5.3. Subtilisin Carlsberg 395
10.5. Effects of Rotational Diffusion on Fluorescence 11.5.4. Domain Motions of Immunoglobulins 396
Anisotropies: The Perrin Equation 366 11.5.5. Effects of Free Probe on Anisotropy
10.5.1. The Perrin Equation: Rotational Decays 397
Motions of Proteins 367 11.6. Frequency-Domain Anisotropy Decays
10.5.2. Examples of a Perrin Plot 369 of Proteins 397
10.6. Perrin Plots of Proteins 370 11.6.1. Apomyoglobin: A Rigid Rotor 397
10.6.1. Binding of tRNA to tRNA Synthetase 370 11.6.2. Melittin Self-Association and
10.6.2. Molecular Chaperonin cpn60 (GroEL) 371 Anisotropy Decays 398
10.6.3. Perrin Plots of an Fob Immunoglobulin 11.6.3. Picosecond Rotational Diffusion
Fragment 371 of Oxytocin 399
10.7. Biochemical Applications of Steady-State 11.7. Hindered Rotational Diffusion in Membranes 399
Anisotropies 372 11.7.1. Characterization of a New
10.7.1. Peptide Binding to Calmodulin 372 Membrane Probe 401
10.7.2. Binding of the Trp Repressor to DNA 373 11.8. Anisotropy Decays of Nucleic Acids 402
10.7.3. Helicase-Catalyzed DNA Unwinding 373 11.8.1. Hydrodynamics of DNA Oligomers 403
10.7.4. Melittin Association Detected from 11.8.2. Dynamics of Intracellular DNA 403
Homotransfer 374 11.8.3. DNA Binding to HIV Integrase Using
10.8. Anisotropy of Membranes and Membrane- Correlation Time Distributions 404
Bound Proteins 374 11.9. Correlation Time Imaging 406
10.8.1. Membrane Microviscosity 374 11.10. Microsecond Anisotropy Decays 408
10.8.2. Distribution of Membrane-Bound 11.10.1. Phosphorescence Anisotropy Decays 408
Proteins 375 11.10.2. Long-Lifetime Metal–Ligand
10.9. Transition Moments 377 Complexes 408
References 378 References 409
Additional Reading on the Application Problems 412
of Anisotropy 380
Problems 381
12. Advanced Anis tropy Concepts
1 11. Tinne-Dependent Anisotropy Decays 12.1. Associated Anisotropy Decay 413
12.1.1. Theory for Associated Anisotropy
11.1. Time-Domain and Frequency-Domain Decay 414
Anisotropy Decays 383 12.1.2. Time-Domain Measurements of
11.2. Anisotropy Decay Analysis 387 Associated Anisotropy Decays 415
1L2.1. Early Methods for Analysis of 12.2. Biochemical Examples of Associated
TD Anisotropy Data 387 Anisotropy Decays 417
11.2.2. Preferred Analysis of TD 12.2.1. Time-Domain Studies of DNA
Anisotropy Data 388 Binding to the Klenow Fragment
11.2.3. Value of ro 389 of DNA Polymerase 417
11.3. Analysis of Frequency-Domain 12.2.2. Frequency-Domain Measurements
Anisotropy Decays 390 of Associated Anisotropy Decays 417
11.4. Anisotropy Decay Laws 390 12.3. Rotational Diffusion of Non-Spherical
11.4.1. Non-Spherical Fluorophores 391 Molecules: An Overview 418
11.4.2. Hindered Rotors 391 12.3.1. Anisotropy Decays of Ellipsoids 419
11.4.3. Segmental Mobility of a Biopolymer- 12.4. Ellipsoids of Revolution 420
Bound Fluorophore 392 12.4.1. Simplified Ellipsoids of Revolution 421
11.4.4. Correlation Time Distributions 393 12.4.2. Intuitive Description of Rotational
11.4.5. Associated Anisotropy Decays 393 Diffusion of an Oblate Ellipsoid 422
PRINCIPLES OF FLUORESCENCI SPECTROSCOPY xxi

12.4.3. Rotational Correlation Times for 13.5.2. RET Imaging of Intracellular Protein
Ellipsoids of Revolution 423 Phosphorylation 459
12.4.4. Stick-versus-Slip Rotational Diffusion 425 13.5.3. Imaging of Rac Activation in Cells 459
12.5. Complete Theory for Rotational Diffusion 13.6. RET and Nucleic Acids 459
of Ellipsoids 425 13.6.1. Imaging of Intracellular RNA 460
12.6. Anisotropic Rotational Diffusion 426 13.7. Energy-Transfer Efficiency from
12.6.1. Time-Domain Studies 426 Enhanced Acceptor Fluorescence 461
12.6.2. Frequency-Domain Studies of 13.8. Energy Transfer in Membranes 462
Anisotropic Rotational Diffusion 427 13.8.1. Lipid Distributions around Gramicidin 463
12.7. Global Anisotropy Decay Analysis 429 13.8.2. Membrane Fusion and Lipid Exchange 465
12.7.1. Global Analysis with Multi-Wavelength 13.9. Effect of 1C2 on RET 465
Excitation 429 13.10. Energy Transfer in Solution 466
12.7.2. Global Anisotropy Decay Analysis with 13.10.1. Diffusion-Enhanced Energy Transfer 467
Collisional Quenching 430 13.11. Representative Ro Values 467
12.7.3. Application of Quenching to Protein References 468
Anisotropy Decays 431 Additional References on Resonance
12.8. Intercalated Fluorophores in DNA 432 Energy Transfer 471
12.9. Transition Moments 433 Problems 472
12.9.1. Anisotropy of Planar Fluorophores
with High Symmetry 435
12.10. Lifetime-Resolved Anisotropies 435 114. Time-Resolved Energy Transfer and
12.10.1. Effect of Segmental Motion on the Conforrnational Distributions of Biopolyrners
Perrin Plots 436
12.11. Soleillet's Rule: Multiplication of Depolarized 14.1. Distance Distributions 477
Factors 436 14.2. Distance Distributions in Peptides 479
12.12. Anisotropies Can Depend on Emission 14.2.1. Comparison for a Rigid and Flexible
Wavelength 437 Hexapeptide 479
References 438 14.2.2. Crossfitting Data to Exclude
Problems 441 Alternative Models 481
14.2.3. Donor. Decay without Acceptor 482
14.2.4. Effect of Concentration of the
113. Energy Transfer D–A Pairs 482
14.3. Distance Distributions in Peptides 482
13.1. Characteristics of Resonance Energy Transfer 443 14.3.1. Distance Distributions in Melittin 483
13.2. Theory of Energy Transfer for a 14.4. Distance-Distribution Data Analysis 485
Donor–Acceptor Pair 445 14.4.1. Frequency-Domain Distance-Distribution
13.2.1. Orientation Factor K2 448 Analysis 485
13.2.2. Dependence of the Transfer Rate on 14.4.2. Time-Domain Distance-Distribution
Distance (r), the Overlap Analysis 487
Integral (J), and T2 449 14.4.3. Distance-Distribution Functions 487
13.2.3. Homotransfer and Heterotransfer 450 14.4.4. Effects of Incomplete Labeling 487
13.3. Distance Measurements Using RET 451 14.4.5. Effect of the Orientation Factor -K2 489
13.3.1. Distance Measurements in oc-Helical 14.4.6. Acceptor Decays 489
Melittin 451 14.5. Biochemical Applications of Distance
13.3.2. Effects of Incomplete Labeling 452 Distributions 490
13.3.3. Effect of K2 on the Possible Range 14.5.1. Calcium-Induced Changes in the
of Distances 452 Conformation of Troponin C 490
13.4. Biochemical Applications of RET 453 14.5.2. Hairpin Ribozyme 493
13.4.1. Protein Folding Measured by RET 453 14.5.3. Four-Way Holliday Junction in DNA 493
13.4.2. Intracellular Protein Folding 454 14.5.4. Distance Distributions and Unfolding
13.4.3. RET and Association Reactions 455 of Yeast Phosphoglycerate Kinase 494
13.4.4. Orientation of a Protein-Bound Peptide 456 14.5.5. Distance Distributions in a Glycopeptide 495
13.4.5. Protein Binding to Semiconductor 14.5.6. Single-Protein-Molecule Distance
Nanoparticles 457 Distribution 496
13.5. RET Sensors 458 14.6. Time-Resolved RET Imaging 497
13.5.1. Intracellular RET Indicator 14.7. Effect of Diffusion for Linked D–A Pairs 498
for Estrogens 458
 CONTENTS

14.7.1. Simulations of FRET for a Flexible 16.3. Tryptophan Emission in an Apolar

Protein Environment 538
D–A Pair 499
14.7.2. Experimental Measurement of D–A 16.3.1. Site-Directed Mutagenesis of a
500 Single-Tryptophan Azurin 538
Diffusion for a Linked D–A Pair
14.7.3. PRET and Diffusive Motions in 16.3.2. Emission Spectra of Azurins with
501 One or Two Tryptophan Residues 539
Biopolymers
14.8. Conclusion 501 16.4. Energy Transfer and Intrinsic Protein
501 Fluorescence 539
References
Representative Publications on Measurement 16.4.1. Tyrosine-to-Tryptophan Energy Transfer
504 in Interferon-y 540
of Distance Distributions
Problems 505 16.4.2. Quantitation of RET Efficiencies
in Proteins 541
16.4.3. Tyrosine-to-Tryptophan RET in
II 5. Energy Transfer to Mulitiple Acceptors in a Membrane-Bound Protein 543
One,Two, orThree Dimensions 16.4.4. Phenylalanine-to-Tyrosine
Energy Transfer 543
15.1. RET in Three Dimensions 507 16.5. Calcium Binding to Calmodulin Using
15.1.1. Effect of Diffusion on FRET with Phenylalanine and Tyrosine Emission 545
Unlinked Donors and Acceptors 508 16.6. Quenching of Tryptophan Residues in Proteins 546
15.1.2. Experimental Studies of RET in 16.6.1. Effect of Emission Maximum on
Three Dimensions 509 Quenching 547
15.2. Effect of Dimensionality on RET 511 16.6.2. Fractional Accessibility to Quenching
15.2.1. Experimental FRET in Two Dimensions 512 in Multi-Tryptophan Proteins 549
15.2.2. Experimental FRET in One Dimension 514 16.6.3. Resolution of Emission Spectra by
15.3. Biochemical Applications of RET with Quenching 550
Multiple Acceptors 515 16.7. Association Reaction of Proteins 551
15.3.1. Aggregation of ß-Amyloid Peptides 515 16.7.1. Binding of Calmodulin to a
15.3.2. RET Imaging of Fibronectin 516 Target Protein 551
15.4. Energy Transfer in Restricted Geometries 516 16.7.2. Calmodulin: Resolution of the
15.4.1. Effect of Excluded Area on Energy Four Calcium-Binding Sites Using
Transfer in Two Dimensions 518 Tryptophan-Containing Mutants 552
15.5. RET in the Presence of Diffusion 519 16.7.3. Interactions of DNA with Proteins 552
15.6. RET in the Rapid Diffusion Limit 520 16.8. Spectral Properties of Genetically Engineered
15.6.1. Location of an Acceptor in Proteins 554
Lipid Vesicles 521 16.8.1. Single-Tryptophan Mutants of
15.6.2. Locaion of Retinal in Rhodopsin Triosephosphate Isomerase 555
Disc Membranes 522 16.8.2. Barnase: A Three-Tryptophan Protein 556
15.7. Conclusions 524 16.8.3. Site-Directed Mutagenesis of
References 524 557
Tyrosine Proteins
Additional References on RET between 16.9. Protein Folding 557
Unlinked Donor and Acceptor 526 16.9.1. Protein Engineering of Mutant
Problems 527 Ribonuclease for Folding Experiments 558
16.9.2. Folding of Lactate Dehydrogenase 559
16. Protein Pluoresce 16.9.3. Folding Pathway of CRABPI 560
16.10. Protein Structure and Tryptophan Emission 560
16.1. Spectral Properties of the Aromatic Amino Acids 530 16.10.1. Tryptophan Spectral Properties
16.1.1. Excitation Polarization Spectra of and Structural Motifs 561
Tyrosine and Tryptophan 531 16.11. Tryptophan Analogues 562
16.1.2. Solvent Effects on Tryptophan Emission 16.11.1. Tryptophan Analogues 564
Spectra 533 16.11.2. Genetically Inserted Amino-Acid
16.1.3. Excited-State Ionization of Tyrosine 534 Analogues 565
16.1.4. Tyrosinate Emission from Proteins 535 16.12. The Challenge of Protein Fluorescence 566
16.2. General Features of Protein Fluorescence 535 References 567
Problems 573
PRHNCIPLES OF FLUORESCENCIE SPECTROSCOPY XXIII

17. Time-Resolved Protein Fluorescence 18.5.1. Excitation Photoselection for

Two-Photon Excitation 612
17.1. Intensity Decays of Tryptophan: 18.5.2. Two-Photon Anisotropy of DPH 612
The Rotamer Model 578 18.6. MPE for a Membrane-Bound Fluorophore 613
17.2. Time-Resolved Intensity Decays of 18.7. MPE of Intrinsic Protein Fluorescence 613
Tryptophan and Tyrosine 580 18.8. Multiphoton Microscopy 616
17.2.1. Decay-Associated Emission Spectra 18.8.1. Calcium Imaging 616
of Tryptophan 581 18.8.2. Imaging of NAD(P)H and FAD 617
17.2.2. Intensity Decays of Neutral Tryptophan 18.8.3. Excitation of Multiple Fluorophores 618
Derivatives 581 18.8.4. Three-Dimensional Imaging of Cells 618
17.2.3. Intensity Decays of Tyrosine and References 619
Its Neutral Derivatives 582 Problems 621
17.3. Intensity and Anisotropy Decays of Proteins 583
17.3.1. Single-Exponential Intensity and
Anisotropy Decay of Ribonuclease T1 584 19. P1u rescence Sensing
17.3.2. Annexin V: A Calcium-Sensitive
Single-Tryptophan Protein 585 19.1. Optical Clinical Chemistry and Spectral
17.3.3. Anisotropy Decay of a Protein with Observables 623
Two Tryptophans 587 19.2. Spectral Observables for Fluorescence Sensing 624
17.4. Protein Unfolding Exposes the Tryptophan 19.2.1. Optical Properties of Tissues 625
Residue to Water 588 19.2.2. Lifetime-Based Sensing 626
17.4.1. Conformational Heterogeneity Can 19.3. Mechanisms of Sensing 626
Result in Complex Intensity and 19.4. Sensing by Collisional Quenching 627
Anisotropy Decays 588 19.4.1. Oxygen Sensing 627
17.5. Anisotropy Decays of Proteins 589 19.4.2. Lifetime-Based Sensing of Oxygen 628
17.5.1. Effects of Association Reactions on 19.4.3. Mechanism of Oxygen Selectivity 629
Anisotropy Decays: Melittin 590 19.4.4. Other Oxygen Sensors 629
17.6. Biochemical Examples Using Time-Resolved 19.4.5. Lifetime Imaging of Oxygen 630
Protein Fluorescence 591 19.4.6. Chloride Sensors 631
17.6.1. Decay-Associated Spectra of Barnase 591 19.4.7. Lifetime Imaging of Chloride
17.6.2. Disulfide Oxidoreductase DsbA 591 Concentrations 632
17.6.3. Immunophilin FKBP59-I: Quenching 19.4.8. Other Collisional Quenchers 632
of Tryptophan Fluorescence by 19.5. Energy-Transfer Sensing 633
Phenylalanine 592 19.5.1. pH and pCO2 Sensing by
17.6.4. Trp Repressor: Resolution of the Two Energy Transfer 633
Interacting Tryptophans 593 19.5.2. Glucose Sensing by Energy Transfer 634
17.6.5. Thermophilic ß-Glycosidase: 19.5.3. Ion Sensing by Energy Transfer 635
A Multi-Tryptophan Protein 594 19.5.4. Theory for Energy-Transfer Sensing 636
17.6.6. Herne Proteins Display Useful 19.6. Two-State pH Sensors 637
Intrinsic Fluorescence 594 19.6.1. Optical Detection of Blood Gases 637
19.6.2. pH Sensors 637
17.7. Time-Dependent Spectral Relaxation of
596 19.7. Photoinduced Electron Transfer (PET) Probes
Tryptophan
598 for Metal Ions and Anion Sensors 641
17.8. Phosphorescence of Proteins
600 19.8. Probes of Analyte Recognition 643
17.9. Perspectives on Protein Fluorescence
600 19.8.1. Specificity of Cation Probes 644
References
605 19.8.2. Theory of Analyte Recognition Sensing 644
Problems
19.8.3. Sodium and Potassium Probes 645
19.8.4. Calcium and Magnesium Probes 647
18. Multiph ton Excitation and Cri icroscopy 19.8.5. Probes for Intracellular Zinc 650
19.9. Glucose-Sensitive Fluorophores 650
18.1. Introduction to Multiphoton Excitation 607 19.10. Protein Sensors 651
18.2. Cross-Sections for Multiphoton Absorption 609 19.10.1. Protein Sensors Based on RET 652
18.3. Two-Photon Absorption Spectra 609 19.11. GFP Sensors 654
18.4. Two-Photon Excitation of a DNA-Bound 19.11.1. GFP Sensors Using RET 654
Fluorophore 610 19.11.2. Intrinsic GFP Sensors 655
18.5. Anisotropies with Multiphoton Excitation 612
 CONTENTS
xxiv

19.12. New Approaches to Sensing 655 21.2.3. DNA Fragment Sizing by

19.12.1. Pebble Sensors and Lipobeads 655 Flow Cytometry 715
19.13. In-Vivo Imaging 656 21.3. DNA Hybridization 715
19.14. Immunoassays 658 21.3.1. DNA Hybridization Measured with
19.14.1. Enzyme-Linked Immunosorbent Assays One-Donor- and Acceptor-Labeled
(ELISA) 659 DNA Probe 717
19.14.2. Time-Resolved Immunoassays 659 21.3.2. DNA Hybridization Measured by
19.14.3. Energy-Transfer Immunoassays 660 Excimer Formation 718
19.14.4. Fluorescence Polarization 21.3.3. Polarization Hybridization Arrays 719
Immunoassays 661 21.3.4. Polymerase Chain Reaction 720
References 663 21.4. Molecular Beacons 720
Problems 672 21.4.1. Molecular Beacons with
Nonfluorescent Acceptors 720
21.4.2. Molecular Beacons with
20. Novel Fluorophores Fluorescent Acceptors 722
21.4.3. Hybridization Proximity Beacons 722
20.1. Semiconductor Nanoparticles 675 21.4.4. Molecular Beacons Based on
20.1.1. Spectral Properties of QDots 676 Quenching by Gold 723
20.1.2. Labeling Cells with QDots 677 21.4.5. Intracellular Detection of mRNA
20.1.3. QDots and Resonance Energy Transfer 678 Using Molecular Beacons 724
20.2. Lanthanides 679 724
21.5. Aptamers
20.2.1. RET with Lanthanides 680 726
21.5.1. DNAzymes
20.2.2. Lanthanide Sensors 681 21.6. Multiplexed Microbead Arrays:
20.2.3. Lanthanide Nanoparticles 682
Suspension Arrays 726
20.2.4. Near-Infrared Emitting Lanthanides 682
21.7. Fluorescence In-Situ Hybridization 727
20.2.5. Lanthanides and Fingerprint Detection 683
21.7.1. Preparation of FISH Probe DNA 728
20.3. Long-Lifetime Metal–Ligand Complexes 683
21.7.2. Applications of FISH 729
20.3.1. Introduction to Metal–Ligand Probes 683
21.8. Multicolor FISH and Spectral Karyotyping 730
20.3.2. Anisotropy Properties of
21.9. DNA Arrays 732
Metal–Ligand Complexes 685
21.9.1. Spotted DNA Microarrays 732
20.3.3. Spectral Properties of MLC Probes 686
21.9.2. Light-Generated DNA Arrays 734
20.3.4. The Energy Gap Law 687
References 734
20.3.5. Biophysical Applications of
Problems 740
Metal–Ligand Probes 688
20.3.6. MLC Immunoassays 691
20.3.7. Metal–Ligand Complex Sensors 694 22. Fluorescence-Liffetime Drnaging Hicroscopy
20.4. Long-Wavelength Long-Lifetime
Fluorophores 695 22.1. Early Methods for Fluorescence-Lifetime
References 697 Imaging 743
Problems 702 22.1.1. FLIM Using Known Fluorophores 744
22.2. Lifetime Imaging of Calcium Using Quin-2 744
22.2.1. Determination of Calcium Concentration
2L DHATechnology from Lifetime 744
21.1. DNA Sequencing 22.2.2. Lifetime Images of Cos Cells 745
705
21.1.1. Principle of DNA Sequencing 22.3. Examples of Wide-Field Frequency-Domain
705
2L1.2. Examples of DNA Sequencing FLIM 746
706
21.1.3. Nucleotide Labeling Methods 22.3.1. Resonance Energy-Transfer FLIM
707
21.1.4. Example of DNA Sequencing 708 of Protein Kinase C Activation 746
21.1.5. Energy-Transfer Dyes for DNA 22.3.2. Lifetime Imaging of Cells Containing
Sequencing Two GFPs 747
709
21.1.6. DNA Sequencing with MR Probes 710 22.4. Wide-Field FLIM Using a Gated-Image
21.1.7. DNA Sequencing Based on Lifetimes Intensifier 747
712
21.2. High-Sensitivity DNA Stains 22.5. Laser Scanning TCSPC FLIM 748
712
21.2.1. High-Affinity Bis DNA Stains 713 22.5.1. Lifetime Imaging of Cellular
21.2.2. Energy-Transfer DNA Stains 715 Biomolecules 750
22.5.2. Lifetime Images of Amyloid Plaques 750
P INCIPLES OF FLUORESCENCE SPECTROSCOPY xxv

22.6. Frequency-Domain Laser Scanning Microscopy 750 24.2. Theory of FCS 800
22.7. Conclusions 752 24.2.1. Translational Diffusion and FCS 802
References 752 24.2.2. Occupation Numbers and Volumes
Additional Reading on Fluorescence-Lifetime in FCS 804
Imaging Microscopy 753 24.2.3. FCS for Multiple Diffusing Species 804
Problem 755 24.3. Examples of FCS Experiments 805
24.3.1. Effect of Fluorophore Concentration 805
24,3.2. Effect of Molecular Weight on
23e Single-Holende Detection Diffusion Coefficients 806
24.4. Applications of FCS to Bioaffinity Reactions 807
23.1. Detectability of Single Molecules 759
24.4.1. Protein Binding to the
23.2. Total Internal Reflection and Confocal Optics 760
Chaperonin GroEL 807
23.2.1. Total Internal Reflection 760
24.4.2. Association of Tubulin Subunits 807
23.2.2. Confocal Detection Optics 761
24.4.3. DNA Applications of FCS 808
23.3. Optical Configurations for SMD 762
24.5. FCS in Two Dimensions: Membranes 810
23.4. Instrumentation for SMD 764
24.5.1. Biophysical Studies of Lateral
23.4.1. Detectors for Single-Molecule Detection 765
Diffusion in Membranes 812
23.4.2. Optical Filters for SMD 766
24.5.2. Binding to Membrane-Bound
23.5. Single-Molecule Photophysics 768
Receptors 813
23.6. Biochemical Applications of SMD 770
24.6. Effects of Intersystem Crossing 815
23.6.1. Single-Molecule Enzyme Kinetics 770
24.6.1. Theory for FCS and Intersystem
23.6.2. Single-Molecule ATPase Activity 770
Crossing 816
23.6.3. Single-Molecule Studies of a
24.7. Effects of Chemical Reactions 816
Chaperonin Protein 771
24.8. Fluorescence Intensity Distribution Analysis 817
23.7. Single-Molecule Resonance Energy Transfer 773
24.9. Time-Resolved FCS 819
23.8. Single-Molecule Orientation and Rotational
24.10. Detection of Conformational Dynamics
Motions 775
in Macromolecules 820
23.8.1. Orientation Imaging of R6G and GFP 777
24.11. FCS with Total Internal Reflection 821
23.8.2. Imaging of Dipole Radiation Patterns 778
24.12. FCS with Two-Photon Excitation 822
23.9. Time-Resolved Studies of Single Molecules 779
24.12.1. Diffusion of an Intracellular
23.10. Biochemical Applications 780
Kinase Using FCS with
23.10.1. Turnover of Single Enzyme Molecules 780
Two-Photon Excitation 823
23.10.2. Single-Molecule Molecular Beacons 782
24.13. Dual-Color Fluorescence Cross-Correlation
23.10.3. Conformational Dynamics of a Spectroscopy 823
Holliday Junction 782
24.13.1. Instrumentation for Dual-Color
23.10.4. Single-Molecule Calcium Sensor 784
FCCS 824
23.10.5. Motions of Molecular Motors 784
24.13.2. Theory of Dual-Color FCCS 824
23.11. Advanced Topics in SMD 784
24.13.3. DNA Cleavage by a
23.11.1. Signal-to-Noise Ratio in Restriction Enzyme 826
Single-Molecule Detection 784
24.13.4. Applications of Dual-Color FCCS 826
23.11.2. Polarization of Single Immobilized 24.14. Rotational Diffusion and Photo Antibunching 828
Fluorophores 786
24.15. Flow Measurements Using FCS 830
23.11.3. Polarization Measurements 24.16. Additional References on FCS 832
and Mobility of Surface-Bound References 832
Fluorophores 786
Additional References to FCS and
23.11.4. Single-Molecule Lifetime Estimation 787
Its Applications 837
23.12. Additional Literature on SMD 788
Problems 840
References 788
Additional References on Single-Molecule
Detection 791 25. Radiative Decay Engineering:
795
Problem Letal-Enhanced Fluorescence
25.1. Radiative Decay Engineering 841
24. Flu rescence Correlation Spectroscopy 25.1.1. Introduction to RDE 841
25.1.2. Jablonski Diagram for Metal-
24.1. Principles of Fluorescence Correlation Enhanced Fluorescence 842
Spectroscopy 798 25.2. Review of Metal Effects on Fluorescence 843
 xxv i CONTENTS

25.3. Optical. Properties of Metal Colloids 845 Appendix HL Fluorescent Liffetirrae Standards
25.4. Theory for Fluorophore-Colloid Interactions 846
25.5. Experimental Results an Metal-Enhanced 1. Nanosecond Lifetime Standards 883
Fluorescence 848 2. Picosecond Lifetime Standards 884
25.5.1. Application of MEF to DNA Analysis 848 3. Representative Frequency-Domain
25.6. Distance-Dependence of Metal-Enhanced Intensity Decays 885
Fluorescence 851 4. Time-Domain Lifetime Standards 886
25.7. Applications of Metal-Enhanced Fluorescence 851
25.7.1. DNA Hybridization Using MEF 853
Appendix IN. Additional Reading
25.7.2. Release of Self-Quenching 853
25.7.3. Effect of Silver Particles an RET 854 1. Time-Resolved Measurements 889
25.8. Mechanism of MEF 855 2. Spectra Properties of Fluorophores 889
25.9. Perspective an RET 856 3. Theory of Fluorescence and Photophysics 889
References 856 4. Reviews of Fluorescence Spectroscopy 889
Problem 859 5. Biochemical Fluorescence 890
6. Protein Fluorescence 890
7. Data Analysis and Nonlinear Least Squares 890
26. Ra.di tive Decay Engirueering: 8. Photochemistry 890
Suriace Plasmon-Coupied Emission 9. Flow Cytometry 890
10. Phosphorescence 890
26.1. Phenomenon of SPCE 861 11. Fluorescence Sensing 890
26.2. Surface-Plasmon Resonance 861 12. Immunoassays 891
26.2.1. Theory for Surface-Plasmon Resonance 863 13. Applications of Fluorescence 891
26.3. Expected Properties of SPCE 865 14. Multiphoton Excitation 891
26.4. Experimental Demonstration of SPCE 865 15. Infrared and NIR Fluorescence 891
26.5. Applications of SPCE 867 16. Lasers 891
26.6. Future Developments in SPCE 868 17. Fluorescence Microscopy 891
References 870 18. Metal-Ligand Complexes and Unusual
Lumophores 891
19. Single-Molecule Detection 891
20. Fluorescence Correlation Spectroscopy 892
Appendix L Corrected Emission Spectra 21. Biophotonics 892
22. Nanoparticles 892
1. Emission Spectra Standards from 300 to 800 nm 873 23. Metallic Particles 892
2. I3-Carboline Derivatives as Fluorescence Standards 873 24. Books an Fluorescence 892
3. Corrected Emission Spectra of 9,10-Diphenyl-
anthracene, Quinine, and Fluorescein 877
4. Long-Wavelength Standards 877 Answers to Problems 893
5. Ultraviolet Standards 878
6. Additional Corrected Emission Spectra 881
References 881 [Index 923

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