Principles of fluorescence spectroscopy:
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| Beschreibung: | XXVI, 954 Seiten Illustrationen, Diagramme 1 CD-ROM ; Extras online [nur bei reprints der 3. edition von 2006] |
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| 100 | 1 | |a Lakowicz, Joseph R. |e Verfasser |4 aut | |
| 245 | 1 | 0 | |a Principles of fluorescence spectroscopy |c Joseph R. Lakowicz |
| 250 | |a Third edition | ||
| 264 | 1 | |a New York, NY |b Springer |c [2006] | |
| 300 | |a XXVI, 954 Seiten |b Illustrationen, Diagramme |e 1 CD-ROM ; Extras online [nur bei reprints der 3. edition von 2006] | ||
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| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
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| 650 | 7 | |a Spectroscopy |2 cabt | |
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Datensatz im Suchindex
| _version_ | 1823947338786275328 |
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| adam_text |
1. Introduction to Fluorescence
1.1. Phenomena of Fluorescence 1
1.2. Jablonski Diagram 3
1.3. Characteristics of Fluorescence Emission 6
1.3.1. The Stokes Shift 6
1.3.2. Emission Spectra Are Typically Independent
of the Excitation Wavelength 7
1.3.3. Exceptions to the Mirror Image Rule 8
1.4. Fluorescence Lifetimes and Quantum Yields 9
1.4.1. Fluorescence Quenching 11
1.4.2. Timescale of Molecular Processes
in Solution 12
1.5. Fluorescence Anisotropy 12
1.6. Resonance Energy Transfer 13
1.7. Steady State and Time Resolved Fluorescence 14
1.7.1. Why Time Resolved Measurements? 15
1.8. Biochemical Fluorophores 15
1.8.1. Fluorescent Indicators 16
1.9. Molecular Information from Fluorescence 17
1.9.1. Emission Spectra and the Stokes Shift 17
1.9.2. Quenching of Fluorescence 18
1.9.3. Fluorescence Polarization or Anisotropy 19
1.9.4. Resonance Energy Transfer 19
1.10. Biochemical Examples of Basic Phenomena 20
1.11. New Fluorescence Technologies 21
1.11.1. Multiphoton Excitation 21
1.11.2. Fluorescence Correlation Spectroscopy 22
1.11.3. Single Molecule Detection 23
1.12. Overview of Fluorescence Spectroscopy 24
References 25
Problems 25
2. Instrumentation for Fluorescence
Spectroscopy
2.1. Spectrofluorometers 27
2.1.1. Spectrofluorometers for Spectroscopy
Research 27
2.1.2. Spectrofluorometers for High Throughput. 29
2.1.3. An Ideal Spectrofluorometer 30
2.1.4. Distortions in Excitation and Emission
Spectra 30
2.2. Light Sources 31
2.2.1. Arc Lamps and Incandescent
Xenon Lamps 31
2.2.2. Pulsed Xenon Lamps 32
2.2.3. High Pressure Mercury (Hg) Lamps 33
2.2.4. Xe Hg Arc Lamps 33
2.2.5. Quartz Tungsten Halogen (QTH) Lamps 33
2.2.6. Low Pressure Hg and Hg Ar Lamps 33
2.2.7. LED Light Sources 33
2.2.8. Laser Diodes 34
2.3. Monochromators 34
2.3.1. Wavelength Resolution and Emission
Spectra 35
2.3.2. Polarization Characteristics of
Monochromators 36
2.3.3. Stray Light in Monochromators 36
2.3.4. Second Order Transmission in
Monochromators 37
2.3.5. Calibration of Monochromators 38
2.4. Optical Filters 38
2.4.1. Colored Filters 38
2.4.2. Thin Film Filters 39
2.4.3. Filter Combinations 40
2.4.4. Neutral Density Filters 40
2.4.5. Filters for Fluorescence Microscopy 41
2.5. Optical Filters and Signal Purity 41
2.5.1. Emission Spectra Taken through Filters 43
2.6. Photomultiplier Tubes 44
2.6.1. Spectral Response of PMTs 45
2.6.2. PMT Designs and Dynode Chains 46
2.6.3. Time Response of Photomultiplier Tubes 47
2.6.4. Photon Counting versus Analog Detection
of Fluorescence 48
2.6.5. Symptoms of PMT Failure 49
2.6.6. CCD Detectors 49
2.7. Polarizers 49
2.8. Corrected Excitation Spectra 51
2.8.1. Corrected Excitation Spectra Using
a Quantum Counter 51
2.9. Corrected Emission Spectra 52
2.9.1. Comparison with Known Emission
Spectra 52
2.9.2. Corrections Using a Standard Lamp 53
2.9.3. Correction Factors Using a Quantum
Counter and Scatterer 53
XV
2.9.4. Conversion between Wavelength and
Wavenumber 53
2.10. Quantum Yield Standards 54
2.11. Effects of Sample Geometry 55
2.12. Common Errors in Sample Preparation 57
2.13. Absorption of Light and Deviation from the
Beer Lambert Law 58
2.13.1. Deviations from Beer's Law 59
2.14. Conclusions 59
References 59
Problems 60
3. Fluorophores
3.1. Intrinsic or Natural Fluorophores 63
3.1.1. Fluorescence Enzyme Cofactors 63
3.1.2. Binding of NADH to a Protein 65
3.2. Extrinsic Fluorophores 67
3.2.1. Protein Labeling Reagents 67
3.2.2. Role of the Stokes Shift in Protein
Labeling 69
3.2.3. Photostability of Fluorophores 70
3.2.4. Non Covalent Protein Labeling
Probes 71
3.2.5. Membrane Probes 72
3.2.6. Membrane Potential Probes 72
3.3. Red and Near Infrared (NIR) Dyes 74
3.4. DNA Probes 75
3.4.1. DNA Base Analogues 75
3.5. Chemical Sensing Probes 78
3.6. Special Probes 79
3.6.1. Fluorogenic Probes 79
3.6.2. Structural Analogues of Biomolecules 80
3.6.3. Viscosity Probes 80
3.7. Green Fluorescent Proteins 81
3.8. Other Fluorescent Proteins 83
3.8.1. Phytofluors: A New Class of
Fluorescent Probes 83
3.8.2. Phycobiliproteins 84
3.8.3. Specific Labeling of Intracellular
Proteins 86
3.9. Long Lifetime Probes 86
3.9.1. Lanthanides 87
3.9.2. Transition Metal Ligand Complexes 88
.10. Proteins as Sensors 88
.11. Conclusion 89
References 90
Problems 94
L Time Domain Lifetime Measurements
4.1. Overview of Time Domain and Frequency
Domain Measurements 98
4.1.1. Meaning of the Lifetime or Decay Time 99
4.1.2. Phase and Modulation Lifetimes 99
4.1.3. Examples of Time Domain and
Frequency Domain Lifetimes 100
4.2. Biopolymers Display Multi Exponential or
Heterogeneous Decays 101
4.2.1. Resolution of Multi Exponential
Decays Is Difficult 103
4.3. Time Correlated Single Photon Counting 103
4.3.1. Principles of TCSPC 104
4.3.2. Example of TCSPC Data 105
4.3.3. Convolution Integral 106
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
4.4.3. Picosecond Dye Lasers 110
4.4.4. Flashlamps 112
4.4.5. Synchrotron Radiation 114
4.5. Electronics for TCSPC 114
4.5.1. Constant Fraction Discriminators 114
4.5.2. Amplifiers 115
4.5.3. Time to Amplitude Converter (TAC)
and Analyte to Digital Converter (ADC) 115
4.5.4. Multichannel Analyzer 116
4.5.5. Delay Lines 116
4.5.6. Pulse Pile Up 116
4.6. Detectors for TCSPC 117
4.6.1. MicroChannel Plate PMTs 117
4.6.2. Dynode Chain PMTs 118
4.6.3. Compact PMTs 118
4.6.4. Photodiodes as Detectors 118
4.6.5. Color Effects in Detectors 119
4.6.6. Timing Effects of Monochromators 121
4.7. Multi Detector and Multidimensional TCSPC 121
4.7.1. Multidimensional TCSPC and
DNA Sequencing 123
4.7.2. Dead Times, Repetition Rates, and
Photon Counting Rates 124
4.8. Alternative Methods for Time Resolved
Measurements 124
4.8.1. Transient Recording 124
4.8.2. Streak Cameras 125
4.8.3. Upconversion Methods 128
4.8.4. Microsecond Luminescence Decays 129
4.9. Data Analysis: Nonlinear Least Squares 129
4.9.1. Assumptions of Nonlinear Least Squares 130
4.9.2. Overview of Least Squares Analysis 130
4.9.3. Meaning of the Goodness of Fit 131
4.9.4. Autocorrelation Function 132
4.10. Analysis of Multi Exponential Decays 133
4.10.1. p Terphenyl and Indole: Two Widely
Spaced Lifetimes 133
4.10.2. Comparison of xR2 Values: F Statistic 133
4.10.3. Parameter Uncertainty: Confidence
Intervals 134
4.10.4. Effect of the Number of Photon Counts 135
4.10.5. Anthranilic Acid and 2 Aminopurine:
Two Closely Spaced Lifetimes 137
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
4.10.6. Global Analysis: Multi Wavelength
Measurements 138
4.10.7. Resolution of Three Closely Spaced
Lifetimes 138
4.11. Intensity Decay Laws 141
4.11.1. Multi Exponential Decays 141
4.11.2. Lifetime Distributions 143
4.11.3. Stretched Exponentials 144
4.11.4. Transient Effects 144
4.12. Global Analysis 144
4.13. Applications of TCSPC 145
4.13.1. Intensity Decay for a Single Tryptophan
Protein 145
4.13.2. Green Fluorescent Protein: Systematic
Errors in the Data 145
4.13.3. Picosecond Decay Time 146
4.13.4. Chlorophyll Aggregates in Hexane 146
4.13.5. Intensity Decay of Flavin Adenine
Dinucleotide (FAD) 147
4.14. Data Analysis: Maximum Entropy Method 148
References 149
Problems 154
5. Frequency Domain Lifetime
Measurements
5.1. Theory of Frequency Domain Fluorometry 158
5.1.1. Least Squares Analysis of Frequency
Domain Intensity Decays 161
5.1.2. Global Analysis of Frequency Domain
Data 162
5.2. Frequency Domain Instrumentation 163
5.2.1. History of Phase Modulation
Fluorometers 163
5.2.2. An MHz Frequency Domain Fluorometer. 164
5.2.3. Light Modulators 165
5.2.4. Cross Correlation Detection 166
5.2.5. Frequency Synthesizers 167
5.2.6. Radio Frequency Amplifiers 167
5.2.7. Photomultiplier Tubes 167
5.2.8. Frequency Domain Measurements 168
5.3. Color Effects and Background Fluorescence 168
5.3.1. Color Effects in Frequency Domain
Measurements 168
5.3.2. Background Correction in Frequency
Domain Measurements 169
5.4. Representative Frequency Domain Intensity
Decays 170
5.4.1. Exponential Decays 170
5.4.2. Multi Exponential Decays of
Staphylococcal Nuclease and Melittin 171
5.4.3. Green Fluorescent Protein: One and
Two Photon Excitation 171
5.4.4. SPQ: Collisional Quenching of a
Chloride Sensor 171
5.4.5. Intensity Decay of NADH 172
5.4.6. Effect of Scattered Light 172
xvii
5.5. Simple Frequency Domain Instruments 173
5.5.1. Laser Diode Excitation 174
5.5.2. LED Excitation 174
5.6. Gigahertz Frequency Domain Fluorometry 175
5.6.1. Gigahertz FD Measurements 177
5.6.2. Biochemical Examples of Gigahertz
FDData 177
5.7. Analysis of Frequency Domain Data 178
5.7.1. Resolution of Two Widely Spaced
Lifetimes 178
5.7.2. Resolution of Two Closely Spaced
Lifetimes 180
5.7.3. Global Analysis of a Two Component
Mixture 182
5.7.4. Analysis of a Three Component Mixture:
Limits of Resolution 183
5.7.5. Resolution of a Three Component
Mixture with a Tenfold Range of
Decay Times 185
5.7.6. Maximum Entropy Analysis of FD Data 185
5.8. Biochemical Examples of Frequency Domain
Intensity Decays 186
5.8.1. DNA Labeled with DAPI 186
5.8.2. Mag Quin 2: A Lifetime Based Sensor
for Magnesium 187
5.8.3. Recovery of Lifetime Distributions from
Frequency Domain Data 188
5.8.4. Cross Fitting of Models: Lifetime
Distributions of Melittin 188
5.8.5. Frequency Domain Fluorescence
Microscopy with an LED Light Source 189
5.9. Phase Angle and Modulation Spectra 189
5.10. Apparent Phase and Modulation Lifetimes 191
5.11. Derivation of the Equations for Phase
Modulation Fluorescence 192
5.11.1. Relationship of the Lifetime to the
Phase Angle and Modulation 192
5.11.2. Cross Correlation Detection 194
5.12. Phase Sensitive Emission Spectra 194
5.12.1. Theory of Phase Sensitive Detection
of Fluorescence 195
5.12.2. Examples of PSDF and Phase
Suppression 196
5.12.3. High Frequency or Low Frequency
Phase Sensitive Detection 197
5.13. Phase Modulation Resolution of Emission
Spectra 197
5.13.1. Resolution Based on Phase or Modulation
Lifetimes 198
5.13.2. Resolution Based on Phase Angles
and Modulations 198
5.13.3. Resolution of Emission Spectra from
Phase and Modulation Spectra 198
References 199
Problems 203
xviii
6. Solvent and Environmental Effects
6.1. Overview of Solvent Polarity Effects 205
6.1.1. Effects of Solvent Polarity 205
6.1.2. Polarity Surrounding a Membrane Bound
Fluorophore 206
6.1.3. Other Mechanisms for Spectral Shifts 207
6.2. General Solvent Effects: The Lippert Mataga
Equation 208
6.2.1. Derivation of the Lippert Equation 210
6.2.2. Application of the Lippert Equation 212
6.3. Specific Solvent Effects 213
6.3.1. Specific Solvent Effects and Lippert Plots . 215
6.4. Temperature Effects 216
6.5. Phase Transitions in Membranes 217
6.6. Additional Factors that Affect Emission Spectra 219
6.6.1. Locally Excited and Internal
Charge Transfer States 219
6.6.2. Excited State Intramolecular Proton
Transfer (ESIPT) 221
6.6.3. Changes in the Non Radiative
Decay Rates 222
6.6.4. Changes in the Rate of Radiative Decay 223
6.7. Effects of Viscosity 223
6.7.1. Effect of Shear Stress on Membrane
Viscosity 225
6.8. Probe Probe Interactions 225
6.9. Biochemical Applications of Environment
Sensitive Fluorophores 226
6.9.1. Fatty Acid Binding Proteins 226
6.9.2. Exposure of a Hydrophobic Surface
on Calmodulin 226
6.9.3. Binding to Cyclodextrin Using a
Dansyl Probe 227
6.10. Advanced Solvent Sensitive Probes 228
6.11. Effects of Solvent Mixtures 229
6.12. Summary of Solvent Effects 231
References 232
Problems 235
7. Dynamics of Solvent and Spectral Relaxation
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) 240
7.2.1. Direct Recording of TRES 240
7.2.2. TRES from Wavelength Dependent
Decays 241
7.3. Spectral Relaxation in Proteins 242
7.3.1. Spectral Relaxation of Labeled
Apomyoglobin 243
7.3.2. Protein Spectral Relaxation around a
Synthetic Fluorescent Amino Acid 244
7.4. Spectral Relaxation in Membranes 245
7.4.1. Analysis of Time Resolved Emission
Spectra 246
7.4.2. Spectral Relaxation of Membrane Bound
Anthroyloxy Fatty Acids 248
CONTENTS
7.5. Picosecond Relaxation in Solvents 249
7.5.1. Theory for Time Dependent Solvent
Relaxation 250
7.5.2. Multi Exponential Relaxation in Water 251
7.6. Measurement of Multi Exponential Spectral
Relaxation 252
7.7. Distinction between Solvent Relaxation
and Formation of Rotational Isomers 253
7.8. Comparison of TRES and Decay Associated
Spectra 255
7.9. Lifetime Resolved Emission Spectra 255
7.10. Red Edge Excitation Shifts 257
7.10.1. Membranes and Red Edge
Excitation Shifts 258
7.10.2. Red Edge Excitation Shifts and
Energy Transfer 259
7.11. Excited State Reactions 259
7.11.1. Excited State Ionization of Naphthol 260
7.12. Theory for a Reversible Two State Reaction 262
7.12.1. Steady State Fluorescence of a
Two State Reaction 262
7.12.2. Time Resolved Decays for the
Two State Model 263
7.12.3. Differential Wavelength Methods 264
7.13. Time Domain Studies of Naphthol Dissociation 264
7.14. Analysis of Excited State Reactions by
Phase Modulation Fluorometry 265
7.14.1. Effect of an Excited State Reaction
on the Apparent Phase and Modulation
Lifetimes 266
7.14.2. Wavelength Dependent Phase and
Modulation Values for an Excited State
Reaction 267
7.14.3. Frequency Domain Measurement of
Excimer Formation 269
7.15. Biochemical Examples of Excited State
Reactions 270
7.15.1. Exposure of a Membrane Bound
Cholesterol Analogue 270
References 270
Problems 275
8. Quenching of Fluorescence
8.1. Quenchers of Fluorescence 278
8.2. Theory of Collisional Quenching 278
8.2.1. Derivation of the Stern Volmer Equation 280
8.2.2. Interpretation of the Bimolecular
Quenching Constant 281
8.3. Theory of Static Quenching 282
8.4. Combined Dynamic and Static Quenching 282
8.5. Examples of Static and Dynamic Quenching 283
8.6. Deviations from the Stern Volmer Equation:
Quenching Sphere of Action 284
8.6.1. Derivation of the Quenching Sphere
of Action 285
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
8.7. Effects of Steric Shielding and Charge on
Quenching 286
8.7.1. Accessibility of DNA Bound Probes
to Quenchers 286
8.7.2. Quenching of Ethenoadenine Derivatives 287
8.8. Fractional Accessibility to Quenchers 288
8.8.1. Modified Stern Volmer Plots 288
8.8.2. Experimental Considerations
in Quenching 289
8.9. Applications of Quenching to Proteins 290
8.9.1. Fractional Accessibility of Tryptophan
Residues in Endonuclease III 290
8.9.2. Effect of Conformational Changes
on Tryptophan Accessibility 291
8.9.3. Quenching of the Multiple Decay
Times of Proteins 291
8.9.4. Effects of Quenchers on Proteins 292
8.9.5. Correlation of Emission Wavelength
and Accessibility: Protein Folding of
ColicinEl 292
8.10. Application of Quenching to Membranes 293
8.10.1. Oxygen Diffusion in Membranes 293
8.10.2. Localization of Membrane Bound
Tryptophan Residues by Quenching 294
8.10.3. Quenchingof Membrane Probes
Using Localized Quenchers 295
8.10.4. Parallax and Depth Dependent
Quenching in Membranes 296
8.10.5. Boundary Lipid Quenching 298
8.10.6. Effect of Lipid Water Partitioning
on Quenching 298
8.10.7. Quenching in Micelles 300
8.11. Lateral Diffusion in Membranes 300
8.12. Quenching Resolved Emission Spectra 301
8.12.1. Fluorophore Mixtures 301
8.12.2. Quenching Resolved Emission Spectra
of the E. Coli Tet Repressor 302
8.13. Quenching and Association Reactions 304
8.13.1. Quenching Due to Specific Binding
Interactions 304
8.14. Sensing Applications of Quenching 305
8.14.1. Chloride Sensitive Fluorophores 306
8.14.2. Intracellular Chloride Imaging 306
8.14.3. Chloride Sensitive GFP 307
8.14.4. Amplified Quenching 309
8.15. Applications of Quenching to Molecular
Biology 310
8.15.1. Release of Quenching upon
Hybridization 310
8.15.2. Molecular Beacons in Quenching
by Guanine 311
8.15.3. Binding of Substrates to Ribozymes 311
8.15.4. Association Reactions and Accessibility
to Quenchers 312
8.16. Quenching on Gold Surfaces 313
8.16.1. Molecular Beacons Based on Quenching
by Gold Colloids 313
xix
8.16.2. Molecular Beacons Based on Quenching
by a Gold Surface 314
8.17. Intramolecular Quenching 314
8.17.1. DNA Dynamics by Intramolecular
Quenching 314
8.17.2. Electron Transfer Quenching in a
Flavoprotein 315
8.17.3. Sensors Based on Intramolecular
PET Quenching 316
8.18. Quenching of Phosphorescence 317
References 318
Problems 327
9. Mechanisms and Dynamics of
Fluorescence Quenching
9.1. Comparison of Quenching and Resonance
Energy Transfer 331
9.1.1. Distance Dependence of RET
and Quenching 332
9.1.2. Encounter Complexes and Quenching
Efficiency 333
9.2. Mechanisms of Quenching 334
9.2.1. Intersystem Crossing 334
9.2.2. Electron Exchange Quenching 335
9.2.3. Photoinduced Electron Transfer 335
9.3. Energetics of Photoinduced Electron Transfer 336
9.3.1. Examples of PET Quenching 338
9.3.2. PET in Linked Donor Acceptor Pairs 340
9.4. PET Quenching in Biomolecules 341
9.4.1. Quenching of Indole by Imidazolium 341
9.4.2. Quenching by DNA Bases and
Nucleotides 341
9.5. Single Molecule PET 342
9.6. Transient Effects in Quenching 343
9.6.1. Experimental Studies of Transient
Effects 346
9.6.2. Distance Dependent Quenching
in Proteins 348
References 348
Problems 351
10. Fluorescence Anisotropy
10.1. Definition of Fluorescence Anisotropy 353
10.1.1. Origin of the Definitions of
Polarization and Anisotropy 355
10.2. Theory for Anisotropy 355
10.2.1. Excitation Photoselection of Fluorophores. 357
10.3. Excitation Anisotropy Spectra 358
10.3.1. Resolution of Electronic States from
Polarization Spectra 360
10.4. Measurement of Fluorescence Anisotropies 361
10.4.1. L Format or Single Channel Method 361
10.4.2. T Format or Two Channel Anisotropies 363
10.4.3. Comparison of T Format and
L Format Measurements 363
XX
10.4.4. Alignment of Polarizers 364
10.4.5. Magic Angle Polarizer Conditions 364
10.4.6. Why is the Total Intensity
Equal to /„ + 2/± 364
10.4.7. Effect of Resonance Energy Transfer
on the Anisotropy 364
10.4.8. Trivial Causes of Depolarization 365
10.4.9. Factors Affecting the Anisotropy 366
10.5. Effects of Rotational Diffusion on Fluorescence
Anisotropies: The Perrin Equation 366
10.5.1. The Perrin Equation: Rotational
Motions of Proteins 367
10.5.2. Examples of a Perrin Plot 369
10.6. Perrin Plots of Proteins 370
10.6.1. Binding of tRNA to tRNA Synthetase 370
10.6.2. Molecular Chaperonin cpn60 (GroEL) 371
10.6.3. Perrin Plots of an Fab Immunoglobulin
Fragment 371
10.7. Biochemical Applications of Steady State
Anisotropies 372
10.7.1. Peptide Binding to Calmodulin 372
10.7.2. Binding of the Trp Repressor to DNA 373
10.7.3. Helicase Catalyzed DNA Unwinding 373
10.7.4. Melittin Association Detected from
Homotransfer 374
10.8. Anisotropy of Membranes and Membrane
Bound Proteins 374
10.8.1. Membrane Microviscosity 374
10.8.2. Distribution of Membrane Bound
Proteins 375
10.9. Transition Moments 377
References 378
Additional Reading on the Application
of Anisotropy 380
Problems 381
11. Time Dependent Anisotropy Decays
11.1. Time Domain and Frequency Domain
Anisotropy Decays 383
11.2. Anisotropy Decay Analysis 387
11.2.1. Early Methods for Analysis of
TD Anisotropy Data 387
11.2.2. Preferred Analysis of TD
Anisotropy Data 388
11.2.3. Value of r0 389
11.3. Analysis of Frequency Domain
Anisotropy Decays 390
11.4. Anisotropy Decay Laws 390
11.4.1. Non Spherical Fluorophores 391
11.4.2. Hindered Rotors 391
11.4.3. Segmental Mobility of a Biopolymer
Bound Fluorophore 392
11.4.4. Correlation Time Distributions 393
11.4.5. Associated Anisotropy Decays 393
CONTENTS
11.4.6. Example Anisotropy Decays of
Rhodamine Green and Rhodamine
Green Dextran 394
11.5. Time Domain Anisotropy Decays of Proteins 394
11.5.1. Intrinsic Tryptophan Anisotropy Decay
of Liver Alcohol Dehydrogenase 395
11.5.2. Phospholipase A2 395
11.5.3. Subtilisin Carlsberg 395
11.5.4. Domain Motions of Immunoglobulins 396
11.5.5. Effects of Free Probe on Anisotropy
Decays 397
11.6. Frequency Domain Anisotropy Decays
of Proteins 397
11.6.1. Apomyoglobin: A Rigid Rotor 397
11.6.2. Melittin Self Association and
Anisotropy Decays 398
11.6.3. Picosecond Rotational Diffusion
ofOxytocin 399
11.7. Hindered Rotational Diffusion in Membranes 399
11.7.1. Characterization of a New
Membrane Probe 401
11.8. Anisotropy Decays of Nucleic Acids 402
11.8.1. Hydrodynamics of DNA Oligomers 403
11.8.2. Dynamics of Intracellular DNA 403
11.8.3. DNA Binding to HIV Integrase Using
Correlation Time Distributions 404
11.9. Correlation Time Imaging 406
11.10. Microsecond Anisotropy Decays 408
11.10.1. Phosphorescence Anisotropy Decays 408
11.10.2. Long Lifetime Metal Ligand
Complexes 408
References 409
Problems 412
12. Advanced Anisotropy Concepts
12.1. Associated Anisotropy Decay 413
12.1.1. Theory for Associated Anisotropy
Decay 414
12.1.2. Time Domain Measurements of
Associated Anisotropy Decays 415
12.2. Biochemical Examples of Associated
Anisotropy Decays 417
12.2.1. Time Domain Studies of DNA
Binding to the Klenow Fragment
of DNA Polymerase 417
12.2.2. Frequency Domain Measurements
of Associated Anisotropy Decays 417
12.3. Rotational Diffusion of Non Spherical
Molecules: An Overview 418
12.3.1. Anisotropy Decays of Ellipsoids 419
12.4. Ellipsoids of Revolution 420
12.4.1. Simplified Ellipsoids of Revolution 421
12.4.2. Intuitive Description of Rotational
Diffusion of an Oblate Ellipsoid 422
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
12.4.3. Rotational Correlation Times for
Ellipsoids of Revolution 423
12.4.4. Stick versus Slip Rotational Diffusion 425
12.5. Complete Theory for Rotational Diffusion
of Ellipsoids 425
12.6. Anisotropic Rotational Diffusion 426
12.6.1. Time Domain Studies 426
12.6.2. Frequency Domain Studies of
Anisotropic Rotational Diffusion 427
12.7. Global Anisotropy Decay Analysis 429
12.7.1. Global Analysis with Multi Wavelength
Excitation 429
12.7.2. Global Anisotropy Decay Analysis with
Collisional Quenching 430
12.7.3. Application of Quenching to Protein
Anisotropy Decays 431
12.8. Intercalated Fluorophores in DNA 432
12.9. Transition Moments 433
12.9.1. Anisotropy of Planar Fluorophores
with High Symmetry 435
12.10. Lifetime Resolved Anisotropies 435
12.10.1. Effect of Segmental Motion on the
Perrin Plots 436
12.11. Soleillet's Rule: Multiplication of Depolarized
Factors 436
12.12. Anisotropies Can Depend on Emission
Wavelength 437
References 438
Problems 441
13. Energy Transfer
13.1. Characteristics of Resonance Energy Transfer 443
13.2. Theory of Energy Transfer for a
Donor Acceptor Pair 445
13.2.1. Orientation Factor k2 448
13.2.2. Dependence of the Transfer Rate on
Distance (r), the Overlap
Integral (J), and t2 449
13.2.3. Homotransfer and Heterotransfer 450
13.3. Distance Measurements Using RET 451
13.3.1. Distance Measurements in a Helical
Melittin 451
13.3.2. Effects of Incomplete Labeling 452
13.3.3. Effect of k2 on the Possible Range
of Distances 452
13.4. Biochemical Applications of RET 453
13.4.1. Protein Folding Measured by RET 453
13.4.2. Intracellular Protein Folding 454
13.4.3. RET and Association Reactions 455
13.4.4. Orientation of a Protein Bound Peptide 456
13.4.5. Protein Binding to Semiconductor
Nanoparticles 457
13.5. RET Sensors 458
13.5.1. Intracellular RET Indicator
for Estrogens 458
xxi
13.5.2. RET Imaging of Intracellular Protein
Phosphorylation 459
13.5.3. Imaging of Rac Activation in Cells 459
13.6. RET and Nucleic Acids 459
13.6.1. Imaging of Intracellular RNA 460
13.7. Energy Transfer Efficiency from
Enhanced Acceptor Fluorescence 461
13.8. Energy Transfer in Membranes 462
13.8.1. Lipid Distributions around Gramicidin 463
13.8.2. Membrane Fusion and Lipid Exchange 465
13.9. Effect of t on RET 465
13.10. Energy Transfer in Solution 466
13.10.1. Diffusion Enhanced Energy Transfer 467
13.11. Representative /?„ Values 467
References 468
Additional References on Resonance
Energy Transfer 471
Problems 472
14. Time Resolved Energy Transfer and
Conformational Distributions of Biopolymers
14.1. Distance Distributions 477
14.2. Distance Distributions in Peptides 479
14.2.1. Comparison for a Rigid and Flexible
Hexapeptide 479
14.2.2. Crossfitting Data to Exclude
Alternative Models 481
14.2.3. Donor Decay without Acceptor 482
14.2.4. Effect of Concentration of the
D A Pairs 482
14.3. Distance Distributions in Peptides 482
14.3.1. Distance Distributions in Melittin 483
14.4. Distance Distribution Data Analysis 485
14.4.1. Frequency Domain Distance Distribution
Analysis 485
14.4.2. Time Domain Distance Distribution
Analysis 487
14.4.3. Distance Distribution Functions 487
14.4.4. Effects of Incomplete Labeling 487
14.4.5. Effect of the Orientation Factor k2 489
14.4.6. Acceptor Decays 489
14.5. Biochemical Applications of Distance
Distributions 490
14.5.1. Calcium Induced Changes in the
Conformation of Troponin C 490
14.5.2. Hairpin Ribozyme 493
14.5.3. Four Way Holliday Junction in DNA 493
14.5.4. Distance Distributions and Unfolding
of Yeast Phosphoglycerate Kinase 494
14.5.5. Distance Distributions in a Glycopeptide. 495
14.5.6. Single Protein Molecule Distance
Distribution 496
14.6. Time Resolved RET Imaging 497
14.7. Effect of Diffusion for Linked D A Pairs 498
xxii
14.7.1. Simulations of FRET for a Flexible
D APair 499
14.7.2. Experimental Measurement of D A
Diffusion for a Linked D A Pair 500
14.7.3. FRET and Diffusive Motions in
Biopolymers 501
14.8. Conclusion 501
References 501
Representative Publications on Measurement
of Distance Distributions 504
Problems 505
15. Energy Transfer to Multiple Acceptors in
One, Two, or Three Dimensions
15.1. RET in Three Dimensions 507
15.1.1. Effect of Diffusion on FRET with
Unlinked Donors and Acceptors 508
15.1.2. Experimental Studies of RET in
Three Dimensions 509
15.2. Effect of Dimensionality on RET 511
15.2.1. Experimental FRET in Two Dimensions. 512
15.2.2. Experimental FRET in One Dimension 514
15.3. Biochemical Applications of RET with
Multiple Acceptors 515
15.3.1. Aggregation of P Amyloid Peptides 515
15.3.2. RET Imaging of Fibronectin 516
15.4. Energy Transfer in Restricted Geometries 516
15.4.1. Effect of Excluded Area on Energy
Transfer in Two Dimensions 518
15.5. RET in the Presence of Diffusion 519
15.6. RET in the Rapid Diffusion Limit 520
15.6.1. Location of an Acceptor in
Lipid Vesicles 521
15.6.2. Locaion of Retinal in Rhodopsin
Disc Membranes 522
15.7. Conclusions 524
References 524
Additional References on RET between
Unlinked Donor and Acceptor 526
Problems 527
16. Protein Fluorescence
6.1. Spectral Properties of the Aromatic Amino Acids. 530
16.1.1. Excitation Polarization Spectra of
Tyrosine and Tryptophan 531
16.1.2. Solvent Effects on Tryptophan Emission
Spectra 533
16.1.3. Excited State Ionization of Tyrosine 534
16.1.4. Tyrosinate Emission from Proteins 535
6.2. General Features of Protein Fluorescence 535
CONTENTS
16.3. Tryptophan Emission in an Apolar
Protein Environment 538
16.3.1. Site Directed Mutagenesis of a
Single Tryptophan Azurin 538
16.3.2. Emission Spectra of Azurins with
One or Two Tryptophan Residues 539
16.4. Energy Transfer and Intrinsic Protein
Fluorescence 539
16.4.1. Tyrosine to Tryptophan Energy Transfer
in Interferon y 540
16.4.2. Quantitation of RET Efficiencies
in Proteins 541
16.4.3. Tyrosine to Tryptophan RET in
a Membrane Bound Protein 543
16.4.4. Phenylalanine to Tyrosine
Energy Transfer 543
16.5. Calcium Binding to Calmodulin Using
Phenylalanine and Tyrosine Emission 545
16.6. Quenching of Tryptophan Residues in Proteins 546
16.6.1. Effect of Emission Maximum on
Quenching 547
16.6.2. Fractional Accessibility to Quenching
in Multi Tryptophan Proteins 549
16.6.3. Resolution of Emission Spectra by
Quenching 550
16.7. Association Reaction of Proteins 551
16.7.1. Binding of Calmodulin to a
Target Protein 551
16.7.2. Calmodulin: Resolution of the
Four Calcium Binding Sites Using
Tryptophan Containing Mutants 552
16.7.3. Interactions of DNA with Proteins 552
16.8. Spectral Properties of Genetically Engineered
Proteins 554
16.8.1. Single Tryptophan Mutants of
Triosephosphate Isomerase 555
16.8.2. Barnase: A Three Tryptophan Protein 556
16.8.3. Site Directed Mutagenesis of
Tyrosine Proteins 557
16.9. Protein Folding 557
16.9.1. Protein Engineering of Mutant
Ribonuclease for Folding Experiments 558
16.9.2. Folding of Lactate Dehydrogenase 559
16.9.3. Folding Pathway of CRABPI 560
16.10. Protein Structure and Tryptophan Emission 560
16.10.1. Tryptophan Spectral Properties
and Structural Motifs 561
16.11. Tryptophan Analogues 562
16.11.1. Tryptophan Analogues 564
16.11.2. Genetically Inserted Amino Acid
Analogues 565
16.12. The Challenge of Protein Fluorescence 566
References 567
Problems 573
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
17. Time Resolved Protein Fluorescence
17.1. Intensity Decays of Tryptophan:
The Rotamer Model 578
17.2. Time Resolved Intensity Decays of
Tryptophan and Tyrosine 580
17.2.1. Decay Associated Emission Spectra
of Tryptophan 581
17.2.2. Intensity Decays of Neutral Tryptophan
Derivatives 581
17.2.3. Intensity Decays of Tyrosine and
Its Neutral Derivatives 582
17.3. Intensity and Anisotropy Decays of Proteins 583
17.3.1. Single Exponential Intensity and
Anisotropy Decay of Ribonuclease T, 584
17.3.2. Annexin V: A Calcium Sensitive
Single Tryptophan Protein 585
17.3.3. Anisotropy Decay of a Protein with
Two Tryptophans 587
17.4. Protein Unfolding Exposes the Tryptophan
Residue to Water 588
17.4.1. Conformational Heterogeneity Can
Result in Complex Intensity and
Anisotropy Decays 588
17.5. Anisotropy Decays of Proteins 589
17.5.1. Effects of Association Reactions on
Anisotropy Decays: Melittin 590
17.6. Biochemical Examples Using Time Resolved
Protein Fluorescence 591
17.6.1. Decay Associated Spectra of Barnase 591
17.6.2. Disulfide Oxidoreductase DsbA 591
17.6.3. Immunophilin FKBP59 I: Quenching
of Tryptophan Fluorescence by
Phenylalanine 592
17.6.4. Trp Repressor: Resolution of the Two
Interacting Tryptophans 593
17.6.5. Thermophilic P Glycosidase:
A Multi Tryptophan Protein 594
17.6.6. Heme Proteins Display Useful
Intrinsic Fluorescence 594
17.7. Time Dependent Spectral Relaxation of
Tryptophan 596
17.8. Phosphorescence of Proteins 598
17.9. Perspectives on Protein Fluorescence 600
References 600
Problems 605
18. Multiphoton Excitation and Microscopy
18.1. Introduction to Multiphoton Excitation 607
18.2. Cross Sections for Multiphoton Absorption 609
18.3. Two Photon Absorption Spectra 609
18.4. Two Photon Excitation of a DNA Bound
Fluorophore 610
18.5. Anisotropies with Multiphoton Excitation 612
xxiv
19.12. New Approaches to Sensing 655
19.12.1. Pebble Sensors and Lipobeads 655
19.13. In Vivo Imaging 656
19.14. Immunoassays 658
19.14.1. Enzyme Linked Immunosorbent Assays
(ELISA) 659
19.14.2. Time Resolved Immunoassays 659
19.14.3. Energy Transfer Immunoassays 660
19.14.4. Fluorescence Polarization
Immunoassays 661
References 663
Problems 672
20. Novel Fluorophores
20.1. Semiconductor Nanoparticles 675
20.1.1. Spectral Properties of QDots 676
20.1.2. Labeling Cells with QDots 677
20.1.3. QDots and Resonance Energy Transfer 678
20.2. Lanthanides 679
20.2.1. RET with Lanthanides 680
20.2.2. Lanthanide Sensors 681
20.2.3. Lanthanide Nanoparticles 682
20.2.4. Near Infrared Emitting Lanthanides 682
20.2.5. Lanthanides and Fingerprint Detection 683
20.3. Long Lifetime Metal Ligand Complexes 683
20.3.1. Introduction to Metal Ligand Probes 683
20.3.2. Anisotropy Properties of
Metal Ligand Complexes 685
20.3.3. Spectral Properties of MLC Probes 686
20.3.4. The Energy Gap Law 687
20.3.5. Biophysical Applications of
Metal Ligand Probes 688
20.3.6. MLC Immunoassays 691
20.3.7. Metal Ligand Complex Sensors 694
20.4. Long Wavelength Long Lifetime
Fluorophores 695
References 697
Problems 702
Zl. DNATechnology
II.1. DNA Sequencing 705
21.1.1. Principle of DNA Sequencing 705
21.1.2. Examples of DNA Sequencing 706
21.1.3. Nucleotide Labeling Methods 707
21.1.4. Example of DNA Sequencing 708
21.1.5. Energy Transfer Dyes for DNA
Sequencing 709
21.1.6. DNA Sequencing with NIR Probes 710
21.1.7. DNA Sequencing Based on Lifetimes 712
:i.2. High Sensitivity DNA Stains 712
21.2.1. High Affinity Bis DNA Stains 713
21.2.2. Energy Transfer DNA Stains 715
CONTENTS
21.2.3. DNA Fragment Sizing by
Flow Cytometry 715
21.3. DNA Hybridization 715
21.3.1. DNA Hybridization Measured with
One Donor and Acceptor Labeled
DNA Probe 717
21.3.2. DNA Hybridization Measured by
Excimer Formation 718
21.3.3. Polarization Hybridization Arrays 719
21.3.4. Polymerase Chain Reaction 720
21.4. Molecular Beacons 720
21.4.1. Molecular Beacons with
Nonfluorescent Acceptors 720
21.4.2. Molecular Beacons with
Fluorescent Acceptors 722
21.4.3. Hybridization Proximity Beacons 722
21.4.4. Molecular Beacons Based on
Quenching by Gold 723
21.4.5. Intracellular Detection of mRNA
Using Molecular Beacons 724
21.5. Aptamers 724
21.5.1. DNAzymes 726
21.6. Multiplexed Microbead Arrays:
Suspension Arrays 726
21.7. Fluorescence In Situ Hybridization 727
21.7.1. Preparation of FISH Probe DNA 728
21.7.2. Applications of FISH 729
21.8. Multicolor FISH and Spectral Karyotyping 730
21.9. DNA Arrays 732
21.9.1. Spotted DNA Microarrays 732
21.9.2. Light Generated DNA Arrays 734
References 734
Problems 740
22. Fluorescence Lifetime Imaging Microscopy
22.1. Early Methods for Fluorescence Lifetime
Imaging 743
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
from Lifetime 744
22.2.2. Lifetime Images of Cos Cells 745
22.3. Examples of Wide Field Frequency Domain
FLIM 746
22.3.1. Resonance Energy Transfer FLIM
of Protein Kinase C Activation 746
22.3.2. Lifetime Imaging of Cells Containing
Two GFPs 747
22.4. Wide Field FLIM Using a Gated Image
Intensifier 747
22.5. Laser Scanning TCSPC FLIM 748
22.5.1. Lifetime Imaging of Cellular
Biomolecules 750
22.5.2. Lifetime Images of Amyloid Plaques 750
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
22.6. Frequency Domain Laser Scanning Microscopy 750
22.7. Conclusions 752
References 752
Additional Reading on Fluorescence Lifetime
Imaging Microscopy 753
Problem 755
23. Single Molecule Detection
23.1. Detectability of Single Molecules 759
23.2. Total Internal Reflection and Confocal Optics 760
23.2.1. Total Internal Reflection 760
23.2.2. Confocal Detection Optics 761
23.3. Optical Configurations for SMD 762
23.4. Instrumentation for SMD 764
23.4.1. Detectors for Single Molecule Detection . 765
23.4.2. Optical Filters for SMD 766
23.5. Single Molecule Photophysics 768
23.6. Biochemical Applications of SMD 770
23.6.1. Single Molecule Enzyme Kinetics 770
23.6.2. Single Molecule ATPase Activity 770
23.6.3. Single Molecule Studies of a
Chaperonin Protein 771
23.7. Single Molecule Resonance Energy Transfer 773
23.8. Single Molecule Orientation and Rotational
Motions 775
23.8.1. Orientation Imaging of R6G and GFP 777
23.8.2. Imaging of Dipole Radiation Patterns 778
23.9. Time Resolved Studies of Single Molecules 779
23.10. Biochemical Applications 780
23.10.1. Turnover of Single Enzyme Molecules. 780
23.10.2. Single Molecule Molecular Beacons 782
23.10.3. Conformational Dynamics of a
Holliday Junction 782
23.10.4. Single Molecule Calcium Sensor 784
23.10.5. Motions of Molecular Motors 784
23.11. Advanced Topics in SMD 784
23.11.1. Signal to Noise Ratio in
Single Molecule Detection 784
23.11.2. Polarization of Single Immobilized
Fluorophores 786
23.11.3. Polarization Measurements
and Mobility of Surface Bound
Fluorophores 786
23.11.4. Single Molecule Lifetime Estimation 787
23.12. Additional Literature on SMD 788
References 788
Additional References on Single Molecule
Detection 791
Problem 795
24. Fluorescence Correlation Spectroscopy
24.1. Principles of Fluorescence Correlation
Spectroscopy 798
XXV
24.2. Theory of FCS 800
24.2.1. Translational Diffusion and FCS 802
24.2.2. Occupation Numbers and Volumes
in FCS 804
24.2.3. FCS for Multiple Diffusing Species 804
24.3. Examples of FCS Experiments 805
24.3.1. Effect of Fluorophore Concentration 805
24.3.2. Effect of Molecular Weight on
Diffusion Coefficients 806
24.4. Applications of FCS to Bioaffinity Reactions 807
24.4.1. Protein Binding to the
Chaperonin GroEL 807
24.4.2. Association of Tubulin Subunits 807
24.4.3. DNA Applications of FCS 808
24.5. FCS in Two Dimensions: Membranes 810
24.5.1. Biophysical Studies of Lateral
Diffusion in Membranes 812
24.5.2. Binding to Membrane Bound
Receptors 813
24.6. Effects of Intersystem Crossing 815
24.6.1. Theory for FCS and Intersystem
Crossing 816
24.7. Effects of Chemical Reactions 816
24.8. Fluorescence Intensity Distribution Analysis 817
24.9. Time Resolved FCS 819
24.10. Detection of Conformational Dynamics
in Macromolecules 820
24.11. FCS with Total Internal Reflection 821
24.12. FCS with Two Photon Excitation 822
24.12.1. Diffusion of an Intracellular
Kinase Using FCS with
Two Photon Excitation 823
24.13. Dual Color Fluorescence Cross Correlation
Spectroscopy 823
24.13.1. Instrumentation for Dual Color
FCCS 824
24.13.2. Theory of Dual Color FCCS 824
24.13.3. DNA Cleavage by a
Restriction Enzyme 826
24.13.4. Applications of Dual Color FCCS 826
24.14. Rotational Diffusion and Photo Antibunching 828
24.15. Flow Measurements Using FCS 830
24.16. Additional References on FCS 832
References 832
Additional References to FCS and
Its Applications 837
Problems 840
25. Radiative Decay Engineering:
Metal Enhanced Fluorescence
25.1. Radiative Decay Engineering 841
25.1.1. Introduction to RDE 841
25.1.2. Jablonski Diagram for Metal
Enhanced Fluorescence 842
25.2. Review of Metal Effects on Fluorescence 843
xxvi
25.3. Optical Properties of Metal Colloids 845
25.4. Theory for Fluorophore Colloid Interactions 846
25.5. Experimental Results on Metal Enhanced
Fluorescence 848
25.5.1. Application of MEF to DNA Analysis 848
25.6. Distance Dependence of Metal Enhanced
Fluorescence 851
25.7. Applications of Metal Enhanced Fluorescence 851
25.7.1. DNA Hybridization Using MEF 853
25.7.2. Release of Self Quenching 853
25.7.3. Effect of Silver Particles on RET 854
25.8. Mechanism of MEF 855
25.9. Perspective on RET 856
References 856
Problem 859
26. Radiative Decay Engineering:
Surface Plasmon Coupled Emission
26.1. Phenomenon of SPCE 861
26.2. Surface Plasmon Resonance 861
26.2.1. Theory for Surface Plasmon Resonance 863
26.3. Expected Properties of SPCE 865
26.4. Experimental Demonstration of SPCE 865
26.5. Applications of SPCE 867
26.6. Future Developments in SPCE 868
References 870
Appendix I. Corrected Emission Spectra
1. Emission Spectra Standards from 300 to 800 nm 873
2. P Carboline Derivatives as Fluorescence Standards 873
3. Corrected Emission Spectra of 9,10 Diphenyl
anthracene, Quinine, and Fluorescein 877
4. Long Wavelength Standards 877
5. Ultraviolet Standards 878
6. Additional Corrected Emission Spectra 881
References 881
CONTENTS
Appendix II. Fluorescent Lifetime Standards
1. Nanosecond Lifetime Standards 883
2. Picosecond Lifetime Standards 884
3. Representative Frequency Domain
Intensity Decays 885
4. Time Domain Lifetime Standards 886
Appendix III. Additional Reading
1. Time Resolved Measurements 889
2. Spectra Properties of Fluorophores 889
3. Theory of Fluorescence and Photophysics 889
4. Reviews of Fluorescence Spectroscopy 889
5. Biochemical Fluorescence 890
6. Protein Fluorescence 890
7. Data Analysis and Nonlinear Least Squares 890
8. Photochemistry 890
9. Flow Cytometry 890
10. Phosphorescence 890
11. Fluorescence Sensing 890
12. Immunoassays 891
13. Applications of Fluorescence 891
14. Multiphoton Excitation 891
15. Infrared and NIR Fluorescence 891
16. Lasers 891
17. Fluorescence Microscopy 891
18. Metal Ligand Complexes and Unusual
Lumophores 891
19. Single Molecule Detection 891
20. Fluorescence Correlation Spectroscopy 892
21. Biophotonics 892
22. Nanoparticles 892
23. Metallic Particles 892
24. Books on Fluorescence 892
Answers to Problems 893
Index 923 |
| adam_txt |
1. Introduction to Fluorescence
1.1. Phenomena of Fluorescence 1
1.2. Jablonski Diagram 3
1.3. Characteristics of Fluorescence Emission 6
1.3.1. The Stokes Shift 6
1.3.2. Emission Spectra Are Typically Independent
of the Excitation Wavelength 7
1.3.3. Exceptions to the Mirror Image Rule 8
1.4. Fluorescence Lifetimes and Quantum Yields 9
1.4.1. Fluorescence Quenching 11
1.4.2. Timescale of Molecular Processes
in Solution 12
1.5. Fluorescence Anisotropy 12
1.6. Resonance Energy Transfer 13
1.7. Steady State and Time Resolved Fluorescence 14
1.7.1. Why Time Resolved Measurements? 15
1.8. Biochemical Fluorophores 15
1.8.1. Fluorescent Indicators 16
1.9. Molecular Information from Fluorescence 17
1.9.1. Emission Spectra and the Stokes Shift 17
1.9.2. Quenching of Fluorescence 18
1.9.3. Fluorescence Polarization or Anisotropy 19
1.9.4. Resonance Energy Transfer 19
1.10. Biochemical Examples of Basic Phenomena 20
1.11. New Fluorescence Technologies 21
1.11.1. Multiphoton Excitation 21
1.11.2. Fluorescence Correlation Spectroscopy 22
1.11.3. Single Molecule Detection 23
1.12. Overview of Fluorescence Spectroscopy 24
References 25
Problems 25
2. Instrumentation for Fluorescence
Spectroscopy
2.1. Spectrofluorometers 27
2.1.1. Spectrofluorometers for Spectroscopy
Research 27
2.1.2. Spectrofluorometers for High Throughput. 29
2.1.3. An Ideal Spectrofluorometer 30
2.1.4. Distortions in Excitation and Emission
Spectra 30
2.2. Light Sources 31
2.2.1. Arc Lamps and Incandescent
Xenon Lamps 31
2.2.2. Pulsed Xenon Lamps 32
2.2.3. High Pressure Mercury (Hg) Lamps 33
2.2.4. Xe Hg Arc Lamps 33
2.2.5. Quartz Tungsten Halogen (QTH) Lamps 33
2.2.6. Low Pressure Hg and Hg Ar Lamps 33
2.2.7. LED Light Sources 33
2.2.8. Laser Diodes 34
2.3. Monochromators 34
2.3.1. Wavelength Resolution and Emission
Spectra 35
2.3.2. Polarization Characteristics of
Monochromators 36
2.3.3. Stray Light in Monochromators 36
2.3.4. Second Order Transmission in
Monochromators 37
2.3.5. Calibration of Monochromators 38
2.4. Optical Filters 38
2.4.1. Colored Filters 38
2.4.2. Thin Film Filters 39
2.4.3. Filter Combinations 40
2.4.4. Neutral Density Filters 40
2.4.5. Filters for Fluorescence Microscopy 41
2.5. Optical Filters and Signal Purity 41
2.5.1. Emission Spectra Taken through Filters 43
2.6. Photomultiplier Tubes 44
2.6.1. Spectral Response of PMTs 45
2.6.2. PMT Designs and Dynode Chains 46
2.6.3. Time Response of Photomultiplier Tubes 47
2.6.4. Photon Counting versus Analog Detection
of Fluorescence 48
2.6.5. Symptoms of PMT Failure 49
2.6.6. CCD Detectors 49
2.7. Polarizers 49
2.8. Corrected Excitation Spectra 51
2.8.1. Corrected Excitation Spectra Using
a Quantum Counter 51
2.9. Corrected Emission Spectra 52
2.9.1. Comparison with Known Emission
Spectra 52
2.9.2. Corrections Using a Standard Lamp 53
2.9.3. Correction Factors Using a Quantum
Counter and Scatterer 53
XV
2.9.4. Conversion between Wavelength and
Wavenumber 53
2.10. Quantum Yield Standards 54
2.11. Effects of Sample Geometry 55
2.12. Common Errors in Sample Preparation 57
2.13. Absorption of Light and Deviation from the
Beer Lambert Law 58
2.13.1. Deviations from Beer's Law 59
2.14. Conclusions 59
References 59
Problems 60
3. Fluorophores
3.1. Intrinsic or Natural Fluorophores 63
3.1.1. Fluorescence Enzyme Cofactors 63
3.1.2. Binding of NADH to a Protein 65
3.2. Extrinsic Fluorophores 67
3.2.1. Protein Labeling Reagents 67
3.2.2. Role of the Stokes Shift in Protein
Labeling 69
3.2.3. Photostability of Fluorophores 70
3.2.4. Non Covalent Protein Labeling
Probes 71
3.2.5. Membrane Probes 72
3.2.6. Membrane Potential Probes 72
3.3. Red and Near Infrared (NIR) Dyes 74
3.4. DNA Probes 75
3.4.1. DNA Base Analogues 75
3.5. Chemical Sensing Probes 78
3.6. Special Probes 79
3.6.1. Fluorogenic Probes 79
3.6.2. Structural Analogues of Biomolecules 80
3.6.3. Viscosity Probes 80
3.7. Green Fluorescent Proteins 81
3.8. Other Fluorescent Proteins 83
3.8.1. Phytofluors: A New Class of
Fluorescent Probes 83
3.8.2. Phycobiliproteins 84
3.8.3. Specific Labeling of Intracellular
Proteins 86
3.9. Long Lifetime Probes 86
3.9.1. Lanthanides 87
3.9.2. Transition Metal Ligand Complexes 88
.10. Proteins as Sensors 88
.11. Conclusion 89
References 90
Problems 94
L Time Domain Lifetime Measurements
4.1. Overview of Time Domain and Frequency
Domain Measurements 98
4.1.1. Meaning of the Lifetime or Decay Time 99
4.1.2. Phase and Modulation Lifetimes 99
4.1.3. Examples of Time Domain and
Frequency Domain Lifetimes 100
4.2. Biopolymers Display Multi Exponential or
Heterogeneous Decays 101
4.2.1. Resolution of Multi Exponential
Decays Is Difficult 103
4.3. Time Correlated Single Photon Counting 103
4.3.1. Principles of TCSPC 104
4.3.2. Example of TCSPC Data 105
4.3.3. Convolution Integral 106
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
4.4.3. Picosecond Dye Lasers 110
4.4.4. Flashlamps 112
4.4.5. Synchrotron Radiation 114
4.5. Electronics for TCSPC 114
4.5.1. Constant Fraction Discriminators 114
4.5.2. Amplifiers 115
4.5.3. Time to Amplitude Converter (TAC)
and Analyte to Digital Converter (ADC) 115
4.5.4. Multichannel Analyzer 116
4.5.5. Delay Lines 116
4.5.6. Pulse Pile Up 116
4.6. Detectors for TCSPC 117
4.6.1. MicroChannel Plate PMTs 117
4.6.2. Dynode Chain PMTs 118
4.6.3. Compact PMTs 118
4.6.4. Photodiodes as Detectors 118
4.6.5. Color Effects in Detectors 119
4.6.6. Timing Effects of Monochromators 121
4.7. Multi Detector and Multidimensional TCSPC 121
4.7.1. Multidimensional TCSPC and
DNA Sequencing 123
4.7.2. Dead Times, Repetition Rates, and
Photon Counting Rates 124
4.8. Alternative Methods for Time Resolved
Measurements 124
4.8.1. Transient Recording 124
4.8.2. Streak Cameras 125
4.8.3. Upconversion Methods 128
4.8.4. Microsecond Luminescence Decays 129
4.9. Data Analysis: Nonlinear Least Squares 129
4.9.1. Assumptions of Nonlinear Least Squares 130
4.9.2. Overview of Least Squares Analysis 130
4.9.3. Meaning of the Goodness of Fit 131
4.9.4. Autocorrelation Function 132
4.10. Analysis of Multi Exponential Decays 133
4.10.1. p Terphenyl and Indole: Two Widely
Spaced Lifetimes 133
4.10.2. Comparison of xR2 Values: F Statistic 133
4.10.3. Parameter Uncertainty: Confidence
Intervals 134
4.10.4. Effect of the Number of Photon Counts 135
4.10.5. Anthranilic Acid and 2 Aminopurine:
Two Closely Spaced Lifetimes 137
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
4.10.6. Global Analysis: Multi Wavelength
Measurements 138
4.10.7. Resolution of Three Closely Spaced
Lifetimes 138
4.11. Intensity Decay Laws 141
4.11.1. Multi Exponential Decays 141
4.11.2. Lifetime Distributions 143
4.11.3. Stretched Exponentials 144
4.11.4. Transient Effects 144
4.12. Global Analysis 144
4.13. Applications of TCSPC 145
4.13.1. Intensity Decay for a Single Tryptophan
Protein 145
4.13.2. Green Fluorescent Protein: Systematic
Errors in the Data 145
4.13.3. Picosecond Decay Time 146
4.13.4. Chlorophyll Aggregates in Hexane 146
4.13.5. Intensity Decay of Flavin Adenine
Dinucleotide (FAD) 147
4.14. Data Analysis: Maximum Entropy Method 148
References 149
Problems 154
5. Frequency Domain Lifetime
Measurements
5.1. Theory of Frequency Domain Fluorometry 158
5.1.1. Least Squares Analysis of Frequency
Domain Intensity Decays 161
5.1.2. Global Analysis of Frequency Domain
Data 162
5.2. Frequency Domain Instrumentation 163
5.2.1. History of Phase Modulation
Fluorometers 163
5.2.2. An MHz Frequency Domain Fluorometer. 164
5.2.3. Light Modulators 165
5.2.4. Cross Correlation Detection 166
5.2.5. Frequency Synthesizers 167
5.2.6. Radio Frequency Amplifiers 167
5.2.7. Photomultiplier Tubes 167
5.2.8. Frequency Domain Measurements 168
5.3. Color Effects and Background Fluorescence 168
5.3.1. Color Effects in Frequency Domain
Measurements 168
5.3.2. Background Correction in Frequency
Domain Measurements 169
5.4. Representative Frequency Domain Intensity
Decays 170
5.4.1. Exponential Decays 170
5.4.2. Multi Exponential Decays of
Staphylococcal Nuclease and Melittin 171
5.4.3. Green Fluorescent Protein: One and
Two Photon Excitation 171
5.4.4. SPQ: Collisional Quenching of a
Chloride Sensor 171
5.4.5. Intensity Decay of NADH 172
5.4.6. Effect of Scattered Light 172
xvii
5.5. Simple Frequency Domain Instruments 173
5.5.1. Laser Diode Excitation 174
5.5.2. LED Excitation 174
5.6. Gigahertz Frequency Domain Fluorometry 175
5.6.1. Gigahertz FD Measurements 177
5.6.2. Biochemical Examples of Gigahertz
FDData 177
5.7. Analysis of Frequency Domain Data 178
5.7.1. Resolution of Two Widely Spaced
Lifetimes 178
5.7.2. Resolution of Two Closely Spaced
Lifetimes 180
5.7.3. Global Analysis of a Two Component
Mixture 182
5.7.4. Analysis of a Three Component Mixture:
Limits of Resolution 183
5.7.5. Resolution of a Three Component
Mixture with a Tenfold Range of
Decay Times 185
5.7.6. Maximum Entropy Analysis of FD Data 185
5.8. Biochemical Examples of Frequency Domain
Intensity Decays 186
5.8.1. DNA Labeled with DAPI 186
5.8.2. Mag Quin 2: A Lifetime Based Sensor
for Magnesium 187
5.8.3. Recovery of Lifetime Distributions from
Frequency Domain Data 188
5.8.4. Cross Fitting of Models: Lifetime
Distributions of Melittin 188
5.8.5. Frequency Domain Fluorescence
Microscopy with an LED Light Source 189
5.9. Phase Angle and Modulation Spectra 189
5.10. Apparent Phase and Modulation Lifetimes 191
5.11. Derivation of the Equations for Phase
Modulation Fluorescence 192
5.11.1. Relationship of the Lifetime to the
Phase Angle and Modulation 192
5.11.2. Cross Correlation Detection 194
5.12. Phase Sensitive Emission Spectra 194
5.12.1. Theory of Phase Sensitive Detection
of Fluorescence 195
5.12.2. Examples of PSDF and Phase
Suppression 196
5.12.3. High Frequency or Low Frequency
Phase Sensitive Detection 197
5.13. Phase Modulation Resolution of Emission
Spectra 197
5.13.1. Resolution Based on Phase or Modulation
Lifetimes 198
5.13.2. Resolution Based on Phase Angles
and Modulations 198
5.13.3. Resolution of Emission Spectra from
Phase and Modulation Spectra 198
References 199
Problems 203
xviii
6. Solvent and Environmental Effects
6.1. Overview of Solvent Polarity Effects 205
6.1.1. Effects of Solvent Polarity 205
6.1.2. Polarity Surrounding a Membrane Bound
Fluorophore 206
6.1.3. Other Mechanisms for Spectral Shifts 207
6.2. General Solvent Effects: The Lippert Mataga
Equation 208
6.2.1. Derivation of the Lippert Equation 210
6.2.2. Application of the Lippert Equation 212
6.3. Specific Solvent Effects 213
6.3.1. Specific Solvent Effects and Lippert Plots . 215
6.4. Temperature Effects 216
6.5. Phase Transitions in Membranes 217
6.6. Additional Factors that Affect Emission Spectra 219
6.6.1. Locally Excited and Internal
Charge Transfer States 219
6.6.2. Excited State Intramolecular Proton
Transfer (ESIPT) 221
6.6.3. Changes in the Non Radiative
Decay Rates 222
6.6.4. Changes in the Rate of Radiative Decay 223
6.7. Effects of Viscosity 223
6.7.1. Effect of Shear Stress on Membrane
Viscosity 225
6.8. Probe Probe Interactions 225
6.9. Biochemical Applications of Environment
Sensitive Fluorophores 226
6.9.1. Fatty Acid Binding Proteins 226
6.9.2. Exposure of a Hydrophobic Surface
on Calmodulin 226
6.9.3. Binding to Cyclodextrin Using a
Dansyl Probe 227
6.10. Advanced Solvent Sensitive Probes 228
6.11. Effects of Solvent Mixtures 229
6.12. Summary of Solvent Effects 231
References 232
Problems 235
7. Dynamics of Solvent and Spectral Relaxation
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) 240
7.2.1. Direct Recording of TRES 240
7.2.2. TRES from Wavelength Dependent
Decays 241
7.3. Spectral Relaxation in Proteins 242
7.3.1. Spectral Relaxation of Labeled
Apomyoglobin 243
7.3.2. Protein Spectral Relaxation around a
Synthetic Fluorescent Amino Acid 244
7.4. Spectral Relaxation in Membranes 245
7.4.1. Analysis of Time Resolved Emission
Spectra 246
7.4.2. Spectral Relaxation of Membrane Bound
Anthroyloxy Fatty Acids 248
CONTENTS
7.5. Picosecond Relaxation in Solvents 249
7.5.1. Theory for Time Dependent Solvent
Relaxation 250
7.5.2. Multi Exponential Relaxation in Water 251
7.6. Measurement of Multi Exponential Spectral
Relaxation 252
7.7. Distinction between Solvent Relaxation
and Formation of Rotational Isomers 253
7.8. Comparison of TRES and Decay Associated
Spectra 255
7.9. Lifetime Resolved Emission Spectra 255
7.10. Red Edge Excitation Shifts 257
7.10.1. Membranes and Red Edge
Excitation Shifts 258
7.10.2. Red Edge Excitation Shifts and
Energy Transfer 259
7.11. Excited State Reactions 259
7.11.1. Excited State Ionization of Naphthol 260
7.12. Theory for a Reversible Two State Reaction 262
7.12.1. Steady State Fluorescence of a
Two State Reaction 262
7.12.2. Time Resolved Decays for the
Two State Model 263
7.12.3. Differential Wavelength Methods 264
7.13. Time Domain Studies of Naphthol Dissociation 264
7.14. Analysis of Excited State Reactions by
Phase Modulation Fluorometry 265
7.14.1. Effect of an Excited State Reaction
on the Apparent Phase and Modulation
Lifetimes 266
7.14.2. Wavelength Dependent Phase and
Modulation Values for an Excited State
Reaction 267
7.14.3. Frequency Domain Measurement of
Excimer Formation 269
7.15. Biochemical Examples of Excited State
Reactions 270
7.15.1. Exposure of a Membrane Bound
Cholesterol Analogue 270
References 270
Problems 275
8. Quenching of Fluorescence
8.1. Quenchers of Fluorescence 278
8.2. Theory of Collisional Quenching 278
8.2.1. Derivation of the Stern Volmer Equation 280
8.2.2. Interpretation of the Bimolecular
Quenching Constant 281
8.3. Theory of Static Quenching 282
8.4. Combined Dynamic and Static Quenching 282
8.5. Examples of Static and Dynamic Quenching 283
8.6. Deviations from the Stern Volmer Equation:
Quenching Sphere of Action 284
8.6.1. Derivation of the Quenching Sphere
of Action 285
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
8.7. Effects of Steric Shielding and Charge on
Quenching 286
8.7.1. Accessibility of DNA Bound Probes
to Quenchers 286
8.7.2. Quenching of Ethenoadenine Derivatives 287
8.8. Fractional Accessibility to Quenchers 288
8.8.1. Modified Stern Volmer Plots 288
8.8.2. Experimental Considerations
in Quenching 289
8.9. Applications of Quenching to Proteins 290
8.9.1. Fractional Accessibility of Tryptophan
Residues in Endonuclease III 290
8.9.2. Effect of Conformational Changes
on Tryptophan Accessibility 291
8.9.3. Quenching of the Multiple Decay
Times of Proteins 291
8.9.4. Effects of Quenchers on Proteins 292
8.9.5. Correlation of Emission Wavelength
and Accessibility: Protein Folding of
ColicinEl 292
8.10. Application of Quenching to Membranes 293
8.10.1. Oxygen Diffusion in Membranes 293
8.10.2. Localization of Membrane Bound
Tryptophan Residues by Quenching 294
8.10.3. Quenchingof Membrane Probes
Using Localized Quenchers 295
8.10.4. Parallax and Depth Dependent
Quenching in Membranes 296
8.10.5. Boundary Lipid Quenching 298
8.10.6. Effect of Lipid Water Partitioning
on Quenching 298
8.10.7. Quenching in Micelles 300
8.11. Lateral Diffusion in Membranes 300
8.12. Quenching Resolved Emission Spectra 301
8.12.1. Fluorophore Mixtures 301
8.12.2. Quenching Resolved Emission Spectra
of the E. Coli Tet Repressor 302
8.13. Quenching and Association Reactions 304
8.13.1. Quenching Due to Specific Binding
Interactions 304
8.14. Sensing Applications of Quenching 305
8.14.1. Chloride Sensitive Fluorophores 306
8.14.2. Intracellular Chloride Imaging 306
8.14.3. Chloride Sensitive GFP 307
8.14.4. Amplified Quenching 309
8.15. Applications of Quenching to Molecular
Biology 310
8.15.1. Release of Quenching upon
Hybridization 310
8.15.2. Molecular Beacons in Quenching
by Guanine 311
8.15.3. Binding of Substrates to Ribozymes 311
8.15.4. Association Reactions and Accessibility
to Quenchers 312
8.16. Quenching on Gold Surfaces 313
8.16.1. Molecular Beacons Based on Quenching
by Gold Colloids 313
xix
8.16.2. Molecular Beacons Based on Quenching
by a Gold Surface 314
8.17. Intramolecular Quenching 314
8.17.1. DNA Dynamics by Intramolecular
Quenching 314
8.17.2. Electron Transfer Quenching in a
Flavoprotein 315
8.17.3. Sensors Based on Intramolecular
PET Quenching 316
8.18. Quenching of Phosphorescence 317
References 318
Problems 327
9. Mechanisms and Dynamics of
Fluorescence Quenching
9.1. Comparison of Quenching and Resonance
Energy Transfer 331
9.1.1. Distance Dependence of RET
and Quenching 332
9.1.2. Encounter Complexes and Quenching
Efficiency 333
9.2. Mechanisms of Quenching 334
9.2.1. Intersystem Crossing 334
9.2.2. Electron Exchange Quenching 335
9.2.3. Photoinduced Electron Transfer 335
9.3. Energetics of Photoinduced Electron Transfer 336
9.3.1. Examples of PET Quenching 338
9.3.2. PET in Linked Donor Acceptor Pairs 340
9.4. PET Quenching in Biomolecules 341
9.4.1. Quenching of Indole by Imidazolium 341
9.4.2. Quenching by DNA Bases and
Nucleotides 341
9.5. Single Molecule PET 342
9.6. Transient Effects in Quenching 343
9.6.1. Experimental Studies of Transient
Effects 346
9.6.2. Distance Dependent Quenching
in Proteins 348
References 348
Problems 351
10. Fluorescence Anisotropy
10.1. Definition of Fluorescence Anisotropy 353
10.1.1. Origin of the Definitions of
Polarization and Anisotropy 355
10.2. Theory for Anisotropy 355
10.2.1. Excitation Photoselection of Fluorophores. 357
10.3. Excitation Anisotropy Spectra 358
10.3.1. Resolution of Electronic States from
Polarization Spectra 360
10.4. Measurement of Fluorescence Anisotropies 361
10.4.1. L Format or Single Channel Method 361
10.4.2. T Format or Two Channel Anisotropies 363
10.4.3. Comparison of T Format and
L Format Measurements 363
XX
10.4.4. Alignment of Polarizers 364
10.4.5. Magic Angle Polarizer Conditions 364
10.4.6. Why is the Total Intensity
Equal to /„ + 2/± 364
10.4.7. Effect of Resonance Energy Transfer
on the Anisotropy 364
10.4.8. Trivial Causes of Depolarization 365
10.4.9. Factors Affecting the Anisotropy 366
10.5. Effects of Rotational Diffusion on Fluorescence
Anisotropies: The Perrin Equation 366
10.5.1. The Perrin Equation: Rotational
Motions of Proteins 367
10.5.2. Examples of a Perrin Plot 369
10.6. Perrin Plots of Proteins 370
10.6.1. Binding of tRNA to tRNA Synthetase 370
10.6.2. Molecular Chaperonin cpn60 (GroEL) 371
10.6.3. Perrin Plots of an Fab Immunoglobulin
Fragment 371
10.7. Biochemical Applications of Steady State
Anisotropies 372
10.7.1. Peptide Binding to Calmodulin 372
10.7.2. Binding of the Trp Repressor to DNA 373
10.7.3. Helicase Catalyzed DNA Unwinding 373
10.7.4. Melittin Association Detected from
Homotransfer 374
10.8. Anisotropy of Membranes and Membrane
Bound Proteins 374
10.8.1. Membrane Microviscosity 374
10.8.2. Distribution of Membrane Bound
Proteins 375
10.9. Transition Moments 377
References 378
Additional Reading on the Application
of Anisotropy 380
Problems 381
11. Time Dependent Anisotropy Decays
11.1. Time Domain and Frequency Domain
Anisotropy Decays 383
11.2. Anisotropy Decay Analysis 387
11.2.1. Early Methods for Analysis of
TD Anisotropy Data 387
11.2.2. Preferred Analysis of TD
Anisotropy Data 388
11.2.3. Value of r0 389
11.3. Analysis of Frequency Domain
Anisotropy Decays 390
11.4. Anisotropy Decay Laws 390
11.4.1. Non Spherical Fluorophores 391
11.4.2. Hindered Rotors 391
11.4.3. Segmental Mobility of a Biopolymer
Bound Fluorophore 392
11.4.4. Correlation Time Distributions 393
11.4.5. Associated Anisotropy Decays 393
CONTENTS
11.4.6. Example Anisotropy Decays of
Rhodamine Green and Rhodamine
Green Dextran 394
11.5. Time Domain Anisotropy Decays of Proteins 394
11.5.1. Intrinsic Tryptophan Anisotropy Decay
of Liver Alcohol Dehydrogenase 395
11.5.2. Phospholipase A2 395
11.5.3. Subtilisin Carlsberg 395
11.5.4. Domain Motions of Immunoglobulins 396
11.5.5. Effects of Free Probe on Anisotropy
Decays 397
11.6. Frequency Domain Anisotropy Decays
of Proteins 397
11.6.1. Apomyoglobin: A Rigid Rotor 397
11.6.2. Melittin Self Association and
Anisotropy Decays 398
11.6.3. Picosecond Rotational Diffusion
ofOxytocin 399
11.7. Hindered Rotational Diffusion in Membranes 399
11.7.1. Characterization of a New
Membrane Probe 401
11.8. Anisotropy Decays of Nucleic Acids 402
11.8.1. Hydrodynamics of DNA Oligomers 403
11.8.2. Dynamics of Intracellular DNA 403
11.8.3. DNA Binding to HIV Integrase Using
Correlation Time Distributions 404
11.9. Correlation Time Imaging 406
11.10. Microsecond Anisotropy Decays 408
11.10.1. Phosphorescence Anisotropy Decays 408
11.10.2. Long Lifetime Metal Ligand
Complexes 408
References 409
Problems 412
12. Advanced Anisotropy Concepts
12.1. Associated Anisotropy Decay 413
12.1.1. Theory for Associated Anisotropy
Decay 414
12.1.2. Time Domain Measurements of
Associated Anisotropy Decays 415
12.2. Biochemical Examples of Associated
Anisotropy Decays 417
12.2.1. Time Domain Studies of DNA
Binding to the Klenow Fragment
of DNA Polymerase 417
12.2.2. Frequency Domain Measurements
of Associated Anisotropy Decays 417
12.3. Rotational Diffusion of Non Spherical
Molecules: An Overview 418
12.3.1. Anisotropy Decays of Ellipsoids 419
12.4. Ellipsoids of Revolution 420
12.4.1. Simplified Ellipsoids of Revolution 421
12.4.2. Intuitive Description of Rotational
Diffusion of an Oblate Ellipsoid 422
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
12.4.3. Rotational Correlation Times for
Ellipsoids of Revolution 423
12.4.4. Stick versus Slip Rotational Diffusion 425
12.5. Complete Theory for Rotational Diffusion
of Ellipsoids 425
12.6. Anisotropic Rotational Diffusion 426
12.6.1. Time Domain Studies 426
12.6.2. Frequency Domain Studies of
Anisotropic Rotational Diffusion 427
12.7. Global Anisotropy Decay Analysis 429
12.7.1. Global Analysis with Multi Wavelength
Excitation 429
12.7.2. Global Anisotropy Decay Analysis with
Collisional Quenching 430
12.7.3. Application of Quenching to Protein
Anisotropy Decays 431
12.8. Intercalated Fluorophores in DNA 432
12.9. Transition Moments 433
12.9.1. Anisotropy of Planar Fluorophores
with High Symmetry 435
12.10. Lifetime Resolved Anisotropies 435
12.10.1. Effect of Segmental Motion on the
Perrin Plots 436
12.11. Soleillet's Rule: Multiplication of Depolarized
Factors 436
12.12. Anisotropies Can Depend on Emission
Wavelength 437
References 438
Problems 441
13. Energy Transfer
13.1. Characteristics of Resonance Energy Transfer 443
13.2. Theory of Energy Transfer for a
Donor Acceptor Pair 445
13.2.1. Orientation Factor k2 448
13.2.2. Dependence of the Transfer Rate on
Distance (r), the Overlap
Integral (J), and t2 449
13.2.3. Homotransfer and Heterotransfer 450
13.3. Distance Measurements Using RET 451
13.3.1. Distance Measurements in a Helical
Melittin 451
13.3.2. Effects of Incomplete Labeling 452
13.3.3. Effect of k2 on the Possible Range
of Distances 452
13.4. Biochemical Applications of RET 453
13.4.1. Protein Folding Measured by RET 453
13.4.2. Intracellular Protein Folding 454
13.4.3. RET and Association Reactions 455
13.4.4. Orientation of a Protein Bound Peptide 456
13.4.5. Protein Binding to Semiconductor
Nanoparticles 457
13.5. RET Sensors 458
13.5.1. Intracellular RET Indicator
for Estrogens 458
xxi
13.5.2. RET Imaging of Intracellular Protein
Phosphorylation 459
13.5.3. Imaging of Rac Activation in Cells 459
13.6. RET and Nucleic Acids 459
13.6.1. Imaging of Intracellular RNA 460
13.7. Energy Transfer Efficiency from
Enhanced Acceptor Fluorescence 461
13.8. Energy Transfer in Membranes 462
13.8.1. Lipid Distributions around Gramicidin 463
13.8.2. Membrane Fusion and Lipid Exchange 465
13.9. Effect of t on RET 465
13.10. Energy Transfer in Solution 466
13.10.1. Diffusion Enhanced Energy Transfer 467
13.11. Representative /?„ Values 467
References 468
Additional References on Resonance
Energy Transfer 471
Problems 472
14. Time Resolved Energy Transfer and
Conformational Distributions of Biopolymers
14.1. Distance Distributions 477
14.2. Distance Distributions in Peptides 479
14.2.1. Comparison for a Rigid and Flexible
Hexapeptide 479
14.2.2. Crossfitting Data to Exclude
Alternative Models 481
14.2.3. Donor Decay without Acceptor 482
14.2.4. Effect of Concentration of the
D A Pairs 482
14.3. Distance Distributions in Peptides 482
14.3.1. Distance Distributions in Melittin 483
14.4. Distance Distribution Data Analysis 485
14.4.1. Frequency Domain Distance Distribution
Analysis 485
14.4.2. Time Domain Distance Distribution
Analysis 487
14.4.3. Distance Distribution Functions 487
14.4.4. Effects of Incomplete Labeling 487
14.4.5. Effect of the Orientation Factor k2 489
14.4.6. Acceptor Decays 489
14.5. Biochemical Applications of Distance
Distributions 490
14.5.1. Calcium Induced Changes in the
Conformation of Troponin C 490
14.5.2. Hairpin Ribozyme 493
14.5.3. Four Way Holliday Junction in DNA 493
14.5.4. Distance Distributions and Unfolding
of Yeast Phosphoglycerate Kinase 494
14.5.5. Distance Distributions in a Glycopeptide. 495
14.5.6. Single Protein Molecule Distance
Distribution 496
14.6. Time Resolved RET Imaging 497
14.7. Effect of Diffusion for Linked D A Pairs 498
xxii
14.7.1. Simulations of FRET for a Flexible
D APair 499
14.7.2. Experimental Measurement of D A
Diffusion for a Linked D A Pair 500
14.7.3. FRET and Diffusive Motions in
Biopolymers 501
14.8. Conclusion 501
References 501
Representative Publications on Measurement
of Distance Distributions 504
Problems 505
15. Energy Transfer to Multiple Acceptors in
One, Two, or Three Dimensions
15.1. RET in Three Dimensions 507
15.1.1. Effect of Diffusion on FRET with
Unlinked Donors and Acceptors 508
15.1.2. Experimental Studies of RET in
Three Dimensions 509
15.2. Effect of Dimensionality on RET 511
15.2.1. Experimental FRET in Two Dimensions. 512
15.2.2. Experimental FRET in One Dimension 514
15.3. Biochemical Applications of RET with
Multiple Acceptors 515
15.3.1. Aggregation of P Amyloid Peptides 515
15.3.2. RET Imaging of Fibronectin 516
15.4. Energy Transfer in Restricted Geometries 516
15.4.1. Effect of Excluded Area on Energy
Transfer in Two Dimensions 518
15.5. RET in the Presence of Diffusion 519
15.6. RET in the Rapid Diffusion Limit 520
15.6.1. Location of an Acceptor in
Lipid Vesicles 521
15.6.2. Locaion of Retinal in Rhodopsin
Disc Membranes 522
15.7. Conclusions 524
References 524
Additional References on RET between
Unlinked Donor and Acceptor 526
Problems 527
16. Protein Fluorescence
6.1. Spectral Properties of the Aromatic Amino Acids. 530
16.1.1. Excitation Polarization Spectra of
Tyrosine and Tryptophan 531
16.1.2. Solvent Effects on Tryptophan Emission
Spectra 533
16.1.3. Excited State Ionization of Tyrosine 534
16.1.4. Tyrosinate Emission from Proteins 535
6.2. General Features of Protein Fluorescence 535
CONTENTS
16.3. Tryptophan Emission in an Apolar
Protein Environment 538
16.3.1. Site Directed Mutagenesis of a
Single Tryptophan Azurin 538
16.3.2. Emission Spectra of Azurins with
One or Two Tryptophan Residues 539
16.4. Energy Transfer and Intrinsic Protein
Fluorescence 539
16.4.1. Tyrosine to Tryptophan Energy Transfer
in Interferon y 540
16.4.2. Quantitation of RET Efficiencies
in Proteins 541
16.4.3. Tyrosine to Tryptophan RET in
a Membrane Bound Protein 543
16.4.4. Phenylalanine to Tyrosine
Energy Transfer 543
16.5. Calcium Binding to Calmodulin Using
Phenylalanine and Tyrosine Emission 545
16.6. Quenching of Tryptophan Residues in Proteins 546
16.6.1. Effect of Emission Maximum on
Quenching 547
16.6.2. Fractional Accessibility to Quenching
in Multi Tryptophan Proteins 549
16.6.3. Resolution of Emission Spectra by
Quenching 550
16.7. Association Reaction of Proteins 551
16.7.1. Binding of Calmodulin to a
Target Protein 551
16.7.2. Calmodulin: Resolution of the
Four Calcium Binding Sites Using
Tryptophan Containing Mutants 552
16.7.3. Interactions of DNA with Proteins 552
16.8. Spectral Properties of Genetically Engineered
Proteins 554
16.8.1. Single Tryptophan Mutants of
Triosephosphate Isomerase 555
16.8.2. Barnase: A Three Tryptophan Protein 556
16.8.3. Site Directed Mutagenesis of
Tyrosine Proteins 557
16.9. Protein Folding 557
16.9.1. Protein Engineering of Mutant
Ribonuclease for Folding Experiments 558
16.9.2. Folding of Lactate Dehydrogenase 559
16.9.3. Folding Pathway of CRABPI 560
16.10. Protein Structure and Tryptophan Emission 560
16.10.1. Tryptophan Spectral Properties
and Structural Motifs 561
16.11. Tryptophan Analogues 562
16.11.1. Tryptophan Analogues 564
16.11.2. Genetically Inserted Amino Acid
Analogues 565
16.12. The Challenge of Protein Fluorescence 566
References 567
Problems 573
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
17. Time Resolved Protein Fluorescence
17.1. Intensity Decays of Tryptophan:
The Rotamer Model 578
17.2. Time Resolved Intensity Decays of
Tryptophan and Tyrosine 580
17.2.1. Decay Associated Emission Spectra
of Tryptophan 581
17.2.2. Intensity Decays of Neutral Tryptophan
Derivatives 581
17.2.3. Intensity Decays of Tyrosine and
Its Neutral Derivatives 582
17.3. Intensity and Anisotropy Decays of Proteins 583
17.3.1. Single Exponential Intensity and
Anisotropy Decay of Ribonuclease T, 584
17.3.2. Annexin V: A Calcium Sensitive
Single Tryptophan Protein 585
17.3.3. Anisotropy Decay of a Protein with
Two Tryptophans 587
17.4. Protein Unfolding Exposes the Tryptophan
Residue to Water 588
17.4.1. Conformational Heterogeneity Can
Result in Complex Intensity and
Anisotropy Decays 588
17.5. Anisotropy Decays of Proteins 589
17.5.1. Effects of Association Reactions on
Anisotropy Decays: Melittin 590
17.6. Biochemical Examples Using Time Resolved
Protein Fluorescence 591
17.6.1. Decay Associated Spectra of Barnase 591
17.6.2. Disulfide Oxidoreductase DsbA 591
17.6.3. Immunophilin FKBP59 I: Quenching
of Tryptophan Fluorescence by
Phenylalanine 592
17.6.4. Trp Repressor: Resolution of the Two
Interacting Tryptophans 593
17.6.5. Thermophilic P Glycosidase:
A Multi Tryptophan Protein 594
17.6.6. Heme Proteins Display Useful
Intrinsic Fluorescence 594
17.7. Time Dependent Spectral Relaxation of
Tryptophan 596
17.8. Phosphorescence of Proteins 598
17.9. Perspectives on Protein Fluorescence 600
References 600
Problems 605
18. Multiphoton Excitation and Microscopy
18.1. Introduction to Multiphoton Excitation 607
18.2. Cross Sections for Multiphoton Absorption 609
18.3. Two Photon Absorption Spectra 609
18.4. Two Photon Excitation of a DNA Bound
Fluorophore 610
18.5. Anisotropies with Multiphoton Excitation 612
xxiv
19.12. New Approaches to Sensing 655
19.12.1. Pebble Sensors and Lipobeads 655
19.13. In Vivo Imaging 656
19.14. Immunoassays 658
19.14.1. Enzyme Linked Immunosorbent Assays
(ELISA) 659
19.14.2. Time Resolved Immunoassays 659
19.14.3. Energy Transfer Immunoassays 660
19.14.4. Fluorescence Polarization
Immunoassays 661
References 663
Problems 672
20. Novel Fluorophores
20.1. Semiconductor Nanoparticles 675
20.1.1. Spectral Properties of QDots 676
20.1.2. Labeling Cells with QDots 677
20.1.3. QDots and Resonance Energy Transfer 678
20.2. Lanthanides 679
20.2.1. RET with Lanthanides 680
20.2.2. Lanthanide Sensors 681
20.2.3. Lanthanide Nanoparticles 682
20.2.4. Near Infrared Emitting Lanthanides 682
20.2.5. Lanthanides and Fingerprint Detection 683
20.3. Long Lifetime Metal Ligand Complexes 683
20.3.1. Introduction to Metal Ligand Probes 683
20.3.2. Anisotropy Properties of
Metal Ligand Complexes 685
20.3.3. Spectral Properties of MLC Probes 686
20.3.4. The Energy Gap Law 687
20.3.5. Biophysical Applications of
Metal Ligand Probes 688
20.3.6. MLC Immunoassays 691
20.3.7. Metal Ligand Complex Sensors 694
20.4. Long Wavelength Long Lifetime
Fluorophores 695
References 697
Problems 702
Zl. DNATechnology
II.1. DNA Sequencing 705
21.1.1. Principle of DNA Sequencing 705
21.1.2. Examples of DNA Sequencing 706
21.1.3. Nucleotide Labeling Methods 707
21.1.4. Example of DNA Sequencing 708
21.1.5. Energy Transfer Dyes for DNA
Sequencing 709
21.1.6. DNA Sequencing with NIR Probes 710
21.1.7. DNA Sequencing Based on Lifetimes 712
:i.2. High Sensitivity DNA Stains 712
21.2.1. High Affinity Bis DNA Stains 713
21.2.2. Energy Transfer DNA Stains 715
CONTENTS
21.2.3. DNA Fragment Sizing by
Flow Cytometry 715
21.3. DNA Hybridization 715
21.3.1. DNA Hybridization Measured with
One Donor and Acceptor Labeled
DNA Probe 717
21.3.2. DNA Hybridization Measured by
Excimer Formation 718
21.3.3. Polarization Hybridization Arrays 719
21.3.4. Polymerase Chain Reaction 720
21.4. Molecular Beacons 720
21.4.1. Molecular Beacons with
Nonfluorescent Acceptors 720
21.4.2. Molecular Beacons with
Fluorescent Acceptors 722
21.4.3. Hybridization Proximity Beacons 722
21.4.4. Molecular Beacons Based on
Quenching by Gold 723
21.4.5. Intracellular Detection of mRNA
Using Molecular Beacons 724
21.5. Aptamers 724
21.5.1. DNAzymes 726
21.6. Multiplexed Microbead Arrays:
Suspension Arrays 726
21.7. Fluorescence In Situ Hybridization 727
21.7.1. Preparation of FISH Probe DNA 728
21.7.2. Applications of FISH 729
21.8. Multicolor FISH and Spectral Karyotyping 730
21.9. DNA Arrays 732
21.9.1. Spotted DNA Microarrays 732
21.9.2. Light Generated DNA Arrays 734
References 734
Problems 740
22. Fluorescence Lifetime Imaging Microscopy
22.1. Early Methods for Fluorescence Lifetime
Imaging 743
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
from Lifetime 744
22.2.2. Lifetime Images of Cos Cells 745
22.3. Examples of Wide Field Frequency Domain
FLIM 746
22.3.1. Resonance Energy Transfer FLIM
of Protein Kinase C Activation 746
22.3.2. Lifetime Imaging of Cells Containing
Two GFPs 747
22.4. Wide Field FLIM Using a Gated Image
Intensifier 747
22.5. Laser Scanning TCSPC FLIM 748
22.5.1. Lifetime Imaging of Cellular
Biomolecules 750
22.5.2. Lifetime Images of Amyloid Plaques 750
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY
22.6. Frequency Domain Laser Scanning Microscopy 750
22.7. Conclusions 752
References 752
Additional Reading on Fluorescence Lifetime
Imaging Microscopy 753
Problem 755
23. Single Molecule Detection
23.1. Detectability of Single Molecules 759
23.2. Total Internal Reflection and Confocal Optics 760
23.2.1. Total Internal Reflection 760
23.2.2. Confocal Detection Optics 761
23.3. Optical Configurations for SMD 762
23.4. Instrumentation for SMD 764
23.4.1. Detectors for Single Molecule Detection . 765
23.4.2. Optical Filters for SMD 766
23.5. Single Molecule Photophysics 768
23.6. Biochemical Applications of SMD 770
23.6.1. Single Molecule Enzyme Kinetics 770
23.6.2. Single Molecule ATPase Activity 770
23.6.3. Single Molecule Studies of a
Chaperonin Protein 771
23.7. Single Molecule Resonance Energy Transfer 773
23.8. Single Molecule Orientation and Rotational
Motions 775
23.8.1. Orientation Imaging of R6G and GFP 777
23.8.2. Imaging of Dipole Radiation Patterns 778
23.9. Time Resolved Studies of Single Molecules 779
23.10. Biochemical Applications 780
23.10.1. Turnover of Single Enzyme Molecules. 780
23.10.2. Single Molecule Molecular Beacons 782
23.10.3. Conformational Dynamics of a
Holliday Junction 782
23.10.4. Single Molecule Calcium Sensor 784
23.10.5. Motions of Molecular Motors 784
23.11. Advanced Topics in SMD 784
23.11.1. Signal to Noise Ratio in
Single Molecule Detection 784
23.11.2. Polarization of Single Immobilized
Fluorophores 786
23.11.3. Polarization Measurements
and Mobility of Surface Bound
Fluorophores 786
23.11.4. Single Molecule Lifetime Estimation 787
23.12. Additional Literature on SMD 788
References 788
Additional References on Single Molecule
Detection 791
Problem 795
24. Fluorescence Correlation Spectroscopy
24.1. Principles of Fluorescence Correlation
Spectroscopy 798
XXV
24.2. Theory of FCS 800
24.2.1. Translational Diffusion and FCS 802
24.2.2. Occupation Numbers and Volumes
in FCS 804
24.2.3. FCS for Multiple Diffusing Species 804
24.3. Examples of FCS Experiments 805
24.3.1. Effect of Fluorophore Concentration 805
24.3.2. Effect of Molecular Weight on
Diffusion Coefficients 806
24.4. Applications of FCS to Bioaffinity Reactions 807
24.4.1. Protein Binding to the
Chaperonin GroEL 807
24.4.2. Association of Tubulin Subunits 807
24.4.3. DNA Applications of FCS 808
24.5. FCS in Two Dimensions: Membranes 810
24.5.1. Biophysical Studies of Lateral
Diffusion in Membranes 812
24.5.2. Binding to Membrane Bound
Receptors 813
24.6. Effects of Intersystem Crossing 815
24.6.1. Theory for FCS and Intersystem
Crossing 816
24.7. Effects of Chemical Reactions 816
24.8. Fluorescence Intensity Distribution Analysis 817
24.9. Time Resolved FCS 819
24.10. Detection of Conformational Dynamics
in Macromolecules 820
24.11. FCS with Total Internal Reflection 821
24.12. FCS with Two Photon Excitation 822
24.12.1. Diffusion of an Intracellular
Kinase Using FCS with
Two Photon Excitation 823
24.13. Dual Color Fluorescence Cross Correlation
Spectroscopy 823
24.13.1. Instrumentation for Dual Color
FCCS 824
24.13.2. Theory of Dual Color FCCS 824
24.13.3. DNA Cleavage by a
Restriction Enzyme 826
24.13.4. Applications of Dual Color FCCS 826
24.14. Rotational Diffusion and Photo Antibunching 828
24.15. Flow Measurements Using FCS 830
24.16. Additional References on FCS 832
References 832
Additional References to FCS and
Its Applications 837
Problems 840
25. Radiative Decay Engineering:
Metal Enhanced Fluorescence
25.1. Radiative Decay Engineering 841
25.1.1. Introduction to RDE 841
25.1.2. Jablonski Diagram for Metal
Enhanced Fluorescence 842
25.2. Review of Metal Effects on Fluorescence 843
xxvi
25.3. Optical Properties of Metal Colloids 845
25.4. Theory for Fluorophore Colloid Interactions 846
25.5. Experimental Results on Metal Enhanced
Fluorescence 848
25.5.1. Application of MEF to DNA Analysis 848
25.6. Distance Dependence of Metal Enhanced
Fluorescence 851
25.7. Applications of Metal Enhanced Fluorescence 851
25.7.1. DNA Hybridization Using MEF 853
25.7.2. Release of Self Quenching 853
25.7.3. Effect of Silver Particles on RET 854
25.8. Mechanism of MEF 855
25.9. Perspective on RET 856
References 856
Problem 859
26. Radiative Decay Engineering:
Surface Plasmon Coupled Emission
26.1. Phenomenon of SPCE 861
26.2. Surface Plasmon Resonance 861
26.2.1. Theory for Surface Plasmon Resonance 863
26.3. Expected Properties of SPCE 865
26.4. Experimental Demonstration of SPCE 865
26.5. Applications of SPCE 867
26.6. Future Developments in SPCE 868
References 870
Appendix I. Corrected Emission Spectra
1. Emission Spectra Standards from 300 to 800 nm 873
2. P Carboline Derivatives as Fluorescence Standards 873
3. Corrected Emission Spectra of 9,10 Diphenyl
anthracene, Quinine, and Fluorescein 877
4. Long Wavelength Standards 877
5. Ultraviolet Standards 878
6. Additional Corrected Emission Spectra 881
References 881
CONTENTS
Appendix II. Fluorescent Lifetime Standards
1. Nanosecond Lifetime Standards 883
2. Picosecond Lifetime Standards 884
3. Representative Frequency Domain
Intensity Decays 885
4. Time Domain Lifetime Standards 886
Appendix III. Additional Reading
1. Time Resolved Measurements 889
2. Spectra Properties of Fluorophores 889
3. Theory of Fluorescence and Photophysics 889
4. Reviews of Fluorescence Spectroscopy 889
5. Biochemical Fluorescence 890
6. Protein Fluorescence 890
7. Data Analysis and Nonlinear Least Squares 890
8. Photochemistry 890
9. Flow Cytometry 890
10. Phosphorescence 890
11. Fluorescence Sensing 890
12. Immunoassays 891
13. Applications of Fluorescence 891
14. Multiphoton Excitation 891
15. Infrared and NIR Fluorescence 891
16. Lasers 891
17. Fluorescence Microscopy 891
18. Metal Ligand Complexes and Unusual
Lumophores 891
19. Single Molecule Detection 891
20. Fluorescence Correlation Spectroscopy 892
21. Biophotonics 892
22. Nanoparticles 892
23. Metallic Particles 892
24. Books on Fluorescence 892
Answers to Problems 893
Index 923 |
| any_adam_object | 1 |
| any_adam_object_boolean | 1 |
| author | Lakowicz, Joseph R. |
| author_facet | Lakowicz, Joseph R. |
| author_role | aut |
| author_sort | Lakowicz, Joseph R. |
| author_variant | j r l jr jrl |
| building | Verbundindex |
| bvnumber | BV021712394 |
| classification_rvk | UH 5870 VG 8750 WC 2600 |
| classification_tum | CHE 808f CHE 243f PHY 513f |
| ctrlnum | (OCoLC)634893023 (DE-599)BVBBV021712394 |
| discipline | Chemie / Pharmazie Physik Biologie Chemie |
| discipline_str_mv | Chemie / Pharmazie Physik Biologie Chemie |
| edition | Third edition |
| format | Kit Book |
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| genre | CD-ROM gnd |
| genre_facet | CD-ROM |
| id | DE-604.BV021712394 |
| illustrated | Not Illustrated |
| index_date | 2024-07-02T15:20:50Z |
| indexdate | 2025-02-13T13:02:11Z |
| institution | BVB |
| isbn | 0387312781 9780387312781 9781489978806 |
| language | English |
| oai_aleph_id | oai:aleph.bib-bvb.de:BVB01-014926185 |
| oclc_num | 634893023 |
| open_access_boolean | |
| owner | DE-29T DE-91G DE-BY-TUM DE-19 DE-BY-UBM DE-703 DE-20 DE-355 DE-BY-UBR DE-83 DE-91S DE-BY-TUM DE-11 DE-M49 DE-BY-TUM DE-706 DE-188 DE-384 |
| owner_facet | DE-29T DE-91G DE-BY-TUM DE-19 DE-BY-UBM DE-703 DE-20 DE-355 DE-BY-UBR DE-83 DE-91S DE-BY-TUM DE-11 DE-M49 DE-BY-TUM DE-706 DE-188 DE-384 |
| physical | XXVI, 954 Seiten Illustrationen, Diagramme 1 CD-ROM ; Extras online [nur bei reprints der 3. edition von 2006] |
| publishDate | 2006 |
| publishDateSearch | 2006 |
| publishDateSort | 2006 |
| publisher | Springer |
| record_format | marc |
| spelling | Lakowicz, Joseph R. Verfasser aut Principles of fluorescence spectroscopy Joseph R. Lakowicz Third edition New York, NY Springer [2006] XXVI, 954 Seiten Illustrationen, Diagramme 1 CD-ROM ; Extras online [nur bei reprints der 3. edition von 2006] txt rdacontent n rdamedia nc rdacarrier Fluorescence cabt Spectroscopy cabt Kinetik (DE-588)4030665-3 gnd rswk-swf Spektrometer (DE-588)4140820-2 gnd rswk-swf Fluoreszenzspektroskopie (DE-588)4017701-4 gnd rswk-swf Organische Verbindungen (DE-588)4043816-8 gnd rswk-swf Fluoreszenz (DE-588)4154818-8 gnd rswk-swf Fluoreszenzspektroskopie (DE-588)4017701-4 s DE-604 Spektrometer (DE-588)4140820-2 s 1\p DE-604 Fluoreszenz (DE-588)4154818-8 s 2\p DE-604 Organische Verbindungen (DE-588)4043816-8 s 3\p DE-604 Kinetik (DE-588)4030665-3 s 4\p DE-604 Erscheint auch als Online-Ausgabe, ebook 978-0-387-46312-4 text/html http://deposit.dnb.de/cgi-bin/dokserv?id=2813203&prov=M&dok_var=1&dok_ext=htm Inhaltstext HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014926185&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 2\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 3\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk 4\p cgwrk 20201028 DE-101 https://d-nb.info/provenance/plan#cgwrk |
| spellingShingle | Lakowicz, Joseph R. Principles of fluorescence spectroscopy Fluorescence cabt Spectroscopy cabt Kinetik (DE-588)4030665-3 gnd Spektrometer (DE-588)4140820-2 gnd Fluoreszenzspektroskopie (DE-588)4017701-4 gnd Organische Verbindungen (DE-588)4043816-8 gnd Fluoreszenz (DE-588)4154818-8 gnd |
| subject_GND | (DE-588)4030665-3 (DE-588)4140820-2 (DE-588)4017701-4 (DE-588)4043816-8 (DE-588)4154818-8 |
| title | Principles of fluorescence spectroscopy |
| title_auth | Principles of fluorescence spectroscopy |
| title_exact_search | Principles of fluorescence spectroscopy |
| title_exact_search_txtP | Principles of fluorescence spectroscopy |
| title_full | Principles of fluorescence spectroscopy Joseph R. Lakowicz |
| title_fullStr | Principles of fluorescence spectroscopy Joseph R. Lakowicz |
| title_full_unstemmed | Principles of fluorescence spectroscopy Joseph R. Lakowicz |
| title_short | Principles of fluorescence spectroscopy |
| title_sort | principles of fluorescence spectroscopy |
| topic | Fluorescence cabt Spectroscopy cabt Kinetik (DE-588)4030665-3 gnd Spektrometer (DE-588)4140820-2 gnd Fluoreszenzspektroskopie (DE-588)4017701-4 gnd Organische Verbindungen (DE-588)4043816-8 gnd Fluoreszenz (DE-588)4154818-8 gnd |
| topic_facet | Fluorescence Spectroscopy Kinetik Spektrometer Fluoreszenzspektroskopie Organische Verbindungen Fluoreszenz |
| url | http://deposit.dnb.de/cgi-bin/dokserv?id=2813203&prov=M&dok_var=1&dok_ext=htm http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=014926185&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT lakowiczjosephr principlesoffluorescencespectroscopy |