Principles of Fluorescence Spectroscopy

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PRINCIPLES OF FLUORESCENCE SPECTROSCOPY xvii 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 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 FD Data ......................................................... 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 CONTENTS 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 ................. 7.2. Measurement of Time-Resolved Emission 239 Spectra (TRES) ........................................................ 240 7.2.1. Direct Recording of TRES............................ 7.2.2. TRES from Wavelength-Dependent 240 Decays ........................................................... 241 7.3. Spectral Relaxation in Proteins................................ 7.3.1. Spectral Relaxation of Labeled 242 Apomyoglobin............................................... 7.3.2. Protein Spectral Relaxation around a 243 Synthetic Fluorescent Amino Acid ............... 244 7.4. Spectral Relaxation in Membranes .......................... 7.4.1. Analysis of Time-Resolved Emission 245 Spectra........................................................... 7.4.2. Spectral Relaxation of Membrane-Bound 246 Anthroyloxy Fatty Acids ............................... 248 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
Page 1 and 2: Principles of Fluorescence Spectros
Page 3 and 4: Joseph R. Lakowicz Center for Fluor
Page 5 and 6: Preface The first edition of Princi
Page 7 and 8: x GLOSSARY OF ACRONYMS DNS dansyl o
Page 9 and 10: xii GLOSSARY OF ACRONYMS TRES time-
Page 11 and 12: xiv GLOSSARY OF MATHEMATICAL TERMS
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Page 17 and 18: xx CONTENTS 10.4.4. Alignment of Po
Page 19 and 20: xxii CONTENTS 14.7.1. Simulations o
Page 21 and 22: xxiv CONTENTS 19.12. New Approaches
Page 23 and 24: xxvi CONTENTS 25.3. Optical Propert
Page 25 and 26: 2 INTRODUCTION TO FLUORESCENCE Figu
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Page 37 and 38: 14 INTRODUCTION TO FLUORESCENCE 1.7
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42 INSTRUMENTATION FOR FLUORESCENCE
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3 Fluorescence probes represent the
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PRINCIPLES OF FLUORESCENCE SPECTROS
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98 TIME-DOMAIN LIFETIME MEASUREMENT
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100 TIME-DOMAIN LIFETIME MEASUREMEN
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238 DYNAMICS OF SOLVENT AND SPECTRA
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278 QUENCHING OF FLUORESCENCE 8.1.
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280 QUENCHING OF FLUORESCENCE Figur
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282 QUENCHING OF FLUORESCENCE ly ev
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290 QUENCHING OF FLUORESCENCE relat
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298 QUENCHING OF FLUORESCENCE σ ar
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320 QUENCHING OF FLUORESCENCE 47. R
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322 QUENCHING OF FLUORESCENCE 113.
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328 QUENCHING OF FLUORESCENCE P8.3.
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330 QUENCHING OF FLUORESCENCE P8.11
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332 MECHANISMS AND DYNAMICS OF FLUO
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10 Measurements of fluorescence ani
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444 ENERGY TRANSFER Figure 13.1. Fl
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446 ENERGY TRANSFER wavelength is e
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448 ENERGY TRANSFER Table 13.1. Cal
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450 ENERGY TRANSFER Figure 13.6. Ab
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452 ENERGY TRANSFER safe for many p
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454 ENERGY TRANSFER Figure 13.12. P
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456 ENERGY TRANSFER Figure 13.16. E
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458 ENERGY TRANSFER Figure 13.21. R
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460 ENERGY TRANSFER Figure 13.24. R
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462 ENERGY TRANSFER tiple acceptors
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464 ENERGY TRANSFER Figure 13.29. A
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466 ENERGY TRANSFER Figure 13.33. F
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468 ENERGY TRANSFER Table 13.3. Rep
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470 ENERGY TRANSFER 55. Clegg RM. 1
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472 ENERGY TRANSFER Tong AK, Jockus
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474 ENERGY TRANSFER Figure 13.39. O
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16 The biochemical applications of
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578 TIME-RESOLVED PROTEIN FLUORESCE
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608 MULTIPHOTON EXCITATION AND MICR
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19 Fluorescence sensing of chemical
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676 NOVEL FLUOROPHORES Figure 20.1.
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678 NOVEL FLUOROPHORES Figure 20.7.
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680 NOVEL FLUOROPHORES Figure 20.12
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698 NOVEL FLUOROPHORES 33. Acherman
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700 NOVEL FLUOROPHORES 103. Demas J
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702 NOVEL FLUOROPHORES 176. Montalt
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21 During the past 20 years there h
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22 Fluorescence microscopy is one o
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758 SINGLE-MOLECULE DETECTION Figur
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766 SINGLE-MOLECULE DETECTION small
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770 SINGLE-MOLECULE DETECTION Table
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786 SINGLE-MOLECULE DETECTION face
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788 SINGLE-MOLECULE DETECTION TCSPC
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790 SINGLE-MOLECULE DETECTION 53. H
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792 SINGLE-MOLECULE DETECTION Ke PC
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794 SINGLE-MOLECULE DETECTION Polar
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862 RADIATIVE-DECAY ENGINEERING: SU
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Appendix I Corrected Emission Spect
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Appendix II Fluorescence Lifetime S
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890 APPENDIX III P ADDITIONAL READI
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892 APPENDIX III P ADDITIONAL READI
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894 ANSWERS TO PROBLEMS A1.4. A. Th
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896 ANSWERS TO PROBLEMS Energy tran
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898 ANSWERS TO PROBLEMS Figure 4.65
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900 ANSWERS TO PROBLEMS expected to
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904 ANSWERS TO PROBLEMS where f is
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906 ANSWERS TO PROBLEMS [BSA] Obser
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908 ANSWERS TO PROBLEMS emission. T
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910 ANSWERS TO PROBLEMS dx rA A (
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912 ANSWERS TO PROBLEMS B. A covale
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920 ANSWERS TO PROBLEMS CHAPTER 24
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Index A Absorption spectroscopy, 12
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Principles of Fluorescence Spectroscopy

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