1. Introduction to Fluorescence 1.1. Phenomena of Fluorescence 1.2. Jablonski Diagram 1.3. Characteristics of Fluorescence Emission 1.4. Fluorescence Lifetimes and Quantum Yields 1.5. Fluorescence Anisotropy 1.6.Resonance Energy Transfer 1.7. Steady-State and Time-Resolved Fluorescence 1.8. Biochemical Fluorophores 1.9. Molecular Information from Fluorescence 1.10. Biochemical Examples of Basic Phenomena 1.11. New Fluorescence Technologies 1.12. Overview of Fluorescence Spectroscopy References Problems 2. Instrumentation for Fluorescence Spectroscopy 2.1. Spectrofluorometers 2.2. Light Sources 2.3. Monochromators 2.4. Optical Filters 2.5. Optical Filters and Signal Purity 2.6 Photomultiplier Tubes 2.7. Polarizers 2.8. Corrected Excitation Spectra 2.9. Corrected Emission Spectra 2.10. Quantum Yield Standards 2.11. Effects of Sample Geometry 2.12. Common Errors in Sample Preparation 2.13. Absorption of Light and Deviation from the Beer-Lambert Law 2.14. Conclusions References Problems 3. Fluorophores 3.1. Intrinsic or Natural Fluorophores 3.2. Extrinsic Fluorophores 3.3.Red and Near-Infrared (NIR) Dyes 3.4. DNA Probes 3.5. Chemical Sensing Probes 3.6. Special Probes 3.7. Green Fluorescent Proteins 3.8. Other Fluorescent Proteins 3.9. Long-Lifetime Probes 3.10. Proteins as Sensors 3.11. Conclusion References Problems 4. Time-Domain Lifetime Measurements 4.l. Overview of Time-Domain and Frequency-Domain Measurements 4.5. Electronics for TCSPC 4.6. Detectors for TCSPC 4.7. Multi-Detector and Multidimensional TCSPC 4.8. Alternative Methods for Time-Resolved Measurements 4.9. Data Analysis: Nonlinear Least Squares 4.10. Analysis of Multi-Exponential Decays 4.11. Intensity Decay Laws 4.12. Global Analysis 4.13. Applications of TCSPC 4.14. Data Analysis: Maximum Entropy Method References Problems 5. Frequency-Domain Lifetime Measurements 5.1. Theory of Frequency-Domain Fluorometry 5.2. Frequency-Domain Instrumentation 5.3. Color Effects and Background Fluorescence 5.4.Representative Frequency-Domain Intensity Decays 5.5. Simple Frequency-Domain Instruments 5.6. Gigahertz Frequency-Domain Fluorometry 5.7. Analysis of Frequency-Domain Data 5.8. Biochemical Examples of Frequency-Domain Intensity Decays 5.9. Phase-Angle and Modulation Spectra 5.10. Apparent Phase and Modulation Lifetimes 5.11. Derivation of the Equations for Phase-Modulation Fluorescence 5.12. Phase-Sensitive Emission Spectra 5.13. Phase-ModulationResolution of Emission Spectra 7.11. Excited-StateReactions 7.12. Theory for aReversible Two-StateReaction 7.13. Time-Domain Studies of Naphthol Dissociation 7.14. Analysis of Excited-StateReactions by Phase-Modulation Fluorometry 7.15. Biochemical Examples of Excited-StateReactions References Problems 8. Quenching of Fluorescence 8.1. Quenchers of Fluorescence 8.2. Theory of Collisional Quenching 8.3. Theory of Static Quenching 8.4. Combined Dynamic and Static Quenching 8.5. Examples of Static and Dynamic Quenching 8.6. Deviations from the Stern-Volmer Equation:Quenching Sphere of Action 8.7. Effects of Steric Shielding and Charge on Quenching 8.8. Fractional Accessibility to Quenchers 8.9. Applications of Quenching to Proteins 8.10. Application of Quenching to Membranes 8.11. Lateral Diffusion in Membranes 8.12. Quenching-Resolved Emission Spectra 8.13. Quenching and AssociationReactions 8.14. Sensing Applications of Quenching 8.15. Applications of Quenching to Molecular Biology 8.16. Quenching on Gold Surfaces 8.17. Intramolecular Quenching 8.18. Quenching of Phosphorescence References Problems 9. Mechanisms and Dynamics of Fluorescence Quenching 9.1. Comparison of Quenching andResonance Energy Transfer 9.2. Mechanisms of Quenching 9.3. Energetics of Photoinduced Electron Transferr 9.4. PET Quenching in Biomoleculesr 9.5. Single-Molecule PET 9.6. Transient Effects in Quenching References Problems 10. Fluorescence Anisotropy 10.1. Definition of Fluorescence Anisotropy 10.2. Theory for Anisotropy 10.3. Excitation Anisotropy Spectra 10.4. Measurement of Fluorescence Anisotropies 10.5. Effects ofRotational Diffusion on Fluorescence Anisotropies: The Perrin Equation 10.6. Perrin Plots of Proteins 10.7. Biochemical Applications of Steady-State Anisotropies 10.8. Anisotropy of Membranes and Membrane-Bound Proteins 10.9. Transition Moments References AdditionalReading on the Application of Anisotropy Problems 11. Time-Dependent Anisotropy Decays 11.1. Time-Domain and Frequency-DomainAnisotropy Decays 11.2. Anisotropy Decay Analysis 11.3. Analysis of Frequency-Domain Anisotropy Decays 11.4. Anisotropy Decay Laws 11.5. Time-Domain Anisotropy Decays of Proteins 11.6. Frequency-Domain Anisotropy Decays of Proteins 11.7. HinderedRotational Diffusion in Membranes 11.8. Anisotropy Decays of Nucleic Acids 11.9. Correlation Time Imaging 11.10. Microsecond Anisotropy Decays References Problems 12. Advanced Anisotropy Concepts 12.1. Associated Anisotropy Decay 12.2. Biochemical Examples of Associated Anisotropy Decays 12.3.Rotational Diffusion of Non-Spherical Molecules: An Overview 12.3.1. Anisotropy Decays of Ellipsoids 12.4. Ellipsoids ofRevolution 12.5. Complete Theory forRotational Diffusion of Ellipsoids 12.6. AnisotropicRotational Diffusion 12.7. Global Anisotropy Decay Analysis 12.8. Intercalated Fluorophores in DNA 12.9. Transition Moments 12.10. Lifetime-Resolved Anisotropies 12.11. Soleillet'sRule: Multiplication of Depolarized Factors 12.12. Anisotropies Can Depend on Emission Wavelength References Problems 13. Energy Transfer 13.1. Characteristics ofResonance Energy Transfer 13.2. Theory of Energy Transfer for a Donor-Acceptor Pair 13.3. Distance Measurements UsingRET 13.4. Biochemical Applications ofRET 13.5.RET Sensors 13.6.RET and Nucleic Acids 13.7. Energy-Transfer Efficiency from Enhanced Acceptor Fluorescence 13.8. Energy Transfer in Membranes 13.9. Effect of "τ2 onRET 13.10. Energy Transfer in Solution 13.11.RepresentativeR0 Values References AdditionalReferences onResonance Energy Transfer Problems 14. Time-Resolved Energy Transfer and Conformational Distributions of Biopolymers 14.1. Distance Distributions 14.2. Distance Distributions in Peptides 14.3. Distance Distributions in Peptides 14.4. Distance-Distribution Data Analysis 14.5. Biochemical Applications of Distance Distributions 14.6. Time-ResolvedRET Imaging 14.7. Effect of Diffusion for Linked D-A Pairs 14.8. Conclusion References Representative Publications on Measurement of Distance Distributions Problems 15. Energy Transfer to Multiple Acceptors in One, Two, or Three Dimensions 15.1.RET in Three Dimensions 15.2. Effect of Dimensionality onRET 15.3. Biochemical Applications ofRET with Multiple Acceptors 15.4. Energy Transfer inRestricted Geometries 15.5.RET in the Presence of Diffusion 15.6.RET in theRapid Diffusion Limit 15.7. Conclusions References AdditionalReferences onRET between Unlinked Donor and Acceptor Problems 16. Protein Fluorescence 16.1. Spectral Properties of the Aromatic Amino Acids 16.2. General Features of Protein Fluorescence 16.3. Tryptophan Emission in an Apolar Protein Environment 16.4. Energy Transfer and Intrinsic Protein Fluorescence 16.5. Calcium Binding to Calmodulin Using Phenylalanine and Tyrosine Emission 16.6. Quenching of TryptophanResidues in Proteins 16.7. AssociationReaction of Proteins 16.8. Spectral Properties of Genetically Engineered Proteins 16.9. Protein Folding 16.10. Protein Structure and Tryptophan Emission 16.11. Tryptophan Analogues 16.12. The Challenge of Protein Fluorescence References Problems 17. Time-Resolved Protein Fluorescence 17.1. Intensity Decays of Tryptophan:TheRotamer Model 17.2. Time-Resolved Intensity Decays of Tryptophan and Tyrosine 17.3. Intensity and Anisotropy Decays of Proteins 17.4. Protein Unfolding Exposes the TryptophanResidue to Water 17.5. Anisotropy Decays of Proteins 17.6. Biochemical Examples Using Time-Resolved Protein Fluorescence 17.7. Time-Dependent SpectralRelaxation of Tryptophan 17.8. Phosphorescence of Proteins 17.9. Perspectives on Protein Fluorescence References Problems 18. Multiphoton Excitation and Microscopy 18.1. Introduction to Multiphoton Excitation 18.2. Cross-Sections for Multiphoton Absorption 18.3. Two-Photon Absorption Spectra 18.4. Two-Photon Excitation of a DNA-Bound Fluorophore 18.5. Anisotropies with Multiphoton Excitation 18.6. MPE for a Membrane-Bound Fluorophore 18.7. MPE of Intrinsic Protein Fluorescence 18.8. Multiphoton Microscopy References Problems 19. Fluorescence Sensing 19.1. Optical Clinical Chemistry and Spectral Observables 19.2. Spectral Observables for Fluorescence Sensing 19.3. Mechanisms of Sensing 19.4. Sensing by Collisional Quenching 19.5. Energy-Transfer Sensing 19.6. Two-State pH Sensors 19.7. Photoinduced Electron Transfer (PET) Probes for Metal Ions and Anion Sensors 19.8. Probes of AnalyteRecognition 19.9. Glucose-Sensitive Fluorophores 19.10. Protein Sensors 19.11. GFP Sensors 19.12. New Approaches to Sensing 19.13. In-Vivo Imaging 19.14. Immunoassays References Problems 20. Novel Fluorophores 20.1. Semiconductor Nanoparticles 20.2. Lanthanides 20.3. Long-Lifetime Metal-Ligand Complexes 20.4. Long-Wavelength Long-Lifetime Fluorophores References Problems 21. DNATechnology 21.1. DNA Sequencing 21.2. High-Sensitivity DNA Stains 21.3. DNA Hybridization 21.4. Molecular Beacons 21.5. Aptamers 21.6. Multiplexed Microbead Arrays:Suspension Arrays 21.7. Fluorescence In-Situ Hybridization 21.8. Multicolor FISH and Spectral Karyotyping 21.9. DNA Arrays References Problems 22. Fluorescence-Lifetime Imaging Microscopy 22.1. Early Methods for Fluorescence-Lifetime Imaging 22.2. Lifetime Imaging of Calcium Using Quin-2 22.3. Examples of Wide-Field Frequency-Domain FLIM 22.4. Wide-Field FLIM Using a Gated-Image Intensifier 22.5. Laser Scanning TCSPC FLIM 22.6. Frequency-Domain Laser Scanning Microscopy 22.7. Conclusions References AdditionalReading on Fluorescence-Lifetime Imaging Microscopy Problem 23. Single-Molecule Detection 23.1. Detectability of Single Molecules 23.2. Total InternalReflection and Confocal Optics 23.3. Optical Configurations for SMD 23.4. Instrumentation for SMD 23.5. Single-Molecule Photophysics 23.6. Biochemical Applications of SMD 23.7. Single-MoleculeResonance Energy Transfer 23.8. Single-Molecule Orientation andRotational Motions 23.9. Time-Resolved Studies of Single Molecules 23.10. Biochemical Applications 23.11. Advanced Topics in SMD 23.12. Additional Literature on SMD References AdditionalReferences on Single-Molecule Detection Problem 24. Fluorescence Correlation Spectroscopy 24.1. Principles of Fluorescence Correlation Spectroscopy 24.2.Theory of FCS 24.4.Applications of FCS to Bioaffinity Reactions 24.5. FCS in Two Dimensions: Membranes 24.6. Effects of Intersystem Crossing 24.7. Effects of ChemicalReactions 24.8. Fluorescence Intensity Distribution Analysis 24.9. Time-Resolved FCS 24.10. Detection of Conformational Dynamics in Macromolecules 24.11. FCS with Total InternalReflection 24.12. FCS with Two-Photon Excitation 24.13. Dual-Color Fluorescence Cross-Correlation Spectroscopy 24.14.Rotational Diffusion and Photo Antibunching 24.15. Flow Measurements Using FCS 24.16. AdditionalReferences on FCS References AdditionalReferences to FCS and Its Applications Problems 25.Radiative Decay Engineering:Metal-Enhanced Fluorescence 25.1.Radiative Decay Engineering 25.2.Review of Metal Effects on Fluorescence 25.3. Optical Properties of Metal Colloids 25.4. Theory for Fluorophore-Colloid Interactions 25.5. ExperimentalResults on Metal-Enhanced Fluorescence 25.6. Distance-Dependence of Metal-Enhanced Fluorescence 25.7. Applications of Metal-Enhanced Fluorescence 25.8. Mechanism of MEF 25.9. Perspective onRET References Problem 26.Radiative Decay Engineering:Surface Plasmon-Coupled Emission 26.1. Phenomenon of SPCE 26.2. Surface-PlasmonResonance 26.3. Expected Properties of SPCE 26.4. Experimental Demonstration of SPCE 26.5. Applications of SPCE 26.6. Future Developments in SPCE References Appendix I. Corrected Emission Spectra 1. Emission Spectra Standards from300 to800 nm 2.13-Carboline Derivatives as Fluorescence Standards 3. Corrected Emission Spectra of9,10-Diphenyl-anthracene, Quinine, and Fluorescein 4. Long-Wavelength Standards 5. Ultraviolet Standards 6. Additional Corrected Emission Spectra References Appendix II. Fluorescent Lifetime Standards 1. Nanosecond Lifetime Standards 2. Picosecond Lifetime Standards 3.Representative Frequency-Domain Intensity Decays 4. Time-Domain Lifetime Standards Appendix III. AdditionalReading 1. Time-Resolved Measurements 2. Spectra Properties of Fluorophores 3. Theory of Fluorescence and Photophysics 4.Reviews of Fluorescence Spectroscopy 5. Biochemical Fluorescence 6. Protein Fluorescence 7. Data Analysis and Nonlinear Least Squares 8. Photochemistry 9. Flow Cytometry 10. Phosphorescence 11. Fluorescence Sensing 12. Immunoassays 13. Applications of Fluorescence 14. Multiphoton Excitation 15. Infrared and NIR Fluorescence 16. Lasers 17. Fluorescence Microscopy 18. Metal-Ligand Complexes and Unusual Lumophores 19. Single-Molecule Detection 20. Fluorescence Correlation Spectroscopy 21. Biophotonics 22. Nanoparticles 23. Metallic Particles 24. Books on Fluorescence Answers to Problems Index
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