荧光分析法原理

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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