1 Introduction to Fluorescence
1.1 Phenomena of Fluorescence
1.2 Jablonski Diagram
1.3 Characteristics of Fluorescence Emission
1.3.1 The Stokes Shift
1.3.2 Emission Spectra Are Typically Independent of the Excitation Wavelength
1.3.3 Exceptions to the Mirror-Image Rule
1.4 Fluorescence Lifetimes and Quantum Yields
1.4.1 Fluorescence Quenching
1.4.2 Timescale of Molecular Processes in Solution
1.5 Fluorescence Anisotropy
1.6 Resonance Energy Transfer
1.7 Steady-State and Time-Resolved Fluorescence
1.7.1 Why Time-Resolved Measurements?
1.8 Biochemical Fluorophores
1.8.1 Fluorescent Indicators
1.9 Molecular Information from Fluorescence
1.9.1 Emission Spectra and the Stokes Shift
1.9.2 Quenching of Fluorescence
1.9.3 Fluorescence Polarization or Anisotropy
1.9.4 Resonance Energy Transfer
1.10 Biochemical Examples of Basic Phenomena
1.11 New Fluorescence Technologies
1.11.1 Multiphoton Excitation
1.11.2 Fluorescence Correlation Spectroscopy
1.11.3 Single-Molecule Detection
1.12 Overview of Fluorescence Spectroscopy
References
Problems
2 Instrumentation for Fluorescence Spectroscopy
2.1 Spectrofluorometers
2.1.1 Spectrofluorometers for Spectroscopy Research
2.1.2 Spectrofluorometers for High Throughput
2.1.3 An Ideal Spectrofluorometer
2.1.4 Distortions in Excitation and Emission Spectra
2.2 Light Sources
2.2.1 Arc Lamps and Incandescent Xenon Lamps
2.2.2 Pulsed Xenon Lamps
2.2.3 High-Pressure Mercury (Hg) Lamps
2.2.4 Xe-Hg Arc Lamps
2.2.5 Quartz-Tungsten Halogen (QTH) Lamps
2.2.6 Low-Pressure Hg and Hg-Ar Lamps
2.2.7 LED Light Sources
2.2.8 Laser Diodes
2.3 Monochromators
2.3.1 Wavelength Resolution and Emission Spectra
2.3.2 Polarization Characteristics of Monochromators
2.3.3 Stray Light in Monochromators
2.3.4 Second-Order Transmission in Monochromators
2.3.5 Calibration of Monochromators
2.4 Optical Filters
2.4.1 Colored Filters
2.4.2 Thin-Film Filters
2.4.3 Filter Combinations
2.4.4 Neutral-Density Filters
2.4.5 Filters for Fluorescence Microscopy
2.5 Optical Filters and Signal Purity
2.5.1 Emission Spectra Taken through Filters
2.6 Photomultiplier Tubes
2.6.1 Spectral Response of PMTs
2.6.2 PMT Designs and Dynode Chains
2.6.3 Time Response of Photomultiplier Tubes
2.6.4 Photon Counting versus Analog Detection of Huorescence
2.6.5 Symptoms of PMT Failure
2.6.6 CCD Detectors
2.7 Polarizers
2.8 Corrected Excitation Spectra
2.8.1 Corrected Excitation Spectra Using a Quantum Counter
2.9 Corrected Emission Spectra
2.9.1 Comparison with Known Emission Spectra
2.9.2 Corrections Using a Standard Lamp
2.9.3 Correction Factors Using a Quantum Counter and Scatterer
2.9.4 Conversion between Wavelength and Wavenumber
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
2.13.1 Deviations from Beers Law
2.14 Conclusions
References
Problems
3 Fluorophores
3.1 Intrinsic or Natural Fhiorophores
3.1.1 Fluorescence Enzyme Cofactors
3.1.2 Binding of NADH to a Protein
3.2 Exlrinsie Fluorophores
3.2.1 Protein-Labeling Reagents
3.2.2 Role of the Stokes Shift in Protein Labeling
3.2.3 Photostability of Fluorophores
3.2.4 Non-Covalent protein-Labeling Probes
3.2.5 Membrane probes
3.2.6 Membrane Potential Probes
3.3 Red and Near-Infrared (NIR) Dyes
3.4 DNA Probes
3.4.1 DNA Base Analogues
3.5 Chemical Sensing Probes
3.6 Special Probes
3.6.1 Fluorogenie Probes
3.6.2 Structural Analogues of Biomolecules
3.6.3 Viscosity Probes
3.7 Green Fluorescent Proteins
3.8 Other Fluorescent Proteins
3.8.1 Phytofluors: A New Class of Fluorescent Probes
3.8.2 Phycobiliproteins
3.8.3 Specific Labeling of Intraeelhilar Proteins
3.9 Long-Lifetime probes
3.9.1 Lanthanides
3.9.2 Transition Metal-Ligand Complexes
3.10 Proteins as Sensors
3.11 Conclusion
References
problems
4 Time-Domain Lifetime Measurements
4.1 Overview of Time-Domain and Frequency-
Domain Measurements
4.1.1 Meaning of the Lifetime or Decay Time
4.1.2 Phase and Modulation Lifetimes
4.1.3 Examples of Time-Domain and Frequency-Domain Lifetimes
4.2 Biopolymers Display Multi-Exponential or Heterogeneous Decays
4.2.1 Resolution of Multi-Exponential Decays Is Difficult
4.3 Time-Correlated Single-Photon Counting
4.3.1 Principles of TCSPC
4.3.2 Example of TCSPC Data
4.3.3 Convolution Integral
4.4 Light Sources for TCSPC
4.4.l Laser Diodes and Light-Emitting Diodes
4.4.2 Femtosecond Titanium Sapphire Lasers
4.4.3 Picosecond Dye Lasers
4.4.4 Flashlamps
4.4.5 Synchrotron Radiation
4.5 Electronics for TCSPC
4.5.1 Constant Fraction Discriminators
4.5.2 Amplifiers
4.5.3 Time-to-Amplitude Converter (TAC) and Analyte-to-Digital Converter (ADC)
4.5.4 Multichannel Analyzer
4.5.5 Delay Lines
4.5.6 Pulse Pile-Up
4.6 Detectors for TCSPC
4.6.1 Microchannel Plate PMTs
4.6.2 Dynode Chain PMTs
4.6.3 Compact PMTs
4.6.4 Photodiodes as Detectors
4.6.5 Color Effects in Detectors
4.6.6 Timing Effects of Monochromators
4.7 Multi-Detector and Multidimensional TCSPC
4.7.1 Multidimensional TCSPC and DNA Sequencing
4.7.2 Dead Times Repetition Rates and Photon Counting Rates
4.8 Alternative Methods for Time-Resolved
Measurements
4.8.1 Transient Recording
4.8.2 Streak Cameras
4.8.3 Upconversion Methods
4.8.4 Microsecond Luminescence Decays
4.9 Data Analysis: Nonlinear Least Squares
4.9.1 Assumptions of Nonlinear Least Squares
4.9.2 Overview of Least-Squares Analysis
4.9.3 Meaning of the Goodness-of-Fit
4.9.4 Autocorrelation Function
4.10 Analysis of Multi-Exponential Decays
4.10.1 p-Terphenyl and Indole: Two Widely
Spaced Lifetimes
4.10.2 Comparison of XR2 Values: F Statistic
4.10.3 Parameter Uncertainty: Confidence
Intervals
4.10.4 Effect of the Number of Photon Counts
4.10.5 Anthranilic Acid and 2-Aminopurine:
Two Closely Spaced Lifetimes
……
5 Frequency-Domain Lifetime Measurements
6 Solvent and Environmental Effects
7 Dynamics of Solvent and Spectral Relaxation
8 Quenching of Fluourescence
9 Mechanisms and Dynamics of Fluorescence Quenching
10 Fluorescence Anisotropy
11 Time-Dependent Anisotropy Decays
12 Advanced Anisotropy Concepts
13 Energy Transfer
14 Time-Resolved Energy Transfer and Conformational Distributions of Biopolymers
15 Energy Transfer to Multiple Acceptors in One, Two, or Three Dimensions
16 Protein Fluorescence
17 Time-Resolved Protein Fluorescence
18 Multiphoton Excitation and microsocopy
19 Fluorescence Sensing
20 Novel Fluorophores
21 DNA Technology
22 Fluorescence-Lifetime Imaging Microscopy
23 Single-Molecule Detection
24 Fluorescence Correlation Spectroscopy
25 Radiative Decay Engineering: Metal-Enhanced Fluorescence
26 Radiative Decay Engineering: Surface Plasmon-Coupled Emission
Appendix I. Corrected Emission Spectra
Appendix II. Fluorescent Lifetime Standards
Appendix III. Additional Reading
Answers to Problems
Index