Principles of Fluorescence Spectroscopy

Transcription

1 Principles of Fluorescence Spectroscopy Third Edition Joseph R. Lakowicz

2 i: , 1999, 1983 SDrinaer Science+Business Media. New York, 1

3 I. Introduction to Fluorescence 2.2. Light Sources Arc Lamps and Incandescent 1.1. Phenomena of Fluorescence... 1 Xenon Lamps Jablonski Diagram Pulsed Xenon Lamps Characteristics of Fluorescence Emission High-Pressure Mercury (Hg) Lamps The Stokes Shift Xe-Hg Arc Lamps Emission Spectra Are Typically Independent Quartz-Tungsten Halogen (QTH) Lamps of the Excitation Wavelength Low-Pressure Hg and Hg-Ar Lamps Exceptions to the Mirror-Image Rule LED Light Sources Fluorescence Lifetimes and Quantum Yields Laser Diodes Fluorescence Quenching Monochromators Timescale of Molecular Processes Wavelength Resolution and Emission in Solution...12 Spectra 1.5. Fluorescence Anisotropy Polarization Characteristics of Steady-State and Time-Resolved Fluorescence Stray Light in Monochromators Why Time-Resolved Measurements? Second-Order Transmission in 1.8. Biochemical Fluorophores...15 Monochromators Fluorescent Indicators Calibration of Monochromators Molecular Information from Fluorescence Optical Filters Emission Spectra and the Stokes Shift Colored Filters Quenching of Fluorescence Thin-Film Filters Fluorescence Polarization or Anisotropy Filter Combinations Resonance Energy Transfer Neutral-Density Filters Biochemical Examples of Basic Phenomena Filters for Fluorescence Microscopy New Fluorescence Technologies Optical Filters and Signal Purity Resonance Energy Transfer...13 Monochromators Multiphoton Excitation Emission Spectra Taken through Filters Fluorescence Correlation Spectroscopy Photomultiplier Tubes Single-Molecule Detection Spectral Response of PMTs Overview of Fluorescence Spectroscopy PMT Designs and Dynode Chains...46 References Time Response of Photomultiplier Tubes...47 Problems Photon Counting versus Analog Detection of Fluorescence Symptoms of PMT Failure Instrumentation for Fluorescence CCD Detectors Spectroscopy 2.7. Polarizers Corrected Excitation Spectra Spectrofluorometers Corrected Excitation Spectra Using Spectrofluorometers for Spectroscopy a Quantum Counter...51 Research Corrected Emission Spectra Spectrofluorometers for High Throughput Comparison with Known Emission An Ideal Spectrofluorometer...30 Spectra Distortions in Excitation and Emission Corrections Using a Standard Lamp...53 Spectra Correction Factors Using a Quantum Counter and Scatterer...53

4 Conversion between Wavelength and Examples of Time-Domain and Wavenumber...53 Frequency-Domain Lifetimes Quantum Yield Standards Biopolymers Display Multi-Exponential or Effects of Sample Geometry Heterogeneous Decays Common Errors in Sample Preparation Resolution of Multi-Exponential Absorption of Light and Deviation from the Decays Is Difficult Beer-Lambert Law Time-Correlated Single-Photon Counting Deviations from Beer's Law Principles of TCSPC Conclusions Example of TCSPC Data References Convolution Integral Problems Light Sources for TCSPC Laser Diodes and Light-Emitting Diodes Femtosecond Titanium Sapphire Lasers Fluorophores Picosecond Dye Lasers Flashlamps Intrinsic or Natural Fluorophores Synchrotron Radiation Fluorescence Enzyme Cofactors Electronics for TCSPC Binding of NADH to a Protein Constant Fraction Discriminators Extrinsic Fluorophores Amplifiers Protein-Labeling Reagents Time-to-Amplitude Converter (TAC) Role of the Stokes Shift in Protein and Analyte-to-Digital Converter (ADC) Labeling Multichannel Analyzer Photostability of Fluorophores Delay Lines Non-Covalent Protein-Labeling Pulse Pile-Up Probes Detectors for TCSPC Membrane Probes Microchannel Plate PMTS Membrane Potential Probes Dynode Chain PMTS Red and Near-Infrared (NIR) Dyes Compact PMTs DNA Probes Photodiodes as Detectors DNA Base Analogues Color Effects in Detectors Chemical Sensing Probes Timing Effects of Monochromators Special Probes Multi-Detector and Multidimensional TCSPC Fluorogenic Probes Multidimensional TCSPC and Structural Analogues of Biomolecules...80 DNA Sequencing Viscosity Probes Dead Times, Repetition Rates, and 3.7. Green Fluorescent Proteins Photon Counting Rates Other Fluorescent Proteins Alternative Methods for Time-Resolved Phytofluors: A New Class of Measurements Fluorescent Probes Transient Recording Phycobiliproteins Streak Cameras Specific Labeling of Intracellular Upconversion Methods Proteins Long-Lifetime Probes... Microsecond Luminescence Decays Data Analysis: Nonlinear Least Squares Lanthanides Assumptions of Nonlinear Least Squares Transition Metal-Ligand Complexes Proteins as Sensors... Overview of Least-Squares Analysis Meaning of the Goodness-of-Fit Conclusion Autocorrelation Function References Problems... Analysis of Multi-Exponential Decays p-terphenyl and Indole: Two Widely Spaced Lifetimes Time-Domain Lifetime Measurements Comparison of xr 2 Values: F Statistic Parameter Uncertainty: Confidence 4.1. Overview of Time-Domain and Frequency- Intervals Domain Measurements Effect of the Number of Photon Counts Meaning of the Lifetime or Decay Time Anthranilic Acid and 2-Aminopurine: Phase and Modulation Lifetimes...99 Two Closely Spaced Lifetimes...137

5 Global Analysis: Multi-Wavelength 5.5. Simple Frequency-Domain Instruments Measurements Laser Diode Excitation Resolution of Three Closely Spaced LED Excitation Lifetimes Gigahertz Frequency-Domain Fluorometry Intensity Decay Laws Gigahertz FD Measurements Multi-Exponential Decays Biochemical Examples of Gigahertz Lifetime Distributions FD Data Stretched Exponentials Analysis of Frequency-Domain Data Transient Effects Resolution of Two Widely Spaced Global Analysis Lifetimes Applications of TCSPC Resolution of Two Closely Spaced Intensity Decay for a Single Tryptophan Lifetimes Protein...I Global Analysis of a Two-Component Green Fluorescent Protein: Systematic Mixture Errors in the Data Analysis of a Three-Component Mixture: Picosecond Decay Time Limits of Resolution... I Chlorophyll Aggregates in Hexane Resolution of a Three-Component Intensity Decay of Flavin Adenine Mixture with a Tenfold Range of Dinucleotide (FAD) Decay Times Data Analysis: Maximum Entropy Method Maximum Entropy Analysis of FD Data References Biochemical Examples of Frequency-Domain Problems Intensity Decays DNA Labeled with DAPI Mag-Quin-2: A Lifetime-Based Sensor 5. Frequency-Domain Lifetime for Magnesium Measurements Recovery of Lifetime Distributions from Frequency-Domain Data Theory of Frequency-Domain Fluorometry Cross-Fitting of Models: Lifetime Least-Squares Analysis of Frequency- Distributions of Melittin Domain Intensity Decays Frequency-Domain Fluorescence Global Analysis of Frequency-Domain Microscopy with an LED Light Source Data Phase-Angle and Modulation Spectra Frequency-Domain Instrumentation Apparent Phase and Modulation Lifetimes History of Phase-Modulation Derivation of the Equations for Phase- Fluorometers Modulation Fluorescence An MHz Frequency-Domain Fluorometer Relationship of the Lifetime to the Light Modulators...I65 Phase Angle and Modulation Cross-Correlation Detection... I Cross-Correlation Detection Frequency Synthesizers Phase-Sensitive Emission Spectra Radio Frequency Amplifiers Theory of Phase-Sensitive Detection Photomultiplier Tubes of Fluorescence Frequency-Domain Measurements Examples of PSDF and Phase 5.3. Color Effects and Background Fluorescence Suppression Color Effects in Frequency-Domain High-Frequency or Low-Frequency Measurements Phase-Sensitive Detection Background Correction in Frequency Phase-Modulation Resolution of Emission Domain Measurements Spectra I Representative Frequency-Domain Intensity Resolution Based on Phase or Modulation Decays Lifetimes Exponential Decays Resolution Based on Phase Angles Multi-Exponential Decays of and Modulations Staphylococcal Nuclease and Melittin Resolution of Emission Spectra from Green Fluorescent Protein: One- and Phase and Modulation Spectra Two-Photon Excitation References SPQ: Collisional Quenching of a Problems Chloride Sensor Intensity Decay of NADH Effect of Scattered Light...172

6 6. Solvent and Environmental Effects 7.5. Picosecond Relaxation in Solvents Overview of Solvent Polarity Effects Theory for Time Dependent Solvent Effects of Solvent Polarity 205 Relaxation Polarity Surrounding a Membrane-Bound Multi-Exponential Relaxation in Water Measurement of Multi-Exponential Spectral Fluorophore Other Mechanisms for Spectral Shifts Relaxation General Solvent Effects: The Lippert-Mataga 7.7. Distinction between Solvent Relaxation Equation and Formation of Rotational Isomers Derivation of the Lippen Equation Comparison of TRES and Decay Associated Application of the Lippert Equation Spectra Specific Solvent Effects Lifetime-Resolved Emission Spectra Specific Solvent Effects and Lippert Plots Red-Edge Excitation Shifts Temperature Effects Membranes and Red Edge 6.5. Phase Transitions in Membranes Excitation Shifts Additional Factors that Affect Emission Spectra Red Edge Excitation Shifts and Locally Excited and Internal Energy Transfer Charge Transfer States Excited-State Reactions Excited State Intramolecular Proton Excited-State Ionization of Naphthol Transfer (ESIPT) Theory for a Reversible Two-State Reaction Changes in the Non-Radiative Steady-State Fluorescence of a Decay Rates Two-State Reaction Changes in the Rate of Radiative Decay Time Resolved Decays for the 6.7. Effects of Viscosity Two-State Model Effect of Shear Stress on Membrane Differential Wavelength Methods Viscosity Time-Domain Studies of Naphthol Dissociation Probe-Probe Interactions Analysis of Excited State Reactions by 6.9. Biochemical Applications of Environment- Phase-Modulation Fluorometry Sensitive Fluorophores Effect of an Excited State Reaction Fatty-Acid-Binding Proteins on the Apparent Phase and Modulation Exposure of a Hydrophobic Surface Lifetimes on Calmodulin Wavelength-Dependent Phase and Binding to Cyclodextrin Using a Modulation Values for an Excited-State Dansyl Probe Reaction Advanced Solvent-Sensitive Probes Frequency-Domain Measurement of Effects of Solvent Mixtures Excimer Formation Summary of Solvent Effects Biochemical Examples of Excited-State References Reactions Problems Exposure of a Membrane Bound Cholesterol Analogue Dynamics of Solvent and Spectral Relaxation References Overview of Excited-State Processes Problems Time-Resolved Emission Spectra Measurement of Time-Resolved Emission Spectra (TRES) Quenching of Fluorescence Direct Recording of TRES TRES from Wavelength-Dependent 8.1. Quenchers of Fluorescence Decays Theory of Collisional Quenching Spectral Relaxation in Proteins Derivation of the Stern-Volmer Equation Spectral Relaxation of Labeled Interpretation of the Bimolecular Apomyoglobin... Quenching Constant Protein Spectral Relaxation around a 8.3. Theory of Static Quenching Synthetic Fluorescent Amino Acid Combined Dynamic and Static Quenching Spectral Relaxation in Membranes Examples of Static and Dynamic Quenching Analysis of Time-Resolved Emission 8.6. Deviations from the Stern-Volmer Equation: Spectra Quenching Sphere of Action Spectral Relaxation of Membrane-Bound Derivation of the Quenching Sphere Anthroyloxy Fatty Acids of Action 285

7 8.7. Effects of Steric Shielding and Charge on Molecular Beacons Based on Quenching Quenching by a Gold Surface Accessibility of DNA-Bound Probes Intramolecular Quenching to Quenchers DNA Dynamics by Intramolecular Quenching of Ethenoadenine Derivatives Quenching Fractional Accessibility to Quenchers Electron-Transfer Quenching in a Modified Stern-Volmer Plots Flavoprotein Experimental Considerations Sensors Based on Intramolecular in Quenching PET Quenching Applications of Quenching to Proteins Quenching of Phosphorescence Fractional Accessibility of Tryptophan References Residues in Endonuclease III Problems Effect of Conformational Changes on Tryptophan Accessibility Quenching of the Multiple Decay 9. Mechanisms and Dynamics of Times of Proteins Fluorescence Quenching Effects of Quenchers on Proteins Correlation of Emission Wavelength 9.1. Comparison of Quenching and Resonance and Accessibility: Protein Folding of Energy Transfer Colicin E Distance Dependence of RET Application of Quenching to Membranes and Quenching Oxygen Diffusion in Membranes Encounter Complexes and Quenching Localization of Membrane-Bound Efficiency Tryptophan Residues by Quenching Mechanisms of Quenching Quenching of Membrane Probes Intersystem Crossing Using Localized Quenchers Electron-Exchange Quenching Parallax and Depth-Dependent Photoinduced Electron Transfer Quenching in Membranes Energetics of Photoinduced Electron Transfer Boundary Lipid Quenching Examples of PET Quenching Effect of Lipid-Water Partitioning PET in Linked Donor-Acceptor Pairs on Quenching PET Quenching in Biomolecules Quenching in Micelles Quenching of Indole by Imidazolium Lateral Diffusion in Membranes Quenching by DNA Bases and Quenching-Resolved Emission Spectra Nucleotides Fluorophore Mixtures Single Molecule PET Quenching-Resolved Emission Spectra 9.6. Transient Effects in Quenching of the E. Coll Tet Repressor Experimental Studies of Transient Quenching and Association Reactions Effects Quenching Due to Specific Binding Distance-Dependent Quenching Interactions in Proteins 348 Sensing Applications of uenching 305 References Chloride-Sensitive Fluorophores Problems Intracellular Chloride Imaging Chloride-Sensitive GFP Fluorescence Anisotropy Amplified Quenching Applications of Quenching to Molecular Definition of Fluorescence Anisotropy Biology Origin of the Definitions of Release of Quenching upon Polarization and Anisotropy Hybridization Theory for Anisotropy Molecular Beacons in Quenching Excitation Photoselection of Fluorophores by Guanine Excitation Anisotropy Spectra Binding of Substrates to Ribozymes Resolution of Electronic States from Association Reactions and Accessibility Polarization Spectra to Quenchers Measurement of Fluorescence Anisotropies Quenching on Gold Surfaces L-Format or Single-Channel Method Molecular Beacons Based on Quenching T-Format or Two-Channel Anisotropies by Gold Colloids Comparison of T-Format and L-Format Measurements...363

8 Alignment of Polarizers Example Anisotropy Decays of Magic-Angle Polarizer Conditions Rhodamine Green and Rhodamine Why is the Total Intensity Green-Dextran Equal to I ii + 21, Time-Domain Anisotropy Decays of Proteins Effect of Resonance Energy Transfer Intrinsic Tryptophan Anisotropy Decay on the Anisotropy of Liver Alcohol Dehydrogenase Trivial Causes of Depolarization Phospholipase Az Factors Affecting the Anisotropy Subtilisin Carlsberg Effects of Rotational Diffusion on Fluorescence Domain Motions of Immunoglobulins Anisotropies: The Perrin Equation Effects of Free Probe on Anisotropy The Perrin Equation: Rotational Decays Motions of Proteins Frequency-Domain Anisotropy Decays Examples of a Perrin Plot of Proteins Perrin Plots of Proteins Apomyoglobin: A Rigid Rotor Binding of trna to trna Synthetase Melittin Self-Association and Molecular Chaperonin cpn60 (GroEL) Anisotropy Decays Perrin Plots of an F ab Immunoglobulin Picosecond Rotational Diffusion Fragment of Oxytocin Biochemical Applications of Steady-State Hindered Rotational Diffusion in Membranes Anisotropies Characterization of a New Peptide Binding to Calmodulin Membrane Probe Binding of the Trp Repressor to DNA Anisotropy Decays of Nucleic Acids Helicase-Catalyzed DNA Unwinding Hydrodynamics of DNA Oligomers Melittin Association Detected from Dynamics of Intracellular DNA Homotransfer DNA Binding to HIV Integrase Using Anisotropy of Membranes and Membrane- Correlation Time Distributions Bound Proteins Correlation Time Imaging Membrane Microviscosity Microsecond Anisotropy Decays Distribution of Membrane-Bound Phosphorescence Anisotropy Decays Proteins Long-Lifetime Metal-Ligand Transition Moments Complexes References References Additional Reading on the Application Problems of Anisotropy Problems Advanced Anisotropy Concepts II. Time-Dependent Anisotropy Decays Associated Anisotropy Decay Time-Domain and Frequency-Domain Theory for Associated Anisotropy Decay 414 Anisotropy Decays Time-Domain Measurements of Anisotropy Decay Analysis Associated Anisotropy Decays Early Methods for Analysis of Biochemical Examples of Associated TD Anisotropy Data Anisotropy Decays Preferred Analysis of TD Time-Domain Studies of DNA Anisotropy Data Binding to the Klenow Fragment Value of r of DNA Polymerase Analysis of Frequency-Domain Frequency-Domain Measurements Anisotropy Decays of Associated Anisotropy Decays Anisotropy Decay Laws Rotational Diffusion of Non-Spherical Non-Spherical Fluorophores Molecules: An Overview Hindered Rotors Anisotropy Decays of Ellipsoids Segmental Mobility of a Biopolymer Ellipsoids of Revolution Bound Fluorophore Simplified Ellipsoids of Revolution Correlation Time Distributions Intuitive Description of Rotational Associated Anisotropy Decays Diffusion of an Oblate Ellipsoid...422

9 Rotational Correlation Times for RET Imaging of Intracellular Protein Ellipsoids of Revolution Phosphorylation Stick-versus-Slip Rotational Diffusion Imaging of Rac Activation in Cells Complete Theory for Rotational Diffusion RET and Nucleic Acids of Ellipsoids Imaging of Intracellular RNA Anisotropic Rotational Diffusion Energy-Transfer Efficiency from Time-Domain Studies Enhanced Acceptor Fluorescence Frequency-Domain Studies of Energy Transfer in Membranes Anisotropic Rotational Diffusion Lipid Distributions around Gramicidin Global Anisotropy Decay Analysis Membrane Fusion and Lipid Exchange Global Analysis with Multi-Wavelength Effect of t 2 on RET Excitation Energy Transfer in Solution Global Anisotropy Decay Analysis with Diffusion-Enhanced Energy Transfer Collisional Quenching I. Representative R Values Application of Quenching to Protein References Anisotropy Decays I Additional References on Resonance Intercalated Fluorophores in DNA Energy Transfer Transition Moments Problems Anisotropy of Planar Fluorophores with High Symmetry Lifetime-Resolved Anisotropies Time-Resolved Energy Transfer and Effect of Segmental Motion on the Conformational Distributions of Biopolymers Perrin Plots Soleillet's Rule: Multiplication of Depolarized Distance Distributions Factors Distance Distributions in Peptides Anisotropies Can Depend on Emission Comparison for a Rigid and Flexible Wavelength Hexapeptide References Crossftting Data to Exclude Problems Alternative Models Donor Decay without Acceptor Effect of Concentration of the 13. Energy Transfer D-A Pairs Distance Distributions in Peptides Characteristics of Resonance Energy Transfer Distance Distributions in Melittin Theory of Energy Transfer for a Distance-Distribution Data Analysis Donor-Acceptor Pair Frequency-Domain Distance-Distribution Orientation Factor K Analysis Dependence of the Transfer Rate on Time-Domain Distance-Distribution Distance (r), the Overlap Analysis 487 Integral (J), and t Distance-Distribution Functions Homotransfer and Heterotransfer Effects of Incomplete Labeling Distance Measurements Using RET Effect of the Orientation Factor K Distance Measurements in a-helical Acceptor Decays Melittin Biochemical Applications of Distance Effects of Incomplete Labeling Distributions Effect of K 2 on the Possible Range Calcium-Induced Changes in the of Distances Conformation of Troponin C Biochemical Applications of RET Hairpin Ribozyme Protein Folding Measured by RET Four-Way Holliday Junction in DNA Intracellular Protein Folding Distance Distributions and Unfolding RET and Association Reactions of Yeast Phosphoglycerate Kinase Orientation of a Protein-Bound Peptide Distance Distributions in a Glvcopeptide Protein Binding to Semiconductor Single-Protein-Molecule Distance Nanoparticles Distribution RET Sensors Time-Resolved RET Imaging Intracellular RET Indicator Effect of Diffusion for Linked D-A Pairs for Estrogens...458

10 Simulations of FRET for a Flexible Tryptophan Emission in an Apolar D-A Pair Protein Environment Experimental Measurement of D-A Site-Directed Mutagenesis of a Diffusion for a Linked D-A Pair Single-Tryptophan Azurin FRET and Diffusive Motions in Emission Spectra of Azurins with Biopolymers One or Two Tryptophan Residues Conclusion Energy Transfer and Intrinsic Protein References Fluorescence Representative Publications on Measurement Tyrosine-to-Tryptophan Energy Transfer of Distance Distributions in Interferon-y Problems Quantitation of RET Efficiencies in Proteins I5. Energy Transfer to Multiple Acceptors in Tyrosine-to-Tryptophan RET in a Membrane-Bound Protein One, Two, or Three Dimensions Phenylalanine-to-Tyrosine Energy Transfer RET in Three Dimensions Calcium Binding to Calmodulin Using Effect of Diffusion on FRET with Phenylalanine and Tyrosine Emission Unlinked Donors and Acceptors Quenching of Tryptophan Residues in Proteins Experimental Studies of RET in Effect of Emission Maximum on Three Dimensions Quenching Effect of Dimensionality on RET Fractional Accessibility to Quenching Experimental FRET in Two Dimensions in Multi-Tryptophan Proteins Experimental FRET in One Dimension Resolution of Emission Spectra by Biochemical Applications of RET with Quenching Multiple Acceptors Association Reaction of Proteins Aggregation of 3-Amyloid Peptides Binding of Calmodulin to a RET Imaging of Fibronectin Target Protein Energy Transfer in Restricted Geometries Calmodulin: Resolution of the Effect of Excluded Area on Energy Four Calcium Binding Sites Using Transfer in Two Dimensions Tryptophan-Containing Mutants RET in the Presence of Diffusion Interactions of DNA with Proteins RET in the Rapid Diffusion Limit Spectral Properties of Genetically Engineered Location of an Acceptor in Proteins Lipid Vesicles Single-Tryptophan Mutants of Locaion of Retinal in Rhodopsin Triosephosphate Isomerase Disc Membranes Barnase: A Three-Tryptophan Protein Conclusions Site-Directed Mutagenesis of References Tyrosine Proteins Additional References on RET between Protein Folding 557 Unlinked Donor and Acceptor Problems... Protein Engineering of Mutant 527 Ribonuclease for Folding Experiments Folding of Lactate Dehydrogenase Protein Fluorescence Folding Pathway of CRABPI Protein Structure and Tryptophan Emission Spectral Properties of the Aromatic Amino Acids Tryptophan Spectral Properties Excitation Polarization Spectra of and Structural Motifs Tyrosine and Tryptophan Tryptophan Analogues Solvent Effects on Tryptophan Emission Tryptophan Analogues Spectra Genetically Inserted Amino-Acid Excited-State Ionization of Tyrosine Analogues Tyrosinate Emission from Proteins The Challenge of Protein Fluorescence General Features of Protein Fluorescence References Problems

11 17. Time-Resolved Protein Fluorescence Excitation Photoselection for Two-Photon Excitation Intensity Decays of Tryptophan: Two-Photon Anisotropy of DPH The Rotamer Model MPE for a Membrane-Bound Fluorophore Time-Resolved Intensity Decays of MPE of Intrinsic Protein Fluorescence Tryptophan and Tyrosine Multiphoton Microscopy Decay-Associated Emission Spectra Calcium Imaging of Tryptophan Imaging of NAD(P)H and FAD Intensity Decays of Neutral Tryptophan Excitation of Multiple Fluorophores Derivatives Three-Dimensional Imaging of Cells Intensity Decays of Tyrosine and References Its Neutral Derivatives Problems Intensity and Anisotropy Decays of Proteins Single-Exponential Intensity and Anisotropy Decay of Ribonuclease T i Fluorescence Sensing Annexin V: A Calcium-Sensitive Single-Tryptophan Protein Optical Clinical Chemistry and Spectral Anisotropy Decay of a Protein with Observables Two Tryptophans Spectral Observables for Fluorescence Sensing Protein Unfolding Exposes the Tryptophan Optical Properties of Tissues Residue to Water Lifetime-Based Sensing Conformational Heterogeneity Can Mechanisms of Sensing Result in Complex Intensity and Sensing by Collisional Quenching Anisotropy Decays Oxygen Sensing Anisotropy Decays of Proteins Lifetime-Based Sensing of Oxygen Effects of Association Reactions on Mechanism of Oxygen Selectivity Anisotropy Decays: Melittin Other Oxygen Sensors Biochemical Examples Using Time-Resolved Lifetime Imaging of Oxygen Protein Fluorescence Chloride Sensors Decay-Associated Spectra of Barnase Lifetime Imaging of Chloride Disulfide Oxidoreductase DsbA Concentrations Immunophilin FKBP59-I: Quenching Other Collisional Quenchers of Tryptophan Fluorescence by Energy-Transfer Sensing Phenylalanine ph and pco, Sensing by Trp Repressor: Resolution of the Two Energy Transfer Interacting Tryptophans Glucose Sensing by Energy Transfer Thermophilic G3-Glycosidase: Ion Sensing by Energy Transfer A Multi-Tryptophan Protein Theory for Energy-Transfer Sensing Herne Proteins Display Useful Two-State ph Sensors Intrinsic Fluorescence Optical Detection of Blood Gases Time-Dependent Spectral Relaxation of ph Sensors Tryptophan Photoinduced Electron Transfer (PET) Probes Phosphorescence of Proteins for Metal Ions and Anion Sensors Perspectives on Protein Fluorescence Probes of Analyte Recognition References Specificity of Cation Probes Problems Theory of Analyte Recognition Sensing Sodium and Potassium Probes Calcium and Magnesium Probes Multiphoton Excitation and Microscopy Probes for Intracellular Zinc Glucose-Sensitive Fluorophores Introduction to Multiphoton Excitation Protein Sensors Cross-Sections for Multiphoton Absorption Protein Sensors Based on RET Two-Photon Absorption Spectra GFP Sensors Two-Photon Excitation of a DNA-Bound GFP Sensors Using RET Fluorophore Intrinsic GFP Sensors Anisotropies with Multiphoton Excitation...612

12 New Approaches to Sensing DNA Fragment Sizing by Pebble Sensors and Lipobeads Flow Cytometry In-Vivo Imaging DNA Hybridization Immunoassays DNA Hybridization Measured with Enzyme-Linked Immunosorbent Assays One Donor- and Acceptor Labeled (ELISA) 659 DNA Probe Time-Resolved Immunoassays DNA Hybridization Measured by Energy-Transfer Immunoassays Excimer Formation Fluorescence Polarization Polarization Hybridization Arrays Immunoassays Polymerase Chain Reaction References Molecular Beacons Problems Molecular Beacons with Nonfluorescent Acceptors Molecular Beacons with 20. Novel Fluorophores Fluorescent Acceptors Hybridization Proximity Beacons Semiconductor Nanoparticles Molecular Beacons Based on Spectral Properties of QDots Quenching by Gold Labeling Cells with QDots Intracellular Detection of mrna QDots and Resonance Energy Transfer Using Molecular Beacons Lanthanides Aptamers RET with Lanthanides DNAzymes Lanthanide Sensors Multiplexed Microbead Arrays: Lanthanide Nanoparticles Suspension Arrays Near-Infrared Emitting Lanthanides Fluorescence In-Situ Hybridization Lanthanides and Fingerprint Detection Preparation of FISH Probe DNA Long-Lifetime Metal-Ligand Complexes Applications of FISH Introduction to Metal-Ligand Probes Multicolor FISH and Spectral Karyotyping Anisotropy Properties of DNA Arrays Metal Ligand Complexes Spotted DNA Microarrays Spectral Properties of MLC Probes Light-Generated DNA Arrays The Energy Gap Law References Biophysical Applications of Problems 740 Metal-Ligand Probes MLC Immunoassays Metal-Ligand Complex Sensors Fluorescence-Lifetime Imaging Microscopy Long-Wavelength Long-Lifetime Fluorophores Early Methods for Fluorescence-Lifetime References Imaging Problems FLIM Using Known Fluorophores Lifetime Imaging of Calcium Using Quin Determination of Calcium Concentration 21. DNA Technology from Lifetime DNA Sequencing Lifetime Images of Cos Cells Principle of DNA Sequencing Examples of Wide-Field Frequency-Domain Examples of DNA Sequencing FLIM Nucleotide Labeling Methods Resonance Energy-Transfer FLIM Example of DNA Sequencing of Protein Kinase C Activation Energy-Transfer Dyes for DNA Lifetime Imaging of Cells Containing Sequencing Two GFPs DNA Sequencing with NIR Probes Wide-Field FLIM Using a Gated-Image DNA Sequencing Based on Lifetimes Intensifier High-Sensitivity DNA Stains Laser Scanning TCSPC FLIM High-Affinity Bis DNA Stains Lifetime Imaging of Cellular Energy-Transfer DNA Stains Biomolecules Lifetime Images of Amyloid Plaques...750

13 22.6. Frequency-Domain Laser Scanning Microscopy Theory of FCS Conclusions Translational Diffusion and FCS References Occupation Numbers and Volumes Additional Reading on Fluorescence-Lifetime in FCS 804 Imaging Microscopy FCS for Multiple Diffusing Species Problem Examples of FCS Experiments Effect of Fluorophore Concentration Single-Molecule Detection Effect of Molecular Weight on Diffusion Coefficients Applications of FCS to Bioaffinity Reactions Detectability of Single Molecules Protein Binding to the Total Internal Reflection and Confocal Optics Chaperonin GroEL Total Internal Reflection Association of Tubulin Subunits Confocal Detection Optics DNA Applications of FCS Optical Configurations for SMD FCS in Two Dimensions: Membranes Instrumentation for SMD Biophysical Studies of Lateral Detectors for Single-Molecule Detection Diffusion in Membranes Optical Filters for SMD Binding to Membrane-Bound Single-Molecule Photophysics Receptors Biochemical Applications of SMD Effects of Intersystem Crossing Single-Molecule Enzyme Kinetics Theory for FCS and Intersystem Single-Molecule ATPase Activity Crossing Single-Molecule Studies of a Effects of Chemical Reactions Chaperonin Protein Fluorescence Intensity Distribution Analysis Single-Molecule Resonance Energy Transfer Time-Resolved FCS Single-Molecule Orientation and Rotational Detection of Conformational Dynamics Motions in Macromolecules Orientation Imaging of R6G and GFP FCS with Total Internal Reflection Imaging of Dipole Radiation Patterns FCS with Two-Photon Excitation Time-Resolved Studies of Single Molecules Diffusion of an Intracellular Biochemical Applications Kinase Using FCS with Turnover of Single Enzyme Molecules Two-Photon Excitation Single Molecule Molecular Beacons Dual-Color Fluorescence Cross-Correlation Conformational Dynamics of a Spectroscopy Holliday Junction Instrumentation for Dual-Color Single-Molecule Calcium Sensor FCCS Motions of Molecular Motors Theory of Dual-Color FCCS Advanced Topics in SMD DNA Cleavage by a Signal-to-Noise Ratio in Restriction Enzyme Single-Molecule Detection Applications of Dual-Color FCCS Polarization of Single Immobilized Rotational Diffusion and Photo Antibunching Fluorophores Flow Measurements Using FCS Polarization Measurements Additional References on FCS and Mobility of Surface-Bound References Fluorophores Additional References to FCS and Single-Molecule Lifetime Estimation Its Applications Additional Literature on SMD Problems References Additional References on Single-Molecule Detection Radiative Decay Engineering: Problem Metal-Enhanced Fluorescence Radiative Decay Engineering Fluorescence Correlation Spectroscopy Introduction to RDE Jablonski Diagram for Metal Principles of Fluorescence Correlation Enhanced Fluorescence Spectroscopy Review of Metal Effects on Fluorescence...843

14 25.3. Optical Properties of Metal Colloids Appendix II. Fluorescent Lifetime Standards Theory for Fluorophore-Colloid Interactions Experimental Results on Metal-Enhanced 1. Nanosecond Lifetime Standards Fluorescence Picosecond Lifetime Standards Application of MEF to DNA Analysis Representative Frequency Domain Distance-Dependence of Metal-Enhanced Intensity Decays Fluorescence Time-Domain Lifetime Standards Applications of Metal-Enhanced Fluorescence DNA Hybridization Using MEF Release of Self-Quenching Appendix III. Additional Reading Effect of Silver Particles on RET Time-Resolved Measurements Mechanism of MEF Spectra Properties of Fluorophores Perspective on RET Theory of Fluorescence and Photophysics References Reviews of Fluorescence Spectroscopy 889 Problem Biochemical Fluorescence Protein Fluorescence Data Analysis and Nonlinear Least Squares Radiative Decay Engineering: 8. Photochemistry Surface Plasmon-Coupled Emission 9. Flow Cytometry Phosphorescence Phenomenon of SPCE Fluorescence Sensing Surface-Plasmon Resonance Immunoassays Theory for Surface-Plasmon Resonance Applications of Fluorescence Expected Properties of SPCE Multiphoton Excitation Experimental Demonstration of SPCE Infrared and NIR Fluorescence Applications of SPCE Lasers Future Developments in SPCE Fluorescence Microscopy References Metal-Ligand Complexes and Unusual Lumophores Single-Molecule Detection Fluorescence Correlation Spectroscopy Appendix I. Corrected Emission Spectra 21. Biophotonics Nanoparticles Emission Spectra Standards from 300 to 800 nm Metallic Particles ß-Carboline Derivatives as Fluorescence Standards Books on Fluorescence Corrected Emission Spectra of 9,10-Diphenylanthracene, Quinine, and Fluorescein Long-Wavelength Standards Answers to Problems Ultraviolet Standards Additional Corrected Emission Spectra References Index...923