Contents. 1. Introduction to Fluorescence. 2. Instrumentation for Fluorescence Spectroscopy

Transcription

1 Contents 1. Introduction to Fluorescence 1.1. Phenomena of Fluorescence Jablonski Diagram Characteristics of Fluorescence Emission The Stokes Shift Emission Spectra Are Typically Independent of the Excitation Wavelength Exceptions to the Mirror-Image Rule Fluorescence Lifetimes and Quantum Yields Fluorescence Quenching Timescale of Molecular Processes in Solution Fluorescence Anisotropy Resonance Energy Transfer Steady-State and Time-Resolved Fluorescence Why Time-Resolved Measurements? Biochemical Fluorophores Fluorescent Indicators Molecular Information from Fluorescence Emission Spectra and the Stokes Shift Quenching of Fluorescence Fluorescence Polarization or Anisotropy Resonance Energy Transfer Biochemical Examples of Basic Phenomena New Fluorescence Technologies Multiphoton Excitation Fluorescence Correlation Spectroscopy Single-Molecule Detection Overview of Fluorescence Spectroscopy References Problems Instrumentation for Fluorescence Spectroscopy 2.1. Spectrofluorometers Spectrofluorometers for Spectroscopy Research Spectrofluorometers for High Throughput An Ideal Spectrofluorometer Distortions in Excitation and Emission Spectra Light Sources Arc Lamps and Incandescent Xenon Lamps Pulsed Xenon Lamps High-Pressure Mercury (Hg) Lamps Xe Hg Arc Lamps Quartz Tungsten Halogen (QTH) Lamps Low-Pressure Hg and Hg Ar Lamps LED Light Sources Laser Diodes Monochromators Wavelength Resolution and Emission Spectra Polarization Characteristics of Monochromators Stray Light in Monochromators Second-Order Transmission in Monochromators Calibration of Monochromators Optical Filters Colored Filters Thin-Film Filters Filter Combinations Neutral-Density Filters Filters for Fluorescence Microscopy Optical Filters and Signal Purity Emission Spectra Taken through Filters Photomultiplier Tubes Spectral Response of PMTs PMT Designs and Dynode Chains Time Response of Photomultiplier Tubes Photon Counting versus Analog Detection of Fluorescence Symptoms of PMT Failure CCD Detectors Polarizers Corrected Excitation Spectra Corrected Excitation Spectra Using a Quantum Counter Corrected Emission Spectra Comparison with Known Emission Spectra Corrections Using a Standard Lamp Correction Factors Using a Quantum Counter and Scatterer xv

2 xvi Conversion between Wavelength and Wavenumber Quantum Yield Standards Effects of Sample Geometry Common Errors in Sample Preparation Absorption of Light and Deviation from the Beer-Lambert Law Deviations from Beer's Law Conclusions References Problems Fluorophores 3.1. Intrinsic or Natural Fluorophores Fluorescence Enzyme Cofactors Binding of NADH to a Protein Extrinsic Fluorophores Protein-Labeling Reagents Role of the Stokes Shift in Protein Labeling Photostability of Fluorophores Non-Covalent Protein-Labeling Probes Membrane Probes Membrane Potential Probes Red and Near-Infrared (NIR) Dyes DNA Probes DNA Base Analogues Chemical Sensing Probes Special Probes Fluorogenic Probes Structural Analogues of Biomolecules Viscosity Probes Green Fluorescent Proteins Other Fluorescent Proteins Phytofluors: A New Class of Fluorescent Probes Phycobiliproteins Specific Labeling of Intracellular Proteins Long-Lifetime Probes Lanthanides Transition Metal Ligand Complexes Proteins as Sensors Conclusion References Problems Time-Domain Lifetime Measurements 4.1. Overview of Time-Domain and Frequency- Domain Measurements Meaning of the Lifetime or Decay Time Phase and Modulation Lifetimes CONTENTS Examples of Time-Domain and Frequency-Domain Lifetimes Biopolymers Display Multi-Exponential or Heterogeneous Decays Resolution of Multi-Exponential Decays Is Difficult Time-Correlated Single-Photon Counting Principles of TCSPC Example of TCSPC Data Convolution Integral Light Sources for TCSPC Laser Diodes and Light-Emitting Diodes Femtosecond Titanium Sapphire Lasers Picosecond Dye Lasers Flashlamps Synchrotron Radiation Electronics for TCSPC Constant Fraction Discriminators Amplifiers Time-to-Amplitude Converter (TAC) and Analyte-to-Digital Converter (ADC) Multichannel Analyzer Delay Lines Pulse Pile-Up Detectors for TCSPC Microchannel Plate PMTs Dynode Chain PMTs Compact PMTs Photodiodes as Detectors Color Effects in Detectors Timing Effects of Monochromators Multi-Detector and Multidimensional TCSPC Multidimensional TCSPC and DNA Sequencing Dead Times, Repetition Rates, and Photon Counting Rates Alternative Methods for Time-Resolved Measurements Transient Recording Streak Cameras Upconversion Methods Microsecond Luminescence Decays Data Analysis: Nonlinear Least Squares Assumptions of Nonlinear Least Squares Overview of Least-Squares Analysis Meaning of the Goodness-of-Fit Autocorrelation Function Analysis of Multi-Exponential Decays p-terphenyl and Indole: Two Widely Spaced Lifetimes Comparison of χ 2 R Values: F Statistic Parameter Uncertainty: Confidence Intervals Effect of the Number of Photon Counts Anthranilic Acid and 2-Aminopurine: Two Closely Spaced Lifetimes

3 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY Global Analysis: Multi-Wavelength Measurements Resolution of Three Closely Spaced Lifetimes Intensity Decay Laws Multi-Exponential Decays Lifetime Distributions Stretched Exponentials Transient Effects Global Analysis Applications of TCSPC Intensity Decay for a Single Tryptophan Protein Green Fluorescent Protein: Systematic Errors in the Data Picosecond Decay Time Chlorophyll Aggregates in Hexane Intensity Decay of Flavin Adenine Dinucleotide (FAD) Data Analysis: Maximum Entropy Method References Problems Frequency-Domain Lifetime Measurements 5.1. Theory of Frequency-Domain Fluorometry Least-Squares Analysis of Frequency- Domain Intensity Decays Global Analysis of Frequency-Domain Data Frequency-Domain Instrumentation History of Phase-Modulation Fluorometers An MHz Frequency-Domain Fluorometer Light Modulators Cross-Correlation Detection Frequency Synthesizers Radio Frequency Amplifiers Photomultiplier Tubes Frequency-Domain Measurements Color Effects and Background Fluorescence Color Effects in Frequency-Domain Measurements Background Correction in Frequency- Domain Measurements Representative Frequency-Domain Intensity Decays Exponential Decays Multi-Exponential Decays of Staphylococcal Nuclease and Melittin Green Fluorescent Protein: One- and Two-Photon Excitation SPQ: Collisional Quenching of a Chloride Sensor Intensity Decay of NADH Effect of Scattered Light xvii 5.5. Simple Frequency-Domain Instruments Laser Diode Excitation LED Excitation Gigahertz Frequency-Domain Fluorometry Gigahertz FD Measurements Biochemical Examples of Gigahertz FD Data Analysis of Frequency-Domain Data Resolution of Two Widely Spaced Lifetimes Resolution of Two Closely Spaced Lifetimes Global Analysis of a Two-Component Mixture Analysis of a Three-Component Mixture: Limits of Resolution Resolution of a Three-Component Mixture with a Tenfold Range of Decay Times Maximum Entropy Analysis of FD Data Biochemical Examples of Frequency-Domain Intensity Decays DNA Labeled with DAPI Mag-Quin-2: A Lifetime-Based Sensor for Magnesium Recovery of Lifetime Distributions from Frequency-Domain Data Cross-Fitting of Models: Lifetime Distributions of Melittin Frequency-Domain Fluorescence Microscopy with an LED Light Source Phase-Angle and Modulation Spectra Apparent Phase and Modulation Lifetimes Derivation of the Equations for Phase- Modulation Fluorescence Relationship of the Lifetime to the Phase Angle and Modulation Cross-Correlation Detection Phase-Sensitive Emission Spectra Theory of Phase-Sensitive Detection of Fluorescence Examples of PSDF and Phase Suppression High-Frequency or Low-Frequency Phase-Sensitive Detection Phase-Modulation Resolution of Emission Spectra Resolution Based on Phase or Modulation Lifetimes Resolution Based on Phase Angles and Modulations Resolution of Emission Spectra from Phase and Modulation Spectra References Problems

4 xviii 6. Solvent and Environmental Effects 6.1. Overview of Solvent Polarity Effects Effects of Solvent Polarity Polarity Surrounding a Membrane-Bound Fluorophore Other Mechanisms for Spectral Shifts General Solvent Effects: The Lippert-Mataga Equation Derivation of the Lippert Equation Application of the Lippert Equation Specific Solvent Effects Specific Solvent Effects and Lippert Plots Temperature Effects Phase Transitions in Membranes Additional Factors that Affect Emission Spectra Locally Excited and Internal Charge-Transfer States Excited-State Intramolecular Proton Transfer (ESIPT) Changes in the Non-Radiative Decay Rates Changes in the Rate of Radiative Decay Effects of Viscosity Effect of Shear Stress on Membrane Viscosity Probe Probe Interactions Biochemical Applications of Environment- Sensitive Fluorophores Fatty-Acid-Binding Proteins Exposure of a Hydrophobic Surface on Calmodulin Binding to Cyclodextrin Using a Dansyl Probe Advanced Solvent-Sensitive Probes Effects of Solvent Mixtures Summary of Solvent Effects References Problems Dynamics of Solvent and Spectral Relaxation 7.1. Overview of Excited-State Processes Time-Resolved Emission Spectra Measurement of Time-Resolved Emission Spectra (TRES) Direct Recording of TRES TRES from Wavelength-Dependent Decays Spectral Relaxation in Proteins Spectral Relaxation of Labeled Apomyoglobin Protein Spectral Relaxation around a Synthetic Fluorescent Amino Acid Spectral Relaxation in Membranes Analysis of Time-Resolved Emission Spectra Spectral Relaxation of Membrane-Bound Anthroyloxy Fatty Acids Picosecond Relaxation in Solvents Theory for Time-Dependent Solvent Relaxation Multi-Exponential Relaxation in Water Measurement of Multi-Exponential Spectral Relaxation Distinction between Solvent Relaxation and Formation of Rotational Isomers Comparison of TRES and Decay-Associated Spectra Lifetime-Resolved Emission Spectra Red-Edge Excitation Shifts Membranes and Red-Edge Excitation Shifts Red-Edge Excitation Shifts and Energy Transfer Excited-State Reactions Excited-State Ionization of Naphthol Theory for a Reversible Two-State Reaction Steady-State Fluorescence of a Two-State Reaction Time-Resolved Decays for the Two-State Model Differential Wavelength Methods Time-Domain Studies of Naphthol Dissociation Analysis of Excited-State Reactions by Phase-Modulation Fluorometry Effect of an Excited-State Reaction on the Apparent Phase and Modulation Lifetimes Wavelength-Dependent Phase and Modulation Values for an Excited-State Reaction Frequency-Domain Measurement of Excimer Formation Biochemical Examples of Excited-State Reactions Exposure of a Membrane-Bound Cholesterol Analogue References Problems Quenching of Fluorescence CONTENTS 8.1. Quenchers of Fluorescence Theory of Collisional Quenching Derivation of the Stern-Volmer Equation Interpretation of the Bimolecular Quenching Constant Theory of Static Quenching Combined Dynamic and Static Quenching Examples of Static and Dynamic Quenching Deviations from the Stern-Volmer Equation: Quenching Sphere of Action Derivation of the Quenching Sphere of Action

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

6 xx Alignment of Polarizers Magic-Angle Polarizer Conditions Why is the Total Intensity Equal to I + 2I Effect of Resonance Energy Transfer on the Anisotropy Trivial Causes of Depolarization Factors Affecting the Anisotropy Effects of Rotational Diffusion on Fluorescence Anisotropies: The Perrin Equation The Perrin Equation: Rotational Motions of Proteins Examples of a Perrin Plot Perrin Plots of Proteins Binding of trna to trna Synthetase Molecular Chaperonin cpn60 (GroEL) Perrin Plots of an F ab Immunoglobulin Fragment Biochemical Applications of Steady-State Anisotropies Peptide Binding to Calmodulin Binding of the Trp Repressor to DNA Helicase-Catalyzed DNA Unwinding Melittin Association Detected from Homotransfer Anisotropy of Membranes and Membrane- Bound Proteins Membrane Microviscosity Distribution of Membrane-Bound Proteins Transition Moments References Additional Reading on the Application of Anisotropy Problems Time-Dependent Anisotropy Decays Time-Domain and Frequency-Domain Anisotropy Decays Anisotropy Decay Analysis Early Methods for Analysis of TD Anisotropy Data Preferred Analysis of TD Anisotropy Data Value of r Analysis of Frequency-Domain Anisotropy Decays Anisotropy Decay Laws Non-Spherical Fluorophores Hindered Rotors Segmental Mobility of a Biopolymer- Bound Fluorophore Correlation Time Distributions Associated Anisotropy Decays Example Anisotropy Decays of Rhodamine Green and Rhodamine Green-Dextran Time-Domain Anisotropy Decays of Proteins Intrinsic Tryptophan Anisotropy Decay of Liver Alcohol Dehydrogenase Phospholipase A Subtilisin Carlsberg Domain Motions of Immunoglobulins Effects of Free Probe on Anisotropy Decays Frequency-Domain Anisotropy Decays of Proteins Apomyoglobin: A Rigid Rotor Melittin Self-Association and Anisotropy Decays Picosecond Rotational Diffusion of Oxytocin Hindered Rotational Diffusion in Membranes Characterization of a New Membrane Probe Anisotropy Decays of Nucleic Acids Hydrodynamics of DNA Oligomers Dynamics of Intracellular DNA DNA Binding to HIV Integrase Using Correlation Time Distributions Correlation Time Imaging Microsecond Anisotropy Decays Phosphorescence Anisotropy Decays Long-Lifetime Metal Ligand Complexes References Problems Advanced Anisotropy Concepts CONTENTS Associated Anisotropy Decay Theory for Associated Anisotropy Decay Time-Domain Measurements of Associated Anisotropy Decays Biochemical Examples of Associated Anisotropy Decays Time-Domain Studies of DNA Binding to the Klenow Fragment of DNA Polymerase Frequency-Domain Measurements of Associated Anisotropy Decays Rotational Diffusion of Non-Spherical Molecules: An Overview Anisotropy Decays of Ellipsoids Ellipsoids of Revolution Simplified Ellipsoids of Revolution Intuitive Description of Rotational Diffusion of an Oblate Ellipsoid

7 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY Rotational Correlation Times for Ellipsoids of Revolution Stick-versus-Slip Rotational Diffusion Complete Theory for Rotational Diffusion of Ellipsoids Anisotropic Rotational Diffusion Time-Domain Studies Frequency-Domain Studies of Anisotropic Rotational Diffusion Global Anisotropy Decay Analysis Global Analysis with Multi-Wavelength Excitation Global Anisotropy Decay Analysis with Collisional Quenching Application of Quenching to Protein Anisotropy Decays Intercalated Fluorophores in DNA Transition Moments Anisotropy of Planar Fluorophores with High Symmetry Lifetime-Resolved Anisotropies Effect of Segmental Motion on the Perrin Plots Soleillet's Rule: Multiplication of Depolarized Factors Anisotropies Can Depend on Emission Wavelength References Problems Energy Transfer Characteristics of Resonance Energy Transfer Theory of Energy Transfer for a Donor Acceptor Pair Orientation Factor κ Dependence of the Transfer Rate on Distance (r), the Overlap Integral (J), and τ Homotransfer and Heterotransfer Distance Measurements Using RET Distance Measurements in α-helical Melittin Effects of Incomplete Labeling Effect of κ 2 on the Possible Range of Distances Biochemical Applications of RET Protein Folding Measured by RET Intracellular Protein Folding RET and Association Reactions Orientation of a Protein-Bound Peptide Protein Binding to Semiconductor Nanoparticles RET Sensors Intracellular RET Indicator for Estrogens RET Imaging of Intracellular Protein Phosphorylation Imaging of Rac Activation in Cells RET and Nucleic Acids Imaging of Intracellular RNA Energy-Transfer Efficiency from Enhanced Acceptor Fluorescence Energy Transfer in Membranes Lipid Distributions around Gramicidin Membrane Fusion and Lipid Exchange Effect of τ 2 on RET Energy Transfer in Solution Diffusion-Enhanced Energy Transfer Representative R 0 Values References Additional References on Resonance Energy Transfer Problems Time-Resolved Energy Transfer and Conformational Distributions of Biopolymers xxi Distance Distributions Distance Distributions in Peptides Comparison for a Rigid and Flexible Hexapeptide Crossfitting Data to Exclude Alternative Models Donor Decay without Acceptor Effect of Concentration of the D A Pairs Distance Distributions in Peptides Distance Distributions in Melittin Distance-Distribution Data Analysis Frequency-Domain Distance-Distribution Analysis Time-Domain Distance-Distribution Analysis Distance-Distribution Functions Effects of Incomplete Labeling Effect of the Orientation Factor κ Acceptor Decays Biochemical Applications of Distance Distributions Calcium-Induced Changes in the Conformation of Troponin C Hairpin Ribozyme Four-Way Holliday Junction in DNA Distance Distributions and Unfolding of Yeast Phosphoglycerate Kinase Distance Distributions in a Glycopeptide Single-Protein-Molecule Distance Distribution Time-Resolved RET Imaging Effect of Diffusion for Linked D A Pairs

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

9 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 17. Time-Resolved Protein Fluorescence Intensity Decays of Tryptophan: The Rotamer Model Time-Resolved Intensity Decays of Tryptophan and Tyrosine Decay-Associated Emission Spectra of Tryptophan Intensity Decays of Neutral Tryptophan Derivatives Intensity Decays of Tyrosine and Its Neutral Derivatives Intensity and Anisotropy Decays of Proteins Single-Exponential Intensity and Anisotropy Decay of Ribonuclease T Annexin V: A Calcium-Sensitive Single-Tryptophan Protein Anisotropy Decay of a Protein with Two Tryptophans Protein Unfolding Exposes the Tryptophan Residue to Water Conformational Heterogeneity Can Result in Complex Intensity and Anisotropy Decays Anisotropy Decays of Proteins Effects of Association Reactions on Anisotropy Decays: Melittin Biochemical Examples Using Time-Resolved Protein Fluorescence Decay-Associated Spectra of Barnase Disulfide Oxidoreductase DsbA Immunophilin FKBP59-I: Quenching of Tryptophan Fluorescence by Phenylalanine Trp Repressor: Resolution of the Two Interacting Tryptophans Thermophilic β-glycosidase: A Multi-Tryptophan Protein Heme Proteins Display Useful Intrinsic Fluorescence Time-Dependent Spectral Relaxation of Tryptophan Phosphorescence of Proteins Perspectives on Protein Fluorescence References Problems Multiphoton Excitation and Microscopy Introduction to Multiphoton Excitation Cross-Sections for Multiphoton Absorption Two-Photon Absorption Spectra Two-Photon Excitation of a DNA-Bound Fluorophore Anisotropies with Multiphoton Excitation Excitation Photoselection for Two-Photon Excitation Two-Photon Anisotropy of DPH MPE for a Membrane-Bound Fluorophore MPE of Intrinsic Protein Fluorescence Multiphoton Microscopy Calcium Imaging Imaging of NAD(P)H and FAD Excitation of Multiple Fluorophores Three-Dimensional Imaging of Cells References Problems Fluorescence Sensing xxiii Optical Clinical Chemistry and Spectral Observables Spectral Observables for Fluorescence Sensing Optical Properties of Tissues Lifetime-Based Sensing Mechanisms of Sensing Sensing by Collisional Quenching Oxygen Sensing Lifetime-Based Sensing of Oxygen Mechanism of Oxygen Selectivity Other Oxygen Sensors Lifetime Imaging of Oxygen Chloride Sensors Lifetime Imaging of Chloride Concentrations Other Collisional Quenchers Energy-Transfer Sensing ph and pco 2 Sensing by Energy Transfer Glucose Sensing by Energy Transfer Ion Sensing by Energy Transfer Theory for Energy-Transfer Sensing Two-State ph Sensors Optical Detection of Blood Gases ph Sensors Photoinduced Electron Transfer (PET) Probes for Metal Ions and Anion Sensors Probes of Analyte Recognition Specificity of Cation Probes Theory of Analyte Recognition Sensing Sodium and Potassium Probes Calcium and Magnesium Probes Probes for Intracellular Zinc Glucose-Sensitive Fluorophores Protein Sensors Protein Sensors Based on RET GFP Sensors GFP Sensors Using RET Intrinsic GFP Sensors

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

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

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

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