PRINCIPLES OF FLUORESCENCE SPECTROSCOPY D Wavenumber,in cm- ty Radiative or natural lifetime Peg Emission center of gravity ts Solvent relaxation time Da(r)Time-resolved emission center of gravity,in A Differential polarized phase angle,differ- cm- ence in phase between the parallel and per- t Decay time pendicular components of the emission t Average lifetime Phase angle at a light modulation frequency Apparent lifetime calculated from the phase angle at a single frequency XGoodness-of-fit parameter,reduced chi- tp Donor decay time or solvent dielectric re- squared laxation time X2 Sum of the squared weighted deviations t Solvent longitudinal relaxation time o Light modulation frequency in radians per mApparent lifetime calculated from the modu- second;2n times the frequency in cycles per lation at a single frequency second
Contents Most sections of this book describe basic aspects of fluorescence spectroscopy.and some sections describe more marked A du an he itted in an introductory c a. The enching(Chapter o)aniso opy 12)and er y transfer 14 and 15)can be skipped in a first re ding. Depending on of the -ader, Ch apte can also be skipped 2.Instrumentation for Fluorescence 1.Introduction to Fluorescence Spectroscopy 2.1.Excitation and Emission Spectra 25 l.l.Phenomenon of Fluorescence·.·.,·. 2.1.A.An Ideal Spectroflu 1.2.Jablofiski Diagram. 2.1.B.Distortionsn meter 1.3.Characteristics of Fluorescence Emission. on and Emission 6 1.3.A.Stokes'Shift. 6 1.3.B.Emission Spectra Are Typically 2.2.Light Sources 28 Independent of the Excitation 2.2.A.Arc and Incandescent Lamps . 8 2.2.B.Solid-State Light Sources Wavelength . 2.3.Monochromators 只 32 2.3.A.Wavelength Resolution and Emission 1.4. Spectra 33 1.4.A.Fluorescence Quenching 2.3.B.Polarization Characteristics of 14.B.Time Scale of Molecular Processes in Monochromators. Solution 。，。，, l.5.Fluorescence Anisotropy.·.,·.·.,· 2.3.C.Stray Light in Monochromators. 34 1.6.Resonance Energy Transfer . 2.3.D.Second-Order Transmission in 1.7.Steady-State and Time-Resolved Fluorescence Monochromators 17.A.Why Time-Resolved Measurements? 2.3.E libration of M 35 1.8.Biochemical Fluorophores 2.4.Optical Filters.· 。·。，。 1.8.A.Fluorescent Indicators 2.4.A.Bandpass Filters. 5 rmation fro 2.4.B.Interference Filters 。,，,。4 Spectra and the Stokes'Shift 17 2.4.C.Filter Combinations 38 1.9.B.Quench ng of Flu 2.4.D.Neutral Density Filters 1.9.C.Fluorescence Polarization or 2.5.Optical Filters and Signal Purity Anis0t00y.,.,.。.,. 2.5.A.Emission Spectra Taken through Filters 40 1.9.D.Resonance Energy Transfer.··. 19 2.6 Photomultinlier Tubes L.l0.Fluorescence Sensing.,.·.··. 26A 1.1l.Summary. 0 2.6.B. Designs ynode Cha References.,.,.,.· 0 2.6.C.Time Response of Photomultiplic 21 Tubes.44
xiv PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 2.6.D.Photon Counting versus Analog 3.6.A.Fluorogenic Probes 78 Detection of Fluorescence 3.6.B.Structural Analogs of Biomolecules. 81 2.6E Symptoms of PMT Failure 46 3.6.C.Viscosity Probes 81 2.6.F.Hybrid Photomultiplier Tubes 47 3.7.Fluorescent Proteins 87 2.6.G.CCD Detectors 47 3.7.A.Phycobiliproteins 82 2.7.Polarizers 47 37B Green Flu ent Protein 84 2.8.Corrected Excitation Spectra 49 3.7.C.Phytofluor A New Class of 2.8.A.Use of a Quantum Counter to Obtain on Spectra 3.8.Long-Lifctim ent Probes 355 2.9.Corrected 2.9.A Comparison with K nide nown Emission 3.8B Transition-Metal-Ligand Complexes Spectra 51 Proteins as Sensors. 88 2.9.B.Correction Factors Obtained by Using 3.10.C0 nclusion.,.,.。. 89 a Standard Lamp 51 Reterences。·.·,··。·········+ 2.9.C.Correction Factors Obtained by Using Problems. 92 a Quantum Counter and Scatterer 5 2.9.D.Conversion between Wavelength and 52 4. Time-Domain Lifetime Measurements 2.10.Quantum Yield Standards 2.11.Effects of Sample Gec 53 2.12 aration 4.1.Overview of Time-Domain and 2.13.Abs0 ion of Light and Deviation from Frequency-Domain Measurements 9 mbert Law 4.1.A.Meaning of the Lifetime or Decay 56 Time 2.13.A.Deviations from Beer's Lav Ph n and Mul ltiphoton Exci ation 77990 and Modulation Lifetime 97 4.1.C Time-Domain and cy-Dom ain Lifetimes 97 Problems 4.2.Biopolymers Display Multiexponential or Heterogeneous Decays 8 4.2.A.Resolution of Multiexponential Decays Is Difficult. 100 3.Fluorophores 4.3.Time-Correlated Single-Photon Counting .101 4.3.A.Principles of TCSPC 101 3.1.Intrinsic or Natural Fluorophores 63 4 3 B Example of TCSPC Data 10i e cofactors 63 4.3.C.Convolution Integral 102 31R Binding of NADH toa Proteir 4.4.Light Sou ces for TCSPC 104 d 4 A Picos nd Dye 104 3.2A DE R 56切 um:Sapphire 10e 3.2.B.Role of the Stokes'Shift in Protein 4.4 mps 107 Labeling. ，。， 4.4.D. 109 3.2.C olid-State Lasers,······· Solvent-Sensitive Probes 71 4.5.Ee ctronics for TCSPC 109 3.2.D.Noncovalent Protein-Labeling Probes 4.5A.Constant Fraction Discriminators 109 3.2.E Membrane Probes 4.5.B.Amplifiers 110 3.2.F Membrane Potential probes 1 4.5.C.Time-to-Amplitude Converter 3.3.Red and Near-Infrared (NIR)Dyes (TAC)Standard and Reversed 33.A Meas nt of Hu 110 with Lasc tation 3.4.DNA Probes 15 CA) 4 5 D Multic E Delay Lines 3.4.A DNA B 77 45 Puls 11 ing Probes 78 4.6. Det for TCS 11 Special Pro 4.6 A.MCP PMTs,.,.,., 111
CONTENTS w 4.6.B.Dynode Chain PMTs 113 46.C.Photodiodes as Detectors Frequency-Domain Lifetime 14 Measurements 4.6.D.Color Effects in Det 14 4.6.E.Timing Effects of Monochromators 116 4.7.Alterative Methods for Time-Resolved 5.1.Theory of Frequency-Domain Fluorometry ·142 Measurements 116 51 A Least-Sou es Analysis of 4.7.A.Pulse Sampling or Gated Detection 116 Intensity Decays 144 4.7.B.Streak Cameras 117 5.1.B.Globa Analysis of 4 7C.Upconversion Methods 118 ency-Domain Data 146 4.8.DataAnalysis 18 5.1.C.Estimation of Parameter Uncertainties 146 4.8.A mptions of Nonlinea 52.Frequency-Domain Instrumentation····147 Least-Squares Analysis 1l9 5.2.A.History of Phase-Modulation 4.8.B.Overview of Least-Squarcs Analysis Fluorometers 147 119 4 8 C.Meaning of the Goodness of Fit.Y 5.2.B.The 200-MHz Frequency-Domain 120 147 4.8 d.autocorrelation function 121 4.9.Analysis of Multiexp 52.C Light Modulators 1d0 ntial de 21 4.9.A and Indole Widely on Detection Spaced Lifetim 12 4.9.B.Comparison of Values- -Frequency Amplifiers -F-Statistic 122 4.9.C.Parameter Uncertainty-Confidence 5.2.G Photomultiplier Tubes 150 5.2.H.Principle of Frequency-Domain Intervals 122 Measurements 4 9 D Effect of the Number of Ph nts 124 151 5.3.Color Effects and Backeround Fluorescence 152 49.E Anth ranilic Acid and 2-Amin 5.3.A.Color Effects in Frequency-Domain -Two Closely Spaced ents 152 Lifetimes 124 5.3.B.Backgro ction in 4.9.F.Global Analysis-Multiwavelength cy-Domai reme 153 Measurements 6.4. 4.9.G.Resolution of Three Closely Spaced 154 Lifer mes 5.4.A.Exponential Decays. 154 4.10.Intensity Decay Laws 9 5.4.B.Effect of Scattered Light.154 4.10.A.Multiexponential Decays I29 5.5.Analysis of Multiexponential Decays 155 4.10.B.Lifetime Distributions 130 5.5.A.Resolution of Two Widely Spaced 4.10.C.Stretched Exponentials 131 Lifetimes 】55 4 10D Transient Effects 131 5.5.B.Resolut n of Two Closely Spaced 4.11.Global Analysis 157 4.12. Repres e Int ensity Decays 55.C. Analysis of a Two-Component 4.12.A.Intensity Decay for a Single Mixtur 4 159 Tryptophan Protein 132 55.D. Analysis of a Three-Component 4.12.B.Green Fluorescent Protein- Mixture Limits of resolution .160 Systematic Errors in the Data 133 55.E Resolution of a Three-component 4.12.C.Erythrosin B-A Picosecond Decay Mixture with a 10-Fold Range of L33 Decay Times .162 ophyll Agg tes in He> ane 5.6.Biochemical Examples of of FAD ency-Domain Intensity Decays 163 cence Decays 5.6.A otein with Th Tryptophan 4.12.G.Subpicosecond Intensity Decays Decay Times.163 4.13.Closing Comments. 136 5.6.B xponenti References. 30 Staphylo coccal Nuclease and Melittin 163 Problems 140 5.6.C.DNA Labeled with DAPI.164
xvi PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 5.6.D.Quin-2-A Lifetime-Based Sensor for 6.3.A.Specific Solvent Effects and Lippert Calcium 165 Plots. .196 56E SPO Collisional quenching of a 64.Temperature Effects .198 Chloride Sensor 165 6.4.A.LE and ICT States of Prodan .200 5.6.F.Green Fluorescent Protein-One-and 6.5.Biochemical Examples Using PRODAN 201 166 6.5.A.Phase Transition in Membranes 01 5.6.G. 6.5B tcin Asso 202 166 6.5.C.Fatty Acid Binding Proteins 202 5.6.H Lifetime Distri ation of 6.6.Biochemical Examples Using Solvent- Photosynthetic Components 167 Sensitive Probes 202 5.6L. Lifetime Distributions of the 6.6.A.Exposure of a Hydrophobic Surface Ca2+-ATPase 167 on Calmodulin .202 5.6.J.Cross Fitting of Models-Lifetime 6.6.B.Binding to Cyclodextrins Using a Distributions of Melittin 168 Dansy!Probe 02 5.6K.Intensity Decay of NaDH 160 6.6.C.Polarityof a Memb 203 5.7.Gigahe rtz freau ney-Dor nain Fluorometry 169 ng Site 5.7.A.Gigah 6.7 lopment of Advanced Solvent-S ensitive ttz ED Me 171 5.7B. 204 t 6.8.Effects of Solvent Mixtures 206 FD Data 172 6.9.Summary of Solvent Effects. 207 5.8.Simple Freque ncy-Domain Instruments 73 References.,.,. 207 5.8.A.Laser Diode Excitation I73 Problems.,. 210 5.8.B.LED Excitation 173 5.9.Phase Angle and Modulation Spectra 175 59 A Resolution of the Two emission Spectra of Tryptophan Using 7.Dynamics of Solvent and Spectral Phase-Modulation Spectra 176 1> Relaxation ivation of the fquations for phase Modulation Flu 7.1.Continuous and Two-State Spectral 5.11.A Relaxation Pha 178 7.2.Measurement of TRES. ，。 213 5.11.B.Cross-Correlation Detecti 180 7.2.A.Direct Recording of TRES 213 5.12.Perspectives on Frequency-Domain 7.2.B.TRES from Wavelength-Dependent Fluorometry 180 Decavs 213 References············· 180 7.3.Biochemical Exan les of TRES Problems 184 73.A a cation in Apomyoglobin 215 7.3.B.TRES of Labeled Membranes 217 7.3.C.Analysis of TRES. 218 6. Solvent Effects on Emission Spectra 7.3.D.Spectral Relaxation in Proteins 220 7.4.Lifetime-Resolved Emission Spectra 527 6.1.Overview of Solvent Effects. 185 75.Picose ond Relaxation in Solve 224 6.1A.Polarity Surrounding a Membrane- 7.5.A ory for Time-Dependent Solvent bound fluorophore 186 axation 6.1.B.Mechanisms for Spectral Shifts 186 7.5.B.Multiexponential Relaxation in Water 225 6.2.General Solvent Effects The Lippert 7.6.Comparison of Continuous and Two-State Equation 181 Rclaxation. 226 6.2.A.Derivation of the Lippert Equation 189 7 6 a Experimental distinction between 6.2.B.Application of the Lippert Equation 191 Continuous and Two-State Relaxation 227 62.CP 193 76 B Phase-Modulation Studies of Solvent 6.3.Sp cific Sol ntEect 194 221