《材料测试技术及方法》课程教学资源（书籍文献）拉曼散射 Principles of Fluorescence Spectroscopy，Second Edition Joseph，R. Lakowicz

Principles of Fluorescence Spectroscopy Second Edition Joseph R. Lakowicz University of Maryland School of Medicine Baltimore, Maryland Kluwer Academic/Plenum Publishers New York, Boston, Dordrecht, London, Moscow

Preface It has been 15 years since publication of the first edition of able probes,and,of course,intrinsic fluorescence from Principles of Fluorescence Spectroscopy.This first vol- proteins.Today the menu of fluorescent probes has ex- ume grew out of a graduate-level course on fluorescence panded manyfold.A wide variety of lipid and protein taught at the University of Maryland.The first edition was probes have been developed,and probes have become written during a transition period in the technology and available with longer excitation and emission wavelengths. applications of fluorescence spectroscopy.In 1983,time- There has been extensive development of cation-sensing resolved measurements were performed using methods probes for use in cellular imaging.The nanosecond barrier which are primitive by today's standards.The dominant of dynamic fluorescence information has been broken by light sources for time-resolved fluorescence wuere the the introduction of long-lifetime probes. ond flashla which t provided reatively wide Another example of the r ansion of fluor d ith rela. is DNA sequencing tively s as pe ed using radio iedatonc6 ailable instrumer cing ha be mp ndhusproidedhmitc trequ The fluo ion on complex time wing to resolved decays.Data analysis was also limited because of the goal of sequencing the human genome ally.whe the lower information content of the experimental data would have expected in 1983 that the gene for the green Much has changed since 1983.The dominant light fluorescent protein could be introduced into cells,with sources are now picosecond dye lasers or femtosecond spontaneous folding and formation of the fully fluorescent Titanium:sapphire lasers.In the case of phase-modulation protein? fluorometry,frequency-domain instrumentation now op- Parts of this book were influenced by a course taught at erates over arange of light modulation frequencies,allow- the Center for Fluorescence Spectroscopy,which has been ing resolution of complex decays.The time resolution in attended by individuals from throughout the world.How- both the frequency and the time domain has been increased ever.the most important factor stimulating the second by the introduction of high-speed microchannel plate edition was the positive comments of individuals who photomultiplier tubes.Data analysis has become increas found value in the first edition many individuals com sophisticated, ause of the vailability of mented on the value of explaining the basic concepts from also b of the heir fundam ental origins.This has me ine incr ed as the nur ctitioeOf sing gl bal an ta si analys capabilitie ignificant in have been extended number of courses at the undergraduate or graduate provide resolution of complex anisotropy d ays,confor. mational distributions,and complex quenching phenom- In this second edition of Principles of Fluores cna. p have atempted to maintain the emphasis Another important change since 1983 has been the ex- on basics,while updating the examples to include more tensive development of fluorescent probes.Early fluores- recent results from the literature.There is a new chapter cent probes were those derived from histochemical providing an overview of extrinsic fluorophores.The dis staining of cells,a limited number of lipid and conjugat- cussion of time-resolved measurements has been ex-

喻 PRINCIPLES OF FLUORESCENCE SPECTROSCOPY panded to two chapters.Quenching has also been ex- gestions.These individuals include felix castellane anded to two chapters Energy transfer and anisotropy Robert E.Dale,Jonathan Dattelbaum,Maurice Eftink pave cach be en expanded to three chapters.There is also a John Gilchrist,Zygmunt Gryezynski,Petr Herman,Gabor new chapter r on fluorescence sensing.To enhance the use Laczko,Li Li,Harriet Lin,Zakir Murtaza,Leah Tolosa fulness of this book as a textbook,each chapter is follow d and Bogumil Zelent.I apologize for any omissions. by a set of problems.Sections which des advanced I also give my special thanks to Dr.Ignacy Gryc vnski pics are indicated as such,to allow these ctions to be and his wife.Kry yna Gryczynska.When I started to write skipped in an introductory course.Glossaries of com this book lgn said"just go and write,don't worry about mathematical symbols are the fig zures"Many of the cellent figr res in this book vided.For thos wanting additional infor were drawn by krystyna,with the valuable sugg III contains a list of re mmended books which e and on Ignacy.Without their dedicated efforts,the book could not various specialized to have been completed in any reasonable period of time In closing.Iwish to ss my appreciation to the many also thank Ms.Suzy Rhinehart for providing a supp individuals who have assisted mo family environment during p naration of this hook Fi of the book but also in the intellectual develor nally.I thank the Nationaln titutes of Health and the ratory.My spe cial thanks go ,to Ms Mar National Science Foundation for support of my labora for her careful aration of th e text.Mary has tory. yp s of al y individ I.R.Lakowicz Proofre chapters and Center for Fluorescence Spectroscopy,Baltimore

Glossary of Acronyms 2.6-ANS 6-Anilinonaphthalene-2-sulfonic acid Bopoyoi MPE Multip asedon1.3.5.7.8- MICT Metal-igan transfer (state) NADH R mide adenine dinucleotid aorom3a4hda2a -BF2,or 4,4- d N-Acetyl- fluoro- NATYTA -tryptoph N-A BODIPY is a trademark of Molecular Near-infrarcd cnz-. oxa-1,3-diazol-4-yl Probes,Inc. NIR CFD Constant fraction discriminator Phenylalaninc Dansyl 5-Dimethylaminonaphthalene-1-sulfonic Phosphatidylcholine acid PMT Photomultiplier tube DAPI 4,6-Diamidino-2-phenylindole POPOP 1,4-Bis(5-phenyloxazol-2-yl)benzene DAS Decay-associated spectra PPD 2,5-Diphenyl-1,3,4-oxadazole DNS-CI Dansyl chloride PPO 2,5-Diphenyloxazole DPH 1,6-Diphenyl-1,3,5-hexatriene Prodan 6-Propionyl-2-(dimethylamino)naphthalene EB Ethidium bromide PSDF Phase-sensitive detection of fluorescence F Single-letter code for phenylalanine RET Resonance energy transfer FAD Flavin adenine dinucleotide So Ground electronic state ED frequency-domain S.First ercited singlet state FISH Fluorescence in situ hybridization SPQ 6-Methoxy-N-(3-sulfopropyl)quinoline FTC nonucloiaeranate scein-5-isothi T First excited triplet state FMN Flavin mo TAC Time-to-amplitude converter energy transfer TCSPC Tim elated single-photon counting TD Time-do ency virus TICT Twisted in mal charg ransfer state HSA um album NS 6- lene-2-sulfonic acid IAEDANS 5-(2 hy）amino TRE (-Tolud yl)n TRITC Time-reso naphthalen 6-)isothiocy IAF 5-(lodo amido)fluoresceir ICT Internal charge transfer (state) trp Tryptophan IRF Instrument response function LE Locally excited (state) Fensowaeaponan MCP Microchannel plate Y Single-letter code for tyrosine MLC Metal-ligand complex,usually of a transi- tion metal (Ru,Rh,or Os)

Glossary of Mathematical Terms A Acceptor or absorption rAnisotropy (sometimes distance in a dis- c Speed of light tance distribution) Co Characteristic acceptor concentration in 7 Average distance in a distance distribution resonance energy transfer r(0)Time-zero anisotropy C(r)Correlation function for spectral relaxation r(t)Anisotropy decay D Donor,diffusion coefficient,or rotational r Distance ofclosest approachbetween donors diffusion coefficient and acceptors in resonance energy transfer, D.or D Rate of rotational diffusion around(displac or fluorophores and quenchers ing)the symmetry axis of an ellipsoid of Fractional amplitudes in a multiexponential E Efficiency of en ro Fundan ntal an isotropy in the absence of rescence rotational diffusion roi a multiexponential ence intervals ni otropy decay F(A)Emission spectrum tropy in an anisotropy decay f Fractional steady-state intensities in a mul- ed anisotropy tiexponential intensity decay Ro Forster distance in resonance energy transfe 8&omandoieoiaapopyneac c Preexponential factors in a multiexponential intensity decay ments B Angle between absorption and emission hw Half-width in a distance or lifetime distribu transition moments tion rRadiative decay rate I()Intensity decay. typically the impulse re y Inverse of the decay time:y=1/t E Dielectric constant or extinction coefficient Rotational Orientation factor in resonance energy trans kr Transfer rate in resonance energy transfer mModulation at a light modulation frequency Ka of the modulated amplitu les of the Polarized components of n Refractive index.when used in consideration Wavelength of solvent effects Emission wavelength N()Number of counts per channel,in time-cor- Maximum emission wavelength related single-photon counting Excitation wavelength Quantum yield Maximum excitation or absorption wave- P(r)Probability function for a distance(r)distri- length for the lowest So5 transition bution 入ma Emission maximum pK.Acid dissociation constant. negative loga Excited-state dipole moment rithm Ground-state dipole moment t

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