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