Principles of Fluorescence Spectroscopy

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PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 239 Figure 7.3. Energy schematic for continuous spectral relaxation. Figure 7.4. Energy schematic for an excited-state reaction. constant k 1 describes the rate at which the F state is transformed into the R state. 7.1.1. Time-Resolved Emission Spectra Excited-state processes result in complex time-dependent decays. The intensity decays depend on the observation wavelength because of the time needed for the F state to Figure 7.5. Schematic of time-resolved emission spectra. I T (t) represents the decay of the total emission. I F (t) and I R (t) are the intensity decays on blue and red sides of the emission spectrum, respectively. The upper insert shows the emission spectra at t = 1, 2, or 3 ns. become an R state or some intermediate state. This situation is illustrated in Figure 7.5. In this figure the solid line shows the intensity decay of the total emission, or the decay that would be observed in the absence of excited-state processes. Now suppose the intensity decay is measured on the short-wavelength side of the total emission. This decay, I F (t), is more rapid than the decay of the total emission, I T (t), because the short-wavelength emission is decaying by both emission and relaxation, which is removing excited fluorophores from the observation wavelength. On the long-wavelength side of the emission the emitting fluorophores are those that have relaxed. Time is needed for the F-state molecules to reach the R state. Even if the F and R states have the same intrinsic decay time, the long-wavelength decay will appear to be slower. Also, at the moment of excitation all the molecules are assumed to be in the F state. No molecules are in the R state until some relaxation has occurred. For this reason one typically observes a rise in the intensity at long wavelengths representing formation of the relaxed state. The rise time is frequently associated with a negative pre-exponential factor that is recovered from the multi-exponential analysis. Following the rise in intensity, the decay I R (t) typically follows the total emission. Another way of understanding the wavelength-dependent decays is to recall that emission is a random event. Some fluorophores emit at earlier times, and
240 DYNAMICS OF SOLVENT AND SPECTRAL RELAXATION Figure 7.6. Normalized time-resolved emission spectra for continuous relaxation (top) and for a two-state process (bottom). some at later times. The decay rate represents the ensemble average. The fluorophores that emit at earlier times tend to have shorter wavelength emission, and those that emit at later times have longer wavelength emission. Suppose that emission spectra could be recorded at any desired instant following the excitation pulse (Figure 7.6) and that the spectra are normalized. First consider a continuous relaxation process (top panel). If the emission spectrum was observed immediately after excitation (t = 1 ns), then a blue-shifted or unrelaxed emission will be observed. If the time of observation is later, then more of the molecules will have relaxed to longer wavelengths, resulting in emission spectra that are progressively shifts to longer wavelengths at longer times. For continuous spectral relaxation the shape of the emission spectra are expected to remain the same. Now consider the ESR model (Figure 7.6, lower panel). At short times the blue-shifted emission would be observed. At long times the emission would be from the reacted fluorophore. At intermediate times emission from both species would be observed. Typically the emission spectrum would be wider at intermediate times due to emission from both forms of the fluorophore. These emission spectra, representing discrete times following excitation, are called the time-resolved emission spectra (TRES). It is technically challenging to determine the TRES, and the molecular interpretation can be equally difficult. 7.2. MEASUREMENT OF TIME-RESOLVED EMISSION SPECTRA (TRES) 7.2.1. Direct Recording of TRES The measurement of TRES is most easily understood using pulse sampling or time-gated detection (Chapter 4). In fact, the first reported TRES were obtained using this method, 4–6 but this method is rarely used at the present time. The sample was 4-aminophthalimide (4-AP) in propanol at –70EC. At this temperature the rate of solvent relaxation is comparable to the decay time. The sample is excited with a brief pulse of light, and the detector was gated on for a brief period (typically
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Principles of Fluorescence Spectros
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Joseph R. Lakowicz Center for Fluor
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Preface The first edition of Princi
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x GLOSSARY OF ACRONYMS DNS dansyl o
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xii GLOSSARY OF ACRONYMS TRES time-
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xiv GLOSSARY OF MATHEMATICAL TERMS
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xvi CONTENTS 2.9.4. Conversion betw
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xviii CONTENTS 6. Solvent and Envir
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xx CONTENTS 10.4.4. Alignment of Po
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xxii CONTENTS 14.7.1. Simulations o
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xxiv CONTENTS 19.12. New Approaches
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xxvi CONTENTS 25.3. Optical Propert
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2 INTRODUCTION TO FLUORESCENCE Figu
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6 INTRODUCTION TO FLUORESCENCE inst
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10 INTRODUCTION TO FLUORESCENCE Fig
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12 INTRODUCTION TO FLUORESCENCE ns,
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14 INTRODUCTION TO FLUORESCENCE 1.7
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24 INTRODUCTION TO FLUORESCENCE due
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28 INSTRUMENTATION FOR FLUORESCENCE
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3 Fluorescence probes represent the
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PRINCIPLES OF FLUORESCENCE SPECTROS
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98 TIME-DOMAIN LIFETIME MEASUREMENT
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100 TIME-DOMAIN LIFETIME MEASUREMEN
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5 In the preceding chapter we descr
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Page 207 and 208: PRINCIPLES OF FLUORESCENCE SPECTROS
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Page 297 and 298: 278 QUENCHING OF FLUORESCENCE 8.1.
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290 QUENCHING OF FLUORESCENCE relat
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292 QUENCHING OF FLUORESCENCE Figur
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298 QUENCHING OF FLUORESCENCE σ ar
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320 QUENCHING OF FLUORESCENCE 47. R
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322 QUENCHING OF FLUORESCENCE 113.
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328 QUENCHING OF FLUORESCENCE P8.3.
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330 QUENCHING OF FLUORESCENCE P8.11
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332 MECHANISMS AND DYNAMICS OF FLUO
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10 Measurements of fluorescence ani
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11 In the preceding chapter we desc
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12 In the preceding two chapters we
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444 ENERGY TRANSFER Figure 13.1. Fl
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446 ENERGY TRANSFER wavelength is e
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448 ENERGY TRANSFER Table 13.1. Cal
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450 ENERGY TRANSFER Figure 13.6. Ab
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452 ENERGY TRANSFER safe for many p
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454 ENERGY TRANSFER Figure 13.12. P
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456 ENERGY TRANSFER Figure 13.16. E
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458 ENERGY TRANSFER Figure 13.21. R
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460 ENERGY TRANSFER Figure 13.24. R
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462 ENERGY TRANSFER tiple acceptors
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464 ENERGY TRANSFER Figure 13.29. A
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466 ENERGY TRANSFER Figure 13.33. F
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468 ENERGY TRANSFER Table 13.3. Rep
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470 ENERGY TRANSFER 55. Clegg RM. 1
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472 ENERGY TRANSFER Tong AK, Jockus
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474 ENERGY TRANSFER Figure 13.39. O
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14 In the previous chapter we descr
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15 In the previous two chapters on
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16 The biochemical applications of
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578 TIME-RESOLVED PROTEIN FLUORESCE
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608 MULTIPHOTON EXCITATION AND MICR
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19 Fluorescence sensing of chemical
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676 NOVEL FLUOROPHORES Figure 20.1.
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678 NOVEL FLUOROPHORES Figure 20.7.
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680 NOVEL FLUOROPHORES Figure 20.12
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698 NOVEL FLUOROPHORES 33. Acherman
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700 NOVEL FLUOROPHORES 103. Demas J
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702 NOVEL FLUOROPHORES 176. Montalt
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21 During the past 20 years there h
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22 Fluorescence microscopy is one o
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758 SINGLE-MOLECULE DETECTION Figur
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766 SINGLE-MOLECULE DETECTION small
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770 SINGLE-MOLECULE DETECTION Table
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786 SINGLE-MOLECULE DETECTION face
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788 SINGLE-MOLECULE DETECTION TCSPC
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790 SINGLE-MOLECULE DETECTION 53. H
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792 SINGLE-MOLECULE DETECTION Ke PC
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794 SINGLE-MOLECULE DETECTION Polar
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24 In the previous chapter we descr
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862 RADIATIVE-DECAY ENGINEERING: SU
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Appendix I Corrected Emission Spect
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Appendix II Fluorescence Lifetime S
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890 APPENDIX III P ADDITIONAL READI
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892 APPENDIX III P ADDITIONAL READI
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894 ANSWERS TO PROBLEMS A1.4. A. Th
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896 ANSWERS TO PROBLEMS Energy tran
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898 ANSWERS TO PROBLEMS Figure 4.65
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900 ANSWERS TO PROBLEMS expected to
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904 ANSWERS TO PROBLEMS where f is
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906 ANSWERS TO PROBLEMS [BSA] Obser
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908 ANSWERS TO PROBLEMS emission. T
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910 ANSWERS TO PROBLEMS dx rA A (
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912 ANSWERS TO PROBLEMS B. A covale
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914 ANSWERS TO PROBLEMS The α i va
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920 ANSWERS TO PROBLEMS CHAPTER 24
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Index A Absorption spectroscopy, 12
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