Principles of Fluorescence Spectroscopy

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224 SOLVENT AND ENVIRONMENTAL EFFECTS Figure 6.34. Effect of solvent polarity on the energies of LE and ICT states. Revised from [79]. cis-stilbene is even more rapid, resulting in very short fluorescence lifetime. Such rotations in the excited state are thought to affect the emission of many other fluorophores. 83 Increase in local viscosity contributes to the increased intensities displayed by many fluorophores when bound to biomolecules. In Chapter 1 we mentioned the viscosity probe CCVJ, which displayed increases in quantum yield with increases in viscosity. Figure 6.37 shows the emission intensities of this probe when bound to an antibody directed against this probe. 84 Binding of CCVJ to the antibody prevents rotation around the ethylene bond and increases the quantum yield. Figure 6.35. Intensity decays of trans-stilbene in methylcyclohexane: isohexane (3:2). Revised from [80]. Figure 6.36. Excited-state isomerization of stilbene. From [81]. Figure 6.37. Effect of anti-CCVJ antibody on the emission of CCVJ. Antibody concentrations are shown in µM. Revised and reprinted with permission from [84]. Copyright © 1993, American Chemical Society.
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 225 Figure 6.38. Chemical structures of viscosity-sensitive fluorophores. The carbon chain on the left is a farnesol group. Revised from [86]. 6.7.1. Effect of Shear Stress on Membrane Viscosity The viscosity of some membranes are thought to be sensitive to the shear stresses created by fluid flow. 85 This possibility was tested using the viscosity probes shown in Figure 6.38. DCVJ was expected to be more water soluble and FCVJ was expected to label cell membranes. The locations of these probes in human epidermal cells were determined by recording images at various heights in the cells (z-axis sectioning). Figure 6.39 shows pseudocolor images of the labeled cells. Blue indicates images recorded in a plane passing through the center of the cells and thus staining of the cytoplasm. Red indicates images recorded in a plane across the top of the cells, and thus staining of the plasma membrane. These images show that FCVJ localizes primarily in the plasma membrane (left) and that DCVJ localizes primarily in the cytoplasm. The fluorescence intensities of DCVJ and FCVJ were examined as the epidermal cells were subjected to shear stress. For DCVJ, which was localized in the cytoplasm, there was no change in intensity (Figure 6.39, lower panel). For FCVJ, which bound to the plasma membrane, there was a large drop in intensity in response to shear stress. The reversibility of the changes with elimination of fluid flow showed that the probes were not being washed out of the cells, indicating a decrease in viscosity. These viscositydependent probes thus provide an optical means to follow changes in the microviscosity of cell membranes in response to stimuli. 6.8. PROBE–PROBE INTERACTIONS In addition to interacting with solvents, fluorophores can interact with each other. One example is excimer formation due to an excited-state complex of two identical fluorophores. Excimer formation is a short-range interaction Figure 6.39. Pseudocolor images of human epidermal cells stained with FCVJ or DCVJ. In the images red indicates labeling of the cell membrane and blue indicates labeling of the cytoplasm. The red and blue lines on the inserts indicates the z-level of the images. The lower panel shows the effect of shear stress on the intensities of cells labeled with FCVJ or DCVJ. Revised from [86]. Courtesy of Dr. Emmanuel Theodorakis from the University of California, San Diego. that requires molecular contact between the fluorophore. In Chapter 1 we described excimer formation by pyrene as a means to measure diffusion in membranes. Excimer formation is beginning to find use in biotechnology. Figure 6.40 shows the use of excimer formation to detect an insertion mutation in DNA. 87–88 A DNA oligomer was synthesized that contained two nearby pyrene residues. The sequence of this probe was mostly complementary to the target sequence, except for the region near the pyrene groups. When bound to the wild-type (WT) sequence, one of the pyrenes intercalated into the double helix and the second remained outside the double helix. Since the pyrenes were not in contact there was no excimer emission. This probe DNA was then hybridized with a insertion mutant (IM) sequence that contained an extra base. This base prevented the pyrene from intercalating into the double helix, resulting in a blue emission from the excimer.
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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 193 and 194: PRINCIPLES OF FLUORESCENCE SPECTROS
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Page 257 and 258: 238 DYNAMICS OF SOLVENT AND SPECTRA
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276 DYNAMICS OF SOLVENT AND SPECTRA
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278 QUENCHING OF FLUORESCENCE 8.1.
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280 QUENCHING OF FLUORESCENCE Figur
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282 QUENCHING OF FLUORESCENCE ly ev
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290 QUENCHING OF FLUORESCENCE relat
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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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