Principles of Fluorescence Spectroscopy

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88 FLUOROPHORES Figure 3.43. Principle of "time-resolved" detection in lanthanide immunoassays. Revised from [174]. to integrate the intensity from the lanthanide. The term "time-resolved" is somewhat of a misnomer, and does not refer to measurement of the decay time. These time-gated immunoassays are essentially steady-state intensity measurements in which the intensity is measured over a period of time following pulsed excitation. The lanthanides do suffer several limitations. One is the need to chelate the lanthanide in order to obtain significant excitation. The requirement often results in multi-step assays, the last step being addition of the chelator. Another difficulty is the absence of polarized emission, so that the lanthanides cannot be used for anisotropy measurements. Figure 3.44. Intensity decay of Ru(bpy) 2 (mcbpy)-PE in DPPG vesicles. Modified From [176]. 3.9.2. Transition Metal–Ligand Complexes Another class of probes with long lifetimes are the transition metal complexes. These are typically complexes of ruthenium (Ru II), rhenium (Re I), or osmium (Os II) with one or more diimine ligands (Figure 3.44). In contrast to the lanthanides, these compounds display molecular fluorescence from a metal-to-ligand charge-transfer state. The transition is partially forbidden, so that the decay times are long. These complexes are highly stable, like covalent bonds, so there is no significant dissociation of the metal and ligands. Transition metal complexes display lifetimes ranging from 10 ns to 10 :s. 175 For example, Ru(bpy) 2 (mcbpy) in Figure 3.44 displayed a decay time near 400 ns when conjugated to proteins and lipids. 177 The MLC probes are highly photostable, and display large Stokes shifts, so that probe–probe interactions are not expected. These molecules are known to display polarized emission (Chapter 20) and are thus useful for measurement of dynamic processes on the microsecond timescale. 3.10. PROTEINS AS SENSORS The use of fluorescence for chemical sensing requires highly specific probes. One approach to obtaining the needed specificity is to rely on proteins that are known to bind the desired analyte. This approach has been used to develop sensing proteins for a variety of analytes. 178–179 One example is a sensor for zinc based on a zinc finger peptide. Zinc fingers are part of transcription factors. These proteins bind zinc with high affinity and specificity, typically to histidine and/or cysteine residues. To make a zinc sensor, the zinc finger amino-acid sequence was modified to contain a covalently linked dansyl group near the middle of the peptide (Figure 3.45). 180 Upon addition of zinc the emission intensity increases, and the emission maxima shift to shorter wavelengths (Figure 3.46). In the absence of zinc the peptide is unfolded, and the dansyl group is exposed to the water. In the presence of zinc the peptide adopts a folded structure that was anticipated to result in shielding the dansyl group from water. Another example of a protein sensor is shown in Figure 3.47. In this case the protein is calmodulin that was genetically modified to contain a cysteine residue at position 109. This residue was labeled with an environmentally sensitive fluorophore. 181 This labeled protein was sensitive to the antipsychotic drug trifluoperazine. The fluorescence of the coumarin label was almost completely quenched upon addition of this drug.
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 89 Figure 3.45. A zinc-sensitive peptide based on the dansyl fluorophore. Reprinted with permission from [180]. Copyright © 1996, American Chemical Society. Figure 3.46. Zinc-dependent emission spectra of the dansyl-labeled zinc finger peptide. From [180]. Figure 3.47. Protein sensor for the antipsychotic drug trifluoperazine. The protein is calmodulin labeled with a fluorophore at a genetically inserted cysteine residue at position 109. From [181]. 3.11. CONCLUSION A diversity of molecules display fluorescence, and numerous interactions and processes can alter the spectral properties. Fluorophores can be covalently attached to macromolecules, or designed to interact with specific ions. Emission can occur from the UV to the NIR, and probes are available with short (ns) and long (:s to ms) lifetimes. The technology of probe chemistry is rapidly changing, and new probes are allowing previously impossible experiments to be performed.
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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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140 TIME-DOMAIN LIFETIME MEASUREMEN
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5 In the preceding chapter we descr
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PRINCIPLES OF FLUORESCENCE SPECTROS
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6 Solvent polarity and the local en
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238 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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Principles of Fluorescence Spectroscopy

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