Principles of Fluorescence Spectroscopy

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PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 621 50. Maiti S, Shear JB, Williams RM, Zipfel WR, Webb WW. 1997. Measuring serotonin distribution in live cells with three-photon excitation. Science 275:530–532. 51. Gostkowski ML, Allen R, Plenert ML, Okerberg E, Gordon MJ, Shear JB. 2004. Multiphoton-excited serotonin photochemistry. Biophys J 86:3223–3229. 52. Konig K. 2000. Multiphoton microscopy in life sciences. J Microsc 200(2):83–104. 53. Blatter LA, Niggli E. 1998. Confocal near-membrane detection of calcium in cardiac myocytes. Cell Calcium 23(5):269–279. 54. Niggli E, Egger M. 2004. Applications of multi-photon microscopy in cell physiology. Front Biosci 9:1598–1610. 55. Fan GY, Fujisaki H, Miyawaki A, Tsay RK, Tsien RY, Ellisman MH. 1999. Video-rate scanning two–photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys J 76:2412–2420. 56. Miyawaki A, Llopis J, Heim R, McCaffery JM, Adams JA, Ikura M, Tsien RY. 1997. Fluorescent indicators for Ca 2+ based on green fluorescent proteins and calmodulin. Nature 388:882–887. 57. Huang S, Heikal AA, Webb WW. 2002. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys J 82:2811–2825. 58. Konig K, Riemann I. 2003. High-resolution multiphoton tomography of human skin with subcellular spatial resolution and picosecond time resolution. J Biomed Opt 8(3):432–439. 59. Bewersdorf J, Hell SW. 1998. Picosecond pulsed two-photon imaging with repetition rates of 200 and 400 MHz. J Microsc 191(1): 28–38. 60. Courtesy of Dr. Ignacy Gryczynski. PROBLEMS Figure 18.27. Steady-state anisotropies of DPPS. From [60]. P18.1. Anisotropies of a Styrene Derivative: Figure 18.26 shows the steady-state anisotropies of 4-Dimethylamino-ω-diphenylphosphinyl-trans-styrene (DPPS) in n-butanol. The anisotropies are higher for two-photon excitation than for one-photon excitation. In both cases the anisotropies are independent of temperature. Explain both results. The difference in anisotropy has been explained in the text, but not the reason for a constant anisotropy at all temperatures. P18.2. In Figure 18.17 the density of datapoints is less with Ti:sapphire excitation (855 nm) than with dye-laser (570 nm) or frequency-doubled dye-laser (285 nm) excitation. Suggest reasons why this is the case. P18.3. Explain the direction of the intensity ratio change shown in Figure 18.21.
19 Fluorescence sensing of chemical and biochemical analytes is an active area of research. 1–8 These efforts are driven by the desire to eliminate the use of radioactive tracers, which are costly to use and dispose of. There is also a need for rapid and low-cost testing methods for a wide range of clinical, bioprocess, and environmental applications. During the past decade numerous methods based on high-sensitivity fluorescence detection have been introduced, including DNA sequencing, DNA fragment analysis, fluorescence staining of gels following electrophoretic separation, and a variety of fluorescence immunoassays. Many of these analytical applications can be traced to the early reports by Undenfriend and coworkers, 9 which anticipated many of today's applications of fluorescence. The more recent monographs 6–8 have summarized the numerous analytical applications of fluorescence. Why is fluorescence rather than absorption used for high-sensitivity detection? Fluorescence is more sensitive because of the different ways of measuring absorbance and fluorescence. Light absorbance is measured as the difference in intensity between light passing through the reference and the sample. In fluorescence the intensity is measured directly, without comparison with a reference beam. Consider a 10 –10 M solution of a substance with a molar extinction coefficient of 10 5 M –1 cm –1 . The absorbance will be 10 –5 per cm, which is equivalent to a percentage transmission of 99.9977%. Even with exceptional optics and electronics, it will be very difficult to detect the small percentage of absorbed light, 0.0023%. Even if the electronics allow measurement of such a low optical density, the cuvettes will show some variability in transmission and surface reflection, which will probably exceed the intensity difference due to an absorbance of 10 –5 . In contrast, fluorescence detection at 10 –10 M is readily accomplished with most fluorometers. This advantage is due to measurement of the fluorescence relative to a dark background, as compared to the bright reference beam in an absorbance meas- Fluorescence Sensing urement. It is relatively easy to detect low levels of light, and the electronic impulses due to single photons are measurable with most photomultiplier tubes. In this chapter we describe the various approaches to fluorescence sensing, which include essentially all the phenomenon discussed in previous chapters. Fluorescence sensing is described mostly within the framework of the medical applications, but it is clear that fluorescence detection is also widely used in biochemical, chemical, environmental and forensic analysis. 19.1. OPTICAL CLINICAL CHEMISTRY AND SPECTRAL OBSERVABLE One long-range goal of fluorescence sensing is noninvasive monitoring of clinically relevant species and physiological parameters (Figure 19.1). A suitable portable device would measure the clinical values of interest, then store and/or transmit them to the physician. At present we are rather distant from the ultimate goal of noninvasive testing with devices similar to cell phones. The limitation is not in optics or electronics, but rather due to the lack of stable and biocompatible methods of in-vivo sensing. It is already possible to measure fluorescence through skin, and the measurements can accurately return pH and ion concentrations. In the near term we are likely to see devices similar to PDAs (Figure 19.2) that contain the chemistry and optics needed to perform clinical assays using body fluid samples. Such portable devices would find widespread usefulness, especially in emergency situations. The sensor array could be exposed to blood, and the results would be immediately available. This concept of rapid point-of-care clinical chemistry is driving the rapid development of numerous fluorescence sensing devices. In the following sections we describe the principles of fluorescence sensing, and illustrate how such devices can provide analytical data. 623
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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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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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Page 585 and 586: PRINCIPLES OF FLUORESCENCE SPECTROS
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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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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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