Principles of Fluorescence Spectroscopy

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840 FLUORESCENCE CORRELATION SPECTROSCOPY PROBLEMS P24.1. Figure 24.8 shows autocorrelation curves for several concentrations of R6G. Assume these concentrations are correct. What is the effective volume (V eff ) of the sample? Assume the ratio for the ellipsoidal volume is µ/s = 4.0. What are the dimensions of the ellipsoid? P24.2. Figure 24.10 shows the autocorrelation function for labeled α-lactalbumin (14,000 daltons) in the absence and presence of GroEL (840,000 daltons). Is the change in diffusion time consistent with complete binding of α-lactalbumin to GroEL? P24.3. Figure 24.21 shows autocorrelation functions for GUVs composed of DLPC or DLPC/DPFC (0.2/ 0.8). Assume the laser beam diameter is 1 µm. Using the same diffusion coefficient, calculate the time it takes a Dil-C20 molecule to diffuse 10 µm. Suppose the beam diameter is increased to 2 µm. How long does it take the molecule to diffuse 10 µm? P24.4. Figure 24.32 shows a correlation function for the opening and closing of a molecular beacon. Assume you have access to a steady-state fluorometer with control of the sample temperature. Suggest a way to separately determine the values of k1 and k2 . P24.5. Figure 24.33 shows a typical configuration for FCS using TIR. Suppose you wanted to measure diffusion coefficients near the interface, and the illuminated spot had a diameter of 5.0 µm. Calculate the volume of the observed solution assuming d = 100 nm. What concentration of fluorophore is needed to obtain approximately 10 fluorophores in the volume? Assume the volume contains a single phospholipid bilayer that contains all the probe molecules. What fraction of the lipids need to be labeled to obtain 10 fluorophores in the volume? The area occupied by a single phospholipid molecule is about 70 Å2 . P24.6. Figure 24.50 shows autocorrelation curves for tubulin with cryptophycin as the sample was diluted. Explain how these data can be used to determine if the complex dissociates upon dilution. Assume all the tubulin substrates contain the TMR label. Figure 24.50. Effect of increasing concentrations of cryptophycin on labeled tubulin dimers. Revised and reprinted with permission from [38]. Copyright © 2003, American Chemical Society.
25 In the preceding chapters we described the wide-ranging applications of fluorescence. All these applications relied upon the spontaneous emission of fluorophores in free space. By free space we mean optically transparent nonconducting media. In these final chapters we describe a new topic called radiative decay engineering (RDE). The term RDE is used because the environment around the fluorophore is modified or engineered to change the radiative decay rate of the fluorophore. In Chapter 1 we showed that the radiative decay rate (Γ) is determined by the extinction coefficient of the fluorophore. Extinction coefficients do not change substantially in different environments. Similarly, the radiative rates remain nearly the same under most conditions. The changes in quantum yield or lifetime displayed by fluorophores in different environments are due to changes in the non-radiative decay rates. In this chapter we describe the effects of conducting metallic silver particles on fluorescence. A fluorophore in the excited state has the properties of an oscillating dipole. The excited fluorophore can induce oscillations of the electrons in the metal. The electric field created by the metal can interact with the excited fluorophore and alter its emission. This interaction is almost certainly bidirectional so that light-induced oscillations in the metal can affect the fluorophore. The interactions of fluorophores with metallic surfaces can have a number of useful effects, including increased quantum yields, increased photostability, increased distances for resonance energy transfer, and decreased lifetimes. These changes can result in increased sensitivity, increased photostability, and decreased interference from unwanted background emission. These effects are called metal-enhanced fluorescence (MEF). The mechanisms of metal-enhanced fluorescence are not yet completely understood, and the applications of MEF Radiative Decay Engineering: Metal-Enhanced Fluorescence are just beginning. Because of the potential importance of RDE and MEF we decided to include chapters on these topics. This is a new topic and the field is changing very rapidly. Hence this chapter represents a summary of the current knowledge in this field. RDE and MEF were developed in this laboratory, and as a result many of the examples are from our own work. 25.1. RADIATIVE DECAY ENGINEERING Prior to describing the unusual effects of metal surfaces on fluorescence it is valuable to describe what we mean by RDE and in particular spectral changes expected for increased radiative decay rates. 25.1.1. Introduction to RDE We typically perform fluorescence measurements in macroscopic solutions, or at least macroscopic in comparison with the size of a fluorophore. The solutions are typically transparent to the emitted radiation. There may be modest changes in refractive index, such as for a fluorophore in a membrane, but such changes have a relatively minor effect on the fluorescence spectral properties. 1–2 In such nearly homogeneous solutions, the fluorophores emit into free space and are observed in the far field. Local effects due to surfaces are not usually observed because of the small size of fluorophores relative to the experimental chamber. The spectral properties of a fluorophore in the excited state are well described by Maxwell's equations for an oscillating dipole radiating into free space and we detect the far-field radiation as light. 841
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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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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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Page 871 and 872: 862 RADIATIVE-DECAY ENGINEERING: SU
Page 881 and 882: Appendix I Corrected Emission Spect
Page 891 and 892: Appendix II Fluorescence Lifetime S
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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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