Principles of Fluorescence Spectroscopy

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PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 867 In the application of SPCE it would be an advantage to use gold rather than silver surfaces. Gold does not tarnish and the chemistry for coating the surface with organic molecules is more highly developed for gold than silver. However, in contrast to silver, gold is known to quench fluorescence. 24–26 Fortunately, gold can be used for SPCE (Figure 26.11). 27 The angular distribution is slightly wider on gold than on silver, which is a result of its different optical properties. The reason gold does not quench in SPCE is because of the longer distances for SPCE as compared to Förster transfer, which is probably the mechanism by which gold quenches fluorescence. SPCE is due mostly to fluorophores within about 200 nm of the surface. This suggests the possibility of selective observation of fluorophores localized near the metal surface. We tested this possibility by adding a "background" fluorophore more distant from the metal. This was accomplished by making the coated silver film part of a 1-mmthick demountable cuvette. The cuvette was filled with a solution of rhodamine 6G in ethanol. PVA is weakly soluble in ethanol and the dyes remained separate for the course of the experiments. The free-space emission was observed at 149E and the SPCE at 77E. The concentrations were chosen so that the free-space emission was dominated by the R6G background (Figure 26.12). Even with this large background signal the SPCE was dominated by S101, which is closer to the metal film. The PVA thickness was only 30 nm, so that some R6G was present within the distance for SPCE, accounting for the remaining intensity of R6G at 550 nm. More effective background suppression can be Figure 26.12. Emission spectra of S101 in PVA with simulated background emission from rhodamine 6G (R6G). The concentration of S101 in the PVA film was about 10 mM and the concentration of R6G in ethanol about 5 µM. From [23]. expected with a thicker PVA or sample film to keep this impurity more distant from the metal film. 26.5. APPLICATIONS OF SPCE Since SPCE occurs for fluorophores near surfaces it will most likely be used with surface-based assays. One expected advantage of SPCE is the suppression of unwanted background because fluorophores distant from the metal surface will not create surface plasmons and SPCE. For this assay the surface-bound antibody was rabbit IgG (Figure 26.13). This surface was incubated with rhodamine-labeled antirabbit IgG and Alexa 647-labeled anti-mouse IgG. The later antibody does not bind to the surface and serves as the unwanted background. Figure 26.14 shows the emission spectra of the freespace and surface-plasmon coupled emission. The freespace emission is dominated by the Alexa 647 background. In contrast, the SPCE is due mostly to the surface-bound antibody. This result shows that SPCE can be used to selectively observe fluorophores near the metal surface and to effectively decrease the amount of background emission. SPCE provides a potentially simple approach for improved performance in a wide variety of surface-bound arrays. Intrinsic biochemical fluorophores such as tryptophan emit at ultraviolet wavelengths. It is possible to imagine many biochemical applications of SPCE occurring at shorter wavelengths. SPCE of UV-emitting fluorophores has Figure 26.13. SPCE immunoassay. Anti-rabbit antibodies (labeled with Rhodamine Red-X) bind to rabbit IgG immobilized on the silver surface. Non-binding anti-mouse antibodies labeled with Alexa Fluor 647 remain in solution. From [28].
868 RADIATIVE-DECAY ENGINEERING: SURFACE PLASMON-COUPLED EMISSION Figure 26.14. Emission spectra of the rhodamine red-X labeled antirabbit antibodies bound to rabbit IgG immobilized on a 50-nm silver mirror surface in the presence of a fluorescent background (antimouse antibodies labeled with Alexa Fluor 647). Top, free-space emission. Bottom, SPCE. From [28]. Figure 26.15. Angle-dependent SPCE of 2-aminopurine (2-AP) at 380 nm with reverse Kretschmann excitation. 2-AP is in a PVA film. Revised and reprinted with permission from [39]. Copyright © 2004, American Chemical Society. been observed with aluminum films. 29–31 Figure 26.15 shows the angle-dependent emission of 2-aminopurine in PVA on a 20-nm-thick aluminum film. Once again SPCE occurs over a narrow angular distribution, but the distribution is wider than that found for silver (Figure 26.9). This wider angular distribution is a result of the optical properties of aluminum and appears to result in a greater angular dispersion of different wavelengths (Figure 26.16). The apparent emission spectra of 2-AP are strongly dependent on the observation angle. 26.6. FUTURE DEVELOPMENTS IN SPCE SPCE offers numerous opportunities for new types of assays and sample configurations. A guiding concept may be the radiating plasmon (RP) model. 32 This model suggests that the optical and geometric properties of the sample can be chosen so that plasmons can radiate effectively and in the desired direction. An example of this approach is for a thin silver grating. Excited fluorophores near such a grating display plasmon-coupled emission through the grating, with different wavelengths appearing at different angles. Plasmon-coupled emission through the grating provides opportunities for novel fluorescence-sensing configurations. Figure 26.17 shows an example where a sensing layer is positioned above the thin film grating. Emission from the sensing layer will couple through the grating and could be observed with Figure 26.16. Free-space (top) and SPCE (bottom) of 2-AP. The emission spectra were recorded at different observation angles. Revised and reprinted with permission from [30]. Copyright © 2004, American Chemical Society.
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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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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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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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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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920 ANSWERS TO PROBLEMS CHAPTER 24
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Index A Absorption spectroscopy, 12
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