Principles of Fluorescence Spectroscopy

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646 FLUORESCENCE SENSING Table 19.4. Spectral and Lifetime Properties of Mg 2+ ,Na + , and K + Probes Excitation a Emission Lifetime (ns) a Probe b λ F (λ B ) [nm] λ F (λ B ) [nm] Q F (Q B ) τ F (τ B ) K D (mM) Mg 2+ Probes Mag-Quin-1 348 (335) 499 (490) 0.0015 (0.009) 0.57 (10.3) 6.7 Mag-Quin-2 353 (337) 487 (493) 0.003 (0.07) 0.84 (8.16) 0.8 Mag-Fura-2 369 (330) 511 (491) 0.24 (0.30) 1.64 (1.72) 1.9 Mag-Fura-5 369 (332) 505 (482) NA c 2.52 (2.39) 2.3 Mag-Indo-1 349 (330) 480 (417) 0.36 (0.59) 1.71 (1.90) 2.7 Mag-Fura-Red 483 (427) 659 (631) 0.012 (0.007) 0.38 (0.35) 2.5 Mg Green 506 532 0.04 (0.42) 0.98 (3.63) 1.0 Mg Orange 550 575 0.13 (0.34) 1.06 (2.15) 3.9 Na + Probes SBFI 348 (335) 499 (490) 0.045 (0.083) 0.27 (0.47) 3.8 SBFO 354 (343) 515 (500) 0.14 (0.44) 1.45 (2.09) 31.0 Na Green 506 535 7-fold d 1.14 (2.38) 6.0 K + Probes PBFI 336 (338) 557 (507) 0.24 (0.72) 0.47 (0.72) 5.1 CD 222 396 (363) 480 (467) 3.7-fold 0.17 (0.71) 0.9 aF and B refer to the free and cation-bound forms of the probes, respectively. bAbbreviations: SBFI, sodium-binding benzofuran isophthalate; SBFO, sodium-binding benzofuran oxazole; PBFI, potassium- binding benzofuran isophthalate. c NA: not available. d QB /Q F = 7. binding benzofuran isophthalate (PBFI) for K + (Table 19.4). While designed to be excitation wavelength-ratiometric probes, these probes suffer several disadvantages. They require UV excitation, which results in substantial Figure 19.46. Representative Na + and K + probes. Sodium Green is a trademark of Molecular Probes Inc. amounts of autofluorescence from cells. The excitation spectra show only minor changes in shape upon binding of Na + and K + to these probes (Figure 19.47). Apparently, the charge densities of these singly charged cations are not suf- Figure 19.47. Excitation (SBFI, PBFI and CD 222) and emission (Sodium Green) spectra of Na + and K + probes in the presence of various concentrations of the ions. Sodium Green is a trademark of Molecular Probes Inc. Data from [144].
PRINCIPLES OF FLUORESCENCE SPECTROSCOPY 647 ficient to result in substantial spectral shifts. In support of this hypothesis, one notices that PBFI, which binds the larger K + ion, shows a smaller spectral shift than that seen for binding for Na + and SBFI. A coumarin-based probe is available for K + : CD222. This probe has a more complex chelating group (Figure 19.46) that is directly connected to the coumarin fluorophore. 145–146 This probe can be excited at longer wavelengths than SBFI and PBFI, and displays larger spectral shifts (Figure 19.47). The dissociation constant for K + binding to CD222 is near 1 mM (Table 19.4). This probe is useful for measurements of extracellular K + , but the binding is too strong for measurements of intracellular K + (Table 19.3). The apparent K D of CD222 for K + is increased in the presence of Na + . As a result, CD222 may be useful for measurements of extracellular K + in blood in the concentration range 3–6 mM. 147 Several other probes with a coumarin fluorophore and a chelator for K + have been described. 148–150 Given the availability of blue and UV LEDs, these probes may soon find use in simple clinical devices. In an effort to avoid cellular autofluorescence, several Na + and K + probes have been developed for longer excitation wavelength. One of these probes is Sodium Green TM , which is a sodium-specific azacrown conjugated on both nitrogens to a dichlorofluorescein (Figure 19.46). Sodium Green can be excited at 488 nm, and displays increasing intensity in the presence of increasing concentrations of Na + (Figure 19.47). Unfortunately, Sodium Green does not display any spectral shifts, so that wavelength-ratiometric measurements are not possible. Furthermore, the analogous probe for potassium has not been reported, so that K + probes are limited in number. The inability to develop wavelength-ratiometric probes for Na + and K + , particularly with long excitation and emission wavelengths, illustrates an advantage of lifetime-based sensing. Sodium Green was found to display a multi-exponential decay, with lifetimes of 1.1 and 2.4 ns in the absence and presence of Na + , respectively. 151 The phase and modulation values (Figure 19.48) are independent of total intensity, allowing the concentration of Na + to be determined even if the probe concentration is unknown. Cation-dependent decay times of SBFI and PBFI have been reported. Unfortunately, SBFI, PBFI, and similar probes display only modest changes in lifetime, 152–154 so that these probes do not seem suitable for lifetime-based sensing of Na + or K + . CD222 does display useful changes in lifetime in response to K + , even in the presence of large amounts of Na + . In this case, lifetime-based sensing of K + using CD222 at the con- Figure 19.48. Sodium-dependent phase (φ) and modulation (m) of Sodium Green. Excitation was at 514 nm, and emission was observed above 530 nm. Data from [151]. Figure 19.49. Representative Ca 2+ and Mg 2+ probes. centration present in blood appears to be possible. In contrast, CD222 does not allow wavelength-ratiometric measurements of K + in the presence of 100 mM sodium. 147 19.8.4. Calcium and Magnesium Probes Calcium probes are perhaps the most widely used intracellular indicators (Figure 19.49). These indicators are based on the BAPTA chelator, which binds Ca 2+ with affinities near 100 nM (Table 19.5). These probes are suitable for measurements of intracellular Ca 2+ , but bind Ca 2+ too tightly for measurement of Ca 2+ in blood or serum, which is near 5 mM (Table 19.3). These probes are often used in fluorescence microscopy, where the local probe concentration is
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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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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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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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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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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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912 ANSWERS TO PROBLEMS B. A covale
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920 ANSWERS TO PROBLEMS CHAPTER 24
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Index A Absorption spectroscopy, 12
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