Dynamics of Solvent and Spectral Relaxation

  • Joseph R. Lakowicz


In the preceding chapter we described the effects of solvents on emission spectra and considered how the solvent-dependent data could be interpreted in terms of the local environment. We assumed that the solvent was in equilibrium around the excited-state dipole prior to emission. Equilibrium around the excited-state dipole is reached in fluid solution because the solvent relaxation times are typically less than 100 ps whereas the decay times are usually 1 ns or longer. However, equilibrium around the excited-state dipole is not reached in more viscous solvents and may not be reached for probes bound to proteins or membranes. In these cases emission occurs during solvent relaxation, and the emission spectrum represents an average of the partially relaxed emission. Under these conditions, the emission spectra display time-dependent changes. These effects are not observed in the steady-state emission spectra but can be seen in the time-resolved data or the intensity decays measured at various emission wavelengths.


Emission Spectrum Spectral Shift Impulse Response Function Intensity Decay Dielectric Relaxation Time 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Bakhshiev, N. G., Mazurenko, Yu. T., and Piterskaya, I. V., 1966, Luminescence decay in different portions of the luminescence spectrum of molecules in viscous solution, Opt. Spectrosc. 21: 307–309.Google Scholar
  2. 2.
    Mazurenko, Yu. T., and Bakshiev, N. K., 1970, Effect of orientation dipole relaxation on spectral, time, and polarization characteristics of the luminescence of solutions, Opt. Spectrosc. 28: 490–494.Google Scholar
  3. 3.
    Bakhshiev, N. K., Mazurenko, Yu. T., and Piterskaya, I. V., 1969, Relaxation effects in the luminescence characteristics of viscous solutions, Akad. Nauk SSSR, Bull. Phys. Sci. 32: 1262–1266.Google Scholar
  4. 4.
    Ware, W. R., Lee, S. K., Brant, G J., and Chow, P. P., 1970, Nanosecond time-resolved emission spectroscopy: Spectral shifts due to solvent-excited solute relaxation, J. Chem. Phys. 54: 4729–4737.Google Scholar
  5. 5.
    Ware, W. R., Chow, E, and Lee, S. K.,1968, Time-resolved nanosecond emission spectroscopy: Spectral shifts due to solvent-solute relaxation, Chem. Phys. Lett. 2: 356–358.Google Scholar
  6. 6.
    Chakrabarti, S. K., and Ware, W. R., 1971, Nanosecond time-resolved emission spectroscopy of 1-anilino-8-naphthalene sulfonate, J. Chem. Phys. 55: 5494–5498.CrossRefGoogle Scholar
  7. 7.
    Easter, J. H., DeToma, R. P., and Brand, L., 1976, Nanosecond time-resolved emission spectroscopy of a fluorescence probe adsorbed to L-a-egg lecithin vesicles, Biophys. J. 16: 571–583.CrossRefGoogle Scholar
  8. 8.
    O’Connor, D. V., and Phillips, D., 1984, Time-Correlated Single Photon Counting, Academic Press, New York, pp. 211–251.CrossRefGoogle Scholar
  9. 9.
    Badea, M. G., and Brand, L., 1979, Time-resolved fluorescence measurements, Methods. Enzymol. 61: 378–425.CrossRefGoogle Scholar
  10. 10.
    Bades, M. G., De Toma, R. P., and Brand, L., 1978, Nanosecond relaxation processes in liposomes, Biophys. J. 24: 197–212.CrossRefGoogle Scholar
  11. 11.
    Brand, L., and Gohlke, J. R., 1971, Nanosecond time-resolved fluorescence spectra of a protein-dye complex, J. Biol. Chem. 246: 2317–2324.Google Scholar
  12. 12.
    Gafni, A., De Toma, R. P., Manrow, R. E., and Brand, L., 1977, Nanosecond decay studies of a fluorescence probe bound to apomyoglobin, Biophys. J. 17: 155–168.CrossRefGoogle Scholar
  13. 13.
    Lakowicz, J. R., and Cherek, H., 1981, Proof of nanosecond time-scale relaxation in apomyoglobin by phase fluorometry, Biochem. Biophys. Res. Commun. 99: 1173–1178.CrossRefGoogle Scholar
  14. 14.
    Lakowicz, J. R., Gratton, E., Cherek, H., Maliwal, B. P., and Laczko, G., 1984, Determination of time-resolved fluorescence emission spectra and anisotropies of a fluorophore-protein complex using frequency-domain phase-modulation fluorometry, J. Biol. Chem. 259: 10967–10972.Google Scholar
  15. 15.
    Pierce, D. W., and Boxer, S. G., 1992, Dielectric relaxation in a protein matrix, J. Phys. Chem. 96: 5560–5566.CrossRefGoogle Scholar
  16. 16.
    Wang, R., Sun, S., Bekos, E. J., and Bright, F. V., 1995, Dynamics surrounding Cys-34 in native, chemically denatured, and silica-adsorbed bovine serum albumin, Anal. Chem. 67: 149–159.CrossRefGoogle Scholar
  17. 17.
    Demchenko, A. P., Apell, H. -J., Stürmer, W., and Feddersen, B., 1993, Fluorescence spectroscopic studies on equilibrium dipole-relaxational dynamics of Na,K-ATPase, Biophys. Chem. 48: 135–147.CrossRefGoogle Scholar
  18. 18.
    Easter, J. H., and Brand, L., 1973, Nanosecond time-resolved emission spectroscopy of a fluorescence probe bound to L-a-egg lecithin vesicles, Biochem. Biophys. Res. Commun. 52: 1086–1092.CrossRefGoogle Scholar
  19. 19.
    Easter, J. H., DeToma, R. P., and Brand, L., 1978, Fluorescence measurements of environmental relaxation at the lipid-water interface region of bilayer membranes, Biochim. Biophys. Acta 508: 27–38.CrossRefGoogle Scholar
  20. 20.
    DeToma, R. P., Easter, J. H., and Brand, L., 1976, Dynamic interactions of fluorescence probes with the solvent environment, J. Am. Chem. Soc. 98: 5001–5007.CrossRefGoogle Scholar
  21. 21.
    Lakowicz, J. R., Cherek, H., Laczko, G., and Gratton, E., 1984, Time-resolved fluorescence emission spectra of labeled phospholipid vesicles, as observed using multifrequency phase-modulation fluorometry, Biochim. Biophys. Acta 777: 183–193.CrossRefGoogle Scholar
  22. 22.
    Parasassi, T., Conti, F., and Gratton, E., 1986, Time-resolved fluorescence emission spectra of laurdan in phospholipid vesicles by multifrequency phase and modulation fluorometry, Cell. Mol. Biol. 32: 103–108.Google Scholar
  23. 23.
    Sommer, A., Paltauf, F., and Hermetter, A., 1990, Dipolar solvent relaxation on a nanosecond time scale in ether phospholipid membranes as determined by multifrequency phase and modulation fluorometry, Biochemistry 29: 11134–11140.CrossRefGoogle Scholar
  24. 24.
    Hutterer, R., Schneider, F. W., Lanig, H., and Hof, M., 1997, Solvent relaxation behaviour of n-anthroyloxy fatty acids in PC-vesicles and paraffin oil: A time-resolved emission spectra study, Biochim. Biophys. Acta 1323: 195–207.CrossRefGoogle Scholar
  25. 25.
    Stryer, L., 1965, The interactions of a naphthalene dye with apomyoglobin and apohemoglobin. A fluorescent probe of non-polar binding sites, J. Mol. Biol. 13: 482–495.CrossRefGoogle Scholar
  26. 26.
    Murakami, H., and Kushida, T., 1994, Fluorescence properties of Zn-substituted myoglobin, J. Lumin. 58: 172–175.CrossRefGoogle Scholar
  27. 27.
    Lakowicz, J. R., and Baiter, A., 1982, Theory of phase-modulation fluorometry for excited state processes, Biophys. Chem. 16: 99–115.CrossRefGoogle Scholar
  28. 28.
    Grinvald, A., and Steinberg, I. Z., 1974, Fast relaxation processes in a protein revealed by the decay kinetics of tryptophan fluorescence, Biochemistry 13: 5170–5178.CrossRefGoogle Scholar
  29. 29.
    Lakowicz, J. R., and Cherek, H., 1980, Dipolar relaxation in proteins on the nanosecond timescale observed by wavelength-resolved phase fluorometry of tryptophan fluorescence, J. Biol. Chem. 255: 831–834.Google Scholar
  30. 30.
    Lakowicz, J. R., and Baiter, A., 1982, Resolution of initially excited and relaxed states of tryptophan fluorescence by differential-wavelength deconvolution of time-resolved fluorescence decays, Biophys. Chem. 15: 353–360.CrossRefGoogle Scholar
  31. 31.
    Lakowicz, J. R., Szmacinski, H., and Gryczynski, I., 1988, Picosecond resolution of indole anisotropy decays and spectral relaxation by 2 GHz frequency-domain fluorometry, Photochem. Photobiol. 47: 31–41.CrossRefGoogle Scholar
  32. 32.
    Szmacinski, H., Lakowicz, J. R., and Gryczynski, I., unpublished observations.Google Scholar
  33. 33.
    Demchenko, A. P., Gryczynski, I., Gryczynski, Z., Wiczk, W., Malak, H., and Fishman, M., 1993, Intramolecular dynamics in the environment of the single tryptophan residue in staphylococcal nuclease, Biophys. Chem. 48: 39–48.CrossRefGoogle Scholar
  34. 34.
    Demchenko, A. P., 1994, Protein fluorescence, dynamics and function: Exploration of analogy between electronically excited and biocatalytic transition states, Biochim. Biophys. Acta 1209: 149164.Google Scholar
  35. 35.
    Vekshin, N., Vincent, M., and Gallay, J., 1992, Excited-state lifetime distributions of tryptophan fluorescence in polar solutions. Evidence for solvent exciplex formation, Chem. Phys. Lett. 199: 459–464.CrossRefGoogle Scholar
  36. 36.
    Vincent, M., Gallay, J., and Demchenko, A. E,1995, Solvent relaxation around the excited state of indole: Analysis of fluorescence lifetime distributions and time-dependence spectral shifts, J. Phys. Chem. 99: 14931–14941.Google Scholar
  37. 37.
    Vincent, M., Gallay, J., and Demchenko, A. P., 1997, Dipolar relaxation around indole as evidenced by fluorescence lifetime distributions and time-dependence spectral shifts, J. Fluoresc. 7: 107S - 1105.CrossRefGoogle Scholar
  38. 38.
    Weber, G., and Lakowicz, J. R., 1973, Subnanosecond solvent relaxation studies by oxygen quenching of fluorescence, Chem. Phys. Lett. 22: 419–423.CrossRefGoogle Scholar
  39. 39.
    Rotkiewicz, K., Grabowski, Z. R., and Jasny, J., 1975, Picosecond isomerization kinetics of excited p-dimethylaminobenzonitriles studied by oxygen quenching of fluorescence, Chem. Phys. Lett. 34: 55–100.CrossRefGoogle Scholar
  40. 40.
    Lakowicz, J. R., and Hogen, D., 1981, Dynamic properties of the lipid-water interface of model membranes as revealed by lifetime-resolved fluorescence emission spectra, Biochemistry 20: 1366 1373.Google Scholar
  41. 41.
    Fleming, G. R., and Cho, M., 1996, Chromophore-solvent dynamics, Annu. Rev. Phys. Chem. 47: 109–134.CrossRefGoogle Scholar
  42. 42.
    Castner, E. W., Maroncelli, M., and Fleming, G. R., 1987, Subpicosecond resolution studies of solvation dynamics in polar aprotic and alcohol solvents, J. Chem. Phys. 86: 1090–1097.CrossRefGoogle Scholar
  43. 43.
    Su, S.-G., and Simon, J. D., 1987, Solvation dynamics in ethanol, J. Phys. Chem. 91: 2693–2696.CrossRefGoogle Scholar
  44. 44.
    Simon, J. D., 1988, Time-resolved studies of solvation in polar media, Acc. Chem. Res. 21: 128–134.CrossRefGoogle Scholar
  45. 45.
    Chapman, C. F., Fee, R. S., and Maroncelli, M., 1995, Measurements of the solute dependence of solvation dynamics in 1-propanol: The role of specific hydrogen-bonding interactions, J. Phys. Chem. 99: 4811–4819.CrossRefGoogle Scholar
  46. 46.
    Kahlow, M. A., Jarzeba, W., Kang, T. J., and Barbara, P. F, 1989, Femtosecond resolved solvation dynamics in polar solvents, J. Chem. Phys. 90: 151–158.CrossRefGoogle Scholar
  47. 47.
    Maroncelli, M., and Fleming, G. R., 1987, Picosecond solvation dynamics of coumarin 153: The importance of molecular aspects of solvation, J. Chem. Phys. 86: 6221–6239.CrossRefGoogle Scholar
  48. 48.
    Jarzeba, W., Walker, G. C., Johnson, A. E., Kahlow, M. A., and Barbara, P. F., 1988, Femtosecond microscopic solvation dynamics of aqueous solutions, J. Phys. Chem. 92: 7039–7041.CrossRefGoogle Scholar
  49. 49.
    Bagchi, B., Oxtoby, D. W., and Fleming, G. R., 1984, Theory of the time development of the Stokes shift in polar media, Chem. Phys. 86:257–267.CrossRefGoogle Scholar
  50. 50.
    Castner, E. W., Fleming, G. R., and Bagchi, B., 1988, Influence of non-Debye relaxation and of molecular shape on the time-dependence of the Stokes shift in polar media, Chem. Phys. Lett. 143: 270–276.CrossRefGoogle Scholar
  51. 51.
    Castner, E. W., Bagchi, B., Maroncelli, M., Webb, S. P., Ruggiero, A. J., and Fleming, G. R., 1988, The dynamics of polar solvation, Ber. Bunsenges. Phys. Chem. 92: 363–372.Google Scholar
  52. 52.
    Bakhshiev, N. G., 1964, Universal intermolecular interactions and their effect on the position of the electronic spectra of molecules in two-component solutions, VII. Theory (general case of an isotropic solution), Opt. Spectrosc. 16: 446–451.Google Scholar
  53. 53.
    Castner, E. W, Fleming, G. R., Bagchi, B., and Maroncelli, M., 1988, The dynamics of polar solvation: Inhomogeneous dielectric continuum models, J. Chem. Phys. 89: 3519–3534.CrossRefGoogle Scholar
  54. 54.
    Piterskaya, I. V., and Bakhshiev, N. G., 1963, Quantitative investigation of the temperature dependence of the absorption and fluorescence spectra of complex molecules, Akad. Nauk SSSR, Bull. Phys. Sci. 27: 625–629.Google Scholar
  55. 55.
    Bushuk, B. A., and Rubinov, A. N., 1997, Effect of specific intermolecular interactions on the dynamics of fluorescence spectra of dye solutions, Opt. Spectrosc. 82: 530–533.Google Scholar
  56. 56.
    Kaatze, U., and Uhlendorf, V., 1981, The dielectric properties of water at microwave frequencies, Z Phys. Chem. N. E. 126: 151–165.CrossRefGoogle Scholar
  57. 57.
    Cole, K. S., and Cole, R. H., 1941, Dispersion and absorption in dielectrics, J. Chem. Phys. 9: 341–351.CrossRefGoogle Scholar
  58. 58.
    Fellner-Feldegg, H., 1969, The measurement of dielectrics in the time domain, J. Phys. Chem. 75: 616–623.CrossRefGoogle Scholar
  59. 59.
    Davidson, D. W., and Cole, R. H., 1951, Dielectric relaxation in glycerol, propylene glycol, and n-propanol, J. Chem. Phys. 19: 1484–1490.CrossRefGoogle Scholar
  60. 60.
    Denny, D. J., and Cole, R. H., 1955, Dielectric properties of methanol and methanol-1-propanol solutions, J. Chem. Phys. 23: 1767–1772.CrossRefGoogle Scholar
  61. 61.
    McDuffie, G. E., and Litovitz, T. A., 1962, Dielectric relaxation in associated liquids, J. Chem. Phys. 37: 1699–1705.CrossRefGoogle Scholar
  62. 62.
    Gard, S. K., and Smyth, C. P., 1965, Microwave absorption and molecular structure in liquids LXII. The three dielectric dispersion regions of the normal primary alcohols, J. Phys. Chem. 69: 1294–1301.CrossRefGoogle Scholar
  63. 63.
    Bamford, C. H., and Compton, R. G., 1985, Chemical Kinetics, Elsevier, New York, 404 pp.Google Scholar
  64. 64.
    Szmacinski, H., Gryczynski, I., and Lakowicz, J. R., 1996, Resolution of multiexponential spectral relaxation of Yt-base by global analysis of collisionally quenched samples, J. Fluoresc. 6: 177–185.CrossRefGoogle Scholar
  65. 65.
    Veselova, T. V., Limareva, L. A., Cherkasov, A. S., and Shirokov, V. I., 1965, Fluorometric study of the effect of solvent on the fluorescence spectrum of 3-amino-N-methylphthalimide, Opt. Spectrosc. 19: 39–43.Google Scholar
  66. 66.
    Lakowicz, J. R., Bevan, D. R., Maliwal, B. P., Cherek, H., and Batter, A., 1983, Synthesis and characterization of a fluorescence probe of the phase transition and dynamic properties of membranes, Biochemistry 22: 5714–5722.CrossRefGoogle Scholar
  67. 67.
    Lakowicz, J. R., Cherek, H., and Bevan, D. R., 1980, Demonstration of nanosecond dipolar relaxation in biopolymers by inversion of apparent fluorescence phase shift and demodulation lifetimes, J. Biol. Chem. 255: 4403–4406.Google Scholar
  68. 68.
    Röcker, C., Heilemann, A., and Fromherz, P., 1996, Time-resolved fluorescence of a hemicyanine dye: Dynamics of rotamerism and resolvation, J. Phys. Chem. 100: 12172–12177.CrossRefGoogle Scholar
  69. 69.
    Knutson, J. R., Walbridge, D. G., and Brand, L., 1982, Decay associated fluorescence spectra and the heterogeneous emission of alcohol dehydrogenase, Biochemistry 21: 4671–4679.CrossRefGoogle Scholar
  70. 70.
    Rudik, K. I., and Pikulik, L. G., 1971, Effect of the exciting light on the fluorescence spectra of phthalimide solutions, Opt. Spectrosc. 30: 147–148.Google Scholar
  71. 71.
    Rubinov, A. N., and Tomin, V.I., 1970, Bathochromic luminescence in solutions of organic dyes at low temperatures, Opt. Spectrosc. 29: 578–580.Google Scholar
  72. 72.
    Galley, W. C., and Purkey, R. M., 1970, Role of heterogeneity of the solvation site in electronic spectra in solution, Proc. Natl. Acad. Sci. U.S.A. 67: 1116–1121.CrossRefGoogle Scholar
  73. 73.
    Itoh, K., and Azumi, T., 1973, Shift of emission band upon excitation at the long wavelength absorption edge, Chem. Phys. Lett. 22: 395399.Google Scholar
  74. 74.
    Azumi, T., Itoh, K., and Shiraishi, H., 1976, Shift of emission band upon the excitation at the long wavelength absorption edge. III. Temperature dependence of the shift and correlation with the time dependent spectral shift, J. Chem. Phys. 65: 2550–2555.CrossRefGoogle Scholar
  75. 75.
    Itoh, K., and Azumi, T., 1975, Shift of the emission band upon excitation at the long wavelength absorption edge. II. Importance of the solute–solvent interaction and the solvent reorientation relaxation process, J. Chem. Phys. 62: 3431–3438.CrossRefGoogle Scholar
  76. 76.
    Kawski, A., Ston, M., and Janie, I., 1983, On the intensity distribution within photoluminescence bands in rigid and liquid solutions, Z Naturforsch. A 38: 322–324.Google Scholar
  77. 77.
    Lakowicz, J. R., and Keating-Nakamoto, S., 1984, Red-edge excitation of fluorescence and dynamic properties of proteins and membranes, Biochemistry 23: 3013–3021.CrossRefGoogle Scholar
  78. 78.
    Demchenko, A. P., 1982, On the nanosecond mobility in proteins: Edge excitation fluorescence red shift of protein-bound 2-(p-toluidinylnaphthalene)-6-sulfonate, Biophys. Chem. 15: 101–109.CrossRefGoogle Scholar
  79. 79.
    Shcherbatska, N. V., van Hoek, A., Visser, A. J. W. G., and Koziol, J., 1994, Molecular relaxation spectroscopy of lumichrome, J. Photochem. Photobiol., A: Chem. 78: 241–246.Google Scholar
  80. 80.
    Demchenko, A. P., and Shcherbatska, N. V., 1985, Nanosecond dynamics of charged fluorescent probes at the polar interface of a membrane phospholipid bilayer, Biophys. Chem. 22: 131–143.CrossRefGoogle Scholar
  81. 81.
    Raudino, A., Guerrera, F., Asero, A., and Rizza, V., 1983, Application of red-edge effect on the mobility of membrane lipid polar head groups, FEBS Lett. 159: 43–46.CrossRefGoogle Scholar
  82. 82.
    Demchenko, A. P., and Ladokhin, A. S., 1988, Temperature-dependent shift of fluorescence spectra without conformational changes in protein: Studies of dipole relaxation in the melittin molecule, Biochim. Biophys. Acta 955: 352–360.CrossRefGoogle Scholar
  83. 83.
    Conti, C., and Forster, L. S., 1974, Non-exponential decay of indole fluorescence—the red-edge effect, Biochem. Biophys. Res. Commun. 57: 1287–1292.CrossRefGoogle Scholar
  84. 84.
    Nemkovich, N. A., Rubinov, A. N., and Tomin, V. I., 1981, Kinetics of luminescence spectra of rigid dye solutions due to directed electronic energy transfer, J Lumin. 23: 349–361.CrossRefGoogle Scholar
  85. 85.
    Milton, J. G., Purkey, R. M., and Galley, W. C., 1978, The kinetics of solvent reorientation in hydroxylated solvents from the exciting-wavelength dependence of chromophore emission spectra, J Chem. Phys. 68: 5396–5403.CrossRefGoogle Scholar
  86. 86.
    Morgenthaler, M. J. E., Meech, S. R., and Yoshihara, K., 1992, The inhomogeneous broadening of the electronic spectra of dyes in glycerol solution, Chem. Phys. Lett. 197: 537–541.CrossRefGoogle Scholar
  87. 87.
    Gakamsky, D. M., Demchenko, A. E, Nemkovich, N. A., Rubinov, A. N., Tomin, V. I., and Shcherbatska, N. V, 1992, Selective laser spectroscopy of 1-phenylnaphthylamine in phospholipid membranes, Biophys. Chem. 42: 49–61.CrossRefGoogle Scholar
  88. 88.
    Weber, G., and Shinitzky, M., 1970, Failure of energy transfer between identical aromatic molecules on excitation at the long wave edge of the absorption spectrum, Proc. Natl. Acad. Sci. U.S.A. 65: 823–830.CrossRefGoogle Scholar
  89. 89.
    Valeur, B., and Weber, G., 1977, Anisotropic rotations in 1-naphthylamine, existence of a red-edge transition moment normal to the ring plane, Chem. Phys. Lett. 45: 140–144.CrossRefGoogle Scholar
  90. 90.
    Valeur, B., and Weber, G., 1978, A new red-edge effect in aromatic molecules: Anomaly of apparent rotation revealed by fluorescence polarization, J. Phys. Chem. 69: 2393–2400CrossRefGoogle Scholar
  91. 91.
    Mantulin, W. W., and Weber, G., 1977, Rotational anisotropy and solvent–fluorophore bonds: An investigation by differential polarized-phase fluorometry, J. Chem. Phys. 66: 4092–4099.CrossRefGoogle Scholar
  92. 92.
    Brand, L., Seliskar, C. J., and Turner, D. C., 1971, The effects of chemical environment on fluorescence probes, in Probes of Structure and Function of Macromolecules and Membranes, B. Chance, C. P. Lee, and J. K. Blaisie (eds.), Academic Press, New York, pp. 17–39.Google Scholar

Copyright information

© Springer Science+Business Media New York 1999

Authors and Affiliations

  • Joseph R. Lakowicz
    • 1
  1. 1.University of Maryland School of MedicineBaltimoreUSA

Personalised recommendations