Fluorescence Anisotropy

  • Joseph R. Lakowicz
Chapter

Abstract

Upon excitation with polarized light, the emission from many samples is also polarized. The extent of polarization of the emission is described in terms of the anisotropy (r). Samples exhibiting nonzero anisotropies are said to display polarized emission. The origin of these phenomena is based on the existence of transition moments for absorption and emission which lie along specific directions within the fluorophore structure. In homogeneous solution the ground-state fluorophores are all randomly oriented. When exposed to polarized light, those fluorophores which have their absorption transition moments oriented along the electric vector of the incident light are preferentially excited. Hence, the excited-state population is not randomly oriented. Instead, there is a somewhat larger number of excited molecules having their transition moments oriented along the electric vector of the polarized exciting light.

Keywords

Fluorescence Anisotropy Rotational Diffusion Transition Moment Rotational Correlation Time Emission Polarizer 
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.
This is a preview of subscription content, log in to check access

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Jabofiski, A., 1960, On the notion of emission anisotropy, Bull. Acad. Pol. Sci. Ser. A 8: 259–264.Google Scholar
  2. 2.
    Weber, G., 1952, Polarization of the fluorescence of macromolecules. I. Theory and experimental method, Biochem. J. 51: 145–155.Google Scholar
  3. 3.
    Weber, G., 1966, Polarization of the fluorescence of solutions, in Fluorescence and Phosphorescence Analysis, D. M. Hercules (ed.), John Wiley amp; Sons, New York, pp. 217–240.Google Scholar
  4. 4.
    Selényi, P., 1939, Wide-angle interferences and the nature of the elementary light sources, Phys. Rev. 56: 477–479.CrossRefGoogle Scholar
  5. 5.
    Lakowicz, J. R., and Gryczynski, I., 1997, Multiphoton excitation of biochemical fluorophores, in Topics in Fluorescence Spectroscopy, Volume 5, Nonlinear and Two-Photon-Induced Fluorescence, J. R. Lakowicz (ed.), Plenum Press, New York, pp. 87–144.Google Scholar
  6. 6.
    Weber, G., 1972, Use of fluorescence in biophysics: Some recent developments, Annu. Rev. Biophys. Bioeng. 1: 553–570.CrossRefGoogle Scholar
  7. 7.
    Shinitzky, M., and Barenholz, Y., 1974, Dynamics of the hydrocarbon layer in liposomes of lecithin and sphingomyelin containing dicetylphosphate, J. Biol. Chem. 249: 2652–2657.Google Scholar
  8. 8.
    Michl, J., and Thulstrup, E. W., 1986, Spectroscopy with Polarized Light, VCH Publishers, New York.Google Scholar
  9. 9.
    Albinsson, B., Kubista, M., Nordén, B., and Thulstrup, E. W., 1989, Near-ultraviolet electronic transitions of the tryptophan chromophore: Linear dichroism, fluorescence anisotropy, and magnetic circular dichroism spectra of some indole derivatives, J. Phys. Chem. 93: 6646–6655.CrossRefGoogle Scholar
  10. 10.
    Albinsson, B., Eriksson, S., Lyng, R., and Kubista, M., 1991, The electronically excited states of 2-phenylindole, Chem. Phys. 151: 149–157.CrossRefGoogle Scholar
  11. 11.
    Kubista, M., Akerman, B., and Albinsson, B., 1989, Characterization of the electronic structure of 4’,6-diamidino-2-phenylindole, J. Am. Chem. Soc. 111: 7031–7035.CrossRefGoogle Scholar
  12. 12.
    Vincent, M., de Foresta, B., Gallay, J., and Alfsen, A., 1982, Nanosecond fluorescence anisotropy decays of n-(9-anthroyloxy) fatty acids in dipalmitoylphosphatidylcholine vesicles with regard to isotropic solvents, Biochemistry 21: 708–716.CrossRefGoogle Scholar
  13. 13.
    Weber, G., 1960, Fluorescence-polarization spectrum and electronic-energy transfer in tyrosine, tryptophan and related compounds, Biochem. J. 75: 335–345.Google Scholar
  14. 14.
    Valeur, B., and Weber, G., 1977, Resolution of the fluorescence excitation spectrum of indole into the 1La and 1Lb excitation bands, Photochem. Photobiol. 25: 441–444.CrossRefGoogle Scholar
  15. 15.
    Eftink, M. R., Selvidge, L. A., Callis, P. R., and Rehms, A. A., 1990, Photophysics of indole derivatives: Experimental resolution of La and Lb transitions and comparison with theory, J. Phys. Chem. 94: 3469–3479.CrossRefGoogle Scholar
  16. 16.
    Yamamoto, Y., and Tanaka, J., 1972, Polarized absorption spectra of crystals of indole and its related compounds, Bull. Chem. Soc. Jpn. 65: 1362–1366.CrossRefGoogle Scholar
  17. 17.
    Suwaiyan, A., and Zwarich, R., 1987, Absorption spectra of substituted indoles in stretched polyethylene films, SpectrochimActa, Part A 41A: 605–609.CrossRefGoogle Scholar
  18. 18.
    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
  19. 19.
    Kawski, A., 1992, Fotoluminescencja Rortworów, Wydawnictwo Naukowe PWN, Warsaw, p. 306.Google Scholar
  20. 20.
    Jabofiski, A., 1970, Anisotropy of fluorescence of molecules excited by excitation transfer, Acta Phys. Pol. A 38: 453–458. 42.Google Scholar
  21. 21.
    Baumann, J., and Fayer, M. D., 1986, Excitation transfer in disordered two-dimensional and anisotropie three-dimensional systems: Effects of spatial geometry on time-resolved observables, J. Chem. Phys. 85: 4087–4107.CrossRefGoogle Scholar
  22. 22.
    Teale, E W. J., 1969, Fluorescence depolarization by light scattering in turbid solutions, Photochem. Photobiol. 10:363–374. 44.Google Scholar
  23. 23.
    Lentz, B. R., 1979, Light scattering effects in the measurement of membrane microviscosity with diphenylhexatriene, Biophys. J. 25: 489–494.CrossRefGoogle Scholar
  24. 24.
    Berberan-Santos, M. N., Nunes Pereira, E. J., and Martinho, J. M. G., 1995, Stochastic theory of molecular radiative transport, J. Chem. Phys. 103: 3022–3028.CrossRefGoogle Scholar
  25. 25.
    Nunes Pereira, E. J., Berberan-Santos, M. N., and Martinho, J. M. G., 1996, Molecular radiative transport. II. Monte-Carlo simulation, J. Chem. Phys. 104: 8950–8965.CrossRefGoogle Scholar
  26. 26.
    Perrin, E, 1929, La fluorescence des solutions. Induction moléculaire. Polarisation et durée d’émission. Photochimie, Ann. Phys. Ser. 10 12: 169–275.Google Scholar
  27. 27.
    Perrin, E, 1926, Polarisation de la lumière de fluorescence. Vie moyenne des molécules dans l’état excité, J. Phys. Radium V, Ser. 67: 390–401.Google Scholar
  28. 28.
    Perrin, E, 1931, Fluorescence. Durée élémentaire d’émission lumineuse, Conférences d’Actualités Scientifiques et Industrielles XXII: 2–41.Google Scholar
  29. 29.
    Weber, G., 1953, Rotational Brownian motion and polarization of the fluorescence of solutions, Adv. Protein Chem. 8: 415–459.CrossRefGoogle Scholar
  30. 30.
    Yguerabide, J., Epstein, H. E, and Stryer, L., 1970, Segmental flexibility in an antibody molecule, J. Mol. Biol. 51:573–590. 50.Google Scholar
  31. 31.
    Weber, G., 1952, Polarization of the fluorescence of macromolecules. II. Fluorescence conjugates of ovalbumin and bovine serum albumin, Biochem. J. 51: 155–167.Google Scholar
  32. 32.
    Laurence, D. J. R., 1952, A study of the absorption of dyes on bovine serum albumin by the method of polarization of fluorescence, Biochem. J. 51: 168–180.Google Scholar
  33. 33.
    Gottlieb, Y. Ya., and Wahl, E, 1963, Étude théorique de la polarisation de fluorescence des macromolécules portant un groupe émetteur mobile autour d’un axe de rotation, J. Chim. Phys. 60: 849–856.Google Scholar
  34. 34.
    Ferguson, B. Q., and Yang, D. C. H., 1986, Methionyl-tRNA synthetase induced 3’-terminal and delocalized conformational transition in tRNAtMe1: Steady-state fluorescence of tRNA with a single fluorophore, Biochemistry 25: 529–539.CrossRefGoogle Scholar
  35. 35.
    Buchner, J., 1996, Supervising the fold: Functional principals of molecular chaperones, FASEB J. 10: 10–19.Google Scholar
  36. 36.
    Ellis, R. J., 1996, The Chaperonins, Academic Press, New York.Google Scholar
  37. 37.
    Braig, K., Otwinowski, Z., Hegde, R., Bolsvert, D. C., Joachimiak, A., Horwich, A. L., and Sigler, R B., 1994, The crystal structure of the bacterial chaperonin GroEL at A, Nature 371: 578–586.CrossRefGoogle Scholar
  38. 38.
    Gorovits, B. M., and Horowitz, P. M., 1995, The molecular chaperonin cpn60 displays local flexibility that is reduced after binding with an unfolded protein, J. Biol. Chem. 270: 13057–13062.CrossRefGoogle Scholar
  39. 39.
    Lim, K., Jameson, D. M., Gentry, C. A., and Herron, J. N., 1995, Molecular dynamics of the anti–fluorescein 4–4–20 antigen–binding fragment. 2. Time–resolved fluorescence spectroscopy, Biochemistry 34: 6975 – 6984.CrossRefGoogle Scholar
  40. 40.
    Lukas, T. J., Burgess, W. H., Prendergast, E G., Lau, W., and Watterson, D. M., 1986, Calmodulin binding domains: Characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase, Biochemistry 25: 1458–1464.CrossRefGoogle Scholar
  41. 41.
    Malencik, D. A., and Anderson, S. R., 1984, Peptide binding by calmodulin and its proteolytic fragments and by troponin C, Biochemistry 23: 2420–2428.CrossRefGoogle Scholar
  42. 42.
    LeTilly, V., and Royer, C. A., 1993, Fluorescence anisotropy assays implicate protein-protein interactions in regulating trp repressor DNA binding, Biochemistry 32: 7753–7758.CrossRefGoogle Scholar
  43. 43.
    Runnels, L. W., and Scarlata, S. F., 1995, Theory and application of fluorescence homotransfer to melittin oligomerization, Biophys. J. 69: 1569–1583.CrossRefGoogle Scholar
  44. 44.
    Shinitzky, M., Dianoux, A. C., Gifler, C., and Weber, G., 1971, Microviscosity and order in the hydrocarbon region of micelles and membranes determined with fluorescence probes I. Synthetic micelles, Biochemistry 10: 2106–2113.CrossRefGoogle Scholar
  45. 45.
    Cogen, U., Shinitzky, M., Weber, G., and Nishida, T., 1973, Micro-viscosity and order in the hydrocarbon region of phospholipid and phospholipid-cholesterol dispersions determined with fluorescent probes, Biochemistry 12: 521–528.CrossRefGoogle Scholar
  46. 46.
    Thulborn, K. R., Tilley, L. M., Sawyer, W. H., and Treloar, E E., 1979, The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipid bilayers, Biochim. Biophys. Acta 558: 166–178.CrossRefGoogle Scholar
  47. 47.
    Thulbom, K. R., and Beddard, G. S., 1982, The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers, Biochim Biophys. Acta 693: 246–252.CrossRefGoogle Scholar
  48. 48.
    Lakowicz, J. R., and Weber, G., 1980, Nanosecond segmental mobilities of tryptophan residues in proteins observed by lifetime-resolved fluorescence anisotropies, Biophys. J.32:591–601. Lakowicz, J. R., 49. Maliwal, B. P., Cherek, H., and Baiter, A., 1983, Rotational freedom of tryptophan residues in proteins and peptides, Biochemistry 22: 1741–1752.CrossRefGoogle Scholar
  49. 50.
    Lakowicz, J. R., and Maliwal, B. P., 1983, Oxygen quenching and fluorescence depolarization of tyrosine residues in proteins, J. Biol. Chem. 258: 4794–4801.Google Scholar
  50. 51.
    Eftink, M., 1983, Quenching-resolved emission anisotropy studies with single and multitryptophan-containing proteins, Biophys. J. 43: 323–334.CrossRefGoogle Scholar
  51. 52.
    Lakos, Z., Szarka, A., Koszorús, L., and Somogyi, B.,1995, Quenching-resolved emission anisotropy: A steady state fluorescence method to study protein dynamics, J. Photochem. Photobiol., B: Biol. 27: 55–60.Google Scholar
  52. 53.
    Bentz, J. P., Beyl, P., Beinert, G., and Weill, G., 1973, Simultaneous measurements of fluorescence polarization and quenching: A specially designed instrument and an application to the micro-Brownian motion of polymer chains, Eur. Polym. J. 11: 711–718.CrossRefGoogle Scholar
  53. 54.
    Brown, K., and Soutar, I., 1974, Fluorescence quenching and polarization studies of segmental motion in polystyrene, Eur. Polym. J. 10: 433–437.CrossRefGoogle Scholar
  54. 55.
    Chen, R. F, 1976, Quenching of the fluorescence of proteins by silver nitrate, Arch. Biochem. Biophys. 168: 605–622.Google Scholar
  55. 56.
    Sanyal, G., Charlesworth, M. C., Ryan, R. J., and Prendergast, R G., 1987, Tryptophan fluorescence studies of subunit interaction and rotational dynamics of human luteinizing hormone, Biochemistry 26: 1860–1866.CrossRefGoogle Scholar
  56. 57.
    Soleillet, P., 1929, Sur les paramètres caractérisant la polarisation partielle de la lumière dans les phénomènes de fluorescence, Ann. Phys. Biol. Med. 12: 23–97.Google Scholar
  57. 58.
    Kawski, A., 1986, Fluorescence anisotropy as a source of information about different photophysical processes, in Progress and Trends in Applied Optical Spectroscopy, D. Fassler, K.-H. Feller, and B. Wilhelmi (eds.), Teubner-Texte zur Physik, Vol. 13, Teubner Verlagsgesellschaft, Leipzig, pp. 6–34.Google Scholar
  58. 59.
    Kawski, A., 1993, Fluorescence anisotropy: Theory and applications of rotational depolarization, Crit. Rev. Anal. Chem. 23: 459–529.CrossRefGoogle Scholar
  59. 60.
    Gurinovich, G. P., Sarzhevskii, A. M., and Sevchenko, A. N., 1963, New data on the dependence of the degree of polarization on the wavelength of fluorescence, Opt. Spectrosc. 14: 428–432.Google Scholar
  60. 61.
    Mazurenko, Y. T., and Bakhshiev, N. G., 1970, Effect of orientation dipole relaxation on spectral, time, and polarization characteristics of the luminescence of solutions, Opt. Spectrosc. 28: 490–494.Google Scholar
  61. 62.
    Gakamskii, D. M., Nemkovich, N. A., Rubinov, A. N., and Tomin, V. I., 1988, Light-induced rotation of dye molecules in solution, Opt. Spectrosc. 64: 406–407.Google Scholar
  62. 63.
    Matayoshi, E. D., and Kleinfeld, A. M., 1981, Emission wavelength-dependent decay of the 9-anthroyloxy-fatty acid membrane probes, Biophys. J. 35: 215–235.CrossRefGoogle Scholar
  63. 64.
    Thulstrup, E. W., and Michl, J., 1988, Polarized absorption spectroscopy of molecules aligned in stretched polymers, Spectrochim. Acta, Part A 44: 767–782.Google Scholar
  64. 65.
    Michl, J., and Thulstrup, E. W., 1987, Ultraviolet and infrared linear dichroism: Polarized light as a probe of molecular and electronic structure, Acc. Chem. Res. 20: 192–199.CrossRefGoogle Scholar
  65. 66.
    Van Gurp, M., and Levine, Y. K., 1989, Determination of transition moment directions in molecules of low symmetry using polarized fluorescence. I. Theory, J. Chem. Phys. 90: 4095–4100.CrossRefGoogle Scholar
  66. 67.
    Matsuoka, Y, and Yamaoka, K., 1980, Film dichroism. V. Linear dichroism study of acridine dyes in films with emphasis on the electronic transitions involved in the long-wavelength band of the absorption spectrum, Bull. Chem. Soc. Jpn 53: 2146–2151.CrossRefGoogle Scholar
  67. 68.
    Kawski, A., and Gryczynski, Z., 1986, On the determination of transition-moment directions from emission anisotropy measurements, Z Naturforsch. A 41: 1195–1199.Google Scholar
  68. 69.
    Kawski, A., Gryczynski, Z., Gryczynski, I., Lakowicz, J. R., and Piszczek, G., 1996, Photoselection of luminescent molecules in anisotropic media in the case of two-photon excitation. Part II. Experimental studies of Hoechst 33342 in stretched poly(vinyl alcohol) films, Z. Naturforsch. A 51: 1037–1041.Google Scholar
  69. 70.
    Matsuoka, Y., and Norden, B., 1982, Linear dichroism study of 9-substituted acridines in stretched poly(vinyl alcohol) film, Chem. Phys. Lett. 85:302–306.Google Scholar
  70. 71.
    Holmén, A., Broo, A., Albinsson, B., and B. Nordén, 1997, Assignment of electronic transition moment directions of adenine from linear dichroism measurements, J. Am. Chem. Soc. 119: 122401 2250.Google Scholar
  71. 72.
    Holmén, A., Nordén, B., and Albinsson, B., 1997, Electronic transition moments of 2-aminopurine, J. Am. Chem. Soc. 119:3114–3121.Google Scholar
  72. 73.
    Albinsson, B., Kubista, M., Sandros, K., and Nordén, B., 1990, Electronic linear dichroism spectrum and transition moment directions of the hypermodified nucleic acid base wye, J. Phys. Chem. 94: 4006–4011.CrossRefGoogle Scholar
  73. 74.
    Hall, R. D., Valeur, B., and Weber, G., 1985, Polarization of the fluorescence of triphenylene: A planar molecule with three-fold symmetry, Chem. Phys. Leu. 116(23):202–205.Google Scholar
  74. 75.
    Lakowicz, J. R., and Gryczynski, I., 1997, Multiphoton excitation of biochemical fluorophores, in Topics in Fluorescence Spectroscopy, Volume 5, Nonlinear and Two-Photon-Induced Fluorescence,J. R. Lakowicz (ed.), Plenum Press, New York, pp. 87–144.Google Scholar
  75. 76.
    Callis, P. R., 1997, The theory of two-photon induced fluorescence anisotropy, in Topics in Fluorescence Spectroscopy, Volume 5: Nonlinear and Two-Photon-Induced Fluorescence,J. R. Lakowicz (ed.), Plenum Press, New York, pp. 1–42.Google Scholar
  76. 77.
    Johnson, C. K., and Wan, C., 1997, Anisotropy decays induced by two-photon excitation, in Topics in Fluorescence Spectroscopy, Volume 5, Nonlinear and Two-Photon-Induced Fluorescence, J. R. Lakowicz (ed.), Plenum Press, New York, pp. 43–85.Google Scholar
  77. 78.
    Callis, P. R., 1997, Two-photon induced fluorescence, Annu. Rev. Phys. Chem. 48: 271–297.CrossRefGoogle Scholar
  78. 79.
    Lakowicz, J. R., Gryczynski, I., Gryczynski, Z., Danielson, E., and Wirth, M. J., 1992, Time-resolved fluorescence intensity and anisotropy decays of 2,5-diphenyloxazole by two-photon excitation and frequency-domain fluorometry, J. Phys. Chem. 96: 3000–3006.CrossRefGoogle Scholar
  79. 80.
    Lakowicz, J. R., Gryczynski, I., Kulba, J., and Danielsen, E., 1992, Two photon-induced fluorescence intensity and anisotropy decays of diphenylhexatriene in solvents and lipid bilayers, J. Fluoresc. 2 (4): 247–258.CrossRefGoogle Scholar
  80. 81.
    Gryczynski, I., unpublished observations.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