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Fluorescence Anisotropy

  • Chapter
Principles of Fluorescence Spectroscopy

Abstract

Measurements of fluorescence anisotropy are a powerful tool in biochemical research and medical testing. 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 anisotropy is the existence of transition moments for absorption and emission that lie along specific directions within the fluorophore structure. In homogeneous solution the ground-state fluo-rophores are all randomly oriented. When exposed to polarized light, those fluorophores that have their absorption transition moments oriented along the electric vector of the incident light are preferentially excited. Hence the excited-state population is partially oriented. A significant fraction of the excited molecules have their transition moments oriented along the electric vector of the polarized exciting light.

The emission can become depolarized by a number of processes, the relative importance of which depends upon the sample under investigation. All chromophores have transition moments that occur along a specific direction in the molecular axis. Rotational diffusion changes the direction of the transition moments and is one common cause of depolarization. Anisotropy measurements reveal the average angular displacement of the fluorophore that occurs between absorption and subsequent emission of a photon. This angular displacement is dependent upon the rate and extent of rotational diffusion during the lifetime of the excited state. The rate of rotational diffusion depends on the viscosity of the solvent and the size and shape of the rotating molecule. The rotational rate of fluorophores in solution is dependent upon the viscous drag imposed by the solvent. A change in solvent viscosity will result in a change in fluorescence anisotropy. For small fluorophores in low-viscosity solutions the rate of rotational diffusion is typically faster than the rate of emission. Under these conditions the emission is depolarized and the anisotropy close to zero.

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References

  1. 1. Jablonski A. 1960. On the notion of emission anisotropy. Bull l’Acad Pol Sci, Ser A 8:259–264.

    CAS  Google Scholar 

  2. 2. Weber G, 1952. Polarization of the fluorescence of macromolecules, I: theory and experimental method. Biochem J 51:145–155.

    CAS  Google Scholar 

  3. 3. Weber G, 1966. Polarization of the fluorescence of solutions. In Fluorescence and phosphorescence analysis, pp. 217–240 Ed DM Hercules. John Wiley & Sons, New York.

    Google Scholar 

  4. 4. Selényi P. 1939. Wide-angle interferences and the nature of the elementary light sources. Phys Rev 56:477–479.

    Article  Google Scholar 

  5. 5. Griffiths DJ. 1999. Introduction to electrodynamics. Prentice Hall, Englewood Cliffs, NJ.

    Google Scholar 

  6. 6. Born M, Wolf E. 1980. Principles of optics electromagnetic theory of propagation, interference and diffraction of light. Pergamon Press, New York.

    Google Scholar 

  7. 7. Lakowicz JR, Gryczynski I. 1997. Multiphoton excitation of biochemical fluorophores. In Topics in fluorescence spectroscopy, Vol. 5: Nonlinear and two-photon induced fluorescence, pp. 87–144. Ed JR Lakowicz. Plenum Press, New York.

    Chapter  Google Scholar 

  8. 8. Faucon JF, Lakowicz JR. 1987. Anisotropy decay of diphenylhexa-triene in melittin-phospholipid complexes by multifrequency phasemodulation fluorometry. Arch Biochem Biophys 252(1):245–258.

    Article  CAS  Google Scholar 

  9. 9. Herman P, Konopásek I, Plásek J, Svobodová J. 1994. Time-resolved polarized fluorescence studies of the temperature adaptation in Bacillus subtilisusing DPH and TMA-DPH fluorescent probes. Biochim Biophys Acta 1190:1–8.

    Article  CAS  Google Scholar 

  10. 10. Shinitzky M, Barenholz Y. 1974. Dynamics of the hydrocarbon layer in liposomes of lecithin and sphingomyelin containing dicetylphos-phate. J Biol Chem 249:2652–2657.

    CAS  Google Scholar 

  11. 11. Thompson RB, Gryczynski I, Malicka J. 2002. Fluorescence polarization standards for high-throughput screening and imaging. Biotechnology 32:34–42.

    CAS  Google Scholar 

  12. 12. Michl J, Thulstrup EW. 1986. Spectroscopy with polarized light. VCH Publishers, New York.

    Google Scholar 

  13. 13. Albinsson B, Kubista M, Nordén B, Thulstrup EW. 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.

    Article  CAS  Google Scholar 

  14. 14. Albinsson B, Eriksson S, Lyng R, Kubista M. 1991. The electronically excited states of 2-phenylindole. Chem Phys 151:149–157.

    Article  CAS  Google Scholar 

  15. 15. Kubista M, Åkerman B, Albinsson B. 1989. Characterization of the electronic structure of 4′,6-diamidino-2-phenylindole. J Am Chem Soc 111:7031–7035.

    Article  CAS  Google Scholar 

  16. 16. Vincent M, de Foresta B, Gallay J, 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.

    Article  CAS  Google Scholar 

  17. 17. Mullaney JM, Thompson RB, Gryczynski Z, Black LW. 2000. Green fluorescent protein as a probe of rotational mobility within bacterio-phage T4. J Virol Methods 88:35–40.

    Article  CAS  Google Scholar 

  18. 18. Weber G. 1960. Fluorescence-polarization spectrum and electronic-energy transfer in tyrosine, tryptophan and related compounds. Biochem J 75:335–345.

    CAS  Google Scholar 

  19. 19. Valeur B, Weber G. 1977. Resolution of the fluorescence excitation spectrum of indole into the 1La and 1Lb excitation bands. Photochem Photobiol 25:441–444.

    Article  CAS  Google Scholar 

  20. 20. Eftink MR, Selvidge LA, Callis PR, Rehms AA. 1990. Photophysics of indole derivatives: Experimental resolution of La and Lb transitions and comparison of theory. J Phys Chem 94:3469–3479.

    Article  CAS  Google Scholar 

  21. 21. Shen X, Knutson JR. 2001. Subpicosecond fluorescence spectra of tryptophan in water. J Phys Chem B 105:6260–6265.

    Article  CAS  Google Scholar 

  22. 22. Short KW, Callis PR. 2002. One- and two-photon spectra of jet-cooled 2,3-dimethylindole: 1Lb and 1La assignments. Chem Phys 283: 269–278.

    Article  CAS  Google Scholar 

  23. 23. Yamamoto Y, Tanaka J. 1972. Polarized absorption spectra of crystals of indole and its related compounds. Bull Chem Soc Jpn65:1362–1366.

    Article  Google Scholar 

  24. 24. Suwaiyan A, Zwarich R. 1987. Absorption spectra of substituted indoles in stretched polyethylene films. Spectrochim Acta 43A(5): 605–609.

    CAS  Google Scholar 

  25. 25. Weber G, 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 USA 65(4):823–830.

    Article  CAS  Google Scholar 

  26. 26. Kawski A. 1992. Fotoluminescencja roztworów. Wydawnictwo Naukowe PWN, Warsaw.

    Google Scholar 

  27. 27. Van Der Meer BW, Coker III G, Chen S-YS. 1991. Resonance energy transfer theory and data. Wiley-VCH, New York.

    Google Scholar 

  28. 28. Jablonski A. 1970. Anisotropy of fluorescence of molecules excited by excitation transfer. Acta Phys Pol A 38:453–458.

    CAS  Google Scholar 

  29. 29. Baumann J, Fayer MD. 1986. Excitation transfer in disordered two-dimensional and anisotropic three-dimensional systems: effects of spatial geometry on time-resolved observables. J Chem Phys 85:4087–4107.

    Article  CAS  Google Scholar 

  30. 30. Teale FWJ. 1969. Fluorescence depolarization by light scattering in turbid solutions. Photochem Photobiol 10:363–374.

    Article  CAS  Google Scholar 

  31. 31. Ghosh N, Majumder SK, Gupta PK. 2002. Fluorescence depolarization in a scattering medium: effect of size parameter of a scatterer. Phys Rev E 65:026608-1–6.

    Google Scholar 

  32. 32. Lentz BR. 1979. Light scattering effects in the measurement of membrane microviscosity with diphenylhexatriene. Biophys J 25:489– 494.

    Article  CAS  Google Scholar 

  33. 33. Berberan-Santos MN, Nunes Pereira EJ, Martinho JMG. 1995. Stochastic theory of molecular radiative transport. J Chem Phys 103(8):3022–3028.

    Article  CAS  Google Scholar 

  34. 34. Nunes Pereira EJ, Berberan-Santos MN, Martinho JMG. 1996. Molecular radiative transport, II: Monte-Carlo simulation. J Chem Phys 104(22):8950–8965.

    Article  Google Scholar 

  35. 35. Perrin F. 1929. La fluorescence des solutions: induction moleculaire. Polarisation et duree d'emission. Photochimie. Ann Phys, Ser 10 12:169–275.

    Google Scholar 

  36. 36. Perrin F. 1926. Polarisation de la lumière de fluorescence: vie moyenne des molécules dans l'état excité. J Phys Radium V, Ser 6 7:390–401.

    Article  CAS  Google Scholar 

  37. 37. Perrin F. 1931. Fluorescence: durée élémentaire d’émission lumineuse. Conférences D’Actualités Scientifiques et Industrielles 22:2–41.

    Google Scholar 

  38. 38. Weber G. 1953. Rotational Brownian motion and polarization of the fluorescence of solutions. Adv Protein Chem 8:415–459.

    Article  CAS  Google Scholar 

  39. 39. Yguerabide J, Epstein HF, Stryer L. 1970. Segmental flexibility in an antibody molecule. J Mol Biol 51:573–590.

    Article  CAS  Google Scholar 

  40. 40. Weber G. 1952. Polarization of the fluorescence of macromolecules, II: fluorescence conjugates of ovalbumin and bovine serum albumin. Biochem J 51:155–167.

    CAS  Google Scholar 

  41. 41. Laurence DJR. 1952. A study of the absorption of dyes on bovine serum albumin by the method of polarization of fluorescence. Biochem J 51:168–180.

    CAS  Google Scholar 

  42. 42. Gottlieb YYa, Wahl Ph. 1963. Étude théorique de la polarisation de fluorescence des macromolecules portant un groupe émetteur mobile autour d’un axe de rotation. J Chim Phys 60:849–856.

    Google Scholar 

  43. 43. Ferguson BQ, Yang DCH. 1986. Methionyl-tRNA synthetase induced 3′-terminal and delocalized conformational transition in tRNAfMet: steady-state fluorescence of tRNA with a single fluoro-phore. Biochemistry 25:529–539.

    Article  CAS  Google Scholar 

  44. 44. Buchner J. 1996. Supervising the fold: functional principals of molecular chaperones. FASEB J 10:10–19.

    CAS  Google Scholar 

  45. 45. Ellis RJ. 1996. The chaperonins. Academic Press, New York.

    Google Scholar 

  46. 46. Braig K, Otwinowski Z, Hegde R, Bolsvert DC, Joachimiak A, Horwich AL, Sigler PB. 1994. The crystal structure of the bacterial chaperonin GroEL at 2.8 Å. Nature 371:578–586.

    Article  CAS  Google Scholar 

  47. 47. Gorovits BM, Horowitz PM. 1995. The molecular chaperonin cpn60 displays local flexibility that is reduced after binding with an unfolded protein. J Biol Chem 270(22):13057–13062.

    Article  CAS  Google Scholar 

  48. 48. Lim K, Jameson DM, Gentry CA, Herron JN. 1995. Molecular dynamics of the anti-fluorescein 4-4-20 antigen-binding fragment, 2: time-resolved fluorescence spectroscopy. Biochemistry 34:6975– 6984.

    Article  CAS  Google Scholar 

  49. 49. Lukas TJ, Burgess WH, Prendergast FG, Lau W, Watterson DM. 1986. Calmodulin binding domains: characterization of a phosphory-lation and calmodulin binding site from myosin light chain kinase. Biochemistry 25:1458–1464.

    Article  CAS  Google Scholar 

  50. 50. Malencik DA, Anderson SR. 1984. Peptide binding by calmodulin and its proteolytic fragments and by troponin C. Biochemistry 23: 2420–2428.

    Article  CAS  Google Scholar 

  51. 51. Eftink MR, 1994. The use of fluorescence methods to monitor unfolding transitions in proteins. Biophys J 66:482–501.

    Article  CAS  Google Scholar 

  52. 52. LeTilly V, Royer CA. 1993. Fluorescence anisotropy assays implicate protein–protein interactions in regulating trprepressor DNA binding. Biochemistry 32:7753–7758.

    Article  CAS  Google Scholar 

  53. 53. Banik U, Beechem JM, Klebanow E, Schroeder S, Weil PA. 2001. Fluorescence-based analyses of the effects of full-length recombinant TAF130p on the interaction of TATA box-binding protein with TATA box DNA. J Biol Chem 276(52):49100–49109.

    Article  CAS  Google Scholar 

  54. 54. Hiller DA, Fogg JM, Martin AM, Beechem JM, Reich NO, Perona JJ. 2003. Simultaneous DNA binding and bending by ecoRV endonuclease observed by real-time fluorescence. Biochemistry 42: 14375–14385.

    Article  CAS  Google Scholar 

  55. 55. Taniguchi M, Yoshimi T, Hongo K, Mizobata T, Kawata Y. 2004. Stopped-flow fluorescence analysis of the conformational changes in the GroEL apical domain. J Biol Chem 279(16):16368–16376.

    Article  CAS  Google Scholar 

  56. 56. Xu HQ, Zhang AH, Auclair C, Xi XG. 2003. Simultaneously monitoring DNA binding and helicase-catalyzed DNA unwinding by fluorescence polarization. Nucleic Acids Res 31(14):e70.

    Article  CAS  Google Scholar 

  57. 57. Runnels LW, Scarlata SF. 1995. Theory and application of fluorescence homotransfer to melittin oligomerization. Biophys J 69:1569–1583.

    Article  CAS  Google Scholar 

  58. 58. Shinitky M, Dianoux AC, Gitler C, 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.

    Article  Google Scholar 

  59. 59. Cogen U, Shinitzky M, Weber G, Nishida T. 1973. Microviscosity and order in the hydrocarbon region of phospholipid and phospho-lipid–cholesterol dispersions determined with fluorescent probes. Biochemistry 12:521–528.

    Article  Google Scholar 

  60. 60. Thulborn KR, Tilley LM, Sawyer WH, Treloar FE. 1979. The use of n-(9-anthroyloxy) fatty acids to determine fluidity and polarity gradients in phospholipids bilayers. Biochim Biophys Acta 558: 166–178.

    Article  CAS  Google Scholar 

  61. 61. Thulborn KR, Beddard GS. 1982. The effects of cholesterol on the time-resolved emission anisotropy of 12-(9-anthroyloxy)stearic acid in dipalmitoylphosphatidylcholine bilayers. Biochim Biophy Acta 693:246–252.

    Article  CAS  Google Scholar 

  62. 62. Varma R, Mayor S. 1998. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature 394:798–801.

    Article  CAS  Google Scholar 

  63. 63. Rosell FI, Boxer SG. 2003. Polarized absorption spectra of green fluorescent protein single crystals: transition dipole moment directions. Biochemistry 42:177–183.

    Article  CAS  Google Scholar 

  64. 64. Inoué S, Shimomura O, Goda M, Shribak M, Tran PT. 2002. Fluorescence polarization of green fluorescence protein. Proc Natl Acad Sci USA 99(7):4272–4277.

    Article  CAS  Google Scholar 

  65. Additional Reading on the Application of Anisotropy

    Google Scholar 

Association Reactions

  • Hey T, Lipps G, Krauss G. 2001. Binding of XPA and RPA to damaged DNA investigated by fluorescence anisotropy. Biochemistry 40: 2901–2910.

    Article  CAS  Google Scholar 

Imaging

  • Lidke DS, Nagy P, Barisas BG, Heintzmann R, Post JN, Lidke KA, Clayton AHA, Arndt-Jovin DJ, Jovin TM. 2003. Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET). Biochem Soc Trans 31(5):1020–1027.

    Article  CAS  Google Scholar 

Membranes

  • Gidwani A, Holowka D, Baird B. 2001. Fluorescence anisotropy measurements of lipid order in plasma membranes and lipid rafts from RBL-2H3 mast cells. Biochemistry 40:12422–12429.

    Article  CAS  Google Scholar 

  • Huertas ML, Cruz V, Cascales JJ, Acuña AU, Garcia de la Torre J. 1996. Distribution and diffusivity of a hydrophobic probe molecule in the interior of a membrane: theory and simulation. Biophys J 71:1428–1439.

    Article  CAS  Google Scholar 

  • Sinha M, Mishra S, Joshi PG. 2003. Liquid-ordered microdomains in lipid rafts and plasma membrane of U-87 MG cells: a time-resolved fluorescence study. Eur Biophys J 32:381–391.

    Article  CAS  Google Scholar 

Oriented Systems

  • Fisz JJ, Buczkowski M. 2001. Excited-state orientation-dependent irreversible interconversion and fluorescence depolarization in organized molecular media, I: theory. J Chem Phys 115(15):7051–7060.

    Article  CAS  Google Scholar 

  • Dale RE, Hopkins SC, an der Heide UA, Marszalek T, Irving M, Goldman YE. 1999. Model-independent analysis of the operation of fluorescent probes with restricted mobility in muscle fibers. Biophys J 76:1606–1618.

    Article  CAS  Google Scholar 

RET and Anisotropy

  • Andrews DL, Juzeliunas G. 1991. The range dependence of fluorescence anisotropy in molecular energy transfer. J Chem Phys 95(8): 5513–5518.

    Article  CAS  Google Scholar 

  • Cohen-Kashi M, Moshkov S, Zurgil N, Deutsch M. 2002. Fluorescence resonance energy transfers measurements on cell surfaces via fluorescence polarization. Biophys J 83:1395–1402.

    Article  CAS  Google Scholar 

  • Gaab KM, Bardeen CJ. 2004. Wavelength and temperature dependence of the femtosecond pump-probe anisotropies in the conjugated polymer MEH-PPV: implications for energy-transfer dynamics. J Phys Chem B 108:4619–4626.

    Article  CAS  Google Scholar 

  • Kleima FJ, Hofmann E, Gobets B, van Stokkum IHM, van Grondelle R, Diederichs K, van Amerongen H. 2000. Förster excitation energy transfer in peridinin-chlorophyll-a-protein. Biophys J 78:344–353.

    Article  CAS  Google Scholar 

  • Tanaka F. 1998. Theory of time-resolved fluorescence under the interaction of energy transfer in bichromophoric system: effect of internal rotations of energy donor and acceptor. J Chem Phys 109(3):1084– 1092.

    Article  CAS  Google Scholar 

  • Varnavski OP, Ostrowski J, Bazan GC, Goodson III T. 2002. Energy transfer and fluorescence depolarization in organic dendrimers and branched molecules. Proc SPIE 4464:134–141.

    Article  CAS  Google Scholar 

Surfaces

  • Ishizaka S, Kinoshita S, Nishijima Y, Kitamura N. 2003. Direct observation of molecular recognition mediated by triple hydrogen bonds at a water/oil interface: time-resolved total internal reflection fluorome-try study. Anal Chem 75(22):6035–6042.

    Article  CAS  Google Scholar 

Theory

  • Bajzer Z, Moncrieffe MC, Penzar I, Prendergast FG. 2001. Complex homogeneous and heterogeneous fluorescence anisotropy decays: enhancing analysis accuracy. Biophys J 81:1765–1775.

    Article  CAS  Google Scholar 

  • Bialik CN, Wolf B, Rachofsky EL, Alexander Ross JB, Laws WR. 1998. Dynamics of biomolecules: assignment of local motions by fluorescence anisotropy decay. Biophys J 75:2564–2573.

    Article  CAS  Google Scholar 

  • Bryant J, Bain AJ. 1998. Controlled orientational photoselection via three-pulse excitation. Chem Phys Lett 286:121–130.

    Article  CAS  Google Scholar 

  • Fisz JJ. 1998. Flexible fluorophores as the probes for monitoring the structural and dynamical properties of organized molecular media. Chem Phys Lett 297:409–418.

    Article  CAS  Google Scholar 

  • Fisz JJ. 1996. Polarized fluorescence spectroscopy of two-ground- and two-excited-state systems in solutions. Chem Phys Lett 262:495–506.

    Article  CAS  Google Scholar 

  • Tanaka F. 2000. Effects of internal rotations on the time-resolved fluorescence in a bichromophoric protein system under the energy transfer interaction. J Fluoresc 10(1):13–19.

    Article  CAS  Google Scholar 

  • Tanaka F, Mataga N. 1998. Analytical theory of time-resolved fluorescence anisotropy and dynamic Stokes shift of polar solute molecules based on continuum model for solvent. Chem Phys 236:277–289.

    Article  CAS  Google Scholar 

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Editors and Affiliations

  1. Center for Fluorescence Spectroscopy, University of Maryland School of Medicine, Baltimore, MD, 21201, USA

    Joseph R. Lakowicz

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(2006). Fluorescence Anisotropy. In: Lakowicz, J.R. (eds) Principles of Fluorescence Spectroscopy. Springer, Boston, MA. https://doi.org/10.1007/978-0-387-46312-4_10

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