Two-Photon Fluorescence Polarization Anisotropy Decay on Highly Diluted Solutions by Phase Fluorometry

Abstract

We report here the phase-sensitive measurements of two-photon fluorescence polarization anisotropy (FPA) on highly diluted solutions. We first describe the characterization of the response of the two-photon microscope to the light polarization and its test by means of measurements of the FPA on rhodamine 6G as a function of the viscosity. Further, we report the study of the FPA at high dilutions on a globular protein, the beta-lactoglobulin B (BLG) labeled with Alexa 532. The number of molecules (from 0.4 to 17 molecules per excitation volume) is measured by means of fluorescence correlation spectroscopy (FCS). The average rotational and translational diffusion coefficients measured with the FPA and FCS methods are in good agreement with the protein size and do not show a substantial dependence on the protein concentration, despite the very low signal (≌3 times the background) observed for highly diluted solutions.

Keywords

Fluorescence polarization anisotropy Fluorescence correlation spectroscopy Two-photon excitation Multifrequency phase fluorometer

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References

Brand

Eggeling

Zander

Drexhage

K. H.

Seidel

C. A. M.

, J. Phys. Chem. 101, 4313 (1997).

Eggeling

Fries

J. R.

Brand

Gunther

Seidel

C. A. M.

, Proc. Natl. Acad. Sci. USA 95, 1556 (1998).

Schaffer

Volkmer

Eggeling

Subramaniam

Striker

Seidel

C. A. M.

, J. Phys. Chem. 103, 331 (1999).

Watkins

A. N.

Ingersoll

C. M.

Baker

G. A.

Bright

F. V.

, Anal. Chem. 70, 3384 (1998).

Lakowicz

J. R.

, “Fluorescence Polarization”, in Principles of Fluorescence Spectroscopy (Plenum Press, New York and London, 1991), p. 131.

Tsien

R. Y.

Wagonner

, “Fluorophores for Confocal Microscopy”, in Handbook of Biological Confocal Microscopy (Plenum Press, New York and London, 1995), 2nd ed., Chap. 16, p. 267.

Beechem

J. M.

Gratton

, Proc. SPIE-Int. Soc. Opt. Eng. 909, 70 (1988).

Steiner

R. F.

, “Fluorescence Anisotropy: Theory and Applications”, in Topics in Fluorescence Spectroscopy: Principles, Lakowicz

J. R.

Ed. (Plenum Press, New York and London, 1991), Vol. 2.

Thompson

N. L.

, “Fluorescence Correlation Spectroscopy”, in Topics in Fluorescence Spectroscopy: Techniques, Lakowicz

J. R.

Ed. (Plenum Press, New York and London, 1991), Vol. 1.

10.

Berland

K. M.

P. T. C.

Gratton

, Biophys. J. 68, 694 (1995).

11.

Chirico

Olivini

Beretta

, Appl. Spectrosc. 54, 1084 (2000).

12.

Bevington

P. R.

, Data Reduction and Error Analysis for the Physical Sciences (McGraw Hill, New York, 1992), Chap. 11, p. 204.

13.

Press

W. H.

Teukolsky

S. A.

Vetterling

W. T.

Flannery

B. P.

, Numerical Recipes in C: The Art of Scientific Computing (Cambridge University Press, Cambridge, 1993), 2nd ed.

14.

Windegren

Mets

Rigler

, J. Phys. Chem. 99, 13368 (1995).

15.

Lakowicz

J. R.

, “Fluorescence Polarization”, in Principles of Fluorescence Spectroscopy (Plenum Press, New York and London, 1991), p. 125.

16.

Laurence

T. A.

Chemla

D. S.

Weiss

, J. Phys. Chem. B 103, 6839 (1999).

17.

Axelrod

, Biophys. J. 26, 557 (1979).

18.

CRC Handbook of Chemistry and Physics, Weast

R. C.

Ed. (The Chemical Rubber Co., Cleveland, Ohio, 1968–1969), 49th ed.

19.

Volkmer

Subramaniam

Birch

D. J.

Jovin

T. M.

, Biophys. J. 78, 1589 (2000).

20.

Lakowicz

J. R.

, “Fluorescence Polarization”, in Principles of Fluorescence Spectroscopy (Plenum Press, New York and London, 1991), p. 166.

21.

De la Torre

J. G.

Huertas

M. L.

Carrasco

, Biophys. J. 78, 719 (2000).

22.

Baldini

Beretta

Chirico

Franz

Maccioni

Mariani

Spinozzi

, Macromolecules 32, 6128 (1999).

23.

Eigen

Rigler

, Proc. Natl. Acad. Sci. USA 91, 5740 (1994).