European Biophysics Journal

, Volume 22, Issue 3, pp 169–175 | Cite as

Fluorescence correlation spectroscopy with high count rate and low background: analysis of translational diffusion

  • R. Rigler
  • Ü. Mets
  • J. Widengren
  • P. Kask
Article

Abstract

An epi-illuminated microscope configuration for use in fluorescence correlation spectroscopy in bulk solutions has been analyzed. For determining the effective sample dimensions the spatial distribution of the molecule detection efficiency has been computed and conditions for achieving quasi-cylindrical sample shape have been derived. Model experiments on translational diffusion of rhodamine 6G have been carried out using strong focusing of the laser beam, small pinhole size and an avalanche photodiode in single photon counting mode as the detector. A considerable decrease in background light intensity and measurement time has been observed. The background light is 40 times weaker than the fluorescence signal from one molecule of Rh6G, and the correlation function with signal-to-noise ratio of 150 can be collected in 1 second. The effect of the shape of the sample volume on the autocorrelation function has been discussed.

Key words

Fluorescence correlation spectroscopy Fluorescence intensity fluctuations Translational diffusion Epifluorescence microscope Silicon photon counter 

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References

  1. Aragón SR, Pecora R (1976) Fluorescence correlation spectroscopy as a probe of molecular dynamics. J Chem Phys 64:1791–1803Google Scholar
  2. Ehrenberg M, Rigler R (1974) Rotational brownian motion and fluorescence intensity fluctuations. Chem Phys 4:390–401Google Scholar
  3. Elson EL, Magde D (1974) Fluorescence correlation spectroscopy. I. Conceptual basis and theory. Biopolymers 13:1–27Google Scholar
  4. Koppel DE (1974) Statistical accuracy in fluorescence correlation spectroscopy. Phys Rev A 10:1938–1945Google Scholar
  5. Koppel DE, Axelrod D, Schlessinger J, Elson EL, Webb WW (1976) Dynamics of fluorescence marker concentration as a probe of mobility. Biophys J 16:1315–1329Google Scholar
  6. Marquardt DW (1963) An algorithm for least-squares estimation of non-linear parameters. J Soc Ind Appl Math 11:431–441MATHGoogle Scholar
  7. Palmer III AG, Thompson NL (1989) Optical spatial intensity profiles for high order autocorrelation in fluorescence spectroscopy. Appl Opt 28:1214–1220Google Scholar
  8. Qian H, Elson EL (1991) Analysis of confocal laser-microscope optics for 3-D fluorescence correlation spectroscopy. Appl Opt 30:1185–1195Google Scholar
  9. Rigler R, Widengren J (1990) Ultrasensitive detection of single molecules by fluorescence correlation spectroscopy. Bioscience 3:180–183Google Scholar
  10. Rigler R, Mets Ü (1993) Diffusion of single molecules through a Gaussian laser beam. SPIE Proceedings. Laser Applications in Life Sciences. Sept. 1992 Jyväskylä, Finland (in press)Google Scholar
  11. Rigler R, Widengren J, Mets Ü (1992) Interactions and kinetics of single molecules as observed by fluorescence correlation spectroscopy. In: Wolfbeis OS (ed) Fluorescence spectroscopy. New methods and applications. Springer, Berlin Heidelberg New York, pp 13–24Google Scholar
  12. Schneider MB, Webb WW (1981) Measurement of submicron laser beam radii. Appl Opt 20:1382–1388Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • R. Rigler
    • 1
  • Ü. Mets
    • 1
    • 2
  • J. Widengren
    • 1
  • P. Kask
    • 2
  1. 1.Department of Medical BiophysicsKarolinska InstituteStockholmSweden
  2. 2.Estonian Academy of SciencesInstitute of Chemical Physics and BiophysicsTallinnEstonia

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