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European Biophysics Journal

, Volume 41, Issue 12, pp 1055–1064 | Cite as

Simultaneous diffusion and brightness measurements and brightness profile visualization from single fluorescence fluctuation traces of GFP in living cells

  • Victor V. Skakun
  • Ruchira Engel
  • Jan Willem Borst
  • Vladimir V. Apanasovich
  • Antonie J. W. G. Visser
Original Paper

Abstract

Fluorescence correlation spectroscopy (FCS) and photon-counting histogram (PCH) analysis use the same experimental fluorescence intensity fluctuations, but each analytical method focuses on a different property of the signal. The time-dependent decay of the correlation of fluorescence fluctuations is measured in FCS yielding, for instance, molecular diffusion coefficients. The amplitude distribution of these fluctuations is calculated by PCH analysis yielding information about the molecular brightness of fluorescent species. Analysis of both FCS and PCH results in the molecular concentration of the sample. Using a previously described global analysis procedure we report here precise, simultaneous measurements of diffusion constants and brightness values from single fluorescence fluctuation traces of green-fluorescent protein (GFP, S65T) in the cytoplasm of Dictyostelium cells. The use of a polynomial profile in PCH analysis, describing the detected three-dimensional shape of the confocal volume, enabled us to obtain well fitting results for GFP in cells. We could visualize the polynomial profile and show its deviation from a Gaussian profile.

Keywords

Green fluorescent protein Fluorescence correlation spectroscopy Photon-counting histogram analysis Fluorescence intensity distribution analysis Global analysis of ACF and PCD Dictyostelium cells 

Supplementary material

249_2012_864_MOESM1_ESM.doc (741 kb)
Supplementary material 1 (DOC 741 kb)

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Copyright information

© European Biophysical Societies' Association 2012

Authors and Affiliations

  • Victor V. Skakun
    • 1
  • Ruchira Engel
    • 2
    • 3
  • Jan Willem Borst
    • 2
  • Vladimir V. Apanasovich
    • 1
  • Antonie J. W. G. Visser
    • 2
  1. 1.Department of Systems Analysis and Computer SimulationBelarusian State UniversityMinskBelarus
  2. 2.Laboratory of Biochemistry, and Microspectroscopy CentreWageningen UniversityWageningenThe Netherlands
  3. 3.Department of ImmunopathologySanquin Blood Supply FoundationAmsterdamThe Netherlands

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