Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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Record number 433330
Title Simultaneous diffusion and brightness measurements and brightness profile visualization from single fluorescence fluctuation traces of GFP in living cells.
Author(s) Skakun, V.V.; Engel, R.; Borst, J.W.; Apanasovich, V.V.; Visser, A.J.W.G.
Source European Biophysics Journal 41 (2012)12. - ISSN 0175-7571 - p. 1055 - 1064.
DOI https://doi.org/10.1007/s00249-012-0864-y
Department(s) Biochemistry
EPS-1
Publication type Refereed Article in a scientific journal
Publication year 2012
Keyword(s) photon-counting histogram - intensity distribution analysis - correlation spectroscopy - molecular brightness - protein - oligomerization - distributions - excitation - level - dimer
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.
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