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

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.