Abstract
Typically, fluctuation correlation spectroscopy (FCS) data acquisition cards measure the number of photon events per time interval (i.e., bin)—time mode. Commercial FCS cards combine the bins through hardware in order to calculate the autocorrelation function. Such a design therefore does not yield the time resolved photon sequence, but only the autocorrelation of that sequence. A different acquisition method which measures the number of time intervals between photon events has been implemented—photon mode. This method takes advantage of the fact that in FCS the rate of photon counts is much less than the frequency of the clock that is used to determine the temporal location of the photons. By using this new mode of data acquisition, the current card design allows for 25 ns time resolution. The data acquisition card can operate in both time and photon mode and yields the time resolved sequence of photon arrivals in both cases. Therefore, the data is available for analysis by any method(s), such as but not limited to, autocorrelation, photon counting histogram, and higher order autocorrelation.
Highlights
Fluctuation correlation spectroscopyFCSis a technique used to obtain kinetic information through analysis of the stochastic fluctuations in a molecule’s fluorescence.[1]
Since the excitation volume is known, the correlation resulting from the time it takes for a dye to cross this volume intrinsically yields a diffusion coefficient
Any property which causes fluctuations in the fluorescence intensity of the dye at a rate fast enough to occur during the fraction of time when the dye is in the excitation volume, will be captured by the autocorrelation function
Summary
Fluctuation correlation spectroscopyFCSis a technique used to obtain kinetic information through analysis of the stochastic fluctuations in a molecule’s fluorescence.[1]. Any property which causes fluctuations in the fluorescence intensity of the dye at a rate fast enough to occur during the fraction of time when the dye is in the excitation volume, will be captured by the autocorrelation function. Such processes as rotational diffusion,[2,3] triplet state formation,[4] and chemical reactions[5–9] can all be measured using this technique. The electronics of the commercial correlator boards determine the autocorrelation function by a time resolved binning of the photon counts.[10]. The width of the time bin determines the temporal resolution of the autocorrelation function
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