Quantification of fluctuations from fluorescence correlation spectroscopy experiments in reaction-diffusion systems

CECILIA VILLARRUEL; SILVINA PONCE DAWSON

Physical Review E

American Physical Society

Lugar: New York; Año: 2020 vol. 102 p. 5240701 - 5240715

2470-0045

Fluorescence Correlation Spectroscopy} (FCS) is commonly used to estimate diffusion coefficients and reaction rates in various systems. In FCS the fluorescence coming from a small volume is recorded and the autocorrelation function (ACF) of the fluctuations about its mean are subsequently computed. The ACF decays with one or various timescales, depending on the system. Scaling out the fluctuations due to the emission process, the timescales of the ACF can be associated to changes in the number of fluorescent molecules in the volume. Fitting the ACF with theoretical expressions, diffusion coefficients and reaction rates can be drawn from the timescales. In deriving the theoretical ACF for the molecule number fluctuations, it is usually assumed that, if several species interact among themselves, their numbers are instantaneously uncorrelated and that they obey Poisson statistics. In this paper we study the ACF for a reaction-diffusion system with three species, one of which is fluorescent when bound to another. We then compare the theoretical results with those derived from experiments performed in solutions with Ca2+, a single-wavelength Ca2+ dye and a Ca2+ buffer.} We find discrepancies between the FCS theory and the experiments that can be solved if the assumptions on the instantaneous correlations} are modified. Furthermore, we show that it is possible to derive the diffusion coefficients of the non-fluorescent} species (Ca2+ and the buffer in the current case) because the initial correlations do not behave as it is usually assumed in the theoretical derivation of the molecule number ACF. Our studies lead to the important conclusion that it is due to the effect of the interactions between the various species that occur during the acquisition time interval that transport properties of the non-fluorescent species can be estimated from FCS experiments performed in reaction-diffusion systems.