Fluorescence Correlation Spectroscopy experiments using single-wavelength calcium dyes

CECILIA VILLARRUEL; LORENA SIGAUT; SILVINA PONCE DAWSON

Conferencia; XLVII Reunión Anual de la Sociedad Argentina de Biofísica; 2018

Fluorescence Correlation Spectroscopy (FCS) is an optical technique that is commonly used to estimate diffusion coefficients and reaction rates in various systems. In FCS the fluorescence coming from a small volume is collected. Determining the timescales over which the fluorescence fluctuations decay it is possible to quantify the processes that affect the transport of the observed molecules across the observation volume. To this end the autocorrelation function (ACF) of the fluctuations about the mean fluorescence are computed. In the case of single-wavelength calcium dyes fluorescence fluctuations are both due to the diffusive transport of the observed molecules and to the reactions with the calcium ions present in the sample. Namely, these dyes increase their fluorescence intensity several orders of magnitude upon calcium binding. In this talk I will describe how the ACF should be interpreted in such a case. Comparing with experiments performed in solution, I will show that it is possible to infer from these experiments the free diffusion coefficient of the calcium ions and of other species that affect their transport even if they are not fluorescent. Results on experiments performed in intact cells will also be presented.