FCS experiments to infer calcium dye properties.

CECILIA VILLARRUEL; LORENA SIGAUT; MARIA LAURA PONCE; SILVINA MATHA PONCE DAWSON

Villa Carlos Paz, Córdoba

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofisica; 2013

Sociedad Argentina de Biofisica

Optical techniques provide a relatively non-invasive means to study intracellular calcium signals. Although the fluorescence amplitude increases with the free calcium concentration, it is only linearly related to the calcium-bound dye concentration. Therefore, it is key to know the reaction rates and diffusion coefficients of the dyes to extract quantitative information on the free calcium concentration that underlies the signals observed. In this work we show how Fluorescence Correlation Spectroscopy (FCS) experiments can be used to estimate these parameters from fits to the autocorrelation function that is obtained from the data. We first present an analytic approximation to this function that is valid for a ?fast? calcium-dye reaction. We then present the results of numerical simulations with which we analyze the limits of applicability of this approximation and the errors introduced in the estimation of some parameters when the reaction is not fast enough. We subsequently discuss briefly the results of its application to FCS experiments performed in solution using calcium and a dye. Finally, we show the results of experiments performed in oocytes with which we determine the factor by which the coefficients estimated in solution should be multiplied to obtain those that hold in the cytoplasm.