Ca signals are ubiquitous. One of the key factors for their versatility is the variety of spatio-temporal distributions that the cytosolic Ca can display. In most cell types Ca signals not only depend on Ca entry from the extracellular medium but also on Ca release from internal stores, a process which is in turn regulated by cytosolic Ca itself. The rate at which Ca is transported, the fraction that is trapped by intracellular buffers, and with what kinetics are thus key features that affect the time and spatial range of action of Ca signals. The quantification of Ca diffusion in intact cells is quite challenging because the transport rates that can be inferred using optical techniques are intricately related to the interaction of Ca with the dye that is used for its observation and with the cellular buffers. In this paper, we introduce an approach that uses Fluorescence Correlation Spectroscopy (FCS) experiments performed at different conditions that in principle allows the quantification of Ca diffusion and of its reaction rates with unobservable (non-fluorescent) Ca buffers. To this end, we develop the necessary theory to interpret the experimental results and then apply it to FCS experiments performed in a set of solutions containing Ca, a single wavelength Ca dye, and a non-fluorescent Ca buffer. We show that a judicious choice of the experimental conditions and an adequate interpretation of the fitting parameters can be combined to extract information on the free diffusion coefficient of Ca and of some of the properties of the unobservable buffer. We think that this approach can be applied to other situations, particularly to experiments performed in intact cells.

Fluorescence correlation spectroscopy (FCS) is commonly used to estimate diffusion and reaction rates. In FCS the fluorescence coming from a small volume is recorded and the autocorrelation function (ACF) of the fluorescence fluctuations is computed. Scaling out the fluctuations due to the emission process, this ACF can be related to the ACF of the fluctuations in the number of observed fluorescent molecules. In this paper the ACF of the molecule number fluctuations is studied theoretically, with no approximations, for a reaction-diffusion system in which the fluorescence changes with binding and unbinding. Theoretical ACFs are usually derived assuming that fluctuations in the number of molecules of one species are instantaneously uncorrelated to those of the others and obey Poisson statistics. Under these assumptions, the ACF derived in this paper is characterized only by the diffusive timescale of the fluorescent species and its total weight is the inverse of the mean number of observed fluorescent molecules. The theory is then scrutinized in view of previous experimental results which, for a similar system, gave a different total weight and correct estimates of other diffusive timescales. The total weight mismatch is corrected by assuming that the variance of the number of fluorescent molecules depends on the variance of the particle numbers of the other species, as in the variance decomposition formula. Including the finite acquisition time in its computation, it is shown that the ACF depends on various timescales of the system and that its total weight coincides with the one obtained with the variance decomposition formula. This calculation implies that diffusion coefficients of nonobservable species can be estimated with FCS experiments performed in reaction-diffusion systems. Ways to proceed in future experiments are also discussed.

Many cell signaling pathways involve the diffusion of messengers that bind and unbind to and from intracellular components. Quantifying their net transport rate under different conditions then requires having separate estimates of their free diffusion coefficient and binding or unbinding rates. In this paper, we show how performing sets of fluorescence correlation spectroscopy (FCS) experiments under different conditions, it is possible to quantify free diffusion coefficients and on and off rates of reaction-diffusion systems. We develop the theory and present a practical implementation for the case of the universal second messenger, calcium (Ca^{2+}) and single-wavelength dyes that increase their fluorescence upon Ca^{2+} binding. We validate the approach with experiments performed in aqueous solutions containing Ca^{2+} and Fluo4 dextran (both in its high and low affinity versions). Performing FCS experiments with tetramethylrhodamine-dextran in Xenopus laevis oocytes, we infer the corresponding free diffusion coefficients in the cytosol of these cells. Our approach can be extended to other physiologically relevant reaction-diffusion systems to quantify biophysical parameters that determine the dynamics of various variables of interest.

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