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FCS experiments to quantify Ca2+ diffusion and its interaction with buffers.

Ca2+ signals are ubiquitous. One of the key factors for their versatility is the variety of spatio-temporal distributions that the cytosolic Ca2+ can display. In most cell types Ca2+ signals not only depend on Ca2+ entry from the extracellular medium but also on Ca2+ release from internal stores, a process which is in turn regulated by cytosolic Ca2+ itself. The rate at which Ca2+ 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 Ca2+ signals. The quantification of Ca2+ 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 Ca2+ 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 Ca2+ diffusion and of its reaction rates with unobservable (non-fluorescent) Ca2+ 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 Ca2+, a single wavelength Ca2+ dye, and a non-fluorescent Ca2+ 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 Ca2+ 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.