Quantitative aspects of calcium fluorimetry

Ca(2+) indicator dyes by necessity are Ca(2+) chelators, because it is the binding of Ca(2+) to dye molecules that induces the change in fluorescence on which the Ca(2+) signal is based. As chelators, once introduced into a cell, they contribute to cellular Ca(2+) buffering. It has been a question of much debate to what extent this added Ca(2+) buffer (exogenous Ca(2+) buffer) changes Ca(2+) homeostasis and the signals of interest. I discuss this problem here, emphasizing the distinction between the influence of the dyes on amplitudes (which may be not so severe) and on the dynamics of Ca(2+) signals (which may be drastic). Once the Ca(2+)-buffering action of dyes relative to intrinsic Ca(2+) buffers is understood for a given preparation, Ca(2+) dyes can be used as very versatile tools for studying both Ca(2+) concentrations and Ca(2+) fluxes. I describe in detail some of my own experiences in calibrating the indicator dye Fura-2. These refer exclusively to experiments in which the dye is loaded into the cell via a patch pipette because acetoxymethyl ester loading introduces problems that very often prohibit precise quantitative conclusions.