Quantitative Aspects of Calcium Fluorimetry

Abstract

Ca2+ indicator dyes by necessity are Ca2+ chelators, because it is the binding of Ca2+ to dye molecules that induces the change in fluorescence on which the Ca2+ signal is based. As chelators, once introduced into a cell, they contribute to cellular Ca2+ buffering. It has been a question of much debate to what extent this added Ca2+ buffer (exogenous Ca2+ buffer) changes Ca2+ 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 Ca2+ signals (which may be drastic). Once the Ca2+-buffering action of dyes relative to intrinsic Ca2+ buffers is understood for a given preparation, Ca2+ dyes can be used as very versatile tools for studying both Ca2+ concentrations and Ca2+ 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.