A new calibration equation for ratiometric fluorescent ion indicators: Application to fura-2

The absolute values of intracellular ion concentrations as monitored by specific fluorescent indicators are determined by using calibration curves obtained underin vitro andin vivo conditions. In the derivation of the calibration curve by Grynkiewicz et al [(1985)J. Biol. Chem 260, 3440] it is implicitly assumed that the observed fluorescence signal is directly related to the concentrations of the free dye and the dye-ion complex in the ground state. We modified the calibration equation so that ion binding and dissociation in the excited state are taken into account. The extended calibration equation assumes the knowledge of the rate constants in the excited state. Expressions for the calibration curve assuming the absence or presence of an excited-state reaction are compared for the Ca(2+) indicator Fura-2. The excited-state rate constants are determined by global compartmental analysis of time-resolved fluorescence decays of Fura-2 collected at various excitation and emission wavelengths using different Ca(2+) concentrations. It is found that for Fura-2 there is negligible interference of the excited-state reaction so that the original calibration can used.