Spectra of intracellular Fura-2.

In the theory of measurement of calcium ion activity by determination of Fura-2 fluorescence at two excitation wavelengths, the accuracy of the result depends upon the accuracy both of the sample measurements and of the calibration measurements which are made on calcium-bound and free dye. Two factors underlie adequate calibration and accuracy. The first is the elimination of systematic error due to spectral shifts arising from the intracellular environment felt by the dye. To this end, detailed comparisons between complete spectra of both calcium-bound and calcium-free Fura-2 can be used to help separate spectral effects due to light absorption by cellular constituents versus polarity and viscosity of the intracellular milieu. The second major factor which determines accuracy is the experimental uncertainty (in both sample and calibration measurements). For samples in which the ratio of bound to free dye is large, the uncertainty in the ratio is also large, even when it is expressed as a percentage of the ratio itself. The errors in calibration measurements impact on the accuracy of the method primarily through the measurements made at wavelengths which are off the spectral peaks of the bound or free dye, since these are the least accurate. In order to obtain a guide to the choice of wavelengths and estimation of the reliability of results, a mathematical expression is derived for the dependence of the accuracy of the method on the accuracy of both sample and calibration measurements.