MEASURING [CA2- THEORETICAL APPROACH TO THE RATIO METHOD(] WITH FLUORESCENT INDICATORS )

In this work we present a theoretical analysis of the ratio method, a widely used technique for measuring intracellular calcium concentratio n, [Ca2+](i), in isolated cells. From the ratio of fluorescence measur ed at two different excitation or emission wavelengths, [Ca2+](i) may be estimated from the equation: [Ca2+](i) = K-d . beta . (R - R-min)/( R-max - R). From this equation we determined the method sensitivity sh owing that its maximum is located at [Ca2+] = K-d . beta . (R-min/R-ma x)(1/2), i.e. for [Ca2+] < K-d . beta. We also analyzed the error prop agation due to inaccuracies in the calibration parameters. The fluores cence phenomenon was described, aiming at providing a basis for the mi croscopic interpretation of the method and giving physical meaning to the calibration parameters. In this sense beta, is shown to depend not only on the set-up, but also on the spectrum of the indicator for the particular sample studied. A new approach to estimate beta with highe r accuracy is also proposed. Experimentally obtained beta values using this approach were not statistically different from those determined as F-min2/F-max2. A graphical interpretation of the method is presente d to provide users of fluorescence systems with a simple technique to help understand equipment performance and design.