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|Type:||Artigo de periódico|
|Title:||Measuring [Ca2+] with fluorescent indicators: theoretical approach to the ratio method|
|Abstract:||In this work we present a theoretical analysis of the ratio method, a widely used technique for measuring intracellular calcium concentration, [Ca2+](i), in isolated cells. From the ratio of fluorescence measured 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 showing that its maximum is located at [Ca2+] = K-d . beta . (R-min/R-max)(1/2), i.e. for [Ca2+] < K-d . beta. We also analyzed the error propagation due to inaccuracies in the calibration parameters. The fluorescence phenomenon was described, aiming at providing a basis for the microscopic 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 higher 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 presented to provide users of fluorescence systems with a simple technique to help understand equipment performance and design.|
|Appears in Collections:||Artigos e Materiais de Revistas Científicas - Unicamp|
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