Calculation of [Ca²⁺] with excitation at two wavelengths

Reference: Grynkiewicz et al, 1985

Fura-2 Excitation Spectra

F1, F2: Fluorescence Intensity at λ1 and λ2 R: Ratio (F1/F2). Rmax: Maximum Ratio when all dyes are bound. Rmin: Minimum Ratio when all dyes are free. Sf: Proportionality Coefficient (Free) Sb: Proportionality Coefficient (Ca2+-bound) Cf: Free Dye Concentration Cb: Ca2+-bound Dye Concentration Kd: Dissociation Constant

Since (3), (4) =>