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) =>