Assume that in a ratiometric imaging experiment with fluorescein as described in Section 11.5, fluorescence counts of F49 0 = 1080 and F 440 = 216 are detected with excitation wavelengths 490 nm and...

Assume that in a ratiometric imaging experiment with fluorescein as described in Section 11.5, fluorescence counts of
_F490 = 1080 and F₄₄₀
= 216 are detected with excitation wavelengths 490 nm and 440 nm, respectively, resulting in a ratio of R490/440 = F₄₉₀/F₄₄₀
= 5. Now assume that these counts are not corrected for background fluorescence, and further assume that the same background level is observed at the two excitation wavelengths. Denoting the background fluorescence count by F_b, calculate the background-corrected ratio R₄₉₀/440,C = (F₄₉₀
− F_b)/(F440 − F_b) for different values of F_b
and assess the error in pH measurement based on the curve in Fig. 11.12(b).