Fig. 2.

KMC validation of the forward model. FRET_R was calculated from KMC simulations of a system consisting of one CFP donor and one YFP acceptor. CFP and YFP were represented as dimensionless points that correspond to the centers of the fluorophore. FRET_R was computed with varying distance between fluorophores, donor excitation rate k_d^X/k_d^F, and donor/acceptor excitation rate ratio k_da = k_d^X,430/k_a^X,430 (circles and squares). k_d^F was set (35) at 0.4 nm⁻¹. The YFP photobleaching ratio during the acquisition of the CFP image was set at 0.3. In the regime of low excitation and fast de-excitation (k_d^X/k_d^F < 0.05), the value of FRET_R predicted by the forward model (lines) was in excellent agreement with KMC simulations (error analysis in supplemental Fig. S2).