Fig. 2.

Time-resolved EEM analysis sequence of three-color FRET. The analysis is performed on a channel-by-channel basis, with each channel involving only at most one unknown lifetime parameter. EEM channels in the illustration are color-coded by their decay models. The analysis first obtains the longest wavelength acceptor (fluorophore No. 3) lifetime τ₃ in ê₃₃, then finds quenched lifetime ${\tau }_2^{123}$ of fluorophore No. 2 in FRET channel ê₂₃. The percentage of quenched fluorophore, P₂ is then calculated from fluorophore 2 EEM channel ê₂₂. The FRET channel ê₁₂ is next, which yields the quenched lifetime ${\tau }_1^{123}$of fluorophore 1. The percentage of quenched fluorophore No. 1, P₁ is extracted from the EEM channel ê₁₁, and finally the FRET channel ê₁₃ serves as a verification of the time-resolved EEM analysis.