Figure 6.

Uncertainties of the condition probability matrix may propagate to errors of the donor–acceptor distances in particular for minorly populated states. Limitations of the homogeneous approximation illustrated by simulations of a typical time-resolved experiment with three discrete FRET states. (A) Simulated time-resolved fluorescence decay histograms with 100 000 photons in peak (bin width 14.1 ps) using an experimental recorded IRF with a fwhm of 250 ps of a system with three discrete donor states 4 ns (80%), 2.5 ns (14%), and 0.5 ns (6%), and three discrete FRET states 40 Å (30%), 45 Å (50%), and 60 Å (20%) (R₀ = 50 Å, k₀^–1 = 4 ns). The 40 and 45 Å state are associated with the donor lifetime of 4 ns; the 60 Å state is associated with the donor lifetimes 2.5 and 0.5 ns. The conditional probability matrix [ξ^(i,j)] and the corresponding values of the [x_DA^(i,j)] matrix are shown as numbers in the tables. (B) The analysis result using the correct model and the inappropriate homogeneous model are shown on the top and bottom, respectively. The weighted residuals (w.res.) of both models are indistinguishable. To the right the recovered distances and fractions are plotted in a bar diagram.