Figure 11.

Influence of the donor decay function on FLIM-FRET evaluation. On the left mixtures of decays (black curve) are shown that are separated in the lifetime decays (gray curves) of the contributing states of the putative donor on the right. For donor fluorophores that show a monoexponential fluorescence decay, a bi-exponential fit is required for data evaluation to consider donor fraction that transfer energy to the acceptor and the donor fraction that emits directly (A). If the donor already shows a biexponential behavior fluorescence decay behavior, four contributing fractions of donor have to be considered for the fit of the lifetime decay (B). The fit also provides information on the fraction of donor that undergoes energy transfer to the acceptor.