FRET photobleaching controls

In FRET photobleaching controls, selectively destroy the FRET acceptor's ability to absorb and test whether FRET donor fluorescence increases. Under unchanged excitation and detection, complete loss of transfer, and an intact FRET donor,This is a destructive comparison. Monitor acceptor loss, donor-only samples exposed to the bleaching illumination, and an unbleached reference region or time trace. Incomplete bleaching, donor damage, movement or label replenishment violate the comparison. During ordinary recordings, gradual unequal bleaching can instead produce an apparent change of FRET without a biological change.^[ref]