Acceptor photobleaching analysis is available when a bleaching event is found in the image metadata. In this case the FRET efficiency can be calculated the following way:

E = (Dpost – Dpre)/Dpost

D is the donor signal and subscripts refer to pre- and post-bleach images.

In all cases the FRET values are calculated for every pixel that is considered “valid” and then the mean value is calculated and presented in the table. In case of acceptor photobleaching, the efficiency is also calculated from the pixel-averaged Dpre and Dpost signals.