= FRET photobleaching controls
{c}
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>,
$$
E=1-\frac{I_{D,\rm before}}{I_{D,\rm after}}.
$$
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.<Advanced Fluorescence Microscopy Techniques—FRAP, FLIP, FLAP, FRET and FLIM by Hellen C. Ishikawa-Ankerhold and others>[§3.4.1.1–3.4.1.2: donor recovery, sample damage, acceptor replenishment and photoconversion artifacts]{ref}
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