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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]

Photobleaching is an irreversible loss of fluorescent capacity caused by illumination. It changes the number of active fluorophores over experimental time; fluorescence lifetime concerns the delay after an individual excitation. Reversible dark states and ordinary return to the ground state are different processes.

In a stipulated population-loss model, let $T$ denote elapsed illumination time and $t$ denote delay after a pulse. If active population declines as $e^{-k_{\mathrm{b}}T}$ while surviving fluorophores retain the same decay rates, then $I(t;T)=A_0{e}^{-k_{\mathrm{b}}T}{e}^{-t/\tau }$. Brightness falls while $\tau$ stays fixed. This example does not assert that all bleaching follows exponential decay: chemical changes and selective loss from a mixture can also change the observed decay shape. The existing FRET photobleaching controls illustrate why deliberate bleaching requires careful interpretation.