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The FRET donor lifetime is the mean time spent in the excited state. For the constant-rate model of FRET efficiency,After a sufficiently brief excitation pulse, the FRET donor's fluorescence follows exponential decay, $I(t)=I(0)e^{-t/\tau }$. With the illustrative values ${\tau }_D=4$ nanoseconds and ${\tau }_{DA}=2$ nanoseconds, the inferred FRET efficiency is $1/2$. Multiplying $I(0)$ changes brightness without changing this decay time.

The lifetime comparison requires the same nontransfer decay rates in the reference and paired samples. Changes in the FRET donor's environment or a different nonradiative loss can also shorten the FRET donor lifetime. Heterogeneous FRET efficiencies or slowly varying FRET dipole orientation factors need an appropriate decay model; substituting any fitted average lifetime into the single-pair formula can mislead. ^[ref]

FRET optical crosstalk includes FRET donor emission leaking into the FRET acceptor detection band and direct excitation of the FRET acceptor by the nominal FRET donor excitation light. Measure donor-only, acceptor-only and unlabeled preparations with the same fluorescence microscope settings to separate these contributions and background.^[ref]

An explicit linear measurement model makes the controls' purpose clear. Let $D$ be background-subtracted donor-band counts under donor excitation, $A$ acceptor-band counts under acceptor excitation, and $F$ acceptor-band counts under donor excitation. Donor-only and acceptor-only preparations determine leakage coefficients $a$ and $b$. Thenis a corrected FRET sensitized emission signal under the assumed linear, fixed optical response. In a synthetic example $D=100$, $A=80$, $a=0.10$, $b=0.05$ and $F=34$ give $S=20$. Even a nontransferring sample would give $F=14$ from these two contaminants. Correcting that signal still does not convert $S$ into FRET efficiency without a calibration of excitation, yields and detection.

A biological comparison also needs a pairing-negative preparation and a known transferring positive preparation. Match label abundance and localization where possible; changing concentration can change encounter frequency as well as brightness. Check that attaching the labels preserves the protein function being investigated. Otherwise the measurement can faithfully report a perturbation introduced by the labels themselves.