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The Förster distance is the separation giving FRET efficiency $E=1/2$ for the specified pair and conditions. It is a property of the optical pair and its environment, rather than a universal length. In the usual pair model,where ${\kappa }^2$ is the FRET dipole orientation factor, $Q_D$ the FRET donor quantum yield, $J$ the FRET spectral overlap integral, and $n$ the medium's refractive index. The omitted proportionality constant depends on the unit convention for $J$.^[ref]

If FRET efficiency is known and those assumptions hold, inversion givesThe derivative explains why errors become troublesome near $E=0$ or $E=1$. An illustrative pair with $R_0=4$ nanometres and another with $R_0=6$ nanometres both give $E=1/2$ at their different Förster distances: knowing FRET efficiency alone does not determine $r$.

A heterogeneous population adds another ambiguity. In a synthetic example with equal weights per FRET donor excitation, efficiencies $0.1$ and $0.9$ average to $0.5$, but their separations are $9^{1/6}{R}_0$ and $9^{-1/6}{R}_0$. Their average separation is approximately $1.068R_0$, whereas inverting the average FRET efficiency gives $R_0$. A measured emission ratio has additional detection weights; it need not equal even this excitation-weighted average. The transferable lesson is that an average readout does not uniquely specify a distribution of molecular separations.

Förster resonance energy transfer is a nonradiative transfer of energy from an excited state of a FRET donor to a nearby FRET acceptor, through coupling of their electric dipoles. The transfer step does not emit a photon that the FRET acceptor subsequently absorbs. It competes with the FRET donor's own fluorescence and other routes out of its excited state. A fluorescent FRET acceptor can then emit fluorescence; an absorbing but nonfluorescent FRET acceptor can still receive the transferred energy.^[ref]

The striking use of FRET is to turn a change in molecular proximity into an optical readout. Proteins carrying a FRET donor and a FRET acceptor may change the readout when they associate, as in the chemotaxis FRET assay; two labels on one protein may report a change in its shape. Interpretation depends on the FRET spectral overlap, FRET dipole orientation factor and labeling geometry, as well as separation. A change in FRET is therefore evidence about proximity under specified conditions, rather than a direct measurement of an arbitrary distance.^[ref]

For a single pair with fixed Förster distance $R_0$ and label separation $r$, the FRET efficiency isAt $r=R_0$, half the FRET donor excitations transfer energy. At $r=R_0/2$, the model gives $E=64/65\approx 98.46\%$; at $r=2R_0$, it gives $E=1/65\approx 1.54\%$. The sixth power makes FRET especially useful near the Förster distance, with little sensitivity to further separation once transfer is already negligible. These are model calculations, not measurements of a particular pair.

A FRET acceptor receives the excited state energy. Its absorption must support FRET spectral overlap with the FRET donor. FRET sensitized emission requires a fluorescent FRET acceptor; measuring a shortened FRET donor lifetime does not.

FRET spectral overlap quantifies overlap between the FRET donor's emission and the FRET acceptor's absorption as functions of wavelength $\lambda$. With normalized donor emission $f_D$ and acceptor molar absorption coefficient ${\epsilon }_A$, one convention isMatching two emission colors is insufficient: the receiving absorption matters.^[ref]