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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.

The FRET dipole orientation factor describes the geometry of the two transition electric dipoles relative to their separation. Let ${\hat{\mathbf{\mu }}}_D$, ${\hat{\mathbf{\mu }}}_A$ and $\hat{\mathbf{r}}$ be their unit vectors. ThenFor a separation along $z$, parallel electric dipoles along $z$ give ${\kappa }^2=4$, parallel ones along $x$ give $1$, and one along $x$ with the other along $y$ gives $0$. Thus rotating a pair can change FRET efficiency without changing its separation.

The familiar value ${\kappa }^2=2/3$ requires suitable rapid, independent orientation averaging during the FRET donor lifetime. Random orientations across a population of slowly rotating fluorescent proteins do not establish that dynamic condition. ^[ref]

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]