FRET Efficiency Calculator

FRET is a non-radiative energy transfer process between two fluorophores — a donor and an acceptor — when they are in close proximity (typically 1–10 nm). The excited donor transfers energy directly to the acceptor via dipole–dipole coupling, without emitting a photon. Because efficiency depends on the sixth power of the inter-fluorophore distance, FRET is an exquisitely sensitive 'molecular ruler' used throughout biochemistry and cell biology.

FRET efficiency E is given by E = 1 / (1 + (R/R₀)⁶), where R is the actual donor–acceptor distance and R₀ is the Förster radius. When R = R₀ the efficiency is exactly 50%. Efficiency approaches 100% as R decreases well below R₀, and approaches 0% as R greatly exceeds R₀.

R₀ is the donor–acceptor distance at which FRET efficiency equals 50%. It is calculated from spectroscopic properties of the pair: R₀ (nm) = 0.2108 × (n⁻⁴ · ΦD · κ² · J)^(1/6), where n is the refractive index of the medium, ΦD is the donor quantum yield, κ² is the orientation factor, and J is the spectral overlap integral in M⁻¹cm⁻¹nm⁴. Typical R₀ values for common fluorescent protein pairs range from ~4 to 7 nm.

For freely rotating fluorophores — the most common assumption in solution-based FRET experiments — κ² = 2/3 ≈ 0.667. If the dipoles are fixed and parallel, κ² = 1; if collinear, κ² = 4. In practice, the random-orientation assumption (κ² = 2/3) is standard unless fluorophore mobility is known to be restricted.

For aqueous buffers and most biological imaging conditions, n ≈ 1.33 (pure water) to 1.40 (cytoplasm). A value of 1.33 is the standard default. If your experiment uses glycerol-based mounting media or cell interiors with high protein content, a slightly higher value (1.36–1.40) may be more appropriate.

J(λ) quantifies the degree of spectral overlap between the donor's emission spectrum and the acceptor's absorption spectrum, weighted by wavelength to the fourth power. A larger J means a larger R₀ and more efficient energy transfer at a given distance. J is typically reported in units of M⁻¹cm⁻¹nm⁴, and for common fluorescent protein pairs it falls in the range of 10¹³–10¹⁶ M⁻¹cm⁻¹nm⁴.

FRET efficiency is most sensitive to distance in the range of roughly 0.5×R₀ to 2×R₀. Outside this range, efficiency is either near 100% or near 0% and changes very little with distance. This makes FRET particularly powerful as a molecular ruler for distances of approximately 2–10 nm, ideal for probing protein–protein interactions, conformational changes, and membrane dynamics.

Common experimental approaches include measuring the decrease in donor fluorescence intensity or lifetime in the presence of the acceptor (donor quenching), measuring the increase in acceptor emission (sensitized emission), acceptor photobleaching FRET (recovering donor fluorescence after bleaching the acceptor), and fluorescence lifetime imaging (FLIM-FRET). Each method has trade-offs in sensitivity, quantitation accuracy, and required instrumentation.