FRET, FRAP & FLIM
Energy transfer from an excited donor to an acceptor a few nanometres away, read here as sensitised acceptor emission with three channels and bleed-through corrections, or as donor recovery after acceptor photobleaching.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
FRET
Energy transfer from an excited donor to an acceptor a few nanometres away, read here as sensitised acceptor emission with three channels and bleed-through corrections, or as donor recovery after acceptor photobleaching.
How it differs from the standard method
- Single-chain biosensors fix the donor:acceptor ratio at 1:1 and report conformational change.
- Intermolecular FRET needs both expression ratios measured and corrected.
- Choose bright, photostable pairs with a large R0 (e.g. mTurquoise2–mVenus).
1Measuring what an image cannot show
Light microscopy resolves about 200 nm, but proteins interact over a few nanometres and move in seconds. FRET, FRAP and FLIM get at those scales indirectly, by measuring how fluorescence behaves rather than where it is.
E = 1/(1 + (r/R0)⁶)
R0, the Förster radius, is the distance of 50% transfer, about 5–6 nm for fluorescent-protein pairs. Efficiency changes steeply between about 0.5 and 1.5 R0 and is negligible beyond 10 nm.
Mobile fraction and t½
The plateau tells how much of the protein exchanges; the half-time tells how fast. For pure diffusion into a circular spot of radius w, t½ ≈ 0.88 w²/4D.
Lifetime, not intensity
A lifetime does not depend on concentration or excitation intensity, which is why FLIM-FRET is often the most robust FRET readout: E = 1 − τ_DA/τ_D.
Measuring bleaches
All three methods spend photons, and each exposure bleaches the sample a little; the analysis has to correct for it or the acquisition design has to keep it negligible.
2FRET by intensity: three cubes and two controls
Sensitised emission is measured with three images: donor excitation and donor emission, acceptor excitation and acceptor emission, and donor excitation with acceptor emission (the FRET channel). The FRET channel is contaminated: donor emission spills into it (bleed-through) and the acceptor is excited directly at the donor wavelength.
3FRAP: bleach fast, watch gently, normalise twice
A good FRAP experiment bleaches a defined spot deeply and quickly compared with the recovery (otherwise molecules diffuse during the bleach and the spot is wider and shallower than intended), then monitors at low power with frames several times faster than t½, for long enough to reach the plateau. The raw curve is distorted twice: each monitoring frame bleaches the whole cell a little, and the bleach itself removes a share of the cell's fluorescent pool.
4FLIM: counting single photons in time
Time-correlated single photon counting (TCSPC) times each detected photon against the laser pulse that excited it and builds a decay histogram per pixel. Precision is set by the number of photons: roughly τ/√N, so a lifetime map needs on the order of a thousand photons per pixel.
Key terms
Practise these as flashcards →Donor–acceptor distance at which FRET efficiency is 50%; set by spectral overlap, donor quantum yield, orientation (κ²) and refractive index.
Acceptor fluorescence caused by energy transfer from an excited donor, measured in the FRET channel after correcting for bleed-through and direct excitation.
Donor emission detected in the acceptor or FRET channel.
The share of molecules that exchange into a bleached region over the experiment; the rest are immobile on that timescale.
Dividing the bleached-region intensity by a reference region, each relative to its pre-bleach level, to correct acquisition bleaching and pool loss.
The average time a fluorophore stays excited before emitting, a few nanoseconds for fluorescent proteins; FRET shortens the donor's.
Time-correlated single photon counting: timing individual photons relative to laser pulses to build decay curves.
A fit-free representation of decays as points (g, s); single exponentials lie on a semicircle and mixtures inside it.
When to use it
- Testing whether two proteins come within a few nanometres, or reading a conformational biosensor's activity in living cells: FRET.
- Measuring how freely a protein moves or how long it stays bound to a structure: FRAP.
- Quantifying interactions independently of expression levels, or mapping environment-sensitive lifetimes: FLIM.
Compared with alternatives
Detects proximity of two endogenous proteins (≈ 40 nm) in fixed cells with antibodies, with no fusions needed.
Measures binding in solution through rotational mobility, without cells or imaging.
Biochemical evidence for a complex from lysates; prefer it when spatial information is not needed.
Resolves positions down to tens of nanometres directly, rather than inferring proximity from energy transfer.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Energy transfer from an excited donor to an acceptor a few nanometres away, read here as sensitised acceptor emission with three channels and bleed-through corrections, or as donor recovery after acceptor photobleaching.
- Single-chain biosensors fix the donor:acceptor ratio at 1:1 and report conformational change.
- Intermolecular FRET needs both expression ratios measured and corrected.
- Choose bright, photostable pairs with a large R0 (e.g. mTurquoise2–mVenus).