Centre Optical Instrumentation Laboratory

Fluorescence Resonance Energy Transfer Protocol

For Fluorescence Resonance Energy Transfer (FRET) to occur it must satisfy 5 criteria (Periasamy and Day 1999)

(i) The Emission spectra of one fluorophore (Donor) must significantly overlap the excitation spectra of another fluorophore (Acceptor)
(ii) Donor and Acceptor must be aligned so that an acceptor dipole can be induced by the donor
(iii) The Donor/Acceptor separation can be no greater than 10nm
(iv) The Donor has a high quantum yield
(v) The Donor is saturated by the acceptor
FRET diagram

Assuming that these criteria have all been met then energy transfer ET will occur and is described by the Forster equation.


FRET Equation

R = Donor-Acceptor separation distance
Ro = Donor-Acceptor separation distance at which FRET efficiency is 50%

FRET Advantages/Disadvantages

Advantages

Disadvantages

Relatively cheap to implement
Only works if fluorophores are in correct orientation
Measurements are obtained very rapidly
Size of probes can introduce problems
Very good at measuring changes in distance
Gives no information about which probe moves
Free fluorophores can mask energy transfer
FRET pair labelling needs to be bright with the donor completely saturated by acceptor
Can be pH sensitive
If it doesn't work, it doesn't tell you anything, as you have no idea whether it failed due to incorrect orientation, free fluorophores etc or whether the proteins are simply too far apart.


There are generally 3 commonly used approaches for measuring FRET.

  • Decreased donor lifetime - measured using Fluorescence Lifetime Imaging Microscopy (FLIM) which requires expensive secondary equipment.

  • Decrease in Donor emission intensity - known as donor quenching, measured by acceptor photobleaching

  • Increase in Acceptor emission intensity - known as sensitized emission


Practical guides to FRET

Fluorescence Lifetime FRET

Acceptor photobleaching FRET

Sensitized Emission FRET