Fluorescence resonance energy transfer (FRET)

About this technique

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Fluorescence resonance energy transfer (FRET) is an interaction between the electronic excited states of two dye molecules. Excitation energy is transferred from a donor molecule to an acceptor molecule without emission of a photon from the donor. FRET depends on the inverse sixth power of the intermolecular separation, making it useful over distances comparable with the dimensions of biological macromolecules, which are typically 2 nm to 50 nm. The efficiency with which this energy transfer is conducted can be used as a “molecular ruler” to determine distances between molecules. This powerful technique allows us to study molecular interactions within cells in live and fixed conditions. There are several conditions that need to be met before FRET will occur:

• donor and acceptor molecules must be in close proximity (typically 1–10 nm).
• absorption (excitation) spectrum of the acceptor must overlap fluorescence emission spectrum of the donor.
• donor and acceptor transition dipole orientations must be approximately parallel.

When FRET occurs the donor fluorescence is diminished and the acceptor fluorescence is increased. The amount of these intensity changes is determined by the FRET efficiency which is directly proportional to the distance between any pair of fluorophores. While this is an incredibly powerful technique care must be taken in setting up the experiment.

• This technique can be used on both wide-field and confocal microscopes for examining either fixed or living tissue.
• Selection of efficient dye pairs is critical.
• When calculating FRET results, excitation cross talk, bleed-through emission, background fluorescence and the possibility of bleaching must all be taken into account.
• Appropriate control preparations are essential.