Fluorescence resonance energy transfer (FRET) has extended fluorescence microscopy beyond simple subcellular co-localization to a technique that is sensitive to molecular conformation, association, and separation distances on the nm range. Recent advances in FRET allow the possibility of real-time and in vivo imaging. The basis of FRET can be traced to the work of Theodor Förster (Förster, 1965). Along with FRAP, FRET exploits photobleaching to obtain information. FRET is based on the energy transfer between the electronic excited states of donor and acceptor molecules without the emission of a photon. This results from long-range dipole-dipole coupling. The absorption spectrum of the acceptor must overlap the fluorescence emission of the donor and the two molecules must be in close proximity. Homo-FRET, in which the donor and acceptor molecules are the same, is detected by fluorescence depolarization while hetero-FRET is detected by acceptor sensitization or donor quenching. While both molecules can be fluorophores, use of nonfluorescent acceptor molecules eliminates background fluorescence in hetero-FRET.

Applications:

1. Receptor/ligand interactions
2. Structure and conformation of proteins and nucleic acids
3. Assembly of protein complexes
4. Immunoassays
5. Transport of lipids
6. Single molecule studies (Foeldes-Papp, Zeno - Review 3 below)

Pros:

1. new photosensitive tags can now be used for intracellular labeling (Schultz2010, Gautier2008)
2. useful for real-time activity and interaction measurements
   

Cons:

1. Although required separation distances have been tabularized, the actual values may vary some due to local environmental factors.