Förster Resonance Energy Transfer (FRET) measurements
Despite the limited resolution of light microscopes the molecular vicinity of molecules can be tested by exploiting a physical phenomenon, Förster Resonance Energy Transfer (FRET). FRET occurs when a donor fluorophore (D) is brought into close vicinity (< 10 nm) to a suitable acceptor molecule (A). In this case, the donor can transfer its excitation energy to the acceptor, which in turn can emit its excitation energy as fluorescence light.
When FRET occurs, both the intensity and the lifetime of the donor fluorescence decrease, whereas the intensity of the acceptor emission increases. This offers different approaches for the determination of FRET efficiency. One is to detect changes in the fluorescence intensity of the donor or the acceptor emission, the other is to measure changes in the fluorescence lifetime of the donor molecule.
By determining the FRET efficiency information about the distance and relative orientation of donor and acceptor fluorophores can be obtained. Apart from observing intermolecular interactions also intramolecular conformational changes can be investigated.