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FLIM FRET - Fluorescence Resonance Energy Transfer

The FLIM-FRET method

A typical application of FLIM is FLIM-FRET. FRET is a well-established technique to study molecular interactions. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well. The SP8 FALCON system together with the integrated FRET analyzer provides FRET-efficiency and binding maps.

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The FRET effect

In a FRET experiment the potential binding partners are labeled with spectrally distinct fluorophores in such a way that the emission spectrum of the donor molecule overlaps the excitation spectrum of the acceptor molecule. If both interaction partners are in close contact at a distance of only a few nanometers, the excited donor can transfer its energy to the acceptor. In turn, the acceptor emits a fluorescence photon and the fluorescence lifetime of the donor molecule decreases.

Motivation for FLIM-FRET

Intensity-based FRET methods are quite susceptible to variations in expression level or molecule diffusion inherent in the sample. This also applies to external influences such as sample movements and excitation fluctuation. Regarding this, FLIM-FRET is a great advantage as it is calibrated internally. It allows FRET measurements independent of such disturbances. If FRET occurs a shortening of donor lifetime is observed. This value is a measure for the FRET-efficiency. The combination of FLIM-FRET with SP FLIM can be used to improve data quality and to enhance precision of quantitative data.