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 Leica TCS SP8 SMD system together with the advanced FRET analysis tools within SymPhoTime Software from PicoQuant form a solid base to perform such studies with a high quality.
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.
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.