Many signal transduction events in cells are mediated by protein-protein or protein-RNA interactions. Changes in protein activity are often mediated by a change in protein conformation. Such interactions and conformational changes can be monitored by Förster resonance energy transfer (FRET) between donor and acceptor fluorophores attached to the molecules of interest. The most reliable method for FRET detection in live cells is fluorescence lifetime imaging (FLIM). FLIM-FRET is based on detecting changes in fluorescence lifetime of the donor fluorophore and is independent of donor and acceptor concentrations in the cell.
This instrument will allow 2P-based FLIM-FRET imaging in live thick-tissue specimens such as brain tissue slices. The methods require expression of genetically-encoded fluorophores.
The rig is equipped with a Becker & Hickl single-channel FLIM detector and workstation for time-correlated single photon counting (TCSPC). 2P excitation is achieved using a Coherent Chameleon Vision II laser. The FLIM system is integrated with an upright microscope (Leica SP5) for confocal/2P imaging.