Two-photon excitation energy transfer microscopy

Fluorescence resonance energy transfer (FRET) imaging is a unique tool used to visualize the spatiotemporal dynamics of protein-protein interactions in living cells. We used FRET to study the dimerization of the pituitary-specific transcription factor of Pit-1 fused with blue fluorescent protein (BFP, donor) and green fluorescent protein (GFP, acceptor). Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. The energy transfer in the conventional fluorescence microscopy was less efficient due to photobleaching of the BFP-Pit-1 donor molecules. In our studies we developed two-photon (2p) excitation energy transfer microscopy, where the photobleaching of blue fluorescent protein was considerably reduced. This 2p-FRET imaging system was used to acquire the donor and acceptor images from a living HeLa cell nucleus. We selected 732 nm from the tunable Verdi pumped ti: sapphire laser, in a way that only excites the BFP-Pit-1 and not the GFP-Pit-1 proteins. The efficiency of the 2p-FRET signal increased to 30% compared to the conventional FRET imaging, which clearly demonstrates that there is considerable reduction in photobleaching of donor molecules in the 2p-FRET microscopy.