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Proceedings Paper

Two-photon excitation energy transfer microscopy
Author(s): Ammasi Periasamy
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Paper Abstract

Fluorescence resonance energy transfer (FRET) imaging is a unique tool used to visualize the spaciotemporal 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 flourescent protein and green fluorescent protein. 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 excitation energy transfer microscopy, where the photobleaching of blue flourescent protein was considerably reduced. This 2p-FRET imaging system was used to acquire the donor and acceptor images for 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 percent compared to the conventional FRET imaging, which clearly demonstrates that there is considerable reduction in photobleaching of donor molecules in the 2p-FRET microscopy.

Paper Details

Date Published: 27 April 2000
PDF: 6 pages
Proc. SPIE 3921, Optical Diagnostics of Living Cells III, (27 April 2000); doi: 10.1117/12.384224
Show Author Affiliations
Ammasi Periasamy, Univ. of Virginia (United States)

Published in SPIE Proceedings Vol. 3921:
Optical Diagnostics of Living Cells III
Daniel L. Farkas; Daniel L. Farkas; Robert C. Leif, Editor(s)

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