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Proceedings Paper

Two-photon FLIM-FRET microscopy for protein localization
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Paper Abstract

Intensity based FRET visualizes protein molecules in living cells and tissues. Lifetime techniques, however, will demonstrate the dynamic functional activity of the protein molecules, because its signal does not depend on changes in fluorophore concentration or excitation intensity. If a laser pulse excites a large number of similar molecules with a similar local environment, and as long as no energy is transferred to another molecule, the lifetime is the “natural fluorescence lifetime”. If energy is transferred, however, the actual fluorescence lifetime is less than the natural lifetime, because an additional path for de-excitation is present. With the occurrence of FRET, strong energy transfer results in extreme quenching of the donor fluorescence and a decrease in the fluorescence lifetime. In this paper we will explain the development of the two-photon FLIM-FRET microscopy with our existing multiphoton microscopy and demonstrate the change in donor lifetime by photobleaching the acceptor molecules in living cells.

Paper Details

Date Published: 21 June 2004
PDF: 9 pages
Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); doi: 10.1117/12.538312
Show Author Affiliations
Ye Chen, W. M. Keck Ctr. for Cellular Imaging/Univ. of Virginia (United States)
Ammasi Periasamy, W. M. Keck Ctr. for Cellular Imaging/Univ. of Virginia (United States)


Published in SPIE Proceedings Vol. 5323:
Multiphoton Microscopy in the Biomedical Sciences IV
Ammasi Periasamy; Peter T. C. So, Editor(s)

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