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Proceedings Paper

Multiphoton FLIM: a reliable FRET detection tool in cell biological applications
Author(s): Ramanujan V. Krishnan; Eva Biener; Victoria E. Centonze; Arieh Gertler; Brian A. Herman
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Paper Abstract

Fluorescence lifetime imaging microscopy (FLIM) using multiphoton excitation is emerging as a reliable quantitative tool for measuring fluorescence resonance energy transfer (FRET) in living cells. By virtue of being free from spectroscopic artifacts encountered in conventional FRET detection methods, multiphoton FLIM methods offer the advantages of high spatial and temporal resolution, faster data acquisition and data analysis. We compare the FRET results obtained by two different methods namely (i) multiphoton excitation lifetime-based FRET and (ii) single photon excitation intensity-based acceptor photobleaching FRET. Using the same biological samples, we apply these two different methods in understanding the growth hormone receptor dimerization kinetics at the cell surface of human embryonic kidney cells. We conclude that the multiphoton FLIM using the streak-camera approach provides the best ability to monitor FRET in dynamic situations where high temporal and spatial resolution are required with minimal photodamage/phototoxicity.

Paper Details

Date Published: 21 June 2004
PDF: 8 pages
Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); doi: 10.1117/12.528050
Show Author Affiliations
Ramanujan V. Krishnan, Univ. of Texas Health Science Ctr. (United States)
Eva Biener, Institute of Biochemistry/Hebrew Univ. of Jerusalem (Israel)
Victoria E. Centonze, Univ. of Texas Health Science Ctr. (United States)
Arieh Gertler, Institute of Biochemistry/Hebrew Univ. of Jerusalem (Israel)
Brian A. Herman, Univ. of Texas Health Science Ctr. (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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