Multiphoton FLIM: a reliable FRET detection tool in cell biological applications

Ramanujan V. Krishnan; Eva Biener; Victoria E. Centonze; Arieh Gertler; Brian A. Herman

doi:10.1117/12.528050

21 June 2004 Multiphoton FLIM: a reliable FRET detection tool in cell biological applications

Ramanujan V. Krishnan, Eva Biener, Victoria E. Centonze, Arieh Gertler, Brian A. Herman

Proceedings Volume 5323, Multiphoton Microscopy in the Biomedical Sciences IV; (2004) https://doi.org/10.1117/12.528050
Event: Biomedical Optics 2004, 2004, San Jose, CA, United States

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.

Citation Download Citation

Ramanujan V. Krishnan, Eva Biener, Victoria E. Centonze, Arieh Gertler, and Brian A. Herman "Multiphoton FLIM: a reliable FRET detection tool in cell biological applications", Proc. SPIE 5323, Multiphoton Microscopy in the Biomedical Sciences IV, (21 June 2004); https://doi.org/10.1117/12.528050

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