Comparison of FRET microscopy imaging techniques for studying protein-protein interactions in living cells using FRET standards

Yuansheng Sun; Soo-Ah Seo; Sydney Provence; Ammasi Periasamy

doi:10.1117/12.842186

26 February 2010 Comparison of FRET microscopy imaging techniques for studying protein-protein interactions in living cells using FRET standards

Yuansheng Sun, Soo-Ah Seo, Sydney Provence, Ammasi Periasamy

Author Affiliations +

Proceedings Volume 7569, Multiphoton Microscopy in the Biomedical Sciences X; 75690Z (2010) https://doi.org/10.1117/12.842186
Event: SPIE BiOS, 2010, San Francisco, California, United States

Abstract

Förster resonance energy transfer (FRET) microscopy is a powerful tool to localize protein-protein interactions in living specimens. Various FRET microscopy imaging techniques have been established and are generally categorized into intensity-based and lifetime-based methods. Based on the detection of the acceptor sensitized emission, we have developed the FRET imaging methodologies that can be applied in combination of wide-field, conventional confocal or spectral microscopy. All these FRET microscopy methods have the capability to interpret the change in proximity between the donor and the acceptor through measuring the apparent energy transfer efficiency (E). However, to answer what subtle change of E can be detected and to link correctly FRET data to biological information, the imaging techniques have to be well calibrated. In this regard, we compare various FRET microscopy methods and assess their utilities using several genetic ("FRET standard") constructs where Cerulean and Venus fluorescent proteins are tethered by different amino acid linkers.

Citation Download Citation

Yuansheng Sun, Soo-Ah Seo, Sydney Provence, and Ammasi Periasamy "Comparison of FRET microscopy imaging techniques for studying protein-protein interactions in living cells using FRET standards", Proc. SPIE 7569, Multiphoton Microscopy in the Biomedical Sciences X, 75690Z (26 February 2010); https://doi.org/10.1117/12.842186

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