Single molecule FRET using the FRET pair DRONPA/PhotoActivable mCherry

Viviane Devauges; Elena Ortiz-Zapater; Christina Efthymiou; Melanie Keppler; Jody Barbeau; Daniel R. Matthews; James Monypenny of Pitmilly; Paul R. Barber; Daniel Rolfe; Tony Ng; Simon M. Ameer-Beg

doi:10.1117/12.2002470

22 February 2013 Single molecule FRET using the FRET pair DRONPA/PhotoActivable mCherry

Viviane Devauges, Elena Ortiz-Zapater, Christina Efthymiou, Melanie Keppler, Jody Barbeau, Daniel R. Matthews, James Monypenny of Pitmilly, Paul R. Barber, Daniel Rolfe, Tony Ng, Simon M. Ameer-Beg

Author Affiliations +

Proceedings Volume 8590, Single Molecule Spectroscopy and Superresolution Imaging VI; 85900A (2013) https://doi.org/10.1117/12.2002470
Event: SPIE BiOS, 2013, San Francisco, California, United States

Abstract

Photoswitchable and photoactivable proteins Dronpa and PhotoActivable mCherry (PA-mCherry) respectively, were used in order to perform FRET (Förster Resonance Energy Transfer) imaging at the single molecule level, using a FRET standard construct consisting of Dronpa and PA-mCherry separated by seven amino acids expressed in cells. Given Dronpa’s complex photophysical properties and the existence of a preswitched emissive state, irradiation conditions at 491 and 405 nm were optimised. We discuss strategies for observing FRET at the single molecule level with photoactivatable proteins by monitoring modifications in the donor and acceptors emissive states.

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Viviane Devauges, Elena Ortiz-Zapater, Christina Efthymiou, Melanie Keppler, Jody Barbeau, Daniel R. Matthews, James Monypenny of Pitmilly, Paul R. Barber, Daniel Rolfe, Tony Ng, and Simon M. Ameer-Beg "Single molecule FRET using the FRET pair DRONPA/PhotoActivable mCherry", Proc. SPIE 8590, Single Molecule Spectroscopy and Superresolution Imaging VI, 85900A (22 February 2013); https://doi.org/10.1117/12.2002470

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