A time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo

Poland, Simon P. and Krstajic, Nikola and Coelho, Simao and Tyndall, David and Walker, Richard J. and Devauges, Viviane and Morton, Penny E. and Nicholas, Nicole S. and Richardson, Justin and Li, David Day-Ue and Suhling, Klaus and Wells, Claire M. and Parsons, Maddy and Henderson, Robert K. and Ameer-Beg, Simon M. (2014) A time-resolved multifocal multiphoton microscope for high speed FRET imaging in vivo. Optics Letters, 39 (20). pp. 6103-6106. ISSN 0146-9592

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    Abstract

    Imaging the spatio-temporal interaction of proteins in vivo is essential to understanding the complexities of biological systems. The highest accuracy monitoring of protein-protein interactions is achieved using FRET measured by fluorescence lifetime imaging with measurements taking minutes to acquire a single frame, limiting their use in dynamic live cell systems. We present a diffraction limited, massively parallel, time-resolved multifocal multiphoton microscope capable of producing fluorescence lifetime images with 55 ps time-resolution giving improvements in acquisition speed of a factor of 64. We present demonstrations with FRET imaging in a model cell system and demonstrate in vivo FLIM using a GTPase biosensor in the zebrafish embryo.