A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

Poland, Simon P. and Krstajic, Nikola and Monypenny, James and Coelho, Simao and Tyndall, David and Walker, Richard J. and Devauges, Viviane and Richardson, Justin and Dutton, Neale and Barber, Paul and Li, David Day-Uei and Suhling, Klaus and Ng, Tony and Henderson, Robert K. and Ameer-Beg, Simon M. (2015) A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging. Biomedical Optics Express, 6 (2). pp. 277-296. ISSN 2156-7085

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    Abstract

    We demonstrate diffraction limited multiphoton imaging in a massively parallel, fully addressable time-resolved multi-beam multiphoton microscope capable of producing fluorescence lifetime images with sub-50ps temporal resolution. This imaging platform offers a significant improvement in acquisition speed over single-beam laser scanning FLIM by a factor of 64 without compromising in either the temporal or spatial resolutions of the system. We demonstrate FLIM acquisition at 500 ms with live cells expressing green fluorescent protein. The applicability of the technique to imaging protein-protein interactions in live cells is exemplified by observation of time-dependent FRET between the epidermal growth factor receptor (EGFR) and the adapter protein Grb2 following stimulation with the receptor ligand. Furthermore, ligand-dependent association of HER2-HER3 receptor tyrosine kinases was observed on a similar timescale and involved the internalisation and accumulation or receptor heterodimers within endosomes. These data demonstrate the broad applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.