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

Simon P. Poland (Lead / Corresponding author), Nikola Krstajić, James Monypenny, Simao Coelho, David Tyndall, Richard J. Walker, Viviane Devauges, Justin Richardson, Neale Dutton, Paul Barber, David Day-Uei Li, Klaus Suhling, Tony Ng, Robert K. Henderson, Simon M. Ameer-Beg

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    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.

    Original languageEnglish
    Pages (from-to)277-296
    Number of pages20
    JournalBiomedical Optics Express
    Issue number2
    Publication statusPublished - 1 Feb 2015


    • fluorescence lifetime imaging
    • CMOS SPADs
    • multiphoton microscopy


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