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

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Poland, Simon P. and Krstajić, 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 (https://doi.org/10.1364/BOE.6.000277)

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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.

ORCID iDs

Poland, Simon P., Krstajić, Nikola, Monypenny, James, Coelho, Simao, Tyndall, David, Walker, Richard J., Devauges, Viviane, Richardson, Justin, Dutton, Neale, Barber, Paul, Li, David Day-Uei ORCID logoORCID: https://orcid.org/0000-0002-6401-4263, Suhling, Klaus, Ng, Tony, Henderson, Robert K. and Ameer-Beg, Simon M.;
  • Item type:Article
    ID code:51046
    Dates:
    Date
    Event
    1 February 2015
    Published
    6 January 2015
    Published Online
    30 November 2014
    Accepted
    Notes:This paper was published in Biomedical Optics Express and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://www.opticsinfobase.org/boe/abstract.cfm?uri=boe-6-2-277. Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law.
    Subjects:Science > Physics > Optics. Light
    Department:Faculty of Science > Strathclyde Institute of Pharmacy and Biomedical Sciences
    Depositing user: Pure Administrator
    Date deposited:13 Jan 2015 16:14
    Last modified:16 Dec 2024 05:07
    Related URLs:
    URI:https://strathprints.strath.ac.uk/id/eprint/51046
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