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

Biomedical Optics Express
Simon P PolandS M Ameer-Beg

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.

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Citations

Sep 16, 2015·Optics Letters·Nikola KrstajićRobert K Henderson
Oct 28, 2015·Biomedical Optics Express·Anthony TsikourasQiyin Fang
Sep 15, 2015·Optics Express·Marina PopleteevaAlessandro Esposito
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Nov 17, 2015·IEEE Transactions on Bio-medical Engineering·Hongqi YuDavid Day-Uei Li
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Dec 1, 2017·Nature Communications·Natalia FiliChristopher P Toseland
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Nov 13, 2019·Methods and Applications in Fluorescence·Herbert Schneckenburger
Nov 11, 2019·International Journal of Molecular Sciences·Ruth LebenRaluca A Niesner
Oct 28, 2019·Light, Science & Applications·Claudio BruschiniEdoardo Charbon
May 1, 2019·Scientific Reports·Elena PerevedentsevaChia-Liang Cheng
Apr 1, 2017·Optics Letters·Claire A MitchellSimon M Ameer-Beg
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Dec 4, 2020·Nature Protocols·Simao CoelhoKatharina Gaus
May 1, 2021·Sensors·Andrea Chiuri, Federico Angelini
Sep 22, 2021·ACS Nano·Adam J Bowman, Mark A Kasevich

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