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A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging
journal contribution
posted on 2023-06-08, 19:32 authored by Simon P Poland, Nikola Krstajic, 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-BegWe 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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Publication status
- Published
File Version
- Published version
Journal
Biomedical Optics ExpressISSN
2156-7085Publisher
Optical Society of AmericaExternal DOI
Issue
2Volume
6Page range
277-296Department affiliated with
- Engineering and Design Publications
Full text available
- No
Peer reviewed?
- Yes
Legacy Posted Date
2015-01-14First Compliant Deposit (FCD) Date
2015-01-14Usage metrics
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