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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-Beg
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.

History

Publication status

  • Published

File Version

  • Published version

Journal

Biomedical Optics Express

ISSN

2156-7085

Publisher

Optical Society of America

Issue

2

Volume

6

Page range

277-296

Department affiliated with

  • Engineering and Design Publications

Full text available

  • No

Peer reviewed?

  • Yes

Legacy Posted Date

2015-01-14

First Compliant Deposit (FCD) Date

2015-01-14

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