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

TY - JOUR

T1 - A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

AU - Poland, Simon P.

AU - Krstajić, Nikola

AU - Monypenny, James

AU - Coelho, Simao

AU - Tyndall, David

AU - Walker, Richard J.

AU - Devauges, Viviane

AU - Richardson, Justin

AU - Dutton, Neale

AU - Barber, Paul

AU - Day-Uei Li, David

AU - Suhling, Klaus

AU - Ng, Tony

AU - Henderson, Robert K.

AU - Ameer-Beg, Simon M

PY - 2015/2/1

Y1 - 2015/2/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84942366918&partnerID=8YFLogxK

U2 - 10.1364/BOE.6.000277

DO - 10.1364/BOE.6.000277

M3 - Article

AN - SCOPUS:84942366918

SN - 2156-7085

VL - 6

SP - 277

EP - 296

JO - Biomedical Optics Express

JF - Biomedical Optics Express

IS - 2

ER -