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

Simon P. Poland; Nikola Krstajić; 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

doi:10.1364/BOE.6.000277

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

Simon P. Poland (Lead / Corresponding author), Nikola Krstajić, 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

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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.

Original language	English
Pages (from-to)	277-296
Number of pages	20
Journal	Biomedical Optics Express
Volume	6
Issue number	2
DOIs	https://doi.org/10.1364/BOE.6.000277
Publication status	Published - 1 Feb 2015

Keywords

fluorescence lifetime imaging
CMOS SPADs
multiphoton microscopy

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10.1364/BOE.6.000277

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Poland, S. P., Krstajić, N., Monypenny, J., Coelho, S., Tyndall, D., Walker, R. J., Devauges, V., Richardson, J., Dutton, N., Barber, P., Li, D. D.-U., Suhling, K., Ng, T., Henderson, R. K., & Ameer-Beg, S. M. (2015). A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging. Biomedical Optics Express, 6(2), 277-296. https://doi.org/10.1364/BOE.6.000277

@article{5edfce7a0baf414da36851fab704dacd,

title = "A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging",

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.",

keywords = "fluorescence lifetime imaging, CMOS SPADs, multiphoton microscopy",

author = "Poland, {Simon P.} and Nikola Krstaji{\'c} and James Monypenny and Simao Coelho and David Tyndall and Walker, {Richard J.} and Viviane Devauges and Justin Richardson and Neale Dutton and Paul Barber and Li, {David Day-Uei} and Klaus Suhling and Tony Ng and Henderson, {Robert K.} and Ameer-Beg, {Simon M.}",

note = "The authors would like to thank: The Biotechnology and Biological Sciences Research Council UK (BB/I022074/1 and BB/I022937/1); STMicroelectronics (Imaging Division, Edinburgh, UK); Cancer Research UK, Engineering and Physical Sciences Research Council (UK), Medical Research Council (UK) and Department of Health (UK), KCL/UCL Comprehensive Cancer Imaging Centre; Dimbleby Cancer Care endowment fund to King{\textquoteright}s College London and The Royal Society. The Megaframe project has been supported by the European Community within the Sixth Framework Programme IST FET Open, http://www.megaframe.eu.",

year = "2015",

month = feb,

day = "1",

doi = "10.1364/BOE.6.000277",

language = "English",

volume = "6",

pages = "277--296",

journal = "Biomedical Optics Express",

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Poland, SP, Krstajić, N, Monypenny, J, Coelho, S, Tyndall, D, Walker, RJ, Devauges, V, Richardson, J, Dutton, N, Barber, P, Li, DD-U, Suhling, K, Ng, T, Henderson, RK & Ameer-Beg, SM 2015, 'A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging', Biomedical Optics Express, vol. 6, no. 2, pp. 277-296. https://doi.org/10.1364/BOE.6.000277

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 - Li, David Day-Uei

AU - Suhling, Klaus

AU - Ng, Tony

AU - Henderson, Robert K.

AU - Ameer-Beg, Simon M.

N1 - The authors would like to thank: The Biotechnology and Biological Sciences Research Council UK (BB/I022074/1 and BB/I022937/1); STMicroelectronics (Imaging Division, Edinburgh, UK); Cancer Research UK, Engineering and Physical Sciences Research Council (UK), Medical Research Council (UK) and Department of Health (UK), KCL/UCL Comprehensive Cancer Imaging Centre; Dimbleby Cancer Care endowment fund to King’s College London and The Royal Society. The Megaframe project has been supported by the European Community within the Sixth Framework Programme IST FET Open, http://www.megaframe.eu.

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.

KW - fluorescence lifetime imaging

KW - CMOS SPADs

KW - multiphoton microscopy

U2 - 10.1364/BOE.6.000277

DO - 10.1364/BOE.6.000277

M3 - Article

C2 - 25780724

SN - 2156-7085

VL - 6

SP - 277

EP - 296

JO - Biomedical Optics Express

JF - Biomedical Optics Express

IS - 2

ER -

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

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Keywords

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