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

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

Research output: Contribution to journalArticle

54 Citations (Scopus)

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.
LanguageEnglish
Pages277-296
Number of pages20
JournalBiomedical Optics Express
Volume6
Issue number2
Early online date6 Jan 2015
DOIs
Publication statusPublished - 1 Feb 2015

Fingerprint

Optical Imaging
microscopes
high speed
proteins
life (durability)
fluorescence
cells
Proteins
temporal resolution
acquisition
Ligands
Endosomes
adapters
Receptor Protein-Tyrosine Kinases
ligands
Green Fluorescent Proteins
Epidermal Growth Factor Receptor
tyrosine
stimulation
Lasers

Keywords

  • arrays
  • diffractive optics
  • fluorescence microscopy
  • microscopy
  • nonlinear microscopy

Cite this

Poland, S. P., Krstajic, N., Monypenny, J., Coelho, S., Tyndall, D., Walker, R. J., ... 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
Poland, Simon P. ; Krstajic, Nikola ; Monypenny, James ; Coelho, Simao ; Tyndall, David ; Walker, Richard J. ; Devauges, Viviane ; Richardson, Justin ; Dutton, Neale ; Barber, Paul ; Li, David Day-Uei ; Suhling, Klaus ; Ng, Tony ; Henderson, Robert K. ; Ameer-Beg, Simon M. / A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging. In: Biomedical Optics Express. 2015 ; Vol. 6, No. 2. pp. 277-296.
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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.",
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note = "Date of Acceptance: 30/11/2014 This paper was published in Biomedical Optics Express and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://www.opticsinfobase.org/boe/abstract.cfm?uri=boe-6-2-277. Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law.",
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Poland, SP, Krstajic, 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

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging. / Poland, Simon P.; Krstajic, Nikola; Monypenny, James; Coelho, Simao; Tyndall, David; Walker, Richard J.; Devauges, Viviane; Richardson, Justin; Dutton, Neale; Barber, Paul; Li, David Day-Uei; Suhling, Klaus; Ng, Tony; Henderson, Robert K.; Ameer-Beg, Simon M.

In: Biomedical Optics Express, Vol. 6, No. 2, 01.02.2015, p. 277-296.

Research output: Contribution to journalArticle

TY - JOUR

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

AU - Poland, Simon P.

AU - Krstajic, 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 - Date of Acceptance: 30/11/2014 This paper was published in Biomedical Optics Express and is made available as an electronic reprint with the permission of OSA. The paper can be found at the following URL on the OSA website: http://www.opticsinfobase.org/boe/abstract.cfm?uri=boe-6-2-277. Systematic or multiple reproduction or distribution to multiple locations via electronic or other means is prohibited and is subject to penalties under law.

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.

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KW - arrays

KW - diffractive optics

KW - fluorescence microscopy

KW - microscopy

KW - nonlinear microscopy

UR - http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-6-2-277

U2 - 10.1364/BOE.6.000277

DO - 10.1364/BOE.6.000277

M3 - Article

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EP - 296

JO - Biomedical Optics Express

T2 - Biomedical Optics Express

JF - Biomedical Optics Express

SN - 2156-7085

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