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.
authors.affiliation: Division of Cancer Studies, Guy's Campus, Kings College, London, UK ; Randall Division of Cell and Molecular Biophysics, Guy's Campus, Kings College, London, UK.
authors.forename: Simon P
authors.initials: SP
authors.lastname: Poland
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh, UK.
authors.forename: Nikola
authors.initials: N
authors.lastname: Krstajić
authors.affiliation: Division of Cancer Studies, Guy's Campus, Kings College, London, UK ; Randall Division of Cell and Molecular Biophysics, Guy's Campus, Kings College, London, UK.
authors.forename: James
authors.initials: J
authors.lastname: Monypenny
authors.affiliation: Division of Cancer Studies, Guy's Campus, Kings College, London, UK ; Randall Division of Cell and Molecular Biophysics, Guy's Campus, Kings College, London, UK.
authors.forename: Simao
authors.initials: S
authors.lastname: Coelho
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh, UK.
authors.forename: David
authors.initials: D
authors.lastname: Tyndall
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh, UK ; Photon-Force Ltd., Edinburgh, UK.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Walker
authors.affiliation: Division of Cancer Studies, Guy's Campus, Kings College, London, UK ; Randall Division of Cell and Molecular Biophysics, Guy's Campus, Kings College, London, UK.
authors.forename: Viviane
authors.initials: V
authors.lastname: Devauges
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh, UK ; Photon-Force Ltd., Edinburgh, UK.
authors.forename: Justin
authors.initials: J
authors.lastname: Richardson
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh, UK.
authors.forename: Neale
authors.initials: N
authors.lastname: Dutton
authors.affiliation: Gray Institute for Radiation Oncology & Biology, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Oxford OX3 7DQ UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Barber
authors.affiliation: Strathclyde Institute of Pharmacy and Biomedical Sciences, 161 Cathedral Street, Glasgow, G4 0RE, UK.
authors.forename: David Day-Uei
authors.initials: DD
authors.lastname: Li
authors.affiliation: Department of Physics, King's College London, Strand, London, UK.
authors.forename: Klaus
authors.initials: K
authors.lastname: Suhling
authors.affiliation: Division of Cancer Studies, Guy's Campus, Kings College, London, UK ; Randall Division of Cell and Molecular Biophysics, Guy's Campus, Kings College, London, UK ; UCL Cancer Institute, Paul O'Gorman Building, University College London, London WC1E 6DD, UK.
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
authors.affiliation: Institute for Integrated Micro and Nano Systems, School of Engineering, University of Edinburgh, Edinburgh, UK.
authors.forename: Robert K
authors.initials: RK
authors.lastname: Henderson
authors.affiliation: Division of Cancer Studies, Guy's Campus, Kings College, London, UK ; Randall Division of Cell and Molecular Biophysics, Guy's Campus, Kings College, London, UK.
authors.forename: Simon M
authors.initials: SM
authors.lastname: Ameer-Beg
keywords: (040.1240) Arrays; (090.1970) Diffractive optics; (170.2520) Fluorescence microscopy; (180.0180) Microscopy; (180.4315) Nonlinear microscopy