Multifocal Multiphoton Fluorescence Lifetime Imaging for high speed protein-protein interaction monitoring by FRET in live cells and tissues.

Simon Ameer-Beg, Simon P. Poland, Nikola Krstajic, James Moneypenny, James Levitt, Viviane Devauges, Jakub Nedbal, Robert Knight, Tony Ng, Robert Henderson

Research output: Contribution to conferencePaperpeer-review

Abstract / Description of output

Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique for high resolution imaging of
the functional spatio-temporal dynamics in situ. Forster resonance energy transfer (FRET) is, by far, the
most extensively studied technique for observation of protein-protein homo- and hetero-dimer
interactions in intact cells(1,2). For intermolecular FRET, a key benefit of performing donor FLIM (when
compared to intensity based ratiometric techniques), is that fluorescence-lifetime measurements of donor
emission are independent of acceptor concentration and is thus suited to imaging studies in intact
cells(3,4). Multiphoton microscopy confers additional advantages in terms of inherent three dimensional
sectioning and enhanced depth penetration for in vivo imaging(5-8). However, the data acquisition rate
for FLIM is a significant limitation in current implementations of laser scanning microscopy.
In this paper 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 unparalleled imaging rates 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. Finally we demonstrate
the applicability of this new technique to in vivo imaging in zebrafishThese data demonstrate the broad
applicability of this novel FLIM technique to the spatio-temporal dynamics of protein-protein interaction.
Original languageEnglish
Publication statusPublished - Jun 2015
EventCongress of International Society for the Advancement of Cytometry (CYTO) - Glasgow, United Kingdom
Duration: 26 Jun 201530 Sept 2015


ConferenceCongress of International Society for the Advancement of Cytometry (CYTO)
Country/TerritoryUnited Kingdom


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