Abstract

Quantitative measurement of protein-protein interaction is important for many biological processes, including cell growth, intercellular communication, gene expression and apoptosis. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET can be measured by changes in fluorescence lifetime of the fluorophors by Fluorescence lifetime imaging microscopy (FLIM) in live cells. Combined with confocal imaging, FLIM can achieve 3-dimensional optical sectional capability and resolve sub-cellular structures. The change in lifetime is inversely proportional to the ratio of bound to non-bound proteins. Time-resolved conventional confocal scanning microscopy is inherently slow and not suitable for rapid imaging applications. We present a 32×32 multiplexing confocal microscope, equipped with a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. The multiplexing setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. We used this multiplexing confocal FLIM system to measure Bcl-2 family proteins interactions in live cells and are able to capture a 240×240 μm FOV multi-channel confocal FLIM images in less than 1.5s. Protein binding affinities are estimated by measuring the changes in FRET as function of acceptor to donor ratio.

© 2019 SPIE/OSA

PDF Article
More Like This
Imaging Protein-Protein Interactions By Multiphoton FLIM

S. M. Ameer-Beg, N. Edme, M. Peter, P. R. Barber, T. Ng, and B. Vojnovic
5139_180 European Conference on Biomedical Optics (ECBO) 2003

Time Resolved Multiphoton Imaging Of The Interaction Between The PKC and NFκB Signalling Pathways.

Penny E. Morton, Tony C. Ng, Sarah A. Roberts, Borivoj Vojnovic, and Simon M. Ameer-Beg
5139_216 European Conference on Biomedical Optics (ECBO) 2003

FLIM-FRET of Cell Signalling in Chemotaxis

Sean Warren, Christopher Kimberley, Anca Margineanu, Romain Laine, Christopher Dunsby, Matilda Katan, and Paul French
MTh1C.5 Optical Molecular Probes, Imaging and Drug Delivery (OMP) 2013

References

You do not have subscription access to this journal. Citation lists with outbound citation links are available to subscribers only. You may subscribe either as an OSA member, or as an authorized user of your institution.

Contact your librarian or system administrator
or
Login to access OSA Member Subscription