Time-lapse FRET microscopy using fluorescence anisotropy

Matthew, D.R., Carlin, L.M. , Ofo, E., Barber, P.R., Vojnovic, B., Irving, M., Ng, T. and Ameer-Beg, S.M. (2010) Time-lapse FRET microscopy using fluorescence anisotropy. Journal of Microscopy, 237(1), pp. 51-62. (doi: 10.1111/j.1365-2818.2009.03301.x) (PMID:20055918)

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Abstract

We present recent data on dynamic imaging of Rac1 activity in live T‐cells. Förster resonance energy transfer between enhanced green and monomeric red fluorescent protein pairs which form part of a biosensor molecule provides a metric of this activity. Microscopy is performed using a multi‐functional high‐content screening instrument using fluorescence anisotropy to provide a means of monitoring protein–protein activity with high temporal resolution. Specifically, the response of T‐cells upon interaction of a cell surface receptor with an antibody coated multi‐well chamber was measured. We observed dynamic changes in the activity of the biosensor molecules with a time resolution that is difficult to achieve with traditional methodologies for observing Förster resonance energy transfer (fluorescence lifetime imaging using single photon counting or frequency domain techniques) and without spectral corrections that are normally required for intensity based methodologies.

Item Type:Articles
Additional Information:Daniel Matthews and Leo Carlin are supported by UKEPSRC grant 12 (EP/C546105/1). Enyinnaya Ofo is a recipient of both a Wellcome Trust Clinical Training Fellowship and the Dimbleby Cancer Care Award. Paul Barber and Borvoj Vojnovic are supported by Cancer Research UK (C133/A/1812). Simon Ameer-Beg and Tony Ng are supported by an endowment fund from Dimbleby Cancer Care to King’s College London.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Carlin, Dr Leo
Authors: Matthew, D.R., Carlin, L.M., Ofo, E., Barber, P.R., Vojnovic, B., Irving, M., Ng, T., and Ameer-Beg, S.M.
College/School:College of Medical Veterinary and Life Sciences > School of Cancer Sciences
Journal Name:Journal of Microscopy
Publisher:Wiley
ISSN:0022-2720
ISSN (Online):1365-2818
Published Online:10 December 2009

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