Time-lapse FRET microscopy using fluorescence anisotropy

D. R. Matthews, L. M. Carlin, E. Ofo, P. R. Barber, B. Vojnovic, M. Irving, T. Ng, S. M. Ameer-Beg

Research output: Contribution to journalArticlepeer-review

37 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)51 - 62
Number of pages12
JournalJournal of Microscopy
Volume237
Issue number1
Early online date26 Aug 2009
DOIs
Publication statusPublished - Jan 2010

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