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Time‐lapse FRET microscopy using fluorescence anisotropy
Author(s) -
MATTHEWS D.R.,
CARLIN L.M.,
OFO E.,
BARBER P.R.,
VOJNOVIC B.,
IRVING M.,
NG T.,
AMEERBEG S.M.
Publication year - 2010
Publication title -
journal of microscopy
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.569
H-Index - 111
eISSN - 1365-2818
pISSN - 0022-2720
DOI - 10.1111/j.1365-2818.2009.03301.x
Subject(s) - förster resonance energy transfer , microscopy , biosensor , fluorescence , fluorescence anisotropy , fluorescence in the life sciences , biophysics , resolution (logic) , chemistry , anisotropy , fluorescence microscope , materials science , nuclear magnetic resonance , optics , nanotechnology , physics , computer science , biology , artificial intelligence
Summary 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.