Open AccessSteady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane
Author(s) -
Viviane Devauges,
Daniel R. Matthews,
Justin Aluko,
Jakub Nedbal,
James A. Levitt,
Simon P. Poland,
Oana Coban,
Gregory Weitsman,
James Monypenny,
Thomas Ng,
Simon AmeerBeg
Publication year - 2014
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0110695
Subject(s) - förster resonance energy transfer , total internal reflection fluorescence microscope , fluorescence lifetime imaging microscopy , fluorescence anisotropy , fluorescence , live cell imaging , materials science , biophysics , chemistry , optics , physics , biology , cell , biochemistry
We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.