title: Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.
abstract: 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.
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Viviane
authors.initials: V
authors.lastname: Devauges
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Matthews
authors.affiliation: Department of Physics, King's College London, London, United Kingdom.
authors.forename: Justin
authors.initials: J
authors.lastname: Aluko
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Jakub
authors.initials: J
authors.lastname: Nedbal
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: James A
authors.initials: JA
authors.lastname: Levitt
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Simon P
authors.initials: SP
authors.lastname: Poland
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Oana
authors.initials: O
authors.lastname: Coban
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Gregory
authors.initials: G
authors.lastname: Weitsman
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: James
authors.initials: J
authors.lastname: Monypenny
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom; UCL Cancer Institute, University College London, London, United Kingdom.
authors.forename: Tony
authors.initials: T
authors.lastname: Ng
authors.affiliation: Richard Dimbleby Cancer Research Laboratory, Division of Cancer Studies and Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.
authors.forename: Simon M
authors.initials: SM
authors.lastname: Ameer-Beg
mesh_terms: D015374:Biosensing Techniques; D002462:Cell Membrane / Q000378:metabolism*; D005454:Fluorescence Polarization / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D061986:MCF-7 Cells; D057054:Molecular Imaging / Q000379:methods*; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000378:metabolism
chemical_list: D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins