Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

被引：10

作者：

Devauges, Viviane ^{[1
,2
]}

Matthews, Daniel R. ^{[1
,2
]}

Aluko, Justin ^{[3
]}

Nedbal, Jakub ^{[1
,2
]}

Levitt, James A. ^{[1
,2
]}

Poland, Simon P. ^{[1
,2
]}

Coban, Oana ^{[1
,2
]}

Weitsman, Gregory ^{[1
,2
]}

Monypenny, James ^{[1
,2
]}

Ng, Tony ^{[1
,2
,4
]}

Ameer-Beg, Simon M. ^{[1
,2
]}

机构：

[1] Kings Coll London, Div Canc Studies, Richard Dimbleby Canc Res Lab, London WC2R 2LS, England

[2] Kings Coll London, Randall Div Cell & Mol Biophys, London WC2R 2LS, England

[3] Kings Coll London, Dept Phys, London WC2R 2LS, England

[4] UCL, UCL Canc Inst, London, England

来源：

PLOS ONE | 2014年 / 9卷 / 10期

基金：

英国生物技术与生命科学研究理事会; 英国工程与自然科学研究理事会; 英国医学研究理事会;

关键词：

INTERNAL-REFLECTION FLUORESCENCE; RESONANCE ENERGY-TRANSFER; NUMERICAL-APERTURE OBJECTIVES; LIVING CELLS; IMMUNOLOGICAL SYNAPSE; BREAST-CANCER; RHO-GTPASE; LIVE CELLS; STRUCTURED ILLUMINATION; POLARIZATION MICROSCOPY;

D O I：

10.1371/journal.pone.0110695

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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 Forster 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.

引用

页数：16

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