2014
DOI: 10.1371/journal.pone.0110695
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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 arrangemen… Show more

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Cited by 10 publications
(3 citation statements)
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“…The g factor accounts for any systemic differences introduced by the optics or detectors into the parallel vs. perpendicular channel and this was calibrated in our system by measurement of fluorescein, a fast-rotating molecule whose emission polarization should be isotropic. Depolarization effects caused by use of a high numerical aperture objective were taken-into-account by incorporation of an additional correction factor into r that was derived from referencing eGFP anisotropy values acquired with a 60X water objective to eGFP anisotropy values acquired with a ×10 air objective 50 . After application of this correction factor to FAIM measurements acquired in live DiVA cells expressing free eGFP we find that in our system the fluorescence anisotropy value of eGFP in the absence of homo-FRET ( r eGFP ) is 0.32.…”
Section: Methodsmentioning
confidence: 99%
“…The g factor accounts for any systemic differences introduced by the optics or detectors into the parallel vs. perpendicular channel and this was calibrated in our system by measurement of fluorescein, a fast-rotating molecule whose emission polarization should be isotropic. Depolarization effects caused by use of a high numerical aperture objective were taken-into-account by incorporation of an additional correction factor into r that was derived from referencing eGFP anisotropy values acquired with a 60X water objective to eGFP anisotropy values acquired with a ×10 air objective 50 . After application of this correction factor to FAIM measurements acquired in live DiVA cells expressing free eGFP we find that in our system the fluorescence anisotropy value of eGFP in the absence of homo-FRET ( r eGFP ) is 0.32.…”
Section: Methodsmentioning
confidence: 99%
“…Conversely, time-resolved fluorescence anisotropy can detect FRET between identical fluorescent proteins. In fact, this technique has been used in living cells to study the effect of protein dimerization and aggregation on the cell functioning ( 19 , 21 , 22 , 23 ). Although oligomerization in the biological milieu may involve a mixture of monomers and dimers, as well as higher-order oligomers, at various distances and orientations, and does not typically involve a covalent linker, the short FRET component in the anisotropy decay is a qualitative tell-tale sign of the occurrence of oligomerization.…”
Section: Introductionmentioning
confidence: 99%
“…(v) Fluorescence anisotropy of the acceptor is typically very low if excited through the donor by FRET allowing quantification of the FRET efficiency [4,26].…”
Section: Theory Of Fret In Briefmentioning
confidence: 99%