Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.
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 imagin...
Saved in:
| Main Authors: | , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Public Library of Science (PLoS)
2014-01-01
|
| Series: | PLoS ONE |
| Online Access: | https://doi.org/10.1371/journal.pone.0110695 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849726732547915776 |
|---|---|
| author | Viviane Devauges Daniel R Matthews Justin Aluko Jakub Nedbal James A Levitt Simon P Poland Oana Coban Gregory Weitsman James Monypenny Tony Ng Simon M Ameer-Beg |
| author_facet | Viviane Devauges Daniel R Matthews Justin Aluko Jakub Nedbal James A Levitt Simon P Poland Oana Coban Gregory Weitsman James Monypenny Tony Ng Simon M Ameer-Beg |
| author_sort | Viviane Devauges |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-0bbb5efb929c4e98a3a43983744b3d78 |
| institution | DOAJ |
| issn | 1932-6203 |
| language | English |
| publishDate | 2014-01-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS ONE |
| spelling | doaj-art-0bbb5efb929c4e98a3a43983744b3d782025-08-20T03:10:06ZengPublic Library of Science (PLoS)PLoS ONE1932-62032014-01-01910e11069510.1371/journal.pone.0110695Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane.Viviane DevaugesDaniel R MatthewsJustin AlukoJakub NedbalJames A LevittSimon P PolandOana CobanGregory WeitsmanJames MonypennyTony NgSimon M Ameer-BegWe 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.https://doi.org/10.1371/journal.pone.0110695 |
| spellingShingle | Viviane Devauges Daniel R Matthews Justin Aluko Jakub Nedbal James A Levitt Simon P Poland Oana Coban Gregory Weitsman James Monypenny Tony Ng Simon M Ameer-Beg Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. PLoS ONE |
| title | Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. |
| title_full | Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. |
| title_fullStr | Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. |
| title_full_unstemmed | Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. |
| title_short | Steady-state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of FRET at the plasma membrane. |
| title_sort | steady state acceptor fluorescence anisotropy imaging under evanescent excitation for visualisation of fret at the plasma membrane |
| url | https://doi.org/10.1371/journal.pone.0110695 |
| work_keys_str_mv | AT vivianedevauges steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT danielrmatthews steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT justinaluko steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT jakubnedbal steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT jamesalevitt steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT simonppoland steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT oanacoban steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT gregoryweitsman steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT jamesmonypenny steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT tonyng steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane AT simonmameerbeg steadystateacceptorfluorescenceanisotropyimagingunderevanescentexcitationforvisualisationoffretattheplasmamembrane |