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Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation
Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map...
Autores principales: | , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
WILEY-VCH Verlag
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660788/ https://www.ncbi.nlm.nih.gov/pubmed/23184449 http://dx.doi.org/10.1002/jbio.201200185 |
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author | Alibhai, Dominic Kelly, Douglas J Warren, Sean Kumar, Sunil Margineau, Anca Serwa, Remigiusz A Thinon, Emmanuelle Alexandrov, Yuriy Murray, Edward J Stuhmeier, Frank Tate, Edward W Neil, Mark A A Dunsby, Chris French, Paul M W |
author_facet | Alibhai, Dominic Kelly, Douglas J Warren, Sean Kumar, Sunil Margineau, Anca Serwa, Remigiusz A Thinon, Emmanuelle Alexandrov, Yuriy Murray, Edward J Stuhmeier, Frank Tate, Edward W Neil, Mark A A Dunsby, Chris French, Paul M W |
author_sort | Alibhai, Dominic |
collection | PubMed |
description | Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z ′ factors exceeding 0.6 are realised for this FLIM FRET assay. [Image: see text] Fluorescence lifetime plate map with representative images of high and low FRET cells and corresponding dose response plot. |
format | Online Article Text |
id | pubmed-3660788 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | WILEY-VCH Verlag |
record_format | MEDLINE/PubMed |
spelling | pubmed-36607882013-05-22 Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation Alibhai, Dominic Kelly, Douglas J Warren, Sean Kumar, Sunil Margineau, Anca Serwa, Remigiusz A Thinon, Emmanuelle Alexandrov, Yuriy Murray, Edward J Stuhmeier, Frank Tate, Edward W Neil, Mark A A Dunsby, Chris French, Paul M W J Biophotonics Editor's Choice Fluorescence lifetime measurements can provide quantitative readouts of local fluorophore environment and can be applied to biomolecular interactions via Förster resonant energy transfer (FRET). Fluorescence lifetime imaging (FLIM) can therefore provide a high content analysis (HCA) modality to map protein-protein interactions (PPIs) with applications in drug discovery, systems biology and basic research. We present here an automated multiwell plate reader able to perform rapid unsupervised optically sectioned FLIM of fixed and live biological samples and illustrate its potential to assay PPIs through application to Gag protein aggregation during the HIV life cycle. We demonstrate both hetero-FRET and homo-FRET readouts of protein aggregation and report the first quantitative evaluation of a FLIM HCA assay by generating dose response curves through addition of an inhibitor of Gag myristoylation. Z ′ factors exceeding 0.6 are realised for this FLIM FRET assay. [Image: see text] Fluorescence lifetime plate map with representative images of high and low FRET cells and corresponding dose response plot. WILEY-VCH Verlag 2013-05 2012-11-27 /pmc/articles/PMC3660788/ /pubmed/23184449 http://dx.doi.org/10.1002/jbio.201200185 Text en © 2013 The Authors. J. Biophotonics published by WILEY-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. http://creativecommons.org/licenses/by/2.5/ Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. |
spellingShingle | Editor's Choice Alibhai, Dominic Kelly, Douglas J Warren, Sean Kumar, Sunil Margineau, Anca Serwa, Remigiusz A Thinon, Emmanuelle Alexandrov, Yuriy Murray, Edward J Stuhmeier, Frank Tate, Edward W Neil, Mark A A Dunsby, Chris French, Paul M W Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation |
title | Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation |
title_full | Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation |
title_fullStr | Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation |
title_full_unstemmed | Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation |
title_short | Automated fluorescence lifetime imaging plate reader and its application to Förster resonant energy transfer readout of Gag protein aggregation |
title_sort | automated fluorescence lifetime imaging plate reader and its application to förster resonant energy transfer readout of gag protein aggregation |
topic | Editor's Choice |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3660788/ https://www.ncbi.nlm.nih.gov/pubmed/23184449 http://dx.doi.org/10.1002/jbio.201200185 |
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