FLIM, FRET and high content analysis

Edwin Garcia; Wenjun Guo; Sunil Kumar; Frederik Görlitz; Hugh Sparks; Yuriy Alexandrov; Ian Munro; Douglas J. Kelly; Sean Warren; George Chennell; Alessandro Sardini; David Carling; Peter Thorpe; Christopher Dunsby; Paul M. W. French

doi:10.1117/12.2547517

2 March 2020 FLIM, FRET and high content analysis

Edwin Garcia, Wenjun Guo, Sunil Kumar, Frederik Görlitz, Hugh Sparks, Yuriy Alexandrov, Ian Munro, Douglas J. Kelly, Sean Warren, George Chennell, Alessandro Sardini, David Carling, Peter Thorpe, Christopher Dunsby, Paul M. W. French

Author Affiliations +

Proceedings Volume 11244, Multiphoton Microscopy in the Biomedical Sciences XX; 1124417 (2020) https://doi.org/10.1117/12.2547517
Event: SPIE BiOS, 2020, San Francisco, California, United States

Abstract

Fluorescence lifetime imaging (FLIM) provides a means to contrast different molecular species and to map variations in the local fluorophore molecular environment, including to read out Förster resonant energy transfer (FRET), e.g. to assay protein interactions or genetically expressed FRET biosensors. We have implemented wide-field time-gated FLIM in a modular open automated microscopy platform for high content analysis (HCA). To demonstrate its relevance to drug discovery, we have demonstrated the capability of our openFLIM HCA platform to assay interactions of low copy number endogenous proteins in yeast cells labelled with fluorescent proteins. We have also demonstrated the capability of multiwell plate FLIM assays to provide readouts of a FRET biosensor in 2-D and 3-D cell cultures.

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Edwin Garcia, Wenjun Guo, Sunil Kumar, Frederik Görlitz, Hugh Sparks, Yuriy Alexandrov, Ian Munro, Douglas J. Kelly, Sean Warren, George Chennell, Alessandro Sardini, David Carling, Peter Thorpe, Christopher Dunsby, and Paul M. W. French "FLIM, FRET and high content analysis", Proc. SPIE 11244, Multiphoton Microscopy in the Biomedical Sciences XX, 1124417 (2 March 2020); https://doi.org/10.1117/12.2547517

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KEYWORDS

Fluorescence lifetime imaging

Fluorescence resonance energy transfer

Biosensors

Proteins

Luminescence

Data acquisition

Confocal microscopy

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