5 March 2008 A high-content screening platform utilizing polarization anisotropy and FLIM microscopy
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Abstract
An automated high-content screening microscope has been developed which uses fluorescence anisotropy imaging and fluorescence lifetime microscopy to identify Förster resonant energy transfer between eGFP and mRPF1 in drug screening assays. A wide-field polarization resolved imager is used to simultaneously capture the parallel and perpendicular components of both eGFP and mRFP1 fluorescence emission to provide a high-speed measurement of acceptor depolarization. Donor excited state lifetime measurements performed using laser scanning microscopy is then used to determine the FRET efficiency in a particular assay. A proof-of-principle assay is performed using mutant Jurkat human T-cells to illustrate the process by which FRET is first identified and then quantified by our high-content screening system.
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Daniel R. Matthews, Simon M. Ameer-Beg, Paul Barber, Glenn P. Pierce, Robert G. Newman, Boris Vojnovic, Leo M. Carlin, Melanie D. Keppler, Tony Ng, Klaus Suhling, Malcolm Irving, "A high-content screening platform utilizing polarization anisotropy and FLIM microscopy", Proc. SPIE 6859, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI, 685919 (5 March 2008); doi: 10.1117/12.763310; https://doi.org/10.1117/12.763310
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