A high-content screening platform utilizing polarization anisotropy and FLIM microscopy

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

doi:10.1117/12.763310

5 March 2008 A high-content screening platform utilizing polarization anisotropy and FLIM microscopy

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

Author Affiliations +

Proceedings Volume 6859, Imaging, Manipulation, and Analysis of Biomolecules, Cells, and Tissues VI; 685919 (2008) https://doi.org/10.1117/12.763310
Event: SPIE BiOS, 2008, San Jose, California, United States

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.

Citation Download Citation

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, and 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); https://doi.org/10.1117/12.763310

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