A high-content screening platform utilizing polarization anisotropy and FLIM microscopy
Matthews DR., Ameer-Beg SM., Barber P., Pierce GP., Newman RG., Vojnovic B., Carlin LM., Keppler MD., Ng T., Suhling K., Irving M.
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. © 2008 Copyright SPIE - The International Society for Optical Engineering.