28 February 2011 An automated wide-field time-gated optically sectioning fluorescence lifetime imaging multiwell plate reader for high-content analysis of protein-protein interactions
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Abstract
We describe an optically-sectioned FLIM multiwell plate reader that combines Nipkow microscopy with wide-field time-gated FLIM, and its application to high content analysis of FRET. The system acquires sectioned FLIM images in <10 s/well, requiring only ~11 minutes to read a 96 well plate of live cells expressing fluorescent protein. It has been applied to study the formation of immature HIV virus like particles (VLPs) in live cells by monitoring Gag-Gag protein interactions using FLIM FRET of HIV-1 Gag transfected with CFP or YFP. VLP formation results in FRET between closely packed Gag proteins, as confirmed by our FLIM analysis that includes automatic image segmentation.
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Dominic Alibhai, Dominic Alibhai, Sunil Kumar, Sunil Kumar, Douglas Kelly, Douglas Kelly, Sean Warren, Sean Warren, Yuriy Alexandrov, Yuriy Alexandrov, Ian Munro, Ian Munro, James McGinty, James McGinty, Clifford Talbot, Clifford Talbot, Edward J Murray, Edward J Murray, Frank Stuhmeier, Frank Stuhmeier, Mark A. A. Neil, Mark A. A. Neil, Chris Dunsby, Chris Dunsby, Paul M. W. French, Paul M. W. French, } "An automated wide-field time-gated optically sectioning fluorescence lifetime imaging multiwell plate reader for high-content analysis of protein-protein interactions", Proc. SPIE 7904, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII, 79041Q (28 February 2011); doi: 10.1117/12.875135; https://doi.org/10.1117/12.875135
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