An automated wide-field time-gated optically sectioning fluorescence lifetime imaging multiwell plate reader for high-content analysis of protein-protein interactions

Dominic Alibhai; Sunil Kumar; Douglas Kelly; Sean Warren; Yuriy Alexandrov; Ian Munro; James McGinty; Clifford Talbot; Edward J Murray; Frank Stuhmeier; Mark A. A. Neil; Chris Dunsby; Paul M. W. French

doi:10.1117/12.875135

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

Dominic Alibhai, Sunil Kumar, Douglas Kelly, Sean Warren, Yuriy Alexandrov, Ian Munro, James McGinty, Clifford Talbot, Edward J Murray, Frank Stuhmeier, Mark A. A. Neil, Chris Dunsby, Paul M. W. French

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Proceedings Volume 7904, Three-Dimensional and Multidimensional Microscopy: Image Acquisition and Processing XVIII; 79041Q (2011) https://doi.org/10.1117/12.875135
Event: SPIE BiOS, 2011, San Francisco, California, United States

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, Sunil Kumar, Douglas Kelly, Sean Warren, Yuriy Alexandrov, Ian Munro, James McGinty, Clifford Talbot, Edward J Murray, Frank Stuhmeier, Mark A. A. Neil, Chris Dunsby, and 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); https://doi.org/10.1117/12.875135

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KEYWORDS

Fluorescence resonance energy transfer

Fluorescence lifetime imaging

Luminescence

Data modeling

Proteins

Picosecond phenomena

Image segmentation

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