Investigating Bcl-2 family protein-protein interactions using a high-speed multiplexing confocal FLIM microscope

Nehad Hirmiz; Anthony Tsikouras; Elizabeth J. Osterlund; Morgan Richards; Jessica Kun; David W. Andrews; Qiyin Fang

doi:10.1117/12.2526906

31 July 2019 Investigating Bcl-2 family protein-protein interactions using a high-speed multiplexing confocal FLIM microscope

Nehad Hirmiz, Anthony Tsikouras, Elizabeth J. Osterlund, Morgan Richards, Jessica Kun, David W. Andrews, Qiyin Fang

Author Affiliations +

Proceedings Volume 11076, Advances in Microscopic Imaging II; 1107617 (2019) https://doi.org/10.1117/12.2526906
Event: European Conferences on Biomedical Optics, 2019, Munich, Germany

Abstract

Quantitative measurement of protein-protein interaction is important for many biological processes, including cell growth, intercellular communication, gene expression and apoptosis. Förster Resonance Energy Transfer (FRET) provides a molecular level ruler to measure the distance, within a few nanometers, between two proteins. FRET can be measured by changes in fluorescence lifetime of the fluorophors by Fluorescence lifetime imaging microscopy (FLIM) in live cells. Combined with confocal imaging, FLIM can achieve 3-dimensional optical sectional capability and resolve sub-cellular structures. The change in lifetime is inversely proportional to the ratio of bound to non-bound proteins. Time-resolved conventional confocal scanning microscopy is inherently slow and not suitable for rapid imaging applications. We present a 32×32 multiplexing confocal microscope, equipped with a 64×32 time-gated single-photon avalanche photodiode (SPAD) sparse array detector. The multiplexing setup allows the use of the sparse array with high frame rate and sub-nanosecond time-gating to achieve high throughput FLIM acquisition. We used this multiplexing confocal FLIM system to measure Bcl-2 family proteins interactions in live cells and are able to capture a 240×240 μm FOV multi-channel confocal FLIM images in less than 1.5s. Protein binding affinities are estimated by measuring the changes in FRET as function of acceptor to donor ratio.

Citation Download Citation

Nehad Hirmiz, Anthony Tsikouras, Elizabeth J. Osterlund, Morgan Richards, Jessica Kun, David W. Andrews, and Qiyin Fang "Investigating Bcl-2 family protein-protein interactions using a high-speed multiplexing confocal FLIM microscope", Proc. SPIE 11076, Advances in Microscopic Imaging II, 1107617 (31 July 2019); https://doi.org/10.1117/12.2526906

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