Highly Multiplexed Confocal Fluorescence Lifetime Microscope Designed for Screening Applications

Abstract

Protein-protein interactions can be measured in live cells, at nanometer scale, using Fluorescence Lifetime Imaging Microscopy (FLIM) enabled Forster Resonance Energy Transfer (FRET). There are growing interests in exploring protein-protein interactions in drug discovery applications. Traditional single point confocal microscopes, however, are slow and unsuited to small molecule screening, especially when combined with FLIM-FRET. We developed a 32 × 32 multiplexed confocal microscope, which employs a single-photon avalanche photodiode array with time gating capabilities for rapid FLIM acquisition. It has been demonstrated that such multiplexing technique can capture a 960 × 960 pixel multi-channel confocal fluorescence lifetime images in less than 1.5 seconds. Binding curves of two Bcl-2 family proteins: Bcl-XL and Bad were generated in live cells imaging experiments. The results show that the small molecule inhibitor A-1131852 is a more effective compound for disrupting Bcl-XL binding to Bad than ABT-263, which demonstrated the feasibility of screening of protein-protein interactions in high density well-plates.