Challenge: Studies of intracellular protein-protein interactions (PPI) is emerging as an important drug discovery tool. Since many relevant targets can only be affected by drugs candidates in living cells, specific screening methods are implemented using sophisticated microscopy systems. A way to measure these interactions in cellulo is through the implementation of Förster Resonance Energy Transfer (FRET) combined with fluorescence lifetime imaging microscopy (FLIM) methods. However, FLIM-FRET data have so far fallen short of the ultimate goal of generating intracellular binding curves, essential for properly identifying potential hits.

Solution: The project aims to develop a method to identify drug candidates that interfere with PPI by using a newly built microscope with the ability to dynamically image interactions of fluorescently-labeled proteins within living cells. For their studies the research team implement both fluorescence lifetime imaging and resonant energy transfer together with spectral
detection to reach unsurpassed combination of spatial and temporal resolutions. Their unique method allows for the generation of protein-protein binding curves necessary to evaluate the efficacy of drug candidates.

Achievements/Impacts: To overcome the limitations typically encountered in high-content screening (HCS), the research team conceived, developed, and validated the performances of a new FLIM hyperspectral confocal microscope, so-called INO F-HS microscope. They demonstrated that this system allowed full 384 wells plate screening based on FRET-FLIM binding curves in less than 8 hours through a highly parallelized, automated, and robust time correlated single photon counting (TCSPC) scheme as well as full integration to a hyperspectral microscope. In association with this INO F-HS microscope, a user-friendly software for rapid analysis was developed. Through this technology, researchers demonstrated an outstanding mechanical stability and repeatability of measurement, as well as generating data on specific PPIs not possible with other modalities. This new instrument not only provides the pharmaceutical industry a new way to accelerate the screening for drugs based on PPI but also translate the research towards personalized medicine where a cocktail of drugs can be evaluated and tailored to individual patients.