Project Summary High-content automated fluorescence imaging is a broadly useful cell-level bioassay platform technology that can be used to interrogate biological systems in the context of drug discovery, and genetic perturbations and to characterize physiologically relevant complex culture systems like patient-derived organoids. High-Content Screening (HCS) and High-Content Analysis (HCA) are methods that combine high-throughput automated fluorescence imaging and machine vision to extract spatially resolved, quantitative single-cell data used in drug discovery and biological research. In the past 5 years, there has been a confluence of significant advancements in high-content imaging hardware, precision patient modeling using organoids, and deep- learning analysis tools that have significantly increased the utility of these methods. Typically, mammalian cells are plated in multi-well dishes and are then incubated with substances, and after an incubation period, the cellular morphology, and biomarkers of interest can be probed using fluorescent markers to quantitate the effect and to gain information into the mechanism of action. HCS is one of the main modalities of phenotypic screening wherein thousands of compounds/conditions can be tested to deliver a single-cell and multivariate readout toward the identification of compounds/conditions with therapeutic potential in cell-based disease models. Conventional fluorescence microscopy is low throughput and generally yields qualitative results to support other experimental methodologies. HCS addresses both the bottleneck in data acquisition through automation and analysis with machine vision software used to identify cells and extract biomarker intensity and morphologic features for further analysis. Additionally, the quality of the high-content microscope, optics, and robotic control directly influence the types of experiments that can be achieved with the more advanced microscopes being capable of higher throughput, live-cell analysis, drug dispensing, and higher resolution confocal fluorescence images. The Yokogawa CellVoyager 8000 is a state-of-the-art high-content imaging platform that has 4-cameras, spinning disc confocal with water-immersion objective lenses that can accommodate fixed or live cells in a wide array of samples/formats. The U-M Center for Drug Repurposing has extensive experience in the development of highly physiologically-relevant in vitro disease models, high- content assay development, and in conducting large-scale high throughput screening. There is currently no laser-based high-content imaging microscope at U-M. The addition of the Yokogawa CellVoyager 8000 will significantly enhance drug discovery, drug repurposing, and quantitative biological imaging efforts at U-M.