A microscale sorting cytometer for cell-based screens

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DESCRIPTION (provided by applicant): We propose the development of a cytometer that combines microscopic imaging with automated sorting. The cytometer addresses two steps in cell-based genetic screens: observation of phenotypes and retrieval of "mutants". Our goal is to be able to sort cells where the sort parameter is based upon the rich information available from microscopy: morphology, intracellular fluorescence, dynamics. Our cytometer is essentially an "active" coverslip that uses an array of reversible addressable traps created with semiconductor technology to hold individual cells in place for observation and release selected cells. The traps use an electrical analogue of optical tweezers--termed dielectrophoresis--to transiently hold the adherent cells in place until they attach to the substrate. After attachment, the cytometer is a passive chip that fits under a conventional upright fluorescence microscope. After live-cell assays have been performed, individual cells in selected traps can be released. The specific aims of our research are to (1) create trapping arrays of increasing complexity, starting with a 4x4 array to verify function, a 20x20 array to characterize sorting, and ending in a 100x100 array for use in an library screen; (2) create the control systems to provide constant temperature, pH, and oxygen tension on-chip coupled with automated microscopy; (3) investigate effects of our cytometer on cell health and perform a phenotype screen.