Monitoring physiology of individual cardiomyocytes in high throughput has not been reported. The inability to perform high throughput physiological measurements limits many basic and applied studies, including the use of stem cell derived cardiomycoytes in cardiotoxicity testing. Current automated cardiotoxicity tests have poor predictive value because they use tumor cell lines engineered with single channels (e.g. hERG), and physiologically relevant tests are reserved for few candidates during the relatively late stages of development. The poor biological relevance of these models contributes to the high failure rate of drug candidates before FDA approval and even after commercialization. We have automated recording from myocytes for Calcium Transients, but are still limited by use of electrode devices for pacing that prevents miniaturization beyond 96- well format. Furthermore, Action Potential measurement, the most relevant physiological parameter in excitable cells, is still reserved to low throughput analysis. We propose several conceptual advances to solve these problems by developing a miniaturized, cell-based optogenetic pacing device for high throughput analysis of human Induced Pluripotent Stem Cell (hIPSC)-derived cardiomyocytes in an automated platform for cell-by-cell cytometric analysis of cardiomyocyte physiology. We will also develop automatic segmentation/analysis of Action Potentials (AP) through fluorescent voltage probes and post-recording tracking to identify the same cells after fixation and immunostaining analysis. Calcium Transient (CT) analysis, already developed in a previous SBIR contract, will converge with AP and post-recording tracking to generate single cell multiparametric measurement of all these endpoints conducted in High Throughput. Extensive evaluation will be conducted with drugs that alter AP through different mechanisms to validate the platform. Preliminary data show that stable cell lines expressing the light-triggered protein Channelrhodopsin-2 (ChR2) will electrically couple to cardiomyocytes, allowing optically controlled stimulation of AP without disruption of normal cardiomyocyte physiology. Membrane AP can be recorded in cardiomyocytes through voltage probes and are suitable to image segmentation analysis. Automatic CT measurement and hIPSC-derived cardiomyocytes are an effective model to test cardiotoxic effects of reference drugs. The Aims will advance the use of fluorescent probes to measure action potential, calcium flux and cell characteristics in response to the stimulation. Cardiomyocyte physiology will be quantified by image analysis software that records and analyzes single-cell AP and CT in relation to cardiac subtype or specific protein expression. The software will segment the images into single cell recordings, thus all measurements and data analysis will be on a cell-by- cell basis. The format will be evaluated for 384- and 1536-well to conduct screening on hundreds of cells per individual data point (e.g. compound tested), allowing throughput of tens to hundreds of thousands of datapoints in a single screen by the end of the funding period. Channel openers and blockers will be tested to validate the platform. The platform will find applications in basic and applied research, including regenerative medicine research and drug development/safety testing.