High-throughput heart rate sensing in freely moving zebrafish using a multi-well plate-based fluorescence microscope.

The measurement of zebrafish heart rates is important for studying cardiovascular diseases and advancing drug discovery. However, current imaging methods either allow tracking of freely moving zebrafish at insufficient resolution for heart segmentation or require mounting and anesthesia to confine the specimen. Here, we introduce a high-throughput method to simultaneously track and segment dozens of freely moving zebrafish from embryonic to juvenile stages. We use a 48-camera microscope to capture multi-well fluorescence zebrafish videos from a macroscopically large field-of-view with co-designed machine learning software performing semi-automatic tracking and fluorescence analysis. To demonstrate the effectiveness of our method, we tracked the heart rates of Tg(myl7:GCaMP)s878 transgenic zebrafish across different experimental conditions of temperature changes and drug trials with distinct results. Our robust tracking capabilities of freely swimming fish enable novel, to the best of our knowledge, long-term in vivo fluorescence imaging experiments, allowing for new developmental questions to be analyzed.