High-Resolution, Parallelized Imaging of Freely Swimming Zebrafish with a Gigapixel Microscope

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Significance: High-throughput optical microscopy is currently transforming the research fields of genetics, drug discovery and neuroscience. Large-scale optical assays now routinely use thousands of high-resolution images to offer critical insights into the human body, our brain and the diseases that affect us. Today's optical microscopes, however, are still far from ideal. Due to challenges with large lens design, no standard microscope can capture more than 50 megapixels per image snapshot, which makes it impossible to simultaneously image at cellular-resolution over a multi-centimeter viewing area (field of view, FOV). For screening and monitoring zebrafish in vivo, this resolution/FOV tradeoff is a critical bottleneck: each organism must be constrained or paralyzed to image at high resolution, freely swimming organisms can only be viewed at low resolution, and no setups yet can monitor multiple swimming zebrafish at cellular resolution in parallel. Proposal: Optical Wavefront Laboratories, LLC (OWL) has developed a new microscope that overcomes these limitations. Its Phase I “micro-camera array microscope” prototype (the MCAM-1) consists of 24 micro-camera units and associated electronics to capture sub-cellular resolution images over an entire large petri dish (0.24 gigapixel images). In Phase II, OWL will produce a market-ready product, the MCAM-2, with improved specifications and software for acquiring both bright-field and fluorescence videos. The MCAM-2 will significantly improve the efficiency of high-throughput microscope screening, reduce the complexity of current setups, and enable completely new biological experiments (e.g., SA3). SA1: Optimize MCAM-2 hardware: OWL will create a market-ready MCAM-2 device that achieves 6 µm resolution imaging across an 120 cm2 FOV at 8 frames/sec (fps). Software options will allow video imaging rates to approach 24 fps over a reduced area. The MCAM-2 offers 15-20X more pixels per image (0.3 gigapixels) than top competing microscopes. SA2: Develop electronics and software for high-speed digital tracking: Working with the Engert Lab at Harvard, OWL will dramatically reduce the amount of data saved by the MCAM using automated digital tracking. This new software will segment each larva from images and discard all residual pixels, decreasing memory requirements by 100X and facilitating 30 fps single-organism video tracking. In addition, OWL will add several image analysis functions to its current Python software interface (e.g. 3D position, eye position, tail curvature) offering state-of-the-art accuracy (<5% error, 3-10 min.). SA3: Demonstrate fluorescence imaging of neural activity: Working with the Naumann Lab at Duke University, OWL will improve the MCAM's sensitivity and accuracy of fluorescence detection. Dedicated hardware add-ons (an excitation source and emission filter array) will provide a fluorescence image signal-to-noise ratio of 15-25 in stationary and freely moving transgenic larvae. Calibrated videos of freely swimming transgenic larvae with pan-neuronal GCaMP6s expression will verify the MCAM-2 can non-invasively measure neural activity in >10 organisms simultaneously during natural interactions. SA4: Conduct user trials and gather feedback: OWL will provide MCAM-2 prototypes to 5 research groups for detailed feedback via questionnaires over a 3-month trial. OWL will then incorporate comments into a finalized product. The outcome of this Phase II project will be a flagship MCAM-2 device and software ready for medium-scale production.