Ramona Optics Inc

A Parallelized Imaging Platform for Accurate and Efficient Long-term Assessment of Brain Organoid Development - Significance: High-content Microscopy Is Rapidly Accelerating Biological Research and Drug Development. Unfortunately, Existing Imaging Systems Fail to Efficiently Image Three-dimensional Disease Models, Such as Organoid Cultures, at the Throughput Required by Clinical and Commercial Researchers. Generating Mature Brain Organoids, in Particular, Is a Complex Process That Requires Months of Intensive and Costly Maintenance With Regular Imaging to Assess Health and Structure. One of the Primary Drawbacks of Standard Microscopes Is Their Need to Step-and-scan Across a Well Plate, Which Can Take Minutes to Image All Wells in Two Dimensions, and an Hour or More for 3D Capture. This Not Only Leads to Cumbersome Experimentation With a Limited Number of Specimens, But Also Prevents Current High-content Experiments From Measuring Short-term (< 1 Hour) 3D Morphological Dynamics, Such as Neural Activity. Proposal: Ramona Optics Is Developing a New Imaging Platform for Rapid High-content 3D Screening of Brain Organoids to Effectively Assess of Dozens of Well Plates/day Across at Least 90 Days of Differentiation. We Will Also Develop Image Analysis Software to Identify, Measure and Track Organoid Volume and 3D Morphology Over Time. These New Capabilities Should Significantly Expedite Imaging Workflow and Yield Novel Insights Into Organoid Development Within High-content Experimentation. After Carefully Testing the 3D Imaging Performance of Our Parallelized Microscope and Assessing Higher-resolution Optics in This Phase I Proposal, We Aim to Enhance Performance With Fluorescence Capabilities in a Phase Ii Effort, Focused on Live-cell Whole-plate Recording of Neural Activity in Large Numbers of Brain Organoids. SA1: Parallelized Microscope Hardware Improvement: Ramona Optics Will Produce New Parallelized Microscope Hardware to Simultaneously Image 96 Full Wells (8X12 Fov) at 3 ΜM and 39 ΜM Half-pitch Lateral and Axial Resolution, Respectively. We Will Also Develop Hardware and Firmware Capable of Axial Scanning Across 1500 ΜM at 20 ΜM Step Size (75 Axial Slices) to Produce a Full Well Plate Focal Stack in 7.5 Sec., >100X Less Time Than What State-of-the-art Scanning High-content Microscopes Require. SA2: Automated Organoid Analysis Software: We Will Produce Software to Map Acquired Whole- Plate Axial Scan Measurements Into Per-organoid 3D Measurements and Associated Statistics in <45 Sec. We Will Also Create a User Interface to Longitudinally Track Per-organoid Measurements. SA3: Longitudinal Testing and Verification of 3D Brain Organoid Shape/size: With the Stein Lab at Unc, We Will Evaluate the Platform for High-content Longitudinal Measurement of Brain Organoid Shape/size. We Will Image 20 Plates (96 Organoids Each) Daily for 90 Days and Aim for 95% Correlation With Current Standard Measurement Methods Across a Variety of Organoid Morphologies and Sizes. We Will Also Assess 3D Scan Specs With Higher Resolution Optics (0.8 and 0.5 Μm/pixel) and Fluorescence.