SBIR-STTR Award

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 in parallel multiple swimming zebrafish at cellular resolution. Proposal: Optical Wavefront Laboratories, LLC (OWL) will develop and test a new microscope to overcome these limitations. It will capture sub-cellular resolution images over an 80 cm2 area (an entire large petri dish). This unique micro-camera array microscope (MCAM) is designed to form 0.25 gigapixel images, 10X more pixels per image than the top competing microscopes. This architecture can directly scale into the multi-gigapixel regime. The MCAM 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: Finalize MCAM hardware: OWL will complete a prototype MCAM device (currently under construction) consisting of 24 micro-camera units and associated electronics for 8 µm resolution imaging across an 80 cm2 FOV at 1 frame per second. Unlike any competing technology, our unique optical design will produce gigapixel-scale snapshot microscope images. Milestone: Completed MCAM hardware to deliver image data to a desktop computer at 0.25 gigapixels/sec. SA2: Develop MCAM software: A software program will transform the data captured by the MCAM system into standard digital images. The software program will also allow the end user to control and adjust basic imaging parameters (exposure time, contrast, and frame rate). Milestone: A standard Windows desktop computer program to directly control the MCAM and produce and save images (.bmp or .jpg format, 0.25 gigapixels/image) from an attached MCAM device. SA3: Test the MCAM's effectiveness in imaging freely swimming zebrafish: Working with the Engert Lab at Harvard University, OWL will demonstrate the MCAM's benefits in a behavioral imaging experiment to measure the visual function of >10 freely swimming zebrafish (larval stage) simultaneously across an 80 cm2 swim arena at 8 µm resolution. Milestones: 1) Confirm the MCAM accurately measures eye position for stationary zebrafish larvae embedded in agar. 2) Demonstrate the MCAM can measure eye position from 10 freely swimming zebrafish across an 80 cm2 swim area continuously (1 measurement/sec. per larvae). Results will be compared to two top-of-the-line single-lens microscopes. After successfully demonstrating the MCAM provides 10X more pixels per image than current microscopes, OWL plans to use the new device for non-invasive and parallelized in vivo fluorescent imaging of neural activity in multiple freely swimming zebrafish at video frame rates. This will be the subject of a Phase II proposal.

Public Health Relevance Statement:
Current microscopes cannot form images that offer cellular-scale resolution over an area larger than a few square centimeters, which fundamentally limits our ability to monitor the detailed movements of living systems. In this project, Optical Wavefront Laboratories, LLC (OWL) will develop a new micro-camera array microscope (MCAM) to overcome these limitations and offer cellular-level detail over an area the size of a large petri dish. We will finalize the production of an MCAM prototype capable of 8 µm resolution imaging over an 80 cm2 area and apply it to monitor the visual system of multiple freely swimming zebrafish in parallel at cellular resolution for the first time. !

Project Terms:
Adolescence; Affect; Agar; Animal Model; Animals; Architecture; Area; Behavior; Behavioral; Biological; Biological Assay; Brain Diseases; computer program; Computer software; Computers; Data; design; Devices; digital; digital imaging; drug discovery; Effectiveness; Electronics; experimental study; Eye; fluorescence imaging; Genetic; high resolution imaging; Human body; Image; improved; in vivo; insight; Laboratories; Larva; lens; Measurement; Measures; Mechanics; Microscope; microscopic imaging; Microscopy; Monitor; Movement; Neurosciences; Optics; Organism; Paralysed; Phase; Positioning Attribute; Production; programs; prototype; relating to nervous system; Research; Resolution; Sampling; Scanning; screening; Small Business Innovation Research Grant; Swimming; System; Technology; Testing; Time; Universities; Variant; Vision; Visual system structure; Whole Organism; Writing; Zebrafish

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.

Thesaurus Terms:
Affect; Algorithms; Area; Behavior; Behavioral; Benchmarking; Bioimaging; Biological; Biological Assay; Brain; Brain Diseases; Calcium Indicator; Calibration; Commercialization; Computer Software; Custom; Data; Design; Detection; Development; Devices; Digital; Discipline; Drug Discovery; Electronics; Experimental Study; Eye; Feedback; Fluorescence; Fluorescence Imaging; Future; Genetic; Goals; High Resolution Imaging; Hindbrain; Human Body; Image; Image Analysis; Image Processing; Imaging System; Improved; In Vivo; In Vivo Imaging; Insight; Laboratories; Larva; Lens; Light; Measures; Memory; Methods; Microscope; Microscopic Imaging; Microscopy; Monitor; Movement; Neurons; Neurosciences; Nobel Prize; Noise; Non-Invasive Imaging; Optical Imaging; Optics; Organism; Outcome; Paralysed; Performance; Phase; Positioning Attribute; Prevent; Process; Production; Prototype; Published Comment; Pythons; Questionnaires; Relating To Nervous System; Research; Residual State; Resolution; Sales; Screening; Sensor; Series; Signal Transduction; Social Interaction; Source; Sparrows; Speed; Swimming; System; Tail; Technology; Time; Tool; Transgenic Organisms; Universities; Zebrafish;