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

使用十亿像素显微镜对自由游动的斑马鱼进行高分辨率并行成像

• 批准号: 9789387
• 负责人: Mark Harfouche
• 金额: $ 73.84万
• 依托单位: RAMONA OPTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-07-15 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9789387
• 关键词: 3-Dimensional; Affect; Algorithms; Area; Behavior; Behavioral; Benchmarking; Biological; Biological Assay; Brain; Brain Diseases; Calibration; Computer software; Custom; Data; Detection; Development; Devices; Discipline; Electronics; Eye; Feedback; Fluorescence; Future; Genetic; Goals; Human body; Image; Image Analysis; Laboratories; Larva; Light; Measures; Memory; Methods; Microscope; Microscopy; Monitor; Movement; Neurons; Neurosciences; Nobel Prize; Noise; Optics; Organism; Outcome; Paralysed; Performance; Phase; Positioning Attribute; Process; Production; Published Comment; Pythons; Questionnaires; Research; Residual state; Resolution; Sales; Series; Signal Transduction; Social Interaction; Source; Sparrows; Speed; Swimming; System; Tail; Technology; Time; Transgenic Organisms; Universities; Zebrafish; bioimaging; calcium indicator; commercialization; design; digital; drug discovery; experimental study; fluorescence imaging; high resolution imaging; hindbrain; image processing; imaging system; improved; in vivo; in vivo imaging; insight; lens; microscopic imaging; non-invasive imaging; optical imaging; prevent; prototype; relating to nervous system; screening; sensor; tool

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.

意义：高通量光学显微镜当前正在转化遗传学的研究领域，药物发现 和神经科学。现在，大规模的光学测定通常使用数千张高分辨率图像来提供关键见解 进入人体，我们的大脑和影响我们的疾病。但是，当今的光学显微镜仍然远非理想。 由于大型镜头设计的挑战，没有标准显微镜可以捕获每个图像快照超过50兆像素， 这使得不可能简单地在蜂窝分辨率上图像在多厘米查看区域（视场， fov）。为了在体内筛查和监测斑马鱼，该分辨率/FOV权衡是一个关键的瓶颈：每个生物体 必须在高分辨率下被限制或瘫痪以形象，自由游泳生物只能以低分辨率查看 而且尚无设置可以并行监测细胞分辨率的多个游泳斑马鱼。建议：光波前 Laboratories，LLC（OWL）开发了一种克服这些局限性的新显微镜。它的第一阶段“微型摄像机阵列 显微镜”原型（MCAM-1）由24个微型摄像机单元和相关电子设备组成，以捕获亚细胞 在整个大型培养皿（0.24吉米像素图像）上的分辨率图像。在第二阶段，猫头鹰将生产出市场上的 产品，MCAM-2，具有改进的规格和软件，可鉴定明亮场和荧光视频。 MCAM-2将显着提高高通量显微镜筛选的效率，降低电流的复杂性 设置，并启用全新的生物学实验（例如SA3）。 SA1：优化MCAM-2硬件：OWL将创建一个可实现6 µm分辨率的市场就绪MCAM-2设备 在8帧/秒（FPS）上跨120 cm2 FOV进行成像。软件选项将允许视频成像率接近24 fps 减少的区域。 MCAM-2比顶级竞争显微镜多于每个图像（0.3吉普赛素）的像素多15-20倍。 SA2：开发用于高速数字跟踪的电子和软件：与哈佛的Engert实验室合作， OWL将使用自动数字跟踪大大减少MCAM节省的数据量。这个新软件 将从图像中分割每个幼虫并丢弃所有残差像素，使记忆要求减少100倍，并促进 30 FPS单生物视频跟踪。此外，OWL将在其当前的Python中添加多个图像分析功能 软件接口（例如3D位置，眼睛位置，尾曲率）提供最先进的精度（误差<5％，3-10分钟）。 SA3：演示神经活动的荧光成像：与杜克大学的Naumann Lab合作， 猫头鹰将提高MCAM的荧光检测灵敏度和准确性。专用硬件附加组件（令人兴奋的 源和发射过滤器阵列）将在固定和游离的荧光图像信号噪声比率为15-25 移动转基因幼虫。带有泛神经GCAMP6S表达的免费游泳转基因幼虫的校准视频将 在自然相互作用期间，验证MCAM-2可以同时测量> 10种生物的神经活动。 SA4：进行用户试验并收集反馈：OWL​​将为5个研究小组提供MCAM-2原型 通过问卷调查的详细反馈在3个月的试验中。然后，猫头鹰将将评论纳入最终产品。 该第二阶段项目的结果将是一种旗舰MCAM-2设备和软件，可用于中等规模生产。