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

基本信息

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
项目类别：
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

项目摘要

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：高通量光学显微镜目前正在改变遗传学、药物发现的研究领域大规模光学分析现在通常使用数千张高分辨率图像来提供重要的见解。然而，今天的光学显微镜仍远未达到理想状态。由于大镜头设计的挑战，没有标准显微镜可以捕获超过 50 兆像素的每张图像快照，这使得不可能在多厘米的观察区域（视野、对于活体斑马鱼的筛选和监测，这种分辨率/FOV 权衡是一个关键瓶颈：每个生物体。必须限制或瘫痪才能以高分辨率成像，自由游动的生物体只能以低分辨率查看，目前还没有任何装置可以以细胞分辨率并行监测多个游泳的斑马鱼提案：光学波前。 Laboratories, LLC (OWL) 开发了一种新型显微镜，克服了其第一阶段的“微型相机阵列”。 “显微镜”原型（MCAM-1）由 24 个微型相机单元和相关电子设备组成，用于捕获亚细胞在第二阶段，OWL 将生产出整个大型培养皿的分辨率图像（0.24 十亿像素图像）。产品 MCAM-2 具有改进的规格和软件，用于采集明场和荧光视频。 MCAM-2将显着提高高通量显微镜筛选的效率，降低当前的复杂性设置，并启用全新的生物实验（例如 SA3）。 SA1：优化 MCAM-2 硬件：OWL 将创建一款可立即上市的 MCAM-2 器件，可实现 6 µm 分辨率在 120 cm2 FOV 内以 8 帧/秒 (fps) 进行成像软件选项将使视频成像速率接近 24 fps。与顶级竞争显微镜相比，MCAM-2 每张图像的像素增加了 15-20 倍（0.3 十亿像素）。 SA2：开发用于高速数字跟踪的电子设备和软件：与哈佛大学 Engert 实验室合作， OWL 将使用自动数字跟踪功能显着减少 MCAM 保存的数据量。将从图像中分割每个幼虫并丢弃所有残留像素，将内存需求减少 100 倍并促进 30 fps 单一生物体视频跟踪此外，OWL 还将为其当前的 Python 添加多个图像分析功能。软件界面（例如 3D 位置、眼睛位置、尾部曲率）提供最先进的精度（<5% 误差，3-10 分钟）。 SA3：展示神经活动的荧光成像：与杜克大学 Naumann 实验室合作， OWL 将提高 MCAM 荧光检测的灵敏度和准确性专用硬件附加组件（激发）。源和发射滤光片阵列）将在静止和自由状态下提供 15-25 的荧光图像信噪比具有泛神经元 GCaMP6 表达的自由游动转基因幼虫的校准视频。验证 MCAM-2 可以在自然相互作用期间同时非侵入性测量 > 10 个生物体的神经活动。 SA4：进行用户试验并收集反馈：OWL 将向 5 个研究小组提供 MCAM-2 原型 OWL 将通过为期 3 个月的试用期调查问卷提供详细反馈，并将意见纳入最终产品中。该第二阶段项目的成果将是一款旗舰级 MCAM-2 设备和软件，可用于中等规模生产。