BRAIN CONNECTS: Center for a pipeline of high throughput integrated volumetric electron microscopy for whole mouse brain connectomics

项目摘要： 使用高通量集成体积分析的全小鼠脑连接组学中心 电子显微镜（HIVE） 脑功能的结构基础的两个基本组成部分是细胞类型组成和脑功能。 这些细胞之间的接线图。在过去十年中，在以下方面取得了范式转变的进展： 了解大脑的细胞类型组成。现在是时候系统地揭示大脑的线路了 图，并将其放置到细胞类型的上下文中。关于C. elegans 和果蝇彻底改变了对这些系统中细胞类型和电路功能的理解。 透射电子显微镜（TEM）一直引领着这场革命的进展， 在某些领域进行技术改进，在IARPA MICRON 在一个项目中，HIVE团队的成员建立了一个完整的管道来对mm 3进行切片和成像，并创建了数据 处理、重建和分析基础设施，使细胞和连接可分析。结果是 在任何系统中具有最大EM水平细胞重建的数据集，其中神经元包含超过 14,000个输入和15,000个输出。我们通过应用严格的结构化科学过程来实现这一目标， 是艾伦研究所团队科学方法的标志。 在这个项目中，我们的目标是改善我们的管道，开发关键技术，以应对挑战， 放大到整个小鼠大脑并与细胞类型相关联。我们的建议将准备和部分整个 半球，以120 nm分辨率对其成像，并在皮质内以突触分辨率成像高达10 mm 3 基底神经节丘脑回路提供了对该回路内回路机制的关键见解。完成 为此，我们需要改进管道的各个方面，同时保持集成测试的一致性 以确保各个部分能够协同工作。我们将标准化整个小鼠脑染色方案， 将连续切片的自动化推进到收集整个小鼠大脑的连续TEM切片。 连续切片倾斜TEM断层扫描的发展将允许将EM成像扩展到整个小鼠大脑， 多尺度，利用连续切片TEM的再成像能力。我们将开发开源数据 处理工具，以降低分割成本，同时提高准确性，并与 全球可访问的实时校对和分析平台。最后，我们将整合我们的数据， 与基因表达相关的形态重建，使我们能够创建一个完整的细胞类型图谱， 和连通性。我们的传播战略将进一步扩大我们的影响， 科学和教育界。

Project Summary: Center for whole mouse brain connectomics using high-throughput integrated volumetric electron microscopy (HIVE) Two fundamental components of the structural basis of brain function are cell type composition and the wiring diagram between those cells. Over the past decade there has been paradigm-shifting progress in understanding cell type composition of the brain. Now it’s time to systematically uncover the brain’s wiring diagram and place it into the context of cell types. Knowledge about the complete connectomes in C. elegans and Drosophila have revolutionized the understanding of cell types and circuit function in those systems. Transmission Electron Microscopy (TEM) has consistently led progress in that revolution and has the potential to scale up to the entire mouse brain with technical improvements in certain areas. During the IARPA MICrONS project, members of the HIVE team built a complete pipeline to section and image the mm3 and created the data processing, reconstruction and analysis infrastructure to make cells and connections analyzable. The result was a dataset with the largest EM level reconstructions of cells in any system, with neurons containing more than 14,000 inputs and 15,000 outputs. We accomplished this by applying a rigorous structured science process that is a hallmark of the Allen Institute’s team science approach. In this project we aim to improve our pipeline, developing critical technologies to tackle the challenges of scaling up to the whole mouse brain and linking to cell types. Our proposal will prepare and section an entire hemisphere, image it at 120 nm resolution, and image up to 10 mm3 at synaptic resolution within the Cortical Basal Ganglia Thalamic loop to provide key insights into circuit mechanisms within this circuit. To accomplish this, we will need to improve all individual aspects of the pipeline, while maintaining consistent integration tests that ensure that the pieces work together. We will standardize a whole mouse brain staining protocol and advance the automation of serial sectioning to collection of serial TEM sections across a whole mouse brain. Developments of serial section tilt TEM tomography will allow to scale EM imaging to a whole mouse brain at multiple scales, leveraging the re-imaging capacity of serial section TEM. We will develop open source data processing tools to bring down the cost of segmentation and while improving accuracy and integrating with a real-time globally accessible proofreading and analysis platform. Finally, we will integrate our data with full morphology reconstructions linked to gene expression, allowing us to create an integrated atlas of cell types and connectivity. Our dissemination strategy will further amplify our impact by democratizing access for both the scientific and educational community.