"Grid-Tape": A High-Throughput Platform for Brain Connectomics and Nanoscale Structural Analysis

“Grid-Tape”：用于脑连接组学和纳米级结构分析的高通量平台

• 批准号: 9255254
• 负责人: Ryan M Smith
• 金额: $ 17.42万
• 依托单位: LUXEL CORPORATION
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-06-07 至 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9255254
• 关键词: Address; Alzheimer' s Disease; Area; Autistic Disorder; Brain; Collection; Communities; Data Collection; Data Set; Development; Diagnostics Research; Drosophila melanogaster; Electron Beam; Electron Microscopy; Electrons; Equipment; Evaluation; Face; Film; Foundations; Goals; Image; Imaging technology; Individual; Institutes; Intuition; Investigation; Ions; Length; Maps; Methods; Microtome - medical device; Microtomy; Modernization; Mus; Nature; Network-based; Neurodegenerative Disorders; Neurons; Neurosciences; Noise; Phase; Plant Roots; Process; Production; Protocols documentation; Research; Research Personnel; Resolution; Sampling; Scanning; Scanning Electron Microscopy; Schizophrenia; Signal Transduction; Speed; Standardization; Synapses; Technology; Thinness; Tissue imaging; Tissues; Transmission Electron Microscopy; Universities; Vacuum; base; brain tissue; cost; data acquisition; design; hydrophilicity; improved; manufacturing process; manufacturing scale-up; microscopic imaging; millimeter; nanoscale; nervous system disorder; prototype; sample collection; scale up; tool; transmission process

The wiring diagram of brain circuits is one of the foundational and fundamental questions of modern neuroscience. Extracting a circuit-sized volume at synaptic resolution requires large-scale, high-throughput imaging of many thin sections of brain tissue. The immediate goal of researchers is to capture and map a cubic millimeter of cortex at synaptic resolution, requiring the collection and imaging of 25,000 tissue sections for a single dataset. The slow speed of tissue sectioning and imaging remains a major limitation to the field of connectomics, requiring many years to acquire a dataset of this size. A number of research intuitions (e.g. Harvard University, Janelia Research Campus, Allen Institute) are actively seeking solutions for higher- throughput imaging. Scanning electron microscopy (SEM) offers fast, reliable sample sectioning and collection via Automatic Tape-collecting Ultra-Microtome (ATUM-SEM), as well as automated in-vacuum sample cutting via Serial-Block-Face (SBF-SEM) or Focused Ion Beam (FIB-SEM). However, SEM has intrinsically poor resolution and signal level, due to the serial nature of the scanning electron beam pixel collection. In comparison, camera-based transmission electron microscopy (TEM) allows fast, simultaneous collection of millions of pixels at higher resolution than SEM, but is limited by slow handling of individual sample supports. To overcome this throughput limitation, researchers at Harvard University are developing a tape sample substrate for TEM called “GridTape”, comprising thin-film-covered slots in a tape form that is compatible with commercially available ATUM equipment. Sections are imaged via an in-column reel-to-reel TEM imaging stage. Harvard has chosen Luxel as their partner for the initial investigation and demonstrated ~70X speed improvement in pickup of thin sections of mouse and Drosophila melanogaster brains cut with an ATUM. The proposed project will optimize and scale up GridTape to meet the urgent unmet needs of large scale connectomics research. The technical challenges include reel-to-reel thin film lamination, film breakage, wrinkling, and image background noise. The specific aims of this proposal are: (1) Develop a pilot manufacturing process for the production of GridTape prototypes having the number of imaging slots required by researchers (5,000-16,000) in a continuous length, with production throughput of 2,000 slots/day; (2) Optimize GridTape specifications to meet end-user requirements, reducing damaged slot rate to <1%; and (3) Connectomics field evaluation, acquiring datasets on GridTape prototypes have >10,000 slots and a quantitative comparison between GridTape and SEM datasets demonstrating pickup speed and imaging throughput at least equal to existing practice. Phase II will address manufacturing scale-up with greater slot counts and lower cost per slot.

脑回路接线图是现代科学的基础和根本问题之一 以突触分辨率提取电路大小的体积需要大规模、高通量。 研究人员的直接目标是捕获并绘制一个立方体的图像。 毫米级的皮质突触分辨率，需要收集 25,000 个组织切片并对其进行成像 单一数据集的组织切片和成像速度慢仍然是该领域的主要限制。 连接组学，需要很多年才能获得如此规模的数据集。 哈佛大学、珍妮莉亚研究园、艾伦研究所）正在积极寻求更高层次的解决方案 扫描电子显微镜 (SEM) 提供快速、可靠的样品切片和收集。 通过自动胶带收集超薄切片机 (ATUM-SEM) 以及自动真空样品切割 通过串行块面 (SBF-SEM) 或聚焦离子束 (FIB-SEM) 然而，SEM 本质上较差。 由于扫描电子束像素收集的串行性质，分辨率和信号电平。 相比之下，基于相机的透射电子显微镜（TEM）可以快速、同步地收集 数百万像素的分辨率比 SEM 更高，但受到单个样品支撑处理缓慢的限制。 为了克服这种吞吐量限制，哈佛大学的研究人员正在开发一种磁带样本 用于 TEM 的基底称为“GridTape”，由薄膜覆盖的带状槽组成，与 商用 ATUM 设备通过柱内卷轴 TEM 成像对切片进行成像。 哈佛大学选择 Luxel 作为初步调查的合作伙伴，并展示了约 70 倍的速度。 改进了用 ATUM 切割的小鼠和果蝇大脑薄片的拾取能力。 拟议项目将优化和扩展 GridTape，以满足大规模的迫切未满足的需求 连接组学研究的技术挑战包括卷对卷薄膜层压、薄膜破损、 该提案的具体目标是：（1）开发一个试点。 生产具有所需成像槽数量的 GridTape 原型的制造过程 由研究人员 (5,000-16,000) 组成，连续长度，生产量为 2,000 个插槽/天 (2) 优化GridTape规格以满足最终用户需求，将损坏槽位率降低至<1%；以及(3) 连接组学现场评估，在 GridTape 原型上获取数据集有超过 10,000 个插槽和一个 GridTape 和 SEM 数据集之间的定量比较展示了拾取速度和成像 吞吐量至少等于现有实践，第二阶段将通过更大的槽位来解决制造规模扩大问题。 计数并降低每个插槽的成本。