BRAIN CONNECTS: Multi-beam transmission electron microscopy of iteratively milled semi-thick tissue sections

大脑连接：迭代研磨半厚组织切片的多束透射电子显微镜

• 批准号: 10669305
• 负责人: Andreas Schaefer
• 金额: $ 167.88万
• 依托单位: PAUL SCHERRER INSTITUT PSI
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2026-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10669305
• 关键词: 3-Dimensional; Address; Algorithms; Back; Biological; Biological Process; Brain; Collection; Development; Diamond; Electromagnetic Fields; Electron Microscope; Electron Microscopy; Electrons; Failure; Film; Hour; Human; Image; Ions; Magnetism; Manuals; Microtomy; Morphologic artifacts; Mus; Neurons; Organ; Pyramidal Cells; Research Personnel; Resolution; Sampling; Scanning; Scanning Electron Microscopy; Series; Silicon; Solid; Surface; Synapses; System; Techniques; Thick; Thinness; Tissue Sample; Tissues; Transmission Electron Microscopy; Visualization; Yin; automated segmentation; improved; microscopic imaging; millimeter; nanometer resolution; neuronal circuitry; reconstruction; segmentation algorithm; transmission process

Project Summary/Abstract Volume electron microscopy is the only technique to-date that provides both sufficient resolution (<20 nm) and sufficient field of view (>100 μm) for the dense reconstruction of neuronal wiring diagrams. Currently, there exist two systems that have already delivered mm3-sized synaptic resolution electron microscopy stacks: Multi-beam scanning electron microscopy(Eberle et al. 2015; Ren and Kruit 2016) (mSEM) and Gridtape-based automated transmission electron microscopy(Yin et al. 2020; Maniates-Selvin et al. 2020) (Gridtape-TEM). In mSEM, the sample is scanned with up to 91 parallel beams and an image is formed by low energy secondary electrons that are generated during scanning. Gridtape-TEM detects transmitted electrons with one or multiple fast cameras simultaneously. Both techniques currently rely on collecting and imaging thousands of ultrathin serial sections (30 - 40 nm) that are being cut with a diamond knife on an ultramicrotome. For mSEM, the sections are either collected using an automated tape collecting ultramicrotome or directly onto silicon wafers. For Gridtape-TEM, the sections are collected onto an electron-transparent film in millimeter-sized apertures on Gridtape. However, serial collection of ultrathin sections is delicate and inherently prone to failures and artifacts such as section loss, folds and cracks or knife marks. More than 50% of the errors of today’s state-of-the-art automated neuron segmentation algorithms can be attributed to missing information due to serial-sectioning. As a consequence, more than 40 hours of manual segmentation proofreading by human experts are currently required to accurately reconstruct a single cortical pyramidal cell. Some of the remaining automated segmentation issues can certainly be addressed by improving the underlying algorithms. But in order to scale dense automated neuronal circuit reconstructions to whole mouse brains with about 70 million neurons, it is necessary to significantly reduce the experimental artifacts. The collection of semi-thin sections with a thickness around 100 - 500 nm has been proposed as a much more robust alternative to ultrathin sectioning. In order to maintain or even increase the resolution in Z, these semi-thin sections could be iteratively milled and scanned in the case of mSEM or a series of images at different tilt angles could be acquired in the case of Gridtape-TEM. Here we propose to combine the commercially available multi-beam scanning transmission electron microscope FASTEM from Delmic with iterative broad ion beam milling of semi-thin sections (BIB-mSTEM). First, hundreds of semi-thin sections will be collected directly onto scintillator plates using the commercially available MagC magnetic collection system. Subsequently, these sections will be iteratively thinned and imaged by going back and forth between broad ion beam milling and imaging with FASTEM. For each section, this will produce a series of iteratively milled TEM projection images that can be used to reconstruct a high-resolution 3d stack of each section. BIB-mSTEM will be substantially more robust and reliable than mSEM and Gridtape-TEM based workflows: In contrast to Gridtape-TEM, the sections are collected onto a solid substrate and not on a fragile support film. In contrast to mSEM, BIB-mSTEM forms the image from high energy transmitted electrons that are much less sensitive to local electromagnetic fields and milling-induced irregular surface topography than low energy secondary electrons.

项目概要/摘要 体积电子显微镜是迄今为止唯一能够提供足够分辨率（<20 nm）和足够分辨率的技术。 用于神经布线图密集重建的视场（>100μm）目前，存在两种系统。 已经交付了 mm3 大小的突触分辨率电子显微镜堆栈：多束扫描电子 显微镜（Eberle et al. 2015；Ren and Kruit 2016）（mSEM）和基于 Gridtape 的自动传输电子 显微镜（Yin 等人，2020 年；Maniates-Selvin 等人，2020 年）（Gridtape-TEM），在 mSEM 中，使用高达 91 的倍率扫描样品。 平行光束和图像是由扫描过程中产生的低能量二次电子形成的。 目前，Gridtape-TEM 通过一台或多台快速相机同时检测传输电子。 依靠收集和成像数千个用金刚石刀切割的超薄连续切片 (30 - 40 nm) 对于 mSEM，使用自动胶带收集超薄切片机收集切片或 对于 Gridtape-TEM，切片被收集到电子透明薄膜上。 Gridtape 上的毫米级孔径然而，超薄切片的连续收集是脆弱的并且本质上容易发生。 故障和伪影，例如截面丢失、折叠和裂纹或刀痕，占当今错误的 50% 以上。 最先进的自动神经分割算法可归因于由于信息缺失 因此，人类专家需要进行 40 多个小时的手动分割校对。 目前需要精确地重建单个皮质锥体细胞，其中一些剩余是自动化的。 分割问题当然可以通过改进底层算法来解决，但为了扩展密集。 对包含约 7000 万个神经元的整个小鼠大脑进行自动神经回路重建，有必要 显着减少实验伪影，收集厚度约为 100 - 500 nm 的半薄切片。 已被提议作为超薄切片的更强大的替代方案，以维持甚至增加切片的厚度。 Z 轴分辨率，这些半薄切片可以在 mSEM 或一系列图像的情况下迭代铣削和扫描 在 Gridtape-TEM 的情况下可以获取不同倾斜角度的数据。这里我们建议将商业化的数据结合起来。 Delmic 提供的多光束扫描透射电子显微镜 FASTEM，具有迭代宽离子束 首先，将直接收集数百个半薄切片到半薄切片上。 随后，将使用市售 MagC 磁性收集系统的闪烁体板。 通过在宽离子束铣削和 FASTEM 成像之间来回反复进行细化和成像。 每个部分，这将产生一系列迭代研磨的 TEM 投影图像，可用于重建 每个部分的高分辨率 3D 堆栈将比 mSEM 更加稳健和可靠。 基于 Gridtape-TEM 的工作流程：与 Gridtape-TEM 相比，切片收集在固体基质上，而不是收集在 与 mSEM 不同，BIB-mSTEM 通过高能透射电子形成图像。 与低能量相比，对局部电磁场和铣削引起的不规则表面形貌的敏感度要低得多 二次电子。