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.

项目摘要/摘要 体积电子显微镜是唯一提供足够分辨率（<20 nm）和足够分辨率的技术 视场（>100μm），用于神经元接线图的密集重建。目前，存在两个系统 已经传递了MM3大小的突触分辨率电子显微镜堆栈：多光束扫描电子 显微镜（Eberle等人，2015; Ren and Kruit 2016）（MSEM）和基于Gridtape的自动变速器电子电子 显微镜（Yin等，2020； Maniates-Selvin等，2020）（Gridtape-Tem）。在MSEM中，将样品扫描，最多为91 平行梁和图像是由扫描过程中产生的低能二级电子产品形成的。 Gridtape-Tem简单地检测到一个或多个快速相机的传输电子。目前这两种技术 依靠收集和成像成千上万的超薄串行部分（30-40 nm），它们被用钻石刀切割 在超大型机构上。对于MSEM，该部分要么使用自动胶带收集超级机动体或 直接进入硅波。对于栅格-TEM，将部分收集到电子透明膜上 毫米大小的孔。但是，超薄部分的串行收集是微妙的，固有的容易 故障和人工制品，例如截面损失，折叠和裂缝或刀具。当今错误的50％以上 最先进的自动神经元细分算法可以归因于由于缺失的信息 串行剖面。结果，人类专家的40多个小时的手动细分校对是 目前需要准确重建单个皮质锥体细胞。剩下的一些自动化 细分问题当然可以通过改进基础算法来解决。但是为了扩展密集 自动化的神经元电路重建约7,000万个神经元，有必要 显着减少实验伪影。厚度约为100-500 nm的半薄部分的集合 已被认为是超薄截面的更强大的替代品。为了维护甚至增加 在z中的分辨率，这些半薄的部分可以在MSEM或一系列图像的情况下进行迭代铣削和扫描 在Gridtape-Tem的情况下，可以以不同的倾斜角度获取。在这里，我们建议将商业结合起来 可用的多光束扫描传输电子显微镜FASTEM来自Delmic，带有迭代宽离子束 半薄部分的铣削。首先，将直接收集数百个半薄的部分 使用市售的MAGC磁收集系统的闪烁板板。随后，这些部分将是 迭代地稀释和成像，通过在宽离子束铣削和使用FastEM成像之间来回成像。为了 每个部分，这都会产生一系列迭代铣削的TEM投影图像，可用于重建A 每个部分的高分辨率3D堆栈。 Bib-Mstem将比MSEM更健壮和可靠，并且 基于网状tem-Tem的工作流：与Gridtape-TEM相比，将部分收集到实心基板上，而不是在固体基板上 脆弱的支持电影。与MSEM相反，围嘴形成来自高能传输电子的图像 对局部电磁场和铣削引起的不规则表面地形的敏感性要小得多 二级电子产品。