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

项目总结/摘要 体积电子显微镜是迄今为止唯一既提供足够的分辨率（<20 nm）又提供足够的透射率的技术。 视野（>100 μm），用于神经元布线图的密集重建。目前，存在两种系统， 我已经交付了毫米3大小的突触分辨率电子显微镜堆栈：多束扫描电子显微镜 显微镜（Eberle et al. 2015; Ren and Kruit 2016）（mSEM）和基于Gridtape的自动透射电子显微镜 显微镜（Yin等人2020; Maniates-Selvin等人2020）（Gridtape-TEM）。在mSEM中，样品扫描高达91 平行束，并且图像由在扫描期间产生的低能量二次电子形成。 Gridtape-TEM同时用一个或多个快速相机检测透射电子。这两种技术目前 依靠收集和成像成千上万的连续切片（30 - 40 nm），这些切片正在用金刚石刀切割 在超薄切片机上对于mSEM，使用自动带收集超薄切片机收集切片，或 直接在硅晶片上。对于Gridtape-TEM，将切片收集到电子透明膜上， 网格胶带上毫米大小的小孔然而，连续收集切片是微妙的，并且固有地倾向于 故障和伪影，如部分损失、折叠和裂缝或刀痕。今天超过50%的错误 现有技术的自动神经元分割算法可归因于丢失信息， 连续切片因此，由人类专家进行的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。剩下的一些自动化 分割问题当然可以通过改进底层算法来解决。但为了扩大密度 自动神经回路重建到整个小鼠大脑约7000万个神经元，有必要 大大减少了实验假象。厚度约为100 - 500 nm的半薄切片的集合 已经被提出作为一种比分段更稳健的替代方案。为了保持甚至增加 在Z中的分辨率下，这些半薄切片可以在mSEM或一系列图像的情况下被迭代地研磨和扫描 在不同的倾斜角的情况下，可以获得的Gridtape-TEM。在这里，我们建议将商业上的联合收割机 来自Delmic的具有迭代宽离子束的可用多束扫描透射电子显微镜FASTEM 半薄切片的研磨（BIB-mSTEM）。首先，数百个半薄切片将被直接收集到 使用市售MagC磁收集系统的闪烁体板。随后，这些部分将 通过在宽离子束铣削和用FASTEM成像之间来回进行迭代地薄化和成像。为 每个部分，这将产生一系列迭代研磨的TEM投影图像，可用于重建一个 每个部分高分辨率3D叠加。BIB-mSTEM将比mSEM更加稳健和可靠， 基于Gridtape-TEM的工作流程：与Gridtape-TEM相反，切片收集在固体基底上，而不是在 易碎的支撑膜。与mSEM相比，BIB-mSTEM从高能量透射电子形成图像， 对局部电磁场和铣削引起的不规则表面形貌的敏感性远低于低能量 次级电子

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.