Integrated ultramicrotome for serial block face scanning electron microscopy (SBF-SEM)

生物医学电子显微镜(EM)的最新技术发展旨在以高分辨率的3D(体积EM)显示结构(例如，细胞或组织样本)的地形背景。通过对大样本体积的分析，可以揭示生物在空间复杂性上的“连接体”。我们申请了一台集成在扫描电子显微镜柱中的超微切割机，用于通过连续块面扫描电子显微镜(SBF-SEM)进行自动3D分析。该系统将补充场发射阴极扫描电子显微镜(FEG-SEM)，自2019年以来由柏林Charité-Universityätsmediz的核心设施电子显微镜(CFEM)运行。超微切割机安装在一张桌子上，可以通过FEG-SEM柱的前面进行整合。通过交换工作台，FEG-SEM既可以在常规模式下操作，也可以在SBF-SEM模式下操作。在扫描电子显微镜中生成3D图像数据集的基本方法是基于从样品表面机械去除薄片的原理，每次去除后交替成像样品表面。在SBF-SEM中，这种从样品表面的去除是通过超微切割(即用金刚石刀)的原理实现的。在正确对齐之后，原始数据的获取将自动运行。最大空间分辨率分别为10-15(x/y)和25-50 nm(Z)。最大样本体积约为1 mm(Z)，面积约为0.5 x 0.5 mm(x/y)。根据柏林Charité-Universityätsmediz CFEM的用户政策，为SBF-SEM研究而升级的显微镜将向所有感兴趣的用户提供。此外，它还将纳入柏林大学联盟(BUA)的研究基础设施。

Recent technical developments in biomedical electron microscopy (EM) aim at visualizing structures (e.g. cells or tissue samples) in their topographic context at high resolution in 3D (volume EM). The analysis of large sample volumes allows to reveal biological "connectomes" in their spatial complexity.We apply for an ultramicrotome, integrated into the column of a scanning electron microscope, for automated 3D analyses by serial block face scanning electron microscopy (SBF-SEM). The system will complement a scanning electron microscope with field emission cathode (FEG-SEM) operated since 2019 by the Core Facility Electron Microscopy (CFEM) of Charité - Universitätsmedizin Berlin. The ultramicrotome is mounted on a table and can be integrated via the front of the FEG-SEM column. By exchange of the table, the FEG-SEM can either be operated in conventional mode or in SBF-SEM mode.An essential approach for generating 3D image datasets in SEM is based on the principle of mechanical removal of thin slices from the sample surface, alternating to imaging the sample surface after each removal. In SBF-SEM, this removal from the sample surface is realized by the principle of ultramicrotomy (i.e. with a diamond knife). After proper alignment, the acquisition of raw data runs automatically. The maximum spatial resolution is about 10-15 (x/y) and 25-50 nm (z), respectively. The maximum sample volume is in the order of about 1 mm (z) over an area of about 0.5 x 0.5 mm (x/y).The microscope, upgraded for SBF-SEM investigations, will be made available to all interested users according to the user policy of the CFEM of Charité - Universitätsmedizin Berlin. Furthermore, it will be embedded into the research infrastructure of the Berlin University Alliance (BUA).