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3D（体积EM）的高分辨率在其地形背景下可视化结构（例如细胞或组织样本）。对大量样本的分析可以揭示生物“连接体”的空间复杂性。我们申请了一个集成在扫描电子显微镜柱中的超微组，用于串行块面扫描电子显微镜（SBF-SEM）的自动3D分析。该系统将补充自2019年以来由charit<s:1> - Universitätsmedizin柏林核心设施电子显微镜（CFEM）运行的带场发射阴极（fg - sem）的扫描电子显微镜。超微显微镜安装在一个桌子上，可以通过FEG-SEM柱的前部集成。通过交换表，FEG-SEM可以在常规模式或SBF-SEM模式下工作。在SEM中生成三维图像数据集的一个基本方法是基于从样品表面机械去除薄片的原理，每次去除后交替对样品表面进行成像。在SBF-SEM中，这种从样品表面的去除是通过超显微切割原理实现的（即使用金刚石刀）。经过适当的校准后，原始数据的采集将自动运行。最大空间分辨率分别约为10-15 （x/y）和25-50 nm (z)。在约0.5 x 0.5 mm （x/y）的面积上，最大样本量约为1mm (z)。该显微镜，升级为SBF-SEM调查，将提供给所有感兴趣的用户根据慈善<s:1> - Universitätsmedizin柏林CFEM的用户政策。此外，它将嵌入到柏林大学联盟（BUA）的研究基础设施中。

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).