Kuipers Jeroen, Kalicharan Ruby D, Wolters Anouk H G, van Ham Tjakko J, Giepmans Ben N G
Cell Biology, UMC Groningen.
Clinical Genetics, Erasmus MC Rotterdam.
J Vis Exp. 2016 May 25(111):53635. doi: 10.3791/53635.
Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.
大规模二维电子显微镜（EM），即纳米切割术，是纳米级分辨率电子显微镜在整个组织层面的应用。我们和其他研究人员之前已将大规模EM应用于人类皮肤、胰岛、组织培养物以及完整的斑马鱼幼虫（1 - 7）。在此，我们描述一种适用于整个组织层面扫描EM的通用方法，用于无偏检测亚细胞和分子特征。纳米切割术被用于研究健康的和神经退行性病变的斑马鱼大脑。我们的方法基于标准化的EM样本制备方案：用戊二醛和锇固定，接着进行环氧树脂包埋、超薄切片，并将超薄切片安装在单孔网格上，随后用铀和铅进行后染色。使用连接到外部大面积扫描发生器的扫描电子显微镜（SEM）并采用扫描透射电子显微镜（STEM）采集大规模二维EM镶嵌图像。大规模EM图像通常大小约为5 - 50 G像素，使用可缩放的HTML文件能获得最佳观看效果，这些文件可在任何网络浏览器中打开，类似于在线地理HTML地图。该方法可应用于（人类）组织、整个动物的横截面以及组织培养物（1 - 5）。在此，我们在一个非侵入性神经元消融模型中分析了斑马鱼大脑。我们在单个数据集中可视化组织、细胞和亚细胞变化，这些变化可在包括神经元和小胶质细胞（大脑中的巨噬细胞）在内的各种细胞类型中进行量化。此外，纳米切割术有助于在同一组织上实现EM与光学显微镜（CLEM）（8）的关联，因为先前使用荧光显微镜成像的大表面积随后可进行大面积EM成像，从而得到组织的纳米解剖结构（纳米切割术）。总之，纳米切割术能够以在整个组织层面可量化的方式无偏检测EM水平的特征。
