CIL:50152, Homo sapiens, hela. In Cell Image Library

Cultured cells and tissues were prepared as previously described using a combination of glutaraldehyde fixation, ferrocyanide reduced osmium tetroxide postfixation, thiocarbohydrazide and osmium tetroxide liganding, followed by en bloc uranyl acetate and lead aspartate staining (Deerinck et al., 2010; Williams et al., 2011; Ngo et al., 2016). Cultured cells were also stained for DNA using click-chemistry as previously described (Ngo et al., 2016). Briefly, HeLa cells were incubated overnight in media containing 10 micromolar 5-ethynyl-2'-deoxyuridine (EdU, Life Technologies, Waltham, MA, USA) and the following day, copper-mediated click-chemistry (Click-iT Cell Reaction Kit, Invitrogen, Waltham, MA, USA) was used to attach dibromofluorescein-azide (1 micromolar) to the EdU incorporated into cellular DNA during replication, which was then used to photooxidise diaminobenzidine into a reaction product prior to treatment with osmium tetroxide (Ngo et al., 2016). Cultured cells were also prepared using a genetically targeted ascorbate peroxidase in order to stain the endomembrane system (Martell et al., 2012). Epoxy embedded samples were mounted to aluminium pins (Gatan) using either silver epoxy (Ted Pella, Redding CA) or cyanoacrylic adhesive, or mounted on a custom designed tip-tilt holder and sputter coated with a thin layer of Au/Pd prior to block-face imaging. HaLa cells with DAB-labeled DNA imaged at high-vacuum on SBEM using Focal Charge Compensation device to eliminate charging artifacts. No alignment was performed on mrc stack of images. Slice-by-Slice movie was generated with ImageJ.

培养细胞与组织的制备参照既往文献方法，采用戊二醛固定、亚铁氰化还原锇酸后固定、硫代卡巴肼与锇酸配位结合，随后进行整体块染乙酸铀酰与天冬氨酸铅染色（Deerinck等，2010；Williams等，2011；Ngo等，2016）。培养细胞亦采用点击化学（click-chemistry）法进行DNA染色，操作流程同前（Ngo等，2016）。简言之，将HeLa细胞置于含10 μmol/L 5-乙炔基-2'-脱氧尿苷（5-ethynyl-2'-deoxyuridine, EdU，美国马萨诸塞州沃尔瑟姆市Life Technologies公司）的培养基中孵育过夜；次日采用铜介导的点击化学法（Click-iT细胞反应试剂盒，美国马萨诸塞州沃尔瑟姆市Invitrogen公司），将1 μmol/L二溴荧光素叠氮连接至复制过程中掺入细胞DNA的EdU，随后在经锇酸处理前，利用该标记物将二氨基联苯胺（DAB）光氧化为反应产物（Ngo等，2016）。此外，研究亦采用基因靶向的抗坏血酸过氧化物酶对培养细胞进行处理，以标记内膜系统（Martell等，2012）。环氧包埋的样品可通过银胶（美国加利福尼亚州雷丁市Ted Pella公司）或氰基丙烯酸酯粘合剂固定于铝制样品钉（Gatan），亦可安装于定制的倾转支架上，并在块面成像前采用金钯（Au/Pd）薄层溅射镀膜。带有DAB标记DNA的HeLa细胞在扫描块面电子显微镜（SBEM）下，于高真空环境中借助聚焦电荷补偿装置消除充电伪影后完成成像。未对mrc格式图像栈进行配准操作。采用ImageJ软件生成逐帧切片电影。