CIL:50162, 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.

HeLa cells with DNA labeled with diaminiobenzidine, imaged at high-vacuum on SBEM using Focal Charge Compensation device to eliminate charging artifacts. No alignment was performed on mrc stack of images.