Figure 4.

Studies of viral ROs in whole cell 3D SEM reconstructions. (A) Focused ion beam SEM (FIB-SEM) segmented volume of a whole human pulmonary epithelial Calu-3 cell at 24 h after infection with SARS-CoV-2 (adapted from [40] with permission). The large-scale data showed how a large network of DMVs (red) and endoplasmic reticulum (ER) (green) spreads throughout the cell, while the resolution was sufficient to resolve small membrane connections between the elements of this network. Other segmented cellular features include Golgi membranes (blue), mitochondria (brown), and nucleus (grey). (B) Whole-cell serial blockface SEM (SBF-SEM) of a CVB3-infected Vero E6 cell at 6 h post-infection. (Top) A slice through the volume displaying abundant ROs in the perinuclear area of the cell. Individual membrane bilayers could not be resolved in the images due to the limited SEM lateral resolution. Despite this, single-membrane, double-membrane and multilamellar ROs could be distinguished by the thickness of the stained membranes (inset: white, hatched and black arrowheads, respectively). (Bottom) Corresponding segmented volume exposing the heterogenous distribution of differently sized RO clusters (multicolored) around the nucleus (blue) and within the cell (beige, semitransparent). Images adapted from [59]. Scale bars, 2 µm.