Serial block-face (SBF) scanning electron microscopy (SEM) is used for imaging the entire internal ultrastructure of cells, tissue samples or small organisms. We developed a workflow for SBF SEM of adherent cells, such as Giardia parasites and HeLa cells, attached to the surface of a plastic culture dish, which preserves the interface between cells and plastic substrate. Cells were embedded in situ on their substrate using silicone microwells and were mounted for cross-sectioning which allowed SBF imaging of large volumes and many cells. In total we provide 10 data sets with image series from SBF SEM of Giardia and HeLa cells prepared with protocol variants to improve the workflow. A detailed description of the methods and the data set is provided in the download container. Data set 05 comprises an image series of 500 images recorded of a HeLa cell adhered to the plastic substrate of a culture dish. SBF SEM was done at a section interval of 50 nm using the DBS detector of the SEM at low vacuum (0.4 mbar). Original pixel size was 6 nm. The data folder contains the raw image files, processed image files (see data set description for details of the processing), a video file of a processed image file series.