Figure 1

The S. aureus Asp23 protein and the serial section microscopy workflow. (a) Scheme of Asp23 membrane localization. AmaP is a small protein encoded within the same operon as Asp23. CM, cytoplasmic membrane. (b) Cellular localization of Cerulean-tagged Asp23 by fluorescence microscopy. In the wild type (i) Asp23 appears to be evenly distributed below the cell membrane. In the amaP mutant (ii), Asp23-Cer oligomerizes to ring shaped structures. Scale bar, 1 µm. (c) Purified Asp23 forms long telephone cord-like structures in vitro. Scale bar, 200 nm. (d) Serial section microscopy work flow. After fixation and resin embedding, cells are sectioned and subsequently labeled with the primary and secondary antibody for antigen detection. For fluorescence microscopy, a fluorescence conjugated antibody is used, while for electron microscopy the secondary antibody is linked to a gold particle. Following serial image acquisition of different sections, the software program Reconstruct is used to stack, align and segment the images to generate the final model. (e) Zoom in of an EM grid showing serial section of S. aureus. Scale bars from left to right micrograph as indicated below images. (f) Example of an entire single cell followed over 13 consecutive sections of 50 nm thickness. Segmentation is shown for the 400 nm plane. Gold particles were highlighted for better visualization in sections showing entire cells. Original contrast is shown in the 500 nm plane zoom in. Scale bar, 200 nm.