Figure 7: Cryo-STET analysis of a T. gondii tachyzoite. A-F) These images are 25 nm thick virtual sections extract for a cryo-STET reconstruction of an entire T. gondii tachyzoite cell. Several intracellular structures are visible in the reconstruction. At the top, the micronemes (Mic) next to the vacuole (Va) and some dense granules (Dg). Rhoptries (Rh) are clearly identified thanks to their strong contrast, and the mitochondria (Mit) is also visible, yet with lighter contrast. The plasma membrane (Pm) and the internal membrane complex (Im) are resolved at several locations. Scale bar is 500 nm.
Cryo-STET has been used to investigate the 3D structure of various bacterial and eukaryotic cells (including unicellular parasites)16,36–38. To present the cryo-STET method, we estimated that the best would be to apply the method on a new type of sample previously unpublished. Figure 7 shows the ultrastructure of aT. gondii tachyzoite cell. This cell is one of the thickest cells studied using cryo-STET and is almost 2 µm thick and 3.5 µm long. Such a thickness might represent the current limit for cryo-STET studies. Despite the thickness of this sample, several internal structural elements are clearly visible with a fair amount of details. At the top, the tip of the cell is packed with micronemes (Mic) identified by their typical shape and texture, a vacuole (Va) and some dense granules (Dg) (Fig. 7B and C). In this dataset, the conoid is unfortunately outside of the field of view, it would otherwise be visible above the micronemes. Going down to the other side of the cell, the rhoptries (Rh) are particularly visible thanks to their usual high contrast. Next to the rhoptries, it is possible to spot a long vesicle-like structure slightly darker than the cytoplasm, which likely corresponds to the mitochondria (Mit) (Fig. 7F). At the periphery of the cell, and visible on most of the reconstruction slices, the plasma membrane (Pm) and the internal membrane complex (Im) are resolved. Again, the potential of STET, especially under cryo-conditions, is tremendous compared to what can be achieved in conventional TEM which would have required the use of many sections.