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sample previously unpublished.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.