Figure 6: STET analysis of a T. brucei cell posterior part. A-H) These images are 10 nm thick virtual sections extracted from a STET reconstruction of a 500 nm thick resin section. This volume contains the mature- and pro-basal bodies (mBB and pBB), the kinetoplast (Kin) and a large portion of the flagellar pocket (FP). Other structural elements can be recognized, the microtubule quartet (MTq) and the basal plate (BP) from which the central pair of microtubules (CP) emerges. Scale bar is 200 nm.
Cryo-STET principle & applications
Cryo-STET was developed in 2014 with the pioneering work of Wolfet al. 13. Up to now, less than a handful of laboratories have implemented cryo-STET, which might illustrate the difficulty of setting up the method. However, a recent video-based article explains very well how to perform the data collection34 hopefully inspiring researchers to use the method and apply it to different biological systems. As with cryo-TEM and cryo-STEM methods, the specimens studied in cryo-STET are cryo-fixed, which protects and maintains the sample native state. Cryo-STET is a 3D imaging method, which can theoretically be used to observe samples thicker than the ones studied in room-temperature STET. The reason is that room-temperature STET observes chemically fixed and heavy metal-stained samples, which then contain atoms heavier than the original organic ones, reducing the electron mean free path and their penetration through matter. In cryo-STET, only typical organic atoms are present (mainly H, C and O), allowing the observation of samples as thick as 1 µm and beyond35. Then, in terms of volume of information, cryo-STET exceeds by far what is possible in cryo-TET (by one order in magnitude). Because cryo-STET can observe samples slightly thicker than 1 µm, the exploration of basically any unicellular eukaryotic parasite species becomes possible (e.g. Trypanosoma sp. , Leishmania sp. and Toxoplasma sp.). Yet, as explained above, cryo-STET is not a trivial method. The successful collection of cryo-STET data requires first a correct understanding of the concepts associated with STEM imaging, which differ from those associated with TEM imaging.