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.