Figure
4: STEM-in-SEM images of cell subcellular structures. A) Overall
view of cytoplasm showing a Golgi apparatus (G) and some mitochondria in
the bottom left corner. B) Type A virus infected cell showing a viral
factory (box and insert). Samples were EPON resin-embedded and sections
were cut to a 100 nm nominal thickness. Observation performed on a JEOL
IT800 at working distance 6 mm and 8 kV. Scale bar is 200 nm.
Cryo-STEM principle & applications
As introduced above, cryo-STEM combines the benefits of STEM
(amplitude contrast and potential to observe thick specimens) and
cryo-methods (hydrated specimen in its native state). A cryo-STEM
advantage over cryo-TEM is the delayed apparition of radiation damages
caused by the electron beam13. This is an advantage,
especially if multiple images are collected as it is the case in
tomography workflows. Cryo-STEM even enables chemical imaging,
allowing the study of metal distribution in proteins such as the
distribution of Fe and Zn in ferritin24. This has
the potential to study the atomic content of specimen using
compositional contrast25. In summary, cryo-STEM is a
very interesting alternative to conventional cryo-TEM for the study of
biological samples and, as above-mentioned, it is best adapted to
study thick samples.
An example of the contrast increase in cryo-STEM is presented in
Figure 5, it focuses on the observation of nucleic acids encapsulated
in lipid nanoparticles (LNPs) and frozen in presence of 10% sugars
(excipients used in pharmaceutical products). In conventional cryo-TEM
the contrast is low because of the similar chemical composition of
sugars (H, C, O) and nucleic acids-LNPs (H, C, O, P) (Fig. 5A). The
use of STEM increases the contrast even in presence of sugars (Fig.
5B). Thus, cryo-STEM helps and allows the observation of different
types of organic materials (here sugars and nucleic acid LNPs). This
example on LNPs can be extended to larger specimens such as bits of
cells thin enough to allow cryo-STEM imaging. Indeed, in the
literature, cryo-STEM has been used on different types of eukaryotic
cells of varying thickness14,15.