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.