Figure 1: Sample preparation methods for parasitology samples suitable for scanning transmission electron microscopy. The sample (top), can be either chemically-fixed and resin-embedded (top-left) or cryo-fixed (top-right). The resin-embedding step generates a sizeable resin block several millimetre-thick which must be thinned-down before being imaged at the electron microscope (bottom-left). Cryo-fixed samples which are thin enough to be imaged directly at the electron microscope are usually cryo-fixed using plunge-freezing. But if they are too thick they must be frozen using high-pressure freezing which generates an ice block several hundreds of micrometre-thick which must be thinned-down before being imaged at the electron microscope (bottom-right). For each type of prepared sample, 2D or 3D structural information can be recovered, it only depends on the imaging method used.
One of the main limitations of TEM is the thickness of the samples that can be observed with the typical acceleration voltage of up to 300kV, which is about 300 nm. Therefore, biological samples are typically embedded in resin and sliced into ultrathin sections of less than 100 nm. STEM is an alternative to TEM3. STEM allows to image thicker samples than TEM, in particular thicker resin sections4. This can be advantageous as it requires the observation of fewer sections for the same sample size. It should be notated that in TEM not the original structures are observed but heavy-metals binding to proteins, lipids and other cellular components. In addition, due to fixation, dehydration and resin embedding, structures are often denatured. This limits access to high-resolution structural information required for accurate studies of the intricate and fragile cellular structures.
In cryo-electron microscopy, the sample is rapidly cooled down and fixed under cryogenic conditions in a liquid cryogenic fluid (e.g. liquid ethane) (Fig. 1, top-right)5. Rapid freezing is required to prevent the formation of ice crystals inside and outside the cells which can be deleterious for the sample integrity and affect the observation under the electron microscope. Samples that are thin enough (< 2-3 µm) such as single-cell organisms can be cryo-fixed directly on an electron microscopy grid using plunge-freezing in liquid ethane cooled-down by liquid nitrogen6. Thicker samples including unicellular or multicellular organisms are preferably frozen under high-pressure (~2000 bars) at liquid nitrogen temperature7. As no fixative is used and samples remain hydrated, cryo-electron microscopy is the method of choice to study samples in their native state. Yet, this does not lift the thickness requirements of TEM. This is the reason why thick cryo-fixed cells are thinned-down using cryo-sectioning8 or cryo-focused ion beam (FIB) milling9 (Fig. 1, bottom-right). These sections/lamellae can be observed either using cryo-TEM or cryo-TEM tomography (cryo-TET)10. Combined with averaging computational methods known as sub-tomogram averaging, 3D maps of proteins can be generated in-situ at sub-nanometre resolution11,12.
Cryo-STET was first developed in 2014 at the Weizmann Institute of Science13. This pioneering work represented a great step forward as it allows the observation of micrometre-thick samples in their native state by combining STEM and cryo-conditions14,15. In 2020, cryo-STET was used to characterise the 3D organisation of the flagellar attachment zone staples in Trypanosoma brucei 16. Because the method is relatively new and is likely to be further improved, the potential of cryo-STET is important, especially in the field of parasitology as the thickness of samples studied fits particularly well within the method capacities and limitations.
The main goal of this review is to familiarise the parasitology readership with the basis of STEM imaging in 2D and 3D workflows. Several applications are presented to outline what can be achieved with the STEM methods. Then, the latest developments and applications in cryo-STET are presented. This overview should provide the readers with a good sense of how they could potentially benefit from using STEM methods.