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.