3D scanning transmission electron microscopy approaches for
parasitology
Images produced in scanning transmission electron microscopy and in
conventional TEM contain structural information of the inside of the
object of interest because they are projection images, as opposed to
surface images. Projection images can be combined to reconstruct
biological objects by computational approaches thus providing access to
the 3D structure. When projection images are acquired by tilting the
sample around a single axis the computational approach is called
tomography26.
STET principle & applications
Historically, STEM was mostly used in the field of material sciences,
even though there were some early uses of the technique to measure the
amino acids content of proteins, thanks to the atomic mass sensitivity
of the method27,28. In biology, STEM became
increasingly popular when the community realised that it could be used
to study thick sections of cells embedded in
resin4,17,29,30. STET of biological samples was then
developed to explore cellular and sub-cellular structures of samples
embedded in thick resin sections19,31.
In STET, the sample is tilted at different angles inside the electron
microscope and, for each tilt angle an image is collected. In the
microscope, the sample cannot be tilted up to ±90° because its apparent
thickness would be virtually infinite (at this angle the electrons would
have to traverse the whole diameter of the grid). Therefore, the angular
collection is incomplete. In the Fourier space, this creates a wedge
(called the missing-wedge) which does not contain any
information32. A typical tilt range is between -60° to
+60°, using 2° or 3° increments, but some studies collect images with
tilts up to ±70°. Collecting images at higher tilt angles can be useful
to fill in and reduce the missing-wedge. During the experiment, the
sample must be tracked so that the same field of view is imaged during
the whole data collection and focusing must be performed to avoid
collecting out-of-focus images. It is particularly important in STEM to
collect images in focus otherwise they suffer a strong
blurriness32. This is particularly true if high
convergence semi-angles are used (depth of focus limited to a few tens
of nm) but can be mitigated by using low convergence semi-angles (depth
of focus up to a few microns). After data collection, the 2D projections
must be aligned to a common origin, which can be done by using gold
beads as fiducial markers or fiducials-free reference mathematical
methods. Then, dedicated algorithms are used to reconstruct the 3D
volumes using the aligned 2D projections. The details of these steps are
out of the scope of this review but can be found in the
literature26.
Resin sections up to 1 µm-thick can be studied using STET. Because the
field of view is usually greater than 1 µm², the total volume available
in a single 3D reconstruction is greater than 1 µm3,
which corresponds to about the volume of small prokaryote, or using
parasitology standards, the whole flagellar pocket of a T. bruceicell19. In comparison, the sample volume usually
captured in conventional TEM ranges between 0.1 and 0.3
µm3 (for 100 and 300 nm-thick resin sections,
respectively, and provided that the field-of-view is 1 µm²).
Early STET works in biology were performed using the ADF detector as it
was often employed in material science studies4,17. In
tomography, the sample is tilted and its apparent thickness increases
inversely proportional to the cosine of the tilt angle. This means that
at ±60°, the apparent thickness of a sample is twice its nominal
thickness. Because of this, multiple inelastic scattering occurs,
leading to the undesirable detection of light elements on the ADF or
HAADF detector. This, in turn, results in the collection of noisy
ADF/HAADF images. For the observation of thick samples, more meaningful
images are obtained using the BF detector29,31.
In parasitology, STET has so far only been used on a limited number of
organisms19,29. A video article describing the use of
STET on T. brucei thick sections is also
available33. In this review, we introduce another
case, also involving T. brucei . In this example, STET has been
performed on a 500 nm-thick resin section of T. brucei , showing
structural details next to the mature- and pro-basal bodies, between the
flagellar pocket and the kinetoplast of the cell (Figure 6). This is the
same region as the one shown in Figure 3D. Each image corresponds to a
virtual 10 nm-thick slice within the reconstruction and is separated
from the next one by a 20 nm-thick gap. The entire pro-basal body is
present in this 500 nm-thick section. The microtubule triplets are
clearly visible, with microtubules A, B and C well-delineated. The
flagellum is slightly tilted inside the reconstruction as we can see it
appearing from the top of the image and disappearing from the bottom. It
extends from the mature basal body, which is almost fully contained in
the resin section. The penultimate image corresponds to the centre of
the flagellum as the central pair is visible above the basal plate. The
microtubule quartet is visible on the third and fourth images (Fig. 6C
and D). The microtubules of the quartet are then directed towards the
anterior part of the cell and are visible on the left side of the
flagellar pocket membrane in the fifth and sixth images (Fig. 6E and F).
On the bottom part of the images, the large kinetoplast is easily
recognised thanks to its specific shape and texture. The disk of the
kinetoplast DNA is particularly well defined. Other details could be
described in this dataset, such as some thin strings connecting the
kinetoplast and the mature basal body or the collarette around the
flagellum membrane in the flagellar pocket (Fig. 6F). However, what we
want to point out here is the large volume of structural information one
can get in using STET on thick resin sections. Using a 200 kV STEM
capable electron microscope, 750 nm-thick resin sections can routinely
be studied.