Figure caption
Figure 1: Current approaches in scanning transmission
electron microscopy suitable for parasitology. The sample (top), can
be either chemically-fixed and resin-embedded (top-left) or cryo-fixed
(top-right). The resin-embedding step generates a thick block which must
be thinned-down before being imaged at the electron microscope
(bottom-left). Cryo-fixed samples can be thin enough (plunge-frozen) to
be imaged in cryo-STEM. But if they are too thick (high-pressure
freezing) they must be cut.
Figure 2: Schematics of the electron beam optical path in STEM.The beam is focused at the sample level, forming a probe that is scanned
over the sample in a raster. The size of the probe is determined by the
convergence semi-angle (\(d=\lambda/\alpha\)). The depth of field is
also dependent on the convergence semi-angle
(\(\Delta z\ =\ 1.77\ \lambda/\alpha 2\)). The camera length value can
be modified to change the collection angles of the different detectors
(a unique camera length for all the detectors).
Figure 3: TEM and STEM images of resin-embedded sample
sections. A and B) Respectively TEM and STEM images of extracellular
flagellum (Fla) portions and flagellum attachment zones (Faz). The
position of microtubules doublets (MTd) and sub-pellicular microtubules
(sMT) is indicated. C and D) Respectively TEM and STEM images ofT. brucei posterior intracellular organisation showing the
kinetoplast (Kin), the flagellar pocket (FP) and the mature- and
pro-basal bodies (mBB and pBB). The sections observed in TEM are 100 nm
thick, whereas the sections observed in STEM are 500 nm thick. Scale bar
is 200 nm.
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.
Figure 5: Conventional cryo-TEM and cryo-STEM images
acquired on the same grid of nucleic acid LNPs on a drug product with
10% sugars. A) Conventional cryo-TEM image. B) Cryo-STEM image
showing in addition to lipid bilayer, the phase segregation between
nucleic acid (n.a.) and water (w) in background vitreous ice and within
LNPs. Arrows point at lipid bilayers of LNP. Images were acquired on a
Glacios microscope operated at 200KV using a Falcon 4 camera for the
conventional cryo-TEM and a Panther STEM detector for the cryo-STEM
image.
Figure 6: STET analysis of a T. brucei cell posterior
part. A-H) These images are 10 nm thick virtual sections extracted
from a STET reconstruction of a 500 nm thick resin section. This volume
contains the mature- and pro-basal bodies (mBB and pBB), the kinetoplast
(Kin) and a large portion of the flagellar pocket (FP). Other structural
elements can be recognized, the microtubule quartet (MTq) and the basal
plate (BP) from which the central pair of microtubules (CP) emerges.
Scale bar is 200 nm.
Figure 7: Cryo-STET analysis of a T. gondii tachyzoite.A-F) These images are 25 nm thick virtual sections extract for a
cryo-STET reconstruction of an entire T. gondii tachyzoite cell.
Several intracellular structures are visible in the reconstruction. At
the top, the micronemes (Mic) next to the vacuole (Va) and some dense
granules (Dg). Rhoptries (Rh) are clearly identified thanks to their
strong contrast, and the mitochondria (Mit) is also visible, yet with
lighter contrast. The plasma membrane (Pm) and the internal membrane
complex (Im) are resolved at several locations. Scale bar is 500 nm.