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 of T. bruceiposterior 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. BF STEM images were
collected on a JEOL 2200FS. Scale bar is 200 nm.
STEM can be used to image thick resin sections of biological
samples18. Examples showing the differences between
TEM and STEM images are presented in Figure 3. Thanks to the use of
heavy atom contrasting agents during sample preparation, the contrast of
resin section images in STEM is particularly high, even when thick
sections are imaged. This is due to the substantial amplitude contrast
of heavy atoms. In STEM of thick resin sections, the resin areas have a
more homogeneous texture compared to TEM on thin resin sections. In the
TEM image (Fig. 3A), the densities of the microtubules (sub-pellicular
and axonemal ones) appear shorter, and the details are sharp because it
is a projection of a thin section. In the STEM image (Fig. 3B), the
microtubules appear longer as a thicker section is imaged. This
demonstrates that STEM allows obtaining information over a greater depth
compared to conventional thin resin sections used in TEM. In STEM, it is
possible to study the kinetoplast, the mature- and pro-basal bodies and
a large portion of the flagellum inside the flagellar pocket all at once
(Fig. 3D). Being able to observe an organelle as thick as the flagellum
over its entire depth, without the need to cut or thin it down, offers
the possibility to better understand how the organelle and its
constituents are organised. Since many structural details can be
observed at once, it allows measuring the dimensions of the organelle
components and their distances with much more accuracy than what would
be possible using a collection of serial resin sections. Indeed, during
cutting, i) a thin amount of matter is lost between consecutively sliced
sections and ii) accurate registration of serial sections is not trivial
and prone to measurement errors. In a previous work, STEM of thick resin
sections allowed to build the first 3D organisational map of the
flagellar pocket in T. brucei 19. Using thicker
sections can also be advantageous in observation and/or counting of gold
particles in gold-immunolabelled resin sections as it increases
statistical measurements.
STEM-in-SEM principle & applications
STEM-in-SEM consists in using a scanning electron microscope (SEM) to
obtain images similar to those obtained in a TEM. Because it is based on
the use of low voltages (5 to 30 kV instead of > 100 kV
used in TEM) it is also known as “low voltage STEM”. In SEM,
transmitted electrons are typically not collected because the design of
the microscope does not allow it since the original purpose of a SEM is
to collect backscattered and secondary electrons. The STEM-in-SEM
approach was initially proposed in 1972 by designing a tailored SEM
stage20 to obtain transmission images on material
sciences specimens. Evolution in the design of the specimen holder
allowed to perform DF STEM-in-SEM imaging21. These new
specimen holders were used in biology to study the internalisation of
gold-nanoparticles in bivalve tissues in Spurr resin sections up to 400
nm-thick22 and the structure of myoblasts in 400
nm-thick EPON sections23. These works demonstrate the
potential of STEM-in-SEM for the observation of eukaryotic cells and
tissues. Allowing the observation of semi-thin sections (< 400
nm) removes the need for ultrathin sections (< 100 nm) which
simplifies the ultramicrotomy process to access subcellular structures.
To present more examples of the STEM-in-SEM method, we illustrate the
capability of this technic to resolve subcellular details on another
sample in Figure 4. In these 100 nm-thick resin sections, fine details
can be observed. Organelles such as the Golgi apparatus (Fig. 4A) or
viral factories (Fig. 4B) can be clearly identified. Therefore,
STEM-in-SEM could be an interesting alternative to the more conventional
TEM for histology, cell biology, and basic structural biology.