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.