Applications of SBF-SEM

The SBF-SEM method is most usable for histological specimen in smaller size range than in micro-CT or SPIM -methods. SBF-SEM has been successfully utilized in imaging, for instance, cell populations, axons, cell ultrastructure and structures of different cell organelles in nanometre scale. The lateral resolution it can achieve is approximately 5 nanometres and the axial resolution is maximized with slice thickness of 20–25 nanome-tres (Peddie & Collinson 2014; Smith & Starborg 2018).

As an example of using SBF-SEM in imaging larger volumes, Svensson et al. (2017) studied the tendon structure of a mouse by cutting it into 100 nm thick slices between

electron microscopy imaging. They achieved to differentiate collagen fibrils of the tendon and study the length of them. However, the fibrils of a human tendon, which they also attempted to image, turned out to be too long of a research topic since they did not achieve desired resolution and the fibril tracking became hard due to movement of indi-vidual fibrils across the field of view.

Brain tissue and nerves are suitable research topics of SBF-SEM because they are more conductive compared to other tissue types. Hence, the nervous tissue does not require any special preparation to make the tissue more conductive in order to remove charging which moreover causes artefacts to the images. For instance, Kornfeld et al. (2017) stained three axons and SBF-SEM-reconstructed them to study the connectivity of them for better understanding of a bird’s ability to chirp which seems to be a similar process than a human’s ability to form phonemes with very precise timing. Despite that SBF-SEM was not able to find every computationally estimated synaptic connection between the cells, they still managed to discover one sixth of them. They could also study the corre-lation between the distance from the cell’s soma and the amount of both inhibitory and excitatory synapses based on the SBF-SEM data.

An example of using SBF-SEM to form a 3D reconstruction at cellular and ultracellular level, Pinali et al. (2013) presented the structure of cardiac myocytes of a rat and a sheep in three dimensions imaged with SBF-SEM. The attained resolution of their imaging sys-tem was 15 nm at lateral axes and the slice thickness was 50 nm which was suitable for both studying the sarcoplasmic reticulum and t-tubule structure within the myocytes. In addition, by adjusting the cutting depth and the lateral resolution to be bigger, they could also present the cellular structure of the myocardium. The second example of SBF-SEM application by Vihinen et al. (2013) demonstrated the volumetric structure of the endo-plasmic reticulum (ER) of the human hepatoma cells. The ER was stained in accordance with a conventional SBF-SEM heavy-metal staining protocol which improved the resolu-tion so that the ER could be visualized in detail. However, the ribosomes on the ER surface were not able to be visualized with SBF-SEM, while another imaging technique, electron tomography, succeeded to illustrate both the smooth ER and ribosomes. They state that the main potential of the SBF-SEM is to image many cells at a time with reso-lution that reaches the size of the cell organelles which enables, among other things, the determination of the amounts of different cell organelles. In addition, SBF-SEM was poorer method compared to electron tomography when membrane connections were researched. Figure 5 shows the structure of ER. On top left, there is an overall view of the cells, and on bottom right, the ER is shown in yellow (ER sheets) and lilac (ER tu-bules). On the right, the ER structure is presented in yellow in 3D.

Figure 5. The ER of human hepatoma cells imaged by SBF-SEM. a) An overall structure of the cells. b) The 3D structure of ER sheets in yellow. c) The 3D structure of ER sheets in yellow and ER tubules in lilac. (Vihinen et al. 2013) SBF-SEM is usually performed to soft tissue types since it makes the cutting and the controlling of conductivity more simple. However, Goggin et al. (2020) used it to visualize osteocytes in order to understand the mechanical signals between them, osteoblasts and osteoclasts in more detail because the bone development and resorption were not fully understood. In addition, the most used high-resolution method in bone imaging, mi-cro-CT, was not able to visualize the osteocytes in ultracellular level. The group was able to reconstruct the 3D structure of a osteocyte and the distribution of them in bone matrix with higher resolution than in micro-CT which they estimated to be useful in further un-derstanding of mechanical interactions between bone cells, for instance, in modelling the development of osteoporosis. The resolution of SBF-SEM was approximately 10 nm lat-erally and 25 nm axially, whereas micro-CT can usually reach axial and lateral resolution of 1–10 μm. Another advantage of SBF-SEM was that the obtained images were spon-taneously aligned well to each other because of the way of imaging the stationary block face instead of multiple slices separately. However, the destructiveness, complex prep-aration process and non-isotropic voxel data that is generated during SBF-SEM ap-peared to be the drawbacks compared to micro-CT in bone tissue imaging.

In addition to cellular and sub-cellular imaging achieved with SBF-SEM, it can also be applied to even smaller structures, such as chromosomes. For instance, a survey by

Chen et al. (2017) covered 3D imaging of a human cell during prophase in which the chromatin condenses into chromosomes that consist of two adjacent chromatids, or at least the studies in two dimensions suggest that. When they performed 3D reconstruction and studied the chromosomes, most of them observed the principle mentioned above.

In addition, the chromosomes had a clear 3D structure and the bigger ones resembled letters S or C and the smaller ones had a structure more similar with letter X. However, either a small amount of the chromosomes broke down during the preparation stage or the SBF-SEM system was not able to slice those chromosomes since the volume recon-struction could not be completed for them. All in all, SBF-SEM is applicable to nuclear and chromosomal research since the obtained results were compatible with current knowledge.

The SBF-SEM technology is constantly evolving and the quality of the obtained images is increased further. In addition, the scale of suitable tissue types and sizes is constantly expanded. The greatest disadvantages of the method are the destructiveness of it and the maximum sample size of approximately 1000–10 000 000 μm³ which is mostly lim-ited by the measurement setup and the size of ultramicrotome. (Peddie & Collinson 2014) There are also other 3D electron microscopy techniques available, such as serial section transmission electron microscopy in which the tissue is sliced beforehand and the slices are imaged separately. Compared with such techniques, SBF-SEM is more easy to use since the imaging system operates automatically after adjustments and ob-tained images are more easily aligned during the reconstruction process since the sta-tionary block is imaged.