Urany-Less Low Voltage Transmission Electron Microscopy: A Powerful Tool for Ultrastructural Studying of Cyanobacterial Cells.

Sample preparation protocols for conventional high voltage transmission electron microscopy (TEM) heavily rely on the usage of staining agents containing various heavy metals, most commonly uranyl acetate and lead citrate. However high toxicity, rising legal regulations, and problematic waste disposal of uranyl acetate have increased calls for the reduction or even complete replacement of this staining agent. One of the strategies for uranyless imaging is the employment of low-voltage transmission electron microscopy. To investigate the influence of different imaging and staining strategies on the final image of cyanobacterial cells, samples stained by uranyl acetate with lead citrate, as well as unstained samples, were observed using TEM and accelerating voltages of 200 kV or 25 kV. Moreover, to examine the possibilities of reducing chromatic aberration, which often causes issues when imaging using electrons of lower energies, samples were also imaged using a scanning transmission electron microscopy at 15 kV accelerating voltages. The results of this study demonstrate that low-voltage electron microscopy offers great potential for uranyless electron microscopy.

Biodistribution and Cellular Internalization of Inactivated SARS-CoV-2 in Wild-Type Mice.

Despite the growing list of identified SARS-CoV-2 receptors, the human angiotensin-converting enzyme 2 (ACE2) is still viewed as the main cell entry receptor mediating SARS-CoV-2 internalization. It has been reported that wild-type mice, like other rodent species of the Muridae family, cannot be infected with SARS-CoV-2 due to differences in their ACE2 receptors. On the other hand, the consensus heparin-binding motif of SARS-CoV-2's spike protein, PRRAR, enables the attachment to rodent heparan sulfate proteoglycans (HSPGs), including syndecans, a transmembrane HSPG family with a well-established role in clathrin- and caveolin-independent endocytosis. As mammalian syndecans possess a relatively conserved structure, we analyzed the cellular uptake of inactivated SARS-CoV-2 particles in in vitro and in vivo mice models. Cellular studies revealed efficient uptake into murine cell lines with established syndecan-4 expression. After intravenous administration, inactivated SARS-CoV-2 was taken up by several organs in vivo and could also be detected in the brain. Internalized by various tissues, inactivated SARS-CoV-2 raised tissue TNF-α levels, especially in the heart, reflecting the onset of inflammation. Our studies on in vitro and in vivo mice models thus shed light on unknown details of SARS-CoV-2 internalization and help broaden the understanding of the molecular interactions of SARS-CoV-2.

Hexapole corrector for LVEM.

In recent years, aberration correction has slowly become standard in high-end conventional transmission electron microscopy (50-200 kV). However, the integration of correctors to low voltage transmission systems (5-25 kV) has proved to be difficult. The hexapole corrector based on permanent magnet technology seems to be a promising solution for the correction of the primary spherical aberration. Especially if the compact dimensions and low complexity are to be preserved. However, the high importance of chromatic aberration with respect to the microscope resolution still remains a serious obstacle. It must be taken into account when the design is made. The following presented concept is intended exclusively for STEM mode to avoid additional chromatic deterioration caused by electron passage through the sample. The design of the key segment (transfer lens doublet) is discussed in detail, including its compensation systems, which guarantees proper alignment.

Aberration correction for low voltage optimized transmission electron microscopy.

Further development of low voltage electron microscopy leads to an aberration correction of the device in order to improve its spatial resolution. The integration of a corrector to a desktop transmission electron microscope with exclusively low-voltage design seems to be a challenging task. The benefits and potential of the Rose hexapole corrector implemented to such a system are critically considered in this paper. The feasibility of miniaturized corrector suitable for desktop LVEM is especially discussed, including the aspect of corrector contribution to chromatic aberration that appears to be crucial. Optimal corrector parameters and resolution limits of such a system are proposed. •Improved spatial resolution•Spherical aberration correction•Permanent magnet transfer lenses.