Microscopy Conference 2023

Prof.

Session chairs

Virus diagnosis can be easily optimized by the combination of electron microscopy and molecular biology

LS 6: Pathology, pathogens and diagnostics

LS 7: Advances in sample preparation

Abstract text (incl. figure legends and references) Introduction: In past, EM was involved with the detection and identification of a broad range of viruses some of which have impact on human, veterinary and wildlife health. However, since pathogen identification was performed mainly by molecular biology the use of diagnostic EM declined radically. With whole genome sequencing and metagenomic approaches the perspectives for molecular pathogen diagnostics changed from a primer-based testing system to a kit-independent diagnostic tool [Metzker, M.L. (2010): Genetic 11:31-45]. But molecular biology can still detect only genetic components of a pathogen whereas EM gives you a morphological picture of the virus. In the present study the method of EM virus screening followed by PCR described by Johnson at al. 2006 [J. Virol. Methods. 134:92-98] was further developed. Material &amp; methods: Viruses of different genera were investigated by negative staining in a TEM (Zeiss EM 906) at 80kv. Negative staining with 0,5% UA, 0,5% PTA or 4% NdAC was performed with pure or inactivated samples. Samples were provided from feces, cell supensions and biopsy material. Some samples were inactivated with 4% PFA; final PFA-concentration was 2%. For some samples the enrichment method by ultracentrifugation at 84000g (Beckmann airfuge) was used. The stained grids were exposed to the EM beam for 30-180min. Some grids were also stored 1 week before further processing. After EM analysis, virus RNA or DNA was obtained from the virus particles adhering to the 400 mesh copper grid(s) by placing the grid(s) into a vial and adding 180µl lysis buffer. The grid(s) were incubated in lysis buffer for 10min. Then 200µl of PBS was added and the solution with the grid(s) was vortexed for 1 min. The grid(s) was/were then removed before the sample was processed for nucleic acid amplification. The further steps of (RT-)PCR were routinely performed. Results: Virus detection with EM combined with PCR was successful – in nearly all samples viruses could be detected by both methods. Since it is not possible to quantify the correct amount of viruses on a grid we will only present here a qualitative result (yes/no decision). Exposure to the electron beam does not significantly affect the diagnosis. Storage of grids for a short time (1 week) before processing to the lysis buffer also had no influence except for a slight decrease in viral copies. Enrichment by airfuge or eludation of more than one grid in 180µl lysis buffer naturally resulted in an increase of viral copies. Virus nucleic acids could be detected by PCR in pure or PFA inactivated samples. Viruses were also detected by PCR after using different stains. All used staining solutionsdid not inhibit essentially the amplification process. This is important in so far as NdAC (Neodymium (III)acetate), not toxic and easy to use, is an excellent stain for negative staining [Kuipers J. &amp; Giepmans B, 2020. Histochemistry and Cell Biology 153:271-277]. It replaces the toxic and radioactive uranyl acetate. Conclusion: Molecular biologists and electron microscopists should be aware that their methods bear a lot of uncertainties in their technical procedure. The results show that a combined EM/Molecular Biology method based on the same sample material eluted from a grid can be performed in an easy and fast procedure. Thus - with the simultanous use of both methods - diagnosis can be confirmed and false negative or positive diagnostic results can be prevented.

Poster

Mag.

Univ.-Prof. Dr.

Univ.Prof. Mag. Dr.rer.nat.

Ultrastructural + mechanical properties of spidersilk or why do Schwann cells like spider silk?

Abstract text (incl. figure legends and references) Spider silk (SPSI) has been established as one of nature&quot;s most fascinating materials due to its unique properties. A remarkable application of the SPSI is its use in reconstructive medicine as nerve guidance structure/filament for nerve regeneration [1]. The Schwann cells (SCs), which are a crucial part of the nerve regeneration process adhere to SPSI and migrate along it to support axonal elongation [2]. SPSI degrades without inflammatory response or physiological pH changes. However, the interaction between the SCs and the silk and by that the SPSI properties, that promote SC adhesion are still unclear. The aim of this project is to elucidate material properties of SPSI, that are crucial for its unique performance in nerve regeneration. Not all spider silks show the same medical success, and we believe that properties such as composition, ultrastructure, and mechanical behaviour have a pronounced influence on the acceptance of SPSI by SCs. Therefore, by combining experiments consisting of in vitro studies and the material characterization of various SPSIs, the properties, which are responsible for the advanced success of SPSI in nerve regeneration, will be clarified. Figure 1. Force-displacement curves and SEM micrographs of the native and autoclaved N. edulis MAG SPSI [1] Radtke, C., et al., Spider Silk Constructs Enhance Axonal Regeneration and Remyelination in Long Nerve Defects in Sheep. PLOS ONE, 2011. 6(2): p. e16990. [2] Kornfeld T, et al. Spider silk nerve graft promotes axonal regeneration on long distance nerve defect in a sheep model, Biomaterials, 2021 Feb 2;271:120692. doi: 10.1016/j.biomaterials.2021.120692. [3] Riekel, C., et al., Nanoscale X-Ray Diffraction of Silk Fibers, Frontiers in Materials, 2019. 6(315).

Initiation of human cytomegalovirus secondary envelopment requires the gM/gN glycoprotein complex and its palmitoylation

LS 3: Imaging of large volumes and plastic section tomography

Abstract text (incl. figure legends and references) The human cytomegalovirus (HCMV) belongs to the family of herpesviruses. As all herpesviruses, its complex maturation pathway includes two distinct envelopment processes in the host cell: primary envelopment on the way from the nucleus into the cytoplasm, and secondary envelopment in the cytoplasmic viral assembly complex (cVAC), the process that provides the virions with their final envelope. HCMV glycoprotein M (gM) forms a protein complex with glycoprotein N (gN), whose precise function in viral morphogenesis is poorly understood. Both proteins are highly conserved among herpesviruses and presumably exert similar functions in assembly and egress, with the gM/gN complex being essential in HCMV. Ultrastructural analyses using TEM imaging and STEM tomography of high-pressure frozen and freeze substitution were applied in combination with viral mutant and siRNA knockdown experiments to clarify at which step of secondary envelopment the gM/gN complex is involved. In wild-type virus infected cells, most capsids at the cVAC are enveloped and only a few partially tegumented but naked capsids can be found. Furthermore, naked and enveloped capsids are homogeneously distributed throughout the entire cVAC area/volume. In contrast, TEM of a palmitoylation-deficient gN mutant virus (TB-gNmutC123S) showed few enveloped virions at the cVAC, however, many partially tegumented and not enveloped nucleocapsids accumulated in the outer region of the cVAC. The centre of the cVAC was devoid of capsids. Detailed analysis of the various stages of secondary envelopment revealed that most nucleocapsids in TB-gNmutC123S-infected cells either did not contact cellular membranes at all or, if they did, showed no sign of membrane curvature that marks the initiation of secondary envelopment. In addition, large protein aggregates were found in and adjacent to the cVAC, surrounded by numerous naked, partially tegumented capsids. This ultrastructural phenotype was accompanied with a pronounced release defect. A very similar phenotype concerning capsid distribution, defect in secondary envelopment, protein aggregates and release defect was found in wild-type virus infected cells transfected with siRNA against gM. Furthermore, wild-type virus infection after inhibition of palmitoylation by 2-BP resulted in an altered nucleocapsid distribution in the outer region of the cVAC, as observed in the TB-gNmutC123S mutant. Taken together, our results show that the gM/gN complex and its palmitoylation are essential for the initiation of secondary envelopment of partially tegumented capsids at the cVAC.

Comparative ultrastructural studies of conidia and blastospores of the entomopathogenic fungus Metarhizium brunneum

Abstract text (incl. figure legends and references) Introduction For biological control of economically important herbivorous insects, entomopathogenic fungi are efficiently used. They are established as an eco-friendly control tool in organic farming. Products of Metarhizium brunneum are successfully used against several pest insects, such as Western Corn Rootworm (Diabrotica virgifera), Japanese Beetle (Popillia japonica), Black Wine Weevil (Otiorhynchus sulcatus), and thrips (Frankliniella occidentalis) (https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/active-substances/?event=search.as). This fungus can be produced on selective solid and liquid artificial media, resulting in different spore types. On solid media, areal conidia are formed, while in liquid media often so-called blastospores are produced. Objectives Conidia and blastospores differ in their physiology, e. g. germination of blastospores is much faster compared to conidia. Ultrastructure of both spore types was investigated in transmission electron microscopy to elucidate differences in their metabolism and will be presented in several micrographs. Materials &amp; methods For best results of observing ultrastructure of conidia and blastospores, the two spore types had to be fixed and embedded in different ways. Conidia were fixed in 3.5% glutaraldehyde, 2% osmium tetroxide and embedded in Methacrylate, while blastospores were fixed in 4% glutaraldehyde, 2% osmium tetroxide and embedded in Epon. Materials and methods protocol with detailed information will be provided. Results Studies on the ultrastructure of the two spore types illustrated that blastospores are in a physiologically active state and therefore capable of immediate germination, while conidia are in a kind of dormant condition. In contrast to conidia, blastospores have numerous mitochondria, many ribosomes and endoplasmic reticulum as well as densely enriched storage substances (Fig. 1). Fig. 1. Micrograph of an ultrathin section of a Metarhizium brunneum blastopore. Active metabolism is obvious by numerous ribosomes (r), mitochondria (m), droplets of storage substances (o = oil, l = lipid) and by endoplasmic reticulum (er), n = nucleus. Perspective Production of blastospores of M. brunneum in liquid media is fast, and enables a high number of spore yield. One advantage of blastospores is their ability to germinate within a few hours. Therefore, penetration through insect cuticle and killing of its host can proceed in shorter time compared to conidia. Entomopathogenic fungi are susceptible to UV-light, high temperatures and dryness. The application of M. brunneum blastospores could therefore be of great advantage for biocontrol of pest insects.

Ultrastructural investigation of samples from monkeypox virus and coronavirus infected patients using thin section electron microscop

LS 4: Image analysis of large data sets

Abstract text (incl. figure legends and references) The recent outbreak of monkeypox and the ongoing COVID-19 pandemic emphasize the importance of studying infectious diseases as one of the major threats of human health. While molecular diagnostics is sensitive, specific and efficient in the diagnosis of viruses, immunohistochemistry and in-situ hybridization techniques allow the localization of virus protein or nucleic acids in patient samples by using special probes at the light microscopy level and its correlation with classical histopathology. However, all of these methods only detect either molecules of viruses or effects of virus infection and not the intact virus itself which is the infectious unit. Electron microscopy (EM) is the only technique which is capable to directly identify virus particles and their precursor structures in patient samples by using their typical morphological features (size, shape, special surface pattern) [1]. The reliable identification of viruses in patient samples is important to discriminate between direct and indirect effects caused by the infection and, thus, for the understanding of the pathogenesis of the disease, which, at the end, is the basis for a better treatment of patients. Thin section EM of patient samples (e.g. from biopsies or autopsies) offer the possibility to localize virus particles in the cellular context of the affected tissue and to correlate it with (histo-) pathological findings. However, investigation of the complex ultrastructure of human tissue and identification of virus particles need experience which seems to vanish as the many mis-interpretations of putative coronavirus particles published during the last years demonstrate [2, 3]. Here, I present the strategies (e.g. sampling, sample preparation for EM, microscopy) which we have applied to study the ultrastructural pathology of coronavirus and monkeypox virus infection in patient material (see Figure) and what we have learned from the results [e.g. 4, 5]. Figure Caption: (A) Mature monkeypox virus particles in the cytoplasm of a fibroblast in the lamina propria mucosae of the colon. Different section profiles of the complex mature virus particles are seen, e.g. a side view (arrowhead) showing the dumbbell-shaped core and a view in top-down direction (arrow) which reveals the brick-shaped form of the virus particle. (B) Part of an isolated naso-pharyngeal ciliated mucosa cell which is regularly found in extracted swab material used for PCR-testing. Coronavirus particles are seen in the extracellular space (arrows) among the base of the cilia and within membrane-bound compartments (arrowheads) of the cytoplasm. Scale bars = 200 nm. References: [1] Miller (1986) Journal of Electron Microscopy Technique (doi.org/1002/jemt.1060040305) [2] Dittmayer et al. (2020) The Lancet 20 (doi.org/10.1016/ S0140&shy;6736(20)32079&shy;1) [3] Krasemann et al. (2022) eBioMedicine (doi.org/10.1016/j. ebiom.2022.104193) [4] Meinhardt et al. (2021) Nature Neuroscience (doi.org/10.1038/s41593-020-00758-5) [5] Cortese et al. (2022) Virchows Archive (doi.org/10.1007/s00428-022-03308-5)

PS 21
Poster session LS 6: Pathology, pathogens and diagnostics

Appointment

Session chairs

Programme

PS 21Poster session LS 6: Pathology, pathogens and diagnostics

Appointment

Session chairs

Programme

PS 21
Poster session LS 6: Pathology, pathogens and diagnostics