Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution

High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

Insights at the molecular level into the formation of oxo-bridged trinuclear uranyl complexes

Reaction of 1,3,4,6-tetra-O-acetyl-N-(2-hydroxy)-naphthylidene glucosamine (HL(Ac)) with uranyl acetate in ethanol leads to formation of dinuclear [(UO2)2(L)2] (1). In a second step 1 is quantitatively transferred into the trinuclear oxo-bridged complex...

The first report of Tomato brown rugose fruit virus in tomato in Norway

In May 2021, tomato (Solanum lycopersicum L.) plants with necrosis and ringspot symptoms were observed in a farm greenhouse at Sundbyfoss, Norway. This greenhouse production focused on ecological growing of several tomato varieties (i.e., “Blush Tiger”, “Sailor’s Luck”, “Evil Oliver”, etc.) with compost for local customers. The presence of tomato brown rugose fruit virus (ToBRFV) was suspected due to the symptoms in the diseased plants. Sap inoculation was carried out with extracts from 100 mg symptomatic tomato fresh leaves in 0.03 M PBS (phosphate buffered saline, pH 7.0), and inoculation on three Nicotiana tabacum cv. Xanthi plants at the 4-6 leaf stage grown in an experimental greenhouse. The test plants showed local necrotic lesions after 3-5 days. Two symptomatic leaf samples, one from tomato and the other from a test plant, were analysed by transmission electron microscope (TEM) with the help of negative staining. The samples were treated with 0.1 M PBS (pH 7.3) first and placed on carbon-coated EM grids and stained with 2% uranyl acetate before TEM observation. Rigid rod-shaped viral particles, typical of tobamovirus particls (around 300 nm) were observed. Total RNA was isolated from two tomato leaves showing symptoms using a Norgen Plant/Fungi RNA kit (Norgen Biotek, Canada). One-step reverse-transcription (RT)-PCR with specific primers ToBRFV-F/ToBRFV-R for ToBRFV (Alkowni et al. 2019), which amplified a 560-bp fragment, was performed. The sequence obtained by Sanger sequencing from the amplicon (535 nt) showed 99.8% nt identity with ToBRFV isolate PV-1241 (NCBI accession no. MZ202349) from DSMZ (Leibniz Institute, German) and was deposited in the GenBank database under the accession number OK358628. The infection was also confirmed with duplex real-time RT-PCR test for ToBRFV using CaTa28 and CSP1325 primers and probe (ISF 2020; EPPO, PM7/146 2021). In addition, eight tomato samples from the same farm greenhouse were collected according to cultivars, location in the greenhouse and symptoms before eradication and disinfection: four of the tomato samples were confirmed positive for ToBRFV with one-step RT-PCR as described above. A surveillance program for ToBRFV in commercial greenhouse production has been carried out in 2021 in Norway. Around 4000 tomato plants from 18 commercial tomato growers were tested. No positive ToBRFV samples have been detected. However, unregulated tomato seeds from abroad and self-propagation of tomato by private gardeners and hobby growers is a potential threat to commercial production of tomato in Norway. To our knowledge, this is the first report of ToBRFV associated with tomato in Norway and in the Nordic countries.

Improved analysis method of neuromuscular junction in Drosophila larvae by transmission electron microscopy

The Drosophila neuromuscular junction is an excellent model for neuroscience research. However, the distribution of neuromuscular junctions is very diffuse, and it is not easy to accurately locate during ultrathin sectioning, which seriously interferes with the ultrastructural analysis under electron microscopy that only has a small field of view. Here, we reported an efficient method for acquiring the ultrastructural picture of neuromuscular junctions in Drosophila larva under electron microscopy. The procedure was as follows: first, the larval sample of body wall muscle was placed between the metal mesh and was dehydrated with alcohol and infiltrated with epoxy resin to prevent the sample from curling or bending, after it was dissected and fixed into thin slices. Second, the sample was embedded in resin into a flat sheet to facilitate the positioning of the muscles. Third, carefully and gradually remove the excess resin and the cuticle of the larvae, cut off both ends of the special body segment, and trim the excess specific muscles according to the recommended ratio of trimming muscles, which would reduce the workload exponentially. At last, the trimmed sample were prepared into serial about 1000 ultrathin sections that was about total 80 microns thickness, and 30–40 sections were gathered into a grid to stain with lead citrate and uranyl acetate. This method could also be applied to the other small and thin samples such as the Drosophila embryo, ventral nerve cord and brain.

Ultrastructural pathology of bioprosthetic heart valves with infectious endocarditis

Aim. To study the ultrastructure of mitral bioprosthetic heart valves (BHVs) which failed due to infective endocarditis.Materials and Methods. Here we examined 7 ethylene glycol diglycidyl ether-treated xenopericardial BHVs excised during repeated BHV replacement because of prosthetic endocarditis. After being fixed in formalin and postfixed in osmium tetroxide, BHVs were dehydrated and stained in uranyl acetate with the subsequent embedding into epoxy resin, grinding, polishing, and lead citrate counterstaining. Upon the sputter coating with carbon, we visualised the BHV microanatomy by means of backscattered scanning electron microscopy at 15 kV voltage.Results. The extracellular matrix underwent degradation and disintegration resulting in loosening, fragmentation, and reduction in the electron density of collagen and elastin fibers. We observed a number of recipient cells (macrophages, multinucleated giant cells, neutrophils, endothelial cells and smooth muscle cells) within the BHVs. The highest number of cells was localized on the valve surfaces. The localization of the recipient cells on the ventricular and atrial surfaces was different. The central part of the valves was abundantly populated by macrophages.Conclusion. Prosthetic endocarditis is accompanied by the migration of recipient cells into the BHV structure, which is the consequence of surface and extracellular matrix disintegration.

Elemental analysis of valvular and atherosclerotic calcification

Aim. To analyze the topographic patterns of valvular and atherosclerotic calcification growth.Methods. Dysfunctional aortic valves (n = 18) and atherosclerotic plaques (n = 20) were fixed in formalin, postfixed in 1% osmium tetroxide, consecutively stained by 2% osmium tetroxide and 2% uranyl acetate, and embedded into epoxy resin (Epon) with the further grinding and polishing ofthe samples. Upon the counterstaining by lead citrate and sputter coating with carbon, samples were visualized by backscattered scanning electron microscopy. Elemental analysis was conducted via energy-dispersive X-ray spectroscopy. Measurement of Ca/P ratio within the mineral deposits was carried out employing a pool table principle (i.e., in the center of the deposit, in the near and far circumferences (clockwise), and in control regions around the mineral deposit). Topographic patterns of calcifications were modeled using the correlation analysis. Results. Significant correlation was revealed between the Ca/P ratio in the deposit center and in the near and far circumferences of deposit in both in valvular (r = 0,35-0,78 - near circumference; r = 0,63-0,69 - far circumference) and atherosclerotic mineral deposits (r = 0,37-0,56 - near circumference; r = 0,48-0,63 - far circumference), suggesting the hierarchical growth of cardiovascular calcification around the initial nucleation sites.Conclusion. Valvular and atherosclerotic calcifications development is concentric.