Veleta digital camera

Exosomes prepared for electron microscopy were further isolated by sucrose gradient centrifugation as previously described49 (link). Fractions with a density of 1.10 to 1.20 g/ml were pooled from the MalaEx samples harvested from the cultures with the different pH values and aliquots of 3 μl were added to a grid with a glow discharged carbon coated supporting film for 3 minutes. The excess solution was soaked off by filter paper, the grid was rinsed in 5 μL distilled water for 10 seconds, stained with 2% uranyl acetate in water for 10 seconds and then air-dried. The samples were examined in a Hitachi 7700 electron microscope (Tokyo, Japan) at 80 kV and images were taken by a Veleta digital camera (Olympus Soft Imaging Solutions, GmbH, Münster, Germany).

hTert fibroblast were seeded on sapphire discs and simultaneously transfected with CTRL and MCM2 siRNAs (as described before). Cells were grown to confluence and serum starved for another 48 h. Sapphire discs containing transfected cells were high-pressure frozen without previous chemical fixation using the Wohlwend HPF Compact 01 (Engineering Office M. Wohlwend GmbH, Switzerland) as described in (17 (link)). Freeze-substitution was facilitated by immersion in acetone pre-chilled to −90°C containing 0.2% osmium tetroxide, 0.1% uranyl acetate and 5% to enhance membrane contrasting (all reagents: Merck, Germany) in a computer-assisted substitution apparatus. In there the samples were brought to 0°C over a time of 16 h. Thereafter, the samples were acclimatized to room temperature and Epon (Fluka, Germany) embedded. 60 nm sections were cut using a Leica Ultracut UCT ultra-microtome with a diamond knife (Diatome, Switzerland) and collected on copper grids. Finally, sections were post-stained using lead citrate. Imaging of ultra-thin sections was performed on a JEM-1400 transmission electron microscope (Jeol GmbH, Germany) at an acceleration voltage of 120 kV. To record the images a Veleta digital camera (Olympus Soft Imaging Solutions GmbH, Germany) with a resolution of 2024 Å–2024 pixel and the iTEM software (Olympus Soft Imaging Soluions GmbH, Münster, Germany) were used.

Perfusion fixed aortas were harvested from 16-wk-old, 24-wk-old, and 36-wk-old normal Db/db (n = 4) and diabetic db/db (n = 4) mice, fixed in 4% paraformaldehyde in PBS for 24–48 h, and transferred to 70% ethanol until paraffin embedding. Staining for pentachrome was performed according to standard protocols on 16-wk-old samples (Hinton et al., 2006 (link); Levay et al., 2008 (link)). Picrosirius red staining was performed as previously described on 16-, 24-, and 36-wk-old samples (Junqueira et al., 1979 (link); Trask et al., 2010 (link)). Images were analyzed using ImageJ (NIH) for elastin and collagen expression, which was normalized to medial cross-sectional area (mCSA). Other sections of thoracic aortas from 16-wk-old mice were fixed in 2.5% glutaraldehyde in Millonig’s phosphate buffer for 24–48 h and then stored in Millonig’s buffer until embedding and sectioning. Transmission electron microscopy (TEM) images calibrated and captured using a JEOL JEM-1400 TEM (JEOL Ltd. Tokyo, Japan) equipped with a Veleta digital camera (Olympus Soft Imaging Solutions GmbH, Munster, Germany) as previously described (Tonniges et al., 2016 (link)). Aortic collagen fibril diameters (150–550 per animal) were determined by measuring the length of the shortest diameter of collagen cross-sections using ImageJ as previously described (Tonniges et al., 2016 (link)).

Bacteriophage EC151 was isolated from a human feces sample, and bacteriophage isolation and propagation were performed as described previously [16 (link)]. The study was approved by the Local Ethics Committee of the Center for Personalized Medicine in Novosibirsk, Russia; Protocol #2, 12.02.2019.
The bacterial host range for phage EC151 was examined by spotting serial phage dilutions onto freshly prepared lawns of bacteria, as described previously [17 (link)]. Eighty bacterial strains from the Collection of Extremophile Microorganisms and Type Cultures of the ICBFM SB RAS were used for host range examination. The list of strains is given in Table S1.
A phage sample was prepared for electron microscopy as described previously [15 (link)]. A drop of phage EC151 suspension was adsorbed for 1 min on a copper grid covered with formvar film; the excess liquid was removed, and the grid was contrasted on a drop of 1% uranyl acetate for 5–7 s. An electron micrograph of phage EC151 particles was obtained using a JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan). Digital images were collected using a side-mounted Veleta digital camera (Olympus SIS, Hamburg, Germany).

A carbon-coated copper grid was overlaid with a drop of phage suspension for 1 min and then stained with 1% uranyl acetate for 5–7 s. A JEM 1400 transmission electron microscope (JEOL, Tokyo, Japan) was used to obtain electron microscopy images of StM171. Digital images were collected using a side-mounted Veleta digital camera (Olympus SIS, Hamburg, Germany). The capsid diameter, tail length, and tail width of virions were measured using ImageJ (version 1.51) [26 (link)], and average sizes were calculated using Microsoft excel 365.