Quemesa digital camera

Sterilized discs (diameter 6 mm) were punched from transparent Melinex film (Plano, Wetzlar, Germany), treated with 70% ethanol for 30 min, dried, and placed into the wells of a microtiter plate. Cells were seeded (3 × 10⁵ cells/well) in F-12 K medium with 5% serum and cultured on the discs. After 24 h, the medium was replaced by serum-free F-12 K medium containing 100 µg/mL of either PEBCA or PEBCA CBZ. Particle uptake was then continued for 4 and 16 h under cell culture conditions (37 °C, 5% CO₂). Then, the cells were fixed with 2.5% glutardialdehyde in 0.1 M sodium phosphate buffer for 60 min. Cells were washed in PBS, post-fixed in 1% OsO₄, stained en bloc with uranium acetate (1%), and embedded in Epon 812 (Sigma-Aldrich, Taufkirchen, Germany) as described earlier [20 (link)]. Isolated PEBCA particles were embedded in warm agar dissolved in phosphate-buffered saline (PBS) containing 3% bovine serum albumin and cooled on ice. Small pieces of the hardened mixture were embedded in Epon 812 and processed as described for the cells. Thin sections (50–60 nm) of all preparations were cut with a Reichert Ultracut microtome and viewed with a Tecnai G2 electron microscope at 120 kV. Digital images were taken with a Quemesa digital camera (Olympus Soft Imaging Solutions, Münster, Germany).

A 10 μL drop of EV in PBS was applied to nitrocellulose carbon-coated PELCO® Cu grids (Ted Pella Inc., Redding, CA, USA) and incubated for 1 min. Liquid was removed by touching the top of the grid with filter paper. This grid was exposed to a 10 μL drop of 2% uranyl acetate, followed by 10 s incubation with further removal of the moisture by touching the grid with the filter paper. Samples were examined in a transmission electron microscope, JEOL JEM-1400 TEM (JEOL, Tokyo, Japan) attached to an Olympus Quemesa digital camera and analyzed by iTEM software (Olympus Soft Imaging Solutions GmbH, Munster, Germany).

The samples were prepared as described by Nikitin et al. [25 (link)] and examined either in a transmission electron microscope JEOL JEM-1400 TEM (JEOL, Japan) operating at 80 kV, or JEOL JEM-2100 (JEOL, Japan) operating at 200 kV. Images were recorded on an Olympus Quemesa digital camera under the control of iTEM software (Olympus Soft Imaging Solutions GmbH, Germany). The length and width of viral and virus-like particles were measured manually from digital prints using scientific image manipulation software ImageJ (National Institutes of Health, USA). To obtain the 3D reconstructions, the images were captured with an Ultrascan 1000XP 4x4 pixel CCD camera (Gatan) at x40,000 magnification with an underfocus of 1.5–2.8 um.