Veleta side mounted camera

All reagents for microscopic studies were purchased from EMS (Hatfield, PA, USA).
Samples of fixed cell cultures and spheroids were washed from paraformaldehyde with Hank’s balanced solution and were postfixed with 1% osmium tetraoxide solution for 1 h, dehydrated in ethanol and acetone according to the standard method, and then embedded in an epon-araldite mixture to obtain hard blocks.
Ultrathin and semithin sections were prepared on an ultramicrotome EM UC7 (Leica, Wetzlar, Germany) using a diamond knife (Diatome, Nidau, Switzerland). The semithin sections of spheroids were stained with Azur II and were examined in a Leica DM 2500 light microscope (Leica, Wetzlar, Germany) to choose an area for ultrathin sectioning. Ultrathin sections were contrasted with 2% water solutions of uranyl acetate and lead citrate and examined in a JEM 1400 TEM (JEOL, Japan). Digital images were collected using a Veleta side-mounted camera (EM SIS, Muenster, Germany).

Samples of EVs (pellets) were fixed with 4% paraformaldehyde at 4 °C for 24 h, postfixed with 1% OsO₄, dehydrated in graded ethanol and acetone, and embedded in an Araldite–epon mixture. Ultrathin sections (65–75 nm) from obtained hard blocks were prepared on an ultramicrotome EM UC7 (Leica, Wetzlar, Germany) using a diamond knife (Diatome, Nidau, Switzerland). Ultrathin sections were contrasted with water solutions of uranyl acetate and lead citrate and examined in a JEM 1400 TEM (JEOL, Tokyo, Japan). Digital images were collected using a Veleta side-mounted camera (EM SIS, Muenster, Germany).

Ultrathin sections from all obtained blocks were prepared on an ultramicrotome EM UC7 (Leica, Wetzlar, Germany) using a diamond knife (Diatome, Nidau, Switzerland), and contrasted with 2% water solutions of uranyl acetate and lead citrate; some sections were left unstained. The sections were examined in a JEM 1400 TEM (JEOL, Tokyo, Japan). Digital images were collected by a Veleta side-mounted camera (EM SIS, Muenster, Germany). All measurements were made using the iTEM software version 5.2 (EM SIS, Muenster, Germany).
From each block, 20–25 ultrathin sections were obtained, which were examined in TEM. Each ultrathin section contained at least 1000 differently cut cells. Ultrathin sections have a thickness of 70–80 nm and can pass through cells in different planes, leading to an “imaginary” polymorphism of S. aureus cells. We analyzed cells and their structures on cross sections only. The thickness of the cell wall and cell membrane was measured only on sections perpendicular to these structures.