Helios 660 g3 uc fib sem

For scanning transmission electron microscopy (STEM) examination, 1 × 10⁷ THP-1 cells were seeded in RPMI into T75 cell culture flasks. Four groups were prepared: macrophages (control, 1), macrophages with 0.5 mg ml⁻¹ Zn2-SrBG/ceria (2), Macrophages with internalized MRSA (3), macrophages with internalized MRSA and 0.5 mg ml⁻¹ Zn2-SrBG/ceria (4) as described above. Cells were detached from the flask using trypsin-EDTA and a cell scraper, pelleted by centrifugation (500g, 5 min, 20 °C) and resuspended in 1 ml paraformaldehyde. After fixation at 4 °C overnight, the cells were spun down at 1500g for 5 min and stained with 2% osmium tetroxide in 0.1 M sodium cacodylate buffer. After drying in an ethanol gradient series, the cells were embedded in epoxy resin (EPON 812, Sigma-Aldrich). Ultrathin sections were either imaged on an FEI Helios 660 G3 UC FIB/SEM at 25 kV or contrasted with lead citrate (Reynolds 1963) before observation by TEM. Elemental distribution maps were recorded from these ultrathin sections in scanning transmission mode on a Talos F200X TEM (Super-X EDS, 4 detector configuration, FEI, USA) at an accelerating voltage of 200 kV. Samples were mounted on a double-tilt holder and fixed using a molybdenum ring and clamp. The data were processed using the software Velox 2.9 (FEI, USA).