Auriga field emission sem

Scanning electron microscopy (SEM) was used to characterize bacterial cell morphology and μSiM-CA bacterial propagation as previously described (Masters et al., 2019a (link), 2020 (link)). For cell morphology characterization, S. aureus cultures were grown overnight, and then subcultured and seeded onto poly-L-lysine-coated glass coverslips for 6 h before rinsing bacterial cells and fixating with 2.5% glutaraldehyde/4% paraformaldehyde in 0.1 M cacodylate buffer overnight. Similarly, μSiM-CA membranes were incubated for 6 h, as described above, and fixed with 2.5% glutaraldehyde/4% paraformaldehyde in 0.1 M cacodylate buffer overnight. Samples were postfixed in 1% osmium tetroxide, dehydrated in a graded series of ethanol to 100%, and critical point dried in a Tousimis CPD (Rockville, MD, United States). Samples were sputter coated with gold and imaged using a Zeiss Auriga Field Emission SEM (Jena, Germany) for quantification of cell diameters or qualitative assessment of bacterial propagation. ImageJ, specifically Fiji (Schindelin et al., 2012 (link)), was used to measure the maximum cell diameter across six separate SEM images per cell type, where a minimum of 20 cells were measured in each image.

Immature DCs complexed to platelets (day 5) were allowed to adhere onto poly‐L‐lysine–coated coverslips for 2 h and were subsequently placed into 0.1 M sodium cacodylate–buffered 2.5% glutaraldehyde at 4°C for overnight fixation. The cells on the cover glasses were post‐fixed using the same buffer in 1.0% osmium tetroxide and then transitioned through a graded series of ethanol to 100% (×2), then through a graded series of 100% ethanol/hexamethyldisilazane (HMDS) and finally into three changes of pure HMDS. The last change was allowed to evaporate off of the cover glasses overnight in a fume hood. The cover glasses were then mounted onto aluminum stubs and sputter coated with gold. Imaging was performed using a Zeiss Auriga field emission SEM (Carl Zeiss AG, Oberkochen, Germany).