Orius 830

Concentrated bacteria grown in liquid broth were directly filled into 3 nm carriers (Leica, Germany) and vitrified with Leica EM HMP100. Freeze substitution was carried out as previously described (Bleck et al., 2010 (link)). The infiltration at -90°C lasted for 48 h before the temperature was raised with 5°C per h until reaching -30°C. Samples were kept at -30°C for 3 h before the staining solution was washed out 3 times and replaced with acetone. The bacteria were infiltrated with increasing epoxy Embed812-DER 736 resin (EMS, Hatfield, PA) concentration (1:2. 1:1, 2:1 and 1:0) for 2 h each before final exchange to 100% epoxy resin overnight was done and polymerized at 60°C for 48 h. Epoxy resin embedded bacteria were sectioned with a Leica FC6 ultramicrotome and 45° diamond knife (Diatome, Nidau, Switzerland) into 80 nm thin sections, which were placed on formvar coated copper grids and counterstained with lead citrate. The samples were examined with a Jeol 1230 TEM operating at 80 kV at 40,000x magnification. Micrographs were recorded with Gatan Orius 830 with 2048 x 2048 pixels charge-coupled device (CCD) camera using the Digital Micrograph software.

NPs were characterized by TEM using a JEOL JEM-1400 Plus (Jeol, Akishima-shi, Japan), with LaB6 thermionic source with an acceleration voltage of 120 kV. Images were acquired using a Gatan CCD camera Orius 830 (2048 × 2048 active pixels). The particle size distribution was obtained by manual measurement of the diameter of at least 500 NPs using ImageJ software.