Orius 10.7 megapixel

Bacterial strains were grown overnight for 16 h in the stated medium at 37 °C. Fixation and processing was performed using conventional procedures for TEM on bacterial samples. Briefly, ~2 mL of culture was spun down and fixed in 50 μL of 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) for 1 h at room temperature, followed by post-fixation in 1% osmium tetroxide for 1 h at 4 °C and then 1% uranyl acetate overnight. Dehydration through ethanol was followed by acetonitrile and finally embedding in Embed 812 (EMS #14120). Imaging of thin sections was performed on a JEOL1400 TEM operating at 120 kV and 10,000X with a Gatan Orius 10.7 megapixel CCD camera. All steps from post-fixation to sectioning were conducted by staff at the Stanford Cell Sciences Imaging Facility. To measure cell wall thickness, images showing a section through the midplane of a bacterial cell were chosen and distances were quantified using ImageJ.

After protein insertion into semi-permeabilised cells, cells were fixed with 4% formaldehyde/PBS for 20 min, washed in PBS, and quenched with 50 mM glycine/PBS for 5 min. After washing in PBS, unspecific binding sites were blocked with 3%BSA/ 5% FBS/ PBS for 20 min followed by incubation with anti-opsin antibodies for 1h. Fluoro-nanogold, which is a 1.4 nm nanogold particle and Alexa Flour 488 fluorophore coupled to an affinity-purified Fab′ fragment (Nanoprobes, Cat. 7202) was used as a secondary probe in a 1:300 dilution. Specific labelling of sUBXD8op insertion sites as seen with conventional immunofluorescence was verified by examining a parallel sample by fluorescence microscopy. For ultrastructural characterization specimens were fixed in 4% PFA/ 2% glutaraldehyde/ 0.1M Cacodylate. Gold-particles were enhanced for 6 min using GoldEnhance EM Plus (Nanoprobes) followed by post-fixation in 1% OsO₄ for 1h and en bloc staining with 1% uranyl acetate over night. After dehydration samples were embedded into Epon resin and 80 nm ultra-thin sections collected on carbon-coated copper grids. Contrasting was performed with a 1:1 mix of 3% uranyl acetate and acetone for 30 sec followed by lead citrate staining (0.2%) for 3 min. Specimens were examined with a Joel, JEM1400 transmission electron microscope equipped with a Gatan Orius 10.7 megapixel CCD camera at 120kV.