Veleta side mounted ccd camera

L. monocytogenes cells were fixed, sedimented and washed (0.05 M HEPES buffer, pH 7.2) as described above. Washed pellets were embedded in low melting point agarose (3%, mixed 1:1 with the sample; Sigma, Germany), trimmed into small pieces (about 1 × 1 mm) and fixed in 2.5% glutardialdehyde in 0.05 M HEPES buffer, pH 7.2. Samples were postfixed in osmium tetroxide (1% in water) for 60 min and 2 h in uranyl acetate (2% in water) at room temperature and dehydrated in an ethanol series (30, 50, 70, 90, 96% and absolute ethanol for 20 min). Samples were infiltrated with a mixture (1:1) of ethanol and resin (LR White hard grade; Science Services, Germany), embedded in pure resin and finally polymerized at 60°C overnight. Ultrathin sections of about 60–70 nm were cut with an ultramicrotome (Leica Microsystems, Germany), poststained with uranyl acetate followed by lead citrate. Sections were examined using a JEM-2100 transmission electron microscope (Jeol, Japan) at 200 KV. Images were recorded at full resolution of 2k × 2k with a Veleta side-mounted CCD camera (Olympus SIS, Germany).

Purified C9orf72-SMCR8 was diluted to approximately 10 μg/ml and applied to glow-discharged carbon-coated electron microscope grids (Pure C 200 mesh Cu grid, Ted Pella, Redding, California, USA) and stained with 2% (w/v) uranyl acetate. Data were acquired on a Tecnai Spirit electron microscope (Thermo Fisher Scientific) operated at 120 kV, with a nominal magnification of 135,000x corresponding to 3.04 Å/pixel. Images were recorded with a Veleta side-mounted CCD camera (Olympus, Tokyo, Japan) with varying defocus (−1 μm to −2.0 μm).