Oneview 4k camera

Mouse backskin was spread on whatman paper and immersion fixed in 2% formaldehyde, 2% glutaraldehyde in 0,1 M sodium cacodylate buffer (Applichem) for 48 h at 4 °C.
For epon embedding, fixed tissue was washed with 0,1 M sodium cacodylate buffer, incubated with 2%OsO₄ (Science Services) in 0,1 M cacodylate buffer for 2 h at 4 °C, and washed three times with 0,1 M cacodylate buffer. Subsequently, tissue was dehydrated at 4 °C using ascending ethanol series for 15 min 50%, overnight 70%, 15 min 90%, 3 × 15 min 100%, 15 min 50% ethanol/propylene oxide and 2 × 15 min 100% propylene oxide. Tissue was infiltrated for 2 h with 50% epon in propylene oxide (Sigma Aldrich), 2 h 75% epon in propylene oxide, overnight 100% epon and finally 2 h with fresh epon at RT. Tissue was transferred into embedding moulds and cured for 72 h at 60 °C. Ultrathin sections of 70 nm were cut using an ultramicrotome (Leica Microsystems, UC6) and a diamond knife (Diatome, Biel, Switzerland) and stained with 1.5% uranyl acetate for 15 min at 37 °C and lead citrate solution for 4 min. Images were acquired using a JEM-2100 Plus Transmission Electron Microscope (JEOL) operating at 80 kV equipped with a OneView 4 K camera (Gatan).

Purified phage particles at a concentration of 10⁹ PFU/mL were deposited on 300 mesh carbon-coated copper grids (Agar Scientific, United Kingdom) and negatively stained with 1% uranyl acetate (pH 4) as follows: the surface of the copper grids were ionized for 2 min immediately prior to sample deposition. 5 μL of phage lysate were spotted on the surface of the grid and allowed to stand for 1 min. This was followed by 30 sec negative staining and subsequent air drying of the grid. Visualization was performed using a JEOL JEM-1400Plus TEM, operated at 120 kV (pixel size = 0.1 nm), equipped with a Gatan OneView 4K camera with automatic drift correction.

Extracted mitochondria were treated based on (Unger et al, 2017) (link), but fixed in suspension using 1.5% glutaraldehyde, 3% formaldehyde, and 2.5% sucrose in 0.1M sodium cacodylate buffer o/n at 4°C. Mitochondria were spun down into a pellet at 13,000g in a 1.5-ml microfuge tube. The fragile pellet was washed carefully three times with ddH₂O and postfixed with 1% osmium tetroxide for 1 h at 4°C. The pellet was washed four times with ddH₂O and incubated in 0.5% uranyl acetate overnight at 4°C. The pellet was washed three times in ddH₂O and embedded in 2% low-melting agarose, which was cut into small pieces of 1-mm edge length using a razor blade. Agar pieces were dehydrated for 15 min using ascending ethanol concentrations of 50%, 70%, 90%, 2× 100%, and 2× propylene oxide at 4°C. Pieces were infiltrated with Epon/propylene oxide 1:1 overnight at 4°C and pure Epon for 6 h at RT and embedded into BEEM capsules with conical tip (#69913-01; Science Services) and cured for 48 h at 60°C. Images were acquired using a OneView 4K camera (Gatan) mounted on a Jem-2100Plus (Jeol) transmission electron microscope operating at 200 kV. Large montages of 100 images were acquired using SerialEM (Mastronarde, 2003 (link)).