Leo 912 ab energy filter transmission electron microscope

Wild-type TIE-1 and Phb mutants grown with 3-hydroxybutyrate with either N₂ or NH₄Cl as a nitrogen source was used as representative samples for TEM. Briefly, 5 mL planktonic cell suspensions were centrifuged at 6000×g for 5 min. followed by primary fixation by resuspending the cell pellets in 2% formaldehyde and 2.5% glutaraldehyde in 0.05 M sodium cacodylate buffer (pH 7.2) for ~45 min. at room temperature. Cell pellets were agar encapsulated followed by primary fixation for ~20 min. Polymerized agar was cut into small cubes and were subjected to secondary fixation for ~5 h followed by acetone dehydration and resin infiltration. Ultrathin sections (~70 nm) were cut on a Reichert Ultracut UCT ultramicrotome (Leica, Buffalo Grove, IL, USA), mounted on copper grids (FCFT300-CU-50, Electron Microscopy Sciences, Hatfield, PA, USA), and counterstained with lead citrate for 8 min⁷⁵ . The sample was imaged with a LEO 912 AB Energy Filter Transmission Electron Microscope (Zeiss, Oberkochen, Germany). Images were acquired with iTEM software (ver. 5.2) (Olympus Soft Imaging Solutions GmbH, Germany) with a TRS 2048 × 2048k slow-scan charge-coupled device (CCD) camera (TRÖNDLE Restlichtverstärkersysteme, Germany). Each TEM image was acquired at ×10,000 magnification and 1.37 nm pixel resolution.