Tecnai f30 electron microscope

The purified Holliday junction complex was checked for homogeneity by screening negatively-stained samples on a Morgagni electron microscope (FEI, Hillsboro, Oregon). For cryo preparation, we applied 3.0 μL of complex solution to a C-flat 1.2/1.3 Cu grid (400 mesh) (Protochips, Raleigh, NC), which had been glow discharged at 20 mA for 30 s. Grids were plunge-frozen with a Vitrobot Mark II (FEI) at 85% humidity, offset -2, blot time 7 s. Images were recorded on a Tecnai F30 electron microscope (FEI) operated at 300 kV and using a liquid-nitrogen cooled 626 cryo-specimen holder (Gatan Inc., Pleasanton, CA). We used the semi-automated acquisition program SerialEM (Mastronarde, 2005 (link)) to record 1359 movies with a Falcon II direct detector (FEI) at an underfocus set between 2.7 – 4.2 μm. Each movie consisted of 25 frames, collected in a 2 s exposure of 35.5 electrons/Ų. The nominal magnification was 78,000x, corresponding to a magnification of 100,000x on the detector and 1.4 Å pixel size on the specimen. Movie frames were aligned and summed using motioncorr (Li et al., 2013 (link)).

For EM analysis, the embryos were high-pressure frozen (HPM010 AbraFluid), using 20% dextran as cryoprotectant. The embryos were pierced with a needle in a cryo-microtome chamber (Leica EM FC6) at −160 °C to facilitate freeze substitution^{53 (link)}. Embryos were then freeze-substituted (EM-AFS2 - Leica Microsystems) with 0.3% Uranyl Acetate (UA), 0.3% Glutaraldehyde and 3% water in acetone at −90 °C for 48 h. The temperature was then raised to −45 °C at 2 °C/h and samples were further incubated for 16 h. After rinsing in acetone, the samples were infiltrated in Lowicryl HM20 resin, while raising the temperature to −25 °C and left to polymerize under UV light for 48 hours at −25 °C and for further 9 hours while the temperature was gradually raised to 20 °C(5 °C/h). 70 nm cross sections were cut from the polymerized resin block in the region of the embryo cauterization and picked up on formvar coated slot grids. Tiled 2D images were acquired with a FEI Tecnai F30 electron microscope. Images were then stitched using Etomo (Imod software package^{54 (link)}).

Briefly, livers harvested from both WT and Ubl4A KO newborn pups (~6 h after birth) were fixed overnight in 0.1 M sodium cacodylate buffer containing 4% PFA. After fixation, the tissues were incubated in 0.1 M sodium cacodylate buffer with 1% osmium tetroxide for 1 h, followed by staining with 1% uranyl acetate in maleate buffer for 1 h. The samples were further processed and subjected to transmission electron microscopy at the Advanced Electron Microscopy Facility at the University of Chicago with an FEI Tecnai F30 electron microscope.

X-ray diffraction (XRD) patterns were recorded on an X-ray diffractometer (Rigaku SmartLab) with Cu Kα radiation (λ = 0.15418 nm) at a voltage of 45 kV and a current of 200 mA. Transmission electron microscope (TEM) images were obtained by using FEI Tecnai F30 electron microscope (300 kV). High-angle annular dark-field scanning TEM (HAADF-STEM) and energy-dispersive X-ray (EDX) elemental mapping were obtained by using FEI Talos TEM. TEM samples were prepared by placing a drop of the nanoparticle colloidal solution onto a carbon-coated Cu grid under ambient conditions. The metal compositions and loading of catalysts were measured by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7700x).

Equiaxial-grained nanotwinned Cu foil samples were synthesized by means of pulsed electrodeposition8 (link). The as-deposited Cu foils have dimensions of 20 mm × 10 mm × 50 μm.
TEM specimens were thinned with double-jet electropolishing in a solution of 25% phosphoric acid, 25% ethanol and 50% distilled water at 263 K. TEM observations were conducted using a FEI Tecnai F30 electron microscope operating at 300 kV with a spatial resolution of 0.20 nm and a FEI Titan aberration-corrected electron microscope operating at 300 kV with a spatial resolution of 0.08 nm. An in situ TEM investigation was conducted using a Gatan model 654 single-tilt straining holder at room temperature19 (link)22 (link). Time-resolved bright-field TEM and HRTEM images were taken during the intermittent straining. During the in situ tensile experiment, dislocation activities are normally observed under the loading with a tensile displacement of up to 15–20 μm. The thin area of the sample, where most of the tensile stress is loaded and is thus presumably the deformed area under the tensile loading, is ∼2–2.5 mm in length (Supplementary Fig. 18). Therefore, the apparent strain can be roughly estimated by the ratio of the tensile displacement to the length of the deformed area as ∼1%.