Jem 2200fs electron microscope

MSNs were diluted to 100 μg/mL and 10 µL were dropped on to copper grids and maintained at ambient temperature for 10 min to allow for samples to dry. Grids were imaged at 200 keV using a DE-20 (Direct Detector Inc.) direct electron detector camera. The energy selecting slit was set to 20 eV and the microscope had a field emission electron source and omega-type electron energy filter to remove inelastically scattered electrons from the image formation. A DE-20 camera was used to collect images in movie mode with a frame rate of 25 frames/second. After image collection, frame alignment was performed using the DE_process_frames.py script provided by Direct Electron Inc. Images were collected at 40,000× magnification and the pixel size on the specimen scale corresponded to 1.5 Å/pixel. For cryo-electron microscopy analysis, LCMSNs were prepared as above and vitrified using an automatic plunge freezer (Leica). LCMSN solution was added to a C-flat grid (Protochips, Inc) with 2 μm holes and blotted with filter paper. Grid was flash frozen in liquid ethane and stored in liquid nitrogen until being transferred to a JEM 2200FS electron microscope (JEOL) and imaged as described above.

The surface microstructure of the Nb-Ti thin-film alloys along the library was characterised with a field emission scanning electron microscope (FE-SEM, Zeiss Leo 1550 VP, Jena, Germany). Images were acquired at a 3 kV acceleration voltage using the in-lens detector. With these experimental conditions, the surface grains of the Nb-Ti alloys could be best observed.
To shed light on the structure and chemistry of the specimens at the nanoscale, cross-sectional transmission electron microscopy (TEM) was applied. Characterisation was performed using a JEOL JEM-2200FS electron microscope (JEOL, Tokyo, Japan) operated at 200 kV. The TEM was fitted with an in-column Omega filter and a CMOS-based camera, TemCam-XF416 (TVIPS, Gauting, Germany). Images were captured utilising zero-loss filtering. Cross-sectional lamellae were prepared via focused ion beam (FIB) milling (CrossBeam 1540 XB, Zeiss, Germany). Before cutting, the samples were covered with an electron beam-stimulated Pt deposit, followed by an ion-stimulated Pt sacrificial layer to protect the surface. For qualitative elemental analysis, energy-dispersive X-ray spectroscopy (EDX) was performed in scanning (S)TEM mode utilising an X-Max^N 80 T detector from Oxford Instruments (UK). The data were processed with dedicated Aztec Version 4.0 software.

The human proteasome α7 subunit (PSMA3 [P25788]; residues 1–255) was expressed and purified as described previously [23 (link),24 (link)]. The sample was dissolved in a buffer containing 20 mM Tris-HCl (pH 8.0) and 150 mM NaCl. An aliquot (2.5 μL) of sample solution was applied onto R1.2/1.3 MO 200 mesh holey grids (Quantifoil Micro Tools, Jena, Germany) coated with thin carbon membranes and pre-treated with glow discharge using a plasma ion bombarder (PIB-10, Vacuum Device, Mito, Japan) for 30 s. The grid was blotted for 4 s with a force level of 7 at 4 °C and 95% humidity, and then it was flash frozen in liquid ethane using a Vitrobot Mark IV system (Thermo Fischer Scientific, Hillsboro, OR, USA). The vitreous ice sample grid was maintained at liquid-nitrogen temperature within a JEM2200FS electron microscope (JEOL Ltd., Tokyo, Japan) using a side-entry Gatan 626 cryo-transfer holder (Gatan Inc., Pleasanton, CA, USA), and it was imaged using a field-emission gun operated at 200 kV and an in-column (Omega-type) energy filter operating in zero-energy-loss mode with a slit width of 20 eV. A total of 100 images were collected on a direct-detector CMOS camera (DE20, Direct Electron, LP, San Diego, CA, USA) at a nominal magnification of 40,000×, corresponding to 1.42 Å per pixel on the specimen.

The size and structure of the trCLN3-L4 nanoconstructs were analyzed by negative stain electron microscopy. Samples were prepared using negative staining^{74 (link)}. In brief, carbon coated grids (Quantifoil Micro Tools GmbH, Jena, Germany, 200 mesh) were glow discharged to render the surface hydrophilic prior to applying samples. 10 µL of an aqueous solution of trCLN3-L4 were applied to the grid. Afterward excess solution was carefully blotted off using filter paper followed by three times washing with ddH₂O. In the final step, grids were stained with negative staining reagent by placing them (plastic side down) on a 10 µL drop of freshly prepared 2% (v/v) uranyl formiate aqueous staining solution. TEM micrographs were recorded using a JEOL JEM 2200 FS electron microscope (JEOL, Japan) operated at 200 kV. The size of the micelles measured on the TEM images could typically be observed in a range between 20 and 25 nm.