Talos electron microscope

Negative stain TEM data were collected on well-dried specimens using a Talos electron microscope (FEI), operated at 200 kV acceleration. Images were recorded using the EPU system (Thermo Fisher Scientific, MA USA) at room temperature using a 4096 × 4096 pixel CMOS camera Ceta16M (FEI), at a magnification of x 57,000 (pixel size 1.83 Å), 1 s exposure per micrograph. FLAG-FADD complexes were manually picked from 620 micrographs yielding a dataset of 3200 particles, as summarized in Supplementary Table 1. The single-particle analysis was then performed using RELION^{27 (link),52 (link)} and initial models generated and refined in CryoSPARC^{53 (link)}, as summarized in Supplementary Figs. 4, and 7.

We report the properties of the annealed thin films. Their morphologies and thicknesses were investigated by scanning electron microscopy (SEM) from Zeiss and Bruker and atomic force microscopy (AFM) from Bruker. Chemical analysis of thin films were performed by SEM combined with energy dispersive X-ray spectroscopy (SEM-EDS) on a Zeiss Merlin system (beam energy: 15 keV). X-ray diffraction spectra were recorded in the glancing incidence mode on a Malvern Panalytical (Empyrean model) diffractometer with incidence angle of 0.8°. The morphologies of the annealed nanotubes were investigated by both SEM and transmission electron microscopy (TEM), chemical element distribution was examined by scanning transmission electron microscopy (STEM) combined with energy dispersive X-ray spectroscopy (STEM-EDS). The TEM and STEM experiments were carried out using an FEI Talos electron microscope operated at 200 kV. The thicknesses of the NTs were extracted from EDS elemental 2D maps of Ga and Ni using the software Velox, as previously discussed.^{48 (link)} The diffraction pattern was simulated using the software JEMS, for the Ni : Fe crystal structure with atomic ratio 75 : 25.

The consortium was visualized in mid-stationary phase (12 h incubation, MMSY, 0.6 mM SMX) by Cryo-Transmission Electron Microscopy (Cryo-TEM) for morphological characterization. Briefly, a 4 μl aliquot of the overnight grown liquid culture was adsorbed onto a holey carbon-coated grid (Lacey, Tedpella, USA), blotted with Whatman 1 filter paper and vitrified into liquid ethane at − 180 °C using a vitrobot (FEI, USA). Frozen grids were transferred onto a Talos Electron microscope (FEI, USA) using a Gatan 626 cryo-holder (GATAN, USA). Electron micrographs were recorded at an accelerating voltage of 200 kV using a low-dose system (20 to 40 e−/Å2) and keeping the sample at − 175 °C. Defocus values were − 3 to 6 μm. Micrographs were recorded on 4 K × 4 K Ceta CMOS camera. The cell size, and periplasmic and cell wall thickness were measured with Fiji from the ImageJ platform [101 (link)]. For Transmission Electron Microscopy (TEM) analyses, 4 μl aliquot of the sample was adsorbed onto a glow-discharged carbon film-coated copper grid, and subsequently negatively stained with 2% uranyl acetate. Images were recorded using Philips CM200FEG electron microscope operating at 200 kV on TemCam-F416 CMOS camera (TVIPS, Germany).

Purified SARS-CoV-2 spike samples in 10mM Hepes pH7.2 and 150mM NaCl (3 μL at 0.62–0.75mg/ml) were kept at 4°C until their application onto QUANTIFOIL R 1.2/1.3 Cu:300-mesh grids (QUANTIFOIL). The grids were vitrified using a Leica GP automatic vitrification robot (Leica). Chamber conditions were set at 10°C and 95% relative humidity. Grids were glow discharged for 30 seconds prior to application of the samples. Data were collected on a FEI Talos electron microscope operated at 200 kV and images recorded on a FEI Falcon III detector operating in electron counting mode. A total of 4,841 and 2,492 movies were recorded for [S:A222V + S:D614G] and S:D614G, respectively; at a calibrated magnification of 120,000x, yielding a pixel size of 0.85 Å on the specimen. Each movie comprises 60 frames with an exposure rate of 0.54 e-/Ų per frame, with a total exposure time of 20 s and an accumulated exposure of 32.4 e-/Ų. Data acquisition was performed with EPU Automated Data Acquisition Software for Single Particle Analysis (Thermo Fisher Scientific) at -0.3 μm to -3.5 μm defocus.

For cryo-TEM analysis, nPMVs and native EVs were concentrated (≥10¹² particles/mL) using Amicon Ultra-4 filters (10 kDa MWCO, 20-30 min, 4000 × g, 4 °C, Merck). Samples were adsorbed onto a carbon-coated grid (Lacey, Ted Pella, CA) and vitrified by rapid transfer into liquid ethane using a Leica GP plunger (Leica, Wetzlar, Germany). Frozen grids were transferred into a Talos electron microscope (FEI, Hillsboro, OR) using a Gatan 626 cryo-holder. Electron micrographs were recorded at an accelerating voltage of 200 kV and a nominal magnification of ×73,000, using a low-dose system (20 e⁻/Ų) and keeping the sample at low temperature.
For cryo-TEM tomography, samples were premixed with gold fiducials (10 nm) and adsorbed onto carbon-coated grids as described above. Tilt series were acquired from −60° to +60° in 3° steps in consecutive order. The total dose received by 61 images corresponds to ~200 e⁻/Ų. Segmentation, generation of a nPMV density map, and sample visualization were performed^{125 (link)}. The final rendering of the nPMV model was created with Blender V3.3 (Blender Foundation, Amsterdam, Netherlands).