Cm200 microscope

Next, the defocus-dependence of astigmatism was examined using live images of carbon film obtained on our CM200 microscope at 200 kV and FEI Titan Krios at 300 kV, and recorded on a Gatan US4000 CCD and K2 Summit camera, respectively. We minimized the objective lens astigmatism at small, medium and large defocuses using our s²stigmator tool, then increased or decreased defocus from the starting defocus used for astigmatism correction. At each defocus, ten images were collected and the defocuses, astigmatisms were calculated to obtain their mean and root-mean-square deviation (RMSD) values. The same experiment was repeated on different days to explore the reproducibility of defocus-dependence of astigmatism.

Ten microliters of the Hcp1 sample diluted to 0.01 mg/ml was deposited onto glow-discharged copper 400 mesh grids with a continuous carbon film (Agar Scientific). After incubation for 2 min, the sample was washed twice with water before being stained with 2% (wt/vol) uranyl acetate for 1 min. Two hundred micrographs were collected with a 4 k by 4 k TVIPS camera on an FEI CM200 microscope operating at 200 kV at a magnification of 30,000× with a defocus range of −1.5 to −3.0 μm. All subsequent data processing steps were performed with crypSPARC v3.0.1 (52 (link)). The contrast transfer function parameters for each micrograph were calculated with CTFFIND4 (53 (link)). Approximately 1,500 particles were manually picked to generate representative two-dimensional (2D) class averages for automated particle picking of the entire data set. A total of 85,598 particles were autopicked and extracted using a 256- by 256-pixel-size box. After several rounds of 2D classification, 56,350 particles were kept in good classes showing top and side views.

For Cryo-EM, freshly isolated EVs, PK-treated and non-treated, were plunge frozen as previously described30 (link), using a Vitrobot Mk2 (FEI; Eindhofen, The Netherlands). Images were acquired using the TVIPS EMMENU 3.0 software and a TVIPS TemCam F224 camera on a FEI CM200 microscope operated at 200kV in low dose mode. For negative stain electron microscopy, PK-treated and non-treated vesicles were put on formvar and carbon coated electron microscopy grids (Cu; 200 mesh) for 5 min. Samples were washed (3x using PBS) and then fixed using 2.5% glutaraldehyde in PBS. After two further washes in filtered water, the samples were stained using 2% uranyl acetate for 1.5 min. Images were taken using a SiS Morada CCD-camera (Olympus, Münster, Germany) on a LEO 912AB Omega electron microscope (Carl Zeiss NTS, Jena, Germany) operated at 120kV.