Gif 2002 post column energy filter

Tomography was performed on a Tecnai G2 Polara transmission electron microscope (FEI, Eindhoven, Netherlands) equipped with a field emission gun operated at 300 kV, a GIF 2002 post-column energy filter (Gatan, Pleasanton, CA), and either a 2048 × 2048 Gatan slow scan CCD camera or a 3838 × 3710 Gatan K2 Summit direct detection camera (only Figures 7 and 11A–E). Tilt-series acquisition was controlled by SerialEM software (Mastronarde, 2005 (link)) under low-dose conditions (∼100 e/Ų cumulative dose). CCD images were recorded at 2° tilt increments, with −8 μm to −12 μm defocus, at 31,500×, 42,000×, and 52,500× magnifications (pixel sizes of 9.6 Å, 7.1 Å, and 5.7 Å). 52,500× was the highest possible magnification that still prevented excessive electron damage to the sample. K2 images were recorded at 2° tilt increments, with −5 μm defocus, at 11,800× and 14,500× magnifications (pixel sizes of 4.2 Å and 3.4 Å). The dataset for this study consisted of 43 tomograms from 27 FIB lamella preparations.

A 3 μL aliquot of the EV fractions was pipetted onto a glow-discharged holey carbon-coated copper electron microscopy grid (C-flat, Protochips). The drop was blotted, and the sample was vitrified by plunging it into liquid ethane (−183 °C). Projection images were recorded on a Tecnai F30 Polara TEM (FEI) operated at 300 kV and equipped with a GIF2002 post column energy filter (Gatan) operated in zero loss mode at defocus settings between −4 μm to −6 μm [33 (link)].

Throughout this study intact, untreated and unfixed parasitized erythrocytes (at the trophozoite stage) were examined. Cryo-preparation and imaging was done as described10 (link). Briefly, whole, intact parasitized erythrocytes were applied on to a holey EM grid and plunge frozen in liquid ethane cooled down with liquid nitrogen. The grids were investigated using a cryo-electron microscope (Polara, FEI, equipped with a field-emission gun and a Gatan GIF2002 post-column energy filter (Gatan), and operating at an accelerating voltage of 300 kV; FEI). The tomographic tilt series were recorded on a Gatan multi-scan charge-coupled device camera (Gatan) at × 18,000 nominal magnification, and typically consisted of 50–60 images collected in increments of 2° at a low electron dose of cumulatively 6,000–8,000 electrons per nm². Three-dimensional (3D) tomographic reconstructions were generated by weighted back-projection of aligned series of tilted images, using EM, TOM and IMOD image-processing software packages10 (link). Resulting tomograms were analysed using the AMIRA volume-processing software (TGS Europe S.A.) for surface rendering and visualization. Actin filaments were tracked and their lengths were measured, using the AMIRA volume processing software (TGS Europe S.A.). Six tomograms were analysed for each condition.