Cryo-EM Imaging of Plasmodium Sporozoites

Plasmodium berghei sporozoites were isolated from the midgut of infected Anopheles stephensi mosquitoes by dissection and placed in RPMI media supplemented with BAS onto holey EM carbon grids, incubated for 10–20 minutes and rapidly frozen after removal of excess liquid. The frozen grids were mounted in a cryo-holder and imaged by electron microscopy as described previously^{45 (link),46 (link)}. We focused on imaging nuclei from sporozoites, which are located centrally within the sporozoites with a shift towards the rear end of the cell. Tilt series of low-dose images were recorded using a Phillips CM 300 at a magnification of 51.000 and an objective lens defocus of −5 to −15 μm or a FEI Polara at a magnification of 18.000 and a defocus of −10 μm; both operated at 300 kV and equipped with a field emission gun and a Gatan post column energy filter (GIF 2002) with 2k × 2k pixel CCD camera (Gatan). Tomographic reconstructions were calculated by weighted back-projection using the EM-image processing package^{47 (link)} or IMOD^{48 (link)}. In total 10 tomographic reconstructions of the nuclei were performed.