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Figure 4
Focused ion beam cryo-milling of herpesvirus-infected cells. Cryo-EM/ET of lamellae produced by focused ion beam (FIB) milling with the eBIC FEI Scios dual-beam scanning electron microscope (SEM). (a) Screenshot of the FIB-SEM acquisition software shortly before milling a lamella into a plunge-frozen porcine kidney cell grown on electron-microscopy grids and infected for 10 h with herpesvirus PrVΔUS3 (muliplicity of infection 10). Several imaging modalities support efficient milling, e.g. SEM for targeting an appropriate cell specimen (I), FIB imaging for planning and controlling lamella geometry (II), an in-column detector to provide material-specific contrast to check for a protective platinum coat on the sample (III) and an infrared live camera to monitor the cryostage (IV). (b) FIB image of the completed lamella through the cell depicted in (a) as viewed from the milling angle (18°). (c) The same lamella as in (b) imaged via SEM from the built-in angle of 52° between the electron and ion beams. A low electron acceleration voltage allows the observation of cellular details. (d) Low-magnification cryo-EM projection image at 0° of the lamella depicted in (b) and (c). Before milling, the leading edge was protected from erosion by the gallium ion beam by a platinum layer (black asterisk; ice contamination is shown by white asterisks). Denser objects in the cell led to curtaining (cytoplasmic lipid body; arrowhead). (e) Cryo-ET slice of a tomogram taken in the area marked by a white square in (d), lamella thickness 130 nm. Visible within the nucleoplasm (nuc) are nucleocapids at different stages of maturation: spherical assemblies of scaffolding protein (1), procapsids (2), partly DNA-filled (3) and nuclear C-capsids (4), which subsequently bud into nucleoplasmic reticulum (NR) forming nuclear egress complex (arrow)-lined capsid-containing vesicles in the perinuclear space (5).

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