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. 2020 Mar 9;9:e52286. doi: 10.7554/eLife.52286

Figure 3. Automated sequential cryoFIB milling results in high-quality lamellae and cryotomograms.

(A) CryoTEM overview image of a lamella (session H) containing three yeast cells. Red mark indicates the area imaged in (B). Bar, 5 µm. (B) A 22.85 nm thick slice through a cryo-tomogram of a yeast cell (session H) [indicated by red mark in (A)]. The tomogram shows a nuclear pore complex (NPC), nuclear envelope (NE), microtubules (MT), cytoplasm (CP), cytoplasmic ribosomes (R), mitochondria (MIT) and a putative vacuole (V). Bar, 200 nm. (C) Top view of the microtubule indicated by dashed red box in (B). From a single slice through the tomogram it is possible to identify 13 protofilaments that make up the microtubule. (D) Cross-section of the tomogram showing the microtubule in (C) inside the generated lamella. From this view, we also determined the lamella thickness to be ~284 nm. (E) Shown is a 14 nm thick slice through a cryo-tomogram of a septum between two cyanobacteria cells (session F). The thickness of the lamella was determined to be ~208 nm. Arrowheads indicate septal junctions. The inset shows a magnified view of the septal junction indicated by a red arrowhead. Other cellular features include cytoplasmic membranes (CM), phycobilisomes (PB), thylakoid membranes (TM) and septal peptidoglycan (PG). Bars, 100 nm and 25 nm (inset). (F) Subtomogram average generated by extracting 343 septal junction particles from nine tomograms and performing fivefold symmetrization. Shown are longitudinal and perpendicular slices (thickness 0.68 nm) and a surface rendering of the symmetrized average. The observed characteristic structural modules were similar to a recent study that applied manual cryoFIB milling (Weiss et al., 2019) (also see Figure 3—figure supplement 1). Bars, 25 nm.

Figure 3.

Figure 3—figure supplement 1. Comparison of data quality between manual and automated milling.

Figure 3—figure supplement 1.

Shown is a Fourier Shell Correlation (FSC) curve (red) for the septal junction subtomogram average shown in Figure 3F (resulting from automated milling). The second curve (blue) results from a dataset published previously (Weiss et al., 2019) (resulting from manual milling) and was calculated with the same number of randomly selected subvolumes after fivefold symmetrization (n = 1715). Both approaches result in a comparable resolution estimate.
Figure 3—figure supplement 1—source data 1. Source data for Figure 3—figure supplement 1.