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. Author manuscript; available in PMC: 2021 Jun 1.
Published in final edited form as: Nat Protoc. 2020 May 13;15(6):2041–2070. doi: 10.1038/s41596-020-0320-x

Figure 8∣. A representative data of the cellular cryo-ET using protocol described here.

Figure 8∣

Left panel (a-c): SEM images (top view) of vitrified cells. The cell in the center is shown before a, and after b, cryo-FIB milling; image c, presents a zoomed in image of b. Middle panel (d-f): Images of the same cell taken with the FIB. The cell is shown before (d) and after (e) cryo-FIB milling; a 45° rotation of the cell is displayed in the image (f). Right panel (g,h): TEM images of vitrified mammalian cells before (g) and after (h) milling. The image in g illustrates the very limited areas accessible to tomography due to the cell's thickness. Some level of ice contamination (white asterisks) on the sample and even on the lamella is expected and acceptable. The deposited platinum layer is distinguishable from the vitrified sample on the front edge of the milled lamellae (b, c, f, h). It is indicated by the white arrowheads. N: nucleus. i, The example tomogram taken from milled vitrified mammalian cells (lamella thickness: 150 nm). j, k, The corresponding segmented data showing various cellular organelles and cytoskeletons. The subtomogram average of ribosomes from the tomogram is replaced in the segmented data. Annotations of the organellae are shown below. Scale bars (a) 40 μm, (b) 40 μm, (c) 5 μm, (d) 10 μm, (e) 5 μm, (f-h) 10 μm, (i) 200 nm.