Fig. 2.

FIB lamella milling of frozen-hydrated D. discoideum. (A) FIB micrograph of a frozen-hydrated cell embedded in a thin layer of ice and attached to the holey carbon support film (view in the direction of the incident ion beam). In order to prepare an in situ lamella, an upper and lower pattern for site-specific milling is defined (white dotted rectangles). (B) Corresponding region after FIB milling yielding the lamella (white arrowheads) which is supported on the sides by the remaining bulk ice material. (C) SEM top view of the same region showing the prepared area, having a size of approximately 25 μm². (D) TEM micrograph of the boxed region from (C) taken after cryotransfer of the lamella into the TEM. The high pass filtered image of the thinned region exhibits a view into the cell’s cytoplasm. On the right side of the micrograph the milling edge can be recognized, containing putative gallium droplets (white asterisks) responsible for curtaining streaks (white arrowheads; showing direction of milling). (E) Slice from tomographic reconstruction of the boxed region in (D). Traversing microtubules (white arrowheads) and various interconnecting ducts and cisternae structures can be recognized. [Scale bars, (A–C) 5 μm, (D) 1 μm, (E) 200 nm.]