(A) Cross-section diagram of a Chlamydomonas cell. The chloroplast is shown in color. Labels: (1) flagellum, (2) cell wall, (3) eyespot, (4) nucleus, (5) plastoglobule, (6) thylakoids, (7) chloroplast envelope, (8) starch granule, (9) pyrenoid, (10) chloroplast stroma, (11) plasma membrane. (B–E) Preparation of FIB lamellas in the dual-beam microscope. (B) Top-view scanning electron microscopy (SEM) image of Chlamydomonas cells frozen onto the holey carbon of a 200 mesh grid. (C) The same cells imaged with the FIB (secondary electron detection) at the milling angle of 9°. (D) Schematic of a finished lamella. The direction of FIB milling is from left to right. (E) SEM top-view of the lamella milled from the cells shown in B and C. The direction of FIB milling is from right to left. (F) TEM high-defocus montaged overview of the same lamella. Unbinned pixel size: 30.1 Å. (G) Slice from a tomographic volume acquired at the position indicated by the yellow box in F. From left to right, RuBisCO complexes of the pyrenoid (py) are surrounded by a starch sheath (ss), followed by thylakoids (th), a starch granule (sg), and the chloroplast envelope (ce). Outside of the chloroplast, Golgi stacks and rough endoplasmic reticulum can be seen. Both cytoplasmic and chloroplast ribosomes are also visible. The tomogram was 2× binned. Unbinned pixel size: 9.6 Å. Scale bars: 10 μm in B–C, 5 μm in E–F, 500 nm in G. Figure accompanied by Video 1.