Process of cryo-electron tomography. (A) Plunge freezing for cryo-electron tomography and microscopy. Left: before blotting (EM grid with mounted specimen solution is shown in the inset of the top panel). Center: after blotting. Right: after plunging. Upper panels: freezing apparatus (Gatan Cp3). Middle panels: schematic diagrams to describe the side view of the grid and the specimen. Grey: holey carbon membrane. Brown: cupper mesh. Bottom panels: Chlamydomonas flagella and cells before blotting and after plunging. The specimen condition after blotting cannot be observed with the current instruments. (B) Electron micrographs and a tomogram. A fiducial gold marker is shown by arrows. (C) Specific image analysis strategy of subtomogram averaging in our research on cilia, based on periodicity.