FIB-SEM analyses show the change of mitochondrial volume and morphology in early germarium. (A) A violin plot showing the distribution of individual mitochondrial volume in germarium 2-cell (n = 808), 4-cell (n = 2,992) and 16-cell (n = 4,782) cysts. The dashed lines indicate the median volume. (B and C) The volume (V) versus surface area (SA) of each mitochondrion from regions 1 (B) and 2A (C) in wild-type ovaries. The dashed blue lines represent the relationship between V and SA of perfect spheres, which have the lowest SA/V ratio among all shapes. For a given volume, the lower SA/V reflects more rounded shape. Note that more mitochondria in region 2A shift toward the blue line, compared with those in 2-cell cyst at region 1. (D and E) Frequency distribution of mitochondrial volume in region 1 (n = 600) and region 2A (n = 3,949) of wild-type germarium (D), and those in region 2A of wild-type and Fis1 knockdown germarium (n = 3,509; E). The mean voxel intensity was applied to filter out the light-stained objects, and only the densely stained objects were included in the analyses.