3D analysis of mitochondrial fission stages during schizogony. (A) 3D visualization of mitochondrial segmentations based on thresholding of the mito-mScarlet signal in Arivis image analysis software. Smaller images in the top row are a single slice of the Z-stack with anti-GAP45 labeling (IMC, green), DAPI (DNA, blue) and mito-mScarlet (magenta), and a maximum intensity projection of the mito-mScarlet signal. The larger bottom picture is a 3D visualization of the segmented mitochondrial signal. The color of the mitochondrial fragment represents the size of this fragment, as is shown in the color bar at the bottom. Two representative parasites are depicted for each of the four segmentation stages defined in Fig. 4. Scale bars, 2 μm. (B) Boxplot indicating the number of nuclei per parasite in the different segmentation stages. Two-sided t test was performed; **, P < 0.01; ***, P < 0.001. (C) Boxplot indicating the number of mitochondrial fragments per parasite in the different segmentation stages. Two-sided t test was performed; ***, P < 0.001. (D) Boxplot indicating the size of the mitochondrial fragments in the different segmentation stages. A total of 40 schizonts were analyzed, pre-segmentation (n = 6), early-segmentation (n = 9), mid-segmentation (n = 15), and late-segmentation (n = 10).