Extended Data Fig. 1: Workflow for SBF-SEM analysis and 3D reconstruction.
(a) Worms were frozen with a high-pressure freezing machine (Leica EM ICE) and then freeze-substituted in a Leica EM AFS2 system using osmium-thiocarbohydrazide-osmium (OTO). After positioning the targeted worms with a Versa 510 X-Ray microscope (XRM-510, Zeiss), the samples were trimmed and mounted on SBF-SEM pin. The specimens were sputter coated with gold-palladium and imaged using a Gemini scanning electron microscope (Zeiss) equipped with a 3View2XP and OnPoint backscatter detector (Gatan). (b) Serial SBF-SEM images were segmented using Amira software to reconstruct 3D models of plasma membrane (blue), mitochondria (green), MOs (orange) and nucleus (yellow) of spermatids and mitophers (bright blue). (c-e) The SBF-SEM images and bar graph revealing that individual mitophers contain a single mitochondrion. The EM image displayed in (c) denotes four boxed regions, indicating the locations with mitophers. An enlargement of the boxed region in (c) is presented in (d), which depicts three mitophers located in the extracellular space of spermatids in the male germline, and only one single mitochondrion was observed in each of these three mitophers (d). The mitochondrial number of each mitopher was evaluated through 3D reconstruction of the EM images, and a total of 311 mitophers from three males were quantified (e). For all of these mitophers, each of them carries only one single mitochondrion (e). Source numerical data in (e) are available in source data.
