Extended Data Fig. 2: Fluorescent signals indicate the mitochondrial, plasma membrane, MOs and nucleus appearance under ODT imaging.

(a-i) The 3D microscopic images presenting three different angles of the spermatid displayed in Fig. 2a(i)-(vi) (0.00 s). The images in (d-f) and (g-i) were obtained by rotating images in (a-c) 40-degrees clockwise and 184-degrees counterclockwise along the y-axis, respectively. The spermatids were marked by GFP::CATP-4 (green, plasma membrane) and MTDR (yellow, mitochondria). The rose dashed lines distinguish two separate spermatids. These images indicate that there were no extracellular structures nearby the spermatids before mitopherogenesis (0.00 s). (j-l) Images showing colocalization of MTDR (red) with the large, round, bright dots (white) under ODT imaging mode. The yellow arrows indicate several typical mitochondria. (m-o) Images demonstrating the colocalization of the nucleus-located spe-11p::mCherry::histone with the largest, round, and bright-white dots (in the center of the cell) observed in ODT images. Red arrow, nucleus. (p-r) Images displaying the cell shape of the spermatid captured by ODT imaging mode. The overall white signals within the spermatids detected by ODT imaging (q) depict the cell border, as demonstrated by the localization of GFP::CATP-4 (p). PM, plasma membrane. (s-u) Images showing that GFP::CATP-4-marked MOs colocalize with the lighter white signals in the spermatozoon head (u). After sperm activation, the GFP::CATP-4 signals localizes to the fused MOs as previously described²⁰. Yellow arrow, mitochondria; blue dashed lines, cell border; green arrow, MOs in focal plane; purple arrow, MOs off the focal plane. (v) A representative TEM image of a spermatozoon. Yellow arrow, mitochondria; red arrow, nucleus; blue dashed lines, cell border; green arrow, MOs in focal plane; purple arrow, MOs off the focal plane. (w,x) Images of the spermatid corresponding to Fig. 2f at Z-12 focal plane indicating that the budding mitopher and the cell body remain connected at 277.2 s (w) and 278.4 s (x). The yellow box highlights the region where the budding mitopher (blue arrow) connects with the spermatid, and the enlarged images are shown in the bottom left corner. Scale bar, 500 nm (inset). Three biologically independent experiments were performed in (j–u).