Visualizing the dynamics of hemi-fusion, fusion pore opening and vesicular-content release with STED microscopy. (A–C) STED XZ images PH (green, labeling inner leaflet) and A532 (red, in the bath) (A, PH and A532 appear in the W-profile at the same time), PH-earlier (B, PH appears before A532, indicating hemi-fusion, and A532 appears later, indicating fusion pore has opened. (C) Ω-profile labeled with PH only, indicating hemi-fusion. Labeled times are relative to depol_1s onset. Cytosol, PM, bath, and coverslip location are labeled in A. (D) STED XZ images of PH and A532 taken at 50-nm intervals along the Y-axis, showing a W-profile with a visible pore (Pore_v). (E) Upper left, W-profile at Y=250 nm from panel D; upper right, the fluorescence profile of the dotted line across the pore with W_H (full-width-half-maximum) labelled. Lower, XY images reconstructed from panel D at a Z-focal plane across the pore (dotted line in upper panel). Inset, drawing of reconstructed plane (gray). (F) PH-labelled W-profile fluorescence (F_PH, normalized to baseline), A532 spot fluorescence (F₅₃₂), Porev WH, and sampled images at times indicated with lines showing a pore undergoing rapid opening and slow constriction. (G) Left, PH and dynamin 2-mTurquoise2 (Dyn2, pseudo-color) XZ-images at different Y-axis location showing dynamin surrounding the pore. Right, XY-reconstructed images across the pore (dotted line). (H–I) Pore-dynamics control content release: F_PH, F_FFN (FFN511 fluorescence), and PH/FFN511 images for Ω-profile with Pore_v (H) or without a visible pore (Pore_noV, I).