SIM reveals intracellular 3D distributions of individual actin filaments and vesicles labeled with GFP-synaptophysin in growth cones. (A) Left, F-actin (magenta) and GFP-synaptophysin (GFP-Syp; green) in a glutaraldehyde-fixed growth cone of a neuroblastoma NG108–15 cell. Right, enlarged image of the region marked by the dashed box on the left. The focus plane on the right was 330 nm higher than on the left, which focuses on the bottom of the growth cone. The GFP-Syp signals were detected in the vicinity of the filopodia (arrows in the left panel) and actin bundles (arrowheads in the right panel). (B) Color maps repsenting the height of F-actin from the substrate, in the growth cone shown in (A). Note that the central (C-) domain is obviously thicker than the peripheral (P-) domain. Most actin bundles are red in the C-domain, indicating that they are farther from the substrate. The color/height gradually decreases from the C-domain to the leading edge, suggesting that F-actin bundles are distributed along the dorsal/apical surface, but not along the basal surface, in the growth cone (Nozumi and Igarashi, 2017) (C) Color maps representing the height of GFP-Syp from the substrate in the growth cone shown in (A). By comparing the color map of GFP-Syp with that of F-actin, it is possible to determine which Syp-positive vesicles were close to F-actin bundles, which side of the bundle they were on, and what shape they have (arrowheads in the right panel). (D) SIM images of GFP-Syp accumulation coinciding with F-actin bundling (arrows). The 3D-SIM and live imaging showed Syp assembled in the vicinity of F-actin bundles at the leading edge (Nozumi et al., 2017). Arrowheads: three actin bundles forming into a thicker filopodium.