STORM visualizes distinct actin nanostructures at induced presynapses. (A) Widefield fluorescence image of cultured neurons 2 d after bead seeding at 8 div, labeled for ß2-spectrin (purple), synapsin (orange), and actin (gray). (B) Zooms on a natural synapse at an axon–dendrite contact (top row) and on an induced presynapse at an axon–bead contact (bottom row), corresponding to the highlighted areas in A. Left columns are widefield images of actin (gray), middle channels are overlays of widefield images of ß2-spectrin (purple), synapsin (orange), and actin (gray); right columns are overlays of widefield images of ß2-spectrin (purple) and synapsin (orange) with the STORM image of actin (gray). (C) Zoomed STORM images of the natural synapse (left) and bead-induced presynapse (right). (D) Same image as C, left, with color highlighting of presynaptic actin structures: active zone mesh (red), actin rails (green), perisynaptic corral (blue). (E) Additional representative images of bead-induced presynapses. The first column shows the widefield image of ß2-spectrin (purple), synapsin (orange), and actin (gray). The second column is a zoom of the highlighted area in the first image. The third column shows an overlay of widefield images of ß2-spectrin (purple) and synapsin (orange) with the STORM image of actin (gray). The fourth column is the isolated STORM image of actin (gray). The fifth column is the STORM image of actin with highlighted presynaptic actin structures: active zone mesh (red), actin rails (green), and perisynaptic corral (blue). Scale bars in A, 10 µm; in B–D, 1 µm; in E, 5 µm (first column) and 1 µm (other columns).