Postsynaptic actin impedes the visualization of presynaptic content and organization in hippocampal neurons, even using state-of-the-art super-resolution microscopy. (A–C) Images of neurons after 9 d in vitro (div), stained for synaptophysin (purple), map2 (blue), ß2-spectrin (purple), and actin (gray). (A) Widefield image. (B) Zooms from the highlighted area in A, overlaying the widefield image of synaptophysin, map2, and ß2-spectrin with the STORM image of actin. (C) Zooms from the highlighted area in B, with the top row showing overlayed widefield image of synaptophysin and map2 with the STORM image of actin (gray) and bottom showing the isolated actin STORM image with axon shaft highlighted in orange dashed lines. (D–F) Images of neurons after 9 div, stained for VGAT (green), VGLUT (orange), ß2-spectrin (purple), and actin (gray). (C) Widefield image. (D) Widefield image. (E) Zoom from the highlighted area in D, overlaying widefield images of VGAT, VGLUT, and ß2-spectrin with the STORM image of actin. F, zooms from the highlighted areas in E, with the top row showing overlayed widefield image of VGAT and VGLUT with the STORM image of actin (gray) and the bottom showing the isolated actin STORM image with axon shaft highlighted in orange dashed lines. Scale bars for A and D, 10 µm; for B and E, 2 µm; for C and F, 0.5 µm.