(A–E) Representative confocal (left), STED super-resolution images (center), and power spectral density (PSD) profiles (right) of TSMod::β-spectrin (A) and α-spectrin SPC-1::GFP (B–E) in C. elegans axons in vitro. In panels (A–E), yellow (β-spectrin) and red (α-spectrin) traces show intensity profiles derived from STED images. Scale bar = 1 µm. A single PSD curve (gray) and Lorentzian fit (black) is shown for each STED image. Cells were dissociated from transgenic embryos and cultured on glass coverslips for three days. Similar images were obtained from at least 20 axons examined per condition (genotype, label, treatment) from at least three biological replicates. (A, B) Spectrin in control axons (left, center) and PSD showing strong peaks at 204 and 207 nm for TSMod::β-spectrin and α-spectrin::GFP, respectively. (C) SPC-1::GFP in control neurons treated with the actin depolymerizing agent, Latrunculin A (1 µM) (left, center). The PSD lacks any strong peaks, indicating that this treatment disrupts spectrin periodicity. (D) SPC-1::GFP in unc-70(s1502) β-spectrin null mutant (left, center). As in panel C, the PSD (right) lacks any clear peaks. (E) SPC-1::GFP in unc-70(e524) β-spectrin missense mutant. The PSD has a single strong peak at 185 nm. (F) Average distance between adjacent spectrin labels for TSMod::β-spectrin (top, n = 27) in control neurons; α-spectrin::GFP (middle, n = 31) in control neurons; and SPC-1::GFP (bottom, n = 20) in unc-70(e524). Spacing computed as the inverse distance of the dominant peak in PSD.