Automated detection of periodicity at 190 nm via Stripefinder. (a) Single molecule localizations were generated using Fluosim from particles moving Brownian over the model geometries (top) to generate either “stripy” or random datasets. Middle top: Stripes are apparent in the stripy reconstructions of the localizations, absent for random datasets. Middle bottom: Autocorrelation function (ACF) reveals periodicity of ∼200 nm for stripy datasets and not for random datasets. Bottom: Localizations were analyzed using a Fourier transformation–based custom software (Stripefinder) to calculate a local stripe score that was lower, when no stripes were detected. (b) Stripefinder finds no stripes in localizations of GPI-GFP in CV-1 cells. GPI-GFP was tracked using anti-GFP-NB-conjugated QDs (20,000 frames, 200 Hz). (c) Stripefinder finds stripes in localizations of GPI-GFP in progenitor-derived neuronal cells. GPI-GFP was tracked using anti-GFP-NB-conjugated QDs (5,000 frames, 200 Hz). Scale bars are 500 nm.