A–CAlignment of SSDs of pre‐synaptic RIM1/2 and SSDs of post‐synaptic gephyrin, GlyRs, and GABAARs, respectively. (A1–C1) Distances were measured between the intensity peaks of paired SSDs in rendered dual‐color dSTORM images (yellow line, values given in mean ± SD). RIM1/2 was labeled with Alexa 647 and gephyrin (mAb7a) with Cy3B in (A1). RIM1/2 was labeled with Cy3B, while GlyRs or GABAARs were labeled with Alexa 647 in (B1) and (C1). (A2–C2) Trans‐synaptic alignment of SSDs of gephyrin, GlyRs, GABAARs, and RIM1/2. SSDs were segmented by H‐watershed. Data are plotted as mean ± SD. Number of synapses: n = 48 (A1, A2) from three independent experiments (no treatment), n = 30 (B1, B2) from two experiments with TTX treatment, n = 16 (C1, C2) from two experiments with TTX. Only synapses with side view profiles were included. All images are adjusted to the same scale. Scale bar: 200 nm.