Handling nanoscale molecular information: Illustration by Visual Molecular Dynamics and data analysis by VividSTORM. (A) STORM super-resolution imaging generated localization points, which represent CB₁ cannabinoid receptors visualized by immunofluorescence staining. These localizations all belong to a single GABAergic axon terminal imaged in the CA1 subfield of the hippocampus. Note that other CB₁ receptor-representing localizations belonging to neighboring cellular and subcellular profiles have been excluded from the image for clarity (see Fig. 4 for further details). The individual molecular localizations are illustrated as atoms with the help of the Visual Molecular Dynamics (VMD) software. The color depth encodes the z position of the receptor. All subsequent data analysis were performed by the VividSTORM software. (B) A convex hull was fitted onto the outermost STORM localizations (which become vertices) to approximate the plasma membrane of the axon terminals by exploiting the fact that CB₁ receptors are densely and homogeneously cover the surface of GABAergic boutons. Chemical bonds in VMD are used to illustrate the convex hull edges. (C) The distance of each receptor localizations can be measured from the convex hull faces. Yellow and magenta STORM localizations separate CB₁ receptors along the surface or below the convex hull (80 nm). (D) Receptor density analysis is performed by selecting CB₁ STORM localizations (yellow) and measuring randomly distributed reference points (little green dots) within a certain radius (large yellow sphere) of respective CB₁ localizations. (E) Yellow STORM localizations depict the CB₁ localizations constructing the convex hull. A single presynaptic active zone is visualized by STORM imaging of bassoon-immunostaining (cyan STORM localizations). Because bassoon is located ∼80 nm below the plasma membrane, the STORM localizations representing bassoon coordinates are projected onto the convex hull surface (red STORM localizations) to estimate the active zone position on the axon terminal membrane. (F) Orange lines running along the surface of the convex hull link CB₁ receptors (yellow STORM localizations) with the closest localization on the active zone (red STORM localizations) providing an estimation of the nanoscale distance between the regulatory receptors and the location of their molecular effectors known to be present within the active zone. (G) CB₁-containing GABAergic interneuron terminals form complex synapses and therefore carry multiple active zones. Visualization of all bassoon localizations (cyan STORM localizations) indeed suggest the presence of more than one bassoon cluster. (H) In this particular bouton, 4 bassoon clusters (cyan, pink, orange and red localizations) were identified by DBSCAN, whereas other bassoon localizations identified as noise are depicted as magenta dots. Scale bar is 200 nm; STORM localizations are shown as 17 nm spheres.