(a) To assess whether the local density of STORM labeling on axon terminals is within the dynamic range of the measurement, 10-10 ROIs (30 by 30 confocal pixels each; pixel size = 0.0064 μm2) were placed on the plasma membrane of HEK cells and biocytin-filled axon terminals to measure labeling density. Comparison of the labeling density did not reveal significant difference between boutons of perisomatic and dendritic interneurons (n = 6 cells/cell type, Mann-Whitney U test, p = 0.59, U = 14), and the vast majority of boutons fell within the range defined by the two HEK cells with the highest and lowest expression levels. Graphs show raw data and median±IQR. (b) The NLP values of boutons were averaged for each interneuron, and the mean±SD range of NLP density for interneurons was determined. This range well represents the STORM CB 1 labeling density found on GABAergic axon terminals, as it includes 10 of the 12 interneurons. HEK cells (n = 20) within this range show highly linear positive correlation between intrinsic fluorescence intensity and NLP (Spearman's rank order correlation, p < 0.0001). (c) The distribution of local densities (neighboring localizations within 50 nm 3D distance from each localization point) on the boutons from (a) shows a heavy right tail indicating that the signal is not likely to be saturated. (d) The different size of perisomatic and dendritic boutons is evident regardless of the duration of the imaging. Using the same data set as in Figure 3, the perimeter of a 2D-convex hull fitted on the localization points detected before the given period of imaging was measured, and was plotted as the ratio of the perimeter of dendritic axon terminals over perisomatic boutons (median±IQR of the mean values of 6 cells per cell type is shown). The difference was significant at every time point after 250 cycles (n = 6 cells/cell type, Mann-Whitney U test, p = 0.015, U = 3). (e) Similarly, the finding that axon terminals of dendritic cells had less CB 1 receptors than perisomatic boutons was also independent of the imaging duration after 350 cycles, as the difference was significant at every time point afterwards (n = 6 cells/cell type, Mann-Whitney U test, p = 0.041, U = 5). The same approach was used to compare the number of CB 1 localization points per bouton between interneuron types. (f) The variance of nearest-neighbor distances between CB 1 localization points within boutons was constant in both cell types after 400 cycles into imaging, indicating that running the imaging for up to 1000 cycles for each image provides reliable measurement of CB 1 distribution within GABAergic axon terminals.