dSTORM

Primary mouse hippocampal culturing and fixation of wild-type and AKAP150 CS mice is described above and in detail in Purkey et al. (2018). Neurons were labeled with primary antibodies: rabbit anti-AKAP150 (Brandao et al., 2012; 1:1,000) and mouse anti-PSD95 (Millipore; 1:500). After incubation at room temperature, samples were incubated with secondary antibodies: goat anti-rabbit-CF568, 1:500 and goat anti-mouse-Alexa 647, 1:500 (Rockland, Pottstown, PA, United States).

dSTORM imaging, processing, and analysis has been described in detail (Gookin et al., 2022). Briefly, samples were imaged in a standard dSTORM buffer and imaging was performed on a Zeiss Elyra P.1 TIRF microscope using a Zeiss alpha Plan Apochromat TIRF 63x/1.46 NA oil objective and a tube lens providing an extra factor of 1.6x magnification. Alexa647 and CF568 dyes were imaged in two sequential time-series of approximately 10,000 frames (50 ms integration time) each. Image size was 256 × 256 pixels (pixel size 160 nm). Dark-state conversion and imaging was done using a 100 mW 642 laser (LP 655 emission filter) and a 200 mW 561 laser (BP 570-650 + LP 750 filter). An additional MBS (405/488/561/642) filter was placed in from of the camera, an Andor iXon + 897 EMCCD.

Raw data was processed through a custom written pipeline written in MATLAB (Mathworks, Natick, MA, United States) made up of a number of modular elements: if necessary, the time-series was pre-processed with a temporal filter (Hoogendoorn et al., 2014). Localization of dye emitters was performed using the ThunderSTORM ImageJ plugin (Ovesny et al., 2014). Registration and drift-correction was based on the positions of fiducial beads (Tetraspeck, Thermo Fisher Scientific).

Our coordinate analysis of dSTORM localizations is conceptually similar to methods previously used to classify nanoscale organization at excitatory synapses (Tang et al., 2016), using a local-density calculation, high-density regions (HDRs) were defined by a cutoff determined by randomizing the experimental localizations assuming a uniform distribution across the synaptic region, set at 2.5 standard deviations above the mean of the randomized dataset.