Figure 5.

SMLM Super-resolution Imaging of Synaptic Gephyrin Clusters

(A) Single molecule detections (shown as black dots, or blue in the zoomed images) of recombinant mEos4b-gephyrin expressed in spinal cord neurons in control condition (Ctr) or after forskolin application (For). Arrowheads indicate the positions of small, non-synaptic clusters arising from the repetitive detection of single mEos4b fluorophores. Dense synaptic clusters were identified based on cluster size and detection number in the pointillist images (yellow areas in the zoomed images, see Methods). Clusters with less than 200 detections were not considered (green areas). Scale bar, 1 μm; insert, 100 nm.

(B) Quantification of synaptic cluster areas, detection numbers, and detection densities (n_Ctr = 1,237, n_For = 894 clusters from 32 cells per condition and 3 independent experiments, *p < 0.05, MW test).

(C) dSTORM imaging of endogenous gephyrin labeled with mAb7a and secondary Alexa Fluor 647-coupled antibodies. Cluster analysis was done as in (A), with a lower threshold of 50 detections per cluster. Scale bar, 1 μm; insert, 100 nm.

(D) Quantification of the cluster area and the number of Alexa 647 detections shows a correlated decrease in the forskolin-treated neurons compared with control cells (n_Ctr = 8,162 clusters in 69 cells, n_For = 7,010 clusters in 68 cells, 3 experiments, ***p < 0.001, MW test). Data are shown as 10%, 25%, 50%, 75%, and 90% percentiles and the mean (cross).