Supplementary Figure 3: STED/sptPALM time-lapse imaging and STED laser effect on mEos3.2.

a) STED/sptPALM time-lapse imaging showing GluA1_mEos3.2 trajectories along the dendritic spines with inset showing zoom-in on spine, scale bar 1 µm, inset scale bar 500 nm, two representative images out of 6 independent experiments. b) STED/sptPALM time-lapse imaging showing PSD95_mEos3.2 trajectories along the dendritic spines with inset showing zoom-in on spine, scale bar 1 µm, inset scale bar 500 nm, two representative images out of 6 independent experiments. c) Decay of mEos3.2 during sptPALM/STED time-lapse experiments. Left: at constant 405 nm power without STED images in between (PALM bleaching). Middle: at constant 405 nm power, with STED images in between (PALM + STED bleaching). Right: modulating the 405 nm excitation power from 3 to 80 µW (measured at the sample plane) to compensate for mEos3.2 bleaching. Data points represent mean with SD, N = 5 cells, 2 different preparations. d) Fraction of mobile trajectories for GluA1_mEos3.2 and PSD95_mEos3.2 during three cycles, one-way ANOVA test, N = 12 cells, 6 cultures, N.S = not significant, * p = 0.01, p value), indicating that STED laser is not significantly affecting the dynamics of GluA1 and PSD95 molecules. e) Cumulative and normal distributions of GluA1_mEos3.2 during three cycles, N = 12 cells, 6 cultures. f) Cumulative and normal distributions of PSD95_mEos3.2 during three cycles, N = 12 cells, 6 cultures. g) Cumulative and normal distributions of PSD95_mEos3.2 diffusion coefficients without STED laser, N = 8 cells, 5 cultures. Data points in d, e, f and g represent mean with SEM.