(a) The average mean square displacement (MSD) of Fyn-mEos2 trajectories from individual spines of Tau KO neurons expressing TauWT-GFP (n = 76 spines) or TauP301L-GFP (n = 124 spines). (b) The effective diffusion coefficient computed from the MSD of individual spines. (c) Distribution of the moment scaling spectrum slope (SMSS) of trajectories lasting for at least 20 frames (n = 2282 trajectories from the spines of 11 Tau KO neurons expressing TauWT-GFP and n = 3764 trajectories from the spines of 9 Tau KO neurons expressing TauP301L-GFP). (d) A three-state model showing the inferred apparent diffusion coefficients and state occupancies (state S1, immobile; state S2, confined; state S3, apparently free), with the circle area being proportional to the state occupancy. (e) Comparison of the estimated state occupancies. (f, g) Comparison of average nanodomain diameter (f) and area (g). (h) Comparison of the number of nanodomains per spine. Statistical comparisons were performed using the Kruskal-Wallis test (b), the Kolmogorov-Smirnov test (c) or Student’s t-test (e–h).