The analysis was performed at days in vitro (DIV)11–15 and DIV19–20. (a–d) Representative epifluorescence images of DIV12 (a, c) and DIV20 (b, d) neurons co-expressing mCardinal (a, b) and Fyn-mEos2 (c, d), acquired before photoconversion of mEos2 molecules. Insets are shown at a higher magnification. Scale bars, 1 μm. (e–j) sptPALM imaging was performed at 50 Hz for 320 s (16,000 frames) to construct the maps of the localization intensities (e, f), diffusion coefficients (g, h) and trajectories (i, j) of Fyn-mEos2 molecules. The cooler colors represent higher localization intensities (e, f) and larger diffusion coefficients (g, h), and each trajectory is coded with a different color (i, j). (k–n) Comparison of Fyn mobility parameters with development. (k) Average mean square displacement (MSD) as a function of time. (l) The corresponding area under the MSD curves (AUC). (m) Distribution of diffusion coefficients (D) shown in a semi-log plot. The threshold to distinguish the immobile (Log10[D]≤−1.6) and the mobile (Log10[D]>−1.6) fraction of molecules is indicated with a dashed line. (n) The corresponding immobile fraction. Error bars are standard errors of the mean (SEM). Mean ± SEM values were obtained from n = 31 neurons (DIV11–15) and n = 14 neurons (DIV19–20). Statistical comparisons were performed using the Mann-Whitney U test (l) and Student’s t-test (n).