Figure 3. Lateral diffusion of stx::pHGFP coupled to QDs. A, Examples of trajectories of stx::pHGFP coupled to QDs. The motion can be Brownian (Ai), confined (Aii), or display a pause (Aiii and see text). The gray regions on Aii and Aiii correspond to synapses identified by FM4-64 labeling. Scale bar, 300 nm. B, Average MSD (±SEM) for synaptic (red) and extrasynaptic (blue) trajectories, and for pauses (black). C, Cumulative frequencies of the diffusion coefficients. The color code is the same as in B. The vertical dotted line indicates the threshold for quantum dots considered immobile, as measured on blank coverslips. D, Characteristic length of confinement at synapses (conf) and diameter of the area explored during pauses (median and 25–75% IQR, whiskers: 5 and 95% confidence limits). KS test, **p < 0.01. E, Frequencies (mean ± SEM) of the long, truncated (trunc), and short pauses, at synapses (red) or extrasynaptic regions (blue). Two-tailed Student's t test, ***p < 0.001. F, Example of a “subcompartmental” trajectory. Fi, Trajectory of a synaptic pause. Scale bar, 100 nm. Fii, Two Gaussian clusters of positions were identified by a Gaussian mixture analysis (see Materials and Methods) and are shown here in orange and green. Fiii, Distance of the QD to the center of each cluster (identified in Fii) as a function of time (cluster 1, orange; cluster 2, green). At first, the distance of the QD to the center of cluster 1 is very small compared with that of cluster 2, indicating that QD diffuses within cluster 1. Then, the QD moves to cluster 2 (arrow), as shown by the short distance to cluster 2 relative to cluster 1. Finally, it moves back to cluster 1 (arrow).