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Fig. 9 Dynamics properties of endogenous activated β2-AR and Gαs-coupled GPCR nanoclusters. (A–C) Individual trajectories of the single-particle tracking data of Nb80-mEos2 and Nb37-mEos2 were analyzed using HMM to infer mobility parameters. (A) A three-state model with the inferred apparent diffusion coefficients and state occupancies represented. Each state is illustrated by a colored circle (state 1, immobile, magenta; state 2, intermediate, green; state 3, fast mobile, blue), with the area proportional to the state occupancy. (B) Comparison of apparent diffusion coefficients of each state inferred from Nb80-mEos2 and Nb37-mEos2 trajectories. (C) Comparison of state occupancies inferred from Nb80-mEos2 and Nb37-mEos2 trajectories (n = 28 sets, 34 cells for Nb37-mEos2, n = 19 sets, 31 cells for Nb80-mEos2). Statistics of apparent diffusion coefficient and state occupancy were performed using a Student’s t test with Welch’s correction. (D–N) The dynamics of individual nanoclusters assessed using a combination of Voronoï-based cluster detection and tcPALM analyses in live PC12 cells. (D and J) Representative superresolution images of (D) Nb80-mEos2 and (J) Nb37-mEos2. (E and K) Voronoï diagrams of (E) Nb80-mEos2 and (K) Nb37-mEos2 detections. (F and L) Nanoclusters (black outline) detected by (F) Nb80-mEos2 and (L) Nb37-mEos2 were identified using Voronoï-based tessellation algorithm. (G) Nb80-mEos2 and (M) Nb37-mEos2 trajectories displaying immobile, intermediate, and fast motion patterns inferred using HMM. (H and N) Representative time series of detections from β2-AR nanoclusters detected using (H) Nb80-mEos2 and (N) Nb37-mEos2. (I and O) The distribution of apparent burst times of (I) Nb80-mEos2 and (O) Nb37-mEos2 nanoclusters.