Figure 2.

Antigen stimulation leads to slower and more confined diffusion of IgE-FcεRI receptor complexes. (A) Single-molecule trajectories of IgE-FcεRI complexes on the surface of cells under TIRF illumination before (−Ag) and after (+Ag) stimulation with 1 μg/mL DNP-BSA for 5 min. Tracks shown are accumulated over 1 min, only tracks observed for five or more frames (0.16 s) are displayed, and coloring from blue to red indicates the relative time at which a single probe was observed within the 1 min time frame. (B) MSD curves are generated by averaging over all tracks observed within a 500 frame (16 s) time period at the times during antigen stimulation indicated, as described in Materials and Methods in the Supporting Material. MSD curves are fit to Eqs. 3A and 3B to extract the short- and long-time diffusion coefficients D_S and D_L, respectively. (C and D) Summary of D_S (C) and D_L (D) extracted from MSD curves tabulated from single-molecule trajectories acquired over 500 frames (∼20 s and variable from cell to cell) for 11 distinct cells. Error bars represent the mean ± SE of the 11 live-cell experiments. (E) Confinement as a function of stimulation time, as measured by D_S/D_L from the same 11 live-cell experiments. Error bars represent the mean ± SE. To see this figure in color, go online.