Figure 4.

Slower and more confined diffusion of single receptors correlates with regions of high receptor density. (A) Examples of single-molecule trajectories are shown from the same cell in Fig. 1, A and B, Fig. 2, A and B, and Fig. 4C recorded before and after antigen stimulation. The tracks shown persist for at least 0.5 s for the −1 min (unstimulated) time point and for 1 s for other time points. (B) Short-time diffusion coefficients (D_S) are evaluated from MSD curves tabulated from single-molecule trajectories lasting at least 0.5 s within a 16 s time period and are assembled into histograms. Histograms are normalized by the total number of tracks collected to generate each histogram. (C) Single-molecule trajectories persisting for at least 0.5 s are superimposed on a superresolution image reconstructed from unstimulated data (upper), from data acquired within 1 min of antigen addition (middle), and from data acquired after several minutes of stimulation. Track coloring indicates D_S for each track on a log scale from 10⁻⁵μm²/s (blue) to 1 μm²/s (red). Images on the right are enlargements of the boxed regions in the images on the left. (D) Three-dimensional histograms of D_S versus average receptor density along trajectories lasting at least 0.5 s. The average receptor density for each trajectory is determined by averaging the pixelated grayscale values from the time-averaged reconstructed image over all positions of the trajectory and then normalizing assuming ρ_ave = 200/μm², as described in Materials and Methods in the Supporting Material.