Virtual cell model used to simulate video data and compare single-molecule mobility in different cell regions. (A) Single fluorescent molecules were simulated, moving randomly (D_lat = 0.6 µm² ⋅ s⁻¹) on the surface of a cubic cell (3 × 3 × 3 µm³), which has a cylindrical protrusions 3 µm long. TIRFM imaging conditions, 50 fps, average fluorophore intensity 20 counts ⋅ pixel⁻¹ ⋅ frame⁻¹. The image shows the mobility map (initial slope of the MSD versus dT) for each of the tracked objects (n = 4,320) plotted on top of one another at the optical resolution limit of 250 nm (see Materials and methods). Note that the mobility is lower in the narrow cellular protrusions. (B) Super-resolution (∼10 nm) single-molecule trajectories form a meshwork of thin white lines. Notice that the diameters of the cellular protrusions are faithfully reproduced by the stop-motion, super-resolution tracking procedure (500 nm, 200 nm, and 100 nm). (C) MSD versus dT plots for different regions of the simulated cell. The numbers (1, 2, 3, 4) correspond to the cellular regions shown in A and B.