High-speed imaging reveals corralled GPCR diffusion. (A) A still from a Rhoi3S tracking experiment acquired at 200 fps. (B) Time sequence of the positions of the GPCR tracked in B during the indicated experimental time frame, shown on expanded scale. Black lines represent the boundaries of the cilium. The bottom right plot represents the positions of a Qdot-labeled Rhoi3S fixed with glutaraldehyde (Fig. S1). (C) MSD(τ) analysis (symbols). Black lines represent linear regressions of two plot phases: P₁, the first two points; P₂, τ = 0.1–0.5 s. The slope of P₁ represents diffusion that is least impacted by corral confinement, and that of P₂ represents diffusion that is substantially impeded by the corral boundaries. Green dashed line represents Eq. 1 prediction based on the diffusion coefficient estimated from the P₁ slope. (D) Log–log plot of the results from E. Note that the Eq. 1 prediction is linear, whereas the tracking results have several phases. In A–D, results are representative from a single tracking experiment. (E) Mean diffusion coefficients computed from the slopes of the two phases. Error bars are SEM. n = 18 cells/18 individual cilia obtained in seven individual experiments (Table 2).