Trafficking from membrane to cytoplasm. (A) A representative trajectory of EGFR trafficking from cell membrane to cytoplasm is shown. The green, blue, and red colors represent confined, Brownian, and directed diffusion, respectively. The dark red arrow marks the starting point of the trajectory. The fractions of free diffusion time, confinement time, and active transport time were 24.4%, 73.4%, and 2.2%, respectively. The trajectory can be divided into four phases by visual inspection. (B) Time traces of the three motion classification parameters (α, Δϕ, Λ) is shown. Corresponding periods of the four phases are color-coded in these time traces. (C) Zoom-in views of the four phases, which clearly show the differences in the receptor motion in these phases. In this case, EGFR complex traveled 8.17 μm into the cytoplasm at phase III. (D) Time trace of instantaneous velocity (Vi) is shown. A small peak was noted around 273 s in phase II (inset a) and a large peak took place around 307 s in phase III (inset b), which mark the onset of internalization and the motor-mediated transport, respectively. (E) Histograms of logD0 in phases I–III are provided. (F) Histograms of logVi in phases I–III are provided. The values shown in (E) and (F) represent the means and standard deviations from Gaussian mixture model fitting. The logD0 and logVi of phase III clearly show two distributions. These two distributions are indicated with dotted line and dashed line, respectively. To see this figure in color, go online.