Fig. 1. Analysis of clathrin dynamics in a migrating cell by TIR-FM. (A) A TIR-FM still image (from a sequence taken at 300 milliseconds per frame) demonstrating the presence of numerous dsRed-clathrin basal plasma membrane associated puncta. Scale bar, 5 µm. (B) 12 sequential images taken from an image stream of 200, enlarging region 1 from A. The frames depict the lateral movement of the clathrin punctum marked by the arrowhead. (C) 12 sequential images enlarging region 2 from A. These images depict the disappearance of the clathrin punctum marked by the arrowhead, presumably via internalization. (D) 18 sequential images enlarging region 3 from A, demonstrating the increasing fluorescence at the spot marked by the arrowhead. (E) Quantification of the fluorescence associated with a single disappearing dsRed-clathrin punctum.