Fast FRAP analysis. (A–A′′′) Time-lapse imaging of COS-7 cells transiently expressing GFP–CLIP-170 (every 10th frame is shown). Two GFP–CLIP-170–labeled MT ends are indicated by arrows. In A, one of these is about to traverse a 210 × 210-nm ROI (red rectangles). After 2.25 s (A′′′), this MT end has traversed the ROI. (B) Fluorescence intensity in an ROI of 210 × 210 nm as a GFP–CLIP-170–labeled MT end traverses. (C and G) Mean fluorescence decay of nonbleached, GFP–CLIP-170-labeled (C) or EB3-GFP–labeled (G) MT ends (k_decay is indicated). (D–D′′′) COS-7 cells transiently expressing GFP–CLIP-170 were imaged as in A. An area of 256 × 3 pixels (indicated in D′) was bleached every 7.5 s, occasionally resulting in bleaching of MT end–bound GFP–CLIP-170. Rectangles 1–4 are shown enlarged underneath D–D′′′. Fluorescence intensity was measured in ROIs of 210 × 210 nm (red rectangles). (E) Fluorescence intensity of a bleached GFP–CLIP-170–labeled MT end (black arrow indicates bleach; black line indicates mean fluorescence decay). (F and H) Mean fluorescence recovery of GFP–CLIP-170 (F) and of EB3-GFP (H) on MT ends (k_recovery is indicated).