(A) Experimental design: tracheas are explanted 24 hr post-sulfur dioxide (SO2) injury for continuous time-lapse imaging. (B) Two-photon imaging of trachea epithelium from membrane- GFP, KRT5-H2B-mCherry transgenic mouse. Top image is a stitch of three areas. Bottom image shows displacement vectors over 10 min computed using particle imaging velocimetry (PIV). Scale bar = 50 μm. (C) Computed speed of epithelial cells measured at different time points post-SO2 injury in 22 independent regions from a total of five mice at four different time points (mouse origin is color-coded). Box and whisker plots are superimposed. A two-way ANOVA was run to examine the effect of time post-SO2 injury and different mice on the mean speed determined by PIV. There were 22 ROIs analyzed from five mice over four time points. There was a significant interaction between time and the mean speed, F(2.219, 42.91) = 16.12, p<0.0001, but no significant difference between mouse and mean speed, F(4,17) = 2.193, p=0.113. A Tukey post-hoc test revealed significant pairwise differences between 26 and 50 hr, 26 and 62 hr, 38 and 50 hr, as well as 38 and 62 hr. **p<0.01. (D) Frequency distribution of injury-induced cell movements measured at 26- and 38 hr after injury identifies ‘mover’ and ‘non-mover’ regions.