Quantitative analysis of wound-induced epithelial sheet movement by PIV. (a) Representative example of PIV analysis (PIVlab, MATLAB) of epithelial sheet movement after UV puncture injury of a Tg(krt4:AKT-PH-GFP) larva exhibiting plasma membrane labeling in the suprabasal layer. Green arrows, velocity vectors derived by comparing particle movements between subsequent frames. Red area, extra-tissue area excluded from analysis. Bar, 50 µm. (b) Global PIV analysis of UV-puncture wounded Tg(krt4:AKT-PH-GFP) larvae immersed in solutions of indicated composition (Y = Y27632, Rho-kinase inhibitor, 100 µM). The graph displays the mean of all velocity vector magnitudes within the unmasked field of view as a function of time after injury. (c) Spatial PIV analysis representing the Iso and Hypo datasets from b as spatially resolved 3D plots. (i and ii) Averaged spatial PIV plots of the indicated number of Tg(krt4:AKT-PH-GFP) larvae after UV-laser puncture injury in hypotonic or isotonic medium. Tissue velocities are color-coded (blue to green to yellow to red), and represented as a function of time after injury (x axis) and distance from injury site (y axis). (iii) Differential plot derived by subtraction of the indicated velocimetry plots, and t test filtering of statistically significant differences between groups (unpaired t test, P < 0.05 = significant difference). Statistically significant velocity differences are color-coded (turquoise to pink). Pink, positive values. Turquoise, negative values.