Intravital imaging was performed in the footpad of LysM-eGFP mice 20 min after infection with Listeria monocytogenes (LM). (A) Image sequences illustrate typical neutrophil migration patterns (multi-colored dots). Cell centroids were tracked at successive 42 sec intervals using PicViewer [10], [26]. Blood vessels (red) were labeled by i.v. injection of 1 mg dextran-tetramethylrhodamine 2,000,000 MW 5 min prior to imaging. Neutrophils (eGFP) appear green and collagen fibers in the connective tissue (second harmonic generation signal) appear blue. Scale bar = 20 µm. (B) The plot shows the mean displacement of 50 neutrophil tracks (containing a minimum of 9 and a maximum of 12 time points) from five separate experiments. Error bars represent standard error of the mean (SEM). Neutrophil displacement over time is linear (R² = 0.9959), and slope of the regression line (7.5235 µm/min) yields the interstitial transit rate, a convenient measure of how rapidly the cells migrate through the specific local tissue environment. (C) The Plot shows the median track velocity (8.288 µm/min+/−SD 2.256 µm/min) of the tracks in (B). (D) The meandering index calculated for each track was plotted. This value was obtained by dividing the displacement from the origin (after 8 time steps or 5.6 min) by the track length. The median meandering index equals 0.7583+/−SD of 0.175, which indicates migration has a strong directional bias. (E) The turning angles of neutrophil tracks were determined by calculating the angle change between vectors constructed from each time point (42 sec intervals). Angles are given in absolute values calculated by using the arc sine of the dot product of the two normalized vectors.

Adoptively transferred Vav^NULL (green; CFSE labeled) and WT (red; CMTPX-labeled) neutrophils were imaged by time-lapse 2PM. Intravital imaging was performed in the footpad of WT mice 20 min after infection with LM. Data compiled from four separate experiments is shown. In 2 experiments, dye staining was reversed to exclude potential dye-induced changes in cell motility. (A) Image sequences illustrate typical neutrophil migration patterns (green-dots for Vav^NULL, red-dots for WT neutrophils). Cell centroids were tracked at successive 41 sec intervals using PicViewer. Blood vessels (orange) were labeled by i.v. injection of 1 mg dextran-tetramethylrhodamine 2,000,000 MW 5 min prior to imaging. Neutrophils appear green and reddish respectively and collagen fibers in the connective tissue (second harmonic generation signal) appear blue. Scale bar = 30 µm. (B) The histogram shows the instantaneous velocity distribution of WT neutrophils. The distribution has a bell-shaped symmetrical character with a median velocity of 7.81 µm/min (+/−SD 4.42 µm/min). (C) In contrast to WT neutrophils, the instantaneous velocity distribution of Vav^NULL neutrophils is skewed with a median velocity of 5.22 µm/min (+/−SD 4.31 µm/min). (D) The plot shows the mean displacement of 30 WT and 26 Vav^NULL neutrophil tracks. Error bars represent standard error of the mean (SEM). Neutrophil displacement over time is linear for wild type (R² = 0.997) and Vav^NULL neutrophil tracks (R² = 0.9823). The slope of the regression line yields the interstitial transit rate of 6.09 µm/min for WT neutrophils and 4.19 µm/min for Vav^NULL neutrophils. (E) The Plot shows median track velocities of tracks in (B). The average median track velocity is statistically different (p value = 0.0001) between WT neutrophils (7.261 µm/min+/−SD 2.210 µm/min) and Vav^NULL neutrophils (4.686 µm/min+/−SD 1.738 µm/min). (F) The meandering index was calculated by dividing the cell's displacement from the origin (after 8 time steps or 5.6 min) by the track length. Meandering values for individual tracks were plotted. No statistical differences (p value = 0.525) were observed between WT neutrophils (0.7946+/−SD of 0.156) and Vav^NULL neutrophils (0.7675+/−SD of 0.162). (G) The cell shape index for WT and Vav^NULL neutrophils was calculated as the ratio of the cell's length (from the leading edge to uropod) to the cell's width (taken perpendicular to the axis of migration). WT cells had significantly (p value = 0.0245) more elongated shapes (shape index = 2.266+/−SD 0.51) compared to Vav^NULL neutrophils (shape index = 1.801+/−SD 0.76). Comparisons were made using CFSE-stained WT and Vav^NULL cells.