Time-lapse microscopy of CSΔN and TRAP-VAL sporozoites 10 min after intradermal inoculation (see Video 2, Video 5, and Video 6). (A) Maximum intensity projection over 240 s reveals trajectories of WT, CSΔN, and TRAP-VAL sporozoites (green) moving in the dermis with CD31-labeled vascular endothelia (far red). Scale bars, 50 μm. For panels B–D, every data point represents one video and 18 WT, 25 CSΔN, and 24 TRAP-VAL videos were processed. (B) The time sporozoites spend in proximity to CD31-labeled vessels was quantified by manual tracking of sporozoites and is expressed as percent of motile time (left panel) or total observed time (right panel). Graphs show significant increase in the time CSΔN, but not TRAP-VAL sporozoites spend actively gliding on or around blood vessels. (C) To generate the MSD of sporozoites moving in proximity to CD31-labeled vessels, 313 WT, 352 CSΔN, and 192 TRAP-VAL tracks of sporozoites moving in proximity to CD31-labeled vessels were generated and compared to 163 WT, 121 CSΔN, and 89 TRAP-VAL tracks of sporozoites moving in avascular areas of the dermis. To calculate the MSD of the obtained sporozoite tracks with various duration times, all tracks were uniformly fragmented into tracks of 30 s in duration. MSD plot shows decrease in displacement of mutant sporozoite tracks in proximity to blood vessels. Inset shows slopes of linear regression fitting (all R2 values ≥0.988). (D) Apparent speed of wild-type control and mutant sporozoites in the proximity of blood vessels or in avascular portions of the dermis with lines representing mean values. Dashed line marks the mean value of wild-type control in avascular portions of the dermis. These data reveal a significant decrease in gliding speed of CSΔN and TRAP-VAL sporozoites, once the sporozoite is approaching vasculature, similar to wild-type control sporozoites. (E) Blood and lymphatic vessel invasion events were manually counted. Invasion was classified similar to previously descriptions (Amino et al., 2006), with blood vessel invasion being defined by a sudden increase in speed or visual entry of the blood vessel and disappearance out of the field of view. Lymphatic invasion was characterized by the switch from directed forward movement to sideward drifting with a low velocity (see Videos 7 and Video 8). The proportion of invading sporozoites as percent of total sporozoites in the field of view is shown. In the duration of the acquired 4 min videos, 2.38% and 2.06% of WT sporozoites invaded blood vessels and lymphatic vessels, respectively. This was in sharp contrast to the blood and lymphatic vessel invasion rates of CSΔN (0.23% and 0.26%) and TRAP-VAL (0.05% and 0%); n equals number of videos analyzed.