Video 3

Movie S3. Transient elevations in [Ca2+]i and migratory pauses in thymocytes at 2–3 h versus 12 h of positive selection. Preselection OT1 thymocytes were loaded with the ratiometric calcium indicator dye Indo1LR and allowed to migrate into selecting thymic slices for 2 h and imaged by two-photon microscopy at either 2–3 h (Upper) or 12 h (Lower). Frames were collected every 20 s for 20 min. Individual thymocytes were masked out using Imaris software. Low [Ca2+]i (green) and high [Ca2+]i (red). Graphs indicate the calcium ratio (black), speed (gray), and signaling events of the corresponding movies. Red lines indicate signaling events. Red arrows indicate the trigger events. Blue-shaded regions indicate non-signaling portions of the track during which the 100-s interval speed was >0.6 μm/min and the calcium ratio was <0.2 above the average ratio of the run. The horizontal lines indicate the cutoff for calcium trigger events (black dashed) and the speed cutoff for defining nonsignaling regions of the track (gray dashed).