Time-lapse in vivo imaging of HSCs and exp.HSCs. (A-F) DiD-labeled (A-C) HSCs or (D-F) exp.HSCs were injected into col2.3GFP mice, and 16 to 20 hours later they were imaged every 5 minutes for 5 hours. Shown are representative xyz views of 3-dimensional stacks at the time points indicated, showing a stationary HSC and a migratory exp.HSC. Red, DiD-labeled HSC/exp.HSC; green, osteoblasts. Asterisks and dotted lines in E and F indicate exp.HSC position in previous frame shown and 2-dimensional displacement. Scale bars: 30 µm. (G-H) Gunplot 4.4 software was used to obtain a 3-dimensional representation of (G) HSC and (H) exp.HSC tracks, arranged to overlay the initial point of each track. (I) Proportion ± SE of migratory HSCs (n = 26 pooled from 3 mice containing 8, 8, and 10 cells each) and exp.HSCs (n = 46 pooled from 4 mice containing 7, 8, 13, and 18 cells each) observed (Fisher exact tests, P < .001). (J-L) Analysis of the characteristics of the movement of all HSCs and exp.HSCs observed (n = 26 and 46, respectively). Each dot represents 1 track and shows its (J) displacement (net distance covered from beginning to end of track), (K) mean speed, and (L) linear progression (ratio of displacement and total track length). Red lines indicate the median and the interquartile range (ie, intermediate 50% of values). One off-scale value for displacement (197.215 μm) and mean speed (4.005 μm/min), belonging to the same cell, was not included in the graphs, but was included in the statistical analysis. Differences in the median of these characteristics were tested using pairwise exact Mann-Whitney U-tests with the following results: (J) P = .39; (K) P = .56; and (L) P = .007. Additionally, differences in the variability of these characteristics were tested using the Brown-Forsyth F test: (J) P = .02; (K) P = .04; and (L) P = .02; P ≤ .05 for differences in proportions and medians (but not variance) is indicated by black lines.