Quantitative analysis of clone size and location in 3D and motility in 4D. Volumetric 3D tiled stacks from the spleen shown in Figure 4A were analyzed in a quantitative manner. Cells were computationally extracted both individually (A) and as 32 clones spectrally distinct from neighboring ones (B) using semiautomatic user-interactive segmentation, combining channels and grouping in Imaris software. Distances of the individual clones to the splenic capsule were determined using distance transformation algorithm of Imaris XT. (C) Histogram depicting the distances of 32 clones to the surface of the spleen. Time sequences recorded in the lymph node 14 days after transplantation of Mix 5FPS Lin⁻ HSPCs using resonant scanner were transformed into 4D movies and analyzed using XT module of Imaris software (supplemental Video 16). (D) Example of cells computationally segmented as “spots” objects and the lymph node capsule as a “surface” object. Cells expressing tdTomato, EGFP, or Cerulean were represented by magenta (M), green (G), or white (W) spots, respectively. (E) Distances of individual cells/spots to the surface were plotted according to the FP expressed (M, G, W) for comparison (the lines show the mean). (F) Cell migration trajectories (tracks) over time and displacement vectors can be analyzed. (G) Distances between individual tracks and their displacement in x,y,z directions reveal cell motility heterogeneity in depth (color coded).