Imaging hematopoietic progenitor clonal output in the spleen over time. In a single experiment, Co 5FP Lin⁻ BM cells were transplanted into multiple recipient mice after total body irradiation. Intact spleens were removed at each time point, imaged over large volumes (1.2-mm × 1.6-mm × 100-μm), and are presented as extended focus sections across x, y, and z dimensions; the superficial layers of the capsule were removed computationally, facilitating visibility of the fluorescent cells underneath. (A) At day 4, numerous small clusters of diverse colors are visible under the collagen-rich capsule (SHG, white). (B) At day 19, very large clonal colonies or CFU-S diameter 500-600 μm (x-y) are present, with color homogeneity associated with each CFU, although by this time point there was clonal mixing at the edges of neighboring CFU-S. (C) At day 30, the large CFU-S had disappeared, with only small residual clusters of clonal cells enmeshed by collagen fibers. (D) At day 120, the spleen is repopulated by scattered nonclonal cells indicated by the color diversity and lack of clustering of cells of the same color. Scale bars represent 100 μm (A,C-D), 300 μm (B). Related images shown in supplemental Video 7.