HSPCs within the metaphysis are close to BVs. Homing of HSPCs to the metaphysis is independent of the number and width of BM BV and distance between BVs. (A) Transmission electron micrograph revealing excellent preservation of BVs after perfusion fixation and embedding femurs in resin. RBC indicates red blood cell. Scale bar represents 2 μm. The orientation of BVs within the femur is taken into account for histomorphometric analyses. To obtain cross-sectional profiles of the BVs, femurs are cut transversely through the metaphysis (B) and longitudinally through the diaphysis (C). (B-C) Micrographs represent montages of more than or equal to 48 images captured at high magnification (40× objective, UPlanApo NA 0.85) using the Metamorph “slide scan” function on an Olympus IX81 microscope. Metamorph was used to automatically segment perfusion-cleared individual BVs, which are easily identified at full resolution. Vessels are outlined in red as shown at higher magnification in the insets of panels B and C. Cortical bone and trabeculae are outlined manually in blue. Scale bar represents 0.5 mm. BVs proximal to the growth plate (D) are smaller than those distal from the growth plate (E), where the main central sinus branches into the smaller metaphyseal vessels. Calculation of the distance between individual BVs assumes that most BVs are round and evenly distributed, as exemplified in both panels D and E. BVs are cleared of blood, enabling their clear identification. (E) *Lipid is well preserved because of osmium secondary fixation and cannot be confused with BVs (white areas). The trabecular bone is delineated by a dashed line. Micrographs were captured using a 40× (UPlanApo NA 0.85) objective on an Olympus BX51 microscope fitted with a SPOT RT-SE6 camera. Scale bar represents 50 μm. (B-E) Resin sections (1-μm toluidine blue-stained). (F) Histomorphometric analyses of 1 μm resin sections reveal the distance between BVs within the metaphysis and diaphysis. Each symbol represents individual mice. Data are mean ± SEM. (G) Scanning electron microscopy of vascular casts. Larger-diameter vessels indicative of the early branches of the central sinus are visible below the plane of focus (arrows). The micrograph shown is representative of 5 individual BV casts. (H) Average number of BVs/mm² of cellular marrow is comparable between the metaphysis and the diaphysis. (I) The average widths of BVs in the metaphysis and diaphysis are not significantly different. Symbols represent individual mice. Experimental repeats ≥ 3. Data are shown as mean ± SEM.