Comparison of the vascularization in calvaria and long bones. Structural characterization of the calvarium using in situ imaging of BRA as revealed by an NIR-pamidronate probe. Visualization using an NIR scanner (A) and confocal microscope (B). Scale bar represents 700 μM. (C) A 3D reconstruction of BM vasculature in the calvarium where blood vessels are contrasted using nontargeted Quantum dots. The 3D tiling was applied to acquire 16 × 23 adjacent stacks of 15 steps, covering a 6800-µm/9780-µm/600-µm volume. Scale bar represents 1 mm. (D) Combined BRA and vasculature contrasting obtained after intravenous injection of NIR-pamidronate and IB4-Alexa568, imaged in intact BM. Vasculature can be seen to be in close contact with the bone. Scale bar represents 300 µm. (E) Four-color live tissue imaging combining NIR-pamidronate (endosteum), IB4-Alexa568 (endothelial cells), and FITC-dextran (blood plasma) with simultaneous detection of SHG (bone). (F) A 3D model of bone (SHG, blue), bone endosteum (NIR-pamidronate, purple), and blood vessels contrasted with FITC-dextran (green). (G-J) Ex vivo imaging of long bones (SHG, cyan) after in vivo contrasting with NIR-pamidronate (purple) and IB4-Alexa488 (green). Sagittal section of tibia epiphysis (G) with details of the trabecular region (H). Scale bars represent 200 and 100 µm, respectively. Transverse section of femur metaphysis (I) and diaphysis (J). Scale bar represents 200 µm.