Intravital Imaging of Blood Flow and HSPC Homing in Bone Marrow Microvessels
Two-photon intravital microscopy (2P-IVM) is an advanced imaging technique that allows the visualization of dynamic cellular behavior deeply inside tissues and organs of living animals. Due to the deep tissue penetration, imaging of highly light-scattering tissue as the bone becomes feasible at subcellular resolution.
To better understand the influence of blood flow on hematopoietic stem and progenitor cell (HSPC) homing to the bone marrow (BM) microvasculature of the calvarial bone, we analyzed blood flow dynamics and the influence of flow on the early homing behavior of HSPCs during their passage through BM microvessels. Here, we describe a 2P-IVM approach for direct measurements of red blood cell (RBC) velocities in the BM microvasculature using repetitive centerline scans at the level of individual arterial vessels and sinusoidal capillaries to obtain a detailed flow profile map. Furthermore, we explain the isolation and enrichment of HSPCs from long bones and the transplantation of these cells to study the early homing behavior of HSPCs in BM sinusoids at cellular resolution. This is achieved by high-resolution spatiotemporal imaging through a chronic cranial window using transgenic reporter mice.
Key wordsIntravital imaging Bone marrow microvessels Blood flow velocities Hematopoietic stem cells Stem cell homing Two-photon microscopy
We thank F. Winkler for sharing his expertise on the imaging setup for head immobilization. This work was supported by the Max Planck Society, the University of Münster, the DFG cluster of excellence “Cell in Motion,” and the European Research Council (AdG 339409 AngioBone).
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