This study investigates the localization of haematopoietic stem and progenitor cells (HSPCs) within their niche in living mice. Using high-resolution confocal and two-photon microscopy, the researchers observed HSPCs in the calvarium bone marrow of living mice over time. They found that HSPCs localized to subdomains of bone-marrow microvessels where the chemokine CXCL12 is abundant. Osteoblasts were enmeshed in microvessels, and the relative positioning of HSPCs within this complex tissue was non-random and dynamic. Both cell-autonomous and non-autonomous factors influenced HSPC localization. Different HSPC subsets localized to distinct locations based on their differentiation stage. When physiological challenges drove engraftment or expansion, HSPCs moved closer to bone and osteoblasts. The study provides real-time, single-cell-level observations of processes previously studied only at the organismal level. The study also examined the relationship between HSPCs and the bone marrow vasculature, osteoblasts, and endosteal surface. Bone-marrow vasculature includes arteries that penetrate compact bone and arborize into capillaries that converge into a central sinus. CD31 immunostaining enabled visualization of this vasculature network adjacent to the endosteal surface. The researchers used intravital microscopy to scan a 4 mm × 6 mm region of the mouse calvarium, including the central sinus and surrounding bone marrow cavities. They found that HSPC frequencies in this region were comparable to long bones by immunophenotype and repopulating ability. The study used a combination of confocal and two-photon microscopy to visualize HSPCs at a depth of ~150 μm or 40–60% of the bone marrow cavity in over 75% of measurements. They used second harmonic generation (SHG) microscopy, osteoblast-restricted collagen 1 α promoter (Col2.3–GFP) reporter mice, quantum dots, and flow cytometrically sorted cells to detect HSPCs. They found that HSPCs localized to distinct locations based on their differentiation stage and that their localization was influenced by both cell-autonomous and non-autonomous factors. The study also examined the effects of irradiation and the c-Kit receptor on HSPC localization. HSPCs injected into non-irradiated recipients localized farther than 30 μm from bone and did not engraft. However, when injected into irradiated recipients, HSPCs localized closer to the endosteal surface and engrafted. The study also found that HSPCs injected into WWv mice, which have impaired c-Kit signaling, engrafted and overtook endogenous HSC production of all peripheral blood lineages. The study demonstrated that HSPCs can be tracked in vivo overThis study investigates the localization of haematopoietic stem and progenitor cells (HSPCs) within their niche in living mice. Using high-resolution confocal and two-photon microscopy, the researchers observed HSPCs in the calvarium bone marrow of living mice over time. They found that HSPCs localized to subdomains of bone-marrow microvessels where the chemokine CXCL12 is abundant. Osteoblasts were enmeshed in microvessels, and the relative positioning of HSPCs within this complex tissue was non-random and dynamic. Both cell-autonomous and non-autonomous factors influenced HSPC localization. Different HSPC subsets localized to distinct locations based on their differentiation stage. When physiological challenges drove engraftment or expansion, HSPCs moved closer to bone and osteoblasts. The study provides real-time, single-cell-level observations of processes previously studied only at the organismal level. The study also examined the relationship between HSPCs and the bone marrow vasculature, osteoblasts, and endosteal surface. Bone-marrow vasculature includes arteries that penetrate compact bone and arborize into capillaries that converge into a central sinus. CD31 immunostaining enabled visualization of this vasculature network adjacent to the endosteal surface. The researchers used intravital microscopy to scan a 4 mm × 6 mm region of the mouse calvarium, including the central sinus and surrounding bone marrow cavities. They found that HSPC frequencies in this region were comparable to long bones by immunophenotype and repopulating ability. The study used a combination of confocal and two-photon microscopy to visualize HSPCs at a depth of ~150 μm or 40–60% of the bone marrow cavity in over 75% of measurements. They used second harmonic generation (SHG) microscopy, osteoblast-restricted collagen 1 α promoter (Col2.3–GFP) reporter mice, quantum dots, and flow cytometrically sorted cells to detect HSPCs. They found that HSPCs localized to distinct locations based on their differentiation stage and that their localization was influenced by both cell-autonomous and non-autonomous factors. The study also examined the effects of irradiation and the c-Kit receptor on HSPC localization. HSPCs injected into non-irradiated recipients localized farther than 30 μm from bone and did not engraft. However, when injected into irradiated recipients, HSPCs localized closer to the endosteal surface and engrafted. The study also found that HSPCs injected into WWv mice, which have impaired c-Kit signaling, engrafted and overtook endogenous HSC production of all peripheral blood lineages. The study demonstrated that HSPCs can be tracked in vivo over