Quantification of bone marrow interstitial pH and calcium concentration by intravital ratiometric imaging

The fate of hematopoietic stem cells (HSCs) can be directed by microenvironmental factors including extracellular calcium ion concentration ([Ca²⁺]_e), but the local [Ca²⁺]_e around individual HSCs in vivo remains unknown. Here we develop intravital ratiometric analyses to quantify the absolute pH and [Ca²⁺]_e in the mouse calvarial bone marrow, taking into account the pH sensitivity of the calcium probe and the wavelength-dependent optical loss through bone. Unexpectedly, the mean [Ca²⁺]_e in the bone marrow (1.0 ± 0.54 mM) is not significantly different from the blood serum, but the HSCs are found in locations with elevated local [Ca²⁺]_e (1.5 ± 0.57 mM). With aging, a significant increase in [Ca²⁺]_e is found in M-type cavities that exclusively support clonal expansion of activated HSCs. This work thus establishes a tool to investigate [Ca²⁺]_e and pH in the HSC niche with high spatial resolution and can be broadly applied to other tissue types.