Fig. 4: Quantitative calcium measurements in different cavity types and HSC microenvironment.

a Ratiometric imaging of Rhod-5N/AF488 from BM cavities dominated by bone deposition (D-type), resorption (R-type), or mixed activities (M-type). Bone remodeling is defined by the double bone front staining strategy⁶ based on the Dye 1/Dye 2 ratio, where Dye 1 labels the old bone fronts that has been eroded to various extents. The cross-section view of bone remodeling from a BM cavity is obtained from the x–z or y–z sections of the blue dashed zone, displayed by maximum intensity projection. b, c Quantifications of intravascular and interstitial Rhod-5N/AF488 ratios in D-, M-, R-type cavities and near LT-HSCs, together with the corresponding calcium concentrations converted from ratiometric analyses (n = 25 BM cavities, N = 10 mice, n = 15 LT-HSCs, n = 30 HSPCs). Significant difference in [Ca²⁺]_e was found between M-type vs. D-type cavities (p < 0.0001), HSCs (p = 0.0004), and HSPCs (p < 0.0001); between D-type cavities vs. HSCs and HSPCs (p < 0.0001). d Calcium distribution in the M-type cavities from young (mean = 1.0 mM) and aged (mean = 1.3 mM) animals (N = 4 mice, n = 10 M-type cavities, p < 0.0001). Each data point represents a subregion from a BM cavity. b–d Two-sided Mann–Whitney test. Box and whiskers represent the median, 25 and 75 percentiles, and the 10–90% data range. e A representative image of Rhod-5N/AF488 labeled BM in MDS1^GFP/+;FLT3^Cre mice used to measure interstitial calcium adjacent to GFP+ LT-HSCs. Source data are provided as a Source Data file.