Fig. 1: Ratiometric imaging of bone marrow pH in vivo.
a SNARF-1 fluorescence intensity and ratio images of a single optical section from a z-stack (shown in Supplementary. Video 1). The cell-impermeable SNARF-1 dextran labeled the vasculature and the interstitial space. The BM cells thus appeared as dark objects. b The mean of SNARF-1 (R/G) ratios obtained from vessels located at various distances to endosteum (depths). Without correction, the R/G ratio increased with increasing image depth. After correction for the wavelength-dependent light attenuation, the intravascular R/G ratio became independent of depth (n = 6 BM cavities, mean ± s.d.). c Schematic illustration of the two-step depth correction. Fluorescence signals originated in the BM travel through BM and bone with distinct attenuation coefficients (C1,2) before being collected by the objective lens. d Bone thickness shows varying thickness across the field of view. The corresponding depth map shows varying distances to endosteum from a single z plane. The cross-section view corresponds to an x–z section from the green dashed line. e A pH calibration curve obtained in vitro to convert measured ratios to absolute pH (N = 3 independent experiments). f Ratiometric quantification of intravascular and interstitial pH after the two-step depth correction. Interstitial pH was found to be significantly lower than intravascular pH (p < 0.0001), with 10–90% data points distributed between 7.0 and 7.3. Each data point represents a subregion from a BM cavity (n = 10 BM cavities, N = 2 mice). two-sided Mann–Whitney test. Box and whiskers represent the median, 25 and 75 percentiles, and the 10–90% data range. Source data are provided as a Source Data file.
