Fig. 2: Ratiometric calcium imaging of bone marrow.

a Intravital two-photon fluorescence imaging of BM cavities labeled with Rhod-5N (red) and AF488 (green). The SHG signal from bone is shown in gray. The mask for interstitium and vessels were generated from Rhod-5N images to delineate the vasculature and the interstitial space while excluding areas with low fluorescence signals (i.e., intracellular space that is not labeled by the cell-impermeable dye). Autofluorescent cells are also excluded. V vessels, IS interstitium, O Osteoids. b The mean of Rhod-5N/AF488 ratios obtained from vessels located at various distances to endosteum (depths), showing a consistent increase in the Rhod5-N/AF488 ratios with increasing image depth. Correction for depth attenuation of Rhod-5N and AF488 signals independently yielded intravascular ratios independent of depth (n = 6 BM cavities). c Ratiometric analyses without the two-step depth correction yielded divergent intravascular Rhod-5N/AF488 ratios, while depth corrections minimized variation of intravascular ratios. d Real-time response of the Rhod-5N/AF488 ratio (black circles) during the injection of a calcium chelator, Calcein Blue (blue squares). Each data point represents the mean of 5 subregions from the blood vessels. Mean ± s.d. (s.d. were calculated from all the pixels within the ROI). Source data are provided as a Source Data file.