Characterizing how the microenvironment, or niche, regulates stem cell activity is central to understanding stem cell biology and to developing strategies for therapeutic manipulation of stem cells1. Low oxygen tension (hypoxia) is commonly thought to be a shared niche characteristic in maintaining quiescence in multiple stem cell types2–4. However, support for the existence of a hypoxic niche has largely come from indirect evidence such as proteomic analysis5, expression of HIF-1 and related genes6, and staining with surrogate hypoxic markers (e.g. pimonidazole)6–8. Here we perform direct in vivo measurements of local oxygen tension (pO2) in the bone marrow (BM) of live mice. Using two-photon phosphorescence lifetime microscopy (2PLM), we determined the absolute pO2 of the BM to be quite low (<32 mmHg) despite very high vascular density. We further uncovered heterogeneities in local pO2, with the lowest pO2 (~9.9 mmHg, or 1.3%) found in deeper peri-sinusoidal regions. The endosteal region, by contrast, is less hypoxic as it is perfused with small arteries that are often positive for the marker nestin. These pO2 values change dramatically after radiation and chemotherapy, pointing to the role of stress in altering the stem cell metabolic microenvironment.

The ability to measure oxygen partial pressure (pO2) with high temporal and spatial resolution in three dimensions is crucial for understanding oxygen delivery and consumption in normal and diseased brain. Among existing pO2 measurement methods, phosphorescence quenching is optimally suited for the task. However, previous attempts to couple phosphorescence with two-photon laser scanning microscopy have faced substantial difficulties because of extremely low two-photon absorption cross-sections of conventional phosphorescent probes. Here, we report the first practical in vivo two-photon high-resolution pO2 measurements in small rodents’ cortical microvasculature and tissue, made possible by combining an optimized imaging system with a two-photon-enhanced phosphorescent nanoprobe. The method features a measurement depth of up to 250 µm, sub-second temporal resolution and requires low probe concentration. Most importantly, the properties of the probe allowed for the first direct high-resolution measurement of cortical extravascular (tissue) pO2, opening numerous possibilities for functional metabolic brain studies.

Uncovering principles that regulate energy metabolism in the brain requires mapping of partial pressure of oxygen (PO2) and blood flow with high spatial and temporal resolution. Using two-photon phosphorescence lifetime microscopy (2PLM) and the oxygen probe PtP-C343, we show that PO2 can be accurately measured in the brain at depths up to 300 μm with micron-scale resolution. In addition, 2PLM allowed simultaneous measurements of blood flow and of PO2 in capillaries with less than one-second temporal resolution. Using this approach, we detected erythrocyte-associated transients (EATs) in oxygen in the rat olfactory bulb and showed the existence of diffusion-based arterio-venous shunts. Sensory stimulation evoked functional hyperemia, accompanied by an increase in PO2 in capillaries and by a biphasic PO2 response in the neuropil, consisting of an ‘initial dip’ and a rebound. 2PLM of PO2 opens new avenues for studies of brain metabolism and blood flow regulation.