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.

Background & Aims The gut microbiota is a complex and densely populated community in a dynamic environment determined by host physiology. We investigated how intestinal oxygen levels affect the composition of the fecal and mucosally adherent microbiota. Methods We used the phosphorescence quenching method and a specially designed intraluminal oxygen probe to dynamically quantify gut luminal oxygen levels in mice. 16S rRNA gene sequencing was used to characterize the microbiota in intestines of mice exposed to hyperbaric oxygen, human rectal biopsy and mucosal swab samples, and paired human stool samples. Results Average pO2 values in the lumen of the cecum were extremely low (<1 mmHg). In altering oxygenation of intestines of mice, we observed that oxygen diffused from intestinal tissue and established a radial gradient the extended from the tissue interface into the lumen. Increasing tissue oxygenation with hyperbaric oxygen altered the composition of the gut microbioita in mice. In humans, 16S rRNA gene analyses revealed an increased proportion of oxygen-tolerant organisms of the Proteobacteria and Actinobacteria phyla associated with the rectal mucosa, compared with the feces, indicating an effect of oxygenation on the microbiota. A consortium of asaccharolytic bacteria of the Firmicute and Bacteroidetes phyla, which primarily metabolize peptones and amino acids, was associated primarily with mucus. This could be due to the presence of proteinaceous substrates provided by mucus and the shedding of the intestinal epithelium. Conclusions In an analysis of intestinal microbiota of mice and humans, we observed a radial gradient of microbes linked to distribution of oxygen and nutrients provided by host tissue.

Natural pore-forming proteins act as viral helical coats and transmembrane channels, exhibit antibacterial activity and are used in synthetic systems, such as for reversible encapsulation or stochastic sensing. These diverse functions are intimately linked to protein structure. The close link between protein structure and protein function makes the design of synthetic mimics a formidable challenge, given that structure formation needs to be carefully controlled on all hierarchy levels, in solution and in the bulk. In fact, with few exceptions, synthetic pore structures capable of assembling into periodically ordered assemblies that are stable in solution and in the solid state have not yet been realized. In the case of dendrimers, covalent and non-covalent coating and assembly of a range of different structures has only yielded closed columns. Here we describe a library of amphiphilic dendritic dipeptides that self-assemble in solution and in bulk through a complex recognition process into helical pores. We find that the molecular recognition and self-assembly process is sufficiently robust to tolerate a range of modifications to the amphiphile structure, while preliminary proton transport measurements establish that the pores are functional. We expect that this class of self-assembling dendrimers will allow the design of a variety of biologically inspired systems with functional properties arising from their porous structure.

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.

High-resolution images of oxygen distributions in microheterogeneous samples are obtained by twophoton laser scanning microscopy (2P LSM), using a newly developed dendritic nanoprobe with internally enhanced two-photon absorption (2PA) cross-section. In this probe, energy is harvested by a 2PA antenna, which passes excitation onto a phosphorescent metalloporphyrin via intramolecular energy transfer. The 2P LSM allows sectioning of oxygen gradients with near diffraction-limited resolution, and lifetime-based acquisition eliminates dependence on the local probe concentration. The technique is validated on objects with a priori known oxygen distributions and applied to imaging of pO 2 in cells.