Hypoxia, defined as insufficient oxygen, plays a key role in the pathogenesis of many diseases including, myocardial ischemia, cardiovascular disease, chronic kidney disease, arthritis, and cancer. Hypoxia is a common feature of solid tumors and is associated with resistance to standard therapies, increased metastasis, and poor patient prognosis. Regions of chronic hypoxia occur beyond the diffusion limit of oxygen in metabolically active cancer cells, typically 100 µm, while acute hypoxia can occur within a tumor resulting from changes in red blood cell flux and vascular remodeling. Notably, cycling or intermittent changes in oxygen levels also occurs in tumors and can be exacerbated during therapy. Typically, physiological levels of oxygen (physoxia) are defined as 3-7% O2, tumor hypoxia is defined as <2% O2, and extreme levels of hypoxia (<0.1% O2) termed radiobiological hypoxia, are those most associated with resistance to radiotherapy. Importantly, the biological response differs in an oxygen-dependent manner, for example while the hypoxia-inducible factors (HIFs) are induced at both 2 and <0.1% O2, the DNA damage response or unfolded protein response is restricted to the lower oxygen tension. There is an unmet need for imaging tools that not only detect hypoxia but also inform on the level of oxygen present, because median oxygen concentrations within a tumor can range from 4.2-0.1% O2. One biological response to hypoxia is the production reductase enzymes (cytochrome P450 reductase and nitroreductase) that catalyze oxygen-sensitive bioreductive reactions. These enzymes have been exploited to develop hypoxia sensing pro-drugs and imaging agents, including pimonidazole and EF5 that are routinely used in pre-clinical studies as indicators of hypoxia (Figure ). Both pimonidazole and EF5 contain nitroimidazole groups that are reduced by reductases in hypoxia to generate products that are covalently trapped and can be detected using immunostaining. In contrast, hypoxia sensing fluorescent probes are powerful tools that allow the detection and imaging of hypoxia with high sensitivity and spatiotemporal resolution, but no need for subsequent detection steps involving immunostaining. In general, such probes function by having their emission properties suppressed by addition of a bioreductive group, examples of which include nitroarenes, quinones, azo, or azide groups, which undergo reductase-catalyzed reactions to reveal the fluorophore in an oxygen-dependent manner. Of the hypoxia sensing probes developed to date, only the azo-based probes developed by Piao et al. and Zhang et al. can detect milder levels of hypoxia. These probes are both based on the arylazo motif, which releases positively charged amine motifs that localize in the mitochondria or lysosomes.