Hypoxia—damage to tissue caused by low oxygen levels—can be difficult to detect. Jefferson Chan and coworkers at the University of Illinois, Urbana-Champaign, have developed a small molecule probe they call HyP-1 that improves hypoxia detection by using photoacoustic imaging (Nat. Commun. 2017, DOI: 10.1038/s41467-017-01951-0). In photoacoustic imaging, near-infrared light induces temperature and pressure changes in tissue that result in the production of ultrasound waves. Because sound scatters less in tissue than light does, the sound waves can be used to produce images from deep in tissue. HyP-1 contains an N-oxide trigger that undergoes reduction to the corresponding aniline (red-HyP-1) in the absence of oxygen. This reduction depends on competitive binding of oxygen to the heme iron in various enzymes. Because red-HyP-1 absorbs light at longer wavelengths than HyP-1 does, any photoacoustic signal produced by excitation at those longer wavelengths corresponds exclusively to red-Hyp-1, which indicates hypoxia. The researchers used HyP-1 to detect hypoxia in cultured cells, in tumors in mice, and in hind limb ischemia in mice. Although HyP-1 was designed for photoacoustic imaging, the fact that both it and red-HyP-1 fluoresce in the near-infrared means that the probe pair can also be used for comparative fluorescence imaging.