Dihydroethidium, also called hydroethidine (HE), can be oxidized by reactive species, including superoxide to ethidium that subsequently binds to DNA to produce fluorescence. More recently, the derivative of dihydroethidium bearing a cationic triphenylphosphonium moiety, commonly known as MitoSOX Red or Mito-HE, or more frequently called MitoSOX, has been synthesized and become commercially available (e.g., Thermo Fisher, Waltham, MA USA). This positively charged probe rapidly accumulates in mitochondria, and as such may be used to detect superoxide/ROS production inside mitochondria via fluorometry, microscopy, or flow cytometry (Figure 1). In fact, fluorescence imaging of the dihydroethidium/MitoSOX-stained cells or tissues has been claimed as a selective assay for intracellular and intra-mitochondrial superoxide production [6 (link), 7 (link)], but this claim has received criticism [8 (link)]. Nevertheless, measurement of MitoSOX-derived fluorescence intensity, when the probe is used at appropriate concentrations, seems to be reflective of the levels of mitochondrial total ROS.