Our research group has focused on the reductive cleavage of azo groups under hypoxia and developed NIR probes based on fluorescence resonance energy transfer (FRET) between dicarbocyanines and Black Hole Quencher (BHQ). Although these probes are selectively activated in severe hypoxia, they are not optimal for in cellulo and in vivo applications due to their large molecular size and susceptibility to photobleaching. As another strategy, we have recently developed fluorescence probes that detect hypoxia by incorporating an azo group into an O rhodamine or Si rhodamine (SiR) scaffold. For example, we have developed green-emitting MAR, red-emitting MASR and NIR-emitting azoSiR640 (2,6-diMe azoSiR640), and confirmed that they can detect hypoxia in cultured cells and mouse models. These fluorescence probes initially have no fluorescence due to fluorescence quenching by the azobenzene moiety incorporated in the fluorophore. Upon cleavage of the azo group by reductase-catalyzed reactions that are accelerated in hypoxia, corresponding fluorescent rhodamines are produced, enabling selective detection of hypoxia. However, the oxygen concentration threshold of these rhodamine-based hypoxia probes varies depending on the fluorescent scaffold. For example, MASR and 2,6-diMe azoSiR640 switched on at around 0.1% and 5% oxygen concentration, respectively. These characteristics may be due to differences in the rate of reverse oxidation of the azo moiety depending on the fluorescent scaffold (Figure ). Thus, we considered that the development of NIR probes to selectively detect severe hypoxia of around 1% oxygen concentration might be achieved by using different fluorophores.