To evaluate whether T-azoJSiR640 could visualize hypoxia in living cells, we used it to perform live-cell fluorescence imaging of A549 lung adenocarcinoma cells under various oxygen concentrations. T-azoJSiR640 showed almost no fluorescence under normoxia (Figures )), while it showed significant increase of NIR fluorescence at oxygen concentration around 1％. These results suggested that T-azoJSiR640 was able to selectively detect severe hypoxia. In contrast with the above results, our previously reported 2,6-diMe azoSiR640 was also activated at around 5% oxygen concentration (Figures )). To confirm that the observed fluorescence signals were due to reduction by flavoproteins, we measured fluorescence images in the presence of a NADPH oxidase inhibitor, diphenyleneiodonium chloride (DPI). The fluorescence increase was dramatically suppressed in the presence of DPI, suggesting that T-azoJSiR640 is reduced by flavoproteins such as NADPH-cytochrome P450 reductase.