To determine whether T-azoJSiR640 could detect hypoxia, we first performed an in vitro assay using rat liver microsomes, which contain various reductases such as NADPH-cytochrome P450 reductase. The probe T-azoJSiR640 showed a fluorescence increase only in the presence of rat liver microsomes and NADPH under hypoxia, indicating that the probe could detect hypoxia under reducing conditions catalyzed by microsomal reductases (Figure ).