Building on our previously reported MASR and 2,6-diMe azoSiR640, we designed and developed a novel fluorescence probe for hypoxia by introducing an azobenzene moiety at a free amino group of JSiR (Figure ). Since bulky substitutions at the 2 nd or 6 th positions of the benzene ring in SiR block nucleophilic attack at the 9 th position of the xanthene ring, for example, by water and cysteine containing molecules, we firstly designed a probe possessing carboxylic acid at the 2 nd position of the benzene ring of JSiR. However, this proved synthetically difficult, requiring tedious synthetic and purification steps. So, we next designed a probe possessing a thiophene ring instead of the benzene ring. We synthesized this probe, Tc-azoJSiR640, as shown in Scheme 1, but it proved ineffective due to its poor cellular membrane permeability (Figure ). Since the reason for this was considered to be the presence of the anionic form of the carboxylic acid moiety, we converted it to a methoxycarbonyl group and synthesized T-azoJSiR640. Compared to Tc-azoJSiR640, the membrane permeability of T-azoJSiR640 was apparently improved, as described later. Therefore, we focused on T-azoJSiR640 and evaluated its optical properties and susceptibility to hypoxia.