Photosensitizer rose bengal (RB) reacts with O₂ to generate singlet oxygen ( ¹O₂) under illumination. ¹O₂ reacts with histidine or imidazole to form intermediate products which oxidize RNO. The reaction causes optical absorption of RNO at 440 nm to decrease. It is named the bleaching of RNO. Bleaching of RNO is increased with the increasing of RB illuminated time, which shows that the production of ¹O₂ has grown in the reaction. Bleaching of RNO is promoted by the rising of the concentration of histidine or imidazole. Imidazole plays more evident role in the reaction than histidine does. Bleaching of RNO is decreased by the presence of ¹O₂ quencher NaN₃ or DABCO. ¹O₂ in lettuce thylakoids has been detected by the reaction in strong light. The production of ¹O₂ grows with the increasing of light intensity and illuminated time in lettuce thylakoids.