Detection of Reactive Oxygen Species (ROS) by the Oxidant-sensing Probe 2',7'-dichlorodihydrofluorescein Diacetate in the Cyanobacterium Anabaena Variabilis PCC 7937

The generation of reactive oxygen species (ROS) under simulated solar radiation (UV-B: 0.30Wm(-2), UV-A: 25.70Wm(-2) and PAR: 118.06Wm(-2)) was studied in the cyanobacterium Anabaena variabilis PCC 7937 using the oxidant-sensing fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is a nonpolar dye, converted into the polar derivative DCFH by cellular esterases that are nonfluorescent but switched to highly fluorescent DCF when oxidized by intracellular ROS and other peroxides. The images obtained from the fluorescence microscope after 12h of irradiation showed green fluorescence from cells covered with 295, 320 or 395nm cut-off filters, indicating the generation of ROS in all treatments. However, the green/red fluorescence ratio obtained from fluorescence microscopic analysis showed the highest generation of ROS after UV-B radiation in comparison to PAR or UV-A radiation. Production of ROS was also measured by a spectrofluorophotometer and results obtained supported the results of fluorescence microscopy. Low levels of ROS were detected at the start (0h) of the experiment showing that they are generated even during normal metabolism. This study also showed that UV-B radiation causes the fragmentation of the cyanobacterial filaments which could be due to the observed oxidative stress. This is the first report for the detection of intracellular ROS in a cyanobacterium by fluorescence microscopy using DCFH-DA and thereby suggesting the applicability of this method in the study of in vivo generation of ROS.