Detection of ROS in Cell During Photodynamic Therapy Applying Fluorescence Microscopy

Objective To learn the generation of reactive oxygen species (ROS) during photodynamic reaction induced by hematoporphyrin monomethyl ether (HMME) applying fluorescence microscopy. Methods ECV 304 cells were subcultured for 24 h. HMME was incubated for 4 h with the final concentration of 10 mug/ml. H
₂
DCF-DA was added into it for 30 min with the final concentration of 10 mumol/L. The procedure of light irradiation was carried out during collecting the fluorescent image of DCF. The fluorescent images were collected by CCD fluorescent microscope. The mean fluorescence intensity of DCF in cells and its variety with time were calculated by image analyzing and processing technique. Another group with light-only irradiation was set as control. Results The fluorescence of DCF in ECV 304 cell of HMME-PDT group increased more quickly than that of light-only irradiation group. The fluorescence intensity of the former at 28
^th
second was 5.98 times of that at the 2
^nd
second, but fluorescence intensity of the latter at 60
^th
second was 4.69 times of that at the 2
^nd
second. The site of DCF in ECV 304 cell during the early stage of HMME-PDT seemed to be the same with that during light-only irradiation, and distributed mainly in the cytoplasmic area near karyon. Conclusions The generation of ROS during HMME-PDT was much higher than that during light-only irradiation. And the cytoplastic area near karyon may be the main damage site for the early stage of HMME-PDT.