Preliminary Study On Cellular Photobiological Effect By Reactive Oxygen Species Detection Through Fluorescent Probe Technique

Objective To learn the generation of reactive oxygen species(ROS) during light-only irradiation and during photodynamic reaction induced by hematoporphyrin monomethyl ether (HMME-PDT) with fluorescent microscope and H2DCF-DA.Methods (1) The device for study was determined by comparing the fluorescent images obtained by laser scanning confocal microscope and CCD fluorescent microscope respectively. The mercury-arc lamp of fluorescent microscope was studied to value its possibility to be used as the irradiation light source. Various image analyzing and processing methods were compared to determine which one was the best to be applied in this study. (2) ECV 304 cells were subcultured for 24 h. H2DCF-DA was added into it for 30 min with the final concentration of 10 mol/L, and the other probe MitoFluor Red 589, which distributes in mitochondria specifically, was added simultaneously with the final concentration of 100nmol/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 distributed site of DCF was determined by comparing the fluorescence images of MitoFluor Red 589 and DCF of the same cell. The value of I1/I2 represented the amount of DCF in different cellular regions.(3) HMME was added into it for 4 h with the final concentration of 10 g/ml. MitoFluor Red 589 was unnecessary in this part, and other procedure and operation were similar to the former part. The mean fluorescence intensity of DCF in cells and its variety with time were calculated by image analyzing and processing technique.Results (1) CCD fluorescent microscope was superior to laser scanning confocal microscope in collecting the fluorescent image of DCF. CCD fluorescent microscope was chosen to be the system applied in this research with the consideration of operation, cost and et al. The mercury-arc lamp of fluorescent microscope was fit for the requirement of our study. The wavelength range of the light output was 460-490 nm and the power density was about 100mW/cm in the condition of our study. Organelle-cell fluorescence intensity ratio analysis was more suitable than other methods. (2) The fluorescence of DCF in ECV 304 cell increased slowly during the early stage of light-only irradiation and distributed mainly in mitochondria. The value of I1/I2 in mitochondria region was much higher than that in nucleus region and non-mitochondria cytosol region, and the value of each were 1.949 0.221, 1.113 0.226, 0.725 0.083 respectively, when parameter m was 20%. (3) The site of DCF in ECV 304 cell during the early stage of HMME-PDT seemed to be the same with that of DCF during light-only irradiation, and the fluorescence intensity of the former increased more rapidly than the latter. The fluorescence intensity of the former at 28th second was 5.98 times of that at the 2nd second, but fluorescence intensity of the latter at 60th second was 4.69 times of that at the 2nd second. Conclusions In this study we set up a series of method to detect the intracellular reactive oxygen species with fluorescent microscope and H2DCF-DA, which is simple and reliable. The light irradiation can induce thegeneration of ROS, and the generated site was mainly in mitochondria, which may be associated with its abundant endogenesis photosensitizer. The ROS generated during the photodynamic reaction induced by endogenesis photosensitizer may be the matter that causes the biological effect of low level laser therapy. The generation of ROS in mitochondria during HMME-PDT was much higher than that in other cellular regions. The phenomena indicated the effect of mitochondria inner environment on the velocity of photodynamic reaction. Mitochondria are the main site for the early stage of HMME-PDT.