Fluorescence Image Analysis for Quantification of Active Oxygen Induced by Photochemical Reaction

This study was performed to confirm that activated leukocytes are concerned with Vascular Shut Down effect ; VSD in PhotoDynamic Therapy ; PDT. Hydrogen peroxide ;H2O2, which is a kind of active oxygen that came from monocytes, was made visibility under the confocal laser scanning microscope ;CLSM, and tried to quantify the amount of its formation from the fluorescent intensity with image analysis. Fluorescence images acquired by CLSM were analyzed, and the fluorescence intensity was expressed as gray level, which is graded from 0 to 255. Only the fluorescence derived from a monocyte added Zinc coproporphyrin III ; ZnCP-III just before measurement and with HeNe laser irradiation caused fluorescence distribution move to increase as the time course, while no change of distribution was especially observed in other three conditions (only added Zn CP-III, only irradiated with HeNe laser, Non-treated). The result indicates that photochemical reaction occurred due to excitation of photosensitizer, and active oxygen derived from the reaction stimulated monocytes. The activated monocytes, this time for themselves, generated active oxygen and H2O2 was made visibility by dichlorofluorescin ;DCFH fluorescence method. In conclusion, we confirmed that activated monocytes by photochemical reaction are concerned with VSD effect. Keywords— active oxygen, photochemical reaction, vascular shut down, monocyte, confocal laser scannning microscope