Fluorescence image analysis for quantification of active oxygen induced by photochemical reaction

This study was performed to confirm that activated leukocytes are concerned with the 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 visible under the confocal laser scanning microscope, CLSM, and the amount of its formation quantified from the fluorescent intensity with image analysis. Fluorescence images acquired by CLSM were analyzed, and the fluorescence intensity was expressed as a 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 He-Ne laser irradiation caused the fluorescence distribution to increase with time, while no change of distribution was especially observed in three other conditions (only added Zn CP-III, only irradiated with He-Ne laser, non-treated). The result indicates that a photochemical reaction occurred due to excitation of the photosensitizer, and active oxygen derived from the reaction stimulated monocytes. The activated monocytes, this time for themselves, generated active oxygen and H₂O₂ was made visible by dichlorofluorescein, the DCFH fluorescence method. In conclusion, we confirmed that activated monocytes by photochemical reaction are concerned with the VSD effect.