Use of real-time confocal laser scanning microscopy to study immediate effects of photodynamic activation on photosensitized erythrocytes

With a view towards the design of systems capable of combining the use of chemotherapy and photodynamic therapy in the treatment of cancer and other disorders, it has been proposed that photosensitized erythrocytes might be employed as carriers/vehicles for agents such as cancer chemotherapeutics. In studying the light dependent release of entrapped agents from such a system, the efficacy of light induced release is usually studied by measuring release of an entrapped component into centrifugation supernatants following photoactivation. It has hitherto been extremely difficult to examine what occurs upon immediate irradiation at the microscopic level in real-time. In this study we demonstrate that, using real-time confocal laser scanning microscopy, it is possible to directly observe immediate short-term events occurring during direct irradiation with the visualizing beam. Following irradiation of photosensitized erythrocytes with the visualizing beam from the confocal scanning system, it was noticed that some form of cell-disruptive event occurred. In this study we demonstrate a dose dependent response between this relatively immediate, light induced disruptive event with respect to both irradiation exposure and photosensitizer concentration. We suggest that this system may provide a novel means of observing, at a microscopic level, events occurring in real-time during photodynamic therapy.