Implications of the generation of reactive oxygen species by photoactivated calcein for mitochondrial studies

Calcein is a fluorescent probe that is widely used in studies of cell viabi lity and mitochondrial function by microscopy fluorescence imaging. It was found to have a strong photosensitizing action that prevalently involves th e generation of reactive oxygen species (ROS). The photooxidation propertie s of calcein in solution were studied in the presence of histidine and tryp tophan as oxidizable substrates. The photodegradation of histidine was main ly mediated by singlet oxygen (O-1(2)), as shown by the inhibitory effect o f sodium azide, a specific O-1(2) scavenger. On the other hand, mixed photo sensitization mechanisms were present when tryptophan was used as the targe t of the calcein-stimulated photoprocess. In addition to O-1(2), hydroxyl r adicals and hydrogen peroxide were involved as reactive species, as shown b y using mannitol and catalase as scavengers. The calcein-photosensitized alterations of mitochondria as a potential sour ce of artifacts in confocal microscopy studies of cells were considered. Ir radiation of isolated mitochondria with visible light (500-600 nm) in the p resence of calcein induced opening of the permeability transition (PT) pore . The extent of the mitochondrial membrane photodamage, however, was modula ted by the nature of the calcein environment. Thus, pore opening was trigge red at short irradiation times and low dye concentrations when calcein was dissolved in the bulk medium. On the contrary, calcein concentrated in the matrix space was rather inefficient as photosensitizer even at concentratio ns 10 times higher than those present in the external medium.