Fluorescence imaging studies of the processes leading to photodynamic inactivation of bacteria have been limited due to the small size of microorganisms as well as by the faint fluorescence of most photosensitizers. A versatile method based on highly-sensitive fluorescence microscopy is presented which allows to study, in real time, the incorporation of photosensitizers inside S. aureus upon photodynamic action. The method takes advantage of the fluorescence enhancement of phenothiazine and porphyrin photosensitizers upon entering the bacterial cytosol after the cell wall has been compromised. In combination with typical assays, such as the addition of specific enhancers of reactive oxygen species, it is possible to extract mechanistic information about the pathway of photodynamic damage at the single-cell level. Imaging experiments in deuterated buffer strongly support a Type-I mechanism for methylene blue and a very minor role of singlet oxygen.

UVA radiation plays an important role for adverse reactions in human tissue. UVA penetrates epidermis and dermis of skin being absorbed by various biomolecules, especially endogenous photosensitizers. This may generate deleterious singlet oxygen ( O 2 under UVA irradiation. In conclusion, fatty acids along with peroxidized products are weak endogenous photosensitizers but become strong photosensitizers under continuous UVA irradiation. Since fatty acids and their oxidized products are ubiquitous in living cells and in skin, which is frequently and long-lasting exposed to UVA radiation, this photosensitizing effect may contribute to initiation of deleterious photooxidative processes in tissue.

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