title: Real-time imaging of photodynamic action in bacteria.
abstract: 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.
authors.affiliation: Department of Dermatology, University Hospital Regensburg, 93053, Regensburg, Germany.
authors.forename: Anita
authors.initials: A
authors.lastname: Gollmer
authors.affiliation: Department of Dermatology, University Hospital Regensburg, 93053, Regensburg, Germany.
authors.forename: Ariane
authors.initials: A
authors.lastname: Felgentraeger
authors.affiliation: Department of Dermatology, University Hospital Regensburg, 93053, Regensburg, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Maisch
authors.affiliation: Madrid Institute for Advanced Studies in Nanoscience (IMDEA Nanociencia) and Nanobiotechnology Unit Associated to the National Center for Biotechnology (CSIC), 28049, Madrid, Spain.
authors.forename: Cristina
authors.initials: C
authors.lastname: Flors
mesh_terms: D003600:Cytosol / Q000737:chemistry; D008751:Methylene Blue / Q000737:chemistry; D008856:Microscopy, Fluorescence; D010640:Phenothiazines / Q000737:chemistry; D017319:Photosensitizing Agents / Q000737:chemistry*; D011166:Porphyrins / Q000737:chemistry; D026082:Singlet Oxygen; D013211:Staphylococcus aureus / Q000166:cytology*
chemical_list: D010640:Phenothiazines; D017319:Photosensitizing Agents; D011166:Porphyrins; D026082:Singlet Oxygen; C031637:phenothiazine; D008751:Methylene Blue
keywords: Fluorescence microscopy; Staphylococcus aureus; photodynamic therapy; photosensitizers; reactive oxygen species; singlet oxygen