title: Intrinsic indicator of photodamage during label-free multiphoton microscopy of cells and tissues.
abstract: Multiphoton imaging has evolved as an indispensable tool in cell biology and holds prospects for clinical applications. When addressing endogenous signals such as coherent anti-Stokes Raman scattering (CARS) or second harmonic generation, it requires intense laser irradiation that may cause photodamage. We report that increasing endogenous fluorescence signal upon multiphoton imaging constitutes a marker of photodamage. The effect was studied on mouse brain in vivo and ex vivo, on ex vivo human brain tissue samples, as well as on glioblastoma cells in vitro, demonstrating that this phenomenon is common to a variety of different systems, both ex vivo and in vivo. CARS microscopy and vibrational spectroscopy were used to analyze the photodamage. The development of a standard easy-to-use model that employs rehydrated cryosections allowed the characterization of the irradiation-induced fluorescence and related it to nonlinear photodamage. In conclusion, the monitoring of endogenous two-photon excited fluorescence during label-free multiphoton microscopy enables to estimate damage thresholds ex vivo as well as detect photodamage during in vivo experiments.
authors.affiliation: Clinical Sensoring and Monitoring, Anesthesiology and Intensive Care Medicine, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors.forename: Roberta
authors.initials: R
authors.lastname: Galli
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Dresden, Germany.
authors.forename: Ortrud
authors.initials: O
authors.lastname: Uckermann
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Dresden, Germany; CRTD/DFG-Center for Regenerative Therapies Dresden - Cluster of Excellence, Dresden, Germany.
authors.forename: Elisabeth F
authors.initials: EF
authors.lastname: Andresen
authors.affiliation: Neuropathology, Institute for Pathology, Carl Gustav Carus University Hospital, TU Dresden, Dresden, Germany.
authors.forename: Kathrin D
authors.initials: KD
authors.lastname: Geiger
authors.affiliation: Clinical Sensoring and Monitoring, Anesthesiology and Intensive Care Medicine, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors.forename: Edmund
authors.initials: E
authors.lastname: Koch
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Dresden, Germany.
authors.forename: Gabriele
authors.initials: G
authors.lastname: Schackert
authors.affiliation: Clinical Sensoring and Monitoring, Anesthesiology and Intensive Care Medicine, Faculty of Medicine Carl Gustav Carus, TU Dresden, Dresden, Germany.
authors.forename: Gerald
authors.initials: G
authors.lastname: Steiner
authors.affiliation: Neurosurgery, Carl Gustav Carus University Hospital, TU Dresden, Dresden, Germany; CRTD/DFG-Center for Regenerative Therapies Dresden - Cluster of Excellence, Dresden, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Kirsch
mesh_terms: D000818:Animals; D001921:Brain / Q000166:cytology*; D045744:Cell Line, Tumor; D016616:Cryoultramicrotomy; D005453:Fluorescence; D006801:Humans; D021621:Imaging, Three-Dimensional; D007834:Lasers; D008027:Light; D008819:Mice, Nude; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D013194:Staining and Labeling