title: Carcinogenic damage to deoxyribonucleic acid is induced by near-infrared laser pulses in multiphoton microscopy via combination of two- and three-photon absorption.
abstract: Nonlinear optical imaging modalities (multiphoton excited fluorescence, second and third harmonic generation) applied in vivo are increasingly promising for clinical diagnostics and the monitoring of cancer and other disorders, as they can probe tissue with high diffraction-limited resolution at near-infrared (IR) wavelengths. However, high peak intensity of femtosecond laser pulses required for two-photon processes causes formation of cyclobutane-pyrimidine-dimers (CPDs) in cellular deoxyribonucleic acid (DNA) similar to damage from exposure to solar ultraviolet (UV) light. Inaccurate repair of subsequent mutations increases the risk of carcinogenesis. In this study, we investigate CPD damage that results in Chinese hamster ovary cells in vitro from imaging them with two-photon excited autofluorescence. The CPD levels are quantified by immunofluorescent staining. We further evaluate the extent of CPD damage with respect to varied wavelength, pulse width at focal plane, and pixel dwell time as compared with more pronounced damage from UV sources. While CPD damage has been expected to result from three-photon absorption, our results reveal that CPDs are induced by competing twoand three-photon absorption processes, where the former accesses UVA absorption band. This finding is independently confirmed by nonlinear dependencies of damage on laser power, wavelength, and pulse width.
authors.affiliation: Utrecht University, Debye Institute, Molecular Biophysics, Princetonplein 5, 3508 TA Utrecht, The Netherlands. o.nadyarnykh1@uu.nl
authors.forename: Oleg
authors.initials: O
authors.lastname: Nadiarnykh
authors.forename: Giju
authors.initials: G
authors.lastname: Thomas
authors.forename: Johan
authors.initials: J
authors.lastname: Van Voskuilen
authors.forename: Henricus J C M
authors.initials: HJ
authors.lastname: Sterenborg
authors.forename: Hans C
authors.initials: HC
authors.lastname: Gerritsena
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004247:DNA / Q000737:chemistry / Q000528:radiation effects*; D004249:DNA Damage; D004307:Dose-Response Relationship, Radiation; D007259:Infrared Rays / Q000009:adverse effects; D007834:Lasers / Q000009:adverse effects; D036641:Microscopy, Fluorescence, Multiphoton / Q000009:adverse effects*; D061848:Optical Imaging / Q000009:adverse effects*; D055620:Optical Phenomena; D011740:Pyrimidine Dimers / Q000737:chemistry / Q000528:radiation effects
chemical_list: D011740:Pyrimidine Dimers; D004247:DNA