title: Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 µm wavelength range.
abstract: Light-induced toxicity is a fundamental bottleneck in microscopic imaging of live embryos. In this article, after a review of photodamage mechanisms in cells and tissues, we assess photo-perturbation under illumination conditions relevant for point-scanning multiphoton imaging of live Drosophila embryos. We use third-harmonic generation (THG) imaging of developmental processes in embryos excited by pulsed near-infrared light in the 1.0-1.2 µm range. We study the influence of imaging rate, wavelength, and pulse duration on the short-term and long-term perturbation of development and define criteria for safe imaging. We show that under illumination conditions typical for multiphoton imaging, photodamage in this system arises through 2- and/or 3-photon absorption processes and in a cumulative manner. Based on this analysis, we derive general guidelines for improving the signal-to-damage ratio in two-photon (2PEF/SHG) or THG imaging by adjusting the pulse duration and/or the imaging rate. Finally, we report label-free time-lapse 3D THG imaging of gastrulating Drosophila embryos with sampling appropriate for the visualisation of morphogenetic movements in wild-type and mutant embryos, and long-term multiharmonic (THG-SHG) imaging of development until hatching.
authors.affiliation: Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS UMR 7645, and INSERM U696, Palaiseau, France; Univ. Grenoble Alpes, LIPhy, Grenoble, France; CNRS, LIPhy, Grenoble, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Débarre
authors.affiliation: Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS UMR 7645, and INSERM U696, Palaiseau, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Olivier
authors.affiliation: Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS UMR 7645, and INSERM U696, Palaiseau, France.
authors.forename: Willy
authors.initials: W
authors.lastname: Supatto
authors.affiliation: Laboratory for Optics and Biosciences, Ecole Polytechnique, CNRS UMR 7645, and INSERM U696, Palaiseau, France.
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Beaurepaire
mesh_terms: D000818:Animals; D004331:Drosophila melanogaster / Q000196:embryology*; D004625:Embryo, Nonmammalian; D021621:Imaging, Three-Dimensional; D008027:Light / Q000009:adverse effects*; D036641:Microscopy, Fluorescence, Multiphoton / Q000009:adverse effects* / Q000379:methods*; D055620:Optical Phenomena; D013997:Time Factors
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