Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 mm 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 mm 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. Citation: Débarre D, Olivier N, Supatto W, Beaurepaire E (2014) Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0– 1.2 mm Wavelength Range. PLoS ONE 9(8): e104250.
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Delphine Débarre, Nicolas Olivier, Willy Supatto, Emmanuel Beaurepaire. Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 mm Wavelength Range. PLoS ONE, Public Library of Science, 2014, pp.0104250. ⟨10.1371/journal.pone.0104250⟩. ⟨hal-01079004⟩

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