Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 mm Wavelength Range

Delphine Débarre (1, 2) , Nicolas Olivier (2) , Willy Supatto (2) , Emmanuel Beaurepaire (2)

1 LIPhy - Laboratoire Interdisciplinaire de Physique [Saint Martin d’Hères]
2 LOB - Laboratoire d'optique et biosciences

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.

Domaines: Optique [physics.optics]

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, 2014, pp.0104250. ⟨10.1371/journal.pone.0104250⟩. ⟨hal-01079004⟩