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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  µ m 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.

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