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.
Highlights
Avoiding light-induced toxicity is a fundamental and poorly understood bottleneck in long-term microscopic imaging of live embryos
Live Drosophila or zebrafish embryos can be observed with subcellular three-dimensional (3D) resolution using confocal microscopy, photodamage limits the temporal and spatial resolution that can be achieved without perturbing their development
On third-harmonic generation (THG) images the cellularization front invagination (CFI) contrast arises from the local change in the lipid droplet concentration: during phase 3, droplets accumulate around the CFI, giving rise to a positive contrast, whereas at a later stage, the CFI correspond to a depletion zone within a dense droplet region, thereby inducing a local decrease in the signal
Summary
Avoiding light-induced toxicity is a fundamental and poorly understood bottleneck in long-term microscopic imaging of live embryos. Multiphoton (or nonlinear) microscopy uses excitation wavelengths in the near-infrared (NIR) range (700– 1300 nm), and is attractive for embryo studies because it combines a good penetration depth and a limited perturbation of development compared to confocal microscopy, as shown in [3]. This latter property has been attributed to the reduced one-photon (or linear) absorption of cell components in the NIR range. It is of critical importance to investigate the influence of illumination parameters on embryonic tissue and to define guidelines for safe imaging
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