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Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 µm wavelength range.

Débarre D, Olivier N, Supatto W, Beaurepaire E - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

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Mechanisms of light-induced cell perturbations.Summary of the main processes involved at the onset of photoperturbation during near infrared imaging, as discussed in [13], [14], [43]. See text for more details.
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pone-0104250-g001: Mechanisms of light-induced cell perturbations.Summary of the main processes involved at the onset of photoperturbation during near infrared imaging, as discussed in [13], [14], [43]. See text for more details.

Mentions: In order to analyse the origin of the damage observed in our experiments, we first present a brief summary of the various photo-perturbation effects described in the literature. A wealth of studies has been published on the effects of light on biological tissues, which correspond to very diverse illumination conditions. However, despite major experimental differences in terms of excitation wavelength, illumination time or pulse duration, the described effects share common features and can all be included in a general diagram (figure 1). In short, four main types of experimental conditions have been studied:


Mitigating phototoxicity during multiphoton microscopy of live Drosophila embryos in the 1.0-1.2 µm wavelength range.

Débarre D, Olivier N, Supatto W, Beaurepaire E - PLoS ONE (2014)

Mechanisms of light-induced cell perturbations.Summary of the main processes involved at the onset of photoperturbation during near infrared imaging, as discussed in [13], [14], [43]. See text for more details.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4128758&req=5

pone-0104250-g001: Mechanisms of light-induced cell perturbations.Summary of the main processes involved at the onset of photoperturbation during near infrared imaging, as discussed in [13], [14], [43]. See text for more details.
Mentions: In order to analyse the origin of the damage observed in our experiments, we first present a brief summary of the various photo-perturbation effects described in the literature. A wealth of studies has been published on the effects of light on biological tissues, which correspond to very diverse illumination conditions. However, despite major experimental differences in terms of excitation wavelength, illumination time or pulse duration, the described effects share common features and can all be included in a general diagram (figure 1). In short, four main types of experimental conditions have been studied:

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus