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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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Long-term THG-SHG imaging of Drosophila embryonic development.2D THG-SHG imaging at a wavelength of 1180 nm of a wild-type Drosophila embryo during 36 hours starting from stage 5 up to the larvae stage (i.e. until hatching) and imaged during 3.3 s every 150 s, corresponding to an imaging rate of 2%. (A–C) representative 2D THG-SHG images at different stages of the development, with stage and time after the beginning of the acquisition mentioned in the bottom left. The look-up-table used to represent the SHG and THG signals are displayed below. Scale bar  = 50 µm. See also movie 2 for the full dataset.
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pone-0104250-g004: Long-term THG-SHG imaging of Drosophila embryonic development.2D THG-SHG imaging at a wavelength of 1180 nm of a wild-type Drosophila embryo during 36 hours starting from stage 5 up to the larvae stage (i.e. until hatching) and imaged during 3.3 s every 150 s, corresponding to an imaging rate of 2%. (A–C) representative 2D THG-SHG images at different stages of the development, with stage and time after the beginning of the acquisition mentioned in the bottom left. The look-up-table used to represent the SHG and THG signals are displayed below. Scale bar  = 50 µm. See also movie 2 for the full dataset.

Mentions: First, we studied the perturbation of development as a function of the time between two successive illuminations of the embryo. More precisely, we considered the effect of the percentage of time during which the embryo was illuminated throughout the time-lapse sequence acquisition (duty cycle, or imaging rate). The results are summarized on figure 3, (A) and (B). Above a given threshold ( imaging rate in our conditions), the survival rate of the embryos drops, revealing significant perturbation of development (figure 3(A), black curve). Concurrently, increase in the speed of CFI both in phases 3 and 4 shows that the perturbation of development occurs within a few minutes after the start of the experiment, and can lead to CFI speeds twice as high as in unperturbed embryos (figure 3(B)). However, when the embryo is illuminated at a sustainable rate, i.e. below the perturbation threshold, its development can be imaged entirely up to the larva stage, as demonstrated using simultaneous THG-SHG imaging on figure 4 and Video S2. This movie was recorded with an imaging rate of (imaging time being 3.3s per image). The total imaging time was 36 hours, which corresponds to over a thousand exposures, and the total illumination dose was comparable to that obtained for continuous exposure of the embryo during developmental stage 5 to 8. Nevertheless, we did not observe morphological perturbation or delay in development, indicating that organism-level damage arises beyond a given imaging rate and not simply beyond a given illumination dose.


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)

Long-term THG-SHG imaging of Drosophila embryonic development.2D THG-SHG imaging at a wavelength of 1180 nm of a wild-type Drosophila embryo during 36 hours starting from stage 5 up to the larvae stage (i.e. until hatching) and imaged during 3.3 s every 150 s, corresponding to an imaging rate of 2%. (A–C) representative 2D THG-SHG images at different stages of the development, with stage and time after the beginning of the acquisition mentioned in the bottom left. The look-up-table used to represent the SHG and THG signals are displayed below. Scale bar  = 50 µm. See also movie 2 for the full dataset.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0104250-g004: Long-term THG-SHG imaging of Drosophila embryonic development.2D THG-SHG imaging at a wavelength of 1180 nm of a wild-type Drosophila embryo during 36 hours starting from stage 5 up to the larvae stage (i.e. until hatching) and imaged during 3.3 s every 150 s, corresponding to an imaging rate of 2%. (A–C) representative 2D THG-SHG images at different stages of the development, with stage and time after the beginning of the acquisition mentioned in the bottom left. The look-up-table used to represent the SHG and THG signals are displayed below. Scale bar  = 50 µm. See also movie 2 for the full dataset.
Mentions: First, we studied the perturbation of development as a function of the time between two successive illuminations of the embryo. More precisely, we considered the effect of the percentage of time during which the embryo was illuminated throughout the time-lapse sequence acquisition (duty cycle, or imaging rate). The results are summarized on figure 3, (A) and (B). Above a given threshold ( imaging rate in our conditions), the survival rate of the embryos drops, revealing significant perturbation of development (figure 3(A), black curve). Concurrently, increase in the speed of CFI both in phases 3 and 4 shows that the perturbation of development occurs within a few minutes after the start of the experiment, and can lead to CFI speeds twice as high as in unperturbed embryos (figure 3(B)). However, when the embryo is illuminated at a sustainable rate, i.e. below the perturbation threshold, its development can be imaged entirely up to the larva stage, as demonstrated using simultaneous THG-SHG imaging on figure 4 and Video S2. This movie was recorded with an imaging rate of (imaging time being 3.3s per image). The total imaging time was 36 hours, which corresponds to over a thousand exposures, and the total illumination dose was comparable to that obtained for continuous exposure of the embryo during developmental stage 5 to 8. Nevertheless, we did not observe morphological perturbation or delay in development, indicating that organism-level damage arises beyond a given imaging rate and not simply beyond a given illumination dose.

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