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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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Assessing photoperturbation using THG imaging of cellularization dynamics in Drosophila embryos.Principle of cellular front invagination (CFI) speed measurement: (A), transmitted light imaging (wild-type embryo); (B), two-photon imaging (GFP-moesin-tagged embryo, outlining the cell boundaries); (C) and movie 1, THG imaging (wild-type embryo). The images in (A) – (C) are a zoom over the dorsal equatorial region of different embryos, corresponding approximately to the blue square in (D) on a THG image. Images (A) to (C) share the same scale bars. Top, phase 3 of cellularization; middle, phase 4 of cellularization; bottom, kymographs (YT projections) obtained from the time-lapse XY images, showing the propagation of the CFI over time. The dotted black time indicates the limit between phase 3 and phase 4, and the position of the CFI is indicated by a red (resp. green) line in phase 3 (resp. 4). Kymographs shown here as an example were obtained from time-lapse acquisitions with (A) 2 images/min; (B), 1 image/min; (C), 3 images/min. (E), CFI speed calibration as a function of temperature using transmitted light imaging. Errors bars are the standard deviations from 3 different embryos per temperature point.
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pone-0104250-g002: Assessing photoperturbation using THG imaging of cellularization dynamics in Drosophila embryos.Principle of cellular front invagination (CFI) speed measurement: (A), transmitted light imaging (wild-type embryo); (B), two-photon imaging (GFP-moesin-tagged embryo, outlining the cell boundaries); (C) and movie 1, THG imaging (wild-type embryo). The images in (A) – (C) are a zoom over the dorsal equatorial region of different embryos, corresponding approximately to the blue square in (D) on a THG image. Images (A) to (C) share the same scale bars. Top, phase 3 of cellularization; middle, phase 4 of cellularization; bottom, kymographs (YT projections) obtained from the time-lapse XY images, showing the propagation of the CFI over time. The dotted black time indicates the limit between phase 3 and phase 4, and the position of the CFI is indicated by a red (resp. green) line in phase 3 (resp. 4). Kymographs shown here as an example were obtained from time-lapse acquisitions with (A) 2 images/min; (B), 1 image/min; (C), 3 images/min. (E), CFI speed calibration as a function of temperature using transmitted light imaging. Errors bars are the standard deviations from 3 different embryos per temperature point.

Mentions: In a complementary manner, we have used the dynamics of the cellularization process as a probe of short-term perturbation of development, and in particular of the integrity of the cytoskeleton (fig. 2): indeed, Drosophila embryos during the first stages of development consist of a multinuclear cell (syncytial blastoderm), and the cell membranes separating the nuclei invaginate towards the center of the embryo during cellularization (stage 5 of development as defined in [52]). Cellularization is used as a model for cell cytokinesis [53]. We have measured the speed (see section 6.3) of the cellularization front invagination (CFI) during the latest phases of cellularization (phases 3 and 4, as defined in [54]). First, the speed of CFI was measured using transmitted light microscopy as a function of temperature (figure 2(E), see [5] for more details). These values were used as a control in later experiments. Then, we verified that CFI measurements were consistent when imaging embryos using transmitted-light (TL), 2PEF, and THG. CFI speed measurements were then systematically conducted using the THG images acquired during illumination of unlabelled embryos. One example of typical images of the CFI and the resulting kymographs is shown on figure 2. On THG images the 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. Despite this change in contrast, the signal modulation ensures a precision of typically 0.1 µm/min for the measurement of CFI speed.


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)

Assessing photoperturbation using THG imaging of cellularization dynamics in Drosophila embryos.Principle of cellular front invagination (CFI) speed measurement: (A), transmitted light imaging (wild-type embryo); (B), two-photon imaging (GFP-moesin-tagged embryo, outlining the cell boundaries); (C) and movie 1, THG imaging (wild-type embryo). The images in (A) – (C) are a zoom over the dorsal equatorial region of different embryos, corresponding approximately to the blue square in (D) on a THG image. Images (A) to (C) share the same scale bars. Top, phase 3 of cellularization; middle, phase 4 of cellularization; bottom, kymographs (YT projections) obtained from the time-lapse XY images, showing the propagation of the CFI over time. The dotted black time indicates the limit between phase 3 and phase 4, and the position of the CFI is indicated by a red (resp. green) line in phase 3 (resp. 4). Kymographs shown here as an example were obtained from time-lapse acquisitions with (A) 2 images/min; (B), 1 image/min; (C), 3 images/min. (E), CFI speed calibration as a function of temperature using transmitted light imaging. Errors bars are the standard deviations from 3 different embryos per temperature point.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4128758&req=5

pone-0104250-g002: Assessing photoperturbation using THG imaging of cellularization dynamics in Drosophila embryos.Principle of cellular front invagination (CFI) speed measurement: (A), transmitted light imaging (wild-type embryo); (B), two-photon imaging (GFP-moesin-tagged embryo, outlining the cell boundaries); (C) and movie 1, THG imaging (wild-type embryo). The images in (A) – (C) are a zoom over the dorsal equatorial region of different embryos, corresponding approximately to the blue square in (D) on a THG image. Images (A) to (C) share the same scale bars. Top, phase 3 of cellularization; middle, phase 4 of cellularization; bottom, kymographs (YT projections) obtained from the time-lapse XY images, showing the propagation of the CFI over time. The dotted black time indicates the limit between phase 3 and phase 4, and the position of the CFI is indicated by a red (resp. green) line in phase 3 (resp. 4). Kymographs shown here as an example were obtained from time-lapse acquisitions with (A) 2 images/min; (B), 1 image/min; (C), 3 images/min. (E), CFI speed calibration as a function of temperature using transmitted light imaging. Errors bars are the standard deviations from 3 different embryos per temperature point.
Mentions: In a complementary manner, we have used the dynamics of the cellularization process as a probe of short-term perturbation of development, and in particular of the integrity of the cytoskeleton (fig. 2): indeed, Drosophila embryos during the first stages of development consist of a multinuclear cell (syncytial blastoderm), and the cell membranes separating the nuclei invaginate towards the center of the embryo during cellularization (stage 5 of development as defined in [52]). Cellularization is used as a model for cell cytokinesis [53]. We have measured the speed (see section 6.3) of the cellularization front invagination (CFI) during the latest phases of cellularization (phases 3 and 4, as defined in [54]). First, the speed of CFI was measured using transmitted light microscopy as a function of temperature (figure 2(E), see [5] for more details). These values were used as a control in later experiments. Then, we verified that CFI measurements were consistent when imaging embryos using transmitted-light (TL), 2PEF, and THG. CFI speed measurements were then systematically conducted using the THG images acquired during illumination of unlabelled embryos. One example of typical images of the CFI and the resulting kymographs is shown on figure 2. On THG images the 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. Despite this change in contrast, the signal modulation ensures a precision of typically 0.1 µm/min for the measurement of CFI speed.

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