Mitigating Phototoxicity during Multiphoton Microscopy of Live Drosophila Embryos in the 1.0–1.2 µm Wavelength Range
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{"title"=>"Mitigating phototoxicity during multiphoton microscopy of live drosophila embryos in the 1.0-1.2 μm wavelength range", "type"=>"journal", "authors"=>[{"first_name"=>"Delphine", "last_name"=>"Débarre", "scopus_author_id"=>"7004436970"}, {"first_name"=>"Nicolas", "last_name"=>"Olivier", "scopus_author_id"=>"56732670000"}, {"first_name"=>"Willy", "last_name"=>"Supatto", "scopus_author_id"=>"7801684070"}, {"first_name"=>"Emmanuel", "last_name"=>"Beaurepaire", "scopus_author_id"=>"7006318230"}], "year"=>2014, "source"=>"PLoS ONE", "identifiers"=>{"pmid"=>"25111506", "doi"=>"10.1371/journal.pone.0104250", "sgr"=>"84905842354", "scopus"=>"2-s2.0-84905842354", "issn"=>"19326203", "pui"=>"373746937"}, "id"=>"3229af97-172d-3c29-964c-01031989a8b5", "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.", "link"=>"http://www.mendeley.com/research/mitigating-phototoxicity-during-multiphoton-microscopy-live-drosophila-embryos-1012-%CE%BCm-wavelength-ra", "reader_count"=>49, "reader_count_by_academic_status"=>{"Professor > Associate Professor"=>1, "Researcher"=>21, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>14, "Student > Postgraduate"=>2, "Student > Master"=>4, "Student > Bachelor"=>3, "Professor"=>2}, "reader_count_by_user_role"=>{"Professor > Associate Professor"=>1, "Researcher"=>21, "Student > Doctoral Student"=>2, "Student > Ph. D. Student"=>14, "Student > Postgraduate"=>2, "Student > Master"=>4, "Student > Bachelor"=>3, "Professor"=>2}, "reader_count_by_subject_area"=>{"Engineering"=>6, "Biochemistry, Genetics and Molecular Biology"=>1, "Agricultural and Biological Sciences"=>16, "Neuroscience"=>1, "Physics and Astronomy"=>18, "Chemistry"=>5, "Immunology and Microbiology"=>1, "Medicine and Dentistry"=>1}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>6}, "Neuroscience"=>{"Neuroscience"=>1}, "Chemistry"=>{"Chemistry"=>5}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>1}, "Physics and Astronomy"=>{"Physics and Astronomy"=>18}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>1}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>16}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>1}}, "reader_count_by_country"=>{"Netherlands"=>1, "Italy"=>1, "Switzerland"=>1, "Germany"=>1}, "group_count"=>3}

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/1631278"], "description"=>"<p>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.</p>", "links"=>[], "tags"=>["developmental biology", "embryology", "embryo development", "Imaging techniques", "In vivo imaging", "microscopy", "Light microscopy", "optical microscopy", "photoperturbation", "thg", "imaging", "cellularization"], "article_id"=>1135554, "categories"=>["Biological Sciences"], "users"=>["Delphine Débarre", "Nicolas Olivier", "Willy Supatto", "Emmanuel Beaurepaire"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0104250.g002", "stats"=>{"downloads"=>4, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Assessing_photoperturbation_using_THG_imaging_of_cellularization_dynamics_in_Drosophila_embryos_/1135554", "title"=>"Assessing photoperturbation using THG imaging of cellularization dynamics in <i>Drosophila</i> embryos.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-11 03:42:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/1631282"], "description"=>"<p>(A,B) Cumulative effects and wavelength dependence. (A), embryo survival rate and (B), CFI speed (see <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104250#pone-0104250-g002\" target=\"_blank\">figure 2</a>) as a function of excitation wavelength and illumination rate. (C,D) Effect of the spatial spreading of the illuminated planes. (C) embryo survival rate, and (D) CFI rate, for volume (red) and single plane (black) imaging. Volume imaging was achieved by acquiring 2,3,4,6 or 18 images (3.2 s per image) axially separated by 2 <i>µ</i>m every 60 s, whereas for single plane imaging the same number of images where acquired always in the same plane (see cartoons in (C)). The survival rate is not significantly decreased when the embryo is continuously illuminated provided that the imaging rate in each plane is kept low (1 image/minute). (E,F) Influence of the pulse duration on development photoperturbation. THG efficiency, scaling as P<sup>3</sup>/, is kept constant. (E), embryo survival rate, and (F), CFI speed, as a function of imaging rate. All error bars are the standard deviation of the mean over 11 measurements for each data point.</p>", "links"=>[], "tags"=>["developmental biology", "embryology", "embryo development", "Imaging techniques", "In vivo imaging", "microscopy", "Light microscopy", "optical microscopy", "imaging", "light-induced", "perturbation", "multiphoton", "embryos"], "article_id"=>1135558, "categories"=>["Biological Sciences"], "users"=>["Delphine Débarre", "Nicolas Olivier", "Willy Supatto", "Emmanuel Beaurepaire"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0104250.g003", "stats"=>{"downloads"=>2, "page_views"=>14, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Influence_of_imaging_parameters_on_light_induced_perturbation_during_multiphoton_imaging_of_Drosophila_embryos_at_1_18_181_m_/1135558", "title"=>"Influence of imaging parameters on light-induced perturbation during multiphoton imaging of <i>Drosophila</i> embryos at 1.18 <i>µ</i>m.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-11 03:42:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/1631316"], "description"=>"<p>(A) 3D dynamic visualisation of half a wild-type <i>Drosophila</i> embryo during gastrulation. Imaging conditions: 57 s per 3D stack, 2 min between successive stacks, 750 nm/pixel lateral sampling, 2 <i>µ</i>m/pixel axial sampling, 1180 nm excitation wavelength, 100 fs pulses. (A), sagittal view (through the ventral furrow). Left, cellularization (stage 5); right, gastrulation (stage 8). Red arrow, CFI. Green arrow, cephalic furrow. (B) and movie 3, ventral furrow formation visualised over a transverse slice of a half-embryo image acquired in successive frontal (coronal) planes. (C) THG imaging of the ventral side of Sna- (left) and wild-type (right) <i>Drosophila</i> embryos during gastrulation. Imaging conditions: 40 s per 3D stack, 750 nm/pixel lateral sampling, 3 <i>µ</i>m/pixel axial sampling, 3 <i>µ</i><i>s</i>/pixel integration time, 30 planes by stack, continuous imaging, 1180 nm excitation wavelength, 100 fs pulses. See also movie 4.</p>", "links"=>[], "tags"=>["developmental biology", "embryology", "embryo development", "Imaging techniques", "In vivo imaging", "microscopy", "Light microscopy", "optical microscopy", "label-free", "characterization", "gastrulation", "movements", "wild-type", "mutant"], "article_id"=>1135578, "categories"=>["Biological Sciences"], "users"=>["Delphine Débarre", "Nicolas Olivier", "Willy Supatto", "Emmanuel Beaurepaire"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0104250.g005", "stats"=>{"downloads"=>7, "page_views"=>23, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_3D_label_free_characterization_of_gastrulation_movements_in_wild_type_and_mutant_Drosophila_embryos_/1135578", "title"=>"3D, label-free characterization of gastrulation movements in wild-type and mutant <i>Drosophila</i> embryos.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-11 03:42:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/1631317"], "description"=>"<p>Illumination parameters used in this work.</p>", "links"=>[], "tags"=>["developmental biology", "embryology", "embryo development", "Imaging techniques", "In vivo imaging", "microscopy", "Light microscopy", "optical microscopy"], "article_id"=>1135579, "categories"=>["Biological Sciences"], "users"=>["Delphine Débarre", "Nicolas Olivier", "Willy Supatto", "Emmanuel Beaurepaire"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0104250.t001", "stats"=>{"downloads"=>5, "page_views"=>9, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Illumination_parameters_used_in_this_work_/1135579", "title"=>"Illumination parameters used in this work.", "pos_in_sequence"=>0, "defined_type"=>3, "published_date"=>"2014-08-11 03:42:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/1631267"], "description"=>"<p>Summary of the main processes involved at the onset of photoperturbation during near infrared imaging, as discussed in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104250#pone.0104250-Karu1\" target=\"_blank\">[13]</a>, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104250#pone.0104250-Hockberger1\" target=\"_blank\">[14]</a>, <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104250#pone.0104250-Vogel1\" target=\"_blank\">[43]</a>. See text for more details.</p>", "links"=>[], "tags"=>["developmental biology", "embryology", "embryo development", "Imaging techniques", "In vivo imaging", "microscopy", "Light microscopy", "optical microscopy", "light-induced"], "article_id"=>1135548, "categories"=>["Biological Sciences"], "users"=>["Delphine Débarre", "Nicolas Olivier", "Willy Supatto", "Emmanuel Beaurepaire"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0104250.g001", "stats"=>{"downloads"=>2, "page_views"=>11, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Mechanisms_of_light_induced_cell_perturbations_/1135548", "title"=>"Mechanisms of light-induced cell perturbations.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-11 03:42:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/1631295"], "description"=>"<p>2D THG-SHG imaging at a wavelength of 1180 nm of a wild-type <i>Drosophila</i> 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 <i>µ</i>m. See also movie 2 for the full dataset.</p>", "links"=>[], "tags"=>["developmental biology", "embryology", "embryo development", "Imaging techniques", "In vivo imaging", "microscopy", "Light microscopy", "optical microscopy", "thg-shg", "imaging", "embryonic"], "article_id"=>1135570, "categories"=>["Biological Sciences"], "users"=>["Delphine Débarre", "Nicolas Olivier", "Willy Supatto", "Emmanuel Beaurepaire"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0104250.g004", "stats"=>{"downloads"=>1, "page_views"=>15, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Long_term_THG_SHG_imaging_of_Drosophila_embryonic_development_/1135570", "title"=>"Long-term THG-SHG imaging of <i>Drosophila</i> embryonic development.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2014-08-11 03:42:50"}
  • {"files"=>["https://ndownloader.figshare.com/files/1631347", "https://ndownloader.figshare.com/files/1631348", "https://ndownloader.figshare.com/files/1631349", "https://ndownloader.figshare.com/files/1631350"], "description"=>"<div><p>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 <i>Drosophila</i> 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 <i>µ</i>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 <i>Drosophila</i> 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.</p></div>", "links"=>[], "tags"=>["developmental biology", "embryology", "embryo development", "Imaging techniques", "In vivo imaging", "microscopy", "Light microscopy", "optical microscopy", "mitigating", "phototoxicity", "multiphoton", "embryos", "wavelength"], "article_id"=>1135599, "categories"=>["Biological Sciences"], "users"=>["Delphine Débarre", "Nicolas Olivier", "Willy Supatto", "Emmanuel Beaurepaire"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0104250.s001", "https://dx.doi.org/10.1371/journal.pone.0104250.s002", "https://dx.doi.org/10.1371/journal.pone.0104250.s003", "https://dx.doi.org/10.1371/journal.pone.0104250.s004"], "stats"=>{"downloads"=>6, "page_views"=>18, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Mitigating_Phototoxicity_during_Multiphoton_Microscopy_of_Live_Drosophila_Embryos_in_the_1_0_1_2_181_m_Wavelength_Range/1135599", "title"=>"Mitigating Phototoxicity during Multiphoton Microscopy of Live <i>Drosophila</i> Embryos in the 1.0–1.2 <i>µ</i>m Wavelength Range", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2014-08-11 03:42:50"}

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