Photoporation of Biomolecules into Single Cells in Living Vertebrate Embryos Induced by a Femtosecond Laser Amplifier
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{"title"=>"Photoporation of biomolecules into single cells in living vertebrate embryos induced by a femtosecond laser amplifier", "type"=>"journal", "authors"=>[{"first_name"=>"Yoichiroh", "last_name"=>"Hosokawa", "scopus_author_id"=>"55565357200"}, {"first_name"=>"Haruki", "last_name"=>"Ochi", "scopus_author_id"=>"35234621400"}, {"first_name"=>"Takanori", "last_name"=>"Iino", "scopus_author_id"=>"36663610900"}, {"first_name"=>"Akihiro", "last_name"=>"Hiraoka", "scopus_author_id"=>"54580955300"}, {"first_name"=>"Mikiko", "last_name"=>"Tanaka", "scopus_author_id"=>"55502235900"}], "year"=>2011, "source"=>"PLoS ONE", "identifiers"=>{"issn"=>"19326203", "scopus"=>"2-s2.0-81155131676", "pui"=>"362925617", "doi"=>"10.1371/journal.pone.0027677", "isbn"=>"1932-6203 (Electronic)\r1932-6203 (Linking)", "sgr"=>"81155131676", "pmid"=>"22110717"}, "id"=>"3653d509-708f-3ad7-a362-e838fb4ba2de", "abstract"=>"Introduction of biomolecules into cells in living animals is one of the most important techniques in molecular and developmental biology research, and has potentially broad biomedical implications. Here we report that biomolecules can be introduced into single cells in living vertebrate embryos by photoporation using a femtosecond laser amplifier with a high pulse energy and a low repetition rate. First, we confirmed the efficiency of this photoporation technique by introducing dextran, morpholino oligonucleotides, or DNA plasmids into targeted single cells of zebrafish, chick, shark, and mouse embryos. Second, we demonstrated that femtosecond laser irradiation efficiently delivered DNA plasmids into single neurons of chick embryos. Finally, we successfully manipulated the fate of single neurons in zebrafish embryos by delivering mRNA. Our observations suggest that photoporation using a femtosecond laser with a high pulse energy and low repetition rate offers a novel way to manipulate the function(s) of individual cells in a wide range of vertebrate embryos by introduction of selected biomolecules.", "link"=>"http://www.mendeley.com/research/photoporation-biomolecules-single-cells-living-vertebrate-embryos-induced-femtosecond-laser-amplifie", "reader_count"=>26, "reader_count_by_academic_status"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>8, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Student > Master"=>3, "Student > Bachelor"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_user_role"=>{"Unspecified"=>1, "Professor > Associate Professor"=>3, "Researcher"=>8, "Student > Doctoral Student"=>1, "Student > Ph. D. Student"=>6, "Student > Postgraduate"=>1, "Student > Master"=>3, "Student > Bachelor"=>1, "Lecturer > Senior Lecturer"=>1, "Professor"=>1}, "reader_count_by_subject_area"=>{"Engineering"=>3, "Unspecified"=>1, "Biochemistry, Genetics and Molecular Biology"=>2, "Agricultural and Biological Sciences"=>8, "Medicine and Dentistry"=>2, "Physics and Astronomy"=>10}, "reader_count_by_subdiscipline"=>{"Engineering"=>{"Engineering"=>3}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>2}, "Physics and Astronomy"=>{"Physics and Astronomy"=>10}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>8}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>2}, "Unspecified"=>{"Unspecified"=>1}}, "reader_count_by_country"=>{"United Kingdom"=>2, "Germany"=>1}, "group_count"=>1}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/711442"], "description"=>"<p>(A) Experimental setup for targeted introduction of biomolecules with femtosecond laser irradiation. Specifications are described in <a href=\"http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027677#s4\" target=\"_blank\">Materials and Methods</a>. (B) Schematic representation of targeted delivery to single cells of zebrafish embryos. FITC-MO, FITC-dextran, or mini-ruby dextran was injected into the chorion cavity of anesthetized embryos mounted in methylcellulose solution. The femtosecond laser pulse train was focused on the surface of single cells. (C) FITC-MO was detected in three targeted single cells (arrowheads). Inset: FITC-MO was localized to the nucleus (N) of the targeted cell. (D) Success rate of delivery of FITC-MO into 28-hpf zebrafish epithelial cells. (E, F) Success rate of delivery of mini-ruby dextran into 25- to 26-hpf (E) and 30-hpf (F) zebrafish epithelial cells. (G) Mini-ruby dextran was detected in a single targeted cell (arrowhead) of a 25- to 26-hpf zebrafish embryo. Inset: Magnification of the targeted cell. (H) At 24 h after introduction of FITC-dextran, FITC fluorescence was detected in newly divided cells (arrowheads). n, number of individual experiments. Scale bars: 250 µm (C, G); 50 µm (insets in C, G); 100 µm (H).</p>", "links"=>[], "tags"=>["photoporation", "mos", "dextran", "cells", "zebrafish"], "article_id"=>381800, "categories"=>["Developmental Biology"], "users"=>["Yoichiroh Hosokawa", "Haruki Ochi", "Takanori Iino", "Akihiro Hiraoka", "Mikiko Tanaka"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027677.g001", "stats"=>{"downloads"=>1, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_NIR_fs_Laser_Photoporation_of_MOs_or_dextran_into_single_cells_of_zebrafish_embryos_/381800", "title"=>"NIR-fs Laser Photoporation of MOs or dextran into single cells of zebrafish embryos.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-11-16 00:30:00"}
  • {"files"=>["https://ndownloader.figshare.com/files/711662"], "description"=>"<p>Mini-ruby dextran was injected into the neural tube of mouse (A, B) and shark (C, D) embryos, and the femtosecond laser was used to irradiate targeted single neurons. (A) Schematic of targeted delivery to single neurons of E9 mouse embryos. (B) Lateral view of the head region in bright field. (B′) Higher magnification of the midbrain region shown in B. Dextran was identified in a single neuron (white arrowheads). (C) Side view of a cultured stage 29 shark embryo prior to the photoporation. Red arrowhead indicates the spot where the laser was focused. (D) Dorsal view of the head region. Dextran was identified in a single neuron (white arrowheads). (D′) Higher magnification of the hindbrain region shown in D. h, heart; hb, hindbrain; mb, midbrain. Scale bars: 200 µm (B, B′, D′); 2 mm (C); 1 mm (D).</p>", "links"=>[], "tags"=>["biomolecules", "neurons"], "article_id"=>382024, "categories"=>["Developmental Biology"], "users"=>["Yoichiroh Hosokawa", "Haruki Ochi", "Takanori Iino", "Akihiro Hiraoka", "Mikiko Tanaka"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027677.g003", "stats"=>{"downloads"=>4, "page_views"=>6, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Targeted_introduction_of_biomolecules_into_single_neurons_of_mouse_and_shark_embryos_/382024", "title"=>"Targeted introduction of biomolecules into single neurons of mouse and shark embryos.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-11-16 00:33:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/711784"], "description"=>"<p>Targeted introduction of dn<i>PKA</i> mRNA induced floor plate marker gene expression in single dorsal neurons of zebrafish embryos. (A) Schematic illustration of dn<i>PKA</i> mRNA delivery to single dorsal neurons of zebrafish embryos. dn<i>PKA</i> mRNA was injected into the cavity between the chorion and the surface of embryo, and dorsal neurons in the neural keel (center) were irradiated at 13–14 hpf with a femtosecond laser pulse (red triangle). Irradiated embryos were fixed at 24 hpf (right panel) and processed for <i>in situ</i> hybridization. (B–E) Expression of the floor plate marker <i>spon1b</i> in embryos injected with control <i>EGFP</i> RNA (B) and dn<i>PKA</i> RNA (C–E) at 24 hpf. (B–D) Lateral view; (E) transverse section. In the control embryo (B), <i>spon1b</i> transcripts were detected only in the floor plate. In the dn<i>PKA</i> RNA-injected embryo, ectopic <i>spon1b</i> signals were observed in individual cells (arrowheads in C, E). (D) Expression of the floor plate marker <i>spon1b</i> (purple) and localization of biotin-dextran (brown) in embryos injected with dn<i>PKA</i> RNA at 24 hpf. Ectopic expression of <i>spon1b</i> was only detected in cells photoporated with biotin-dextran (black arrowhead). Although dextran was also observed in the adjacent dividing cell (open arrowhead), ectopic <i>spon1b</i> expression was barely detected in this cell. CNS, central nervous system; D, dorsal; FP, floor plate; V, ventral. Scale bars: 100 µm (B, C); 50 µm (D, E).</p>", "links"=>[], "tags"=>["targeted", "cells", "zebrafish"], "article_id"=>382140, "categories"=>["Developmental Biology"], "users"=>["Yoichiroh Hosokawa", "Haruki Ochi", "Takanori Iino", "Akihiro Hiraoka", "Mikiko Tanaka"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027677.g004", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Manipulation_of_the_fate_of_targeted_cells_in_zebrafish_embryos_/382140", "title"=>"Manipulation of the fate of targeted cells in zebrafish embryos.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-11-16 00:35:40"}
  • {"files"=>["https://ndownloader.figshare.com/files/711560"], "description"=>"<p>(A) Schematic representation of targeted delivery to single cells of chick embryos. Mini-ruby dextran and pCAGGS-EGFP were injected between the vitelline membrane and the embryo in the New culture system, and the femtosecond laser was focused on targeted cells. (B) Success rate of delivery of mini-ruby dextran into stage 15 chick epithelial cells. (C) At 15 h after introduction, the fluorescence of both mini-ruby dextran (red) and EGFP (green) transcribed from the plasmid vector was detected in an actively dividing cell (white arrowhead). (D) Success rate of delivery of mini-ruby dextran and pCAGGS-EGFP into stage 14–16 chick epithelial cells. (E–G) pCAGGS-RFP was transfected into single targeted cells in chick embryos. DNA was injected into the lumen of the neural tube, and the femtosecond laser was focused on individual cells. (E) At 15 h after introduction, dorsal view shows RFP labeling of targeted neurons (arrowheads). (F–H) At 15 h, transverse sections were visualized with DAPI (blue) and RFP antibody (red). (F) Merged image of anti-RFP (G) and DAPI. RFP was identified in a single neuron and its axon in the neural tube (arrowheads). (H) Higher magnification of transfected neuron in F and G. The nucleus (N) was identified by DAPI. D, dorsal; N, nucleus; NT, neural tube; V, ventral; n, numbers of individual experiments. Scale bars: 50 µm (left in C; E, F, G); 10 µm (right in C); 20 µm (H).</p>", "links"=>[], "tags"=>["photoporation", "dextran", "dna", "cells", "chick"], "article_id"=>381917, "categories"=>["Developmental Biology"], "users"=>["Yoichiroh Hosokawa", "Haruki Ochi", "Takanori Iino", "Akihiro Hiraoka", "Mikiko Tanaka"], "doi"=>"https://dx.doi.org/10.1371/journal.pone.0027677.g002", "stats"=>{"downloads"=>3, "page_views"=>12, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_NIR_fs_Laser_Photoporation_of_dextran_or_DNA_into_single_cells_of_chick_embryos_/381917", "title"=>"NIR-fs Laser Photoporation of dextran or DNA into single cells of chick embryos.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-11-16 00:31:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/361038", "https://ndownloader.figshare.com/files/361103", "https://ndownloader.figshare.com/files/361242", "https://ndownloader.figshare.com/files/361288", "https://ndownloader.figshare.com/files/361360"], "description"=>"<div><p>Introduction of biomolecules into cells in living animals is one of the most important techniques in molecular and developmental biology research, and has potentially broad biomedical implications. Here we report that biomolecules can be introduced into single cells in living vertebrate embryos by photoporation using a femtosecond laser amplifier with a high pulse energy and a low repetition rate. First, we confirmed the efficiency of this photoporation technique by introducing dextran, morpholino oligonucleotides, or DNA plasmids into targeted single cells of zebrafish, chick, shark, and mouse embryos. Second, we demonstrated that femtosecond laser irradiation efficiently delivered DNA plasmids into single neurons of chick embryos. Finally, we successfully manipulated the fate of single neurons in zebrafish embryos by delivering mRNA. Our observations suggest that photoporation using a femtosecond laser with a high pulse energy and low repetition rate offers a novel way to manipulate the function(s) of individual cells in a wide range of vertebrate embryos by introduction of selected biomolecules.</p> </div>", "links"=>[], "tags"=>["photoporation", "biomolecules", "cells", "vertebrate", "embryos", "induced", "femtosecond", "amplifier"], "article_id"=>131382, "categories"=>["Developmental Biology"], "users"=>["Yoichiroh Hosokawa", "Haruki Ochi", "Takanori Iino", "Akihiro Hiraoka", "Mikiko Tanaka"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0027677.s001", "https://dx.doi.org/10.1371/journal.pone.0027677.s002", "https://dx.doi.org/10.1371/journal.pone.0027677.s003", "https://dx.doi.org/10.1371/journal.pone.0027677.s004", "https://dx.doi.org/10.1371/journal.pone.0027677.s005"], "stats"=>{"downloads"=>23, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/Photoporation_of_Biomolecules_into_Single_Cells_in_Living_Vertebrate_Embryos_Induced_by_a_Femtosecond_Laser_Amplifier/131382", "title"=>"Photoporation of Biomolecules into Single Cells in Living Vertebrate Embryos Induced by a Femtosecond Laser Amplifier", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-11-16 00:23:02"}

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Relative Metric

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