A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms
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{"title"=>"A genetically encoded tag for correlated light and electron microscopy of intact cells, tissues, and organisms", "type"=>"journal", "authors"=>[{"first_name"=>"Xiaokun", "last_name"=>"Shu", "scopus_author_id"=>"8410525700"}, {"first_name"=>"Varda", "last_name"=>"Lev-Ram", "scopus_author_id"=>"6701638437"}, {"first_name"=>"Thomas J.", "last_name"=>"Deerinck", "scopus_author_id"=>"7003967948"}, {"first_name"=>"Yingchuan", "last_name"=>"Qi", "scopus_author_id"=>"55446928100"}, {"first_name"=>"Ericka B.", "last_name"=>"Ramko", "scopus_author_id"=>"24071945600"}, {"first_name"=>"Michael W.", "last_name"=>"Davidson", "scopus_author_id"=>"7401858367"}, {"first_name"=>"Yishi", "last_name"=>"Jin", "scopus_author_id"=>"7404457596"}, {"first_name"=>"Mark H.", "last_name"=>"Ellisman", "scopus_author_id"=>"7102020469"}, {"first_name"=>"Roger Y.", "last_name"=>"Tsien", "scopus_author_id"=>"7201933927"}], "year"=>2011, "source"=>"PLoS Biology", "identifiers"=>{"sgr"=>"79955504165", "scopus"=>"2-s2.0-79955504165", "doi"=>"10.1371/journal.pbio.1001041", "isbn"=>"1545-7885 (Electronic)\\r1544-9173 (Linking)", "pui"=>"361688637", "issn"=>"15449173", "pmid"=>"21483721"}, "id"=>"2e4fe17c-4d5f-3294-aa84-1467fabcda31", "abstract"=>"Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce \"miniSOG\" (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.", "link"=>"http://www.mendeley.com/research/genetically-encoded-tag-correlated-light-electron-microscopy-intact-cells-tissues-organisms", "reader_count"=>939, "reader_count_by_academic_status"=>{"Unspecified"=>10, "Professor > Associate Professor"=>83, "Researcher"=>313, "Student > Doctoral Student"=>31, "Student > Ph. D. Student"=>276, "Student > Postgraduate"=>26, "Student > Master"=>58, "Other"=>23, "Student > Bachelor"=>50, "Lecturer"=>12, "Lecturer > Senior Lecturer"=>5, "Professor"=>52}, "reader_count_by_user_role"=>{"Unspecified"=>10, "Professor > Associate Professor"=>83, "Researcher"=>313, "Student > Doctoral Student"=>31, "Student > Ph. D. Student"=>276, "Student > Postgraduate"=>26, "Student > Master"=>58, "Other"=>23, "Student > Bachelor"=>50, "Lecturer"=>12, "Lecturer > Senior Lecturer"=>5, "Professor"=>52}, "reader_count_by_subject_area"=>{"Unspecified"=>21, "Agricultural and Biological Sciences"=>631, "Arts and Humanities"=>1, "Chemistry"=>51, "Computer Science"=>6, "Earth and Planetary Sciences"=>1, "Engineering"=>12, "Environmental Science"=>3, "Biochemistry, Genetics and Molecular Biology"=>90, "Materials Science"=>6, "Mathematics"=>1, "Medicine and Dentistry"=>24, "Neuroscience"=>54, "Pharmacology, Toxicology and Pharmaceutical Science"=>1, "Physics and Astronomy"=>26, "Psychology"=>1, "Social Sciences"=>2, "Immunology and Microbiology"=>8}, "reader_count_by_subdiscipline"=>{"Materials Science"=>{"Materials Science"=>6}, "Medicine and Dentistry"=>{"Medicine and Dentistry"=>24}, "Social Sciences"=>{"Social Sciences"=>2}, "Physics and Astronomy"=>{"Physics and Astronomy"=>26}, "Psychology"=>{"Psychology"=>1}, "Mathematics"=>{"Mathematics"=>1}, "Unspecified"=>{"Unspecified"=>21}, "Environmental Science"=>{"Environmental Science"=>3}, "Pharmacology, Toxicology and Pharmaceutical Science"=>{"Pharmacology, Toxicology and Pharmaceutical Science"=>1}, "Arts and Humanities"=>{"Arts and Humanities"=>1}, "Engineering"=>{"Engineering"=>12}, "Chemistry"=>{"Chemistry"=>51}, "Neuroscience"=>{"Neuroscience"=>54}, "Earth and Planetary Sciences"=>{"Earth and Planetary Sciences"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>8}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>631}, "Computer Science"=>{"Computer Science"=>6}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>90}}, "reader_count_by_country"=>{"Republic of Singapore"=>2, "United States"=>59, "Portugal"=>2, "Russia"=>2, "Sweden"=>2, "Netherlands"=>2, "South Korea"=>1, "Austria"=>3, "Korea (South)"=>2, "China"=>1, "Brazil"=>4, "France"=>12, "Japan"=>5, "United Kingdom"=>9, "Switzerland"=>6, "Spain"=>3, "India"=>1, "Canada"=>5, "Czech Republic"=>1, "Belgium"=>1, "Taiwan"=>1, "Denmark"=>2, "Israel"=>2, "Australia"=>1, "Germany"=>21}, "group_count"=>24}

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/785394"], "description"=>"<p>Confocal fluorescence images of miniSOG-targeted endoplasmic reticulum (A), Rab5a (B), zyxin (C), tubulin (D), β-actin (E), α-actinin (F), mitochondria (G), and histone 2B (H) in HeLa cells; scale bars, 10 µm.</p>", "links"=>[], "tags"=>["proteins", "organelles", "localization", "microscopic"], "article_id"=>455757, "categories"=>["Biotechnology", "Biochemistry", "Neuroscience"], "users"=>["Xiaokun Shu", "Varda Lev-Ram", "Thomas J. Deerinck", "Yingchuan Qi", "Ericka B. Ramko", "Michael W. Davidson", "Yishi Jin", "Mark H. Ellisman", "Roger Y. Tsien"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001041.g002", "stats"=>{"downloads"=>0, "page_views"=>8, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MiniSOG_labeled_proteins_and_organelles_exhibit_correct_localization_at_the_light_microscopic_level_/455757", "title"=>"MiniSOG-labeled proteins and organelles exhibit correct localization at the light microscopic level.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-05 01:35:57"}
  • {"files"=>["https://ndownloader.figshare.com/files/785527"], "description"=>"<p>(A) Schematic diagram of how miniSOG produces EM contrast upon blue-light illumination. Spin states are depicted by the arrows. ISC, intersystem crossing. Correlated confocal fluorescence (B,F,J), transmitted light (C,G,K), and electron microscopic (D,E,H,I,L,M) imaging of a variety of proteins. (B–E) HeLa cells expressing miniSOG labeled α-actinin. Arrows denote correlated structures. (F–I) Histone 2B. Panel H is a 3 nm thick computed slice from an electron tomogram. Panel I is a high magnification thin section electron micrograph showing labeled chromatin fibers near the nuclear envelope (arrows) and a nuclear pore (arrowhead). (J–M) Mitochondrial targeted miniSOG. Panels J and K show a confocal image prior to photooxidation and a transmitted light image following photooxidation, respectively. The differential contrast generated between a transfected (arrows) and non-transfected cell (arrowheads) is evident. Bars B–D, 1 micron; E, 200 nm; F–H, 2 microns; I, 100 nm; J–L, 5 microns; M, 200 nm.</p>", "links"=>[], "tags"=>["produces", "correlated", "fluorescence", "em", "localization", "labeled", "proteins"], "article_id"=>455881, "categories"=>["Biotechnology", "Biochemistry", "Neuroscience"], "users"=>["Xiaokun Shu", "Varda Lev-Ram", "Thomas J. Deerinck", "Yingchuan Qi", "Ericka B. Ramko", "Michael W. Davidson", "Yishi Jin", "Mark H. Ellisman", "Roger Y. Tsien"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001041.g003", "stats"=>{"downloads"=>2, "page_views"=>17, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MiniSOG_produces_correlated_fluorescence_and_EM_contrast_with_correct_localization_of_labeled_proteins_and_organelles_/455881", "title"=>"MiniSOG produces correlated fluorescence and EM contrast with correct localization of labeled proteins and organelles.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-05 01:38:01"}
  • {"files"=>["https://ndownloader.figshare.com/files/393833", "https://ndownloader.figshare.com/files/393846", "https://ndownloader.figshare.com/files/393859", "https://ndownloader.figshare.com/files/393862", "https://ndownloader.figshare.com/files/393869", "https://ndownloader.figshare.com/files/393876", "https://ndownloader.figshare.com/files/393883", "https://ndownloader.figshare.com/files/393888", "https://ndownloader.figshare.com/files/393892", "https://ndownloader.figshare.com/files/393903", "https://ndownloader.figshare.com/files/393909", "https://ndownloader.figshare.com/files/393915", "https://ndownloader.figshare.com/files/393924", "https://ndownloader.figshare.com/files/393927", "https://ndownloader.figshare.com/files/393929"], "description"=>"<div><p>Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce “miniSOG” (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from <em>Arabidopsis</em> phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy.</p> </div>", "links"=>[], "tags"=>["genetically", "encoded", "correlated", "electron", "microscopy", "organisms"], "article_id"=>137766, "categories"=>["Biotechnology", "Biochemistry", "Neuroscience"], "users"=>["Xiaokun Shu", "Varda Lev-Ram", "Thomas J. Deerinck", "Yingchuan Qi", "Ericka B. Ramko", "Michael W. Davidson", "Yishi Jin", "Mark H. Ellisman", "Roger Y. Tsien"], "doi"=>["https://dx.doi.org/10.1371/journal.pbio.1001041.s001", "https://dx.doi.org/10.1371/journal.pbio.1001041.s002", "https://dx.doi.org/10.1371/journal.pbio.1001041.s003", "https://dx.doi.org/10.1371/journal.pbio.1001041.s004", "https://dx.doi.org/10.1371/journal.pbio.1001041.s005", "https://dx.doi.org/10.1371/journal.pbio.1001041.s006", "https://dx.doi.org/10.1371/journal.pbio.1001041.s007", "https://dx.doi.org/10.1371/journal.pbio.1001041.s008", "https://dx.doi.org/10.1371/journal.pbio.1001041.s009", "https://dx.doi.org/10.1371/journal.pbio.1001041.s010", "https://dx.doi.org/10.1371/journal.pbio.1001041.s011", "https://dx.doi.org/10.1371/journal.pbio.1001041.s012", "https://dx.doi.org/10.1371/journal.pbio.1001041.s013", "https://dx.doi.org/10.1371/journal.pbio.1001041.s014", "https://dx.doi.org/10.1371/journal.pbio.1001041.s015"], "stats"=>{"downloads"=>3, "page_views"=>21, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Genetically_Encoded_Tag_for_Correlated_Light_and_Electron_Microscopy_of_Intact_Cells_Tissues_and_Organisms/137766", "title"=>"A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-04-05 02:09:26"}
  • {"files"=>["https://ndownloader.figshare.com/files/785719"], "description"=>"<p>(A) The green fluorescence of miniSOG reveals gap junctions and transporting vesicles. (B) Electron microscopy indicates negatively stained structures of appropriate size and spacing to be gap junction channels (arrows). (C) Studs on the membranes of trafficking vesicles suggest single connexons. The arrowhead points to two dots with a center-to-center distance ∼14 nm. (D) A high-quality immunogold image showing a randomly labeled fraction of densely packed Cx43 gap junctions. This figure is reproduced from Figure 4D of Gaietta et al. <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001041#pbio.1001041-Gaietta1\" target=\"_blank\">[9]</a>. (E) A cartoon showing miniSOG-labeled Cx43 gap junctions. Bar A, 10 microns; B–D, 100 nm.</p>", "links"=>[], "tags"=>["cx43", "forms"], "article_id"=>456081, "categories"=>["Biotechnology", "Biochemistry", "Neuroscience"], "users"=>["Xiaokun Shu", "Varda Lev-Ram", "Thomas J. Deerinck", "Yingchuan Qi", "Ericka B. Ramko", "Michael W. Davidson", "Yishi Jin", "Mark H. Ellisman", "Roger Y. Tsien"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001041.g004", "stats"=>{"downloads"=>1, "page_views"=>7, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MiniSOG_tagged_Cx43_forms_gap_junctions_/456081", "title"=>"MiniSOG-tagged Cx43 forms gap junctions.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-05 01:41:21"}
  • {"files"=>["https://ndownloader.figshare.com/files/785858"], "description"=>"<p>(A) Confocal fluorescence image of miniSOG targeted to the mitochondria in body wall muscles of <i>C. elegans</i>. (B–C) Thin section EM images of a portion of <i>C. elegans</i> showing a subset of labeled mitochondria in the body wall muscle (arrow) and adjacent unlabeled mitochondria in a different cell type (arrowheads). (D–E) Ultrastructural localization of miniSOG-labeled synaptic cell-adhesion molecules (SynCAMs) in cultured cortical neurons. (D) SynCAM1 fusion reveals uniform membrane labeling at the presynaptic apposition (arrow). (E) SynCAM2 fusion shows postsynaptic membrane labeling (pointed by arrow). Ultrastructural details including synaptic vesicles and nerve terminal substructure were well preserved in both (D) and (E). (F–G) Ultrastructural localization of miniSOG-labeled synaptic cell-adhesion molecule 2 (SynCAM2) in intact mouse brain. (A) A large area (∼14 µm × 14 µm) of one of the tissue sections imaged by serial block-face scanning electron microscopy. (B) Enlargement of the region boxed in (A) reveals postsynaptic membrane labeling (pointed by arrow) apposing a presynaptic bouton containing vesicles. Ultrastructural details including synaptic vesicles and membrane-bound structures of synapses were well preserved and easily recognizable (e.g. arrowhead in the upper left). Bar A, 50 microns; B–C, 500 nm; D–E, 500 nm; F, 2 microns; G, 500 nm.</p>", "links"=>[], "tags"=>["produces", "fluorescence", "em", "reveals", "localization", "synaptic", "adhesion", "molecules"], "article_id"=>456217, "categories"=>["Biotechnology", "Biochemistry", "Neuroscience"], "users"=>["Xiaokun Shu", "Varda Lev-Ram", "Thomas J. Deerinck", "Yingchuan Qi", "Ericka B. Ramko", "Michael W. Davidson", "Yishi Jin", "Mark H. Ellisman", "Roger Y. Tsien"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001041.g005", "stats"=>{"downloads"=>1, "page_views"=>16, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MiniSOG_produces_fluorescence_and_EM_contrast_in_C_elegans_and_reveals_previously_unknown_localization_of_synaptic_cell_adhesion_molecules_in_mice_/456217", "title"=>"MiniSOG produces fluorescence and EM contrast in <i>C. elegans</i> and reveals previously unknown localization of synaptic cell adhesion molecules in mice.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-05 01:43:37"}
  • {"files"=>["https://ndownloader.figshare.com/files/785264"], "description"=>"<p>(A) Infrared fluorescence of <i>E. coli</i> colonies expressing the fusion proteins before and after irradiation (480±15 nm excitation). (B) Predicted structure of miniSOG by the Swiss-Model structure homology-modeling server <a href=\"http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001041#pbio.1001041-Arnold1\" target=\"_blank\">[52]</a>. (C) Mutations introduced into miniSOG compared to its parent. Numbers in bracket are based on miniSOG protein sequence. (D) Normalized absorbance (blue) and emission (red) spectra. (E) Degradation of ADPA by illumination of miniSOG (red) or free FMN (blue).</p>", "links"=>[], "tags"=>["singlet", "engineered", "photoreceptor"], "article_id"=>455626, "categories"=>["Biotechnology", "Biochemistry", "Neuroscience"], "users"=>["Xiaokun Shu", "Varda Lev-Ram", "Thomas J. Deerinck", "Yingchuan Qi", "Ericka B. Ramko", "Michael W. Davidson", "Yishi Jin", "Mark H. Ellisman", "Roger Y. Tsien"], "doi"=>"https://dx.doi.org/10.1371/journal.pbio.1001041.g001", "stats"=>{"downloads"=>1, "page_views"=>42, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_MiniSOG_a_small_and_efficient_singlet_oxygen_generator_is_engineered_from_a_blue_light_photoreceptor_based_on_protein_crystal_structure_/455626", "title"=>"MiniSOG, a small and efficient singlet oxygen generator, is engineered from a blue light photoreceptor based on protein crystal structure.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-04-05 01:33:46"}

PMC Usage Stats | Further Information

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Relative Metric

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