Cryo-electron microscopy of vitreous sections
Identifieur interne : 000013 ( Pmc/Corpus ); précédent : 000012; suivant : 000014Cryo-electron microscopy of vitreous sections
Auteurs : Ashraf Al-Amoudi ; Jiin-Ju Chang ; Amélie Leforestier ; Alasdair Mcdowall ; Laurée Michel Salamin ; Lars P O. Norlén ; Karsten Richter ; Nathalie Sartori Blanc ; Daniel Studer ; Jacques DubochetSource :
- The EMBO Journal [ 0261-4189 ] ; 2004.
Abstract
Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.
Url:
DOI: 10.1038/sj.emboj.7600366
PubMed: 15318169
PubMed Central: 517607
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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Cryo-electron microscopy of vitreous sections</title><author><name sortKey="Al Amoudi, Ashraf" sort="Al Amoudi, Ashraf" uniqKey="Al Amoudi A" first="Ashraf" last="Al-Amoudi">Ashraf Al-Amoudi</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Chang, Jiin Ju" sort="Chang, Jiin Ju" uniqKey="Chang J" first="Jiin-Ju" last="Chang">Jiin-Ju Chang</name><affiliation><nlm:aff id="O2">Institute of Biophysics, Academy of Sciences, Beijing, China</nlm:aff></affiliation></author><author><name sortKey="Leforestier, Amelie" sort="Leforestier, Amelie" uniqKey="Leforestier A" first="Amélie" last="Leforestier">Amélie Leforestier</name><affiliation><nlm:aff id="O3">Laboratoire de Physique des Solides, CNRS URA002, Université Paris-Sud, Orsay, France</nlm:aff></affiliation></author><author><name sortKey="Mcdowall, Alasdair" sort="Mcdowall, Alasdair" uniqKey="Mcdowall A" first="Alasdair" last="Mcdowall">Alasdair Mcdowall</name><affiliation><nlm:aff id="O4">Centre for Microscopy and Microanalysis, University of Queensland, St Lucia, Australia</nlm:aff></affiliation></author><author><name sortKey="Salamin, Lauree Michel" sort="Salamin, Lauree Michel" uniqKey="Salamin L" first="Laurée Michel" last="Salamin">Laurée Michel Salamin</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Norlen, Lars P O" sort="Norlen, Lars P O" uniqKey="Norlen L" first="Lars P O" last="Norlén">Lars P O. Norlén</name><affiliation><nlm:aff id="O5">Groupe de Physique appliquée, Departement de physique, Université de Genève, Genève, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Richter, Karsten" sort="Richter, Karsten" uniqKey="Richter K" first="Karsten" last="Richter">Karsten Richter</name><affiliation><nlm:aff id="O6">DKFZ, Molecular Genetics (B060), Heidelberg, Germany</nlm:aff></affiliation></author><author><name sortKey="Blanc, Nathalie Sartori" sort="Blanc, Nathalie Sartori" uniqKey="Blanc N" first="Nathalie Sartori" last="Blanc">Nathalie Sartori Blanc</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Studer, Daniel" sort="Studer, Daniel" uniqKey="Studer D" first="Daniel" last="Studer">Daniel Studer</name><affiliation><nlm:aff id="O7">Anatomisches Institut, Postfach, Bern, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Dubochet, Jacques" sort="Dubochet, Jacques" uniqKey="Dubochet J" first="Jacques" last="Dubochet">Jacques Dubochet</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">15318169</idno><idno type="pmc">517607</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC517607</idno><idno type="RBID">PMC:517607</idno><idno type="doi">10.1038/sj.emboj.7600366</idno><date when="2004">2004</date><idno type="wicri:Area/Pmc/Corpus">000013</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000013</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Cryo-electron microscopy of vitreous sections</title><author><name sortKey="Al Amoudi, Ashraf" sort="Al Amoudi, Ashraf" uniqKey="Al Amoudi A" first="Ashraf" last="Al-Amoudi">Ashraf Al-Amoudi</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Chang, Jiin Ju" sort="Chang, Jiin Ju" uniqKey="Chang J" first="Jiin-Ju" last="Chang">Jiin-Ju Chang</name><affiliation><nlm:aff id="O2">Institute of Biophysics, Academy of Sciences, Beijing, China</nlm:aff></affiliation></author><author><name sortKey="Leforestier, Amelie" sort="Leforestier, Amelie" uniqKey="Leforestier A" first="Amélie" last="Leforestier">Amélie Leforestier</name><affiliation><nlm:aff id="O3">Laboratoire de Physique des Solides, CNRS URA002, Université Paris-Sud, Orsay, France</nlm:aff></affiliation></author><author><name sortKey="Mcdowall, Alasdair" sort="Mcdowall, Alasdair" uniqKey="Mcdowall A" first="Alasdair" last="Mcdowall">Alasdair Mcdowall</name><affiliation><nlm:aff id="O4">Centre for Microscopy and Microanalysis, University of Queensland, St Lucia, Australia</nlm:aff></affiliation></author><author><name sortKey="Salamin, Lauree Michel" sort="Salamin, Lauree Michel" uniqKey="Salamin L" first="Laurée Michel" last="Salamin">Laurée Michel Salamin</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Norlen, Lars P O" sort="Norlen, Lars P O" uniqKey="Norlen L" first="Lars P O" last="Norlén">Lars P O. Norlén</name><affiliation><nlm:aff id="O5">Groupe de Physique appliquée, Departement de physique, Université de Genève, Genève, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Richter, Karsten" sort="Richter, Karsten" uniqKey="Richter K" first="Karsten" last="Richter">Karsten Richter</name><affiliation><nlm:aff id="O6">DKFZ, Molecular Genetics (B060), Heidelberg, Germany</nlm:aff></affiliation></author><author><name sortKey="Blanc, Nathalie Sartori" sort="Blanc, Nathalie Sartori" uniqKey="Blanc N" first="Nathalie Sartori" last="Blanc">Nathalie Sartori Blanc</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Studer, Daniel" sort="Studer, Daniel" uniqKey="Studer D" first="Daniel" last="Studer">Daniel Studer</name><affiliation><nlm:aff id="O7">Anatomisches Institut, Postfach, Bern, Switzerland</nlm:aff></affiliation></author><author><name sortKey="Dubochet, Jacques" sort="Dubochet, Jacques" uniqKey="Dubochet J" first="Jacques" last="Dubochet">Jacques Dubochet</name><affiliation><nlm:aff id="O1">Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</nlm:aff></affiliation></author></analytic><series><title level="j">The EMBO Journal</title><idno type="ISSN">0261-4189</idno><idno type="eISSN">1460-2075</idno><imprint><date when="2004">2004</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.</p></div></front></TEI><pmc article-type="other"><pmc-comment>The publisher of this article does not allow downloading of the full text in XML form.</pmc-comment>
<front><journal-meta><journal-id journal-id-type="nlm-ta">EMBO J</journal-id><journal-title>The EMBO Journal</journal-title><issn pub-type="ppub">0261-4189</issn><issn pub-type="epub">1460-2075</issn><publisher><publisher-name>Nature Publishing Group</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">15318169</article-id><article-id pub-id-type="pmc">517607</article-id><article-id pub-id-type="pii">7600366</article-id><article-id pub-id-type="doi">10.1038/sj.emboj.7600366</article-id><article-categories><subj-group subj-group-type="heading"><subject>New EMBO Member's Review</subject></subj-group></article-categories><title-group><article-title>Cryo-electron microscopy of vitreous sections</article-title><alt-title alt-title-type="short">CEMOVIS</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Al-Amoudi</surname><given-names>Ashraf</given-names></name><xref ref-type="aff" rid="O1">1</xref></contrib><contrib contrib-type="author"><name><surname>Chang</surname><given-names>Jiin-Ju</given-names></name><xref ref-type="aff" rid="O2">2</xref></contrib><contrib contrib-type="author"><name><surname>Leforestier</surname><given-names>Amélie</given-names></name><xref ref-type="aff" rid="O3">3</xref></contrib><contrib contrib-type="author"><name><surname>McDowall</surname><given-names>Alasdair</given-names></name><xref ref-type="aff" rid="O4">4</xref></contrib><contrib contrib-type="author"><name><surname>Salamin</surname><given-names>Laurée Michel</given-names></name><xref ref-type="aff" rid="O1">1</xref></contrib><contrib contrib-type="author"><name><surname>Norlén</surname><given-names>Lars P O</given-names></name><xref ref-type="aff" rid="O5">5</xref></contrib><contrib contrib-type="author"><name><surname>Richter</surname><given-names>Karsten</given-names></name><xref ref-type="aff" rid="O6">6</xref></contrib><contrib contrib-type="author"><name><surname>Blanc</surname><given-names>Nathalie Sartori</given-names></name><xref ref-type="aff" rid="O1">1</xref></contrib><contrib contrib-type="author"><name><surname>Studer</surname><given-names>Daniel</given-names></name><xref ref-type="aff" rid="O7">7</xref></contrib><contrib contrib-type="author"><name><surname>Dubochet</surname><given-names>Jacques</given-names></name><xref ref-type="aff" rid="O1">1</xref><xref ref-type="corresp" rid="c1">a</xref></contrib><aff id="O1"><label>1</label>Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, Lausanne, Switzerland</aff><aff id="O2"><label>2</label>Institute of Biophysics, Academy of Sciences, Beijing, China</aff><aff id="O3"><label>3</label>Laboratoire de Physique des Solides, CNRS URA002, Université Paris-Sud, Orsay, France</aff><aff id="O4"><label>4</label>Centre for Microscopy and Microanalysis, University of Queensland, St Lucia, Australia</aff><aff id="O5"><label>5</label>Groupe de Physique appliquée, Departement de physique, Université de Genève, Genève, Switzerland</aff><aff id="O6"><label>6</label>DKFZ, Molecular Genetics (B060), Heidelberg, Germany</aff><aff id="O7"><label>7</label>Anatomisches Institut, Postfach, Bern, Switzerland</aff></contrib-group><author-notes><corresp id="c1"><label>a</label>Laboratoire d'Analyse Ultrastructurale, Bâtiment de Biologie, Université de Lausanne, 1015 Lausanne, Switzerland. Tel.: +41 21 692 42 80; Fax: +41 21 692 41 05; E-mail: <email>jacques.dubochet@lau.unil.ch</email></corresp></author-notes><pub-date pub-type="ppub"><day>15</day><month>09</month><year>2004</year></pub-date><pub-date pub-type="epub"><day>19</day><month>08</month><year>2004</year></pub-date><volume>23</volume><issue>18</issue><fpage>3583</fpage><lpage>3588</lpage><history><date date-type="received"><day>23</day><month>02</month><year>2004</year></date><date date-type="accepted"><day>23</day><month>07</month><year>2004</year></date></history><copyright-statement>Copyright © 2004, European Molecular Biology Organization</copyright-statement><copyright-year>2004</copyright-year><abstract><p>Since the beginning of the 1980s, cryo-electron microscopy of a thin film of vitrified aqueous suspension has made it possible to observe biological particles in their native state, in the absence of the usual artefacts of dehydration and staining. Combined with 3-d reconstruction, it has become an important tool for structural molecular biology. Larger objects such as cells and tissues cannot generally be squeezed in a thin enough film. Cryo-electron microscopy of vitreous sections (CEMOVIS) provides then a solution. It requires vitrification of a sizable piece of biological material and cutting it into ultrathin sections, which are observed in the vitrified state. Each of these operations raises serious difficulties that have now been overcome. In general, the native state seen with CEMOVIS is very different from what has been seen before and it is seen in more detail. CEMOVIS will give its full potential when combined with computerized electron tomography for 3-d reconstruction.</p></abstract><kwd-group><kwd>bacterial envelope</kwd><kwd>desmosome</kwd><kwd>high-pressure freezing</kwd><kwd>intermediate filament</kwd><kwd>vitrification</kwd></kwd-group><counts><page-count count="6"></page-count></counts></article-meta></front><floats-wrap><fig id="f1"><label>Figure 1</label><caption><p>Gram-negative bacteria. (<bold>A</bold>) <italic>Escherichia coli K12</italic> embedded in conventional resin (from <xref ref-type="bibr" rid="b8">Beveridge, 1999</xref>, <xref ref-type="fig" rid="f1">Figure 1</xref>, with permission). (<bold>B</bold>) <italic>Pseudomonas aeruginosa</italic> PAO1 prepared by CEMOVIS (<xref ref-type="bibr" rid="b22">Matias <italic>et al</italic>, 2003</xref>). The cells in exponential growth were put in 20% (wt/vol) dextran (42 kDa) and vitrified in a Leica EMPACT (Vienna, Austria) high-pressure freezer. The sections were cut at −160°C in a Leica UCS/FCS cryomicrotome with a 45° Diatome diamond cryo-knife (Biel, Switzerland). Observation was made at −180°C in a Philips CM12 EM (Eindhoven, The Netherlands) operating at 80 kV and equipped with a Gatan 626 cryo-specimen holder (Warrendale, PA) and a Gatan 600CW Multiscan CCD camera. The total electron dose did not exceed 800 e/nm<sup>2</sup>. Technical details are described in previous publications (<xref ref-type="bibr" rid="b9">Dubochet <italic>et al</italic>, 1988</xref>; <xref ref-type="bibr" rid="b4">Al-Amoudi <italic>et al</italic>, 2002</xref>) or are in preparation. White arrows: cell membrane and outer membrane; black arrows: peptidoglycan layer; the white ellipse marks a region of the nucleoid; small black arrows: 2 nm high-contrast dots identified as portions of DNA filaments seen along their axis. Scale bar: 100 nm.</p></caption><graphic xlink:href="7600366f1"></graphic></fig><fig id="f2"><label>Figure 2</label><caption><p>Cyanobacterium <italic>L. majuscula</italic> prepared by CEMOVIS. (<bold>A</bold>) Low-magnification view. I, II and III are different zones of the extracellular matrix; white asterisk: bulk medium with crevasses. Scale bar: 1 μm. (<bold>B</bold>) High-magnification view of the envelope. CM: cell membrane; P: peptidoglycan; OM: outer membrane; S: putative S-layer; black arrows: additional layers; II and III: zones of the extracellular matrix; black arrowheads: filaments in zone II. Scale bar: 100 nm.</p></caption><graphic xlink:href="7600366f2"></graphic></fig><fig id="f3"><label>Figure 3</label><caption><p>Epidermal desmosome and IFs. (<bold>A</bold>) Desmosome from neonatal mouse epidermis prepared by freeze substitution (from <xref ref-type="bibr" rid="b17">He <italic>et al</italic>, 2003</xref>, from <xref ref-type="fig" rid="f1">Figure 1B</xref>, with permission). (<bold>B</bold>) Human desmosome at midportion of the viable part of forearm epidermis prepared by CEMOVIS. A ca. 200 μm thick layer was sliced with a razor blade and immediately cooled in a Baltec HPM 010 (Balzers, Liechtenstein) high-pressure freezing apparatus. The rest of the preparation was as for <xref ref-type="fig" rid="f2">Figure 2B</xref>. See text for more explanations. (<bold>C</bold>) Same region as in (B) but with IFs oriented along the viewing direction. (<bold>D</bold>) As in (C) but in stratum corneum. The fine structure of the IFs is best resolved in the thinnest sections (inset). (C, D) Modified from <xref ref-type="bibr" rid="b29">Norlén and Al-Amoudi (2004)</xref>, with permission. Section thickness: 70 nm (B, C); less than 50 nm (D). Scale bar: 50 nm (A, B); 20 nm (C, D); 10 nm (inset, D).</p></caption><graphic xlink:href="7600366f3"></graphic></fig><fig id="f4"><label>Figure 4</label><caption><p>Stereo-pair of an 895 bp supercoiled double-stranded DNA molecule. Preparation by vitrification of a thin layer of 200 μg/ml DNA solution. Tilt angle: ±15°. The first exposure is on the right. Electron dose for each micrograph: ca. 2000 e/nm<sup>2</sup>. Scale bar: 50 nm. Courtesy Dr Jan Bednar.</p></caption><graphic xlink:href="7600366f4"></graphic></fig></floats-wrap></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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