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Skeletal muscle triad junction ultrastructure by Focused-Ion-Beam milling of muscle and Cryo-Electron Tomography

Terence Wagenknecht, Chyongere Hsieh, Michael Marko
  • Terence Wagenknecht
    New York State Department of Health, Wadsworth Cente, Empire State Plaza, Albany, NY; Department of Biomedical Sciences, School of Public Health, University at Albany, Albany, NY, United States | terence.wagenknecht@health.ny.gov
  • Chyongere Hsieh
    New York State Department of Health, Wadsworth Cente, Empire State Plaza, Albany, NY, United States
  • Michael Marko
    New York State Department of Health, Wadsworth Cente, Empire State Plaza, Albany, NY, United States

Abstract

Cryo-electron tomography (cryo-ET) has emerged as perhaps the only practical technique for revealing nanometer-level three-dimensional structural details of subcellular macromolecular complexes in their native context, inside the cell. As currently practiced, the specimen should be 0.1- 0.2 microns in thickness to achieve optimal resolution. Thus, application of cryo-ET to intact frozen (vitreous) tissues, such as skeletal muscle, requires that they be sectioned. Cryo-ultramicrotomy is notoriously difficult and artifact-prone when applied to frozen cells and tissue, but a new technique, focused ion beam milling (cryo-FIB), shows great promise for “thinning” frozen biological specimens. Here we describe our initial results in applying cryo-FIB and cryo-ET to triad junctions of skeletal muscle.

Keywords

Cryo-electron microscopy, Cryo-electron tomography, Excitation-Contraction Coupling, Focused-Ion-Beam milling, Skeletal muscle

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Submitted: 2014-11-06 22:30:30
Published: 2015-01-15 09:26:55
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Copyright (c) 2015 Terence Wagenknecht, Chyongere Hsieh, Michael Marko

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