Structural Cell Biology: Preparing Specimens for Cryo-Electron Tomography Using Focused-Ion-Beam Milling

نویسندگان

  • Elizabeth Villa
  • Miroslava Schaffer
  • Ben Engel
  • Jürgen Plitzko
  • Wolfgang Baumeister
چکیده

Cryo-electron microscopy has established itself as a central tool in structural biology, ideally suited to study large macromolecular complexes from in vitro samples. Cryo-electron tomography (CET) can extend the applicability of cryo-electron microscopy to study these complexes in situ. Recent technological advances allow CET to be applied to questions in cell biology that go beyond structural determination, where a quantitative analysis of cellular features is required including molecular distribution and supramolecular architecture. However, the ability of CET to provide three-dimensional landscapes of cells is limited by the thickness of the sample that can be examined in situ with a resolution that is high enough for the identification of molecular species. For intermediate resolution electron microscopes, samples must be of less than 500 nm in thickness. Most cells, specially eukaryotes, exceed the thickness accessible to CET, limiting its routine application to isolated or reconstituted subcellular systems, small prokaryotic cells, or thin peripheral regions of cells.

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تاریخ انتشار 2014