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Integrative Approaches for Cellular Cryo-electron Tomography: Correlative Imaging and Focused Ion Beam Micromachining

MPG-Autoren
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Rigort,  Alexander
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Villa,  Elizabeth
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Bäuerlein,  Felix Jan Benedict
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Engel,  Benjamin D.
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Plitzko,  Jürgen M.
Baumeister, Wolfgang / Molecular Structural Biology, Max Planck Institute of Biochemistry, Max Planck Society;

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Zitation

Rigort, A., Villa, E., Bäuerlein, F. J. B., Engel, B. D., & Plitzko, J. M. (2012). Integrative Approaches for Cellular Cryo-electron Tomography: Correlative Imaging and Focused Ion Beam Micromachining. In Methods in Cell Biology; Vol. 111 (pp. 259-281). San Diego: Academic Press.


Zitierlink: https://hdl.handle.net/11858/00-001M-0000-000E-BB72-D
Zusammenfassung
The application of cryo-electron tomography to cells and tissues is commonly referred to as “cellular tomography,” and enables visualization of the supramolecular architecture of cells in a near-native state. However, in order to access structural features hidden deep inside cellular volumes, it is necessary to use hybrid techniques to identify and localize features of interest and prepare such regions for subsequent analysis by transmission electron microscopy. We present a workflow that integrates different approaches: (1) correlative cryo-fluorescence microscopy to localize features within frozen-hydrated cells, (2) focused ion beam milling to thin these specimens in a targeted manner, and (3) cryo-electron tomography to provide detailed information about the cellular ultrastructure of thinned samples. We describe the combined use of these techniques and the instrumentation required to enable cryo-electron tomography for a vast range of cellular samples.