Protocol

Nanoimaging

Volume 950 of the series Methods in Molecular Biology pp 227-251

Date:

Cellular Nanoimaging by Cryo Electron Tomography

  • Roman I. KoningAffiliated withDepartment of Molecular Cell Biology, Leiden University Medical Center Email author 
  • , Abraham J. KosterAffiliated withDepartment of Molecular Cell Biology, Leiden University Medical Center

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Abstract

Cryo electron tomography is a technique that allows visualization of biological specimens in three dimensions with nanometer resolution. For cryo immobilized life sciences samples it can reveal cellular morphology, the shape of membranous structures, and depict internal macromolecular arrangements and large proteins. Cryo electron tomography is a unique technique in structural biology research because it is the only tool that enables direct visualization of the cellular space at molecular resolution. Here we present the methods that we apply in our lab to perform cellular cryo electron tomography, which require expertise on cell biology for cell growth, physics for electron microscopy, and image processing for reconstruction and 3D visualization. We define the instrumentation, materials, and protocols for cryo electron tomography of whole cells, including cell growth, specimen vitrification, microscope alignments, data acquisition, tomographic image reconstruction, and 3D visualization techniques.

Key words

Cryo electron tomography Cellular microscopy Cryo specimen preparation 3D reconstruction Visualization