Cryo-STEM Tomography of Intact Vitrified Fibroblasts

David Kirchenbuechler; Yael Mutsafi; Ben Horowitz; Smadar Levin-Zaidman; Deborah Fass; Sharon G. Wolf; Michael Elbaum; David Kirchenbuechler; Yael Mutsafi; Ben Horowitz; Smadar Levin-Zaidman; Deborah Fass; Sharon G. Wolf; Michael Elbaum

doi:10.3934/biophy.2015.3.259

AIMS Biophysics

2015, Volume 2, Issue 3: 259-273. doi: 10.3934/biophy.2015.3.259

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Research article Special Issues

Cryo-STEM Tomography of Intact Vitrified Fibroblasts

1.
Department of Materials and Interfaces, Weizmann Institute of Science, Rehovot 7610001;
2.
Department of Structural Biology, Weizmann Institute of Science, Rehovot 7610001;
3.
Electron Microscopy Unit, Chemical Research Support Department, Weizmann Institute of Science, Rehovot 7610001

Received: 26 March 2015 Accepted: 09 July 2015 Published: 12 July 2015

Cryo-tomography of intact, vitrified cells provides a three dimensional view of their structure and organization in a snapshot of the living state. Lacking heavy metal stains, tilt series images are typically produced by defocus phase contrast. Recently, a number of other methods have been introduced for 3D cryo-imaging. These include phase plate imaging, soft X-ray tomography, serial surface imaging using the focused ion beam-scanning electron microscope, and cryo-STEM tomography (CSTET). Here we explain the basis of the STEM setup and demonstrate the capabilities of CSTET to study unfixed, fully hydrated mammalian cells. Numerous cellular features are recognized in CSTET reconstructions, including membranes, vesicles, cytoskeleton, extracellular matrix, coated pits, and ribosomes. STEM signal acquisition configuration is more flexible than defocus phase contrast, and it imposes a much less severe spatial filter on the original images. Because low spatial frequency information is retained, the STEM tomographic reconstruction more faithfully represents the mass density distribution in the specimen.
- 3DEM,
- cryo-tomography,
- scanning transmission electron microscopy,
- cryo-electron tomography,
- CSTET,
- organelles,
- mycoplasma
Citation: David Kirchenbuechler, Yael Mutsafi, Ben Horowitz, Smadar Levin-Zaidman, Deborah Fass, Sharon G. Wolf, Michael Elbaum. Cryo-STEM Tomography of Intact Vitrified Fibroblasts[J]. AIMS Biophysics, 2015, 2(3): 259-273. doi: 10.3934/biophy.2015.3.259

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Abstract

Cryo-tomography of intact, vitrified cells provides a three dimensional view of their structure and organization in a snapshot of the living state. Lacking heavy metal stains, tilt series images are typically produced by defocus phase contrast. Recently, a number of other methods have been introduced for 3D cryo-imaging. These include phase plate imaging, soft X-ray tomography, serial surface imaging using the focused ion beam-scanning electron microscope, and cryo-STEM tomography (CSTET). Here we explain the basis of the STEM setup and demonstrate the capabilities of CSTET to study unfixed, fully hydrated mammalian cells. Numerous cellular features are recognized in CSTET reconstructions, including membranes, vesicles, cytoskeleton, extracellular matrix, coated pits, and ribosomes. STEM signal acquisition configuration is more flexible than defocus phase contrast, and it imposes a much less severe spatial filter on the original images. Because low spatial frequency information is retained, the STEM tomographic reconstruction more faithfully represents the mass density distribution in the specimen.

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