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Novel Electron Tomographic Methods to Study the Morphology of Keratin Filament Networks

Published online by Cambridge University Press:  02 July 2010

Michaela Sailer ,
Katharina Höhn ,
Sebastian Lück ,
Volker Schmidt ,
Michael Beil  and
Paul Walther
Michaela Sailer
Affiliation:
Electron Microscopy Facility, Ulm University, D-89069 Ulm, Germany
Katharina Höhn
Affiliation:
Electron Microscopy Facility, Ulm University, D-89069 Ulm, Germany
Sebastian Lück
Affiliation:
Institute of Stochastics, Ulm University, D-89069 Ulm, Germany
Volker Schmidt
Affiliation:
Institute of Stochastics, Ulm University, D-89069 Ulm, Germany
Michael Beil
Affiliation:
Department of Internal Medicine I, University Hospital Ulm, D-89070 Ulm, Germany
Paul Walther*
Affiliation:
Electron Microscopy Facility, Ulm University, D-89069 Ulm, Germany
*
Corresponding author. E-mail: paul.walther@uni-ulm.de
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Abstract

The three-dimensional (3D) keratin filament network of pancreatic carcinoma cells was investigated with different electron microscopical approaches. Semithin sections of high-pressure frozen and freeze substituted cells were analyzed with scanning transmission electron microscope (STEM) tomography. Preservation of subcellular structures was excellent, and keratin filaments could be observed; however, it was impossible to three-dimensionally track the individual filaments. To obtain a better signal-to-noise ratio in transmission mode, we observed ultrathin sections of high-pressure frozen and freeze substituted samples with low-voltage (30 kV) STEM. Contrast was improved compared to 300 kV, and individual filaments could be observed. The filament network of samples prepared by detergent extraction was imaged by high-resolution scanning electron microscopy (SEM) with very good signal-to-noise ratio using the secondary electron signal and the 3D structure could be elucidated by SEM tomography. In freeze-dried samples it was possible to discern between keratin filaments and actin filaments because the helical arrangement of actin subunits in the F-actin could be resolved. When comparing the network structures of the differently prepared samples, we found no obvious differences in filament length and branching, indicating that the intermediate filament network is less susceptible to preparation artifacts than the actin network.

Keywords

keratin (intermediate) filamentsSEM tomographySTEM tomographypancreaskeratinactinelectron microscopy
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 16 , Issue 4 , August 2010 , pp. 462 - 471
Copyright
Copyright © Microscopy Society of America 2010

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