Comparison of phase contrast transmission electron microscopy with optimized scanning transmission annular dark field imaging for protein imaging

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Abstract

Henderson has already shown that electron microscopy should be superior to X-ray and neutron diffraction for determining protein structure with minimum radiation damage. Since the contrast for a molecule embedded in vitreous ice is very low, it is conceivable that dark field imaging would be superior to bright field phase contrast microscopy. A detailed analysis of contrast and signal/noise for both imaging modes is presented. Annular dark field scanning transmission microscopy gives improved contrast and equivalent signal/noise to phase contrast TEM when the molecule is the same thickness as a vitreous ice embedding medium. For a constant embedding medium thickness of 200Å the contrast is equivalent to phase contrast TEM but the signal/noise is 5 times worse. Even with an efficient detector that only excludes scattering less than 5mrad there is insufficient signal at a dose of 5 electrons/Å2 to produce an image with more than 1 electron/per pixel. For larger molecules (>100Å thick which corresponds to 420kDa for spherical molecules) the weak phase object approximation used to analyse a phase contrast image no longer applies at 100kV. This limit could be extended to about 200Å (about 3MDa) if a 400kV microscope were used.

Original languageEnglish (US)
Pages (from-to)117-124
Number of pages8
JournalUltramicroscopy
Volume96
Issue number1
DOIs
StatePublished - Jul 2003

Fingerprint

phase contrast
Transmission electron microscopy
proteins
Proteins
Scanning
Imaging techniques
transmission electron microscopy
Molecules
scanning
Ice
embedding
molecules
Microscopic examination
ice
microscopy
Electrons
Radiation damage
Neutron diffraction
radiation damage
Electron microscopy

Keywords

  • Phase contrast
  • Protein imaging
  • STEM ADF

ASJC Scopus subject areas

  • Materials Science(all)
  • Instrumentation

Cite this

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title = "Comparison of phase contrast transmission electron microscopy with optimized scanning transmission annular dark field imaging for protein imaging",
abstract = "Henderson has already shown that electron microscopy should be superior to X-ray and neutron diffraction for determining protein structure with minimum radiation damage. Since the contrast for a molecule embedded in vitreous ice is very low, it is conceivable that dark field imaging would be superior to bright field phase contrast microscopy. A detailed analysis of contrast and signal/noise for both imaging modes is presented. Annular dark field scanning transmission microscopy gives improved contrast and equivalent signal/noise to phase contrast TEM when the molecule is the same thickness as a vitreous ice embedding medium. For a constant embedding medium thickness of 200{\AA} the contrast is equivalent to phase contrast TEM but the signal/noise is 5 times worse. Even with an efficient detector that only excludes scattering less than 5mrad there is insufficient signal at a dose of 5 electrons/{\AA}2 to produce an image with more than 1 electron/per pixel. For larger molecules (>100{\AA} thick which corresponds to 420kDa for spherical molecules) the weak phase object approximation used to analyse a phase contrast image no longer applies at 100kV. This limit could be extended to about 200{\AA} (about 3MDa) if a 400kV microscope were used.",
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