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

Research output: Contribution to journalArticle

34 Scopus citations

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

Keywords

  • Phase contrast
  • Protein imaging
  • STEM ADF

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics
  • Instrumentation

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