Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy

By M. Ohi, Ying Li, Yifan Cheng, Thomas Walz. Biological Procedures Online, 2004

688 citations recorded by the source corpus.

Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens.

Subfields: Atomic and Molecular Physics, and Optics, Materials Chemistry, Structural Biology

View Paper: https://www.semanticscholar.org/paper/795e7547beb38b5dee1759344e8a0fbeff76edf8

How researchers see this paper

The researchers whose trusted networks rank it highest, strongest advocate first. Every researcher gets a different order.

In Materials Chemistry

Citations in the corpus

Structural organization of a full-length gp130/LIF-R cytokine receptor transmembrane complex (2008, methodology): “…5, 2008 ª2008 Elsevier Electron Microscopy Receptor complexes were prepared for electron microscopy using the conventional negative staining protocol (Ohi et al., 2004) and imaged at room temperature with a Tecnai T12 electron microscope operated at 120 kV. Images of specimens were recorded using…”
The final steps of integrin activation: the end game (2010, background): “It seems likely that a resolution of some of these hotly debated issues must take into account the relative strengths of negative stain electron microscopy versus electron cryomicroscop”
Complete mapping of mutations to the SARS-CoV-2 spike receptor-binding domain that escape antibody recognition (2020, neutral): “The grids were stained with 0.75% uranyl formate (UF) (Ohi et al., 2004) .”
A Primer to Single-Particle Cryo-Electron Microscopy (2015, neutral): “The images, 2D class averages and 3D maps obtained with vitrified specimens may indicate that the sample requires further improvement to reach near-atomic resolution (‘‘high-resolution’’ sample refinement). sample homogeneity (Ohi et al., 2004).”
CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells (2020, methodology): “A 2D reference-free class-averaging program was then applied.(45) After quality evaluation, particle images from each sample (6681 CD147; 5073 spike(RBD); 12,426 CD147-spike (RBD) complexes) were selected from the corrected micrographs and extracted as 128 × 128-pixel images by using boxer software.”
Three-dimensional EM structure of an intact activator-dependent transcription initiation complex (2009, methodology): “Negatively stained EM specimens were prepared using a carbon-sandwich technique and uranyl-formate stain (20) (see SI Text for details).”
STIM1 Clusters and Activates CRAC Channels via Direct Binding of a Cytosolic Domain to Orai1 (2009, positive): “Electron Microscopy EE-Orai1-His8 alone or copurified with His6-CAD was diluted to a final concentration of 0.01 mg/ml in 20 mM Tris, 150 mM NaCl, and 0.02% DDM buffer and was negatively stained with uranyl formate as described (Ohi et al., 2004).”