Open Access

Negative staining and image classification — powerful tools in modern electron microscopy

Biological Procedures Online6:610023

DOI: 10.1251/bpo70

Received: 16 February 2004

Accepted: 9 March 2004


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preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach.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.

Indexing terms

Negative staining Microscopy, Electron Protein Conformation