Negative Stain Transmission Electron Microscopy

This method is routinely used in screening experiments to assess sample quality. It is also used to answer specific questions concerning, e.g., protein-protein interactions and protein assembly; antibodies can be visualized when bound to their antigen.

The sample is embedded in a layer of a heavy metal salt. This scatters a large number of electrons and looks dark on the images.

  • TEM imaging: Low magnification view of a negatively stained EM sample grid.

  • Grid bars and the perforated carbon layer covering the grid (broken in places).

  • The holes in the perforated film are not empty.

  • One square of the grid mesh.

  • What looks like a hole is in fact thin carbon film.

  • Even the smallest 'holes' are useful - see slider below.

The slider above shows a typical EM grid at increasing magnification. The red boxes indicate the regions enlarged in the next image. The EM-grid was first coated with a perforated carbon layer, and then with a thin carbon film to which the sample was later adsorbed and negatively stained.

The magnification series is continued in the slider below. The thin carbon spanning a hole in the perforated layer and the negatively stained protein complexes on it, become increasingly visible.

  • The thin carbon film is covered by something - lets magnify the region indicated.

  • There is a sheet of something on the thin carbon film.

  • There is a sheet and rod like particles - there are two different proteins on the grid.

  • The rods are tobacco mosaic virus particles. The sheet is a so-called S-layer, the outermost layer of an archaebacterium.

  • The S-layer has a regular structure - it is a naturally occurring two-dimensional crystal and we can visualize its subunits.

Images courtesy of Philippe Ringler, C-CINA.