TEM contrast methods (staining)

In order to visualize a specimen in the TEM, the contrasting regions of electron transparency and electron opacity must be present. As in light microscopy, differences in contrast can be accentuated through the use of a stain. The most commonly used stains in electron microscopy are made up of heavy metal salts. This can be explained by mass-thickness contrast. The interaction of electrons with heavy atoms is stronger than with light atoms. If the thickness is homogeneous, areas in which heavy atoms are concentrated appear with darker contrast than such with light atoms (mass contrast). More electrons are scattered in thick than in thin areas; thus, thick areas appear dark (thickness contrast). However, a thick area with light elements might have the same contrast as a thinner area with heavier atoms. Heavy metal salts have atoms of high atomic weight which are especially good at producing differential electron scattering. The samples, in particular those of organic materials, consist of light elements, such as hydrogen, oxygen, phosphorus, sulphur, etc. They have a low electron- scattering power, resulting in the low contrast in an image of untreated material. Staining by soaking in solutions containing heavy metals, such as tungsten, osmium, lead, or uranium increases electron-scattering power, and results in images with more visible specimen details.

Electron staining falls into one of two categories:

• Positive staining in which contrast is imparted to the specimen itself, and is most common method for tissue sections. The heavy metal is absorbed by tissue or particle itself. The stains for positive staining are osmium tetroxide, uranyl acetate, lead citrate and phosphotungstic acid.

  OsO₄: its atomic weight is 190, and it is sufficient to deflect electrons effectively. Because it reacts more readily with lipids than it does with proteins, osmium tetroxide is used as structure specific positive stain.

  Both uranyl acetate (MW= 422) and lead citrate (MW = 1054) are heavy metal salt stains and are both categorized as general or non-specific stains. Uranyl acetate ions are believed to react with phosphate and amino groups (found in nucleic acids and certain proteins) while lead ions are thought to bind to negatively charged molecules such as hydroxyl groups. Because of this ability to stain different cellular components uranyl acetate and lead citrate are often used in conjunction with one another though not simultaneously.

• Negative staining in which the area surrounding the specimen is given increased electron opacity, while the specimen itself remains more translucent. This method is used to stain suspensions of isolated proteins or macromolecules. The stains, commonly used, include phosphotungstate, silicotungstate, uranyl acetate and ammonium molybdate.

The staining techniques are not desirable for most soft matter systems:

- The structure of the sample may be changed during the preparation process, in particular in soft matter.

- Exposing to different chemicals can result in artifacts, which have to be distinguished from genuine structures within the specimen, particularly in biological samples.