Transmission Electron Microscopy Staining Explained

In Transmission Electron Microscopy (TEM), preparing samples involves cutting them into extremely thin slices, known as ultrathin sections. These sections are typically only tens of nanometers thick. Because biological samples are mostly composed of light elements like carbon, hydrogen, oxygen, and nitrogen, they don't scatter electrons very well. This results in very low contrast when viewed in a TEM.

To overcome this lack of contrast, ultrathin sections need to be stained. Unlike staining in light microscopy which uses colored dyes, staining for TEM uses solutions containing heavy metal ions. These heavy metal ions bind to various structures within the sample. When the electron beam passes through the section, the heavy metal atoms scatter electrons more effectively than the lighter atoms of the biological sample. This differential scattering creates areas of higher and lower electron density, which are then detected to form a high-contrast image.

The most common heavy metal stains used for post-staining ultrathin sections in Transmission Electron Microscopy are:

• Uranyl acetate: This stain contains uranium, a very heavy metal. It binds non-specifically but has an affinity for nucleic acids and proteins. It is often used as an initial stain.
• Lead citrate: This stain contains lead, another heavy metal. It tends to bind more specifically to structures like ribosomes, glycogen granules, and cell membranes. It is typically used after uranyl acetate to further enhance contrast.

Let's look at the other options provided:

• Hematoxylin and eosin: These are standard stains used for staining tissue sections for light microscopy. Hematoxylin stains acidic components (like nuclei) blue/purple, and eosin stains basic components (like cytoplasm and connective tissue) pink. They are not suitable for TEM because they do not contain heavy metals and do not significantly scatter electrons.
• Toluidine blue in 1% borax: Toluidine blue is a basic dye often used to stain semithin sections (thicker than ultrathin sections, typically 0.5 to 1 micrometers) embedded in plastic for light microscopy. It provides good contrast for identifying specific areas before cutting ultrathin sections. Like hematoxylin and eosin, it is not a heavy metal stain and is not used for staining ultrathin sections for TEM viewing.

Therefore, the stains used for staining ultrathin sections specifically for Transmission Electron Microscopy to enhance contrast are uranyl acetate and lead citrate.