Preparing Specimens

Specimen preparation for the Transmission Electron Microscope

Specimen fixation quality is important in EM. Glutaraldehyde is the usual primary fixative and this is normally followed by a second fixative, osmium tetroxide. After chemical fixation, which preserves specimen features in a near life-like state, the specimen is dehydrated in alcohol and infiltrated with liquid plastic monomers (resin) to support the tissue.

After polymerisation, the hard resin block containing the specimen is sectioned. Sections are cut on an instrument called an ultramicrotome using either glass or diamond knives. The ultrathin sections produced float on water in a water bath surrounding the knife and are collected on to the surface of 3mm diameter copper mesh (the specimen grid).

The cell organelles of all human cells would be virtually transparent unless stained with heavy metals that selectively attach to different cell components. Thus, the sections on the grids are stained with heavy metal salts (usually uranyl acetate and lead citrate) to add contrast to the specimen.

A different preparation method is used in virology, where virus particles in a human sample (e.g. faeces) are partially purified and concentrated by ultracentrifugation. The virus suspension is allowed to dry down onto the surface of a grid covered in a thin support film and stained with a heavy metal salt (usually phosphotungstic acid). This stain surrounds and penetrates surface features of viruses, allowing them to stand out from the background.

Tumour cell Histiocytosis X Thin section of a tumour cell showing the characteristic tennis-racket-shaped granules seen in Histiocytosis X.