Tissue Processing: VII. Post Treatment

Abstract

For an image to be seen with an unstained section in the light microscope, different tissue components must have different refractive indices from each other and from the mounting medium. Pieces of tissue, which are predominantly protein, have a refractive index of 1.53–1.54 when fixed with formaldehyde. If they are subsequently infiltrated with a colourless, transparent liquid with the same refractive index as the tissue (e.g. methylsalicylate) they become invisible and no image is seen in the microscope. Contrast can be achieved in three different ways:

• 1. The tissue can be stained. The section becomes visible because light of certain wavelengths is absorbed by the dye or dyes bound to tissue components but is relatively unaffected by the mounting medium. A mounting medium with the same refractive index as protein is now an advantage as this gives an optically clear image

• 2. The tissue can be left unstained, but can be made visible by mounting it in a liquid with a refractive index which differs slightly from that of proteins. The section may then be examined by phase or differential interference contrast microscopy

• 3. A transparent piece of tissue can be made visible by direct microscopy if it is mounted in a liquid with a refractive index substantially different from that of protein. This method is only applicable for low power examinations and it should be noted that boundaries between the tissue and mounting medium appear as sharp lines which have no in vivo correlates