Doubt and certainty in counting

Some of the methods used for counting objects in histological sections are discussed. The method best suited for any particular counting program depends on many variables, which include the level of accuracy required, the type of preparation available for study, the size of the objects to be counted, the thickness of the sections that can be used, the equipment available and the amount of labor that can reasonably be invested. For light and electron microscopy, profile counts are simple and quick for objects that are small relative to section thickness and whose dimensions are readily defined. The 'physical disector' is particularly useful where objects to be counted are large relative to sections thickness, or where their dimensions are unknown or highly variable. For light microscopy, the optical disector is often easier to use. However, it makes more assumptions than the physical disector; some of these can introduce serious bias in the counts, and they are explored. Electron microscopy raises some special problems that relate to the depth of focus, the relatively very thin sections, and the tendency for thin structures that do not span the full thickness of a section to be lost or unrecognizable in some section planes. The importance of recognizing the assumptions that underlie any method of counting and its interpretation is stressed.