Histology is the study of the microscopic structure and function of tissues. Tissue is a general word used to describe the components of animals (and plants), and tissues consist of cells and the surrounding support media (extracellular matrix). Historically, four primary tissue types were categorised (in animals) by grouping together cells with similar form and function: they are epithelial tissue, connective tissue, muscle tissue and nerve tissue. The cells within these categories of tissues may vary in structure and be specialised according to their function and location. Most extracellular matrix is derived from the cells that it surrounds, and its composition is related to its function. For example, a very dense, hard, extracellular matrix is formed by bone cells but, in contrast, the matrix in which blood cells flow is fluid (although most blood cells do not contribute to the fluid that supports them). Various combinations of tissues form organs (e.g. the brain and liver), connecting structures (e.g. ligaments) and packing material around organs (e.g. around the kidney). In addition, various combinations of organs and other structures form systems of the body which together perform related functions (see Chapter 10, Chapter 11, Chapter 12, Chapter 13, Chapter 14, Chapter 15 and Chapter 16).
Introduction to histology
The unaided (good) eye can just about see objects which are 200μm in diameter; very few cells are as big as this, although a very fine hair may be this width. However, there are particular challenges in examining structures smaller than 200μm. Most components of tissues have little colour and contrast, and thus cells and the matrices surrounding them are indistinguishable if light is transmitted through them using a basic light microscope. Indeed, light will only penetrate thin slices of tissues or thin layers of cells growing in vitro. Various types of microscopes and methods of preparing specimens for examination have been developed. Living, isolated, whole cells can be examined using special (phase contrast) light microscopes but contrast is limited and the cells rarely have around them the structures they had in the body. In routine histology, very thin slices of tissues (5–10μm thick) are prepared through which light can penetrate. To achieve sufficient contrast and colours in the tissues so that they may be visualised, dyes or specific chemicals are applied to the slices of tissues. In these specimens, light microscopy can resolve detail of structures about 0.2μm apart. However, by using much thinner slices and electrons instead of light, electron microscopy can resolve detail down to about 0.0002μm. (Note 1mm = 1000μm.)
Numerous advanced techniques, suitable for light and electron microscopy, may be used to identify specific molecules in tissues via their reaction with labelled molecules. The labelled molecule is then detected, e.g. as colour using ordinary light microscopy, as fluorescence by viewing using ultraviolet light or as radioactivity using photographic film. Details of advanced techniques for studying the components of tissues with light and electron microscopes are beyond the scope of this book and the reader is advised to consult other texts. However, we give a brief overview below of basic histological techniques.
If any piece of the living body is removed it begins to degenerate as cell death occurs: this process is referred to as necrosis. In this process, enzymes in cells are released from their normal location and break down the cells and molecules in surrounding areas. Consequently, the precise three-dimensional arrangement of structures within, and surrounding, cells in life disappears. To study the arrangement of molecules, cells, extracellular matrix, tissues and organs as they were in life, necrosis must be prevented and the molecules, cells, etc. must be preserved. There are various methods for preservation, but a standard way is to place samples of tissue in a solution of formaldehyde as rapidly as possible after death or after removing them from a living body. Formaldehyde changes the conformational state of the proteins (and other large molecules) and prevents enzymes from degrading the tissues: this process is known as fixation. This chemical fixation can be compared with the process of boiling an egg in which heat changes the conformational state of the proteins and, with enough boiling, the proteins in the white and yolk of the egg become solid.
Most body tissues are soft in life and only a little harder after they have been fixed in formaldehyde, so they are difficult to slice thinly enough to be examined using a routine microscope. In order to prepare thin slices, tissue samples are impregnated with a substance which makes them solid. The medium used in routine histology to confer rigidity is paraffin wax, which is liquid at about 58°C but solid at room temperature. Wax and water-based tissues are immiscible, so formaldehyde-fixed tissue samples cannot be impregnated directly with wax. Hence, tissue samples are processed through a schedule which removes and replaces the water. This is most easily achieved by transferring the tissue sample through gradually increasing concentrations of alcohol which (at 100%) replaces all the water. Alcohol itself is also immiscible with wax but it is replaced in the tissue sample by processing it through increasing concentrations of a solvent that is miscible with alcohol and wax, e.g. chloroform or xylene. This solvent in turn is removed by placing the tissue sample in several changes of molten wax so that the wax infiltrates the tissue. The sample in the molten wax is then allowed to set and a solid block of wax forms in which the tissue sample is embedded. The tissue is then ready for sectioning. Given the toxicity of the substances used in these processes, appropriate safety precautions must be taken.
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