Fig

Photomicrographs of ::ssi:i- highlighted by spccial Cams and orhcr histologic techniques. . A, Elastin -Mill ot arterv lelastm (arrow) is black} The lumen of the vessel (asterisk), media :m), and adventitia (al are noted. (B) von Kossa stain of epiphyseal growth plate of bone (calcium phosphates ate black). Cartilage (c) and calcified bony ttabeculae (b) are noted, (Si)

Fungal stain of infected heart valve (individual fungal organism designated by arrow), ¡D) Immunohistochemica! staining fo

(actor VlJl-retaied antigen fvon Wi Ik-brand factor), a specific marker for endothelial cells (reaction product dark, endothelial cells designated bv arrows). (F.j Enzyme histochemical staining (or alkaline phosphatase in bovine pericardial bioprostheti heari; - i1 ¡'reaction producrs dark, designated by arrow).

FIG. 8.—continued

eliminates enzymic and immunological reactivity, and kills microorganisms present in tissues.

A 37% solution of formaldehyde is called formalin; thus, 10% formalin is approximately 4% formaldehyde. This solution is the routine fixative in pathology for light microscopy. For TEM and scanning electron microscopy (SEM), giutaralde-hyde preserves structural elements better than formalin. Adequate fixation in formalin and/orglutaraldehyde requires tissue samples less than 1.0 and 0.1 cm, respectively, in largest dimension. For adequate fixation, the volume of fixative into which a tissue sample is placed should generally be at least 5-10 times that of the tissue block.

Dehydration and Embedding

In order to support the specimen during sectioning, specimen water (approximately 70% of tissue mass) must be replaced by paraffin wax or other embedding medium, such as glycolmethacrylate. This is done through several steps, beginning with dehydration of the specimen through increasing concentrations of ethanol (eventually to absolute). However, since alcohol is not tniscibie with paraffin, xylol (an organic solvent) is used as an intermediate solution.

Following dehydration, the specimen is soaked in molten paraffin and placed in a mold larger than the specimen, so that tissue spaces originally containing water, as well as a surrounding cube, are filled with wax. The mold is cooled, and the resultant solid block containing the specimen can then be easily handled.

Sectioning

Tissue specimens are sectioned on a microtome, which has a blade similar to a single-edged razor blade, that is advanced through the specimen block. The shavings are floated on water and picked up on glass slides. Sections for light microscopic analysis must be thin enough to both transmit light and avoid superimposition of various tissue components. Typically sections are approximately 5 ¡xm thick—slightly thicker than a human hair, but thinner than the diameter of most cells. If thinner sections are required {e.g., for TtM analysis, approximately 0.06 fitn thick ultrathin sections are necessary), a harder supporting (embedding) medium (usually epoxy plastic) and a correspondingly harder knife are used (usually diamond;. Section for TTM analysis are cut on an til tram icrotome. Because the conventional paraffin technique requires overnight

FIG. 9, Photomicrographs taken in an electron microscope. [AJ Myocardial biopsy, showing capillary lumen (*). endothelial aril (ej, and heart muscle cell (hm) with sarcomeres (between open arrows), (B) Porcine bioprosthetic heart valve tissue, demonsttarmg fibroblast, including nucleus (nul and cytoplasm (c), surrounded by collagen fibrils (cf). In {Ai bar — 2 ,mii; nl iB) bar = O.r fim

processing, frozen sections can be used to render an immediate diagnosis (e.g., during a surgical procedure that might be modified according to the diagnosis). In this method, the specimen itself is frozen, so that the solidified internal water acts as a support medium, and sections are then cut in a cryostat (i.e., a microtome in a cold chamber). Although frozen sections are extremely useful for immediate tissue examination, the quality of the appearance is inferior to that obtained by conventional fixation and embedding methods.

Staining

Tissue components have no intrinsic contrast and are of fairly uniform optical density. Therefore, in order for tissue to be visible by light microscopy, tissue elements must be distinguished by selective adsorption of dyes (Luna, 1968). Since most stains are aqueous solutions of dyes, staining requires that the paraffin in the tissue section be removed and replaced by water (rehydration). The stain used routinely in histology involves sequential incubation in the dyes hematoxylin and eosin (H&E). Hematoxylin has an alkaline (basic) pH that stains blue-purple; substances stained with hematoxylin are said to be "basophilic" (e.g., cell nuclei). In contrast, substances that stain with eosin, an acidic pigment that colors tissue components pink-red, are said to be "acidophilic" or "eosinophilic" (e.g., cell cytoplasm, collagen). The tissue sections shown in Fig. 3 were stained with hematoxylin and eosin.

Electron Microscopy

Contrast in the electron microscope depends on relative electron densities of tissue components. Sections are stained with salts of heavy metals (osmium, lead, and uranium), which react differentially with different structures, creating patterns of electron density that reflect tissue and cellular architecture. Examples of electron photomicrographs are shown in Fig. 9.

It is often possible to derive quantitative information from routine tissue sections using various manual or computer-aided methods. Morphometric or stereologic methodology, as these techniques are called, can be extremely useful in providing an objective basis for otherwise subjective measurements (Loud and Anversa, 1984).

Three-Dlmenslonal Interpretation

Interpretation of tissue sections depends on the reconstruction of three-dimensional information from two-dimensional observations on tissue sections that are usually thinner than a single cell. Therefore, a single section may yield an unrepresentative view of the whole. A particular structure (even a very simple one) can look very different, depending on the plane of section. Figure 10 shows how multiple sections must be examined to appreciate the actual configuration of an object or a collection of cells.

Special Staining

There are special staining methods for highlighting components that do not stain well with routine stains (e.g., microorganisms) or for indicating the chemical nature or the location of a specific tissue component (e.g., collagen, elastin (Table 4). There are also special techniques for demonstrating the specific chemical activity of a compound in tissues (e.g., enzyme histochemistry). In this case, the specific substrate for the enzyme of interest is reacted with the tissue; a colored product precipitates in the tissue section at the site of the enzyme. In contrast, immunohistochemical staining takes advantage of the immunological properties (antigenicity) of a tissue component to demonstrate its nature and location by identifying sites of antibody binding. Antibodies to the particular tissue constituent are attached to a dye, usually a compound activated by a peroxidase enzyme, and reacted with a tissue section (immunoperoxidase technique), or the antibody is attached to a compound that is excited by a specific wavelength of light (immunofluorescence). Although some antigens and enzymatic activity can survive the conventional histological processing technique, both enzyme activity and immunological reactivity are often largely eliminated by routine fixation and embedding. Therefore, histochemistry and immunohistochemistry are frequently done on frozen sections, although special preservation and embedding techniques now available often allow immunological methods to be carried out on carefully preserved tissue. Special histologic techniques are illustrated in Fig. 8.

Artifacts

Artifacts are unwanted or confusing features in tissue sections that result from errors or technical difficulties in either obtaining, processing, sectioning, or staining the specimen. Recognition of artifacts avoids misinterpretation. The most frequent and important artifacts are autolysis, tissue shrinkage, separation of adjacent structures, precipitates formed by poor buffering or by degradation of fixatives or stains, folds or wrinkles, knife nicks, or rough handling (e.g., crushing) of the specimen.

conclusions

Cells, tissues, and organs are adapted to the complex yet highly organized and regulated activities that make up body functions. Key concepts of biological structure—function correlation include compartmentalization, differentiation, the basic tissues, organs, regeneration following injury, and multicellular communication. Although a wide variety of techniques are available for observing tissue structure and function, the microscopic study of tissue slices, called histology, is the most important tool used to investigate functional tissue architecture in clinical or laboratory investigation.

FIG. 10* Considerations for three-dimensional interpretation of two-dimensional information in relatively thin tissue sections. (A) Sections through a subject in different levels and orientations can give different impressions about its structure, here illustrated for a hard-boiled egg. (B) Sections through uniform structures can be misleading. Examination of the section indicated, shown as lower panel in B, would suggest that there were two populations of cells, one without nuclei, and even that cells of the same size had different-sized nuclei. (Reproduced by permission from A. Ham, Histology, 7th ed. Copyright © 1974 Lippincott.)

FIG. 10* Considerations for three-dimensional interpretation of two-dimensional information in relatively thin tissue sections. (A) Sections through a subject in different levels and orientations can give different impressions about its structure, here illustrated for a hard-boiled egg. (B) Sections through uniform structures can be misleading. Examination of the section indicated, shown as lower panel in B, would suggest that there were two populations of cells, one without nuclei, and even that cells of the same size had different-sized nuclei. (Reproduced by permission from A. Ham, Histology, 7th ed. Copyright © 1974 Lippincott.)

Bibliography

Alberts, B., Bray, D., Lewis, L., Raff, M., Roberts, K., and Watson, J. D. (1989), Molecular Biology of the Cell. 2nd ed., Garland Publ., New York.

Borysenko, M., and Beringer, T. (1989). Functional Histology, 3rd ed, Little, Brown, Boston.

Cormack, D. H. (1987). Ham's Histology, 9th ed. Lippincott, Philadelphia.

Darnell, J., Lodish, H., and Baltimore, D. (1990). Molecular Cell Biology, 2nd ed., Scientific American Books.

Fawcett, D. W. (1986). Bloom and Fawcett's: A Textbook of Histology. Saunders, Philadelphia.

Loud, A. V., and Anversa, P. (1984). Morphometric analysis of biological processes. Lab. Invest. 50: 250—261.

Luna, M. G. (1968). Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology, 3rd ed. McGraw-Hill, New York.

Schoen, F. J. (1989). Interventional and Surgical Cardiovascular Pathology: Clinical Correlations and Basic Principles. Saunders, Philadelphia.

Weiss, L. (1989). Cell and Tissue Biology, 6th ed. Urban and Schwarzenberg, Baltimore, MD.

Wheater, P. R., Burkitt, H. G., and Daniels, V. G. (1987). Functional Histology: A Text and Color Atlas, 2nd ed. Churchill Livingstone, New York.

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