Most fresh tissue specimens are colorless and squishy. They provide little useful information. For scientific or diagnostic purposes, tissue specimens must undergo substantial alteration in preparation for viewing under a microscope.
There are four steps in tissue preparation.
- Fixation stabilizes and preserves the tissue.
- Embedding converts the tissue into a solid form which can be sliced ("sectioned").
- Sectioning (slicing) provides the very thin specimens needed for microscopy.
- Staining provides visual contrast and may help identify specific tissue components.
Most basic histology texts offer a minimal account of basic histological technique. For routine examination of tissues, you probably don't need to know much more.
Artifacts. Be aware that each step of tissue preparation introduces artifacts by altering or distorting the natural appearance of cells.
Some artifacts are unavoidable. Fixation, by its very nature, kills cells and stabilizes dynamic cell processes. Enzyme activity is usually altered. Ions and small molecules are usually washed away.
Some artifacts are intentional, most notably the colors added by staining. The pink and purple colors of H&E staining can become so familiar that they appear "normal", as if they were the natural colors of tissues.
Still other artifacts are accidental. Cells may shrink or swell during fixation. Extracellular spaces may be distorted by compression or stretching. Ripples and wrinkles can be introduced during cutting and handling of sections.
Unintended artifacts can be minimized by optimal procedures -- but optimal procedures are often impractical, especially with human specimens. Ideal tissue preparation preserves cells in a form that resembles the living state, but this ideal is seldom practical with clinical specimens. Often, especially in post mortem (autopsy) material, cells have been dead and deteriorating for several hours before fixation. Therefore, certain artifacts must be appreciated as part of the normal appearance of tissue specimens.
The process of sectioning can introduce still other artifacts.
The most common mode of routine tissue preparation involves fixation with buffered formaldehyde, embedding in paraffin, sectioning into slices about 5 micrometers in thickness, and staining with hematoxylin and eosin.
Modern cell biology uses many tools to reveal cell structures and functions that are not apparent on routine H&E slides. Many of these involve sophisticated reagents based on the specificity of enzymes, immunological antibodies, or gene sequences to label and localize specific proteins or other molecules. Some textbooks present additional detail.
Fresh tissue samples must be preserved for future examination. This process is called fixation, and the resulting specimen is described as fixed.
Boiling an egg and pickling a cucumber represent examples of fixation, in which heat or chemistry stabilizes the organic materials.
A variety of chemicals can be used for fixing histological specimens. Routine fixation often uses a solution of formaldelhyde (formalin) to react with proteins and other organic molecules to stabilize cell structures. This solution is buffered and osmotically balanced to minimize shrinkage, swelling, and other collateral damage.
Ideally, fixation should be accomplished extremely quickly to minimize post-mortem changes in cell structure. Since fixation rate is limited by diffusion, ideal tissue preservation requires that fixative be delivered as closely as possible to each cell. Rapid delivery of fixative can be accomplished either by perfusion or by immersion.
Perfusion involves the delivery of fixative through the circulatory system of living tissue, by direct injection into a major artery. Such a procedure is commonly used with experimental animals but is obviously impractical for obtaining clinical specimens from patients.
Successful fixation by immersion requires very small samples. However, surgical removal of very small tissue samples often entails incidental mechanical damage, especially with punch biopsies.
These constraints on ideal fixation mean that tissue quality may vary across a specimen, with possible distortion near edges (especially with needle or punch biopsies) and with variation in fixation quality (and attendant staining character) in deeper areas (into which fixative diffuses more slowly).
An alternative to chemical fixation is freezing, followed by direct sectioning of the frozen specimen.
Frozen sections are seldom as "pretty" as well-fixed specimens, but they do have certain advantages. Because frozen sections do not require hours for the normal schedule of fixation and embedding, they can provide immediate diagnostic information to a surgeon in the operating room. Frozen sections can also permit analysis of small diffusable molecules or of enzyme activity whose presence would be lost during chemical fixation.
EMBEDDING and SECTIONING
After fixation, tissue specimens are routinely embedded in a solid material which will support very thin sectioning.
To embed a tissue sample, tissue water is replaced first by solvents (such as alcohol and xylene) and then with a liquid such as melted wax (paraffin) or epoxy solution which can be subsequently solidified by cooling or polymerization.
Sectioning is the production of very thin slices from a tissue sample. The tool used for sectioning is called a microtome (tom = to cut, as in appendectomy). A microtome may be as simple as razor blade, or it may be a complex machine costing several tens of thousands of dollars (for producing the ultrathin sections needed for electron microscopy).
- Most laboratory microtomes have the essential machinery of a baloney-slicer:
- a cutting edge (which may be a razor, a heavy knife, a piece of broken glass, or a finely sharpened diamond),
- a specimen holder,
- a screw to advance the specimen toward the blade (ultramicrotomes may use carefully-controlled thermal expansion in lieu of a screw),
- a crank, such that each turn of the crank raises the specimen, advances it, and then lowers it across the blade.
Sections for routine light microscopy are typically 5-10µm (micrometers, microns) in thickness. Exceptionally thin sections may less than 2µm thick. For electron microscopy, sections are typically 50-100 nanometers (millimicrons) in thickness.
Sectioning necessarily reduces the specimen to a two-dimensional representation. Reconstructing the three-dimensional structure of the original sample requires either the "stacking" of multiple images from serial sections, or else judicious use of imagination (3-D visualization). A very small amount of three-dimensional information may be directly visualized under the microscope, by focussing up and down through the thickness of the specimen.
For a further account of 3-D visualization, see here.
Sectioning can certain introduce artifacts.
Among the commonest artifacts, and most distracting for a beginner, are wrinkles. To appreciate why wrinkles form, imagine trying to lay a sheet of wet tissue paper (representing the slice from the sample) flat onto a table (representing the microscope slide). Even with great care, wrinkles sometimes appear. Sometimes wrinkles are "forced" when the tissue section stretches unevenly around structures of differing consistencies.
Another sectioning-related artifact is the disappearance of small structures which fall out of their proper place on the specimen, and the occasional reappearance of such structures at other inappropriate locations. This happens most often when the process of slicing separates a part which is attached only outside the plane of section, such as a hair shaft within a hair follicle. Except in the case of perfect lengthwise slices, the hair shaft will be cut into an oval slice that is not attached to the sides of the hair follicle and may therefore come out (leaving the follicle apparently empty) and then alight somewhere else (as an odd oval structure anywhere on the slide).
Yet other common artifacts are scratches and "chatter". Scratches are caused by flaws or dirt on the cutting edge, and appear as straight slashes or ragged tears across the specimen. "Chatter" is the visible record of knife vibration. The the process of slicing sometimes induces vibrations in the knife edge, which then cause variations in thickness (ripples) in the section. These appear as narrow parallel bands, usually evenly spaced, across a tissue specimen. They are often most evident in areas of smooth texture, such as the colloid in thyroid follicles.
Most cells are essentially transparent, with little or no intrinsic pigment. Even red blood cells, packed with hemoglobin, appear nearly colorless when unstained, unless packed into thick masses. Stains are used to confer contrast, to make tissue components visibly conspicuous. Certain special stains, which bind selectively to particular components, may also be used to identify those structures. But the essential function for staining is simply to make structures easier to see.
NOTE that all stain color is artifactual and does not represent the natural color of the tissue. The same structures may have very different colors with different stains. For example, collagen is pink with H&E but blue or green with trichrome. You should generally use specific aspects of actual structure (location, size, shape, texture) to identify cells and tissues, rather than color. Color can offer additional information if used wisely, but is unreliable by itself.
Routine histology uses the stain combination of hematoxylin and eosin, commonly referred to as H&E.
Hematoxylin is a basic stain with deep purple or blue color. Structures that are stained by basic stains are described as basophilic ("base-loving"). Chromatin (i.e., cell nuclei) and ribosomes are basophilic. With H&E staining, basophilic structures are stained purple.
Eosin is an acidic stain with a red color. Structures stained by acid stains are described as acidophilic (or eosinophilic) and include collagen fibers, red blood cells, muscle filaments, mitochondria. With H&E staining, acidophilic structures are stained red or pink.
- Note that, basophilic cell structures are NOT necessarily acidic; they only happen to stain with basic stains. Likewise for acidophilic structures, which are NOT necessarily basic. Many tissue staining properties are determined by the complex chemistry of proteins and other macromolecules after interactions with fixatives and other processing agents, and defy simple analysis.
- Also note that absolute color intensity on H&E-stained slides can be quite variable, with the same cell structure appearing red on one slide, pink on another, and possibly even blue on yet another. Relative stain intensity on the same slide is a more reliable indicator of acidophilic/basophilic quality than is absolute color, but this can also vary, especially between edges and center of a section.
Remember that nuclei are not really purple and collagen is not really pink. All such stain colors are artifacts, albeit intentional ones.
If an H&E slide shows any colors other than purple/blue and red/pink -- such as yellow or brown -- the additional color is probably due to an intrinsic pigment such as melanin.
Some cell structures do not stain well with aqueous dyes and so routinely appear clear. This is especially so for those which are hydrophobic, containing fat. Included in this category are adipocytes, myelin around axons, and cell membranes of the Golgi apparatus.
Trichrome uses three dyes (hence the name), including one that is specific for the extracellular protein collagen. Depending on the particular stain combination, a trichrome stain may color collagen fibers sky-blue or bright green. The principle use for trichrome is to differentiate collagen from other eosinophilic structures, such as muscle fibers.
Trichrome stains can be especially useful for highlighting an accumulation of scar tissue, as in glomerulosclerosis of the kidney (see WebPath) or cirrhosis of the liver.
PAS (Periodic acid Schiff) is used for glycogen, glycoproteins (such as mucus), and basement membranes (which contain glycoprotein).
Other stains. Be aware that many other stain techniques exist, for special cases. Some of these are classical procedures can yield beautiful results but depend on mysterious art and alchemy for success. Other, more-modern techniques have been rationally designed to exploit recent developments in molecular biology.
In the "classic" category are a number of stains based on metal salts.
A silver-based stain that demonstrates reticular fibers and basement membranes is especially useful for diagnosing certain pathologies of kidney glomeruli.
A variety of silver stains have been very powerful for research into the central nervous tissue. Their only common feature is that silver grains form a dark precipitate on selected structures, with empirical variables determining which structures are visualized.
Some cells have traditional names based on their demonstration with certain stains, such as the "argentaffin cells" (cells with an affinity for silver) and "chromaffin cells" (cells with an affinity for chromium) of the gastrointestinal tract.
In the "modern" category are stains based on the application of particular molecules that can be selectively stained using radioactive labels, enzyme reactions or specific antigen binding. The techniques of autoradiography, enzyme histochemistry and immunocytochemistry often require sections of frozen rather than fixed tissue.
For more on special stains, see the presentation at WebPath.
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Last updated: 15 April 2010 / dgk