EPITHELIUM
The purpose of this laboratory exercise is to give an overview of how various types of epithelia appear in histological section. You will be looking at a host of different tissues and organs. Obviously, it is not the intent of today's assignment to attempt to understand the full complexity of this material. We will return to each of these slides later in the course. Limit yourself to the epithelial structures under study. Often, they will line a surface of associated tissues. Frequently one can locate an epithelium simply by holding a slide up to the light or using the lowest magnification of the microscope. If the slide has been stained with hematoxylin, the high density of epithelial nuclei often reveals the epithelium as a purple line or mass. You should always begin your examination of a slide by naked eye to figure out the orientation of the tissue and what part to examine under the microscope. If you have difficulty in understanding a particular organ, turn to your textbook or atlas.
Objectives:
1. You should be able to locate the epithelium in sections on your slides.
2. You should be able to identify the following in the class slide sets:
| Simple squamous epithelium | Goblet cells | |
| Simple cuboidal epithelium | Basal cells | |
| Simple columnar epithelium | Basement membrane (where visible) | |
| Stratified squamous epithelium | Cilia | |
| Keratinized stratified squamous epithelium | Microvilli (brush border) | |
| Pseudostratified epithelium | ||
3. You should understand how a simple epithelium, as either a covering sheet or a tubule, will appear when sectioned in various orientations.
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Blue Histology |
Slides
to examine
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D-43 | Kidney, simple cuboidal and squamous epithelia (H&E) | |
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D-45 | Kidney, basement membranes (PASH) | |
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D-69 | Epididymis, pseudostratified columnar epithelium (H&E) | |
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D-76 | Bronchus, pseudostratified columnar epithelium (H&E) | |
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D-101 | Esophagus, stratified squamous non-keratinized epithelium (H&E) | |
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D-107 | Duodenum, simple columnar epithelium (H&E) | |
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D-171 | Eyelid, various epithelia (H&E) |
Optional slides
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D-1 | Buccal mucosa scrapings (Pap) | |
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D-48 | Urinary bladder, transitional epithelium (H&E) | |
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D-61 | Vagina, stratified squamous non-keratinized epithelium (H&E) | |
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D-75 | Bronchial tree, pseudostratified columnar epithelium (H&E) | |
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D-161 | Thick skin, stratified squamous keratinized epithelium (H&E) |
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Slide descriptions
D-107 Duodenum: simple columnar epithelium (H&E)
Hold this slide up to the light. The outside surface of the small intestine is smooth. The pink outer layer, visible by eye, is smooth muscle and just internal to it you can discern some veins as tiny bright orange circles. The inner surface is thrown into tall folds which look like fuzzy spikes about 1 cm high in cross-sections. These "plicae" are covered with tiny finger-like projections called villi, each 1-2 mm long. Villi are covered by simple columnar epithelium, which is what we are interested in for this laboratory session
Scan various villi using the 10X objective. The epithelium will be cut in various orientations with different resulting appearances. When sectioned perpendicular to the surface, the cells appear as an orderly single row of tall cells reaching from the basement membrane to the surface (illustration). If the section is oblique the epithelium may appear to have several layers of cells stacked on top of one another - even though the epithelium here is simple (illustration). If the epithelium is sectioned parallel to the surface the cells will appear in a tile pattern (illustration). The individual cells may look square, even though they are columnar. If such sections cut through the basal parts of the cells, where the nuclei are located, every cell will be seen to have a small round nucleus. If the section is higher up the cells will appear to lack nuclei.
This illustrates the importance of choosing an appropriate area of tissue when studying epithelia. Always start off by scanning around and remembering that a simple epithelium may superficially look stratified in some places, but a stratified epithelium cannot be cut in a way that makes it appear to have a single layer of cells. Regardless of the way the epithelium is cut on this slide it is still simple columnar. An old conundrum asks, "If you call a dog's tail a leg, how many feet does it have?" The answer is four because calling a dog's tail a leg does not make it one. Similarly, sectioning simple epithelium obliquely does not make it stratified.
So, spend some time looking at the epithelium cut in various ways. Then examine epithelium cut perpendicularly at 400 X (illustration). A useful relationship to remember is that an epithelium appears thinnest where it has been cut perpendicular to the surface. Most of the cells of the epithelium are of one type, called enterocytes or adsorptive cells. Their purple, oval-shaped nuclei are situated towards the base of the cells. The most distinctive specialization of enterocytes is the brush border of microvilli that covers their free surface, to expand the membrane area for absorption. The microvilli are too small to be seen individually but they form a visible line about one or two microns thick along the surface of the epithelium. You cannot see the basement membrane that the epithelial cells lie on but you can tell where it runs; at the junction between the base of the epithelial cells and the very differently organized connective tissue below them.
A second type of cell, called the goblet cell, is scattered in the epithelium. These cells manufacture and release mucus. Goblet cells have the shape of a wine glass (hence their name). Their apical ends are expanded by masses of stored mucus. Only a narrow stem of cytoplasm extends to the basement membrane. By looking carefully you can see that the nuclei of goblet cells look different from those of enterocytes. They are squeezed into a dark triangle or cone by the plug of mucus above them. Goblet cells are an important cell type of many epithelia. Further downstream, in the large intestine, they dominate the columnar epithelium. In other places columnar mucous epithelium invaginates into the underlying tissue to form mucous glands.
A variable number of lymphocytes invade the epithelium of the gut. They appear as round dark nuclei at any level in the epithelium. When classifying an epithelium as to type, any lymphocytes are ignored as just intruders into the epithelium. They are not epithelial cells, are born elsewhere and eventually will move elsewhere in their job of policing the tissue for pathogenic invaders. The epithelium that lines the gut is considered to be simple columnar regardless of the presence of lymphocytes and of the fact that some goblet cell nuclei may not line up perfectly with those of the enterocytes.
D-43 Kidney: simple cuboidal and squamous epithelia (H&E)
In the gut, the epithelium forms a sheet that covers the surface. An alternative common pattern is for epithelia to form hollow tubules within the organ. These structures still have all of the characteristic features of epithelia: a basal lamina, free surface to a space which eventually connects with the outside and so forth. They just have a different topology.
The kidney is made up almost entirely of long epithelial tubules running in various directions (and cut randomly in your section). All of these epithelia are simple. Most are cuboidal. However, towards the center of the slide, in the "medulla" of the kidney, some tubules have a simple, low columnar epithelium while others are nice examples of a simple squamous epithelium (illustration). You will notice that some epithelia are half way between squamous and cuboidal and can be called either "tall squamous" or low cuboidal.
Some of the tubules in the cortex, the "proximal tubules", have a brush border. Here it is taller than that on enterocytes and the long individual microvilli tend to clump together to give a tattered appearance to the lumen of the tubules.
The outer "cortical" region of the kidney also has circular bodies that are much larger and obviously different from the tubules. These are "renal corpuscles," and their internal masses, glomeruli, are tufts of capillaries. Examine the outer wall which lines the empty space surrounding the glomerulus in a renal corpuscle. It is formed of extremely flattened epithelial cells as an excellent example of the simple squamous epithelium (illustration). Both the cells and their nuclei have approximately the shape of pancakes, and their appearance depends to a large extent upon the angle at which you view them. When cut transversely this epithelium is seen as a very thin layer, and the nuclei appear to be very elongated and dark. (Think of how a fried egg would look in cross section.) Note that here again the shape of the nucleus mirrors the geometry of the cell.
This slide lets you further familiarize yourself with the effects of planes of section. Long tubules obviously will look different when sectioned cross wise and longitudinally. In some parts of the kidney, most notably in the medulla, the tubules are all straight with the same alignment. In other places the tubules are twisted all around like a plate of limp spaghetti. Find an area in the medulla where bundles of straight tubules are cut longitudinally (illustration). This orientation is simple to understand. Now find an area where all of the tubules are in round cross section (illustration). Here all of the tubules must be straight and running in the same direction. Make sure that you agree. If all of the tubules are similarly shaped ovals the tubules again must be straight and running parallel, but cut at an angle (illustration). Where the tubules are convoluted their sections take on all sorts of shapes (illustration). Try to find examples of all of these orientations. Now, answer this question by looking at your slide: Are the renal corpuscles located in regions of convoluted tubules or straight tubules?
D-45, Kidney: basement membranes (PASH).
The kidney has been chosen for demonstrating basement membranes because they are particularly well developed in this organ. This section has been stained with a histochemical reagent (PAS = periodic acid-Schiff) for carbohydrates, a major component of basement membranes. The basement membranes underling the epithelia appear bright magenta (illustration). This includes the ones under the epithelium of the capillaries in the glomerulus. The cells themselves stain lightly because they contain little polysaccharide. PAS does stain the brush border on the cells of proximal tubules (illustration). Cells tend to have a carbohydrate rich layer on their surface called glycocalyx. Usually it is highly attenuated but the great increase in surface area due to the microvilli allows PAS to visibly stain the surface of the proximal tubules. (The same would be true for the brush border of the duodenum, but your slide set does not include a section of that organ stained with PAS)
Basement membrane vs. basal lamina, what is the difference? Almost all epithelia are attached to the underlying connective tissues by a basal lamina. These sheets of extracellular proteins and fibrils are so thin that they can be seen only with an electron microscope. Under many epithelia additional carbohydrate-rich material is appended to the lower face of the basal lamina. If the accumulation is sufficient to be seen under the light microscope it is called a basement membrane. The basal lamina is presumably synthesized by the epithelial cells and the additional underlying material of the basement membrane by the underlying connective tissue cells. Some epithelia do not have a basement membrane but virtually all have a basal lamina.
So, to recapitulate, basement membrane is an old classical histology term for a structure that an epithelium may show, whereas basal lamina is the newer name given to the universal accompaniment of epithelia that was seen only when the electron microscope came into use. The basal lamina has profound influence over the physiology of epithelial cells but in most cases it is not clear that a basement membrane even has a functional significance. You may hear it said that a basement membrane adheres the epithelium to the underlying connective tissue. True enough for the basal lamina but think about it for a minute. Can a thicker layer of glue hold things together better than a thin layer?
D-101 Esophagus: stratified squamous non-keratinized epithelium (H&E)
The inner lining of the esophagus provides an excellent example of a thick stratified squamous epithelium. By the way, squamous means "scale" in Latin. Estimate how many cells are layered between the basement membrane and surface. Note that the deeper cells are not squamous at all. In fact, the basal cells, those touching the basement membrane, are actually columnar, and those above them pass through a zone of polygonal (cuboidal) cells before reaching layers of squamous cells. Remember that stratified epithelia are categorized by their most specialized cells. Those are the cells at the surface. The basal cells are stem cells, capable of undergoing mitosis, and the cells of the middle layers represent intermediate stages of specialization.
Stratified squamous epithelia are quite common in the body, but stratified cuboidal and stratified columnar are not. These rare forms are found only in the most protected wet environment, such as under the eyelid, in ducts of certain glands, and in the male urethra. Most tend to be unstable, and are easily converted either to a stratified squamous epithelium, or to a simple columnar epithelium.
D-171 eyelid: stratified epithelia (H&E)
The eyelid has two surfaces. The outside is covered with skin with keratinized stratified squamous epithelium. The side against the eyeball, called the conjunctiva, has a variable wet epithelium. Turn first to the skin side.
Stratified squamous epithelia can develop a specialization of their surface cells, called keratin, to make them more resistant to stresses. Keratinized stratified squamous epithelium covers the entire outer surface of the body as the epidermis of skin, to prevent desiccation. In the oral cavity, areas of the tongue, palate and gingiva which receive a lot of abrasion become keratinized. In some places the keratin layer may be very thick. Other places are protected by only a thin layer of keratinized cells. To form keratin, the upper layers of cells dispose of all of their organelles, fill up with fibrous proteins and become extremely flattened. The cells are so tightly bound to the ones above and below them that the boundaries are invisible. New cells are continually added to the bottom of the layer of keratin and packets of keratinized cells continually flake off of the surface (e.g. dandruff flakes). The basal layers of cells of keratinized stratified squamous epithelium look similar to their counterparts in unkeratinized stratified squamous epithelium. Only the upper layers are distinctive.
The layer of keratin is easy to see on this slide. It stains darker red than the cells below. As you recall, the eosin dye of H&E stains mainly the proteins of cells. Two factors determine how much dye a particular cell will take up. One is the amount of protein. The other is the solubility of the protein. Unless precautions are taken a lot of the protein can leach out of cells when tissues are fixed and stained. The proteins in keratin happen to be highly insoluble and hence it is almost always easy to tell whether a stratified squamous epithelium is keratinized or not.
Have a good look at the epithelium of this slide and then go back to the esophagus slide for comparison with unkeratinized stratified squamous epithelium. One difference, in addition to the presence or absence of a visible layer of keratin, is that the cells on the surface of unkeratinized epithelia have nuclei. This is a useful practical criterion to use. Another general characteristic is that unkeratinized stratified squamous epithelia usually have more layers of living cells than corresponding keratinized epithelia. At this time, disregard all of the other structures in the deeper regions of the eyelid. The glands and hair follicles are epithelial structures, but complex ones that we will deal with later.
One further comment: Only stratified squamous epithelia can become keratinized. There is no such thing as keratinized simple epithelium or keratinized stratified cuboidal epithelium.
Turn now to the conjunctiva. Its epithelium also is stratified but with only 2-5 layers of cells and no keratin layer. Scan along to see if you can find stratified cuboidal (high mag), low stratified columnar and maybe stratified squamous varieties. You will see numerous - in fact very numerous - lymphocytes just under the epithelium along most of the conjunctiva. These are protective cells of the immune system. The thin wet epithelium of the conjunctiva is not very resistant to pathogens and therefore protective cells accumulate below it.
D-69 Epididymis: pseudostratified columnar epithelium (H&E)
It is best to think of pseudostratified columnar epithelium as a specialized variant of simple columnar epithelium. There are several types of cells whose nuclei lie at different heights in the epithelium, but every cell has a basal process that attaches to the underlying basement membrane. Hence every cell can directly participate in metabolic exchange with nearby capillaries. However, not every cell reaches the surface. In particular, basal cells often do not and serve as potential replacements for the other, more specialized cells.
Hold slide D-69 up to the light. The epididymis is a comma shaped organ that lies alongside the testis. Sperm from the testes are stored and matured there. The epididymis has two sets of ducts, both lined by pseudostratified columnar epithelium (illustration). Focus on the epithelium which lines the duct of the epididymis, the duct with the larger cross sections. This duct is very long and coiled so that it is sectioned many times. Examine the epithelium of one of these sections at 400X power (illustration). The main cells are tall columnar. In addition small cells are crowded next to the basement membrane. The round nuclei of these basal cells lie distinctly below the elongated nuclei of the columnar cells. Thus, this epithelium is pseudostratified. The basal cells are stem cells. They can divide and differentiate into functional columnar cells as the latter get worn out and die. The columnar cells have a very obvious brush border even longer than in the kidney. These are microvilli that are so long that they have been given the special name stereocilia. This is a poor term, left over from before the advent of the electron microscope. These structures have nothing to do with cilia, and their function is to absorb water quickly, as do the microvilli on kidney cells. In case you are wondering what the bluish masses in the centers of some sections of the duct are, they are masses of sperm which are maturing.
The smaller efferent ducts of the epididymis are also composed of pseudostratified epithelium (illustration). The two main cell types are tall ciliated cells and shorter nonciliated secretory cells. The two cell types tend to cluster with their kind (like the two sexes at an Australian cocktail party). Therefore the epithelium varies in height around the tubule. This gives the lumen a star shaped in contrast to the basement membrane which describes a smooth curve.
D-76 Bronchus: pseudostratified columnar epithelium(H&E)
Slide D-76 includes sections through 2 or 3 medium size bronchi, which are branching tubes for conveying air to the lungs. Hold the slide up to the light and identify these bronchi as holes with patches of blue cartilage around them. Under low power, find the epithelium lining their surfaces. Go to 400 X and scan along for a place where the section cuts perpendicular to the surface (illustration). This epithelium is pseudostratified columnar with cilia and mucus cells. The name pseudostratified is appropriate here because the cells are so jumbled that you could have trouble knowing that it was not truly stratified. All of the cells do, indeed, touch the basement membrane. However, many of the tall ones have only a narrow connection to the base. In an epithelium like this the boundaries between cells are pretty well invisible. In such a case, a shrewd histologist infers the shape of the cells from their nuclei. As you have seen, the shape of the nucleus generally reflects that of an epithelial cell. Look at these nuclei. Those of the basal cells closest to the basement membrane are rounded, and so are their cells. In the cells farther up the nuclei are elongate, indicative of columnar cells. If this were a stratified epithelium it would have to be a stratified columnar epithelium. This is possible but highly unlikely. Stratified columnar epithelia are very rare. On the other hand, pseudostratified columnar epithelia are widespread.
Here is one further tiny point concerning nuclear shape. You have seen that some epithelia are in between columnar and cuboidal and can be called either low columnar or tall cuboidal. Which term should we use? I look at the nuclei and if they are round I call the epithelium tall cuboidal, or if elongated, low columnar.
This epithelium contains a half dozen or so different cell types. You should be able to distinguish three; basal cells, cells with cilia on their apical surface and goblet cells which you already saw on a previous slide. Ciliated cells and goblet cells accompany each other throughout the respiratory tract. The one type produces a layer of mucus on the surface of the epithelium to trap dust particles and bacteria: the other moves that layer across the epithelium. As mentioned earlier, the nuclei of goblet cell often are compressed towards the bottom of the cell by the masses of mucus stored in the apex. The ciliated cells have a tall (darkly stained) body which flares out near the surface of the epithelium so that its cilia cover most of the surface area of the epithelium. If you hunt around you should be able to find an area which shows this shape and at the same time gives a good view of the cilia (example).
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Optional slides
D-48 Urinary bladder: transitional epithelium (H&E)
The epithelium of the urinary tract does not fit in the conventional classification scheme for epithelia. It is stratified but the cells do not have a constant shape. When the bladder is distended, the uppermost cells are stretched into a squamous shape (illustration). When the bladder is emptied the cells round up into a cuboidal or columnar configuration. Therefore this epithelium is given its own category called "transitional epithelium". The tissue on slide D-48 was kept stretched while it was fixed and the epithelial cells are flattened out. For comparison the section of bladder on slide D-49 was allowed to contract (illustration). Look at the transitional epithelium on both slides.
You may worry about how to recognize transitional epithelium on a slide if it has the chameleon capability to change from squamous to columnar. Fortunately, transitional epithelium is a specific type of epithelium instead of a general category. It occurs only in the urinary system, and is recognizable by its specific characteristics, not just by the fact that it can change shape. As in all epithelia it is the surface layer of cells which is specialized. These "umbrella cells" are distinctive. They are large, making this the only stratified epithelium (that I am aware of) in which the top layer of cells is bigger than the cells right under them. Another visible specialization is that the umbrella cells tend to stain darker (and slightly more basophilic) than the underlying cells. Also, some of them have two nuclei in them.
We will discuss the specifics of transitional epithelium towards the end of the course when we get to the urinary system. For now the interesting thing about it is that it is a square peg in our round pigeonhole classification scheme. Most of the generalizations that we make in histology have rare exceptions, like this one. Some epithelia do not have free surfaces, and some rare areas are a mosaic of simple and pseudostratified or stratified epithelia. In fact, there is one epithelium, of maybe a quarter of a square inch in total area, that even has blood vessels running in it! Thus, the patterns that the tissues of the body follow are not absolutes, but the result of widespread functional or genetic or developmental generalities. This makes it all the more impressive (to me) of how regular most tissues are. Epithelia can have edges or lack basal lamina or include blood vessels but very rarely do. This is what makes biology so much richer than physics, where patterns are simply dictated by laws that allow no exception. You have to be a Sherlock Holmes instead of a computer robot to understand histology.
D-1 Buccal mucosa scraping (Pap)
Gently scraping the inside of the cheek with a cotton swab detaches cells from the surface of the epithelium. These cells are large and flat with fairly large flat, pale nuclei. Here you can see them face on. In this perspective they look very different from the dark narrow lines seen in cross sections. Why are some cells dark with small nuclei and others larger and paler? (If you cannot figure out why, read the text for the next slide below.)
Scrapings, such as these, have two major uses. One is in Pap smears. Cells taken from the cervix in this manner are examined to detect cancerous and precancerous transformations. Indeed, this slide was stained with a "Pap stain", especially useful for observing abnormal cells. The other modern use is to obtain tissue in a non-invasive way for DNA analysis. For obvious reasons geneticists sample the inner cheek instead of the cervix. It is convenient that the surface cells of nonkeratinized epithelia retain their nuclei, n'est pas?
D-61 Vagina: stratified squamous nonkeratinized epithelium (H&E)
Find the epithelium lining the inside surface of the vagina. It obviously is stratified and, in fact, it is stratified squamous non-keratinized epithelium. In such epithelia even the top most layer of cells have nuclei. Why do you not see nuclei in every surface cell? Compare the appearance of the nuclei at the surface with those in the basal layer. What is the significance of the nuclear changes that occur as the cells migrate up the epithelium and mature? A small, very darkly staining nucleus is described as heteropyknotic. Chromatin that is tightly condensed is called heterochromatin, as distinguished from euchromatin which is dispersed and stains palely. The nuclei of most cell types have grains of heterochromatin, of characteristic size sprinkled within euchromatin. Genes in heterochromatin generally are inactive. Aha, what does this say about cells as they mature in a stratified squamous nonkeratinized epithelium?
The cells at the surface of this epithelium are flattened, but less so than those of the esophagus, D-101. This is because they have stored glycogen, which puffs them out. This polysaccharide does not stain with H&E and is soluble enough to leech out of the cells during the staining procedures.
D-161 Thick skin: stratified squamous keratinized epithelium (H&E)
The skin on the palms of the hands and soles of the feet is called "thick skin " because it has many more layers of cells than in the "thin skin" you looked at before. The band of keratinized cells on the surface is particularly thick. In addition, thick skin has many more nucleated cells, making it easy to distinguish several layers. These "strata" represent successive stages in the differentiation of the keratinocytes from their origin by cell division along the basement membrane to their final keratinized form. We will consider the individual strata in depth in our later session on skin.
D-75 Bronchial tree: pseudostratified squamous epithelium (H&E)
This slide is much like the earlier one D-76 but a bit farther down in the lung. The main difference is that the epithelium is less well preserved on this slide. In some places the cells of the epithelium have come apart (illustration). If you hunt carefully for the right view you can see the shapes of individual cells. Elsewhere the epithelium is intact (and shows up beautifully) or is too degenerated to be informative. The main point is to convince yourself that you can tell that in this pseudostratified epithelium probably every cell indeed extending down to the basement membrane. Ignore everything else on this slide, the blood vessels, lung tissue and even the bloody goop that has leaked into some of the bronchi.
Many of your slides have imperfect preservation or artifacts of fixation, like this one. This is not all bad. Some artifacts are quite useful diagnostically as characteristic of certain tissue types or tissue compositions. You will recognize some tissues, such as cartilage and elastic arteries, in part by looking for particular artifacts.
Whew, that's another lab down.