This slide contains a section of the wall lining the urinary bladder. When you look at the webslide, the lumen of the bladder is at the bottom and the body cavity is at the top of the field. At low power, contrast the appearance of the natural "free edges" at the bottom and top of the section where epithelium lines the bladder lumen and the body cavity, respectively, from the sharp "cut edges" at the left and right of the section where the tissue was cut during specimen preparation. First focus on the top surface of the tissue, and note where a simple squamous epithelium called a mesothelium covers the bladder. This cellular layer is quite thin (less than 5 mm). Note the bulging nuclei and attenuated cytoplasm that forms a continuous sheet that is sharply differentiated from the underlying connective tissue and muscle. Some portion of the specimen has lost its mesothelium, so scan along the top edge of the webslide.
Now examine the lower portion of the slide which is the tissue that lines the lumen of the bladder. Notice the characteristics of the transitional epithelium that is found only in the urinary tract: (1) there are several layers of cells, (2) most of the cells in the outer layer touching the lumen are rounded or polygonal and contain spherical nuclei, (3) in this outer layer of cells there is usually a considerable amount of cytoplasm between the nuclei and the apical plasma membrane. These last two characteristics distinguish this epithelium from stratified squamous epithelium where the outer layer of living cells is flattened and has thin nuclei.
This slide prominently features simple cuboidal epithelium, lining tubules in the kidney. The tubules are cut in all different orientations; look for a region toward the middle of the slide where the tubules are cut more or less in longitudinal section [example] and appear as parallel wavy rows (at 4x magnification). Look for a favorable area where you can see a space (the tubule lumen) lined on either side with simple cuboidal epithelium. Note also that there is very little other tissue between tubules, so that you often see two rows of cuboidal epithelia from adjacent tubules back to back. In other parts of the section, look for tubules in cross-section [example] where the lumen will be surrounded by a circle of cells.
The absorptive surface lining the lumen (bottom of webslide) is thrown into large folds (villi) which increase the absorptive surface area. Scan the slide to observe the orientation of the simple columnar intestinal epithelial cells that line the lumen as they are cut in different planes. Two types of cells are present in this columnar epithelium – absorptive cells with striated borders and secretory goblet cells. Identify the following and understand their fine structure and function:
Striated border (composed of microvilli): what cytoskeletal element is in the core of each villus?
Cell junctions: where would you find tight junctions? desmosomes? hemidesmosomes?
Goblet cells (unicellular glands): What shape are these epithelial cells? What do they secrete?
The lumen of the oviduct (near the center of the slide) is thrown into many folds that are all lined by a simple columnar epithelium. Scan the epithelial surface to find regions where the epithelium is cut in a section perpendicular to the basal lamina. Two distinct types of columnar cells are present in this epithelium, ciliated cells and non-ciliated secretory cells. Measure the height of the cilia and compare to the height of the striated border composed of microvilli in the previous Webslide 32. Can you resolve individual cilia at highest power?
Note that by being able to distinguish the cilia in this slide from the microvilli of the previous Webslide 32 you have learned about the functional properties of the different epithelial surfaces: transport of material (oocytes) in the oviduct and increasing surface area for absorption of food products in the intestine.
The esophagus is lined by a non-keratinized stratified squamous epithelium. This type of epithelium covers some internal surfaces that are kept moist by mucus or other fluids, and the lubrication provided by mucus helps to protect against abrasion. Thus, these epithelia do not need to keratinize to resist desiccation and mechanical wear. Study this type of epithelium [example] and note how the cell morphology changes from the roughly cuboidal in the basal layer to squamous in the apical surface of the epithelium (in this case, the apical surface is the bottom-most edge of the tissue). Recall that stratified epithelia are named based on the morphology of the outermost layer. Unlike keratinizing epithelium, nuclei are still present in most surface cells.
The epidermis of the skin, found at the bottom of the slide, is an example of keratinizedstratified squamous epithelium. This epithelium is found at the surface of the skin and is known as the epidermis. As protection against desiccation, it undergoes a process known as cornification or keratinization. As cells move toward the surface, they differentiate and eventually die, leaving an outermost layer of dead cells filled with keratin [example]. After the cells die, their nuclei break down and fade away, a process called karyolysis, leaving faint "ghosts" or outlines of nuclei that you can observe in some regions [example]. In some slides, the keratinized region is gray, but occasionally it has been penetrated in places by red stain. Note the differences in morphology of the cells as they move toward the surface. In the lower strata, look for the layer of spinous cells (the spines look like little lines between cells, and can be difficult to see) [example]; the spines are sites where desmosomes attach the cells to one another.
The epithelium of the trachea is along the bottom of the slide. Note the different levels of the nuclei in this pseudostratified columnar ciliated epithelium. The elongated nuclei of the columnar ciliated cells are farther from the basal lamina than the rounded nuclei of the short basal cells. Goblet cells can also be observed with their aggregated mucinogen granules near the surface of the epithelium. Observe and measure the dimensions of the cilia. Cilia are involved in transport of material along the epithelial surface. What do they transport in the trachea?
Connective Tissue & Blood
Gartner & Hiatt Atlas (5th ed):
Plates 3-2, -3
Plate 5-1
Text (Junqueira's 12th ed):
Ch 5, Connective Tissue, pp. 86-108
Ch 6, Adipose Tissue, pp. 109-10
Ch 12, Blood, pp. 203-15
Scan around the 63x and 86x slides at high magnification to see the various kinds of blood cells that were discussed in the lecture. Most abundant, of course, are the red blood cells (RBCs) or erythrocytes, which are seen in large numbers everywhere you look. In between the RBCs you should look for small, basophilic fragments which are platelets or thrombocytes [example] that are important in blood clotting. As you continue viewing, you will see occasional white blood cells (leukocytes). Some of the white blood cells may defy identification, often because the cells were damaged during slide preparation, so look for characteristic examples, and ignore the equivocal cells. Refer to the images in your texts and from the lecture and try to find an example of each leukocyte type using the 63x and 86x slides (there's less area to cover in these high-mag slides and the cells present are excellent, although the 63x slide does NOT contain any basophils).
The most common white blood cell is the neutrophil, which has a distinct multi-lobed nucleus (often 3-5 lobes). Also frequently seen are lymphocytes, which are small cells (often as small as RBCs) with a dark nucleus and very little cytoplasm. Another cell type is the monocyte, the largest of the blood cells. It has a large, relatively pale nucleus, and rather clear cytoplasm (granules are usually less apparent than those in the illustration in W). You will also see an occasional eosinophil, with prominent reddish granules filling the cytoplasm, and a nucleus with 2 (or sometimes 3) lobes. The exact color of the granules may vary from slide to slide, depending on how well the slide was prepared. In your particular slides they may be anywhere from bright red to dull brown. The remaining cell type you may see on your slides is the basophil, which is hard to find, since it constitutes less than 1% of the leukocytes (the 86x slide actually has THREE excellent examples). The cytoplasm contains large, irregular granules in a "grape-cluster" appearance that usually stain dark blue or almost black. Basophil nuclei may often appear somewhat oval-shaped, so, at first glance, they may be confused with lymphocytes. However, the presence of the large, dark-staining granules should help you distinguish them; also, remember that basophils are rare.
After you've done some looking on your own, here some quick links showing examples of each type of leukocyte (in order of their normal frequency in a blood smear). USE THESE LINKS ONLY AFTER HAVING TRIED TO FIND THESE CELL TYPES ON YOUR OWN!
For each slide, first locate the surface epithelium and then study the cellular connective tissue layer immediately underlying the epithelium. You will NOT be able to identify all of the cells in these CT regions, but you should be able to identify many of the cells described below.
Lamina propria is the term given to the cellular connective tissue layer lying immediately beneath the moist epithelia that lines the gut and urinary bladder. The lamina propria contains a higher proportion of cells to collagen than found in the dense irregular connective tissue in the skin.
Cellular CT contains some of the white blood cells you examined above in circulating blood, namely lymphocytes (small round cell with little cytoplasm and a round darkly staining nucleus), neutrophils (each with lobulated nucleus and palely staining granules), and eosinophils (lobed or “C”-shaped nucleus with red granules in the cytoplasm). In addition, you should be able to recognize several resident CT cells including:
fibroblasts (each cell is elongated with elongated, darkly staining nucleus and scant cytoplasm),
plasma cells (relatively large ovoid cell with “clockface” or “cartwheel” nucleus at one side of cell and abundant pale staining cytoplasm in the rest of the cell), and
mast cells (round nucleus and cytoplasm full of granules; “fried egg with measles”).
The frequency of each cell type in a given section is variable, but as you search you should be able to find fibroblasts and examples of several of the other cells types in each slide:
Webslide 32 contains many lymphocytes [example] , along withplasmacells [example] and mast cells[example](easiest to find in the nearby dense, irregular connective tissue)
Webslide 98 contains a few excellent examples ofmast cells [example].
Slide UMich 29 is from tissue that was mildly inflamed at the time it was acquired and is therefore particular good for observing neutrophils and plasma cells[example].
With low power, locate the medulla (the interior) of the lymph node. Look for a region characterized by interlacing cords of cells. Macrophages[example] are the biggest, rounded cells that are floating free in the spaces between the cords of cells. Many of the free cells in these medullary sinuses cannot be identified; however, the large rounded cells, with eccentrically placed, vesicular nuclei are the ones you should try to find.
Many of these macrophages contain phagocytosed red blood cells or the brownish breakdown pigment, hemosiderin (which is the result of lysosomal action on the ingested red blood cells.). Be sure you can identify a macrophage and not just a bunch of cells superimposed upon one another.
The "mononuclear phagocyte system" (also called the "reticuloendothelial system" for historic reasons) consists of free and fixed macrophages throughout the body. These cells are important in removing all kinds of debris from the body as well as playing a major role in the immune response.
Slide #30 is from abdominal mesentery (the connective tissue that suspends the viscera within the abdominal cavity), which is an excellent place to find adipose tissue. Locate the spongy areas [example] in between the lymph nodes and vessels in this section. These are fatcells (or adipocytes). In white or unilocular adipose tissue as shown here, lipids are stored as a single, non-membrane bound droplet that is usually extracted during tissue preparation, which is why the cells appear empty. Note that you can't see a nucleus in each adipocyte; think about why this is so (click here for the explanation).
A fatty tissue called brown or multilocular fat, produced during fetal development, has adipocytes that contain multiple fat droplets. Brown fat is important for thermoregulation in newborns and hibernating mammals. In humans, brown fat is widely distributed throughout the body in the first decade of life, but it then disappears except for regions around the kidney, suprarenal glands, aorta, neck and mediastinum. None of our slides of adult tissue shows any brown fat, however this rather unique tissue can be seen in slide H2[example], which is from a developing fetus.Some of the individual fat cells are often broken during tissue preparation, but the overall impression of what the tissue looks like is the important point.
The fine collagenous network that provides support in the bone marrow, lymphatic organs, around individual smooth muscle cells, and beneath most epithelia is composed mainly of Type III collagen, which will pick up eosin stain but is too small to be discerned as fibers. However, this type of collagen has associated carbohydrate moieties that can reduce Ag+ to metallic Ag revealing a network (reticulum) of fine, black fibrils. Hence, these are termed reticular (or, rarely, argyrophilic= "silver loving") fibers. On slide #27 look at the accumulations of darkly stained cells (lymphocytes in a lymph node). Note at high power that fibrils or fibers of any type cannot be readily observed. Now with slide #28, which has been treated with silver nitrate, note how a network of fine black fibrils is present in this same tissue following silver staining. These are reticular fibers, found in skin, muscle, blood vessels, and the stroma of almost every organ in the body.
Please remember that virtually all cells (except for those in the brain and spinal cord) are provided with some degree of support by delicate reticular collagen (reticular fibers) even though that may not be apparent with H&E staining.
The area beneath the stratified squamous epithelium shown in slide 33 is the dermis, which is composed of dense irregular connective tissue. In this section, the fibers clearly predominate. This slide has been stained with iron hematoxylin and eosin so you can see collagenfibers (orange) as well as elastic fibers (purple/black) in the dermis [example]. Note how the diameter of the fibers varies with location. In the region immediately beneath the epidermis you can see how the elastic fibers are interconnected forming an elaborate, delicate net of fibers [example] amongst thin strands of collagen. However, deeper in the dermis, the collagen and elastic fibers are much thicker.
Slide 250 which you used to look at stratified squamous non-keratinizing epithelium is also useful for the study of connective tissue (we will also use this slide to study smooth muscle and peripheral ganglia). Be sure you look at both the H&E and Masson trichrome-stained slides as they provide an excellent opportunity to see how collagen stains in connective tissue when either stain is applied. When we study smooth muscle and peripheral nerve tissue we will come back to this slide to try and distinguish between collagen fibers and fascicles of smooth muscle and/or nerve fibers and ganglia.
Slide 106 has bits of well preserved flexor tendon at the top of the section. Note the regular orientation of the collagen fibers (there's a bit a "waviness" but you should get the idea). You should also observe that there aren't a lot of cells, a characteristic of "dense" connective tissue. The very small cracks between the fibers are just artifacts of shrinkage that occured during tissue preparation. Of course, there are some places where there are breaks in the dense regular connective tissue of the tendon containing loose connective tissue associated with nerves and blood vessels or the occasional bit of adipose tissue.
These slides are examples of regularly arranged sheets (lamellae) of elastin. Slide 36 is stained with aldehyde fuchsin and Masson trichrome (Aldehyde Fuchsin, Fe. Hem. & Mass.), so the elastic lamellae are purple. Slide 88 is stained with H&E where the concentric rings of elastic lamellae are intensely stained with eosin giving a glassy red appearance[example] (one of the few places where elastin is easy to recognize in H&E sections).
Two other locations where elastic fibers can be readily seen in H&E sections are in the lamina propria of the pharynx (slide 152WebScopeImageScope) and the trachea (slide 40 WebScopeImageScope). Unlike the tissue of the aorta which would be classified as "regular," these tissues are obviously irregular but they're mentioned here for the purpose of illustrating how aggregates of elastic fibers appear in H&E-stained sections. Just as in the H&E-stained aorta, the elastic fibers in the pharynx and trachea are glassy and orange-red --they appear as stippled dots because they’ve been cut in cross section.
Cartilage & Bone
Gartner & Hiatt Atlas (5th ed):
Plates 4-1, -2, -3, -4
Text (Junqueira's 12th ed):
Ch 7, Cartilage, pp. 114-20
Ch 8, Bone, pp. 121-39
These slides are good examples of mature hyaline cartilage with its abundant matrix and spaces, lacunae, occupied by cells, chrondocytes, which usually shrink extensively during fixation. The staining of the matrix is variable. Remember that there are abundant type II collagen fibrils in the matrix. However, they are too small to be resolved in the light microscope, so the matrix has an amorphous, glassy (or "hyaline") appearance. The predominately basophilic staining of the matrix in slide #126 [example] reflects preservation of the negatively charged aggrecan molecules in the matrix. Note that the basophilia varies and some interterritorial matrix is eosinophilic reflecting loss (or minimal content) of negative charges, whereas the territorial matrix (the area immediately surrounding each lacuna) is much more basophilic.
Slide 44 is from the pinna of the ear stained with aldehyde fuchsin (stains elastin deep purple) and Masson's trichrome (stains collagen blue). Elastic cartilage can also be readily identified in routine H&E sections as well as shown in slide #44H which is from the epiglottis. Look for the plates of elastic cartilage found just under the glands deep to the respiratory epithelium. Observe that there are chondrocytes within lacunae just as in hyaline cartilage, but note the intensely eosinophilic, fibrillar matrix due to the very high abundance of elastic fibers. As with hyaline cartilage, fibrils of type II collagen are also present, but they cannot be seen in the light microscope. You may also notice that elastic cartilage tends to be more cellular than hyaline cartilage.
This cartilage is named for its textured matrix; it looks fibrous, and in addition lacunae can be seen. Locate the nucleus pulposus (clear area) of the intervertebral disc, then move out to the edge of the section to see fibrocartilage [example]. Note the fibrous texture of the matrix due to the presence of type I collagen fibers in addition to the type II collagen present in all cartilage tissue (type II fibrils are not bundled into fibers large enough to be visible in the light microscope), but note also the distinct chondrocyte lacunae. Also, note that there is no perichondrium in this cartilage.
Prior to sectioning and staining, this sample was soaked in a weak acid solution thus dissolving the mineralized component of the bone matrix but leaving behind all of the organic components (mostly type I collagen). Even though this section is distorted, you should be able to find osteons in various stages of development, lacunae (some with osteocytes), and some hints of canaliculi (literally, "little canals" through which the osteocytes extend fine processes).
This section features a tibia and fibula cut in cross section and offers an excellent opportunity to see very well-preserved osteoblasts[example]. Note that all of the spicules of bone are covered by osteoblasts; some of the osteoblasts are flattened and therefore quiescent whereas other osteoblasts are cuboidal and feature a basophilic cytoplasm indicating that they are actively building bone.
This section shows the bony palate in the roof of the mouth and is also good for seeing osteoblasts that build bone as well as osteoclasts[example] that resorb bone. You may notice areas of bone [example] that are more basophilic and mottled in appearance and with a higher density of osteocytes compared to other regions; this is so-called "woven" or "immature" bone which will eventually be remodeled and replaced with mature or "lamellar" bone.
These "ground sections" were prepared by taking pieces of bone and grinding them with abrasives between two glass plates until they are thin enough to be semi-transparent. First, study the cross section (#51xc). In this sections, the trapped air bends the light giving a dark image; the mineral and matrix generally transmit the light. You should be able to identify osteons and their subdivisions (as in slide 50), interstitial lamellae, Haversian canals and nutrient canals (Volkmann). Note that the latter canals penetrate osteons without causing new lamellae to be laid down around them. Note that Slide 51xc is also an entire cross section of the fibula, so you should try to compare it against Slide 50 discussed above.
Now, look at the longitudinal sections (#51L-EX) of compact bone and try identifying the various structures mentioned above, especially Haversian and Volkmann's canals.
