Part 2: Stains, Cells, and Ultrastructure (EM)
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Hematoxvlin and eosin (H&E) is the most common laboratory stain. Hematoxylin is a blue/purple dye; eosin is red. Nuclear chromatin has a high nudeic acid content and therefore is attracted to the blue, more basic dye (i.e., it is basophilic). Everything else in this picture is relatively neutral in character and takes a wash of eosin.
In a low power view of intestinal wall, rows of epithelial nuclei impart a darker, bluer color to linings of surfaces and glands, as seen to the right of center. The outer, left-hand layers show the pink of muscle cytoplasm. The middle layer of dense, irregular connective tissue shows how brightly collagen fibers can be stained with eosin.
High power of smooth muscle to show that eosinophilic color is mainly due to cytoplasm. Nuclei are quite scattered and have only small, granular clumps of blue heterchromatin. Nucleoli (one or two per nucleus) are stained blue with hematoxylin.
Intestinal wall stained with Mallory's trichrome stain, which specifically colors collagen fibers blue. With this stain the connective tissue layer is clearly distinguished from muscle below and epithelium above, both of which take the pink/purple stain of cytoplasm.
Detail of a group of epithelial cells containing bright red (eosinophilic) secretory granules. Nuclei are dark with hematoxylin.
This organ, the thymus, appears very basophilic in H&E.
At high power, the reason for the basophilia is clear: the thymus is packed with lymphocytes with darkly stained nuclei. Isolated structures such as the whorl of cells in the center, are specifically acidophilic (eosinophilic).
Here are some nerve cells, seen in low power. Their nuclei are pale and vesicular, containing mainly unstained euchromatin. The nucleolus is dark, however, and the cvtoplasm is filled with clumps of darkly stained, basophilic material, implying a content of ribonucleic acid.
Cells take diverse shapes. These are epithelial cords of block-like cells. As always, nucleoli and nuclear heterochromatin stain darkly with hematoxylin.
Blood cells are suspended in fluid plasma and therefore are characteristically round in shape.
Muscle cells are arranged parallel to their direction of contraction and adopt a fusiform or spindle shape. Nuclei are sparse in relation to large amounts of cytoplasm.
In low power, individual muscle cell groups are found to be running in different directions, so that some are cut cross-wise (or transversely) and some are cut lengthwise (longitudinally). Some, of course, are running obliquely and therefore are cut tangentially in relation to their full length.
Nerve cells are typically stellate in shape, with several cytoplasmic extensions or processes. Here again, notice that the cytoplasm of these cells contains dark, basophilic material. In EM, this material will turn out to be abundant rough endoplasmic reticulum, which is associated with protein production. Before leaving this slide, note the many tiny nuclei in the field, in between the two nerve cells. Their size is about equal to the nucleolus of a nerve cell!
Another type of nerve cell, to show again its huge size in relation to the ordinary connective tissue cells around it. Once again, the nucleolus of the nerve cell (lying in the rather small, pink nucleus) is about equal in size to the nuclei of other cells. Look just below the nerve cell (at about the 5:30 position on a clock face) for a small capillary containing a single, quite pink erythrocyte. Figuring that the r.b.c. is about 7.5 microns in diameter, you can estimate the size of the neuron!
Silver staining is useful for a variety of purposes. Here it is used to blacken the reticular fiber network of reticular tissue.
Here silver has been deposited on nerve cells and their delicate processes in the brain.
In this instance, silver has been deposited on the intercellular substance between epithelial cells. You will notice that silver seems particularly useful for viewing very thin, fine structures which become visible when impregnated with grains of silver. Incidentally, this particular view is of the surface of mesothelium (simple squamous epithelium lining body cavities and mesentery).
Now a whole-mount of a small blood vessel has been stained with silver. The thin black vertical lines are reticular fibers running around the outside of the vessel like barrel hoops. The irregular horizontal lines, running parallel to the length of the vessel are the silvered outlines of endothelial cells. The intercellular cement has been stained black, making this surface view of the endothelium look like the pieces of a puzzle interlocked together. Cell nuclei are not visible.
Diagram of a "typical" cell, showing the organelles common to almost all cells of the body. Notice rod-like mitochondria, crescent-shaped Golgi complexes, stacked rough endoplasmic reticulum (called granular reticulum here), a network of smooth endoplasmic reticulum (agranular reticulum), electron-dense lysosomes. The small dots encrusting the rough ER are ribosomes; compare their size with the particles of glycogen, shown as black, irregular clusters scattered among the smooth ER. Notice also that the nucleus contains a nucleolus, very little heterochromatin, and seeming gaps along the nuclear envelope. (We'll get back to other features of this cell when we study liver.)
Transmission electron micrograph of nucleus similar to the one in the previous diagram. The nucleolus is to the left and shows an internal structure. The heterochromatin lies close to the nuclear envelope and is discontinuous at the nuclear pores (arrows). Inset details above and below show that the pores result from a meeting of the inner and outer membranes of the nuclear envelope to form a thin diaphragm for better transport of substances between the nucleus and cytoplasm. Mitochondria and stacks of rough ER are seen outside the nucleus.
Detailed EM of nucleolar structure, showing fibrillar (4), granular (5), and amorphous (3) portions. Associated with the nucleolus are some heterochromatin (l) and euchromatin (2).
View of nuclear envelope fractured between its inner and outer leaflets by the freeze-etch technique. The outside of the nuclear envelope (as it would face the surrounding cytoplasm) is seen along the lower curve. The rest of the view is as if you were inside the envelope, looking toward the leaflet that lies next to the nucleoplasm. Notice the scattered pores.
High power EM showing the two dense and one pale (or lucent) layers of the ordinary cell (or plasma) membrane.
A similar membrane coated with a fuzzy-looking external glycocalvx. Specifically stained with an electron-dense material.
Drawings to show that a basal lamina separates many kinds of cells from the surrounding connective tissue. In EM the portion of the basal lamina referred to as the lamina densa is a thin gray line lying just outside the cell membrane. Notice here that the basal lamina surrounds smooth muscle cells, the endothelium of a blood capillary, a group of nerve fibers, and the epithelium of a secretory acinus. A detail of the acinar cells at the bottom of the screen shows that a glycocalyx lies between the cell membrane and the basal lamina. Two odd points to notice: (1) lymphatic capillaries have no basal lamina surrounding their endothelium, and (2) fat cells do have a basal lamina, which is surprising because these are connective tissue cells and shouldn't seem to need a protective layer between themselves and the surrounding connective tissue ground substance. The true origin of fat cells is open to question.
Diagram of a block-like cell showing the extent of various kinds of cell junctions. A macula is a simple "spot weld". A zonula forms a complete belt of adhesion around the cell. A fascia is a broad, irregular area of adhesion. Notice that the apical surface of the cell has several small cytoplasmic protrusions. They are like microvilli stucturally but are not numerous enough to form a striated or brush border. Such small protrusions are common on cells.
Scanning EM view looking down on the apical surface of a whole sheet of epithelial cells. The long, wavy projections are cilia: the close-cropped ones are microvilli of a brush border.
High power EM of microvilli of a brush border. Notice that they are simple extensions of the apical cytoplasm, with unit membrane continuing over their surface. Very fine actin filaments extend into the microvilli and are rooted in the main mass of cytoplasm below. Angling down the bottom half of the picture is the line of contact between two adjacent cells, each with its own unit membrane. At three points along the way there are specialized junctions: (O) zonula occludens or tight junctions, (A) zonula adherens or intermediate junction, and (D) desmosome or macula adherens. Cytoplasmic filaments (F) are attached to the desmosome, contributing to its density.
Artist's concept of junctional complexes as seen from the interior of a cell, as well as at the line of contact with the next cell. The tonofilaments heading into the desmosomes (MA) are particularly prominent. One hemidesmosome is at the bottom. The continuous bands of zonula occludens and zonula adherens are seen near the top. The disc and the junction labeled GT are gap junctions. Note the width of the intercellular space along its normal length and at the points of various kinds of contacts. The basal lamina is labeled BM. In the lower right drawing notice how zonular contacts can bind groups of cells to each other.
EM detail of several desmosomes, showing the attachment of many tonofilaments (F). Arrows point to the density which typically appears in the intercellular space. The cell membranes of the two neighboring cells are interlocked in a very complex interdigitation here. One cell lies above; the other below. You can follow the undulating course of the intercellular space across the picture.
Freeze-fracture view of one face of a zonula occludens (or tight junction), showing that apposing cell membranes are sealed together in an interlocking pattern of fine ridges rather than a complete band of "glue", so to speak. The net effect is the same, however; substances cannot pass from one side of the band to the other.
EM detail of a tight junction. As the two unit membranes approach each other at upper right, the two outer leaflets merge so that you can see just three dense lines (instead of four) to the left. At the extreme lower left the three lines are becoming four again.
EM of cilia cut longitudinally. (A few microvilli are on the neighboring cell to the left, for a size comparison.) Notice that each cilium is rooted in a barrel-like basal body. The dense lines extending from the basal bodies and up into the cilia are microtubules. The unit membrane of the cell continues up over each cilium.
Detail of cilium cut transversely at various levels of its length. Notice the typical microtubular arrangement of outer nine doublets and two central singles. In the basal bodv (E) this arrangement subtly changes to nine triplets and nothing in the center.
Typical arrangement of cisterns of rough ER in a secretory epithelial cell. A few mitochondria lie at lower left. The presence of ribosomes on the RER, with all their ribonucleic acid content, render them basophilic to stains.
Serous secretory acini showing cytoplasmic basophilia toward their bases where a lot of rough ER lies. The presence of rough ER in such abundance signifies production of protein (in this case, some digestive enzyme). The secretory granules are pale here.
Epithelial cells stained to show a pale negative Golgi (arrow). The Golgi complex typically lies just above the nucleus (toward the lumen) in a secretory cell.
The same epithelium stained with silver to show the presence of the Golgi. Yet another use for silver staining!
EM detail of microtubules, seen as fine parallel lines when cut longitudinally or circles when cut transversely. During mitosis, microtubules form the mitotic spindle, radiating out from the centrioles. In picture 2-66 you see two electron-dense centrioles at right angles to each other (1) and some microtubules radiating into the surrounding cytoplasm (2). A chromosome shows at (3), and the kinetochore region of spindle attachment to the chromosome is at (4). Smooth ER is seen at (5). In picture 2-67, at higher power, a centriole is cut in cross-section (1), showing that it has a microtubular arrangement of nine triplets in a circle. (This, you'll recall, is the same as for the basal bodies of cilia.) The surrounding centrosphere is shown at (2). Other labeled features are: (3) microtubules of the spindle, (4) kinetochore region, (5) chromosomal material, (6) ribosomes.
Two views of some actively absorbing cells. In the lower right inset is a light micrograph of a cross-cut tubule, showing epithelial cells with pale nuclei and very dense basal cytoplasm. Above is an EM detail of the cell bases, showing that the density is due to many infoldings of the cell membrane, plus the presence of many mitochondria. Space (C) is a capillary lumen; notice the very thin strip of endothelial wall along the left margin of the space. Notice also a thin gray line of basal lamina (B) between the endothelium and the tubule epithelium.
