Histology and Pathology Microscopy Resources
Duke University Doctor of Physical Therapy
 

Cartilage & Bone

Gartner & Hiatt Atlas (5th ed):

Plates 4-1, -2, -3, -4
 
Text (Junqueira's 13th ed):
Ch 7, Cartilage, pp. 130-7 (pp. 114-20 in 12th ed.)
Ch 8, Bone, pp. 138-59 (pp. 121-39 in 12th ed.)

 

I. Cartilagenous tissues

A. Hyaline Cartilage

Slide UMich 40: Trachea, human, H&E
[ImageScope] [WebScope]

Slide UMich 126: Trachea & esophagus, human , H&E
[ImageScope][WebScope]

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.

 

II. Elastic cartilage

Slide UMich 44: Ear, pinna, human, aldehyde fuchsin & trichrome
[ImageScope] [WebScope]

Slide UMich 44H: Epiglottis, human, H&E
[ImageScope] [WebScope]

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.


III. Fibrocartilage

Slide UMich 45: Intervertebral disc, human, H&E
[ImageScope] [WebScope]

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.

 

 

II. Bone

Slide UMich 50: Fibula, monkey, decalcified, H&E
[ImageScope] [WebScope]

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).

 

Slide UMich 48: Developing bone (hindlimb), 154mm human embryo, H&E
[ImageScope] [WebScope]

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.

 

Slide UMich 115-N: Developing bone (palate), human, H&E
[ImageScope] [WebScope]

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.

 

 

Ground sections:
Cross section:

Slide UMich 51xc: Fibula, human, ground section
[ImageScope] [WebScope]

Longitudinal section:

Slide UMich 51L-EX: Fibula, human, ground section
[ImageScope] [WebScope]

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.

 

 


Muscle Tissue

Gartner & Hiatt Atlas (5th ed):

Plates 6-1, -6, -8
Text (Junqueira's 12th ed):
Ch 10, Muscle Tissue, pp. 191-211 (pp. 167-84 in 12th ed.)

 

I. Muscle Tissue

As you go through these slides, refer to this schematic drawing showing the key structural features and relative sizes of skeletal, smooth, and cardiac muscle as you would observe them with the 40X objective setting. 

A. SKELETAL MUSCLE

CROSS SECTION

Duke Webslide 0013_N: Skeletal muscle, primate, c.s., H&E
[ImageScope] [WebScope]

At low magnification, note the range of fiber diameters and shapes; note the multiple, peripheral nuclei associated with each fiber (recall that each muscle fiber is a single cell) and numerous blood vessels in the connective tissue surrounding each fiber.  At high magnification, note sizes and shapes of myofibrils within each muscle fiber. Within fibrils, electron microscopy would show an orderly hexagonal filament lattice filling out the many odd shapes.

Learn to distinguish the skeletal muscle fiber nuclei from the more elongated nuclei of capillaries and fibroblasts found in the surrounding connective tissue.

 

LONGITUDINAL SECTION

Slide UMich 58L: Skeletal muscle, human, l.s., H&E
[ImageScope] [WebScope]

This section shows how skeletal muscle appears in longitudinal section. Observe that the cells are long, non-branched cylinders with peripherally located nuclei and prominent cross-striations.

 

Slide UMich 58thin: Skeletal muscle, rabbit, l.s., H&E, 83x
[ImageScope] [WebScope]

This thin section is an ideal specimen in which to observe striations of A and I bands and Z lines. Also, see if you can discern myofibrils in each cell (staggering and longitudinal splitting of cross-banded substance). 

 

 

II. CARDIAC MUSCLE

Duke Webslide 0036_S: Cardiac muscle, human, H&E.
[ImageScope] [WebScope]

Cardiac muscle is arranged as a cross-branched network of fibers, so they will often appear in both cross and longitudinal sections on slides. Refer to this orientation image to see where to look for each in this slide and focus first on the area where fibers are mostly seen in cross section. Note the centrally-located nuclei of the cardiomyocytes surrounded by thick myofibrils, giving the cytoplasm a stippled or punctate appearance. It may be particularly instructive to compare this slide side by side (at the same magnification) with Webslide 0013 above. Note also the more numerous blood vessels filled with RBCs between cardiac muscle fibers.

Next, focus on the areas where longitudinal sections of cardiac muscle fibers may be found.  Note striations, intercalated discs, and branching of the cells --the latter two features are unique to cardiac muscle and therefore particularly helpful in identifying it when viewed in longitudinal section.

You may also notice granules of brownish pigment around many of the nuclei. This pigment is lipofuscin, which is a by-product of lipid oxidation that accumulates with age. Since most cardiomyocytes last for the life of the host, the amount of pigment that accumulates can be quite extensive, but it is generally quite harmless.

An alternate slide to look at is Slide UMich 98-1 [ImageScope] [WebScope], which is slightly thicker so it's not quite as easy to see the striations when viewed in longitudinal section; however, intercalated discs are quite prominent in some places [example]. The increased thickness also makes the punctate appearance of fibers in cross section much more obvious [example].

 

 

 

III. SMOOTH MUSCLE

Slide UMich slide 155: Esophagus-stomach junction, human, l.s., H&E
[ImageScope] [WebScope]

A typical appearance of smooth muscle in paraffin H and E sections is found in this longitudinal section from the gut in which there is an outer longitudinal layer of muscle (so here it is seen in longitudinal section) and an inner circular layer (so here it is in cross section). In addtion, there is a thin layer of longitudinally oriented muscle called the muscularis mucosae found between the cellular connective tissue under the lining epithelium and the denser connective tissue of the submucosa. The stomach (seen on the left side of the slide) actually has an additional layer of muscle (the inner oblique layer) to aid in breaking up food for digestion. Refer to this orientation image to find the various layers of smooth muscle that can be seen.

In the esophagus:

  • muscularis mucosae (shown here in longitudinal section) -this is a strip of smooth muscle adjacent to the epithelium of the esophagus.
  • inner circular layer (shown here in cross section).
  • outer longitudinal layer (shown here in longitudinal section).

In the stomach:

  • innermost "oblique" layer (mostly in longitudinal section here) -this layer is thin and poorly organized, so it tends to appear more as strips of differently oriented smooth muscle next to the prominent middle circular layer.
  • middle circular layer (in cross section here) -this is the most prominent muscle layer in the stomach.
  • outer "longitudinal" layer (some parts are in longitudinal section; others are in cross section) -this layer is also rather thin and poorly organized in the stomach.

Within the smooth muscle, dense red bands indicate where excessive contraction has clumped the contractile material within smooth muscle cells.  Note how clearly nuclei are seen within fibers, and note how in cross section many fibers lack nuclei (remember why?).  Collagen fibers, seen here in submucosa, happen to stain more darkly in this slide.  This makes the contrast with smooth muscle easier to notice.  Note that collagen fibers appear coarser, looser-packed, and more varied in size and direction than smooth muscle fibers.  Note how, among collagen fibers, nuclei are always fewer and lie external to fibers.  Smooth muscle occurs in snug parallel bundles, with more nuclei and with nuclei all internal to fibers.

This slide is also an excellent specimen to test your ability to differentiate smooth muscle from nearby connective tissue and peripheral nerve fibers.

Some additonal slides to test your ability to distinguish tissues are:

Duke Webslide 0098_N: urinary bladder, H&E (try to distinguish smooth muscle from connective tissue)
[ImageScope] [WebScope]

Duke Webslide 0093_N: recto-anal junction, mammal, H&E (try to distinguish the smooth muscle from skeletal muscle)
[ImageScope] [WebScope]

 

 


Nerve Tissue

Gartner & Hiatt Atlas (5th ed):

Plates 7-1, -4, -5
 
Text (Junqueira's 12th ed):
Ch 9, Nerve Tissue, pp. 160-90 (pp. 140-66 in 12th ed.)

 

Overview:

In this section of the laboratory, you will examine nerves, neuronal cell bodies, and ganglia.  It will be helpful to start with a few important definitions:

Nerve fiber  = multicellular, containing both an axon and surrounding myelin sheath.  The axon comes from a single neuron, but the myelin sheath is made by a train of many myelinating Schwann cells.  In the case of unmyelinated axons, the unmyelinated fiber shares each Schwann cell with several other unmyelinated axons.

Nerve =  a bundle or bundles of nerve fibers.

Ganglion = clusters of neuron cell bodies in the peripheral and autonomic nervous systems, as well as associated glial cells and axons.  Therefore, ganglia can be distinguished from peripheral nerves by the presence of neuron cell bodies.

Axons are neuronal processes specialized for electrical impulse conduction.  EMs show a cytoskeleton rich in microtubules (neurotubules) and intermediate filaments (neurofilaments). The organelles associated with protein synthesis are rare in axons, but abundant in neuronal cell bodies, where the membrane channels, ion pumps and synaptic machinery needed in axons and dendrites are synthesized. Axons may be long (spinal cord to foot) or short (many interneurons of CNS).  Myelin is formed by Schwann cells in the PNS and produces segmental insulation interrupted between successive Schwann cells at the nodes of Ranvier, where ion channels and ion pumps are localized and support saltatory impulse conduction.

 

I. Peripheral Nerves and the Myelin Sheath

Duke Webslide 0020_O: Sciatic nerve, c.s, 1.5 um, TB-AF
[ImageScope] [WebScope]

Duke Webslide 0021_O: Sciatic nerve, l.s., 1.5 um, TB-AF
[ImageScope] [WebScope]

The sciatic nerve is a mixed nerve, containing sensory axons from neuron cell bodies in dorsal root ganglia and motor axons from neurons in spinal cord gray matter.  Like all larger peripheral nerves bundles, it is also mixed in the sense of containing both somatic and autonomic nerve fibers.

These thin sections offer an excellent opportunity to see finer structures of nerves. Scan these slides at low power to see the fascicular organization, identifying epineurium (dense connective tissue surrounding the nerve and filling in between nerve bundles or fascicles), perineurium (thin layers of flattened cells immediately surrounding and defining each fascicle), and endoneurium (fine connective tissue within each fascicle between nerve fibers--see your text for orientation).  Around smaller fascicles, the epineurium becomes very thin, leaving the bundle surface visible as the circumferential wrapping of perineurial cells.  Note that the perineurium gives nerve bundles sharply defined boundaries that makes them easy to distinguish from vessels, ducts, CT, and muscle fibers.  The nuclei visible within each nerve bundle include crescent-oval Schwann cell nuclei snugly tangent to nerve fibers, and smaller, denser nuclei belonging to endoneurial fibroblasts or the endothelium of capillaries running through the endoneurium, usually parallel to nerve fibers.

At higher magnification you can see the unstained rings representing individual myelin sheaths surrounding axons.  Unmyelinated nerve fibers (which typically occupy at least 5% of a nerve's cross-section) can be visualized as axons without surrounding myelin sheaths.

 

 

Slide UMich 68: Peripheral nerve, monkey, c.s., H&E
[ImageScope] [WebScope]

Slide UMich 67: peripheral nerve, monkey, l.s., H&E
[ImageScope] [WebScope]

These slides show how nerves appear in typical H&E-stained paraffin sections. These sections are thicker, so the fine details (e.g. distinguishing between Schwann cells and endoneurial fibroblasts) are not so easy to see. However, the distinction between the perineurium and epineurium in slide 68 is quite obvious. Slide 67 is a nerve in longitudinal section, so you should try and find some examples of nodes of Ranvier [example]. You should also start appreciating how nerves differ from the other "linear" structures that you'll encounter on microscope slides such as blood vessels, connective tissue, and muscle tissue.

 

 

 

II. Basic Cell Types of the Central Nervous System and Autonomic Nervous System

Duke Webslide 0034_O (human)Lumbar spinal cord, c.s., H&E
[ImageScope] [WebScope]

This section contains a cross section of the spinal cord somewhat similar to the section shown in your atlas (plate 7-1), although this particular section is lower in the cord where the "central" canal is no longer a single lumen, but is instead broken up into several smaller fluid-filled channels, each lined by columnar ependymal cells [example].  Compare the ependymal nuclei with the neuronal nuclei you observed in previous slides.  Ependymal cells also line the ventricles of the brain that are continuous with the central canal of the spinal cord.

During development, newly formed neurons proliferate adjacent to the canal to form a mantle layer, which becomes the gray matter in the central region of the mature spinal cord. This gray matter occupies a butterfly-shaped cross-section in this mature spinal cord.  In the gray matter, examine the large motor neuron cell bodies of the ventral horn [example] carefully.  Depending on how the cells are cut, you may see the nucleus, nucleolus, and axon hillock, as well as the Nissl bodies (rough ER) filling the cytoplasm.  Note also the smaller nuclei of neuroglial cellNeuropil refers to the regions in gray matter that lie between cell bodies, devoid of nuclei but complexly crowded with neuronal cell processes and synapses. 

The neurons in the gray matter project processes that grow outward to form the fiber tracts of white matter, which carry parallel myelinated axons for long-range interneuronal contacts in CNS.  Note that the white matter in the spinal cord is external to the central butterfly-shaped region of gray matter. 

 

 

 

Slide UMich 65-1N: Spinal cord & dorsal root ganglion, human, Trichrome
[ImageScope] [WebScope]

This slide shows the spinal cord (for comparison to Webslide 034) and, on the right side of the slide, a dorsal root ganglion. Take a closer look at the dorsal root ganglion which is comprised of pseudounipolar neurons that convey sensory information from the periphery (from sensory receptors in the skin, joints and muscles that respond to touch, temperature, pain, stretch) into the central nervous system. In this particular case, the fibers of the dorsal root ganglion cell project into the dorsal horn of the spinal cord and synapse with neurons of the dorsal horn.

In this slide, you will see the large round cell bodies of these primary sensory neurons, called ganglion cells.  The nuclei are large and stain faintly.  The single nucleolus, if it happens to be in the plane of section, stains darkly, giving the nucleus a bull's eye appearance typical of many neurons.  Around each neuronal soma are smaller flattened cells, whose darkly stained nuclei form a ring around the ganglion cells.  These are specialized astroglial cells called satellite cells (not to be confused with satellite cells of skeletal muscle). Because these neurons are pseudounipolar and therefore almost perfectly spherical, the satellite cells are almost always seen as a complete ring around each neuron.

 

 

Slide UMich slide 155: Esophagus-stomach junction, human, l.s., H&E
[ImageScope] [WebScope]

Alternate UMich slide 250: Vagina, human, H&E
[ImageScope] [WebScope]

In Slide 155, you will see autonomic ganglia where axons (whose cell bodies are in the spinal cord) synapse with intrinsic neurons (whose cell bodies are located here in the GI tract).  These ganglia (called the Auerbach's or myenteric plexus) are found all along the GI tract between the circular and longitudinal layers of the outer smooth muscle [example]. Look for the typical neuronal bull's eye nuclei amongst the numerous nerve fibers present in this layer. Like other parts of the peripheral nervous system, these ganglia are covered by a thin connective tissue layer, essentially a perineurium, and the neurons are supported by surrounding glial (satellite) cells. Unlike the dorsal root ganglion cells, however, these neurons are multipolar and much more irregular in shape, so the satellite cells generally do NOT form a complete ring around the neurons. 

Slide UMich 250 features autonomic ganglia that can be found associated with the wall of the reproductive tract, in this particular case the vaginal wall. Look in the upper 1/3 of the section on the right side and you should see rather prominent peripheral nerve fibers and abundant autonomic ganglion cells [example]. Like other neurons, these cells are large with euchromatic nuclei and prominent nucleoli. There are some instances where you might see a complete ring of satellite cells around a neuron, but the overall organization is more chaotic and irregular compared to that which you'll see in dorsal root ganglia.

 

III. Neuromuscular junctions and muscle spindles

Slide UMich 71-2A: Motor end plates, Golgi colloidal gold stain
[ImageScope] [WebScope]

Slide UMich 71-1B: Muscle and muscle spindle, c.s., H&E)
[ImageScope] [WebScope]

Alpha (somatic) motor neurons from the ventral horn of the spinal cord innervate muscle fibers (their effector cells) at specialized synapses called neuromuscular junctions (or motor end plates of skeletal muscles) [example]. These are best visualized with special stains that use heavy metals (gold) to label the nerve fibers or histochemical methods for acetylcholinesterase (an enzyme that hydrolyzes the neurotransmitter used by somatic motor neurons, acetylcholine). Slide #71-2A shows such a similar preparation of motor end plates.   Also, review the diagrams in your atlas (plate 6-3 and graphic 7-2).

The terminal bouton of the motor axon has numerous synaptic vesicles that contain the neurotransmitter, acetylcholine. The terminal bouton lies in a depression in the surface of the muscle fiber, and is separated from it by a gap, the synaptic cleft, of uniform width. The plasma membrane of the muscle fiber is highly folded, and a basal lamina (also called the external lamina), is interposed between the nerve fiber and muscle fiber.  Synaptic transmission of nerve impulses across the synaptic cleft is accomplished by the release of acetylcholine from the synaptic vesicles (by exocytosis) into the synaptic cleft, where it diffuses to the muscle fiber membrane and activates acetylcholine receptors, which trigger membrane depolarization and subsequent muscle contraction.

Neuromuscular spindles are stretch receptor organs that regulate muscle tone via the spinal stretch reflex (Junquiera fig 10-14, p. 179). Look at slide # 71-1B and identify the neuromuscular spindle in the within the perimysium between fascicles in the belly of the muscle [example]. In this preparation, the sensory nerve fibers of the spindle are NOT visible, but the modified skeletal muscle fibers (intrafusal fibers), which are smaller than the muscle fibers proper (extrafusal fibers), are easily visualized -- 2 to 10 fibers are contained in a fluid-filled space within a discrete, external connective tissue capsule. Note the intrafusal fibers are bundled together by a delicate internal capsule that is not so evident in these sections. The sensory receptors (nerve endings) are activated by stretching of the intrafusal fibers, which evokes a reflex contraction of the extrafusal fibers that is driven by large (alpha) somatic motor neurons (located in the ventral horn) in a two-neuron spinal reflex arc.

It is worth noting that, in addition to being stretch receptors, the intrafusal fibers are functional, contractile muscle cells. They are innervated by special (gamma) motor neurons that set the tone of the intrafusal fibers thus modulating sensitivity of the stretch receptor (contraction of the spindle cells makes them more taut and therefore even more sensitive to stretch). This also allows the spindle cells to contract in concert with the extrafusal fibers thus maintaining sensitivity to stretch over the muscle's full range of motion [see explanatory figure].

 

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