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Author: Paul Kwan, Ph.D.

I. Definition

Histology is the study of microscopic anatomy dealing with the structures of cells, tissue and organs in relation to their functions.

The first part of the course deals with basic tissues (a collection of similar cells and the extracellular matrices surrounding them): epithelium; connective tissues, including blood, bone and cartilage; muscles; and nerves.

The second part of the course deals with organs, systemic arrangement of tissues performing a specific function, as of respiration, digestion etc.

Species differences will be included where appropriate. This, however, is not a course in comparative histology.

The course deals mainly with the structural aspects of cells, tissues and organs. It also covers the basic functions of these structures.

Although the students will have extensive course works in physiology, this course tries to mirror the philosophy of Albert Szent-Györgyi, “Structure and function are two sides of the same coin: structure without function is meaningless, and function without structure does not exist”.

II. Methods of Study

The most common way to study tissues in histology is to preserve the material, make a section thin enough for light (or electrons) to pass through it, stain it to give it some contrast, and then observe the tissue under a microscope.

A. Routine Tissue Processing (Paraffin Sections)

Fixation—The preservation of the tissues to be studied

1. Immersion fixation, most common.

Put small pieces of tissue directly into a fixative such as formaldehyde. As the fixative penetrates into the tissue, the material is preserved (‘fixed’). Because the rate of diffusion of the fixative into the tissue is slow, the pieces must be quite small to be preserved properly. Otherwise there may be fixation artifacts (like the center of the piece not properly preserved) in the tissue.

Because of its simplicity, this is the most commonly used method in general histology and pathology.

2. Perfusion fixation, better preservation.

By introducing the fixative into the blood stream, the fixative can reach organs/tissue quite quickly and achieve good fixation. This requires a more elaborate set up and skills (inserting a canula into a blood vessel). Care must be taken to ensure proper pressure and flow of the fixative. Otherwise the tissue may not be fixed well or distorted by too much hydrostatic pressure applied during the procedure. Can produce beautifully preserved material. More commonly used in research projects.

Routine immersion of tissues into an aqueous fixative such as formaldehyde removes soluble compounds such as glycogen. To preserve materials of interest that are water-soluble, a fixative like acetone is used.

3. Dehydration---removal of the aqueous material in the tissues.

In routine processing, the tissue will be embedded in a water insoluble material such as wax. All the water in the tissue must first be removed. This is done by running the tissues through a graded series of alcohol (70%, 95%, 100%). Inadequate removal of water will lead to artifacts.

4. Embedding

The dehydrated tissue is passed through several changes of xylene and then placed in liquified paraffin wax. The paraffin infiltrates the tissue (usually with the help of a vacuum). The tissue is then place in a plastic mold that contains melted paraffin. Upon cooling, this piece of paraffin containing the tissue is called a ‘block’ and is ready to be sectioned into ‘thick’ sections (4-6 micrometers).

5. Sectioning

After trimming away excess material and exposing the tissues, the block is then sectioned with a steel blade. The thickness of each section is about 4 to 6 micrometer (10-6 m). The sections are collected and placed on a glass slide. With the application of mild heat, the paraffin surrounding the tissue melts away and the tissue sticks to the glass.

6. Staining

The sections cut from the blocks are thin enough for light to pass through. However, the material has very little contrast and you cannot make out details when looking at the section under the microscope. Staining the sections with dyes give contrast to the tissues and makes it possible to study fine details.

The most commonly used stains for paraffin sections are hematoxylin and eosin (see later section on stains).

Special stains are used to demonstrate a specific structure or class of compound found in the tissue, e.g. PAS stain for glycogen in liver cells.

7. Cover Slipping

The stained section is covered with a drop of mounting medium such as Permount® and then a thin piece of glass (cover slip) is placed over the medium. When the medium is dry, the section is preserved and protected in a plastic-like medium under a sheet of glass.

B. Plastic Sections

In some cases, after dehydration, the tissue is embedded in epoxy plastic. This very hard material can be cut into ‘semi-thin’ sections (0.5 micrometer) for light microscopy or thin (0.1 micrometer) for electron microscopy.

Plastic sections used for light microscopy show better details than paraffin sections. However, the numbers of stains that can be applied to plastic sections are rather limited.

C. Frozen Sections

To preserve molecules that may not withstand the action of paraformaldehyde and the heat used in paraffin processing, a combination of acetone and low temperature embedding can be used.

First fixation is done by immersing the small pieces of tissues in a low-temperature embedding medium such as OCT®. This syrupy material is put into a little plastic mold and the pieces of tissue put into the medium. The mold is then put into a mixture of acetone and dry ice. The medium freezes quickly into a solid block.

The frozen block is trimmed and then section in a cryostat, which is a refrigerated microtome. The sections are collected on glass slides.

Depending on the protocol, the sections may or may not be fixed briefly with acetone.

The sections are stained and mounted either with a special aqueous mounting medium or, after dehydration, with an organic mounting medium.

D. Electron Microscopy, Routine

Electron microscopy yields much better details than light microscopy and is invaluable in histology.

Fixation for electron microscopy is usually done with glutaraldehyde or paraformaldehyde. The tissue has to be minced into very small pieces so that the fixative can diffuse quickly into the material.

After rinsing away the first fixative, the tissue is usually fixed further with a solution of osmium tetroxide.

After dehydration, the pieces of tissue are embedded in epoxy plastic.

A diamond knife is used to make thin sections from the plastic blocks. The sections are picked up on small copper grids.

Staining of the thin sections usually involve the use of uranyl acetate solution followed by a lead citrate solution. These stains, being of electron-opaque heavy metals, impart contrast to the very thin sections.

Because of the way the tissue is fixed and embedded, very few special staining methods are available for thin sections. The most common one is immunohistochemistry, using gold particles as markers. These particles, bound to structures in the cells, appear as little dark spheres under the electron microscope.

E. Electron Microsopy, Scanning

This uses an electron beam to scan the surface of a structure and gives a three dimensional topographic view of the subject with amazing details.

III. Common Artifacts Encountered with Tissue Sections

Folds --- paraffin sections, after being cut on a steel blade, are placed gently in a bath of warm water. The sections float on the water surface and stretch out like a ribbon. The most common artifact, folds, occurs when the sections did not stretch out completely. This can also happen when the sections are picked up and transferred to glass slides.

Debris --- dirt and debris in the water bath are common and the water surface needs to be cleaned regularly during sectioning.

Knife marks --- nicks in the knife lead to drag marks in the sections.

Shrinkage / swelling --- the tissues may swell or shrink during processing.

Staining artifacts --- sometimes structures can be over-stained or under-stained. Other times the structure may stain inappropriately.

It is important to recognize these artifacts when studying a specimen so as not to make the wrong conclusion or worse, diagnosis.

IV. Routine Stains for Paraffin Sections

The way a tissue component stains depends on the interaction of the component with certain dyes.

The most common stain used in routine paraffin sections is called H&E. This stands for hematoxylin and eosin.

Hematoxylin is a dye extracted from longwood. In routine processes, it stains the nuclei of cells purple/blue.

Eosin is a dye that usually stains the cytoplasm orange-red.

A. Basic/Cationic Dyes

Hematoxylin --- blue/blue-black. Stains heterochromatin in nuclei, rough endoplasmic reticulum, proteoglycans etc.

Methylene blue --- blue. Stains anionic compounds in the cell as well as in the extracellular matrix. Metachromatic with certain polyanions such as those in the granules in mast cells. Also used in semi-thin plastic sections.

Toluidine blue --- blue. Similar to methylene blue.

Azures --- blue. Tissue components that interact with and are “stained” by these dyes are described as being basophilic. For example, DNA in cell nuclei and RNA in the cytoplasm stain bluish with these dyes and you will hear comments such as “ note the basophilia of the chromatin material” or “note the basophilic in the basal cytoplasm”.

Metachromasia (change color) High acid material (polyanion rich) can ‘change’ the ‘normal’ color of a dye (orthochromatic) to a different (metachromatic) color. Mast cell granules, when stained with toluidine blue, show up as purplish droplets. The extracellular matrix of cartilage is also metachromatic due the presence of chondroitin sulfate molecules. This kind of reaction is attributed to the way the dye molecules are bound in proximate array to tissue polyanionic polymers, such as glycosaminoglycans.

B. Acidic/Anionic Dyes

  • Eosin - orange-red/pink
  • Anilin blue - blue
  • Orange G - orange
  • Picric acid - yellow

Tissue components that interact with and are “stained” by these dyes are described as being acidophilic or eosinophilic. For example, certain secretory materials appear bright red/orange and are described as “acidophilic granules in the cytoplasm”. Collagen is a typical eosinophilic substance in the tissue.

C. Mixed Dyes

In routine staining, the dyes are applied in sequence with washes in between. In some cases, however, a mixture of dyes is applied to the tissue.

Romanowsky stains is the best know example of this type of mixed dyes and contain combinations of eosin and methylene blue / azures. They are usually used for the staining of blood or bone marrow smears.

Giemsa stain and Wright stain are common examples of Romanowsky stains.

For more information, see the chapter on Blood.

V. Special Stains and Reactions

A number of stains are commonly encountered in histology. These are used to bring out certain structures / components of interest.

Elastic stains for elastin --- Verhoeff (black), resorcin fuchsin (purple), and orcein (red) --- for walls of blood vessels etc.

Feulgen reaction, a form of Schiff reaction. Used for DNA in cell nuclei. Magenta. The staining is specific for DNA and not RNA. The degree of staining is proportion to the amount of DNA present. This property is employed in the densitometric quantitation of DNA in tissues.

Lipid Stains that dissolve in or stabilize lipid materials --- Sudan black, oil red O, osmium tetroxide (black) --- used for lipid droplets in liver and other tissues. Used with frozen sections because lipids are not extracted as during paraffin processing involving solvents.

Masson trichrome, Mallory’s --- used for connective tissues. Collagen, blue-green. Cytoplasm, red-pink. Nuclei, blue-black.

PAS --- periodic acid-Schiff reaction, demonstrates 1,2-glycol groups present in carbohydrates. Stains polysaccharides, oligosaccharides, and some glycoproteins. Most common example, glycogen in liver and striated muscle cells. Magenta.

Silver --- used for Golgi apparatus, basal lamina, reticular fibers, and neurofibrils. Black-brown.

Gold --- in the form of gold chloride, useful in neurology to demonstrate astrocytes, axons and nerve endings.

Vital stains --- trypan blue, Indian ink--- used for differentiating viable versus dead cells as well as for demonstrating cells with phagocytic capabilities.

VI. Other Methods of Investigation

A. Immunohistochemistry

Detection of specific antigens (usually proteins) in the tissue using antibody and colored markers. Example: presence of oncogene protein products in a specimen. Used extensively in pathology.

B. In situ hybridization

Detection of specific nucleic acid sequences in the tissue using labeled complimentary probes. Example: strain-specific diagnosis of viral infection.

C. Autoradiography

Use of radioactively labeled tags and photographic emulsion to localize compounds in the cell. Example: labeled amino acids to trace the path of protein synthesis and packaging in the exocrine pancreas.

D. Fluorescence Microscopy

Instead of visible light, this technique uses ultraviolet light waves for illuminating the subject. Dyes that fluoresce under UV light are used to stain different object of interest. A routine use is the study of the basement membranes in glomeruli in renal diseases. Some intracellular materials, such as lipofuscin pigment granules in neurons, are autofluorescent and will appear bright under UV light without the application of dyes.

E. Confocal Microscopy

The confocal microscope has a much greater death of field than conventional microscopes. A thicker slide of tissues can be studied and the architecture of the tissue shows up better with confocal microscopy.

VII. 2- and 3-Dimensional Perspectives

The study of routine histology involves the use of many glass microscope slides containing tissue sections. It is important to understand that a section is but a small part of the tissue/organ and may not yield an accurate picture of the tissue/organ being studied. You actually lose a dimension when you take a slice of a three-dimensioned structure (the thickness of a histological section is so small that it usually is insignificant).

For a three-dimensioned structure, sectioning will reduce it to two dimensions. For example, a section of an orange is represented as a slice.

For a two-dimensioned structure, sectioning will reduce it to one dimension. For example, a profile of a sectioned fiber is represented as a dot most of the time.

When examining a histological section, try to visualize what the original organ/tissue looks like in 3 dimensions. This takes understanding and some practice. Another issue to keep in mind is the plane of section. Depending on the plane of section, a familiar shaped structure can take on odd shapes. For example, you generally expect to see a section of a tube to appear as a ‘ring’. However, a section of a tube can appear as a ring, an oval, an eclipse and other shapes depending on the plane of section. Keep this in mind when examining your slides.

Slice a hard boiled egg thinly along the long axis, lay out the slices and look at them. Can you reconstruct the egg if you only have three or four slices? If some slices of the egg you have were cut along the long axis while others were cut along the short axis, how difficult will the job be?

Understanding these concepts will help you make the proper interpretation of the images you are studying and be able to relate to the “real” tissue/organ.