Histology is the study of the structures of cells and tissues. As you learn about the morphology of cells and the architecture of tissues, you will also learn how those structure indicate and facilitate the functions of cells and tissues. Histology focuses on the normal, how cells and tissues should appear in the absence of disease or infection. Understanding the normal will allow you to identify changes that occur to cells and tissues during disease. To help you with this transition, we will introduce some histological images of diseased tissue.
Histology involves careful examination of biological samples in a light microscope. Preparing histological samples requires several steps that determine which structures and features will be seen in the microscope.
Most samples are usually obtained by biopsy from a patient during surgery or who is anesthetized. Samples are processed quickly to minimize degradation of the specimen.
Once removed from the body, all specimens will degrade due to the presence of numerous digestive enzymes in the specimen. To preserve the structure of the specimen, a fixative is applied to inactivate proteins and maintain the integrity of the cells and tissues.
The most common fixative is formaldehyde, also called formalin. Formaldehyde forms chemical cross links between proteins and between nucleic acids. The cross links inactivate most enzymes and preserve the structure of macromolecules.
To view a sample under a light microscope, it must be sectioned into very this sliced (~ 5 microns). Thin slices produce better imaging of the sample in a microscope, but thinly slicing most samples is difficult due to the softness of the sample. Therefore, most samples are embedded in waxy substance such as paraffin which hardens and allows the sample to be sliced into thin sections.
Most biological specimens offer poor visibility under standard light microscopy. To increase visibility and help us identify specific structures, histological samples are stained. The stains bind associate with specific chemical features of the sample and impart a color.
By far, hematoxylin and eosin (H&E) is the most commonly used stain in histology and pathology. Hematoxylin binds negatively charged structures in samples and imparts a blue or purple color to those structures. The largest and most negatively charged structures in cells are nucleic acids such as DNA in chromosomes and RNA in ribosomes. Hematoxylin consequently strongly labels the nucleus and rough endoplasmic reticulum.
In contrast, eosin labels structures with a positive charge and imparts a pinkish color. There are few large structures with strong positive charge in cells, so eosin does not distinctly label all cells as does hematoxylin. Eosin gives the cytoplasm of most cells a pinkish hue and the secretory granules in some cells are labeled by eosin.
There are several additional stains that are used for histological samples. For example, periodic acid–Schiff stain (PAS) labels structure with a lot of carbohydrate (e.g. extracellular matrix, mucus), and is used to diagnose certain diseases, such as glycogen storage diseases. Another common stain is Masson’s trichrome which is often used to label connective tissue. Most of the samples used in the subsequent labs will have been stained with H&E, but samples labeled with a stain will be indicated.
Histological images often resemble modern art with seemingly random swirls of color and no apparent point of focus. However, in most images there are several identifiable structures that can help orient you in the image and make sense of what you are looking at.
Because nuclei of cells are usually strongly labeled by hematoxylin and appear deep blue, they are often the easiest cellular structure to identify. In addition, much can be learned about the identity, function and activity of a cell from the shape and staining pattern of its nucleus.
Although most cells have nuclei that are round or oval, some cells have nuclei with very distinct shapes. Some white blood cells have nuclei that are divided into lobes and the cells can be identified based on the number of lobes. In addition, some cells have nuclei in specific locations within the cell.
All nuclei contain DNA arranged in chromosomes, but the arrangement of the chromosomes within the nucleus can often indicate how actively a cell is transcribing its genes into mRNA. Structurally, chromosomes are often divided into heterochromatin and euchromatin. In heterochromatin, the chromosomes are condensed and often localize to the edge of the nucleus called the nuclear envelope. Genes in heterochromatin are often transcriptionally silent. In euchromatin, the chromosomes are less condensed, tend to be located more centrally within the nucleus and have more genes being transcribed.
Actively dividing cells can be identified by looking at the structure of the nucleus and arrangement of chromosomes. When cells go through mitosis the nuclear envelope breakdown and the chromosomes condense. Eventually, the chromosomes are corralled onto the spindle and then segregated into the two daughter cells. All of these events are visible in histological samples stained by H&E. Estimating the number of dividing or mitotic cells in a tissue is used to help determine whether a tissue has become cancerous.
Most cells have one nucleus and therefore, the number of cells in a tissue can be estimated by counting the number of nuclei. Often a change in the number of nuclei can indicate disease. Increased nuclei (cells) can suggest inflammation or cancer, whereas fewer nuclei can indicate tissue damage and cell death. Becoming familiar with the relative number of cells in the different tissues and organs is important for identifying and diagnosing disease in histological samples.
Within the nucleus lies the nucleolus which can also be seen in histological samples. The nucleolus is the site where ribosomes assemble and contains a high concentration or ribosomal genes and RNA. The nucleolus often localizes to the center of the nucleus and stains deep blue. Cells that are actively synthesizing protein require more ribosomes and consequently, the nucleolus in those cells is more prominent.
Within the cytoplasm the rough endoplasmic reticulum (ER) is readily seen in cells that are secreting large amount of protein. Because the rough ER contains a high concentration of ribosomes, it will stain blue in H&E samples and contrast nicely with the pinkish cytoplasm.
Some secretory cells store their products in vesicles within their cytoplasm. These vesicles are called granules and when stimulated, the cell will allow the granules to fuse with the cell membrane, releasing the contents of the granules to the surrounding environment. Secretory granules are often visible in H&E samples and their color varies depending on the type of cell.
In many histological images, the authors will include a small line of specified length that can be used as a ruler to measure the size of cells and other objects. There is no ruler, however, when looking at samples in a microscope. Conveniently, the body provides its own biological ruler in the form of red blood cells. Red blood cells have a width of about 7 microns and because most samples will have blood vessels, red blood cells are usually available to estimate the size of cells and other structures in histological images.