Introduction to Histology
Histology is the microscopic study of tissues and cells. It involves studying their distinctive microscopic structure, which also helps to elucidate functional and clinical significance of these structures. This field of study is important in medicine as it links physiology, biochemistry and molecular biology to the study of disease.
Histology is also used in understanding the pathogenesis and diagnosis of various diseases. Diseased tissue samples, or biopsies, from the affected organs are usually processed, stained and observed under the microscope. Histology involves the use of both light microscopy and electron microscopy, depending on the requirements of the sample being visualised.
In recent years, the field of histology has greatly advanced, as new practices like protein sequencing, molecular genetics and cloning have provided a greater knowledge and understanding of the ultrastructure of cells and tissues in the human body.
Introduction to Cells and Tissues
Cells are the tiny living units that form the tissues, organs and structures within the body. In turn, the body is composed of different types and varieties of cells to carry out specific functions, but they all have the same basic structure. All cells contain cytoplasm and are surrounded by a membrane, and contain the following structures of organelles:
- Nucleus (contains DNA)
- Mitochondria (produce energy in the form of ATP)
- Ribosomes (synthesise proteins)
- Lysosomes (contain digestive enzymes)
- Endoplasmic reticulum (synthesises protein and lipids)
- Golgi body (folded membranes that process proteins from the endoplasmic reticulum)
- Vesicles (transport materials within the cell)
- Peroxisomes (contain metabolic enzymes)
Body tissues are collections of cells, grouped in the body according to structure and function. In histology, human tissues are separated into four distinct categories:
- Muscular: Muscle tissue is made up of long thin muscle cells called myocytes. Their structure and arrangement allows for muscular contraction.
- Nervous: Nervous tissue forms the nervous system, and is made up of specialised cells called neurons and neuroglial cells. Neurons conduct nerve signals from one to another in the form of electrical impulses.
- Epithelial: Epithelial tissue comprises epithelial cells arranged together in sheets. These sheets serve as protective layers, forming coverings like the skin, and the lining of the small intestine.
- Connective: Connective tissue forms a connective web throughout the body. It fills gaps and connects different organs and body parts, so that the carefully arranged structure of the body can be maintained.
Light microscopes (or optical microscopes), use a combination of visible light and lenses to create a magnified image. In histology, thin sections of tissue are specially prepared and placed under the microscope for observation.
To create thin sections, the tissue is usually first immersed in a fixative solution, which acts as a preservative to stop the sample from degrading. The thin section is then embedded in paraffin wax, which firms it, so that it can be sliced into thin sections. The thin sections are usually between 5 and 8 µm in thickness. The sections are then mounted on a microscope slide, the wax is removed with organic solvent, and then the thin section is rehydrated with diluted alcohol. In instances where a fresh tissue sample is required, for example in surgical biopsies, frozen samples are made. Samples can be snap-frozen using liquid nitrogen, and then stored at -80 degrees Celsius. They are finely sliced inside a freezer using a piece of equipment called a cryostat. In light microscopy, tissue staining is generally required.
One type of optical microscopy is known as wide field microscope. This type of microscopy immerses the specimen in light from either a xenon or mercury source, and the image can be viewed directly. However secondary fluorescence can be produced which may interfere with the resolution of the image.
Another type of optical microscopy is confocal microscopy. This is often referred to as confocal laser scanning microscopy (CLSM). CLSM is used to increase resolution and contrast. This is achieved by adding a spatial pinhole at the confocal plane of the lens. This removes any out of focus light that might be present. CLSM allows for reconstruction of 3D structures from images taken at different depths.
Differential Interference Contrasct Microscopy
A further type of optical microscopy is differential interference contrast microscopy. This is used to improve contrast in unstained or clear samples. This form of microscopy is fairly complex, and uses a process known as interferometry to obtain information regarding the optical path length of a sample. This allows the visualisation of structures that would otherwise be invisible to our eyes.
As cells are generally colourless, they need to be stained so that they can be easily viewed under the microscope. There are four types of stains used in histology. Empirical stains are the most common, but histochemical, enzyme histochemical and immunohistochemical stains can also be used. Details of each stain are listed below:
Empirical: These are simple stains used since many years and discovered by trial and error. They cause differential coloration of the various components of the cells and tissues, allowing their ultrastructure to be viewed more clearly. Common examples of empirical stain are trichrome stains and van Gieson’s stain. Trichrome uses mixture of three different dyes to stain different components of tissues while van Gieson colour muscle tissue yellow, and collagen pink.
Histochemical: This method involves using stains that are specific to a particular molecule within the sample, allowing the chemical components of cells and tissues to be observed under the microscope.
Enzyme Histochemical: As its name suggests, this technique uses a staining agent to identify and locate activity of specific enzymes. For the enzymes to be active, this method requires the use of fresh tissue, which is generally incubated together with the substrate specific to the enzymes being observed.
Immunohistochemical: This uses antibodies to detect specific cell molecules within tissues. The antibodies are often attached to enzymes or immunofluorescence, that will induce a colour change at the site of interest. This method is also important in identifying abnormal cells, such as cancer cells.
Electron microscopy is a more modern form of microscopy than light microscopy and provides much higher magnification. Electron microscopes work by emitting parallel beams of electrons onto the sample being observed. The high magnification capabilities of electron microscopes provide high resolution images, meaning that the tiniest ultrastructures can be seen. Fixation is very important in this type of microscopy, and glutaraldehyde is normally used as the fixative agent. There are two types of electron microscopy; transmission electron microscopy, and scanning electron microscopy.
Transmission Electron Microscopy
Transmission electron microscopy uses very thin sections of tissue. Emitted electrons are absorbed by some parts of the tissue and pass through others. The electrons that penetrate through the tissue sample hit a phosphorescent screen, forming an image. Staining the tissues with chemicals like osmium tetroxide can be used to emphasise the differences in electron uptake of the different structures being visualised.
Scanning Electron Microscopy
Scanning electron microscopy does not require thin sections, instead it uses larger pieces of tissue. One key benefit of this type of microscopy is that it can produce 3-dimensional images. To prepare the tissue, it must first be fixed, before being dried and coated with gold. The specimen is then scanned by a beam of electrons, which creates a 3-dimensional image of the surface structure.
In Situ Hybridisation
In situ hybridization is a technique used to label DNA, RNA, and probes. It enables us to identify the location and density of specific sequences within a tissue sample. In situ hybridisation uses a probe which is labelled (using either radioactive, fluorescent, or antigen-labeled bases), and hybridizes to a target sequence at high temperatures. Hydrolysis takes place, and then excess probe is washed away. The labelled probe is localised and quantified using microscopy, autoradiography, or immunohistochemistry. This technique is very useful in Neuroscience.
Blotting technique, specifically Western Blotting, is a form of immunostaining technique that uses artificial antibodies to determine the presence of a specific type of protein within a sample. During a Western Blot, proteins are separated from one another based on molecular weight using gel electrophoresis. They are then moved to a synthetic membrane via blotting. The membrane is then probed using antibodies that are usually labelled with peroxidase. This catalyses a chemiluminescent reaction. If the required protein is present within the sample, this will be shown by a stained band present on the Western Blot.