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Histology (from Greek ἱστός (histós), cloth and λόγος (lógos), study) is the branch of biology that studies plant and animal tissues.
Histology is applied in medicine, where it plays an important role in pathological anatomy and description of morbid phenomena, also essential for pre- and post-operative analysis in medical and surgical settings.
Histology studies the morphology of tissues, and the cells that compose them, both from a morphological and functional point of view. The essential tool for analysis is the light microscope, which allows direct observation of the tissues that you want to study. To allow adequate observation of the tissues must be processed and treated in various ways: they must be cut into thin strips, to be observed against the light, must be colored in various ways, to be more easily recognizable and distinguishable, and must finally be treated to prevent decomposition and allow the preservation for subsequent analysis. A tissue that has been treated in this way is called a histological preparation.
The field of study of histology is related to that of other important biological and clinical branches including:
Histology had an important precursor in Baron Albrecht von Haller (1708 – 1777), a great figure of scientist, scholar and scholar of the eighteenth century. He was the first to suggest a microscopic structural organization of living bodies, which he traced to invisible “fibers”. The fibers of von Haller were the basic components of organs, and were divided into three basic types: the “structural fibers”, which were to constitute the blood vessels, membranes that can be assimilated to modern connectives, the “irritable fibers”, capable of responding to stimuli, corresponding to muscle tissue and finally the “sensitive fibers”, corresponding to nervous tissue.
Modern histology, however, understood as the study of the cells that make up different tissues, was born only in the mid-nineteenth century, as a result of the cell theory formulated in its first enunciation in 1838 by Matthias Jacob Schleiden and Theodor Schwann. It was only after this revolutionary discovery, which recognized in the cell the fundamental structural and functional unit of living beings, that, also thanks to the progresses made in the field of microscopy, we began to analyze the microscopic structure of organs, and to study and classify the different cell types that compose the various tissues.
Important names in the histology of the mid-nineteenth century were François Magendie and Charles Bell, who focused their studies on nervous tissue and came independently to distinguish sensory nerves from motor nerves in the spinal cord, and Albert von Kölliker, a student of the great anatomist and physiologist Jakob Henle, who studied the organization of muscle tissue both smooth and striated.
Jan Evangelista Purkyně made important contributions to the histology of nervous tissue, so much so that there are many structures that bear his name, and he was the first to describe the anatomy of the neuron. The Spaniard Ramon y Cayal also conducted important studies on nerve tissue, and contributed to formulate what would become known as the “Neuron Theory”, which defined many of the peculiarities of the nerve cell, the pattern of transmission of impulses, and especially the structural and functional unity of the neuron itself. Cayal shared the Nobel Prize for medicine with Camillo Golgi, another fundamental figure in histology, who also studied the nervous system and discovered the cell structure that bears his name.
Histological techniques represent the set of techniques and operations that allow the preparation of a biological tissue for microscopic examination.
Fixation and inclusion
To prevent decomposition, tissues intended for microscopic analysis are treated by a process called fixation. The fixation is made necessary by the fact that, once removed from the organism to which it belongs, the tissues quickly lose their chemical and physical properties, both because of the change in temperature and pH, both for the action of microorganisms that, once removed from the tissue, immediately attack and invade the biological material. By means of fixation it is possible to delay, if not to prevent, these processes, and for this purpose the tissues just taken are treated with chemical compounds such as alcohols and aldehydes, which, in fact, fix the molecules present in the tissue in the chemical state and position in which they are in vivo.
Another very important process for the study of cells is the inclusion: biological tissues, in fact, lose the consistency necessary for their maintenance. They are therefore inserted (included) in stronger materials, which can act as a support. There are several materials suitable for this purpose: kerosene, a waxy compound of lipid nature, used in the preparation of histological preparations for light microscopy (slices of 1-10 microns thick). Since, however, in electron microscopy the thickness must be of the order of Angstrom and depending on the hardness of the sample, for this purpose are used acrylic resins, fluid at room temperature and solidified by means of a curing agent (heat or UV).
Inclusion, therefore, requires the penetration of apolar substances into the cell. However, it consists mainly of water (polar). Before proceeding to the inclusion is therefore necessary to remove the aqueous component using alcohol, which is also insoluble in kerosene: once removed the water, we proceed to the removal of alcohol through substances such as benzene or xylene (mistakenly called benzol and xylol not having hydroxyl groups). This last process is called diaphanisation, as the fabric immersed in xylene becomes transparent. These two substances, benzene and xylene, are soluble in kerosene and therefore inclusion can take place.
For a tissue to be observed under a light microscope, it must be thin enough to allow light to pass through it. To achieve this, prior to microscopic examination, tissues are divided into very thin sections, using an instrument called a microtome or ultramicrotome for electron microscopy sections. Modern microtomes are able to obtain sections with a thickness not exceeding 20-30 µm (1 micrometer = 10-6meters); since these are also, approximately, the measures of a cell, it is possible with a microtome to obtain sections that contain a single layer of cells, thus avoiding that the superimposition of multiple cell layers can disturb the vision.
Another fundamental step to allow the study of tissues under the microscope is the staining. Animal tissues, in fact, are in most cases colorless (because they consist largely of water and have no pigments) and transparent, so as to be almost invisible to the optical microscope. Were therefore discovered or made, since the birth of scientific histology, a series of coloring substances, able to color the cells, or the different parts of a cell, so as to make them immediately visible and distinguishable. Nowadays are known many substances of this type, which can be divided into two major groups according to the mechanisms with which they bind to different cellular components:
- basic dyes, which bind to molecules with acidic characteristics (such as DNA)
- acid dyes, which bind to molecules with basic characteristics (such as most cytoplasmic proteins).
In histological analysis are normally used pairs of basic/acidic dyes, which stain in different ways the different cellular parts: a classic example is the staining with hematoxylin/eosin, one of the most common in the laboratory: hematoxylin, basic, colors the nucleus in blue, eosin, acid, colors the cytoplasm in pink. However there are many other compounds, able to color cellular organelles also very specific.In addition to traditional dyes, in recent years have taken place the techniques of immunohistochemistry to identify and distinguish the different cellular components: these techniques, which are very useful to highlight individual classes of molecules within the cell, involve the use of antibodies properly treated, able to bind and display specific proteins, lipids or carbohydrates.
An alternative technique to fixation and embedding is freezing. In this method, cells are frozen by various techniques and become both resistant to decomposition and sufficiently consistent for analysis. Frozen cells can then be thawed at the time of analysis, or they can be cut while frozen (cryomicrotomy) or after removal of water (freeze-drying or freeze-substitution).
The most widely used technique to perform freezing is exposure to liquid nitrogen, which can instantly bring cells to a temperature of -196 °C. This technique also allows to freeze live cells that, once thawed with the appropriate methods, are still viable. This technique has both advantages and disadvantages. It is disadvantageous because freezing may produce structural damage. In fact, if freezing takes place slowly, the aqueous component is organized in crystals that can distort the original structure; it is like forgetting a plastic bottle containing water in the refrigerator: freezing alters the shape of the bottle. In order to avoid this, it is therefore necessary to use an instantaneous freezing process such as liquid nitrogen.
Among the advantages is the fact that this is a physical technique: there are no chemical substances (such as fixatives) that can react by modifying the structural context of the cell: chemical fixatives, in fact, react in various ways by binding or modifying cellular chemical compounds (in particular carbohydrates and proteins) and can distort them, thus preventing their correct identification at a later stage.
This technique also allows a shortening of the time of preparation of histological preparations, as it avoids the procedure of inclusion in kerosene, which often leads to the loss of much of the lipid component of the tissue, due to dehydration treatments by means of alcohols. This shortening of time can even be used during an operation: if the surgeon has doubts about the diagnosis or wants to have further certainty about it, he can arrange for histological examinations. The sample to be analyzed previously frozen is then sectioned with a particular type of cryostat used in combination with a microtome, in the cutting phase of the histological preparation.
Allowing a resolution power of 0.25 µm, its operating principle is based on the crossing of a histological preparation by a beam of light. For this to occur sharply and clearly, the thickness of the sample must be 4-10 µm. There are three fundamental apparatuses: a support base, an illumination apparatus and a lens system consisting of: condenser, objective and eyepiece. The condenser collimates the light beam on the sample; the objective is near the sample to be observed and allows a magnification generally between 4 and 100 X; the eyepiece is used to observe and allows further magnification (generally from 5 to 15X) of the image produced by the objective. The magnification that allows a microscope is given by the product between that of the objective and that of the eyepiece.
An alternative technique to the analysis of the optical microscope is instead that allowed by the electron microscope, which is essential to study the ultrastructure of cells, as it has a resolution power of about 0.4 nm (1 nanometer = 10-9 meters), and can also resolve large molecules and molecular complexes. In the upper part there is a tungsten foil (cathode) from which depart electrons accelerated towards an anode (located in the lower part) by a potential difference between 80 and 100 kV. Along their path the electrons pass through lenses, which are coils that create magnetic fields that deflect and collimate the electrons on the sample to be observed (whose thickness must be between 600 and 700 Å).
The image obtained by the electron is strictly black and white: it is made up of a series of black and white dots (which originate on a fluorescent screen) and is photographed, since excessive exposure of the biological sample to electrons would damage it. The white dots correspond to cellular areas that allow electrons to pass through them (called electron-crossing), while the black dots correspond to cellular regions that reflect the electrons (called electrondense). Since the image is black and white and any staining of the histological preparation is useless, to emphasize certain cellular structures we proceed to electron staining: by precipitating uranyl acetate on the nucleus, electrons are reflected and the nucleus appears as an electrondense region.
There are two types of electron microscopes: the transmission electron microscope (TEM) and the scanning electron microscope (SEM). The former provides information about the internal structure or ultrastructure of the cell; the SEM provides images of the cell surface. In short, the SEM stands to the TEM as of a person his photograph stands to his x-ray.