Histology
Histology, also known as microscopic anatomy, microanatomy or histoanatomy, is the branch of biology that studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope.
Historically, microscopic anatomy was divided into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, although modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.
Biological tissues
Animal tissue classification
There are four basic types of animal tissues: muscle tissue, nervous tissue, connective tissue, and epithelial tissue. All animal tissues are considered to be subtypes of these four principal tissue types..
Plant tissue classification
For plants, the study of their tissues falls under the field of plant anatomy, with the following four main types:- Dermal tissue
- Vascular tissue
- Ground tissue
- Meristematic tissue
Medical histology
Occupations
The field of histology that includes the preparation of tissues for microscopic examination is known as histotechnology. Job titles for the trained personnel who prepare histological specimens for examination are numerous and include histotechnicians, histotechnologists, histology technicians and technologists, medical laboratory technicians, and biomedical scientists.Sample preparation
Most histological samples need preparation before microscopic observation; these methods depend on the specimen and method of observation.Fixation
Chemical fixatives are used to preserve and maintain the structure of tissues and cells; fixation also hardens tissues which aids in cutting the thin sections of tissue needed for observation under the microscope. Fixatives generally preserve tissues by irreversibly cross-linking proteins. The most widely used fixative for light microscopy is 10% neutral buffered formalin, or NBF.For electron microscopy, the most commonly used fixative is glutaraldehyde, usually as a 2.5% solution in phosphate buffered saline. Other fixatives used for electron microscopy are osmium tetroxide or uranyl acetate.
The main action of these aldehyde fixatives is to cross-link amino groups in proteins through the formation of methylene bridges, in the case of formaldehyde, or by C5H10 cross-links in the case of glutaraldehyde. This process, while preserving the structural integrity of the cells and tissue can damage the biological functionality of proteins, particularly enzymes.
Formalin fixation leads to degradation of mRNA, miRNA, and DNA as well as denaturation and modification of proteins in tissues. However, extraction and analysis of nucleic acids and proteins from formalin-fixed, paraffin-embedded tissues is possible using appropriate protocols.
Selection and trimming
Selection is the choice of relevant tissue in cases where it is not necessary to put the entire original tissue mass through further processing. The remainder may remain fixed in case it needs to be examined at a later time.Trimming is the cutting of tissue samples in order to expose the relevant surfaces for later sectioning. It also creates tissue samples of appropriate size to fit into cassettes.
Embedding
Tissues are embedded in a harder medium both as a support and to allow the cutting of thin tissue slices. In general, water must first be removed from tissues and replaced with a medium that either solidifies directly, or with an intermediary fluid that is miscible with the embedding media.Paraffin wax
For light microscopy, paraffin wax is the most frequently used embedding material. Paraffin is immiscible with water, the main constituent of biological tissue, so it must first be removed in a series of dehydration steps. Samples are transferred through a series of progressively more concentrated ethanol baths, up to 100% ethanol to remove remaining traces of water. Dehydration is followed by a clearing agent which removes the alcohol and is miscible with the wax, finally melted paraffin wax is added to replace the xylene and infiltrate the tissue. In most histology, or histopathology laboratories the dehydration, clearing, and wax infiltration are carried out in tissue processors which automate this process. Once infiltrated in paraffin, tissues are oriented in molds which are filled with wax; once positioned, the wax is cooled, solidifying the block and tissue.Other materials
Paraffin wax does not always provide a sufficiently hard matrix for cutting very thin sections. Paraffin wax may also be too soft in relation to the tissue, the heat of the melted wax may alter the tissue in undesirable ways, or the dehydrating or clearing chemicals may harm the tissue. Alternatives to paraffin wax include, epoxy, acrylic, agar, gelatin, celloidin, and other types of waxes.In electron microscopy epoxy resins are the most commonly employed embedding media, but acrylic resins are also used, particularly where immunohistochemistry is required.
For tissues to be cut in a frozen state, tissues are placed in a water-based embedding medium. Pre-frozen tissues are placed into molds with the liquid embedding material, usually a water-based glycol, OCT, TBS, Cryogen, or resin, which is then frozen to form hardened blocks.
Sectioning
For light microscopy, a knife mounted in a microtome is used to cut tissue sections which are mounted on a glass microscope slide. For transmission electron microscopy, a diamond or glass knife mounted in an ultramicrotome is used to cut between 50 and 150 nanometer thick tissue sections.A limited number of manufacturers are recognized for their production of microtomes, including vibrating microtomes commonly referred to as vibratomes, primarily for research and clinical studies. Additionally, Leica Biosystems is known for its production of products related to light microscopy in the context of research and clinical studies.
Staining
Biological tissue has little inherent contrast in either the light or electron microscope. Staining is employed to give both contrast to the tissue as well as highlighting particular features of interest. When the stain is used to target a specific chemical component of the tissue, the term histochemistry is used.Light microscopy
and eosin is one of the most commonly used stains in histology to show the general structure of the tissue. Hematoxylin stains cell nuclei blue; eosin, an acidic dye, stains the cytoplasm and other tissues in different stains of pink.In contrast to H&E, which is used as a general stain, there are many techniques that more selectively stain cells, cellular components, and specific substances. A commonly performed histochemical technique that targets a specific chemical is the Perls' Prussian blue reaction, used to demonstrate iron deposits in diseases like hemochromatosis. The Nissl method for Nissl substance and Golgi's method are useful in identifying neurons are other examples of more specific stains.