Histology

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Histologic specimen being placed on the stage of an optical microscope. Slide under a microscope.jpg
Histologic specimen being placed on the stage of an optical microscope.
Human lung tissue stained with hematoxylin and eosin as seen under a microscope. Emphysema H and E.jpg
Human lung tissue stained with hematoxylin and eosin as seen under a microscope.

Histology, [help 1] also known as microscopic anatomy or microanatomy, [1] is the branch of biology that studies the microscopic anatomy of biological tissues. [2] [3] [4] [5] Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. [5] [6] Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology , the study of cells, modern usage places all of these topics under the field of histology. [5] In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. [5] [6] In the field of paleontology, the term paleohistology refers to the histology of fossil organisms. [7] [8]

Contents

Biological tissues

Animal tissue classification

There are four basic types of animal tissues: muscle tissue, nervous tissue, connective tissue, and epithelial tissue. [5] [9] All animal tissues are considered to be subtypes of these four principal tissue types (for example, blood is classified as connective tissue, since the blood cells are suspended in an extracellular matrix, the plasma). [9]

Plant tissue classification

Histologic section of a plant stem (Alliaria petiolata). Alliaria petiolata, stalk, cross section, Etzold green.jpg
Histologic section of a plant stem ( Alliaria petiolata ).

For plants, the study of their tissues falls under the field of plant anatomy, with the following four main types:

Medical histology

Histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. [5] [6] It is an important part of anatomical pathology and surgical pathology, as accurate diagnosis of cancer and other diseases often requires histopathological examination of tissue samples. [10] Trained physicians, frequently licensed pathologists, perform histopathological examination and provide diagnostic information based on their observations.

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, [11] 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. [9]

Fixation

Histologic section of a fossilized invertebrate. Ordovician bryozoan. Stigmatella personata thin section.jpg
Histologic section of a fossilized invertebrate. Ordovician bryozoan.

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. [5] [12] Fixatives generally preserve tissues (and cells) by irreversibly cross-linking proteins. [12] The most widely used fixative for light microscopy is 10% neutral buffered formalin, or NBF (4% formaldehyde in phosphate buffered saline). [13] [12] [9]

For electron microscopy, the most commonly used fixative is glutaraldehyde, usually as a 2.5% solution in phosphate buffered saline. [9] Other fixatives used for electron microscopy are osmium tetroxide or uranyl acetate. [9]

The main action of these aldehyde fixatives is to cross-link amino groups in proteins through the formation of methylene bridges (-CH2-), 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. [14] [15]

Selection and trimming

Items used for submitting specimens: (Biopsy) wrap, (biopsy) sponge, (tissue processing) cassette and (biopsy) bag. Biopsy wrap, biopsy sponge and biopsy bag.jpg
Items used for submitting specimens: (Biopsy) wrap, (biopsy) sponge, (tissue processing) cassette and (biopsy) bag.

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. [16]

Embedding

Tissues are embedded in a harder medium both as a support and to allow the cutting of thin tissue slices. [9] [5] In general, water must first be removed from tissues (dehydration) and replaced with a medium that either solidifies directly, or with an intermediary fluid (clearing) that is miscible with the embedding media. [12]

Paraffin wax

Histologic sample being embedded in paraffin wax (tissue is held at the bottom of a metal mold, and more molten paraffin is poured over it to fill it). Tissue processing - Embedding station.jpg
Histologic sample being embedded in paraffin wax (tissue is held at the bottom of a metal mold, and more molten paraffin is poured over it to fill it).

For light microscopy, paraffin wax is the most frequently used embedding material. [12] [13] Paraffin is immiscible with water, the main constituent of biological tissue, so it must first be removed in a series of dehydration steps. [12] Samples are transferred through a series of progressively more concentrated ethanol baths, up to 100% ethanol to remove remaining traces of water. [9] [12] Dehydration is followed by a clearing agent (typically xylene [13] although other environmental safe substitutes are in use [13] ) which removes the alcohol and is miscible with the wax, finally melted paraffin wax is added to replace the xylene and infiltrate the tissue. [9] In most histology, or histopathology laboratories the dehydration, clearing, and wax infiltration are carried out in tissue processors which automate this process. [13] 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. [13] [12]

Other materials

Paraffin wax does not always provide a sufficiently hard matrix for cutting very thin sections (which are especially important for electron microscopy). [12] 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. [12] Alternatives to paraffin wax include, epoxy, acrylic, agar, gelatin, celloidin, and other types of waxes. [12] [17]

In electron microscopy epoxy resins are the most commonly employed embedding media, [9] 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

Histologic sample being cut on a microtome. Tissue processing - Microtome is used to cut a ribbon of 5-micron-thick sections from the paraffin block.jpg
Histologic sample being cut on a microtome.

For light microscopy, a knife mounted in a microtome is used to cut tissue sections (typically between 5-15 micrometers thick) which are mounted on a glass microscope slide. [9] For transmission electron microscopy (TEM), a diamond or glass knife mounted in an ultramicrotome is used to cut between 50 and 150 nanometer thick tissue sections. [9]

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. [18]

Staining

Biological tissue has little inherent contrast in either the light or electron microscope. [17] 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 (and not the general structure), the term histochemistry is used. [9]

Light microscopy

Masson's trichrome staining on rat trachea. Masson's trichrome staining on rat's trachea.jpg
Masson's trichrome staining on rat trachea.

Hematoxylin and eosin (H&E stain) is one of the most commonly used stains in histology to show the general structure of the tissue. [9] [19] Hematoxylin stains cell nuclei blue; eosin, an acidic dye, stains the cytoplasm and other tissues in different stains of pink. [9] [12]

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. [12] A commonly performed histochemical technique that targets a specific chemical is the Perls' Prussian blue reaction, used to demonstrate iron deposits [12] in diseases like hemochromatosis. The Nissl method for Nissl substance and Golgi's method (and related silver stains) are useful in identifying neurons are other examples of more specific stains. [12]

Historadiography

In historadiography, a slide (sometimes stained histochemically) is X-rayed. More commonly, autoradiography is used in visualizing the locations to which a radioactive substance has been transported within the body, such as cells in S phase (undergoing DNA replication) which incorporate tritiated thymidine, or sites to which radiolabeled nucleic acid probes bind in in situ hybridization. For autoradiography on a microscopic level, the slide is typically dipped into liquid nuclear tract emulsion, which dries to form the exposure film. Individual silver grains in the film are visualized with dark field microscopy.

Immunohistochemistry

Recently, antibodies have been used to specifically visualize proteins, carbohydrates, and lipids. This process is called immunohistochemistry, or when the stain is a fluorescent molecule, immunofluorescence. This technique has greatly increased the ability to identify categories of cells under a microscope. Other advanced techniques, such as nonradioactive in situ hybridization, can be combined with immunochemistry to identify specific DNA or RNA molecules with fluorescent probes or tags that can be used for immunofluorescence and enzyme-linked fluorescence amplification (especially alkaline phosphatase and tyramide signal amplification). Fluorescence microscopy and confocal microscopy are used to detect fluorescent signals with good intracellular detail.

Electron microscopy

For electron microscopy heavy metals are typically used to stain tissue sections. [9] Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope. [9]

Specialized techniques

Cryosectioning

Similar to the frozen section procedure employed in medicine, cryosectioning is a method to rapidly freeze, cut, and mount sections of tissue for histology. The tissue is usually sectioned on a cryostat or freezing microtome. [12] The frozen sections are mounted on a glass slide and may be stained to enhance the contrast between different tissues. Unfixed frozen sections can be used for studies requiring enzyme localization in tissues and cells. Tissue fixation is required for certain procedures such as antibody-linked immunofluorescence staining. Frozen sections are often prepared during surgical removal of tumors to allow rapid identification of tumor margins, as in Mohs surgery, or determination of tumor malignancy, when a tumor is discovered incidentally during surgery.

Ultramicrotomy

Green algae under a Transmission electron microscope Chlamydomonas TEM 07.jpg
Green algae under a Transmission electron microscope

Ultramicrotomy is a method of preparing extremely thin sections for transmission electron microscope (TEM) analysis. Tissues are commonly embedded in epoxy or other plastic resin. [9] Very thin sections (less than 0.1 micrometer in thickness) are cut using diamond or glass knives on an ultramicrotome. [12]

Artifacts

Artifacts are structures or features in tissue that interfere with normal histological examination. Artifacts interfere with histology by changing the tissues appearance and hiding structures. Tissue processing artifacts can include pigments formed by fixatives, [12] shrinkage, washing out of cellular components, color changes in different tissues types and alterations of the structures in the tissue. An example is mercury pigment left behind after using Zenker's fixative to fix a section. [12] Formalin fixation can also leave a brown to black pigment under acidic conditions. [12]

History

Santiago Ramon y Cajal in his laboratory. Cajal-va.jpg
Santiago Ramón y Cajal in his laboratory.

In the 17th century the Italian Marcello Malpighi used microscopes to study tiny biological entities; some regard him as the founder of the fields of histology and microscopic pathology. [20] [21] Malpighi analyzed several parts of the organs of bats, frogs and other animals under the microscope. While studying the structure of the lung, Malpighi noticed its membranous alveoli and the hair-like connections between veins and arteries, which he named capillaries. His discovery established how the oxygen breathed in enters the blood stream and serves the body. [22]

In the 19th century histology was an academic discipline in its own right. The French anatomist Xavier Bichat introduced the concept of tissue in anatomy in 1801, [23] and the term "histology" (German : Histologie), coined to denote the "study of tissues", first appeared in a book by Karl Meyer in 1819. [24] [25] [20] Bichat described twenty-one human tissues, which can be subsumed under the four categories currently accepted by histologists. [26] The usage of illustrations in histology, deemed as useless by Bichat, was promoted by Jean Cruveilhier. [27] [ when? ]

In the early 1830s Purkynĕ invented a microtome with high precision. [25]

During the 19th century many fixation techniques were developed by Adolph Hannover (solutions of chromates and chromic acid), Franz Schulze and Max Schultze (osmic acid), Alexander Butlerov (formaldehyde) and Benedikt Stilling (freezing). [25]

Mounting techniques were developed by Rudolf Heidenhain (1824–1898), who introduced gum Arabic; Salomon Stricker (1834–1898), who advocated a mixture of wax and oil; and Andrew Pritchard (1804–1884) who, in 1832, used a gum/isinglass mixture. In the same year, Canada balsam appeared on the scene, and in 1869 Edwin Klebs (1834–1913) reported that he had for some years embedded his specimens in paraffin. [28]

The 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had conflicting interpretations of the neural structure of the brain based on differing interpretations of the same images. Ramón y Cajal won the prize for his correct theory, and Golgi for the silver-staining technique that he invented to make it possible. [29]

Future directions

In vivo histology

Currently there is intense interest in developing techniques for in vivo histology (predominantly using MRI), which would enable doctors to non-invasively gather information about healthy and diseased tissues in living patients, rather than from fixed tissue samples. [30] [31] [32] [33]

See also

Notes

  1. The word histology ( /hɪstˈɒləi/ ) is Neo-Latin using the combining forms of histo- + -logy , yielding "tissue study", from the Greek words ἱστός histos, "tissue", and -λογία , "study".

Related Research Articles

<span class="mw-page-title-main">Electron microscope</span> Type of microscope with electrons as a source of illumination

An electron microscope is a microscope that uses a beam of electrons as a source of illumination. They use electron optics that are analogous to the glass lenses of an optical light microscope to control the electron beam, for instance focusing them to produce magnified images or electron diffraction patterns. As the wavelength of an electron can be up to 100,000 times smaller than that of visible light, electron microscopes have a much higher resolution of about 0.1 nm, which compares to about 200 nm for light microscopes. Electron microscope may refer to:

<span class="mw-page-title-main">Tissue (biology)</span> Group of similar cells performing a specific function

In biology, tissue is an assembly of similar cells and their extracellular matrix from the same embryonic origin that together carry out a specific function. Tissues occupy a biological organizational level between cells and a complete organ. Accordingly, organs are formed by the functional grouping together of multiple tissues.

<span class="mw-page-title-main">Microscope slide</span> Thin, flat piece of glass onto which a sample is placed to be examined under a microscope

A microscope slide is a thin flat piece of glass, typically 75 by 26 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then both are inserted together in the microscope for viewing. This arrangement allows several slide-mounted objects to be quickly inserted and removed from the microscope, labeled, transported, and stored in appropriate slide cases or folders etc.

<span class="mw-page-title-main">Staining</span> Technique used to enhance visual contrast of specimens observed under a microscope

Staining is a technique used to enhance contrast in samples, generally at the microscopic level. Stains and dyes are frequently used in histology, in cytology, and in the medical fields of histopathology, hematology, and cytopathology that focus on the study and diagnoses of diseases at the microscopic level. Stains may be used to define biological tissues, cell populations, or organelles within individual cells.

<span class="mw-page-title-main">Immunostaining</span> Biochemical technique

In biochemistry, immunostaining is any use of an antibody-based method to detect a specific protein in a sample. The term "immunostaining" was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941. However, immunostaining now encompasses a broad range of techniques used in histology, cell biology, and molecular biology that use antibody-based staining methods.

<span class="mw-page-title-main">Histopathology</span> Microscopic examination of tissue in order to study and diagnose disease

Histopathology is the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue micro-fragments.

<span class="mw-page-title-main">Immunohistochemistry</span> Common application of immunostaining

Immunohistochemistry is a form of immunostaining. It involves the process of selectively identifying antigens (proteins) in cells and tissue, by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Albert Hewett Coons, Ernest Berliner, Norman Jones and Hugh J Creech was the first to develop immunofluorescence in 1941. This led to the later development of immunohistochemistry.

Zenker's fixative is a rapid-acting fixative for animal tissues. It is employed to prepare specimens of animal or vegetable tissues for microscopic study. It provides excellent fixation of nuclear chromatin, connective tissue fibers and some cytoplasmic features, but does not preserve delicate cytoplasmic organelles such as mitochondria. Helly's fixative is preferable for traditional dye staining of mitochondria. Zenker's fixative permeabilises the plasma, but not the nuclear membrane. It can therefore be used to selectively stain mitotic cells with antibodies against chromatin

A microtome is a cutting tool used to produce extremely thin slices of material known as sections, with the process being termed microsectioning. Important in science, microtomes are used in microscopy for the preparation of samples for observation under transmitted light or electron radiation.

<span class="mw-page-title-main">H&E stain</span> Histological stain method

Hematoxylin and eosin stain is one of the principal tissue stains used in histology. It is the most widely used stain in medical diagnosis and is often the gold standard. For example, when a pathologist looks at a biopsy of a suspected cancer, the histological section is likely to be stained with H&E.

<span class="mw-page-title-main">Frozen section procedure</span> Rapid histological sectioning procedure

The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery. The technical name for this procedure is cryosection. The microtome device that cold cuts thin blocks of frozen tissue is called a cryotome.

<span class="mw-page-title-main">Fixation (histology)</span> Preservation of biological tissue

In the fields of histology, pathology, and cell biology, fixation is the preservation of biological tissues from decay due to autolysis or putrefaction. It terminates any ongoing biochemical reactions and may also increase the treated tissues' mechanical strength or stability. Tissue fixation is a critical step in the preparation of histological sections, its broad objective being to preserve cells and tissue components and to do this in such a way as to allow for the preparation of thin, stained sections. This allows the investigation of the tissues' structure, which is determined by the shapes and sizes of such macromolecules as proteins and nucleic acids.

<span class="mw-page-title-main">Toluidine blue</span> Chemical compound

Toluidine blue, also known as TBO or tolonium chloride (INN) is a blue cationic (basic) dye used in histology and sometimes clinically.

<span class="mw-page-title-main">Automated tissue image analysis</span>

Automated tissue image analysis or histopathology image analysis (HIMA) is a process by which computer-controlled automatic test equipment is used to evaluate tissue samples, using computations to derive quantitative measurements from an image to avoid subjective errors.

Antigen retrieval is a non-enzymatic pretreatment for immunostaining to reduce or eliminate the chemical modifications caused by formalin fixation, through high temperature heating or strong alkaline solution (non-heating).

<span class="mw-page-title-main">Immunogold labelling</span> Staining technique used in electron microscopy

Immunogold labeling or immunogold staining (IGS) is a staining technique used in electron microscopy. This staining technique is an equivalent of the indirect immunofluorescence technique for visible light. Colloidal gold particles are most often attached to secondary antibodies which are in turn attached to primary antibodies designed to bind a specific antigen or other cell component. Gold is used for its high electron density which increases electron scatter to give high contrast 'dark spots'.

Serial block-face scanning electron microscopy is a method to generate high resolution three-dimensional images from small samples. The technique was developed for brain tissue, but it is widely applicable for any biological samples. A serial block-face scanning electron microscope consists of an ultramicrotome mounted inside the vacuum chamber of a scanning electron microscope. Samples are prepared by methods similar to that in transmission electron microscopy (TEM), typically by fixing the sample with aldehyde, staining with heavy metals such as osmium and uranium then embedding in an epoxy resin. The surface of the block of resin-embedded sample is imaged by detection of back-scattered electrons. Following imaging the ultramicrotome is used to cut a thin section from the face of the block. After the section is cut, the sample block is raised back to the focal plane and imaged again. This sequence of sample imaging, section cutting and block raising can acquire many thousands of images in perfect alignment in an automated fashion. Practical serial block-face scanning electron microscopy was invented in 2004 by Winfried Denk at the Max-Planck-Institute in Heidelberg and is commercially available from Gatan Inc., Thermo Fisher Scientific (VolumeScope) and ConnectomX.

Bouin solution, or Bouin's solution, is a compound fixative used in histology. It was invented by French biologist Pol Bouin and is composed of picric acid, acetic acid and formaldehyde in an aqueous solution. Bouin's fluid is especially useful for fixation of gastrointestinal tract biopsies because this fixative allows crisper and better nuclear staining than 10% neutral-buffered formalin. It is not a good fixative when tissue ultrastructure must be preserved for electron microscopy. However, it is a good fixative when tissue structure with a soft and delicate texture must be preserved. The acetic acid in this fixative lyses red blood cells and dissolves small iron and calcium deposits in tissue. A variant in which the acetic acid is replaced with formic acid can be used for both fixation of tissue and decalcification. The effects of the three chemicals in Bouin solution balance each other. Formalin causes cytoplasm to become basophilic but this effect is balanced by the effect of the picric acid. This results in excellent nuclear and cytoplasmic H&E staining. The tissue hardening effect of formalin is balanced by the soft tissue fixation of picric and acetic acids. The tissue swelling effect of acetic acid is balanced by the tissue shrinking effect of picric acid.

Microscopy with UV Surface Excitation (MUSE) is a novel microscopy method that utilizes the shallow penetration of UV photons excitation. Compared to conventional microscopes, which usually require sectioning to exclude blurred signals from outside of the focal plane, MUSE's low penetration depth limits the excitation volume to a thin layer, and removes the tissue sectioning requirement. The entire signal collected is the desired light, and all photons collected contribute to the image formation.

Microtechnique is an aggregate of methods used to prepare micro-objects for studying. It is currently being employed in many fields in life science. Two well-known branches of microtechnique are botanical (plant) microtechnique and zoological (animal) microtechnique.

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