Histology,[help 1] also known as microscopic anatomy or microanatomy,[1] is the branch
of biology which 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]
Animal tissue classification[edit]
Main article: Anatomy § Animal tissues
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]




Epithelium
o Simple epithelium
 Simple squamous epithelium
 Simple cuboidal epithelium
 Simple columnar epithelium
o Pseudostratified columnar epithelium
o Stratified epithelium
 Stratified squamous epithelium
 Stratified cuboidal epithelium
 Stratified columnar epithelium
 Transitional epithelium
o Multicellular glands
Muscle tissue
o Smooth muscle
o Skeletal muscle
o Cardiac muscle
Connective tissue
o General connective tissue
 Loose connective tissue
 Dense connective tissue
o Special connective tissue
 Cartilage
 Bone
 Hemopoietic
 Blood
 Lymph
Nervous tissue
o Central nervous system
o Peripheral nervous system
o Special receptors
Plant tissue classification[edit]
Histologic section of a plant stem (Alliaria petiolata).
Main article: Plant Anatomy
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[edit]
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[edit]
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[edit]
Most histological samples need preparation before microscopic observation; these methods depend
on the specimen and method of observation.[9]
Fixation[edit]
Main article: Fixation (histology)
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[edit]
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 fixated 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[edit]
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[edit]
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[edit]
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. Prefrozen tissues are placed into molds with the liquid embedding material, usually a water-based
glycol, OCT, TBS, Cryogel, or resin, which is then frozen to form hardened blocks.
Sectioning[edit]
Main article: Microtome
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 50-150 nanometer thick tissue sections.[9]
Staining[edit]
Main article: 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[edit]
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][18] 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[edit]
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[edit]
Main article: 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[edit]
Main article: Electron microscope § Sample preparation
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[edit]
Cryosectioning[edit]
Main article: Frozen section procedure
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[edit]
Green algae under a Transmission electron microscope
Main article: Ultramicrotomy
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[edit]
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[edit]
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.[19][20] 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.[21]
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,[22] and the term
"histology" (German: Histologie), coined to denote the "study of tissues", first appeared in a book
by Karl Meyer in 1819.[23][24][19] Bichat described twenty-one human tissues, which can be subsumed
under the four categories currently accepted by histologists.[25] The usage of illustrations in histology,
deemed as useless by Bichat, was promoted by Jean Cruveilhier.[26][when?]
In the early 1830s Purkynĕ invented a microtome with high precision.[24]
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).[24]
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.[27]
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.[28]