Histology

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A stained histologic specimen, sandwiched between a glass microscope slide and coverslip, mounted on the stage of a light microscope.

Microscopic view of a histologic specimen of human lung tissue stained with hematoxylin and eosin. Histology (compound of the Greek words: "tissue", and - -logia) is the study of the microscopic anatomy of cells and tissues of plants and animals. It is performed by examining cells and tissues commonly by sectioning and staining; followed by examination under a light microscope or electron microscope. Histological studies may be conducted via tissue culture, where live cells can be isolated and maintained in a proper environment outside the body for various research projects. The ability to visualize or differentially identify microscopic structures is frequently enhanced through the use of histological stains. Histology is an essential tool of biology and medicine. Histopathology, the microscopic study of diseased tissue, is an important tool in anatomical pathology, since accurate diagnosis of cancer and other diseases usually requires histopathological examination of samples. Trained medical doctors, frequently board-certified as pathologists, are the personnel who perform histopathological examination and provide diagnostic information based on their observations. The trained scientists who perform the preparation of histological sections are histotechnicians, histology technicians (HT), histology technologists (HTL), medical scientists, medical laboratory technicians, or biomedical scientists. Their field of study is called histotechnology.

Contents
[hide]

1 Histology o 1.1 Fixing 1.1.1 Chemical fixation with formaldehyde or other chemicals 1.1.2 Frozen section fixation o 1.2 Processing - dehydration, clearing, and infiltration o 1.3 Embedding o 1.4 Sectioning o 1.5 Staining 2 Common laboratory stains o 2.1 Alternative techniques 3 History 4 Histological classification of animal tissues 5 Related sciences 6 Artifacts o 6.1 Pre-histology o 6.2 Post-histology 7 See also 8 Notes 9 References 10 External links

[edit] Histology

[edit] Fixing
[edit] Chemical fixation with formaldehyde or other chemicals Main article: Fixation (histology) Chemical fixatives are used to preserve tissue from degradation, and to maintain the structure of the cell and of sub-cellular components such as cell organelles (e.g., nucleus, endoplasmic reticulum, mitochondria). The most common fixative for light microscopy is 10% neutral buffered formalin (4% formaldehyde in phosphate buffered saline). For electron microscopy, the most commonly used fixative is glutaraldehyde, usually as a 2.5% solution in phosphate buffered saline. These fixatives preserve tissues or cells mainly by irreversibly cross-linking proteins. The main action of these aldehyde fixatives is to cross-link amino groups in proteins through the formation of CH 2 (methylene) linkage, in the case of formaldehyde, or by a 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, and can also denature them to a certain extent. This can be detrimental to certain histological techniques. Further fixatives are often used for electron microscopy such as osmium tetroxide or uranyl acetate Formalin fixation leads to degradation of mRNA, miRNA and DNA in tissues. However, extraction, amplification and analysis of these nucleic acids from formalin-fixed, paraffin-embedded tissues is possible using appropriate protocols.[1] [edit] Frozen section fixation Frozen section is a rapid way to fix and mount histology sections. It is used in surgical removal of tumors, and allow rapid determination of margin (that the tumor has been completely removed). It is done using a refrigeration device called a cryostat. The frozen tissue is sliced using a microtome, and the frozen slices are mounted on a glass slide and stained the same way as other methods. It is a necessary way to fix tissue for certain stain such as antibody linked immunofluorescence staining. It can also be used to determine if a tumour is malignant when it is found incidentally during surgery on a patient.

[edit] Processing - dehydration, clearing, and infiltration

The aim of Tissue Processing is to remove water from tissues and replace with a medium that solidifies to allow thin sections to be cut. Biological tissue must be supported in a hard matrix to allow sufficiently thin sections to be cut, typically 5 m (micrometers; 1000 micrometers = 1 mm) thick for light microscopy and 80-100 nm (nanometer; 1,000,000 nanometers = 1 mm) thick for electron microscopy. For light microscopy, paraffin wax is most frequently used. Since it is immiscible with water, the main constituent of biological tissue, water must first be removed in the process of dehydration. Samples are transferred through baths of progressively more concentrated ethanol to remove the water. This is followed by a hydrophobic clearing agent (such as xylene) to remove the alcohol, and finally molten paraffin wax, the infiltration agent, which replaces the xylene. Paraffin wax does not provide a sufficiently hard matrix for cutting very thin sections for electron microscopy. Instead, resins are used. Epoxy resins are the most commonly employed embedding media, but acrylic resins are also used, particularly where immunohistochemistry is required. Thicker sections (0.35m to 5m) of resin-embedded tissue can also be cut for light microscopy. Again, the immiscibility of most epoxy and acrylic resins with water necessitates the use of dehydration, usually with ethanol.

[edit] Embedding
After the tissues have been dehydrated, cleared, and infiltrated with the embedding material, they are ready for external embedding. During this process the tissue samples are placed into molds along with liquid embedding material (such as agar, gelatine, or wax) which is then hardened. This is achieved by cooling in the case of paraffin wax and heating (curing) in the case of the epoxy resins. The acrylic resins are polymerised by heat, ultraviolet light, or chemical catalysts. The hardened blocks containing the tissue samples are then ready to be sectioned. Because Formalin-fixed, paraffin-embedded (FFPE) tissues may be stored indefinitely at room temperature, and nucleic acids (both DNA and RNA) may be recovered from them decades after fixation, FFPE tissues are an important resource for historical studies in medicine. Embedding can also be accomplished using frozen, non-fixed tissue in a water-based medium. Pre-frozen 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.

[edit] Sectioning
Sectioning can be done in limited ways. Vertical sectioning perpendicular to the surface of the tissue is the usual method. Horizontal sectioning is often done in the evaluation of the hair follicles and pilosebaceous units. Tangential to horizontal sectioning is done in Mohs surgery and in methods of CCPDMA. For light microscopy, a steel knife mounted in a microtome is used to cut 10-micrometer-thick tissue sections which are mounted on a glass microscope slide. For transmission electron microscopy, a diamond knife mounted in an ultramicrotome is used to cut 50-nanometer-thick tissue sections which are mounted on a 3-millimeter-diameter copper grid. Then the mounted sections are treated with the appropriate stain. Frozen tissue embedded in a freezing medium is cut on a microtome in a cooled machine called a cryostat.

[edit] 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. Where the underlying mechanistic chemistry of staining is understood, the term histochemistry is used. Hematoxylin and eosin (H&E stain) is the most commonly used light microscopical stain in histology and histopathology. Hematoxylin, a basic dye, stains nuclei blue due to an affinity to nucleic acids in the cell nucleus; eosin, an acidic dye, stains the cytoplasm pink. Uranyl acetate and lead citrate are commonly used to impart contrast to tissue in the electron microscope. Special staining: There are hundreds of various other techniques that have been used to selectively stain cells and cellular components. Other compounds used to color tissue sections include safranin, oil red o, Congo red, fast green FCF, silver salts, and numerous natural and artificial dyes that were usually originated from the development dyes for the textile industry. Histochemistry refers to the science of using chemical reactions between laboratory chemicals and components within tissue. A commonly performed histochemical technique is the Perls Prussian blue reaction, used to demonstrate iron deposits in diseases like hemochromatosis. Histology samples have often been examined by radioactive techniques. In historadiography, a slide (sometimes stained histochemically) is Xrayed. More commonly, autoradiography is used to visualize 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. 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. Digital cameras are increasingly used to capture histological and histopathological image

[edit] Common laboratory stains

Stain Haematoxylin Common use General staining when paired with eosin (i.e. H&E) Nucleus Blue Cytoplasm N/A Red blood cell (RBC) N/A Collagen fibers N/A ER (endoplasmic reticulum)blue Elastic fiberspink Pink Blue Red/pink Pale red Orange/red Blue Red Orange Pink Blue Blue/green Deep blue Muscle fibersred Keratinorange Collagen fiberspink Reticular fibers pink Mast cells granulespurple Cartilageblue/green Specifically stains Nucleic acidsblue

Eosin Toluidine blue

General staining when paired with haematoxylin N/A (i.e. H&E) General staining Blue Black Red

Masson's trichrome Connective tissue stain Mallory's trichrome Connective tissue

stain Cartilageblue Bone matrixdeep blue Muscle fibersred Weigert's elastic stain Heidenhain's AZAN trichrome stain Silver stain Elastic fibers Blue/black N/A Pink N/A Red N/A Blue Cartilageblue Bone matrixblue Reticular fibersbrown/black N/A N/A N/A Nerve fibersbrown/black Neutrophil granulespurple/pink Bluish/purple Bluish/gray Red/pink N/A Eosinophil granulesbright red/orange Basophil granulesdeep purple/violet Platelet granulesred/purple Elastic fibresdark brown Mast cells granulespurple Smooth musclelight blue Glycogen and other carbohydrates magenta Elastic fibersblue/black Muscle fibersred

Distinguishing cells from Red/purple extracellular components Reticular fibers, nerve fibers, fungi N/A

Wright's stain

Blood cells

Orcein stain

Elastic fibres

Deep blue

N/A

Bright red

Pink

Periodic acid-Schiff Basement membrane, stain (PAS) localizing carbohydrates

Blue

N/A

N/A

Pink

Table sourced from Michael H. Ross, Wojciech Pawlina, (2006). Histology: A Text and Atlas. Hagerstown, MD: Lippincott Williams & Wilkins. ISBN 0-7817-5056-3. The Nissl method and Golgi's method are useful in identifying neurons.

[edit] Alternative techniques

Alternative techniques include cryosection. The tissue is frozen using a cryostat, and cut. Tissue staining methods are similar to those of wax sections. Plastic embedding is commonly used in the preparation of material for electron microscopy. Tissues are embedded in epoxy resin. Very thin sections (less than 0.1 micrometer) are cut using diamond or glass knives. The sections are stained with electron dense stains (uranium and lead) so that they can possibly be seen with the electron microscope.

[edit] History
In the 19th century, histology was an academic discipline in its own right. The 1906 Nobel Prize in Physiology or Medicine was awarded to histologists Camillo Golgi and Santiago Ramon y Cajal. They had dueling interpretations of the neural structure of the brain based in differing interpretations of the same images. Cajal won the prize for his correct theory and Golgi for the staining technique he invented to make it possible.

[edit] Histological classification of animal tissues

There are four basic types of tissues: muscle tissue, nervous tissue, connective tissue, and epithelial tissue. All tissue types are subtypes of these four basic tissue types (for example, blood cells are classified as connective tissue, since they generally originate inside bone marrow).

Epithelium: the lining of glands, bowel, skin, and some organs like the liver, lung, and kidney Endothelium: the lining of blood and lymphatic vessels Mesothelium: the lining of pleural and pericardial spaces Mesenchyme: the cells filling the spaces between the organs, including fat, muscle, bone, cartilage, and tendon cells Blood cells: the red and white blood cells, including those found in lymph nodes and spleen Neurons: any of the conducting cells of the nervous system Germ cells: reproductive cells (spermatozoa in men, oocytes in women)

Placenta: an organ characteristic of true mammals during pregnancy, joining mother and offspring, providing endocrine secretion and selective exchange of soluble, but not particulate, blood-borne substances through an apposition of uterine and trophoblastic vascularised parts Stem cells: cells with the ability to develop into different cell types

Note that tissues from plants, fungi, and microorganisms can also be examined histologically. Their structure is very different from animal tissues.

[edit] Related sciences

Cell biology is the study of living cells, their DNA and RNA and the proteins they express. Anatomy is the study of organs visible by the naked eye. Morphology studies entire organisms.

[edit] Artifacts
Artifacts are structures or features in tissue that interfere with normal histological examination. These are not always present in normal tissue and can come from outside sources. Artifacts interfere with histology by changing the tissues appearance and hiding structures. These can be divided into two categories:

[edit] Pre-histology
These are features and structures that have being introduced prior to the collection of the tissues. A common example of these include: ink from tattoos and freckles (melanin) in skin samples.

[edit] Post-histology

Artifacts can result from tissue processing. Processing commonly leads to changes like shrinkage, washing out of particular cellular components, color changes in different tissues types and alterations of the structures in the tissue. Because these are caused in a laboratory the majority of post histology artifacts can be avoided or removed after being discovered. A common example is mercury pigment left behind after using Zenker's fixative to fix a section.

[edit] See also

Medicine portal

Meyer's Histology - a complete online histology course Histology-online Anatomical pathology Automated tissue image analysis Biological staining Geoffrey Bourne Cooperative Human Tissue Network (CHTN) Digital Pathology Arthur Worth Ham Histopathology Important publications in histology (Arthur Worth Ham and David H. Cormack's Histology, for example) Laser capture microdissection Pathology

[edit] Notes
1. ^ Weiss AT, Delcour NM, Meyer A, Klopfleisch R. (2010). "Efficient and Cost-Effective Extraction of Genomic DNA From FormalinFixed and Paraffin-Embedded Tissues". Veterinary Pathology 227 (4): 8348. doi:10.1177/0300985810380399. PMID 20817894.

[edit] References
1. 2. 3. 4. Meyer's Histology - a complete online histology course (2012). (http://meyershistology.moodle.com.au) Merck Source (2002). Dorland's Medical Dictionary. Retrieved 2005-01-26. Stedman's Medical Dictionaries (2005). Stedman's Online Medical Dictionary. Retrieved 2005-01-26. 4,000online histology images (2007). (http://histology-online.com)

[edit] External links

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Human Body Study Section Human Body Index Health Glossary More about this Topic: Introduction to Histology:

What is

Histology ? What is Histopatholo gy? What is Histochemist ry? How to prepare Histology Slides Histology Stains

Not sure what histology is ? See definition of Histology and definition of Histopathology

How to Prepare Histology Slides

How to prepare histology slides is not usually knowledge required for first-level courses in Anatomy & Physiology (e.g. ITEC) and Human Biology (e.g. A-Level in UK). It is, however, useful to have a general awareness of the steps involved in preparing histology slides. It may be useful for school and college students to know a bit about how to prepare histology slides as part of their general appreciation of laboratory biology and techniques used in medical research. Familiarity with terminology used in the discipline of histology is also useful when reading around the subject or communicating with other professionals working in various fields within health sciences e.g. during work experience. The five main stages in the preparation of histology slides are:

Cells and Cell Division

Cell Structure Cell Division (Intro) What is Mitosis ? The Somatic Cell Cycle Diagram of Mitosis

1. 2. 3. 4. 5.

Fixing Processing Embedding Sectioning Staining

Each of these stages is described briefly, as follows:

Fixing

Samples of biological tissue are "fixed" to preserve the cells/tissue in as natural a state as possible and prevent postmortem decay (autolysis and putrefaction). Chemical fixatives are very carefully selected substances whose properties must meet many criteria. Even the most careful fixation alters the sample to a certain

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Glossary Pages

Cell Organelles

extent and may potentially introduce artifacts that can interfere with interpretation of images of the fine detail of cells, incl. all their organelles, that can only be observed using an electron microscope (such fine detail of cells that can only be seen using electron microscopes is called "cellular ultrastructure"). Part of the skill involved in preparing and interpreting histology slides is in maintaining the structure of the sample in as undisturbed a state and possible, minimising the introduction of artifacts and recognising artifacts as such when they are present. Because fixation is usually the first stage in a multistep process to prepare biological material for microscopy or other analysis, the choice of fixation method and specific fixative may depend on the subsequent processing steps appropriate in that particular case. Chemical Fixation. In this case biological structures are preserved (both chemically and structurally) in a state as close to that of the living tissue as possible. This requires a chemical fixative that can stabilise the proteins, nucleic acids and mucosubstances of the tissue by making them insoluble. Frozen Sections Small pieces of tissue (typically 5mm x 5mm x 3 mm) are placed in a cryoprotective embedding medium then snap frozen in isopentane (an alkane) cooled by liquid nitrogen. Tissue is then sectioned in a freezing microtome or cryostat. Sections are then fixed by immersion in a specific fixative or series of fixatives for carefully controlled period of time. Advantages - of fixation by frozen sections

Any Questions ?

Recent health news: Progress towards a diagnostic test for oesophageal cancer - 8 Feb Early bone growth linked to bone density in later life

Disadvantages - of fixation by frozen sections

Give better preservation of

Lack morphological detail

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antigenicity Minimal exposure to fixative Not exposed to the organic solvents

Possibility of biohazard

Processing

Tissue processing is done to remove water from the biological tissues, replacing such water with a medium that solidifies, setting very hard and so allowing extremely thin sections to be sliced. This is important because biological tissue must be supported in an extremely hard solid matrix to enable sufficiently thin sections to be cut. Some typical values are: 5 m thick for light microscopy 5 m (i.e. 5 micrometres) = 0.005 mm = 0.000005 metre 80-100 nm thick for electron microscopy 80-100 nm (i.e. 80-100 nanometres) = 0.00008 mm to 0.0001 mm = 0.00000008 to 0.0000001 metre See the page about Scientific Numbers for more about these units. Removal of water is also referred to as "dehydration".

Embedding

After tissues have been dehydrated and before they can be "sectioned" i.e. sliced very thinly (see the thicknesses mentioned above) they must be secured in a very hard solid block in such a way that the hardened material used to secure all parts of the biological tissues in place is transparent to the optical method used for viewing the finished samples. Different types of embedding techniques and materials are used depending on the

sample being prepared and the other types of processing involved in preparing that particular sample. In general, tissue samples are placed in molds together with liquid embedding material which is then hardened. The result of this stage in the preparation of histology slides is hardened blocks containing the original biological samples together with other substances used so far in the preparation process.

Sectioning

Sectioning an embedded tissue sample is the step necessary to produce sufficiently thin slices of sample that the detail of the microstructure of the cells/tissue can be clearly observed using microscopy techniques (either light microscopy or electron microscopy). Possible orientations at which tissue samples may be sectioned include:

Vertical sectioning perpendicular (i.e. at right-angles) to the surface of the tissue. This is the most common method. Horizontal sectioning is often done for the study of hair follicles and structures that include hairs, hair follicles, arrector pili muscles, and sebaceous glands in general. Such structures are sometimes called "pilosebaceous units". Tangential to horizontal sectioning is done in chemosurgery (also called "Mohs surgery") which is a form of microscopically controlled surgery used to treat certain types of skin cancer.

The method used to actually cut sections from the hardened block of tissue depends on the type of microscopy that will be used to observe it and hence the thickness of sample required. In the case of samples to be studied using light microscopy, a steel

knife mounted in a microtome may be used to cut 10m tissue sections which are then mounted on a glass microscope slide. In the case of samples to be studied using transmission electron microscopy, a diamond knife mounted in an ultramicrotome may be used to cut 50 nm tissue sections which are then mounted on a 3-millimeterdiameter copper grid.

Staining

Finally, the mounted sections are treated with an appropriate histology stain. Why are histology samples stained ? Put another way, what is the purpose of histology stains ? Biological tissue has very little variation in colours/shades when viewed using either an ordinary light (optical) microscope or an electron microscope. Staining biological tissues is done to both increase the contrast of the tissue and also highlight some specific features of interest - depending on the type of tissue and the stain used.

There are many different histology stains. Histology stains are normally selected according to the type of tissue to be observed. Some stains are more widely used than others while some are only used to study very specific types of biological tissue. To continue reading about this topic go on to the page about Histology Stains. For more information see also pages about What is Histology, Histology Stains, Histopathology and the Structure of a Cell. The report Differential Staining With Acid Dyes* may also be of interest. *Copyright Bryan D. Llewellyn. Made available for download from this website for educational purposes within conditions of use stipulating that the text is unchanged and no charge is made. Accessed by us from http://stainsfile.info/StainsFile/downloads.htm

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Histotechniques
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Tissue Processing
Tissues from the body taken for diagnosis of disease processes must be processed in the histology laboratory to produce microscopic slides that are viewed under the microscope by pathologists. The techniques for processing the tissues, whether biopsies, larger specimens removed at surgery, or tissues from autopsy, are described below. The persons who do the tissue processing and make the glass microscopic slides are histotechnologists.

Specimen Accessioning
Tissue specimens received in the surgical pathology laboratory have a request form that lists the patient information and history along with a description of the site of origin. The specimens are accessioned by giving them a number that will identify each specimen for each patient. Specimen accessioning.

Gross Examination
Tissues removed from the body for diagnosis arrive in the Pathology Department and are examined by a pathologist, pathology assistant, or pathology resident. Gross examination consists of describing the specimen and placing all or parts of it into a small plastic cassette which holds the tissue while it is being processed to a paraffin block. Initially, the cassettes are placed into a fixative. Gross specimen examination. Gross specimen examination. When a malignancy is suspected, then the specimen is often covered with ink in order to mark the margins of the specimen. Different colored inks can be used to identify different areas if needed. When sections are made and processed, the ink will mark the actual margin on the slide. Inking a gross specimen for margins.

Fixation - types of fixatives

The purpose of fixation is to preserve tissues permanently in as life-like a state as possible. Fixation should be carried out as soon as possible after removal of the tissues (in the case of surgical pathology) or soon after death (with autopsy) to prevent autolysis. There is no perfect fixative, though formaldehyde comes the closest. Therefore, a variety of fixatives are available for use, depending on the

type of tissue present and features to be demonstrated. There are five major groups of fixatives, classified according to mechanism of action:

Aldehydes Mercurials Alcohols Oxidizing agents Picrates

Aldehydes include formaldehyde (formalin) and glutaraldehyde. Tissue is fixed by cross-linkages formed in the proteins, particularly between lysine residues. This cross-linkage does not harm the structure of proteins greatly, so that antigenicity is not lost. Therefore, formaldehyde is good for immunoperoxidase techniques. Formalin penetrates tissue well, but is relatively slow. The standard solution is 10% neutral buffered formalin. A buffer prevents acidity that would promote autolysis and cause precipitation of formol-heme pigment in the tissues. Glutaraldehyde causes deformation of alpha-helix structure in proteins so is not good for immunoperoxidase staining. However, it fixes very quickly so is good for electron microscopy. It penetrates very poorly, but gives best overall cytoplasmic and nuclear detail. The standard solution is a 2% buffered glutaraldehyde Mercurials fix tissue by an unknown mechanism. They contain mercuric chloride and include such wellknown fixatives as B-5 and Zenker's. These fixatives penetrate relatively poorly and cause some tissue hardness, but are fast and give excellent nuclear detail. Their best application is for fixation of hematopoietic and reticuloendothelial tissues. Since they contain mercury, they must be disposed of

carefully. Alcohols, including methyl alcohol (methanol) and ethyl alcohol (ethanol), are protein denaturants and are not used routinely for tissues because they cause too much brittleness and hardness. However, they are very good for cytologic smears because they act quickly and give good nuclear detail. Spray cans of alcohol fixatives are marketed to physicians doing PAP smears, but cheap hairsprays do just as well. Oxidizing agents include permanganate fixatives (potassium permanganate), dichromate fixatives (potassium dichromate), and osmium tetroxide. They cross-link proteins, but cause extensive denaturation. Some of them have specialized applications, but are used very infrequently. Picrates include fixatives with picric acid. Foremost among these is Bouin's solution. It has an unknown mechanism of action. It does almost as well as mercurials with nuclear detail but does not cause as much hardness. Picric acid is an explosion hazard in dry form. As a solution, it stains everything it touches yellow, including skin.

Fixation - factors affecting fixation

There are a number of factors that will affect the fixation process:

Buffering Penetration Volume Temperature Concentration Time interval

Fixation is best carried out close to neutral pH, in the range of 6-8. Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to prevent excessive acidity. Acidity favors formation of formalin-heme pigment that appears as black, polarizable deposits in tissue. Common buffers include phosphate, bicarbonate, cacodylate, and veronal. Commercial formalin is buffered with phosphate at a pH of 7. Penetration of tissues depends upon the diffusability of each individual fixative, which is a constant. Formalin and alcohol penetrate the best, and glutaraldehyde the worst. Mercurials and others are somewhere in between. One way to get around this problem is sectioning the tissues thinly (2 to 3 mm). Penetration into a thin section will occur more rapidly than for a thick section. The volume of fixative is important. There should be a 10:1 ratio of fixative to tissue. Obviously, we often get away with less than this, but may not get ideal fixation. One way to partially solve the problem is to change the fixative at intervals to avoid exhaustion of the fixative. Agitation of the specimen in the fixative will also enhance fixation. Increasing the temperature, as with all chemical reactions, will increase the speed of fixation, as long as you don't cook the tissue. Hot formalin will fix tissues faster, and this is often the first step on an automated tissue processor. Concentration of fixative should be adjusted down to the lowest level possible, because you will expend less money for the fixative. Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to 4%. Too high a concentration may adversely affect the tissues and produce artefact similar to excessive heat. Also very important is time interval from of removal of tissues to fixation. The faster you can get the tissue and fix it, the better. Artefact will be introduced by drying, so if tissue is left out, please keep it

moist with saline. The longer you wait, the more cellular organelles will be lost and the more nuclear shrinkage and artefactual clumping will occur.

Fixatives - general usage

There are common usages for fixatives in the pathology laboratory based upon the nature of the fixatives, the type of tissue, and the histologic details to be demonstrated. Formalin is used for all routine surgical pathology and autopsy tissues when an H and E slide is to be produced. Formalin is the most forgiving of all fixatives when conditions are not ideal, and there is no tissue that it will harm significantly. Most clinicians and nurses can understand what formalin is and does and it smells bad enough that they are careful handling it. Zenker's fixatives are recommended for reticuloendothelial tissues including lymph nodes, spleen, thymus, and bone marrow. Zenker's fixes nuclei very well and gives good detail. However, the mercury deposits must be removed (dezenkerized) before staining or black deposits will result in the sections. Bouin's solution is sometimes recommended for fixation of testis, GI tract, and endocrine tissue. It does not do a bad job on hematopoietic tissues either, and doesn't require dezenkerizing before staining. Glutaraldehyde is recommended for fixation of tissues for electron microscopy. The glutaraldehyde must be cold and buffered and not more than 3 months old. The tissue must be as fresh as possible and preferably sectioned within the glutaraldehyde at a thickness no more than 1 mm to enhance fixation. Alcohols, specifically ethanol, are used primarily for cytologic smears. Ethanol (95%) is fast and cheap. Since smears are only a cell or so thick, there is no great problem from shrinkage, and since smears are

not sectioned, there is no problem from induced brittleness. For fixing frozen sections, you can use just about anything--though methanol and ethanol are the best.

Tissue Processing
Once the tissue has been fixed, it must be processed into a form in which it can be made into thin microscopic sections. The usual way this is done is with paraffin. Tissues embedded in paraffin, which is similar in density to tissue, can be sectioned at anywhere from 3 to 10 microns, usually 6-8 routinely. The technique of getting fixed tissue into paraffin is called tissue processing. The main steps in this process are dehydration and clearing. Wet fixed tissues (in aqueous solutions) cannot be directly infiltrated with paraffin. First, the water from the tissues must be removed by dehydration. This is usually done with a series of alcohols, say 70% to 95% to 100%. Sometimes the first step is a mixture of formalin and alcohol. Other dehydrants can be used, but have major disadvantages. Acetone is very fast, but a fire hazard, so is safe only for small, hand-processed sets of tissues. Dioxane can be used without clearing, but has toxic fumes. The next step is called "clearing" and consists of removal of the dehydrant with a substance that will be miscible with the embedding medium (paraffin). The commonest clearing agent is xylene. Toluene works well, and is more tolerant of small amounts of water left in the tissues, but is 3 times more expensive than xylene. Chloroform used to be used, but is a health hazard, and is slow. Methyl

salicylate is rarely used because it is expensive, but it smells nice (it is oil of wintergreen). There are newer clearing agents available for use. Many of them are based on limolene, a volatile oil found in citrus peels. Another uses long chain aliphatic hydrocarbons (Clearite). Although they represent less of a health hazard, they are less forgiving with poorly fixed, dehydrated, or sectioned tissues. Finally, the tissue is infiltrated with the embedding agent, almost always paraffin. Paraffins can be purchased that differ in melting point, for various hardnesses, depending upon the way the histotechnologist likes them and upon the climate (warm vs. cold). A product called paraplast contains added plasticizers that make the paraffin blocks easier for some technicians to cut. A vacuum can be applied inside the tissue processor to assist penetration of the embedding agent. The above processes are almost always automated for the large volumes of routine tissues processed. Automation consists of an instrument that moves the tissues around through the various agents on a preset time scale. The "technicon" tissue processor is one of the commonest and most reliable (a mechanical processor with an electric motor that drives gears and cams), though no longer made. Newer processors have computers, not cam wheels, to control them and have sealed reagent wells to which a vacuum and/or heat can be applied. Tissue processor. Automated tissue processor. Tissues that come off the tissue processor are still in the cassettes and must be manually put into the blocks by a technician who must pick the tissues out of the cassette and pour molten paraffin over them. This "embedding" process is very important, because the tissues must be aligned, or oriented, properly

in the block of paraffin. Tissue embedding. Tissue embedding. Alternatives to paraffin embedding include various plastics that allow thinner sections. Such plastics include methyl methacrylate, glycol methacrylate, araldite, and epon. Methyl methacrylate is very hard and therefore good for embedding undecalcified bone. Glycol methacrylate has the most widespread use since it is the easiest to work with. Araldite is about the same as methacrylate, but requires a more complex embedding process. Epon is routinely used for electron microscopy where very thin sections are required. Plastics require special reagents for deydration and clearing that are expensive. For this reason, and because few tissues are plastic embedded, the processing is usually done by hand. A special microtome is required for sectioning these blocks. Small blocks must be made, so the technique lends itself to small biopsies, such as bone marrow or liver.

Sectioning
Once the tissues have been embedded, they must be cut into sections that can be placed on a slide. This is done with a microtome. The microtome is nothing more than a knife with a mechanism for advancing a paraffin block standard distances across it. There are three important necessities for proper sectioning:

(1) a very sharp knife, (2) a very sharp knife, and (3) a very sharp knife. Sectioning with microtome. MPEG movie [672k] demonstrating sectioning technique with microtome. Knives are either of the standard thick metal variety or thin disposable variety (like a disposable razor blade). The former type allows custom sharpening to one's own satisfaction, but is expensive (more than $100 per blade). The latter cost about $1 per blade and are nearly as good. The advantage of the disposable blade becomes apparent when sectioning a block in which is hidden a metal wire or suture. Plastic blocks (methacrylate, araldite, or epon) are sectioned with glass or diamond knives. A glass knife can section down to about 1 micron. Thin sections for electron microscopy (1/4 micron) are best done with a diamond knife which is very expensive ($2500). Microtomes have a mechanism for advancing the block across the knife. Usually this distance can be set, for most paraffin embedded tissues at 6 to 8 microns. The more expensive the microtome ($15,000 to $30,000), the better and longer-lasting this mechanism will be. Sectioning tissues is a real art and takes much skill and practice. Histotechnologists are the artists of the laboratory. It is important to have a properly fixed and embedded block or much artefact can be introduced in the sectioning. Common artefacts include tearing, ripping, "venetian blinds", holes, folding, etc. Once sections are cut, they are floated on a warm water bath that helps remove wrinkles. Then they are picked up on a glass microscopic slide. Picking sections up from water bath. Unstained section on glass slide.

The glass slides are then placed in a warm oven for about 15 minutes to help the section adhere to the slide. If this heat might harm such things as antigens for immunostaining, then this step can be bypassed and glue-coated slides used instead to pick up the sections. Tray of unstained slides in drying oven.

Frozen Sections
At times during performance of surgical procedures, it is necessary to get a rapid diagnosis of a pathologic process. The surgeon may want to know if the margins of his resection for a malignant neoplasm are clear before closing, or an unexpected disease process may be found and require diagnosis to decide what to do next, or it may be necessary to determine if the appropriate tissue has been obtained for further workup of a disease process. This is accomplished through use of a frozen section. The piece(s) of tissue to be studied are snap frozen in a cold liquid or cold environment (-20 to -70 Celsius). Freezing makes the tissue solid enough to section with a microtome. Frozen sections are performed with an instrument called a cryostat. The cryostat is just a refrigerated box containing a microtome. The temperature inside the cryostat is about -20 to -30 Celsius. The tissue sections are cut and picked up on a glass slide. The sections are then ready for staining. Cutting a frozen section.

Staining
The embedding process must be reversed in order to get the paraffin wax out of the tissue and allow water soluble dyes to penetrate the sections. Therefore, before any staining can be done, the slides are "deparaffinized" by running them through xylenes (or substitutes) to alcohols to water. There are no stains that can be done on tissues containing paraffin. The staining process makes use of a variety of dyes that have been chosen for their ability to stain various cellular components of tissue. The routine stain is that of hematoxylin and eosion (H and E). Other stains are referred to as "special stains" because they are employed in specific situations according to the diagnostic need. Slides being stained on automated stainer. Slides being stained on automated stainer. Frozen sections are stained by hand, because this is faster for one or a few individual sections. The stain is a "progressive" stain in which the section is left in contact with the stain until the desired tint is achieved. Staining a frozen section.

H and E staining
Hematoxylin is the oxidized product of the logwood tree known as hematein. Since this tree is very rare nowadays, most hematein is of the synthetic variety. In order to use it as a stain it must be "ripened" or oxidized. This can be done naturally by putting the hematein solution on the shelf and waiting several

months, or by buying commercially ripened hematoxylin or by putting ripening agents in the hematein solution. Hematoxylin will not directly stain tissues, but needs a "mordant" or link to the tissues. This is provided by a metal cation such as iron, aluminum, or tungsten. The variety of hematoxylins available for use is based partially on choice of metal ion used. They vary in intensity or hue. Hematoxylin, being a basic dye, has an affinity for the nucleic acids of the cell nucleus. Hematoxylin stains are either "regressive" or "progressive". With a regressive stain, the slides are left in the solution for a set period of time and then taken back through a solution such as acid-alcohol that removes part of the stain. This method works best for large batches of slides to be stained and is more predictable on a day to day basis. With a progressive stain the slide is dipped in the hematoxylin until the desired intensity of staining is achieved, such as with a frozen section. This is simple for a single slide, but lends itself poorly to batch processing. Eosin is an acidic dye with an affinity for cytoplasmic components of the cell. There are a variety of eosins that can be synthesized for use, varying in their hue, but they all work about the same. Eosin is much more forgiving than hematoxylin and is less of a problem in the lab. About the only problem you will see is overstaining, especially with decalcified tissues.

Coverslipping
The stained section on the slide must be covered with a thin piece plastic or glass to protect the tissue

from being scratched, to provide better optical quality for viewing under the microscope, and to preserve the tissue section for years to come. The stained slide must go through the reverse process that it went through from paraffin section to water. The stained slide is taken through a series of alcohol solutions to remove the water, then through clearing agents to a point at which a permanent resinous substance beneath the glass coverslip, or a plastic film, can be placed over the section. Coverslipping. Coverslipping.

Decalcification
Some tissues contain calcium deposits which are extremely firm and which will not section properly with paraffin embedding owing to the difference in densities between calcium and parffin. Bone specimens are the most likely type here, but other tissues may contain calcified areas as well. This calcium must be removed prior to embedding to allow sectioning. A variety of agents or techniques have been used to decalcify tissue and none of them work perfectly. Mineral acids, organic acids, EDTA, and electrolysis have all been used. Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because they will remove large quantities of calcium at a rapid rate. Unfortunately, these strong acids also damage cellular morphology, so are not recommended for delicate tissues such as bone marrow. Organic acids such as acetic and formic acid are better suited to bone marrow, since they are not as

harsh. However, they act more slowly on dense cortical bone. Formic acid in a 10% concentration is the best all-around decalcifier. Some commercial solutions are available that combine formic acid with formalin to fix and decalcify tissues at the same time. EDTA can remove calcium and is not harsh (it is not an acid) but it penetrates tissue poorly and works slowly and is expensive in large amounts. Electrolysis has been tried in experimental situations where calcium had to be removed with the least tissue damage. It is slow and not suited for routine daily use.

Artefacts in Histologic Sections

A number of artefacts that appear in stained slides may result from improper fixation, from the type of fixative, from poor dehydration and paraffin infiltration, improper reagents, and poor microtome sectioning. The presence of a fine black precipitate on the slides, often with no relationship to the tissue (i.e., the precipitate appears adjacent to tissues or within interstices or vessels) suggests formalin-heme pigment has formed. This can be confirmed by polarized light microscopy, because this pigment will polarize a bright white (and the slide will look like many stars in the sky). Formalin-heme pigment is most often seen in very cellular or bloody tissues, or in autopsy tissues, because this pigment forms when the formalin buffer is exhausted and the tissue becomes acidic, promoting the formation of a complex of heme (from red blood cells) and formalin. Tissues such as spleen and lymph node are particularly prone

to this artefact. Making thin sections and using enough neutral-buffered formalin (10 to 1 ratio of fixative to tissue) will help. If the fixative solution in which the tissues are sitting is grossly murky brown to red, then place the tissues in new fixative. The presence of large irregular clumps of black precipitate on slides of tissues fixed in a mercurial fixative such as B-5 suggests that the tissues were not "dezenkerized" prior to staining. These black precipitates will also appear white with polarized light microscopy. Tissues that are insufficiently dehydrated prior to clearing and infiltration with paraffin wax will be hard to section on the microtome, with tearing artefacts and holes in the sections. Tissue processor cycles should allow sufficient time for dehydration, and final ethanol dehydrant solution should be at 100% concentration. In humid climates, this is difficult to achieve. Covering or sealing the solutions from ambient air will help. Air conditioning (with refrigerants, not with evaporative coolers) will also reduce humidity in the laboratory. Toluene as a clearing agent is more forgiving of poorly dehydrated tissues, but it is more expensive and presents more of a health hazard than other non-xylene clearing agents Though alcohols such as ethanol make excellent fixatives for cytologic smears, they tend to make tissue sections brittle, resulting in microtome sectioning artefacts with chattering and a "venetian blind" appearance. Bubbles under the coverslip may form when the mounting media is too thin, and as it dries air is sucked in under the coverslip. Contamination of clearing agents or coverslipping media may also produce a bubbled appearance under the microscope. Artefact with undezenkerized tissue.

Problems in Tissue Processing

"Floaters" are small pieces of tissue that appear on a slide that do not belong there--they have floated in during processing. Floaters may arise from sloppy procedure on the cutting bench-- dirty towels, instruments, or gloves can have tissue that is carried over to the next case. Therefore, it is essential that you do only one specimen at a time and clean thoroughly before opening the container of the next case. The best way to guard against unrecognized floaters is to always separate like specimens in the numbering sequence. For example, if you have three cases with prostate chips, separate them in accessioning with totally different specimens such as uterus or stomach. That way, if numbers are transposed or labels written wrong or tissue carried over, then you will have an obvious mismatch. Carrying over one prostate to another, or transposing the numbers of identical tissues may never be recognized. If reusable cassettes are employed, you must be aware that tissue may potentially be carried over and appear as "floaters" even several days later, when the cassette is re-used. The problem arises when, during embedding, not all the tissue is removed from the cassette. Then, in the cleaning process, not all of the wax is removed. Then, the next person using the cassette does not pay attention to the fact that there is tissue already in the cassette and puts his specimen in it. The floater that appears on the slide will look well-preserved--it should, because it was processed to paraffin. Always be sure that you properly identify the tissue! This means that you make sure that the patient label on the specimen container matches that of the request slip. An accession number is given to the specimen. This number must appear with the tissue at all times. You must never submit a cassette of tissue without a label. You must never submit a cassette of tissue with the wrong label. Mislabelling or unlabelling of tissues is courting disaster.

Safety in the Lab

The lab should be well-ventilated. There are regulations governing formalin and hydrocarbonds such as xylene and toluene. There are limits set by the Occupational Safety and Health Administration (OSHA) that should not be exceeded. These limits have recently been revised to reduced levels. Every chemical compound used in the laboratory should have a materials safety data sheet on file that specifies the nature, toxicity, and safety precautions to be taken when handling the compound. The laboratory must have a method for disposal of hazardous wastes. Health care facilities processing tissues often contract this to a waste management company. Tissues that are collected should be stored in formalin and may be disposed by incineration or by putting them through a "tissue grinder" attached to a large sink (similar to a large garbage disposal unit). Every instrument used in the laboratory should meet electrical safety specifications (be U.L. approved) and have written instructions regarding its use. Flammable materials may only be stored in approved rooms and only in storage cabinets that are designed for this purpose. Fire safety procedures are to be posted. Safety equipment including fire extinguishers, fire blankets, and fire alarms should be within easy access. A shower and eyewash should be readily available. Laboratory accidents must be documented and investigated with incident reports and industrial accident

reports. Specific hazards that you should know about include:

Bouin's solution is made with picric acid. This acid is only sold in the aqueous state. When it dries out, it becomes explosive. Many reagent kits have sodium azide as a preservative. You are supposed to flush solutions containing sodium azide down the drain with lots of water, or there is a tendency for the azide to form metal azides in the plumbing. These are also explosive. Benzidine, benzene, anthracene, and napthol containing compounds are carcinogens and should not be used. Mercury-containing solutions (Zenker's or B-5) should always be discarded into proper containers. Mercury, if poured down a drain, will form amalgams with the metal that build up and cannot be removed.

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Histology FAQ Staining, Histochemistry and Histotechnology (Frequently Asked Questions)

Dr. John A. Kiernan Department of Anatomy and Cell Biology

The University of Western Ontario London, Canada

FAQ Home > Sectioning, Slide Adhesive, Mounting

Sections coming off slides. Which adhesive? Question. Here is my problem: tissue sections not adhering to the slides. Any hints on solving this problem? Answers. [Textbooks of microtechnique contain recipes for various adhesives: chrome alum-gelatin, Mayer's albumen and starch paste are traditional. More recent methods include giving the glass surface a positive charge by coating with polylysine or reaction with 3-aminopropyltriethoxysilane (APES or TES) to make "silanized" slides. See also the FAQ item on how to prepare silanized slides. There is also an FAQ item about polylysine. Here are some hints from individuals. No. 3 is pertinent to the use of any adhesive or none at all.] 1. We ran into the problem of tissues falling off the slides after about 5 hours of immunohistochemical processing. We seemed to have solved it with Super Frost Plus slides that have some sort of charge on them. Fisher/VWR I think carry them. P. Emry (emry[AT]u.washington.edu) [Pre-prepared silanized slides are commercially available with a variety of trade names.]

2. We go to the expense of using charged slides for everything we do (Plus slides) and nothing really ever floats. If you don't want to go to that expense, we used to use chrom alum-gelatin with fairly good results and only an occasional problem. I personally don't like having chemicals in the waterbath. An exception would be immunos and some frozens for which I would recommend using "Plus" slides regardless. Xylene in paraffin as a cause. I had an interesting thing happen to me once. I worked Saturdays for a while, training a girl at another lab in Histotechnique. The first Saturday we cut, most sections floated to varying degrees, even things like tonsil. The tonsil cut very nicely and seemed well processed. Well, I was supposed to be the person in the know and I was stumped. Took me a while but I finally figured out that they didn't change the processors very often and there was lots of xylene in the paraffin, and I mean lots. Apparently this was the problem, because after changing everything and rotating on a regular basis the problem went away. I just thought I would throw that story in - because this experienced histotech didn't realize that excess xylene in the paraffin could cause problems with adhesion of sections. Marjorie A. Hagerty (mhagerty[AT]emc.org) 3. Here is another possible contribution to the section loss. After picking up the ribbon on the waterbath do you purposefully pull out the water from between the section and the slide? ....you know, using a lap cloth (or whatever absorbant material you keep around) to touch the edge of the paraffin ribbon and soak up the water from under the ribbon. If the edges of the ribbon adhere to the slide but water remains between the section and the slide, when drying occurs, it is possible that not ALL of the water has evaporated from that space. Obviously, if a little water still separates the specimen from the slide (no matter what adhesive material is present), then the less than complete specimen attachment may not be strong enough to make it through the (even gentle) turbulence of the staining process. This negative condition is most often seen when a ribbon is picked up and then the slide is immediately placed flat, horizontally, on th edge of the waterbath. It can also occur, though far less fequently, when the slide is immediately placed vertically against the waterbath or into a slide rack. The vertical positioning, however, does increase the draining of the water as long as the bottom of the ribbon has not fully attached to the slide creating a dam of sorts. Anyway, that's just one more variable for you to consider before perhaps investing in something which may offer no greater adhesive advantage than what you are currently using.

Nancy Klemme (nancy.klemme[AT]sakuraus.com) 4. Nancy is absolutely correct! Even with super adhesives or charged slides, you're liable to lose sections if the interface of the section and the microscope slide's glass is not water-free. This water is also a cause for "nuclear bubbling" artifact. Ken Urban Surgipath Medical Industries, Inc. Richmond Illinois (surgamy[AT]mc.net) 5. I bought 6 slide racks, the ones where slides stand on their ends, each holding 50 slides (Solmedia in the UK). These I keep for coating only. I've also got a couple of deep staining pots, again for coating only. I buy poly-L-lysine from Sigma or make my own gelatin-chrome-alum. Load the racks with slides, clean, I don't trust the manufacturers, wash thoroughly and coat with the coating of choice, dry and box. Couldn't be easier, I make enough in 2 days that will last months. Why be ripped off by the supply houses when for a few P.S./$ you can do it yourself. Ian Montgomery (I.Montgomery[AT]bio.gla.ac.uk) 6. Polylysine has free amine groups that form positively charged ions in water that's less alkaline than about pH 9. Slides are smeared with an aqueous solution of this basic amino acid polymer and then air-dried. This confers a positive charge to the slide's surface when immersed in water. Aminoacid anions(which Predominate in a section of a typical vertebrate animal tissue) are attracted to the polymer that covers the glass. It is a waste of money to buy poly-L-lysine rather than poly-D-lysine or poly-DL-lysine, because the stereochemical form of the amino acid does not affect its ionization. Buy the cheapest. Positively charged slides can also be made in the reaction of an aminoalkylsilane with glass, in the presence of traces of water. It is easy to produce hundreds of "silanized slides in an hour. Alternatively, you can buy the silanized slides, which amounts to paying someone else's employer to do this simple job. John A. Kiernan,

London, Canada (kiernan[AT]uwo.ca) 7. I was satisfied with poly-L-lysine until I tried Superfrost Plus slides. I went from occasionally losing tissue to never losing it...so I vote for Superfrost Plus. Mary Ross (ross.8[AT]osu.edu) Patricia Emry (emry[AT]u.washington.edu)

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Microtome
From Wikipedia, the free encyclopedia Jump to: navigation, search

A microtome used in microscopy. A microtome (from the Greek mikros, meaning "small", and temnein, meaning "to cut") is a sectioning instrument that allows for the cutting of extremely thin slices of material, known as sections. Microtomes are an important device in microscopy preparation, allowing for the preparation of samples for observation under transmitted light or electron radiation. Microtomes use steel, glass, or diamond blades depending upon the specimen being sliced and the desired thickness of the sections being cut. Steel blades are used to prepare sections of animal or plant tissues for light microscopy histology. Glass knives are used to slice sections for light microscopy and to slice very thin sections for electron microscopy. Industrial grade diamond knives are used to slice hard materials such as bone, teeth and plant matter for both light microscopy and for electron microscopy. Gem quality diamond knives are used for slicing thin sections for electron microscopy. Microtomy is a method for the preparation of thin sections for materials such as bones, minerals and teeth, and an alternative to electropolishing and ion milling. Microtome sections can be made thin enough to section a human hair across its breadth, with section thickness between 0.05 and 100 m.

Contents
[hide]

1 History 2 Applications 3 Microtome types o 3.1 Sledge microtome o 3.2 Rotary microtome o 3.3 Cryomicrotome o 3.4 Ultramicrotome o 3.5 Vibrating microtome o 3.6 Saw microtome o 3.7 Laser microtome 4 Microtome knives o 4.1 Knife design and cut types o 4.2 Sectioning 5 See also 6 References

[edit] History

A diagram of a microtome drawn by Cummings in 1770.[1] In the beginnings of light microscope development, sections from plants and animals were manually prepared using razor blades. It was found that to observe the structure of the specimen under observation it was important to make clean reproducible cuts on the order of 100 m, through which light can be transmitted. This allowed for the observation of samples using light microscopes in a transmission mode. One of the first devices for the preparation of such cuts was invented in 1770 by George Adams, Jr. (17501795) and further developed by Alexander Cummings.[2] The device was hand operated, and the sample held in a cylinder and sections created from the top of the sample using a hand crank.[1][3] In 1835, Andrew Prichard developed a table based model which allowed for the vibration to be isolated by affixing the device to the table, separating the operator from the knife.[4]

Occasionally, attribution for the invention of the microtome is given to the anatomist Wilhelm His, Sr. (1865),[5][6] In his Beschreibung eines Mikrotoms (German for Description of a Microtome), Wilhelm wrote: The apparatus has enabled a precision in work by which I can achieve sections that by hand I cannot possibly create. Namely it has enabled the possibility of achieving unbroken sections of objects in the course of research. Other sources further attribute the development to a Czech physiologist Jan Evangelista Purkyn. [7] Several sources describe the Purkyne model as the first in practical use.[8][9] The obscurities in the origins of the microtome are due to the fact that the first microtomes were simply cutting apparatuses, and the developmental phase of early devices is widely undocumented. At the end of the 1800s, the development of very thin and consistently thin samples by microtomy, together with the selective staining of important cell components or molecules allowed for the visualisation of microscope details. [10][11] Today, the majority of microtomes are a knife-block design with a changeable knife, a specimen holder and an advancement mechanism. In most devices the cutting of the sample begins by moving the sample over the knife, where the advancement mechanism automatically moves forward such that the next cut for a chosen thickness can be made. The section thickness is controlled by an adjustment mechanism, allowing for precise control.

[edit] Applications

Microtome (C. Reichert, Vienna, 19051915). The most common applications of microtomes are:

Traditional Histology Technique: tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 50 m (micrometers) thick. From there the tissue can be mounted on a microscope slide, stained with appropriate aqueous dye(s) after prior removal of the paraffin, and examined using a light microscope. Cryosectioning Technique: water-rich tissues are hardened by freezing and cut in the frozen state with a freezing microtome or microtome-cryostat; sections are stained and examined with a light microscope. This technique is much faster than traditional histology (5 minutes vs 16 hours) and is used in conjunction with medical procedures to achieve a quick diagnosis. Cryosections can also be used in immunohistochemistry as freezing tissue stops degradation of tissue faster than using a fixative and does not alter or mask its chemical composition as much. Electron Microscopy Technique: after embedding tissues in epoxy resin, a microtome equipped with a glass or gem grade diamond knife is used to cut very thin sections (typically 60 to 100 nanometers). Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with a transmission electron microscope. This instrument is often called an ultramicrotome. The ultramicrotome is also used with its glass knife or an industrial grade diamond knife to cut survey sections prior to thin sectioning. These survey sections are generally 0.5 to 1 micrometer thick and are mounted on a glass slide and stained to locate areas of interest under a light microscope prior to thin sectioning for the TEM. Thin sectioning for the TEM is often done with a gem quality diamond knife. Complementing traditional TEM techniques ultramicrotomes are increasingly found mounted inside an SEM chamber so the surface of the a block face can be imaged at and then removed with the microtome to uncover the next surface which is ready for imaging. This technique is called Serial Block-Face Scanning Electron Microscopy (SBFSEM). Botanical Microtomy Technique: hard materials like wood, bone and leather require a sledge microtome. These microtomes have heavier blades and cannot cut as thin as a regular microtome. Spectroscopy (especially FTIR or Infrared spectroscopy) Technique: thin polymer sections are needed in order that the infra-red beam will penetrate the sample under examination. It is normal to cut samples to between 20 and 100 micrometres in thickness. For more detailed analysis of much smaller areas in a thin section, FTIR microscopy can be used for sample inspection.

A recent development is the laser microtome, which cuts the target specimen with a femtosecond laser instead of a mechanical knife. This method is contact-free and does not require sample preparation techniques. The laser microtome has the ability to slice almost every tissue in its native state. Depending on the material being processed, slice thicknesses of 10 to 100 m are feasible.

[edit] Microtome types

[edit] Sledge microtome

A sled microtome. A sledge microtome is a device where the sample is placed into a fixed holder (shuttle), which then moves backwards and forwards across a knife. Modern sled microtomes have the sled placed upon a linear bearing, a design that allows for the microtome to readily cut many coarse sections.[12] By adjusting the angles between the sample and the microtome knife, the pressure applied to the sample during the cut can be reduced.[12] Typical applications for this design of microtome are of the preparation of large samples, such as those embedded in paraffin for biological preparations. Typical cut thickness achievable on a sled microtome is between 1 and 60 m.

[edit] Rotary microtome

A rotary microtome of older construction. This instrument is a common microtome design. This device operates with a staged rotary action such that the actual cutting is part of the rotary motion. In a rotary microtome, the knife is typically fixed in a horizontal position. [13] In the figure to the left, the principle of the cut is explained. Through the motion of the sample holder, the sample is cut by the knife position 1 to position 2), at which point the fresh section remains on the knife. At the highest point of the rotary motion, the sample holder is advanced by the same thickness as the section that is to be made, allowing for the next section to be made. The flywheel in many microtomes can be operated by hand. This has the advantage that a clean cut can be made, as the relatively large mass of the flywheel prevents the sample from being stopped during the sample cut. The flywheel in newer models is often integrated inside the microtome casing. The typical cut thickness for a rotary microtome is between 1 and 60 m. For hard materials, such as a sample embedded in a synthetic resin, this design of microtome can allow for good "Semi-thin" sections with a thickness of as low as 0.5 m.

[edit] Cryomicrotome

A cryomicrotome. For the cutting of frozen samples, many rotary microtomes can be adapted to cut in a liquid nitrogen chamber, in a so-called cryomicrotome setup. The reduced temperature allows for the hardness of the sample to be increased, such as by undergoing a glass transition, which allows for the preparation of semi-thin samples.[12] However the sample temperature and the knife temperature must be controlled in order to optimise the resultant sample thickness

[edit] Ultramicrotome

A ribbon of ultrathin sections prepared by room temperature ultramicrotomy, floating on water in the boat of a diamond knife used to cut the sections. The knife blade is the edge at the upper end of the trough of water. An ultramicrotome is a main tool of ultramicrotomy. It can allow for the preparation of extremely thin sections, with the device functioning in the same manner as a rotational microtome, but with very tight tolerances on the mechanical construction. As a result of the careful mechanical construction, the linear thermal expansion of the mounting is used to provide very fine control of the thickness. [12] These extremely thin cuts are important for use with transmission electron microscope (TEM) and Serial Block-Face Scanning Electron Microscopy (SBFSEM), and are sometimes also important for light-optical microscopy.[13] The typical thickness of these cuts is between 40 and 100 nm for transmission electron microscopy and often between 30 and 50 nm for SBFSEM. Thicker sections up to 500 nm thick are also taken for specialized TEM applications or for light microscopy survey sections to select an area for the final thin sections. Diamond knives (preferably) and glass knives are used with ultramicrotomes. To collect the sections they are floated on top of a liquid as they are cut and are carefully picked up onto grids suitable for TEM specimen viewing. The thickness of the section can be estimated by the thin-film interference colors of reflected light that are seen as a result of the extremely low sample thickness. [14]

[edit] Vibrating microtome

The vibrating microtome operates by cutting using a vibrating blade, allowing the resultant cut to be made with less pressure than would be required for a stationary blade. The vibrating microtome is usually used for difficult biological samples. [12] The cut thickness is usually around 30-500 m for live tissue and 10-500 m for fixed tissue.[citation needed]

[edit] Saw microtome

The saw microtome is especially for hard materials such as teeth or bones. The microtome of this type has a recessed rotating saw, which slices through the sample. The minimal cut thickness is approximately 30 m, and can be made for comparatively large samples.[12]

[edit] Laser microtome

See also: Laser microtome

A conceptual diagram of laser microtome operation. The laser microtome is an instrument for contact free slicing.[15] Prior preparation of the sample through embedding, freezing or chemical fixation is not required, thereby minimising the artifacts from preparation methods. Alternately this design of microtome can also be used for very hard materials, such as bones or teeth as well as some ceramics. Dependant upon the properties of the sample material, the thickness achievable is between 10 and 100 m. The device operates using a cutting action of an infra-red laser. As the laser emits a radiation in the near infra-red, in this wavelength regime the laser can interact with biological materials. Through a sharp focussing in the of the probe within the sample, a focal point of very high intensity, up to TW/cm2, can be achieved. Through the non-linear interaction the so-called optical penetration, which the focal region introduces a material separation in a process known as photodisruption. Through the application of very short laser pulse durations, on the order of femtoseconds (1 fs = 1015 s) a pulse of very small energy in the target region be deposited, allowing for precise control of the energy imparted into the sample,

limiting the interaction zone of the cut to under a micrometre. External to this zone the ultra-short beam application time introduces minimal to no thermal damage to the remainder of the sample. The laser radiation is directed onto a fast scanning mirror based optical system which allows for three dimensional positioning of the beam crossover, whilst allowing for beam traversal to the desired region of interest. The combination of high power with a high raster rate allows the scanner to cut large areas of sample in a short time. In the laser microtome the laser-microdissection of internal areas in tissues, cellular structures, and other types of small features is also possible.

[edit] Microtome knives

The design of a microtome knife is dependant upon the material and preparation of the samples, as well as the final sample requirements (e.g. cut thickness and quality).

[edit] Knife design and cut types

Profiles of microtome knives. Generally, knives are characterized by the profile of the knife blade, which falls under the categories of planar concave, wedge shaped or chisel shaped designs. Planar concave microtome knives are extremely sharp, but are also very delicate and are therefore only used with very soft samples. [13] The wedge profile knives are somewhat more stable and find use in moderately hard materials, such as in epoxy or cryogenic sample cutting. Finally, the chisel profile with its blunt edge, raises the stability of the knife, whilst requiring significantly more force to achieve the cut.

For ultramicrotomes, glass and diamond knives are required, the cut breadth of the blade is therefore on the order of a few millimetres and is therefore significantly smaller than for classical microtome knives. Glass knives are usually manufactured by the fracture of glass bars using special "knife-maker" fracturing devices. Glass knives may be used for initial sample preparations even where diamond knives may be used for final sectioning. Glass knives usually have small troughs, made with plastic tape, which are filled with water to allow the sample to float for later collection.[12] Diamond blades may be built into such an existing trough, allowing for the same collection method.

[edit] Sectioning
Prior to cutting by microtomy, biological materials are usually placed in a more rigid fixative, in a process known as embedding. This is achieved by the inflow of a liquid substance around the sample, such as paraffin (wax) or epoxy, which is placed in a mould and later hardened to produce a "block" which is readily cut. The declination is the angle of contact between the sample vertical and knife blade. If the knife blade is at right angles (declination=90) the cut is made directly using a pressure based mode, and the forces are therefore proportionally larger. If however the knife is tilted, the relative motion of the knife is increasingly parallel to sample motion, allowing for a slicing action. This behaviour is very important for large or hard samples The inclination of the knife is the angle between the knife face and the sample. For an optimal result, this angle must be chosen appropriately. The optimal angle depends upon the knife geometry, the cut speed and many other parameters. If the angle is adjusted to zero, the knife cut can often become erratic, and a new location of the knife must be used to smooth this out. If the angle is too large, the sample can crumple and the knife can induce periodic thickness variations in the cut. By further increasing the angle such that it is too large one can damage the knife blade itself.

[edit] See also

Wikimedia Commons has media related to: Microtome

Histology Microscope

[edit] References
1. ^ a b Hill, John (1770). The Construction of Timer, from its early growth; Explained by Microscope, and proven from Experiments, in a great Variety of Kinds.. London. pp. 511, Plate I. 2. ^ Quekett, John (1848). A Practical Treatise on the use of the Microscope. London: Hippolyte Bailliere. pp. 306, Chapter XII (Microtomes and Microtome Knives). 3. ^ Anonymous (1910). "An eighteenth century Microtome". Journal of the Royal Microscopical Society (Oxford, England: The Royal Microscopical Society): 779782. 4. ^ Gilbert Morgan Smith: The Development of Botanical Microtechnique. In: Transactions of the American Microscopical Society 34, Nr. 2. 1915, S. 71129, (PDF-Version of the article) 5. ^ "Wilhelm His". Encyclopdia Britannica. Retrieved 24. Mrz 2009. 6. ^ Loukas, Marios; Pamela Clarke, R. Shane Tubbs, Theodoros Kapos and Margit Trotz (2008). "The His family and their contributions to cardiology". International Journal of Cardiology (Ireland: Elsevier) 123 (2): 7578. doi:10.1016/j.ijcard.2006.12.070. ISSN 01675273. PMID 17433467. Retrieved 24 Mrz 2009. 7. ^ "Histology". msn Encarta. Retrieved 18 March 2009. 8. ^ Detlev Ganten: Handbuch der molekularen Medizin (Handbook of molecular medicine), Springer, ISBN 3540645527, (Google-Books) 9. ^ Werner Gerabek, Bernhard D. Haage, Gundolf Keil, Wolfgang Wegner (2005): Enzyklopdie Medizingeschichte (Encyclopaedia of medical history), Walter de Gruyter, ISBN 3110157144, (Google-Books) 10. ^ Ernst Mayr (2002). [Google-Books Die Entwicklung der biologischen Gedankenwelt. (The evolution of the biological thought )]. Springer. ISBN 3-54043-213-2. 11. ^ Werner Lin, Werner Linb, Jochen Fanghnel: Histologie: Zytologie, allgemeine Histologie, mikroskopische Anatomie. (Histology: Cytology, general Histology, microscopial anatomy) Walter de Gruyter, 1998, ISBN 3-11014-032-2 (Google-Books) 12. ^ a b c d e f g Gudrun Lang (2006). Histotechnik. Praxislehrbuch fr die Biomedizinische Analytik. (Histology : practical textbook for analytical biomedicine). Springer, Wien/New York. ISBN 3-211-33141-7. 13. ^ a b c Klaus Henkel: Das Schneiden mit dem Mikrotom. Mikrobiologische Vereinigung Mnchen e. V., 2006, accessed 15 February 2009

14. ^ Peachey Lee D. (1958). "Thin Sections: A study of section thickness and physical distortion produced during microtomy". J. Biophysic. & Biochem. Cytol. 4 (3): 233242. doi:10.1083/jcb.4.3.233. 15. ^ Holger Lubatschowski 2007: Laser Microtomy, WILEY-VCH Verlag GmbH, Biophotonics, S. 49-51, (PDF). View page ratings Rate this page What's this? Trustworthy Objective Complete Well-written I am highly knowledgeable about this topic (optional) Categories:

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Sledge microtome. 2. Rocking microtome. 3. Rotary microtome. 4. Sliding microtome. 5. Ultra microtome.