Histological technique

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Histological technique (from Gr. Histos "tissue") or histotechnology for short is a collective term in histology for the process of tissue preparation for microscopic examination. The development of these processes played an important role in medical research and science from the end of the 19th to the middle of the 20th century, especially in anatomy and pathology , but also in microbiology . She was accompanied by the improvement of the analog light microscope .

Section for light microscopy.
1 glass slide (76 × 26 mm), 2 cover slip,
3 stained organ section

history

“The vignette shows all the household items of the scientific trifle dealer or microscopist; on the right a simple microscope for preparing small objects, on the left a composite instrument from Amici , next to it tweezers, loupes, knives, needles and the like. ”(Schleiden, 1848).

At the beginning of microscopic nature research, small objects such as frog eggs or razor cuts of objects were examined. Hooke coined the term "cell" in 1665 after examining cork cuts. Other pioneers were Malpighi (1628–1694), Huygens (1629–1695), van Leeuwenhoek (1632–1723). The optics with a small aperture used at the time produced high-contrast images, but with a low resolution. Even Schleiden (1804-1881) and Schwann (1810-1882), the founder of the cell theory, worked with unstained specimens. The latter discovered the cell nucleus .

Towards the end of the 19th century, attempts began to depict cell and tissue structures using staining - with a high point in the first third of the 20th century. The end of this development in histological technology was brought about by the introduction of electron microscopy into cell and tissue research from the middle of the 20th century. Around this time, specific detection methods such as histochemistry, immunohistochemistry or ultraviolet microspectrophotometry were also developed for light microscopy.

Preparation methodology for light microscopy

Thin, translucent sections of an organ sample are a prerequisite for the light microscopic examination in transmitted light. There are only a few cases where incisions can be dispensed with: blood smears , mucous membrane swabs, cell cultures , crushed specimens . For the most common brightfield method, it is also necessary to stain the specimens because the tissue structures hardly have any inherent color, with a few exceptions. The most commonly used processing method for medical samples is paraffin embedding after formaldehyde fixation. In addition, there are many special methods of fixation and embedding that can be selected depending on the examination material, the desired section thickness and the examination objective.

Fixation

The fixation stops the life processes of cells and tissues by denaturing the proteins . The protein precipitating agents used for this prevent structural changes due to autolysis, substance losses during further treatment and post-mortem structural shifts. Suitable fixatives are various aldehydes (e.g. formaldehyde), heavy metal compounds (e.g. sublimate ), organic acids (e.g. acetic acid , picric acid ) and alcohols ( ethanol , methanol ). They denature the proteins through chemical bonds or the unfolding of amino acid chains . This stabilizes structures and switches off enzymatic activities. For rapid saturation and thus preservation that is as realistic as possible, the edge length of the tissue sample should not significantly exceed 1 cm. Another possibility of fixation that is as realistic as possible is perfusion fixation, that is, flushing with fixative solution through the blood vessels. The chemicals used for fixing are then washed out by washing.

The paraffin embedding

Radiographic specimens are required for microscopic examination. The hardening of the tissue through the fixation is not sufficient for the production of sufficiently thin incisions. It is necessary to embed them in cuttable substances: mostly paraffin, for some purposes plastics. The abundant water in the tissues is replaced by the embedding medium. However, since water does not mix with molten paraffin, the exchange must take place via intermediate media. Procedure in detail:

  • Cutting / macroscopic assessment: Relevant areas are cut to a processable size from larger samples (e.g. 20 × 20 × 3 mm).
  • Dehydration: The tissue is gradually soaked with alcohol in increasing concentrations (increasing alcohol series).
  • Intermediate: After dehydration, the tissue must be soaked with a reagent that mixes equally with both alcohol and paraffin . Xylene is mostly used for this.
  • Embedding / infiltration: the tissue is soaked in heated, liquefied paraffin. Spaces previously filled with water are now occupied by the embedding medium.
  • Pouring / blocking: the histotechnician creates small paraffin blocks that contain the tissue. To do this, the initially hot paraffin is allowed to cool and solidify. This gives it the strength it needs to be able to make thin cuts on special equipment.

Other embedding media such as plastic polymers , gelatine , agar or nitrocellulose require different pre- and post-treatment. Tissue embedded in plastic can be used to produce thin and semi-thin sections for light microscopy and ultra-thin sections for electron microscopy .

To cut

The (paraffin) sections, which are usually less than 10 µm thick, are made with a microtome . The sections are drawn onto glass slides.

Freeze cutting, quick cuts

For some purposes, e.g. B. rapid diagnoses during operations, histochemical examinations or preservation of fats, embedding is not applicable (fats are dissolved out by the organic solvents with paraffin embedding). In these cases you can freeze the tissue and then make so-called freeze or quick cuts. If the purpose of the examination permits, prior fixation is recommended. The quality of frozen sections generally does not match that of paraffin sections.

To dye

Hematoxylin-eosin stain: striated muscle cells
Modified azan staining: collagenous connective tissue blue. Epididymis
Masson-Goldner staining: collagen, even fine fibrils are visible in green
Cresyl violet staining:
small blood vessels, plastic incision.
Arteriole (1), venules (2) with confluent capillary (3)

In order to achieve the necessary contrast for an assessment under the microscope, the sections are treated with various dyes. The tissue sample is only occasionally colored in the piece before cutting. Since most color solutions are watery, the paraffin is removed from the section specimens with xylene, followed by alcohol and water.

When it comes to dyes, a distinction is made between basic (color “basophilic” structures) and acidic dyes (color “acidophilic” structures). The physico-chemical mechanism of very few histological staining methods is known. Electrostatic bonds often play a role. Basic, d. H. cationic, positively charged dye groups combine with acidic, anionic, negatively charged groups, e.g. B. the nucleic acids (phosphate groups). Acid, d. H. Anionic, negatively charged dyes are mainly bound to basic, cationic or positive charge groups (amino groups) of the proteins.

There are a variety of dyeing recipes. The combination of a basic and an acidic dye has proven useful for overview displays. In the medical-histological laboratory, hematoxylin , a vegetable dye obtained from the logwood tree ( Haematoxylum campechianum ), is routinely used as the basic dye ; the synthetic acidic dye eosin serves as a counterstain . This routine staining, known as hematoxylin-eosin staining (HE staining), gives a sharp blue-black representation of the basophilic cell nuclei due to the DNA content and a red color of the cytoplasm due to basic proteins.

If three dyes are used, one speaks of trichrome staining . The most common for the representation of connective tissue structures are the azan staining ( Mallory trichrome staining ) ( collagen and mucus blue, cellular structures in red tones), the technically simpler Masson-Goldner trichrome staining (collagen and mucus green, cell nuclei brownish-black, cytoplasmic Structures in shades of red) and the Van Gieson staining (collagen red, cell nuclei black-brown, cytoplasm yellow).

Metachromasia (Greek μετά: after (expresses a change) and χρώμα: color) is understood to mean that some structures take on a different color than the color solution. Only certain dyes produce metachromatism: for example toluidine blue ; while the solution is blue, mast cell granules and mucus are stained red.

With fat-soluble dyes, the coloring of succeeds lipids ( "fat staining"); provided that they were obtained in tissue preparation, e.g. B. by freeze cutting.

Certain structures can be represented by impregnation with silver salts: "Silver impregnation", e.g. B. argyrophilic connective tissue fibers, neurofibrils .

Fluorochroming (fluorescence staining, see fluorescence microscopy ). The secondary fluorescence generated by treatment with fluorescent dyes - fluorochromes (e.g. acridine orange ) - is distinguished from the autofluorescence of certain tissue components . Fluorochrome plays an important role in histochemistry. An example of the application of fluorochrome is the detection of sex chromatin .

Special techniques such as histochemistry, immunohistochemistry or in-situ hybridization ( histological staining ) offer further options for processing the sections .

The dye or reaction solutions are mostly aqueous, so the preparations are dehydrated again using alcohol, xylene or isoparaffins to produce an optically homogeneous permanent preparation and "covered" with a cover slip using a synthetic mounting medium on the slide. Provided that there are no acid residues from the staining in the tissue section, preparations made in this way can be kept for several decades. With the alternative use of an adhesive film coated with a suitable medium, the durability and optical quality are less good.

Blood smears are usually stained according to Pappenheim , mucous membrane smears according to Papanicolaou . These methods use multiple dyes to show cytological details.

In the case of plastic sections, the colorability is usually limited to one dye. However, due to the small thickness (0.5–2 μm), the microscopic image provides good resolution.

Injection preparations

To visualize blood vessels, especially the capillary network, injection preparations can be made with Indian ink or colored gelatine. These solutions are injected into the blood vessel system before the tissue is fixed. After fixation and paraffin embedding, slightly thicker sections are made and the course of the vessels can be followed in the microscope.

Preparation methodology for transmission electron microscopy

In a transmission electron microscope , electrons shine through the object. The tissue samples are prepared for transmission electron microscopy in basically the same way as for light microscopy.

Fixation

The resolving power of the electron microscope, which extends right down to the molecular structure, makes optimal structure maintenance necessary. The edge length of the samples should be less than 3 mm; if possible, perfusion fixation (see above) should be carried out. Solutions of glutaraldehyde and osmium tetroxide have proven themselves as fixatives .

Embedding

After the samples have been dehydrated in alcohol or acetone , they are embedded in plastics, e.g. B. an epoxy resin .

Cutting (ultramicrotomy)

The plastic blocks made from the tissue samples are cut with an ultramicrotome . Glass or diamond knives are used. Section thicknesses between 50 and 100 nm are required. Small copper meshes serve as microscope slides, which dissipate the electrical charge from the electron beam.

Contrast

To increase the contrast between the cell and tissue structures, the thin sections are usually treated with uranium and lead compounds . This contrasting corresponds to the coloring for light microscopy.

Contact radiography

Contact radiography is a technique for examining bone tissue. An X-ray image of a thin section specimen is produced and analyzed. It is used, for example, to study the effect of bone implants on bone tissue.

literature

  • Gudrun Lang: Histotechnology . 2006, Springer-Wien-New York, ISBN 3-211-33141-7
  • Benno Romeis: Microscopic technique , 16th edition 1968, Verlag R. Oldenbourg, Munich-Vienna.
  • Leopold Voss: Histotechnology of the leprous skin , 1910, Hamburg

Individual evidence

  1. Matthias Jacob Schleiden: The plant and its life . Engelmann, Leipzig 1848
  2. T. Casperson: About the chemical structure of the structures of the cell nucleus . Scand. Archive f. Physiol. 73 , Suppl. 8, pp. 1-151, 1936, and references therein. after Romeis
  3. ^ Benno Romeis: Microscopic technique , 16th edition, Verlag R. Oldenbourg Munich-Vienna 1968
  4. ^ Benno Romeis: Microscopic technique , 16th edition, Verlag R. Oldenbourg, Munich-Vienna 1968
  5. Martin Heidenhain : About Mallory's connective tissue staining with carmine and azo carmine as preliminary colors. Journal of Scientific Microscopy and Microscopic Technology, Volume 32, 1915, pp. 361-372. quoted n. B. Romeis
  6. a b c Gudrun Lang (Hrsg.): Histotechnik - practical textbook for biomedical analysis . 2nd, revised and updated edition. Springer, Vienna / New York 2012, ISBN 978-3-7091-1189-5 .