Microtome

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A microtome (from ancient Greek μικρός mikros "small" and τομή tomé "cutting, cutting") is a cutting device with which one can create very thin slices. It is used to produce microscopic specimens that are later to be x-rayed (e.g. biological tissue ). Typical application areas are mainly soft materials and materials , such as in medicine and biology ( Histotechnik ), as well as the analysis of plastics. Biological material is normally hardened by fixation before cutting and then made cuttable by "embedding", i.e. inclusion with a liquid substance ( paraffin , synthetic resin ) that later hardens. Different types of microtome (see below ) are available for creating the sections, depending on the area of ​​application . The thickness of the cuts is significantly less than the diameter of a human hair and is typically 0.1 to 100 µm. The use of a microtome is called a microtomy .

Alternative methods for the production of thin preparations are the production of thin sections for metals, rocks, minerals, bones and teeth, electropolishing for metals and ion thinning .

history

Microtome from Cummings 1770
Microtome (C. Reichert, Vienna, 1905–1915)
Rotary microtome of older design
Cut of a bird

To understand the structure of an object, one has to examine its interior. In the early days of light microscopy , hand cuts were made with razor blades, mostly from plants or parts of animals. In order to recognize the structures of an object very precisely, very thin, even cuts in the order of 10 to 100 µm are required, which can be examined in transmitted light. The devices for making sections were called cutting engines until 1839 , when Jacques Louis Vincent (1770–1841) and Charles Louis Chevalier (1804–1859) coined the term “microtome”.

Probably the first device for making such cuts was made around 1770 by George Adams, Jr. (1750–1795) invented and further developed by Alexander Cumming . It was a hand-held model in which the sample was held in a cylinder and the section thickness (height of the sample) was set with a screw. In 1835 Andrew Pritchard converted the cutter into a table model by attaching it to a table with a clamp and thus being able to operate the knife with both hands.

The first sled microtome was invented by George Adams in 1798 (?). The development of the rotary microtome, however, took place much later (1883 and 1886).

In order to be able to produce thin cuts, other aids such as the double-bladed knife with adjustable blade spacing were developed by Gabriel Gustav Valentin in 1838 . Due to the hardening technology of biological samples and mechanical problems (stability and resharpenability of the blades), this obvious solution, a double-bladed knife, did not lead to the desired success in freehand operation.

Some sources claim that the microtome was invented by the Czech physiologist Jan Evangelista Purkyně . It is reported several times that Purkinje was the first to use the microtome without giving any dates. (See also Jan Evangelista Purkyně # Scientific Research Areas ).

Purkinje himself writes, however, in an essay from 1842: “Attempts have been made several times to invent complex microtomes to achieve and multiply the finest sections.” He describes a “work about the microscope” by Chevalier in which Adams (1770) as inventor and Cumming can be named as the perfecter of the microtome, as well as "finally Custence's more recent times, of which little notice has probably come to Germany". Purkinje continues: “Here in Breslau, Dr. For some time Oschatz was very busy building and perfecting such instruments. Finally, the local skilled Mechanicus Rösselt made one based on his own idea. These instruments may be quite applicable for the rapid factory replication of suites, or even of equal sections for phytotomic preparations, when their use should be more extensive, they seem less suitable for actual research, because fixing the objects takes too much time, and would have to be repeated too often with an examination moving in all directions. "

Furthermore, the anatomist Wilhelm His (1865) is occasionally seen in the literature as the inventor of the microtome . In his description of a microtome from 1870, His writes: “The apparatus allowed me to work with precision that would never have been possible with one hand. He made it possible for me to obtain uninterrupted cutting sequences of the examined objects. ” At the same time, however, he also states that (in the literature) a number of devices for making microscopic sections have (already) been specified and that his device is an extension of a cross-cutter by Professor Hensen. The reason for being named as the inventor may be that Wilhelm His contributed significantly to the broad acceptance of the device with his work.

Together with the microtomes, the preparation technique - consisting of the fixation technique, embedding and staining of preparations - continued to develop. The selective staining of the specimen only leads to useful results if the sample thickness remains constant. This prevented differences in thickness from leading to greater color changes than differences in the sample structure. The creation of very thin and above all uniformly thick sections with a microtome, together with the selective staining of certain cell components or molecules, increased the visibility of microscopic details by at least an order of magnitude at the end of the 19th century.

In the 1870s, Richard Thoma (physician) developed a device for producing wafer-thin histological paraffin sections for microscopic examination. This slide microtome was mass-produced by Rudolf Jung in Heidelberg from 1881 and was used worldwide until the middle of the 20th century (Thoma microtome). Other manufacturers of microtomes were the companies C. Reichert, Vienna and E. Leitz , Wetzlar, whose respective business areas have now all merged into Leica Microsystems GmbH , Wetzlar.

A detailed treatise on the history of the microtome can be found in the review by Gilbert Morgan Smith . There are also numerous historical images of the early devices. Building on this, Krause gives a Eurocentric look at the history of the microtome.

Mechanical microtomes

Most microtomes consist of a knife block with an exchangeable knife, a specimen holder with a sample and a "feed mechanism". Depending on the type of device, the specimen or the knife is moved during cutting, the knife being pressed through the specimen and cutting off a wafer-thin layer due to the wedge effect (section extraction). After each cut, the feed mechanism ensures an automatic shift, the so-called infeed, so that a cut of the same thickness is created in the next cycle. The section thickness can be precisely regulated using a corresponding adjustment mechanism.

Different device types are distinguished depending on the structure. The most important types are described below. The specified section thicknesses are orientation values. The appropriate section thickness depends on the material of the sample, the examination objective and the pretreatment (fixation, embedding, histotechnology).

Sled microtome

Detail of the slide microtome: slide with knife (foreground); cut sample (background)
Sled microtome with fixed specimen and movable knife

In the case of a sled microtome, the specimen is usually firmly fixed on a block carrier, while the knife is moved back and forth on a mostly heavy “sled”. Nowadays the carriage is on a belt mounted on rollers. With many slide microtomes, the knife can be tilted to the cutting direction. This angle is called the declination . This orientation reduces the pressure when cutting compared to a knife positioned crosswise. Typical areas of application are large, soft samples, e.g. B. biological preparations embedded in paraffin. The typical section thickness of the sled microtome is 1 to 60 µm (possibly up to 300 µm).

Alternatively, a variant of the sled microtome is sometimes used, which is called the basic sled microtome . Here the knife is rigidly attached and the sample is pulled through on the slide track under the knife.

Rotary microtome

Rotary microtome with flywheel (right)

The instruments of this type are also known as minot microtomes. Although they are driven by a rotary movement, this is converted into a straight movement, so that the actual cutting movement (which is carried out here by the object) consists of a simple upward and downward movement. In a rotary microtome, the knife is typically arranged horizontally and stationary.

Principle of specimen movement when making a section on a rotary microtome

The basic principle of a cutting process is explained in the sketch below. The downward movement of the sample holder pushes the knife through the sample (position 1 to position 2). The thin section is then on the knife. After the cut has been made, the specimen holder is pulled back slightly so that the specimen does not drag along the knife during the upward movement that now follows. At the highest point of the movement, the sample is delivered, that is, the sample holder is now moved forward so that a thin section of the same section thickness is created during the next downward movement. The cut can either be removed individually from the knife or you wait until several successive cuts have lined up to form a cutting band and then remove them as a band (see picture on the right).

The flywheel can be turned by hand on many microtomes. It also has the advantage that a clean cut is made because the relatively large mass of the flywheel means that differences in the hardness of the specimen do not immediately lead to significant changes in speed in the cut. The rotating flywheel is also integrated in the housing on some newer models. The typical section thickness of the rotary microtome is 1 to 60 µm (possibly up to 300 µm). For hard materials (e.g. embedding in synthetic resins), semi-thin cuts with a thickness in the range of 0.5 µm are possible with good equipment.

Freezing microtome

Cryostat for histotechnology

For sectioning frozen samples, many rotary microtomes can be converted into a so-called freezing or cryomicrotome by adapting a chamber cooled with liquid nitrogen (the sample is practically in an open-top freezer during sectioning). The low temperature is used to increase the hardness of the specimen and thus make it capable of cutting. This mainly affects devices that are suitable for ultramicrotomy or for semi-thin incisions. When creating the sections, both the sample temperature and the knife temperature must be regulated and optimized for the sample material and the section thickness.

In addition, there are also cryostats in histotechnology that are optimized for quick tissue sections and in which the complete microtome is located within the cooling chamber. All work steps from quick freezing to cutting to mounting on a slide take place in the device.

Ultramicrotome

Cutting tape approx. 16 removed ultra-thin cuts (approx. 70 nm thick) on the water surface of a diamond knife
Ultramicrotome for cutting resin-embedded samples for light and electron microscopy

An ultramicrotome is used to produce extremely thin sections and works like a "normal" rotary microtome, but the mechanics are designed for a very fine feed. Instead of a mechanical feed, a feed through the controlled length expansion of the specimen holder by means of heating is also used here. Such extremely thin sections are mainly required for examinations with the transmission electron microscope , and more rarely for light-optical microscopes . The typical thickness of a cut is between 10 and 500  nm . Due to the small thickness of the cuts, it is difficult to remove them directly from the knife. Therefore, the cuts are usually cut on the surface of a liquid (e.g. water) and then fished off. The section thickness and uniformity can be estimated using interference colors.

Vibratome

With vibratoms, the cutting effect is generated by a vibrating blade (e.g. razor blade). The cut is made less by pressure than by moving the blade sideways. Vibratome is mainly used for untreated biological samples. Due to the lower mechanical load, there is no need to embed the sample. Due to the vibration, however, the sectional image is usually significantly worse than with the first-mentioned microtome types. The section thickness is over 30 µm.

Saw microtome

The saw microtome is particularly suitable for very hard material such as B. bones and teeth suitable. With microtomes of this type, a diamond-tipped inner diameter saw rotates, which grinds through the specimen at a defined distance. The minimum section thickness is over 30 µm and therefore only allows comparatively coarse cuts.

Laser microtome

The laser microtome is an instrument for non-contact cutting of samples. In addition to the conventional applications of microtomes, it is particularly suitable for cutting biological tissues in their native state (e.g. liver, kidney, skin, etc.). It is not necessary to prepare the samples by embedding, freezing or chemical fixing. This largely prevents the formation of artifacts . On the other hand, very hard materials such as bones and teeth or even ceramics can be 'cut'. Depending on the properties of the sample material, section thicknesses of 10 to 100 µm are currently  possible.

Principle of the laser microtome

In contrast to mechanically working microtomes, an ultrashort pulse laser is used here as a cutting tool. The laser emits radiation in the near infrared range . In this wavelength range , the laser can penetrate biological tissue, but also other materials, up to a certain depth without visible damage. A strong focus in the interior of the sample results in very high intensities of over one TW / cm² at the focus point . The resulting non-linear interactions lead to what is known as an optical breakthrough, which induces material separation that is limited to the focus. This process is also known as photodisruption. Due to the short pulse duration of a few femtoseconds (1 fs = 10 −15  s), only a very small amount of energy in the range of a few nanojoules is deposited in the sample per pulse . This limits the interaction zone to a diameter of less than one micrometer. Outside this zone, no thermal damage occurs due to the ultra-short interaction times.

The laser beam is deflected by a fast scanner mirror while a three-dimensional positioning unit simultaneously moves the sample back and forth. In combination with a high repetition rate, this procedure enables larger areas to be scanned within a short time.

In addition to the laser microtome, there is also the laser microdissection for cutting out areas within a tissue section, cell smear and the like. Ä. Or for sorting small particles.

Microtome knife

The type of microtome knife used depends on the material and the pretreatment of the sample, as well as the objective of the investigation (e.g. section thickness).

Knife types and sharpening types

Cross-sectional shape of microtome knives with different types of cuts

Relatively heavy steel knives or hard metal knives with thick backs and with different shapes (profile), which are generally identified with the letters A, B, C and D, are traditionally used. The microtome knives of type A and B are extremely sharp due to the plano-concave shape, but also very sensitive and therefore only suitable for very soft samples such as paraffin or foamed material. The wedge shape of the type C cut is significantly more stable and is therefore also used for somewhat harder materials such as synthetic resin or for frozen cuts. With the knife type with shape D, only one side of the knife is sharpened. The front grinding angle of approx. 45 ° increases the stability again, but also makes the knife much blunt. This knife shape is only used for harder materials.

Instead of this classic microtome knife z. B. often used disposable blades to save costs. Some of these are slightly more blunt than the classic microtome knives, but above all they are significantly thinner and therefore more flexible. In the case of harder samples, the knife can therefore vibrate and thus fluctuations in layer thickness in the cut. Disposable blades are therefore mainly used for softer materials.

Glass or diamond knives are required for ultramicrotomes. At a few millimeters, the cutting width of such knives is significantly smaller than that of classic microtome knives. Glass knives are made from glass rods a few millimeters thick by breaking them immediately before use. This creates an extremely smooth and sharp break edge on the narrow side of the glass broken into triangles. Glass knives are typically used for pre-cutting the specimen (trimming). They can be supplemented with a small trough that is filled with water, for example with adhesive tape. As with diamond knives, the individual cuts can then float on the surface of the water.

The sharpness and hardness of the knife are crucial for a good result. Blunted steel knives are ground with special grinding pastes that contain diamond particles. There are special grinding devices for this. Hand sanding on sanding belts and sticks is also possible, but requires a lot of experience.

Cutting angle: declination and inclination

Definition of the term declination in microtomy

The declination is the angle between the direction of the knife edge and the cutting direction (see illustration on the right). With many slide microtomes, it can be set between 90 ° and 160 °. If the knife is positioned transversely (declination = 90 °), the cut is only made by pushing the knife through the specimen. The forces acting on the knife are significantly greater than when the knife is oriented at an angle to the cutting direction (declination 120 ° to 160 °). In the latter case, a relative movement with a portion parallel to the knife edge facilitates the cut. This setting is used especially for large and hard materials. The advantage of the transverse variant is that, with suitable material, cutting bands (multiple cuts in a row) can be created.

Definition of the term inclination in microtomy

The inclination of the knife to the specimen plane is called the inclination. This angle must be selected appropriately for an optimal cutting result. It depends on the exact knife geometry, the specimen, the cutting speed and many other parameters. Typical are angles of inclination at which a small clearance angle of a few degrees remains between the preparation plane and the knife .

If this angle is set too shallow, the knife cuts unevenly, or areas of the lower part of the knife touch the freshly cut surface so that it is smeared.

If, on the other hand, the angle is chosen too large, the knife will “rumble” over the surface and there will be periodic thickness variations in the cut. With an even larger angle of inclination, the lateral load on the cutting edge is extremely high and the knife edge can break off.

Preparation and follow-up of the samples

Biological and other soft materials require extensive pretreatment in order to solidify them and thus make them cutable. The methods of this pretreatment such as fixing and embedding are part of the histotechnology . For embedding, the object is usually completely soaked in a liquid, which is then made to solidify. In this way, the preparation has a fairly uniform strength throughout. Typical embedding media are paraffin , polyethylene glycol , celloidin , gelatin , agar and synthetic resins .

For some investigations the solidification of the material to be cut is achieved by freezing, e.g. B. if embedding would lead to a change in the sample or prevent subsequent coloration. Samples containing water must be shock-frozen at a cooling rate of at least 10,000 K / s ( Kelvin per second) so that the water solidifies in the amorphous state. Otherwise ice crystals will form, which will lead to freezing damage in the material. The sections are then made on freezing microtomes, usually at −20 ° C.

After sectioning, the section must be transferred to a carrier for further processing (e.g. histochemical, immunohistochemical staining). For light microscopic preparations are slides used. Larger paraffin sections are first allowed to float on a surface of water (45 ° C) and smoothed by the surface tension . Then a slide is pushed under the cut at an angle below the surface of the water and then carefully moved upwards. One edge of the cut remains attached to the glass by adhesive forces and is drawn onto the slide. The same principle is also used for ultra-thin sections for electron microscopy , which are too thin and unstable for mechanical lift-off. Here the liquid trough is attached directly to the knife. The cuts form a cutting band (see picture of the ultramicrotome) and are then fished off with a fine metal grid .

application

In histology (tissue theory), the preparation of sections is a basic requirement for examining tissue features. Special freezing microtomes (cryostats) are used, among other things, for quick section diagnostics in order to obtain clarity about the completeness of the removal of a tumor during the operation. Based on the results, a decision will be made on how to proceed with the operation.

In addition, microtomes are used for material analyzes. Examples of this include the light microscopic or spectroscopic examination of layer systems (especially microscopic IR spectroscopy in transmission) or the polarization microscopic examination of spherulites . For transmission electron microscopy , very thin cuts are necessary in order to be able to irradiate them with electrons.

In the ophthalmology be within the scope of refractive surgery known as microkeratomes (a kind Callous plane), or more recently, the femtosecond laser used (also referred to as flap) to a 150 .mu.m thick corneal flap to be cut and thereby expose the underlying layers of the cornea for a Excimerlaseroperation. This device is sometimes called a microtome.

Individual evidence

  1. ^ A b John Hill: The Construction of Timer, from its early growth; Explained by Microscope, and proven from Experiments, in a great Variety of Kinds. 1770, pp. 5–11, Plate I.
  2. ↑ a b c Gretchen L. Humason: Animal tissue techniques . WH Freeman and Company, 1962, p. 43, Chapter 4 (Microtomes and Microtome Knives).
  3. ^ John Quekett: A Practical Treatise on the Use of the Microscope . Hippolyte Bailliere, London 1848, p. 306, Chapter XII (Microtomes and Microtome Knives).
  4. ↑ Anonymous: An eighteenth century microtome . (PDF) In: Journal of the Royal Microscopical Society , 1910, The Royal Microscopical Society, Oxford GB, pp. 779-782.
  5. ^ A b Gilbert Morgan Smith: The Development of Botanical Microtechnique. In: Transactions of the American Microscopical Society 34, No. 2. 1915, pp. 71-129.
  6. ↑ Peter Harting: The microscope. . F. Vieweg & Sohn, Braunschweig 1859, pp. 363–366, section 292.
  7. ↑ Erich Hintzsche : Requirements and development of the microtomy . In: Ciba magazine (Basel) . No. 8 , 1943, pp. 3082-3084 ( PDF ).
  8. ↑ Histology . In: Microsoft Encarta online, March 2009.
  9. ↑ Detlev Ganten: Handbook of molecular medicine . Springer, ISBN 3-540-64552-7 ( Google Books ).
  10. ^ Werner Gerabek, Bernhard D. Haage, Gundolf Keil, Wolfgang Wegner: Enzyklopädie Medizingeschichte . Walter de Gruyter, 2005, ISBN 3-11-015714-4 ( Google Books ).
  11. ↑ See also JE Purkinje: The microtomic squeezer, an indispensable instrument for microscopic examinations. In: Archives for Anatomy, Physiology and Scientific Medicine. 1834, pp. 385-390.
  12. ↑ J. Purkinje: microscope. Application and use in physiological studies . In: Rudolph Wagner (Hrsg.): Short dictionary of physiology with regard to physiological pathology . Second volume. Braunschweig 1844, p. 411-441 (Online version available from Google Books - (The quoted passage is on page 424).
  13. ^ Wilhelm His. In: Encyclopædia Britannica Online. Encyclopædia Britannica, accessed March 24, 2009 .
  14. ↑ Marios Loukas, Pamela Clarke, R. Shane Tubbs, Theodoros Kapos and Margit Sportwetten: The His family and their contributions to cardiology . In: Elsevier (Ed.): International Journal of Cardiology . 123, No. 2, Ireland, 2008, pp. 75-78. doi : 10.1016 / j.ijcard.2006.12.070 .
  15. ^ Wilhelm His: Description of a microtome . (PDF) In: Archive for microscopic anatomy , 6, 1870. Verlag Max Cohen & Sohn, Bonn, pp. 229–232, plate III, doi: 10.1007 / BF02955980 .
  16. ↑ Ole Daniel Enersen: Wilhelm His .
  17. ↑ Ernst Mayr: The development of the biological world of thought. Springer, 2002, ISBN 3-540-43213-2 ( Google Books ).
  18. ↑ Werner Linß, Werner Linb, Jochen Fanghänel: Histology: cytology, general histology, microscopic anatomy. Walter de Gruyter, 1998, ISBN 3-11-014032-2 ( Google Books ).
  19. ^ Richard Thoma, JF Lyon: About a microtome . In: Virchow's Arch. 84, 1881, pp. 189-191.
  20. ↑ Klaus Goerttler (Ed.): Biographical lexicon for the portrait collection of the anatomist Robert Wiedersheim .
  21. ↑ a b c d e f g h i j k l m Klaus Henkel: Cutting with the microtome . Microbiological Association Munich e. V., 2006, accessed Feb. 15, 2009.
  22. ↑ Rudolf Krause: Encyclopedia of microscopic technology . Urban & Schwarzenberg, Berlin 1926 (3rd edition), pp. 1528–1548, Volume II.
  23. ↑ a b c d e f g h i j Gudrun Lang: Histotechnik. Practical textbook for biomedical analysis. Springer, Vienna / New York 2006, ISBN 3-211-33141-7 ( Google Books ).
  24. ↑ Stephen Peters: Frozen section technique . Pathology Innovations (Freeze Section Technique Guide and Videos).
  25. ↑ Holger Lubatschowski 2007: Laser Microtomy . ( Memento of October 9, 2011 in the Internet Archive ) (PDF; 170 kB) WILEY-VCH, Biophotonics, pp. 49–51.
  26. ↑ a b Irene K. Lichtscheidl (Ed.): Light microscopy - theory and application . In: Light microscopy online - theory and application. University of Vienna, accessed on February 15, 2009 (PDF; 897 kB).
  27. ↑ A. Turzynski (Ed.): Schnellschnittdiagnostik . Pathology Lübeck, February 15, 2009.
  28. ↑ T. Kohnen (Ed.): Mikrokeratome . accessed on Feb. 15, 2009 (Schematic description of the work with the microkeratome).
  29. ↑ Internet Media Services, Inc. (Ed.): Understanding LASIK . accessed on Feb. 15, 2009 (description of laser in situ keratomileusis (LASIK), incl. video of an operation, English).
  30. ^ Steven H. Schwartz: Geometrical and Visual Optics. McGraw-Hill, 2002, ISBN 0-07-137415-9 ( Google Books ).

Web links

Commons : Microtome  - album with pictures, videos and audio files
This article was added to the list of excellent articles on March 22, 2009 in this version .