Microtome
A microtome (from the Greek μικρός, micros, " small" and τέμνειν, temnein " cut") is a cutting tool, with which you can create very thin slice preparations. It is used to produce microscopic preparations, which are to be irradiated later ( for example, biological tissue). Typical areas of application are especially soft materials and materials, such as in medicine and biology ( Histology ), as well as the analysis of plastics. Biological material is typically hardened by fixation before cutting and then by " embedding ", ie inclusion with a liquid substance ( paraffin, synthetic resin ), which hardens later made cuttable. For the creation of the cuts are different depending on the application Mikrotomarten (see below ) are available. The thickness of the sections is significantly less than the diameter of a human hair and is typically 0.1 to 100 microns. The use of a microtome, is referred to as microtomy.
Alternative method for the preparation of thin specimens are the production of thin sections for metals, minerals, rocks, bones and teeth, the electropolishing of metals and ion thinning.
- 3.1 kinds of knives and cut types
- 3.2 Cutting angle: declination and inclination
History
In order to understand the structure of an object, one must examine its interior. Were in the early days of light microscopy to -hand cutting with razor blades, usually created by plants or parts of animals. To detect the structures of an object very accurately, very thin, uniform sections in the order of 10 to 100 micrometers are required, which can be examined in transmitted light. The equipment for manufacture of cuts were to 1839 cutting equipment (cutting engine) called up Jacques Louis Vincent ( 1770-1841 ) and Charles Louis Chevalier ( 1804-1859 ) coined the term " microtome ".
Probably the first device for making such cuts was around 1770 by George Adams, Jr. ( 1750-1795 ) invented and developed by Alexander Cumming. It was a hand model, wherein the sample held in a cylinder and the average thickness ( height of the specimen ) was adjusted with a screw. 1835 built Andrew Pritchard the interface device in a table model in order, by attaching it with a clamp at a table, so you could use the knife with both hands.
The first sliding microtome in 1798 (?) Invented by George Adams. The development of the rotary microtomes was, however, until much later (1883 or 1886).
In order to produce thin sections, other tools such as the 1838 double bladed knife with adjustable blade clearance by Gabriel Gustav Valentin were developed. Due to the existing only beginnings in curing technique of biological samples and mechanical problems ( stability and Nachschärfbarkeit the blade ) resulted in this obvious solution, a double-bladed knife, hands-free operation are not successful.
Frequently in the literature as the inventor of the microtome, the anatomist Wilhelm His is viewed (1865 ). In his description of a microtome from 1870 His writes: " The apparatus has allowed me a precision, the work which would never have been possible at the incision with one hand. For he has made it possible for me to gain continuous cut sequences of the investigated objects. " But the same time it also indicates that ( in the literature ) for the preparation of microscopic sections (already ) had been given a number of devices and their device an extension represents a Queer Reaper by Professor Hensen. The reason for naming as inventors may be that Wilhelm His decisively contributed his work to a wide acceptance of the device.
Other sources describe that the microtome, a device for cutting thin proportions of tissues, was invented by the Czech physiologist January Evangelista Purkyně. Multiple is also reported without giving dates, the Purkinje used the microtome first. The lack of clarity regarding the invention of the microtome could on the one hand related to the fact that the first microtomes only as cutting equipment (cutting engine) rather than as microtomes or were the first devices were not considered that, depending on the development stage.
Together with the microtome to the preparation technique developed - consisting of fixation technique, embedding and staining of preparations - always on. The selective staining of the preparation, however, only leads to useful results when the sample thickness remains constant. It prevented so that differences in thickness to larger color changes result as differences in sample structure. The preparation of very thin and evenly mainly thick cut with a microtome preparations had therefore increased the late 19th century by at least an order of magnitude together with the selective staining of certain cell components or molecules, the visibility of microscopic details.
In the 1870s, Richard Thoma (Medic ) developed an apparatus for producing ultra-thin histological paraffin sections for microscopic examination. This sliding microtome was from 1881 by Rudolf Jung series produced in Heidelberg and used until the mid 20th century world ( Thoma microtome ). Other manufacturers of microtomes were the company C. Reichert, Vienna and E. Leitz, Wetzlar, their respective business areas are now all gone up in the Leica Microsystems GmbH, Wetzlar.
A detailed treatise on the history of the microtome can be found in the review article by Gilbert Morgan Smith. There are also many historical figures of the early devices. Based on this Krause is a Euro -centric view of the history of the microtome.
Mechanical microtomes
Most microtomes consist of a knife block with replaceable knife, a specimen holder with sample and a " feed " mechanism. Depending on the device type when cutting moves the sample or the knife, the blade is forced through the specimen and by the wedge effect of a very thin layer cuts ( average production). After each cut, the feed mechanism for an automatic shift, the so-called service provides, so that the next cycle, a section of the same thickness is formed. The section thickness can be adjusted accurately over a corresponding adjustment.
Depending on the structure of different device types can be distinguished. The main types are described below. The average thicknesses specified are guide values represents the sensible section thickness depends on the material of the sample, the aim of analysis and of the pre-treatment ( fixation, embedding, Histology ).
Sliding microtome
When sliding microtome, the specimen is usually firmly fixed on a support block, while the knife is moved to a mostly heavy " slide " back and forth. The slide is now on a mounted on rollers band. The blade can be tilted in many slide microtomes to the cutting direction. This angle is called declination. This orientation is reduced in comparison to a transversely positioned knives pressure during cutting. Typical applications are large, soft samples, such as paraffin-embedded biological specimens. The typical average thickness of the sliding microtome is 1 to 60 microns (possibly up to 300 microns ).
Alternatively can be found partly a variant of the sliding microtome in use, referred to as Grundschlittenmikrotom. Here, the diameter of which is rigidly mounted, and the sample is pulled to the slide rail under the blade.
Rotary microtome
The instruments of this type are also referred to as Minotmikrotom. Although they are driven by a rotational movement, but this is converted into a rectilinear movement, so that the actual cutting movement ( which is here performed by the object) is in a simple up and down movement. When rotary microtome, the knife is typically placed horizontally and fixed.
In the sketch below the basic principle of a cutting operation is explained. By the downward movement of the sample holder, the knife is pushed through the sample ( position 1 to position 2). The thin section is then on the knife. After the cut, the specimen holder is withdrawn slightly so that the sample does not rub on the now following upward movement of the knife along. At the highest point of the movement, the delivery of the sample is carried out, that is, the sample holder is then moved so far to the front, that in the next downward movement of a thin section of the same average thickness. The cut can either be individually removed from the knife or you wait until you have several successive cuts have strung together to form a cut band together and take this as a band from (see image right).
The flywheel can be rotated in many microtomes by hand. It also has the advantage that a clean cut is made, as lead by the relatively large mass of the flywheel differences in the hardness of the sample immediately to significant changes in the speed section. The rotating flywheel is applied to some newer models even in the case. The typical average thickness of the rotary microtome is 1 to 60 microns (possibly up to 300 microns ). For hard materials (eg embedding in resins ) are in good devices Semi -thin sections with a thickness in the range of 0.5 microns is possible.
Freezing microtome
For cutting of frozen samples, many rotary microtomes can by adapting a liquid nitrogen cooled chamber ( the sample is during cutting practice in an open-top freezer) in a so-called freezing or cryomicrotome be rebuilt. The low temperature is used in order to increase the hardness of the sample and thus to make sliceable. This concerns mainly devices which are suitable for the ultra-microtomy or for semi-thin sections. When making your cuts both the sample temperature and the knife temperature must be controlled and optimized for the sample material and the section thickness.
But there are in the Histology and cryostats, which are optimized for rapid tissue sections and the complete microtome is within the cooling chamber. All steps from the rapid freezing, about the cutting up to be mounted on a microscope slide held in the unit.
Ultramicrotome
An ultramicrotome is used to produce extremely thin sections and works like a "normal" rotary microtome, but the mechanism is constructively aligned with a very fine feed. Instead of a mechanical back here is a feed through the controlled linear expansion of the preparations holder in use by heating. These extremely thin cuts are needed primarily for use with the transmission electron microscope, rare even for light-optical microscopes. The typical thickness of a section is between 10 and 500 nm due to the low thickness of the sections is a direct removal from the knife difficult. Therefore, the sections usually on the surface of a liquid (eg water) and then cut fished. The section thickness and uniformity can be estimated via interference colors.
Vibratome
Vibratomen at the cutting action is produced by a vibrating blade (e.g. razor blade ). The cut is less pressure than by the lateral movement of the blade. Vibratome it uses primarily for untreated biological samples. Due to the lower mechanical load can be dispensed to the embedding of the sample. Through the vibration, however, the average image is usually much worse than the former Mikrotomtypen. The average thickness is about 30 microns.
Saw microtome
The Sägemikrotomen is particularly suitable for very hard material such as bone and teeth. In this type microtomes rotates a diamond studded annular saw that grinds at a defined distance through the specimen. The minimum average thickness is about 30 microns and thus allows only relatively coarse cuts.
Laser microtome
The laser microtome is an instrument for non-contact cutting of samples. It is in addition to the conventional applications of microtomes, particularly for cutting of biological tissues in their native state (eg liver, kidney, skin, etc.). A preparation of the sample by embedding, freezing or chemical fixing is not required. Characterized the formation of artefacts is substantially avoided. On the other hand, can be ' cut ' through very hard materials such as bones and teeth, or even ceramics. Depending on the properties of the sample material currently sectional thicknesses of 10 to 100 microns is possible.
In contrast to mechanically operating microtomes serves an ultra -short pulse laser as a cutting tool. The laser emits radiation in the near infrared range. In this wavelength range, the laser biological tissues, but also other materials, which penetrate up to a certain depth without any visible damage. By a strong focusing in the interior of the sample occurs in the focal point to very high intensities of more than a TW / cm ². The consequent non-linear interactions lead to the so-called optical breakdown, which induces a limited focus on the material separation. This process is referred to as photodisruption. The short pulse durations of a few femtoseconds ( 1 fs = 10-15 s ) is deposited only a very small amount of energy in the range of a few nano- Joules per pulse in the sample. This limits the interaction zone to a diameter of less than one micrometer. Outside of this zone occur due to the ultra-short interaction times to no thermal damage.
The laser beam is deflected by a fast scanner mirrors, while a three-dimensional positioning at the same time moving the sample back and forth. In combination with a high repetition rate, this approach allows the scanning of large areas in a short time.
In addition to the laser microtome there is also the laser microdissection to cut out areas within a tissue section, etc., or Zellausstriches for sorting small particles.
Microtome
The type of microtome used is dependent on the material and the pretreatment of the sample, as well as to study target (eg, section thickness ).
Types of knives and cut types
Classically, relatively heavy steel knife or carbide knife with a thick back and with different shapes ( profiles) are used, which are generally identified by the letters A, B, C and D. The microtome cut from type A and B are defined by the plano-concave shape of extremely sharp, but also very sensitive and therefore suitable only for very soft samples, such as paraffin or foamed material. The wedge shape of the cut type C is much more stable and will thus also for some harder materials like resin or frozen sections for use. When blade type with a ground form D only one side of the blade is ground. The front section angles of about 45 ° increases the stability again, but also makes the knife very much duller. This blade shape is used only for harder materials.
Instead of this classical microtome disposable blades are, for example, for cost savings often used. These are sometimes a bit duller than the classical microtome knives, but mostly much thinner and thus more flexible. For harder samples it can therefore vibrations of the blade and thus to variations in layer thickness on average. Once blades are therefore mainly used for softer materials.
For Ultramicrotomes you need glass or diamond knife. The average width of such diameter is significantly smaller than in the classical microtome with a few millimeters. Glass knives are made immediately before use from a few millimeters thick glass rods by crushing. This gives rise to the narrow side of the broken glass into triangles an extremely smooth and sharp broken edge. Glass knives for pre- preparation of ( Antrimmen ) are typically used. They can, for example by adhesive tape, to be complemented by a small trough is filled with water. As with diamond knives the individual sections may then float on the water surface.
Sharpness and hardness of the blade are crucial for a good result. Blunted steel blades are ground with special abrasive pastes containing diamond particles. For this purpose there are special grinding wheels. Hand grinding on abrasive belts and sticks is also possible, but requires a lot of experience.
Cutting angle: declination and inclination
As a declination of angle between the direction of the blade and the cutting direction is referred to ( see figure at right ). It can be set in many slide microtomes between 90 ° and 160 °. The knife is positioned transversely ( declination = 90 °), the cut is made only by the knife is pushed through the sample. Thereby the force acting on the blade forces are significantly greater than if the blade is oriented obliquely to the cutting direction ( declination 120 ° to 160 ° ). In the latter case facilitates relative movement with a portion parallel to the knife edge the cut. This setting is used particularly for large and hard materials. The advantage of the transversely positioned variant is that with suitable material cut bands ( concatenation of several sections ) can be created.
As inclination, inclination of the knife to the specimen plane is called. For best cutting results, this angle must be appropriate selected. It depends on the exact blade geometry, of the sample, the cutting speed, and many other parameters. Are typical inclination angles at which remains a small clearance angle of a few degrees between the specimen plane and knife.
This angle is set too low, the knife cuts unevenly, or areas of the lower part of the blade contacting the freshly cut surface, so that it is smeared.
If the angle is too large, however, so " overwhelmed " the knife over the surface and leads to periodic variations in thickness on average. At even larger inclination angle, the lateral load on the cutting edge is extremely large, and it may lead to outbreaks of the knife edge.
Preparation and evaluation of the samples
Biological and other soft materials require an elaborate pretreatment to solidify it and thereby make sliceable. The methods of treatment such as fixation and embedding are part of Histology. Embedding the object is usually completely impregnated with a liquid which is subsequently solidified. In this way, the compound will receive throughout fairly uniform strength. Typical embedding media are paraffin, polyethylene glycol, celloidin, gelatin, agar, and synthetic resins.
For some studies, the solidification of the material to be cut is achieved by freezing, for example, if an embedding in a change in the sample cause or prevent a subsequent staining would. Water-containing samples should be snap-frozen at a cooling rate of at least 10,000 K / s ( Kelvin per second), to solidify the water in the amorphous state. Otherwise, ice crystals form, leading to freezing damage in the material. The sections are then made to Gefriermikrotomen, usually at -20 ° C.
After cutting, the cut for further processing (for example, histochemical, immunohistochemical staining ) must be transferred to a carrier. For light microscopic preparations slides are used. Larger paraffin sections is allowed to first on a water surface ( 45 ° C) float and smooth by the surface tension. Then, a slide is pushed below the water surface at an angle under the cut and then gently moved up. The section remains with an edge by adhesive forces depend on the glass and is thereby mounted on the slide. The same principle is also used for ultra-thin sections for electron microscopy are too thin and fragile to a mechanical lift. Here the liquid trough is mounted directly on the blade. The cuts form a cut strip (see picture ultramicrotome ) and then with a fine metal mini-networks fished ( engl. grid).
Application
In histology ( histology ) the making of cuts is a prerequisite for the study of tissue characteristics. Special Gefriermikrotome ( cryostats ) are among other things used for frozen section diagnosis in order to obtain clarity on the completeness of the removal of a tumor during surgery. Based on the results will be decided on how to proceed with the operation.
In addition, microtomes are used for material analysis. Here, for example, may be mentioned (especially microscopic IR spectroscopy in transmission ) or the polarization microscopic examination of spherulites the light microscopic or spectroscopic examination of layered systems. For transmission electron microscopy very thin sections are necessary to irradiate with these electrons can.
In ophthalmology called microkeratome (a type of microkeratome ) or, more recently, femtosecond lasers are used in the context of refractive surgery (also called flap) to cut and thereby expose the underlying corneal layers for a Excimerlaseroperation a 150 micron thick corneal flap. This device is sometimes referred to as a microtome.