Histology studies biological tissues that are preserved carefully, usually by embedding them in paraffin wax. These methods of careful preservation maintain relationships between cells and their various components and also helps in passing illuminating radiation through them.
Since most biological tissues are optically dense, these require thin slicing so that the microscopic details of each cell can be easily studied. The mechanical device, microtome, helps achieve the thin slicing of the tissues for studying under a microscope.
Micro means small, and tome means cut in Greek. The instrument has sharp blades or a microtome knife for making the thin slice. Traditionally, the microtomes helped in free-hand sectioning of the tissues using a sharp razor.
Whereas modern microtomes are precise instruments for cutting thin sections uniformly. With time one can cut fragile and translucent areas using the device. Generally, a microtome has a knife, base, and sample holder.
It has many different types based on operating mode and knife material; manual, automated, semi-automated microtome with steel, diamond, glass, tungsten carbide, or sapphire knife. The instrument is mainly used in histological and pathological studies and analyses.
Table of Contents
Parts of Microtome
The instrument has mainly three parts; the base/body of the microtome, the knife in the knife holder, and the tissues/material holder.
- Base/body: The body/base of the microtome helps to keep the instrument upright. It has a part for the attachment of the knife holder. It has a scale where the distance for movement of the material holder is set.
- Knife and knife holder: The knife holder holds the knife fixed on the body. The knife/blade can be made up of diamonds, glass, or metals. It is of different types.
- Tissue/material holder: The material holder holds the specimen. Unlike any other cutting appliance, it is the movable part of the microtome. The cutting action can be vertical or horizontal, and the holder moves at a pre-selected distance in the body.
Types of Knife and Its Angle
The materials used in making of the knife are either stainless steel, diamond, tungsten, sapphire, or glass. Likewise, its profile also decides the types of a knife; plano-concave, biconcave, wedge, and tool edge knives.
Based on the material used for construction
- Steel knife: The material used for this kind of knife is from high-quality carbon or tool-grade steel. The steel should be rust-resistant and heated to harden the edge. The hardening of the edge of the steel determines its sharpness.
- Tungsten knife: These knives are made of tungsten carbide. These are non-magnetic and 100 times harder than that steel. These types are highly brittle. It is resistant to wear and can make up to 30,000 serial sections of undecalcified bone embedded in methacrylate after each sharpening.
- Diamond knife: The knife is made of gen quality diamonds. The blades are expensive but very durable due to their hardness. The knife is used for cutting very thin sections.
- Sapphire knife: The knife is produced from a single piece of sapphire created artificially from alumina monocrystal under computer-controlled thermal conditions. It is harder than glass and tungsten, which ensures its durability. The knife can only cut smaller-sized blocks, and the edge is limited to 11 mm.
- Glass knife: It is used in ultramicrotomy. Its cutting edge is placed against/across the thickness of the glass. It is also called the Ralph knife, and different profiles of the knife are used for cutting sections from various embedded materials. The knives are hard but brittle. However, the knife is susceptible to storage for an extended period. So, it should be treated before use.
- Disposable knife: The knife is made of stainless steel and has replaced conventional knives. Teflon coating is preferred for cryostats.
- Non-corrosive knife: The non-corrosive blade is used for cryo-microtome and is made of heat-treated stainless steel, free from all impurities. It has 12-15% chromium.
Based on the profile
- Plano-concave knife: The knife has one plane surface, and another surface is concave. The biconcave side has two hollow ground surfaces, and both surfaces are incredibly sharp. The blade is used for cutting soft, celloidin-embedded material or foam compounds. The knife does not apply to hard substances. The knife should be placed obliquely to the object when cutting sections.
- Biconcave knife: It is similar to the plano-concave knife, only with a thicker back. It is used for cutting hard materials. The blade is placed obliquely to the material being sectioned. The degrees of concavity vary highly.
- Wedge knife: This knife is more rigid than plano-concave and biconcave knives. It is used for cutting rigid materials. It has an extra thick wedge at the tip; it cannot grind as sharp as a plano-concave and biconcave knife. It helps cut thin specimens of the cryostat, frozen, and paraffin-embedded specimens. The cutting plane should be placed transverse to the object.
- Tool edge knife: It helps cut hard material because its stability is excellent. The blade has the least sharpness of all the types. The knife’s cutting edge is made by grinding a bevel on each side of plano-concave, biconcave, and wedge knives or the angled surface of a tool edge knife. The face of the bevel encloses a sharper angle than the main surfaces of the blade.
Types of Microtome
Microtomes are available in different types depending on the manufacturing companies used. Based on the operating mode, there are three types; manual, semi-automatic, and automatic microtome.
The microtome that requires human intervention for operation is the manual microtome. All stages of this kind of microtome are manual.
There are different types of manual microtomes; rocking, rotary, freezing, vibrating, sledge, sliding, cryostat, saw, hand, and ultra microtomes. It is simple to use and has three moving parts;
It has the oldest design and is relatively cheaper than other types of the microtome. It is named due to its rocking action due to cross-arm presence and is exclusively applicable in slicing paraffin blocks. Here the tissue moves through the arc as the material holder moves towards the knife (Heiffor knife) that is rigidly attached to the holder. The thinly cut section comes out curved due to the presence of a slightly biconcave knife.
Since the tissue section comes out as curved, it may lack flat surfaces for viewing. Although highly reliable, lightweight, and low maintenance, their lightweight might lead to unstableness and vibration while operating it. Nowadays, the rotary microtome significantly replaces the rocking microtome.
It is the most commonly used instrument in routine laboratories, and the blade is kept horizontally. The paraffin block with tissue moves up and down with the help of a rotatory handle in the microtome. One rotation of the handle moves the block down; another moves it down. This action helps in cutting the tissue as thin as a ribbon. The rotary microtome can be automated or semi-automated by adjusting and controlling the block’s movement and the knife’s angle.
The section comes out very thin (2-3 μm) with suitable quality ribbons. The microtome is highly stable and can easily cut various types of tissue. However, the microtome is not ideal for cutting large blocks of tissue. Likewise, it is expensive and leads to accidents as the knife faces up.
This kind of microtome is only applicable in sectioning rigid botanical material. Although thin tissue sections can be obtained from plant cells, thin sections from animal tissues are difficult to obtain.
This microtome is applicable for producing thin slices from unfixed, unprocessed, or not frozen tissue samples from animal and botanical sources. It is assumed to replace the hand microtome. Its name is derived front he high-speed vibration used by the safety blade, which can increase the cutting speed. The vibration speed is adjusted by altering the electrical voltage applied to the knife. The fresh tissue sample is immersed in the fluid to prevent the tearing of the material and dissipate heat produced during vibration.
This instrument is used to cut sections of large blocks of tissue. Here the block is fixed in a static position within a steel carriage. The carriage slides backward and forward against a fixed horizontal knife. The knife is large (usually 24 cm long) and wedge-shaped, reducing the vibration and requiring less sharpening. The knife holders are adjustable and tiltable to the desired angle of the knife to the block. The whole instrument is heavy and hence very stable. The only disadvantage of this microtome is; it is relatably slow than the rocking or rotary microtome.
In this type of microtome, cutting occurs by placing it in a deep freeze cabinet. The cabinet has a double glass window and a door for passing the samples in and out. The inside of the cabinet has a fluorescent light and a fan. The fan helps to provide proper cool air circulation. The temperature inside the cabinet is kept between -10ºC to -40ºC using liquid nitrogen. It is used as an alternative for freezing microtomes. It became popular after developing fluorescent antibody staining techniques for making thin sections of fresh frozen tissue free of ice crystals.
This instrument obtains ultrathin (40-100 nano micron) tissue sections for the transmission electron microscope. This microtome uses a diamond, sapphire, or glass knife. The extensive tissue sections are cut into small blocks (1×1 mm size) and then cut using an ultramicrotome under an optical microscope. After the cut, each section is floated to a water bath adjacent to the knife edge.
The advanced mechanisms used in this type of microtome are thermal and mechanical. In the thermal mechanism, the tissue sample is heated in a bifurcated metal strip to induce expansion. In mechanical advancement, a microprocessor couples with a precise stepping motor. The motor helps provide even cutting of the sections and for constant reproducibility.
In this instrument, the knife moves horizontally against a fixed block. The movement progresses in an inclined plane. It was designed for cutting celloidin-embedded sections and paraffin-embedded sections.
This type of microtome is applicable for cutting thin slices of frozen tissue samples. The instrument is connected to the CO2 by a flexible metal tube and attached to one of the working tables. This microtome differs from another type because the knife/blade moves, and the tissue holder stays static. The tissue block moves slightly in a pre-set amount at the end of each cut.
Although this microtome helps cut thin sections of frozen specimens, it cannot generate consistent, high-quality thin sections of tissues.
The instrument does not require solid CO2 or liquid nitrogen to provide the cooling effects; It has two different kinds of metals in opposite directions. When electric current flows through them, one metal helps to generate heat, and the other benefits in losing the generated heat.
Much human intervention is not required to operate this type of microtome. One must feed the information about size and direction to a computer for cutting thin slices of tissues.
Ultra-thin, computerized, and laser microtomes are some kinds of the commercially available automatic microtome.
This type of microtome has an advanced thermostatic switch, cyro-scalpel, cyroplate, and semiconductor freezing. It cuts slices in the range of 1-25 μm. It can carry out both freezing and routine paraffin sectioning. The microtome operates once the technical staff feeds data (the required size and the number of sections needed). The cryo-scalpel and cyroplate temperatures range from 0ºC to -18ºC and -10ºC to -40ºC, respectively.
This kind of microtome helps in cutting biological specimens using a laser. This type helps obtain non-contact sectioning without thermal damage and gets precise slices. An infrared laser beam with a short pulse duration is used because there is almost no heat generation. Depending on its type, the processed tissue is sliced in about 5-100 μm before processing.
Uses of Microtome
Microtome is used in different laboratories to study other tissues’ histopathology (animal and plant). Different types of microtomes are used in various laboratories for different purposes.
- In ophthalmology, sledge microtome helps study tissue sections of the eyes.
- Sliding microtome helps in sectioning brain tissues better by this type of microtome.
- Cryomicrotome is used in nerve biopsy.
- Rocking, rotary, hand, and vibrating microtome is helpful in general biological laboratories for studying tissue sections embedded in paraffin. It is also used in performing a small biopsy.
- Sledge microtome is also helpful in the study of tissues embedded in celloidin.
- Electron microscopy requires slices as thin as ten nano micrometers, so the ultramicrotome helps achieve pieces as thin as 5-10 nano microns.
Sectioning of tissue using blades and knives may prove to be difficult because of the preservation methods used. Some tissues are preserved in paraffin blocks, which can be hard to cut into thin pieces. Likewise, frozen specimens prove to be very difficult to cut. The use of microtomes may help in achieving thin slices of biological samples.
Besides helping cut thin slices of biological specimens, its different types carry different advantages. The advantages of different kinds of microtomes are as follows:
- Laser microtome helps obtain non-contact processing of the tissue and cut the tissue in its native forms without thermal damage. It also enables secure sub-micrometer precision, is less time-consuming, and has fewer artifacts.
- Rocking microtome is easy to operate, requires low maintenance, and is relatively inexpensive.
- Sledge microtome helps in cutting hard and large tissue.
- Like sledge microtome, sliding microtome helps in cutting large tissues. It is easy to operate and inexpensive. It is also applicable for tissue embedded in celloidin.
- Ultramicrotome helps obtain ultra-thin slices of the tissue specimens for observation under an electron microscope.
- Cryomicrotome and freezing microtome help obtain a thin slice of the freshly frozen specimens.
Disadvantages of Microtome
Although the microtome is a handy instrument, microtome has some disadvantages depending on the type of microtome used. Some of them are:
- Some microtomes are expensive. Cryostat, Ultramicrotome, and laser microtome are some of the expensive instruments. One must be mindful of purchasing the right kind of microtome.
- Some are lightweight so that they can be unsteady. The rocking microtome is one of the lightest microtomes, which can cause vibration, which leads to uneven tissue sectioning.
- Most of them require a skilled workforce. Since many commonly used microtomes are manually operated, the technical staff managing these must be experienced to obtain even thin sections.
- Without much care, the blades of the microtome may lead to accidents. Sharpening and fixing of edges may lead to accidents in the laboratory. The accidents can be avoided if the procedure of tissue sectioning is done carefully.
- Dey, P. (2018). Tissue Microtomy: Principle and Procedure. In: Basic and Advanced Laboratory Techniques in Histopathology and Cytology. Springer, Singapore. https://doi.org/10.1007/978-981-10-8252-8_5
- Mohammed, Faraz & Thapasum Fairozekhan, Arishiya & Mohamed, Shamaz. (2012). Microtomes and microtome knives. Annals of Dentistry. 19. 62-65. 10.22452/adum.vol19no2.4.
- Donald B. McMillan, Richard J. Harris, Introduction, Editor(s): Donald B. McMillan, Richard J. Harris, An Atlas of Comparative Vertebrate Histology, Academic Press, 2018, Pages ix-xxix, ISBN 9780124104242, https://doi.org/10.1016/B978-0-12-410424-2.00018-4. (https://www.sciencedirect.com/science/article/pii/B9780124104242000184)