Microtome

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A microtome (from the Greek mikros, meaning "small", and temnein, meaning "to cut") is a cutting tool used to produce extremely thin slices of material known as sections. Important in science, microtomes are used in microscopy, allowing for the preparation of samples for observation under transmitted light or electron radiation.

Contents

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 histological sections of animal or plant tissues for light microscopy. 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 tough plant matter for both light microscopy and for electron microscopy. Gem-quality diamond knives are also 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 50  nm and 100  μm.

History

A diagram of a microtome drawn by Cummings in 1770. Cummings 1774 Microtome.jpg
A diagram of a microtome drawn by Cummings in 1770.

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 μm, 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. (1750–1795) 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 Purkyně. [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.

Applications

Microtome (C. Reichert, Vienna, 1905-1915). Microtome1905.JPG
Microtome (C. Reichert, Vienna, 1905–1915).

The most common applications of microtomes are:

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 μm are feasible.

Sectioning intervals can be classified mainly into either:

Types

Sledge

A sled microtome Sledge microtome.jpg
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 the microtome to readily cut many coarse sections. [13] By adjusting the angles between the sample and the microtome knife, the pressure applied to the sample during the cut can be reduced. [13] 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 sledge microtome is between 1 and 60 μm.

Rotary

A rotary microtome of older construction Microtome-1.jpg
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 vertical position. [14]

Principle of sample movement for making a cut on a rotary microtome Microtome principle.svg
Principle of sample movement for making a cut on a rotary microtome

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 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 μm. For hard materials, such as a sample embedded in a synthetic resin, this design of microtome can allow good "semi-thin" sections with a thickness of as low as 0.5 μm.

Cryomicrotome

A cryomicrotome Cryostat microtome.jpg
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 the hardness of the sample to be increased, such as by undergoing a glass transition, which allows the preparation of semi-thin samples. [13] However the sample temperature and the knife temperature must be controlled in order to optimise the resultant sample thickness.

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. Microtome-ultras.jpg
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 allows 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. [13]

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. [14] 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. [15]

Vibrating

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. [13] The cut thickness is usually around 30–500 μm for live tissue and 10–500 μm for fixed tissue. [16]

A variation on the vibrating microtome is the Compresstome microtome. [17] The Compresstome uses a specimen syringe or "lipstick-like" tube to hold the tissue. [18] The tissue specimen is completely embedded in agarose (a polysaccharide), and the tissue is slowly and gently pressed out of the tube for the vibrating blade to cut. The device operates in the following way: the end of the specimen tube where the tissue emerges is slightly narrower than the loading end, which allows gentle "compression" of the tissue as it comes out of the tube. The slight compression prevents shearing, uneven cutting, and vibration artifacts from forming. Note that the compression technology does not damage or affect the tissue being sectioned.

There are several advantages of the Compresstome microtome: 1) the agarose embedding provides stability to the entire specimen on all sides, which prevents uneven slicing or shearing of tissue; 2) the compression technology gently compresses tissue for even cutting, so that the blade doesn't push against the tissue; 3) faster sectioning than most vibrating microtomes; and 4) it cuts tissue from older or more mature animals well to provide healthier tissues. [19]

Saw

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 μm and can be made for comparatively large samples. [13]

Laser

A conceptual diagram of laser microtome operation Laser-microtome-schematic.png
A conceptual diagram of laser microtome operation

The laser microtome is an instrument for contact-free slicing. [20] Prior preparation of the sample through embedding, freezing or chemical fixation is not required, thereby minimizing 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. Dependent upon the properties of the sample material, the thickness achievable is between 10 and 100 μm.

The device operates using a cutting action of an infrared laser. As the laser emits a radiation in the near infrared, in this wavelength regime the laser can interact with biological materials. Through sharp focusing 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 of the optical penetration in the focal region a material separation in a process known as photo-disruption is introduced. By limiting the laser pulse durations to the femtoseconds range, the energy expended at the target region is precisely controlled, thereby 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 three-dimensional positioning of the beam crossover, whilst allowing 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.

Knives

A diamond knife blade used for cutting ultrathin sections (typically 70 to 350 nm) for transmission electron microscopy. Diamond Knife Blade Edge.jpg
A diamond knife blade used for cutting ultrathin sections (typically 70 to 350 nm) for transmission electron microscopy.
The cutting edge of a disposable blade for a microtome under a microscope. Odnorazovoe lezvie dlia mikrotoma.jpg
The cutting edge of a disposable blade for a microtome under a microscope.

The selection of microtome knife blade profile depends upon the material and preparation of the samples, as well as the final sample requirements (e.g. cut thickness and quality).

Design and cut types

Profiles of microtome knives. Microtome-knife-profile.svg
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. [14] 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. [13] Diamond blades may be built into such an existing trough, allowing for the same collection method.

Sectioning

Prior to cutting by microtome, 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 mold 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 the knife is tilted, however, 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.

See also

Related Research Articles

Electron microscope Type of microscope with electrons as a source of illumination

An electron microscope is a microscope that uses a beam of accelerated electrons as a source of illumination. As the wavelength of an electron can be up to 100,000 times shorter than that of visible light photons, electron microscopes have a higher resolving power than light microscopes and can reveal the structure of smaller objects. A scanning transmission electron microscope has achieved better than 50 pm resolution in annular dark-field imaging mode and magnifications of up to about 10,000,000× whereas most light microscopes are limited by diffraction to about 200 nm resolution and useful magnifications below 2000×.

Histology Study of the microscopic anatomy of cells and tissues of plants and animals

Histology, also known as microscopic anatomy or microanatomy, is the branch of biology which studies the microscopic anatomy of biological tissues. Histology is the microscopic counterpart to gross anatomy, which looks at larger structures visible without a microscope. Although one may divide microscopic anatomy into organology, the study of organs, histology, the study of tissues, and cytology, the study of cells, modern usage places all of these topics under the field of histology. In medicine, histopathology is the branch of histology that includes the microscopic identification and study of diseased tissue. In the field of paleontology, the term paleohistology refers to the histology of fossil organisms.

Transmission electron microscopy Technique in microscopy

Transmission electron microscopy (TEM) is a microscopy technique in which a beam of electrons is transmitted through a specimen to form an image. The specimen is most often an ultrathin section less than 100 nm thick or a suspension on a grid. An image is formed from the interaction of the electrons with the sample as the beam is transmitted through the specimen. The image is then magnified and focused onto an imaging device, such as a fluorescent screen, a layer of photographic film, or a sensor such as a scintillator attached to a charge-coupled device.

Microscope slide Thin, flat piece of glass onto which a sample is placed to be examined under a microscope

A microscope slide is a thin flat piece of glass, typically 75 by 26 mm and about 1 mm thick, used to hold objects for examination under a microscope. Typically the object is mounted (secured) on the slide, and then both are inserted together in the microscope for viewing. This arrangement allows several slide-mounted objects to be quickly inserted and removed from the microscope, labeled, transported, and stored in appropriate slide cases or folders etc.

Histopathology Microscopic examination of tissue in order to study and diagnose disease

Histopathology refers to the microscopic examination of tissue in order to study the manifestations of disease. Specifically, in clinical medicine, histopathology refers to the examination of a biopsy or surgical specimen by a pathologist, after the specimen has been processed and histological sections have been placed onto glass slides. In contrast, cytopathology examines free cells or tissue micro-fragments.

Cryostat

A cryostat is a device used to maintain low cryogenic temperatures of samples or devices mounted within the cryostat. Low temperatures may be maintained within a cryostat by using various refrigeration methods, most commonly using cryogenic fluid bath such as liquid helium. Hence it is usually assembled into a vessel, similar in construction to a vacuum flask or Dewar. Cryostats have numerous applications within science, engineering, and medicine.

Humberto Fernández-Morán

Humberto Fernández-Morán Villalobos was a Venezuelan research scientist born in Maracaibo, Venezuela, renowned for inventing the diamond knife or scalpel, significantly advancing the development of electromagnetic lenses for electron microscopy based on superconducting technology, and many other scientific contributions.

Glass knife

A glass knife is a knife with a blade made of glass, with a fracture line forming an extremely sharp cutting edge.

Ultramicrotomy is a method for cutting specimens into extremely thin slices, called ultra-thin sections, that can be studied and documented at different magnifications in a transmission electron microscope (TEM). It is used mostly for biological specimens, but sections of plastics and soft metals can also be prepared. Sections must be very thin because the 50 to 125 kV electrons of the standard electron microscope cannot pass through biological material much thicker than 150 nm. For best resolutions, sections should be from 30 to 60 nm. This is roughly the equivalent to splitting a 0.1 mm-thick human hair into 2,000 slices along its diameter, or cutting a single red blood cell into 100 slices.

Frozen section procedure Rapid histological sectioning procedure

The frozen section procedure is a pathological laboratory procedure to perform rapid microscopic analysis of a specimen. It is used most often in oncological surgery. The technical name for this procedure is cryosection.

Laser capture microdissection

Laser capture microdissection (LCM), also called microdissection, laser microdissection (LMD), or laser-assisted microdissection, is a method for isolating specific cells of interest from microscopic regions of tissue/cells/organisms.

Thin section Thin slice of a material prepared for microscopic examination

In optical mineralogy and petrography, a thin section is a thin slice of a rock, mineral, soil, pottery, bones, or even metal sample, prepared in a laboratory, for use with a polarizing petrographic microscope, electron microscope and electron microprobe. A thin sliver of rock is cut from the sample with a diamond saw and ground optically flat. It is then mounted on a glass slide and then ground smooth using progressively finer abrasive grit until the sample is only 30 μm thick. The method uses the Michel-Lévy interference colour chart to determine thickness, typically using quartz as the thickness gauge because it is one of the most abundant minerals.

Laser microtome

The laser microtome is an instrument used for non-contact sectioning of biological tissues or materials. It was developed by the Rowiak GmbH, a spin-off of the Laser Centre, Hannover.

Diamond knife

A diamond knife is a very sharp knife in which the edge is made from diamond, invented by Humberto Fernández-Morán in 1955. Diamond knives are used for medical and scientific applications where an extremely sharp and long-lasting edge is essential. The knives are very expensive to purchase, depending on the quality and size of the knife; in addition the knives must be professionally sharpened as the edge dulls.

Vibratome

A vibratome is an instrument used to cut thin slices of material. It is similar to a microtome but uses a vibrating blade to cut through tissue. The vibration amplitude, the speed, and the angle of the blade can all be controlled. Fixed or fresh tissue pieces are embedded in low gelling temperature agarose. The resulting agarose block containing the tissue piece is then attached to a metal block and sectioned while submerged in a water or buffer bath. Individual sections are then collected with a fine brush and transferred to slides or multiwell plates for staining.

Low-voltage electron microscope (LVEM) is an electron microscope which operates at accelerating voltages of a few kiloelectronvolts or less. Traditional electron microscopes use accelerating voltages in the range of 10-1000 keV.

Nanoknife

A nanoknife is a carbon nanotube-based prototype compression cutting tool intended for sectioning of biological cells. Working principle is similar to that of a 'cheese slicer', a nanometer-thin individual carbon nanotube strung between two tungsten needles would allow sectioning of very thin slices of biological matter for imaging under an electron microscope. Tests are currently being performed by scientists at Virginia Tech, CU-Boulder and other universities. A successful development of this new tool will allow scientists and biologists to make 3D images of cells and tissues for electron tomography, which typically requires samples less than ~300 nanometers in thickness. In 2009, the nano-knife was used to create indentation marks on biological cell plasticizer. The whole cutting process is currently limited by electron charging of polymeric specimen in the SEM, which makes it difficult to observe any small cut or mark as the carbon nanotube is pressed against the specimen.

Serial block-face scanning electron microscopy is a method to generate high resolution three-dimensional images from small samples. The technique was developed for brain tissue, but it is widely applicable for any biological samples. A serial block-face scanning electron microscope consists of an ultramicrotome mounted inside the vacuum chamber of a scanning electron microscope. Samples are prepared by methods similar to that in transmission electron microscopy (TEM), typically by fixing the sample with aldehyde, staining with heavy metals such as osmium and uranium then embedding in an epoxy resin. The surface of the block of resin-embedded sample is imaged by detection of back-scattered electrons. Following imaging the ultramicrotome is used to cut a thin section from the face of the block. After the section is cut, the sample block is raised back to the focal plane and imaged again. This sequence of sample imaging, section cutting and block raising can acquire many thousands of images in perfect alignment in an automated fashion. Practical serial block-face scanning electron microscopy was invented in 2004 by Winfried Denk at the Max-Planck-Institute in Heidelberg and is commercially available from Gatan Inc., Thermo Fisher Scientific (VolumeScope) and ConnectomX.

Microtechnique is an aggregate of methods used to prepare micro-objects for studying. It is currently being employed in many fields in life science. Two well-known branches of microtechnique are botanical (plant) microtechnique and zoological (animal) microtechnique.

Cryotome is a microtome device that cold cuts thin blocks of frozen tissue. It is used on tissue that has been removed from the body. The device is designed for rapid microscopic analysis of the tissue. Another type of microtome, called ultracryotome, which is very similar device to crytome, can cut ultrathin blocks if tissue, and that tissue can be observed by transmission electron microscopy. The cutting thickness of ultracryotome is about dozens of nanometers. The ultrastructural properties can be studied without embedding of the tissue, and so the molecular conservation is better.

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