Microtome

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, with the process being termed microsectioning. Important in science, microtomes are used in microscopy for the preparation of samples for observation under transmitted light or electron radiation.

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

[[File:Cummings 1774 Microtome.jpg|thumb|150px|A diagram of a microtome drawn by Cummings in 1770 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.

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.

Occasionally, attribution for the invention of the microtome is given to the anatomist Wilhelm His, Sr. (1865).

In his (German for Description of a Microtome), Wilhelm wrote:

Other sources further attribute the development to a Czech physiologist Jan Evangelista Purkyně. Several sources describe the Purkyne model as the first in practical use.

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 microscopic details.

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

thumb|Microtome (C. Reichert, Vienna, 1905–1915)

The most common applications of microtomes are:

Traditional Histology Technique: tissues are fixed, dehydrated, cleared, and embedded in melted paraffin, which when cooled forms a solid block. The tissue is then cut in the microtome at thicknesses varying from 2 to 50 μm. From there the tissue can be mounted on a microscope slide, stained with appropriate aqueous dye(s) after removal of the paraffin, and examined using a light microscope. and for DNA sensing applications.

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:

Serial sectioning: obtaining a continuous ribbon of sections from a paraffin block and using all for slides.
Step sections: collected at specified depths in the block.

Precision cut kidney slices

Precision-cut kidney slices refer to thin sections of the kidney tissue that are prepared using a microtome to study kidney functions, drug metabolism or disease processes. Researchers use these slices to study the impact of substances on renal function. This includes drug metabolism and the effects of toxic substances.

Types

Sledge

thumb|left|A sled microtome used for [[thin section of sea ice]]

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. By adjusting the angles between the sample and the microtome knife, the pressure applied to the sample during the cut can be reduced.

thumb|left|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

thumb|left|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.

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.

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. 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/cm<sup>2</sup>, 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

thumb|A diamond knife blade used for cutting ultrathin sections (typically 70 to 350 nm) for transmission electron microscopy

thumb|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

thumb|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. is undergoing a significant transformation due to rapid advancements in laboratory automation and precision engineering. Modern laboratories are adopting advanced microtome systems such as rotary microtomes, cryostat microtomes, and ultramicrotomes to enhance tissue sample preparation.

References

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