Microtomes-Various types of Microtomes, their Working Principle Author：pankaj | Size：1.27 M | View：1084 | Page：28Microtomes-Various types, their Working Principle and MaintenanceINTRODUCTIONMicrotome (Greek “mikros-”small & temnin- “to cut”) Is a sectioning instrument allowing cutting of extremely thin slices of material. Using Steel, Glass or Diamond Blades, depending upon the specimen to be slic...
Microtomes-Various types, their Working Principle and MaintenanceINTRODUCTIONMicrotome (Greek “mikros-”small & temnin- “to cut”) Is a sectioning instrument allowing cutting of extremely thin slices of material. Using Steel, Glass or Diamond Blades, depending upon the specimen to be sliced and the desired thickness of sections being cut. Microtomy is the method of using Microtomes for sectioning Tissues including bones to a thickness of 0.05-100µm.HISTORYIn 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 microscopically it was important to make clean reproducible cuts through which light can be transmitted. This allowed for the observation of samples using light microscopes in a transmission mode. First device for preparation of such cut sections was invented by George Adams Jr. in 1770 and further developed by Alexander Cummings. 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 called cutting engine. Andrew Prichard in 1835 developed table based model to reduce vibrations. Occasionally, attribution for the invention of the microtome is given to the anatomist Wilhelm His, Sr. (1865). This apparatus enabled a precision in work, by which enabled the possibility of achieving unbroken sections. 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. 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. Microtome (C. Reichert, Vienna, 1905–1915).Microtome types 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 sledge microtomes have the sled placed upon a linear bearing, a design that allows for 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. 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 sled microtome is between 1 and 60 µm. Rocking microtome Sledge microtome Sliding microtomeRotary microtome A Rotary microtome of older construction, 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 horizontal position. PRINCIPALThe principle of the cut is explained through the motion of the sample holder, The sample is cut by the knife 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 for 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 for good "Semi-thin" sections with a thickness of as low as 0.5 µm. 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 for the hardness of the sample to be increased, such as by undergoing a glass transition, which allows for the preparation of semi-thin samples. However the sample temperature and the knife temperature must be controlled in order to optimise the resultant sample thickness CryomicrotomeUltramicrotome A ribbon of ultrathin sections prepared by ultramicrotomy, at room temperature, floating on water in the boat of a diamond knife is used to cut the E.M. sections. The Glass blade is the cutting edge at the upper end of the trough of water. An ultramicrotome can allow for 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. Ultramicrotome Ultramicrotomy These extremely thin cuts are important for use with transmission electron microscope (TEM) and Scanning Electron Microscopy (SEM). The typical thickness of these cuts is between 40 and 100 nm for TEM and often between 30 and 50 nm for SEM. The thickness of the cut is also controlled by the quality of knife, with the thinner specimens, 70 nm and less, generally being cut with special gem-quality diamond knives and thicker sections with histology gemstone knives or glass knives. 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 interference colors of transmitted light that are seen as a result of the extremely low sample thickness. Vibrating microtomeThe 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. The cut thickness is usually around 30-500 µm for live tissue and 10-500 µm for fixed tissue.Saw microtome The saw microtome is especially for hard materials such as teeth or bones. The microtome of this type has a rotating saw, which slices through the sample. The minimal cut thickness is approximately 30 µm, and can be made for comparatively large samples.Laser microtome Is an instrument for contact free slicing. Prior preparation of the sample through embedding, freezing or chemical fixation is not required, thereby minimising the artefacts from preparation methods. Alternately this design of microtome can also be used for very hard materials, such as bones or teeth. Dependant 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 infra-red laser. As the laser emits a radiation in the near infra-red, in this wavelength regime the laser can interact with biological materials. The ultra-short beam application time introduces minimal to no thermal damage to the remainder of the sample.APPLICATIONSTraditional Histology technique: tissues are hardened by replacing water with paraffin. The tissue is then cut in the microtome at thicknesses varying from 2 to 50 µm (micrometers) thick. From there the tissue can be mounted on a microscope slide, stained with appropriate aqueous dye(s) after prior removal of the paraffin, and examined using a light microscope. Cryosectioning : water-rich tissues are hardened by freezing and cut in the frozen state with a freezing microtome or Cryostat. Sections are stained and examined with a light microscope. This technique is much faster than traditional histology (5 minutes vs 16 hours) and is used in conjunction with Surgical procedures to achieve a quick diagnosis. Cryosections can be used in Immunohistochemistry, as freezing tissue stops degradation of tissue faster than using a fixative and does not alter or mask its chemical. Electron Microscopy: after embedding tissues in epoxy resin, a microtome equipped with a glass or diamond knife is used to cut very thin sections (typically 60 to 100 nanometers). Sections are stained with an aqueous solution of an appropriate heavy metal salt and examined with TEM. This instrument is often called an ultramicrotome.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. MaintenanceAt the time of purchase it should be ensured to get Guarantee/ Warranty/AMC from the supplier of the instrument. A log book must be maintained under the supervision of Lab. In-charge. Instrument should be used gently and carefully. Cleaning and covering the instrument after use is mandatory. THANK YOU