Microtome and microtomy Microtomy is the means by which tissue can be sectioned and attached to a slide so that examination

can be done by microscope. Microtome. Is a mechanical devices for cutting thin uniform slices of tissue (sections).

Modern microtomes are precision instruments designed to cut uniformly thin sections of a variety of materials for detailed microscopic examination. For light microscopy, where magnifications can reach up to 1800x, the thickness of a section can vary between 1 and 10 microns (thin sections). For electron microscopy, where magnifications of several hundred thousands are possible, the thickness of a section is usually of the order of 10 nanometres (ultra-thin sections). Parts of microtome All microtomes consist of three main parts: Base (microtome body) Knife attachment and knife Material or tissue holder

With most microtomes a section is cut by advancing the material holder towards the knife whilst the knife is held rigidly in place, material holder is moved after each cut. The distance moved is pre-selected using a scale setting on the microtome body and usually extends between 0.5 and 50 microns on microtomes ,cutting thin sections from less than 60 nm to over 500 nm on machines cutting ultra thin sections. Types of microtomes and their uses 1. HAND MICROTOME The successful use of a hand microtome is limited to sectioning intrinsically rigid botanical material. It is difficult to obtain thin, even sections of animal tissues.

2. ROTARY MICROTOME Machines of this sort are general purpose microtomes for cutting semi-thin to thin sections for light microscopy. The microtome operation is based upon the rotary action of a hand wheel activating the advancement of a block towards a rigidly held knife. The block moves up and down in a vertical plane in relation to the knife and therefore cuts flat sections. Available machines range from lightweight, rotary microtomes suitable for cutting paraffin wax embedded material in a continuous ribbon to heavy duty,motor driven instruments used with a slow, continuous speed and retracting advance movement to section hard material embedded in synthetic resin.

The rotary microtome can also be found in most cryostats for cutting frozen sections. Section thickness settings range from 0.5m to 60m on most machines.

Sections of paraffin wax embedded tissues are normally cut within the range 3 to 5m whilst resin sections are between 0.5 to 1m. Rotary microtomes are especially suited to cutting sections using disposable steel knives.

3. SLEDGE MICROTOME These are designed for cutting large blocks of paraffin and resin embedded material including whole organs, for light microscopy. The knife holding clamps allow the knife to be offset to the direction of cut, a major advantage when sectioning large, hard blocks. The microtome, which is very heavy for stability and not usually subject to vibration, can also be used to cut materials from various industrial applications (wood, plastics, textile fibres).

They are not suitable for cutting very hard resins such as araldite because of the risk of vibration. 4.ULTRAMICROTOME

The ultramicrotome is used to prepare ultrathin sections for light and electron microscopy.

Very small samples of tissue are usually embedded in hard resin before cutting. It has been reported that sections can be cut as thin as 10 nanometres. The cutting stroke is motor driven to provide a regular, smooth motion for sections of even thickness and constant reproducibility. Knives are usually made from glass, diamond or sapphire. The block is brought to the knife edge under microscopical control and as each section is cut it is floated on to a water bath adjacent to the knife edge. 5. CRYOSTAT The cryostat commonly consists of a microtome contained within a refrigerated chamber, the temperature of which can be maintained at a preset level. A recent innovation has the body of the microtome positioned outside the refrigerated chamber. The cryostat usually contains a rotary microtome although some portable units utilize a rocking microtome.

With the object, object holder and knife all at the same temperature and all other conditions for cutting the material optimal, sections as thin as 1 micron are possible. 6. SAW MICROTOME

Saw microtomes will cut sections from very hard material such as undecalcified bone, glass or ceramics. The samples, commonly embedded in resins, are moved extremely slowly against a diamond coated saw rotating at approximately 600 rpm. Sections of 20 m or greater are possible providing the saw blade is in perfect condition. Very thin sections are not possible. 7.FREEZING MICROTOME

The freezing microtome is equipped with a stage upon which tissue can be quickly frozen using either liquid carbon dioxide, from a cylinder, or a low temperature recirculating coolant. Some cooling systems also allow the knife to be cooled at the same time. The cutting action of the freezing microtome differs from those described previously as in this case the knife is moved whilst the tissue block remains static. The block moves by a pre-set amount, in microns, at the end of each cut.

Consistent, high quality, thin sections are very difficult to obtain with this type of microtome.

8. VIBRATING MICROTOME

Originally conceived as a microtome which could produce high quality sections of fresh, unfixed material from animal or botanical sources and to replace the hand microtome. The name of the instrument derives from the high speed vibration produced in a safety razor blade to provide the cutting power. The amplitude of vibration is adjusted by altering electrical voltage applied to the 'knife'.

Different degrees of vibration are required to produce sections from varying densities of material. To prevent tearing, soft material is cut whilst immersed in a fluid which also aids in dissipating heat produced at the vibrating edge of the razor as it cuts.

9. ROCKING MICROTOME

Produced in large numbers early in the 20th century.

Designed only for cutting paraffin sections, the tissue moves through an arc as it advances towards the knife (the slightly biconcave heiffor knife is used) which is held rigid causing the sections to be cut in a curved plane. Very thin sections are difficult to obtain and one major disadvantage is a limit to the size of block which can be cut. Because of the lightness of the frame the microtome has a tendency to move during cutting. The rocking microtome has largely been replaced by the more precise rotary microtome although it is re-appearing in portable cryostats. Now mainly used for botanical applications sections of 5 m in thickness. 10. LASER MICROTOME The laser microtome has been designed to slice biological tissue and various materials with high precision. The cutting process is performed by ultra short laser pulses in the near infrared region, inducing an optical breakdown below the surface of the material.

Biological Tissue can be processed in its native state without prior fixation and embedding. This is of great importance for many biological applications such as immunohistological investigations. Working principle of the laser microtome. A laser induced plasma is used to cut the tissue without any mechanical forces applied. The near infrared radiation of the laser penetrates the tissue up to one millimetre. Microtome knives STEEL KNIVES Steel microtome knives are manufactured from high quality carbon steel which is heat treated to harden the edge. The steel should be free from impurities, contain anti-corrosives and be rust-resistant. The best knives are those that are fully hardened.. Those which are only surface hardened lose the cutting edge very quickly once the hardened area is removed through repeated re-sharpening. NON-CORROSIVE KNIVES FOR CRYOSTATS These are manufactured from hardened, heat treated stainless steel free from all impurities and containing 12 to 15% chromium. DISPOSABLE BLADES The edge of disposable blades can be coated with platinum or chromium to enhance strength and prolong cutting life. Teflon coated blades are particularly suitable for use in cryostats as these offer reduced cutting resistance and minimal friction.

The smaller, thinner disposable blade also reaches cryostat chamber temperature more rapidly than a conventional knife minimizing time delay during blade exchanges or temperature adjustments. TUNGSTEN CARBIDE Knives manufactured from high quality tungsten carbide are non corrosive, practically non magnetic and 100 times harder than hardened tool steel. The knives have excellent resistance to wear but are brittle because of their extreme hardness and should be handled carefully.

Up to 30,000 serial sections of undecalcified bone embedded in methacrylate per sharpening has been reported. GLASS KNIVES Used for ultramicrotomy. Can be used with a rotary microtome. A commercial glass knife maker is recommended to ensure consistency and reproducibility of the knife edge. Glass knives are hard but brittle and care is required with their handling. These knives deteriorate with storage due to changes in the 'flow' or 'strain' of the glass and from oxidation impurities remaining in the hardened glass after manufacture. DIAMOND KNIVES Diamond knives are manufactured from gem quality diamonds without flaws. Although this makes them very expensive the knives are extremely durable, because of the hardness factor of the diamond, and are used primarily for cutting very thin, resin sections. Profile of steel knives Profile A: plano concave Recommended for use on sledge- type microtome and certain rotary microtomes. Profile B: Plano concave (very concave) Used for celloidin work, since the blade is comparatively thin and would vibrate if used to cut a harder material. Profile C: Plane -Wedge Shaped Commonly used for cutting sections from paraffin wax embedded material, frozen sections, cryostat sections and for small, synthetic resin embedded material. Profile D: Bi - concave Recommended for paraffin section cutting on rocking and sledge- type microtomes. Angles of Knives Bevel angle: usually vary between 27-32 degrees. Smaller the bevel angle sharper is the knife, however too small bevel angle permits elastic distortion of the edge.

Rake and clearance angles: high rake angle and low clearance angle gives less compression to the tissue block and produces a smooth plastic flow type during sectioning. High rake angles suitable for soft tissues and need to be reduced for harder tissues MICROTOMY PART-2 Sharpening steel HONING After prolong use, or after cutting very hard tissue, the cutting edge of the knife becomes so damaged that stropping will not re-sharpen it. Small pieces of metal should be removed which will give a jagged edge, causing lines or even tears in sections during cutting. A straight cutting edge and the correct bevel must be restored by grinding the knife on a hone. Manual: Coarse honing can also be performed manually using various types of hones :belgian black vein arkansas aloxite tam o shanter scotch hone carborundum Plate glass Honing KNIFE SHARPENING Automatic:In most instances the knife is held rigid whilst being moved against a rotating drum, rotating wheels or a lapping plate. Abrasive compounds of various grades, from coarse to fine, move against the knife edge. The knife blade is automatically turned at preset intervals so that each side is evenly sharpened. Atomatic spencer microtome knife sharpener knives

STROPPING Stropping polishes the knife edge and removes fine metal burs retained along the edge after honing. Leather, coated with a fine powder or a lapping stick coated with a diamond paste containing industrial diamonds of less than 0.5 m are effective for this purpose. It is important that very little pressure is used while stropping. Lubricants Lubricants are essential with these methods. It is best to use a non aqueous lubricant (thin lubricating oil) in order to prevent the knife edge from rusting. If an aqueous lubricant is used the knife should be thoroughly cleaned after the sharpening and coated with a fine film of oil. Soap, detergent, glycerol and water soluble oils have all been used as lubricants. Abrasives Abrasive powders various agents act as abrasives including carborundum (silicon carbide), alumina (aluminium Oxide), magnesium oxide, chromium oxide and iron oxide. Abrasive powders are supplied in a variety of grade sizes. Coarse powders are effective for restoring damaged edges whilst finer powders are used for polishing PARAFFIN SECTIONING

METHOD: Set the blocks on to a cold surface to harden the face to be cut (a refrigerated cold plate or ice). Avoid prolonged cooling and very cold surfaces as both can cracks the surface of the paraffin wax block. Install a sharp, trimming knife in the microtome and set the correct clearance angle, normally 2 to 5o. Trim a paraffin wax block, with a sharp blade, so that the sides are parallel and 2 to 3 mm of wax surrounds the tissue. Fit the trimmed block into the block holder and orientate so the edge offering least resistance meets the knife edge first. Advance the block until it just touches the knife edge. Coarse cut the block at 15 m until the full face has been trimmed.

Return the trimmed block to the cold surface for 1 to 2 minutes. Set the advance feed to the desired thickness (3 to 5 m for most purposes). Remove any debris associated with coarse cutting from the knife edge with alcohol. (Xylene should not be used as it often leaves an oily remnant on the knife to which cut sections will stick). Install a fresh, sharp knife in the microtome or move the previous knife to a new, unused area. Re-install the cold block in the microtome and cut a series (or ribbon) of sections at the required thickness. Section compression is minimised by using a sharp knife set to the correct clearance angle. The ribbon is separated from the knife edge with a moist camel hair brush and pulled across the surface of a warm water bath. Section expansion will compensate for the compression caused when cutting. The temperature of the water should be approximately 10C below the melting point of the wax used in the block. Wrinkles in the section can be removed along with small air bubbles trapped beneath the wax, carefully with a moist camel haired brush or metal probe (although the latter may damage to the section). Wrinkles usually develop because different tissue components expand at different rates as the section warms on the surface of the water. Sections can be separated whilst floating on the water with gentle pressure from the tips of forceps. Sections are collected on to a clean glass slide. The slide is held vertical and mostly beneath the surface of the water. The section is apposed to the slide which, when lifted from the water, draws the section with it. Slides are dried for a minimum of 10 minutes to ensure the section is firmly attached. A hot plate set just above the melting point of the wax or a hot air oven are both effective. For delicate tissues more prolonged drying in a hot air oven at a lower temperature may prove beneficial.

Overnight drying at 37C is necessary for maximum section adhesion. Slides left in the open for drying will accumulate dust. Factors for production of good sections The correct clearance angle is important. The clearance angle is that between the knife edge bevel and the block. . Hard tissues can be soaked in Mollifex overnight and cut in usual way next morning. To avoid fragmentation of tissue , the block should be coated with celloidin between sections. PROBLEMS AND CAUSES Crooked or uneven ribbons Edges of knife and block are not parallel The four sides of the block are not trimmed parallel The knife edge is not regularly sharpened Impurities in the wax causing dense areas Varying densities in the tissue Wrinkled or compressed sections Blunt knife Warm block or knife Clearance angle too great Micrometer set too thin for wax hardness Loose knife

Thick and thin sections Loose knife or loose block Block too large for the microtome in use Knife clearance angle too fine Tissue or wax excessively hard

Areas of calcification present in tissue Ribbons split vertically or scratch line appearance Small nicks in the knife edge Hard material embedded in the wax (grit, dirt, crystals, talc) Hard material in the tissue (calcium,crystals, sutures) Dirt on the knife edge Sections crumble or tear Tissue is incompletely dehydrated Clearing not done properly Paraffin wax denatured from excessive heating Blunt knife Wrong melting point wax used for the conditions - wax too soft Wax has crystallised from slow cooling Wax is contaminated with clearing agent or water Sections show areas of varying thickness (chatters) Loose knife or block Knife clearance angle too great Excessively hard tissue or wax Areas of calcification present within tissue Sections adhere to block on upstroke of microtome Knife clearance angle too fine Blunt knife Dirty knife edge or dirty underside of block Sections will not ribbon Knife clearance angle too fine or too great

Dirty knife edge Wax surface too cold Sections roll up on cutting Blunt knife Knife clearance angle too great Micrometer set too great Wrong melting point wax for the conditions Sections disintegrate on water bath Water temperature too high Wax contaminated with clearing agent References Theory and practice of histological techniques, Bancroft. DJ, Gamble. M; fifth edition, 2002, Edinburgh, Churchill Livingstone. Culling FA, Allison RT. Cellular Pathology technique 4thedition. www.google images.com