TISSUE SECTIONING

Trimming

The Microtome

• Sharp razor - earliest form of microtomy

• Modern microtomes are precision instruments designed to cut uniformly thin sections of a variety of materials for detailed microscopic examination.– 1,800x, the thickness of a section (1

and 10 microns or thin sections – For EM, several hundred thousands

magnification (10 nm or ultra-thin sections)

Parts of the Microtome

1. Base (microtome body)

2. Knife attachment and knife

3. Material or tissue holder

Types of Microtome

• ROTARY MICROTOME – general purpose microtomes (also

found in cryostat)

Types of Microtome

• ROTARY MICROTOME – for cutting semi-thin to thin sections

for light microscopy– Rotary action of a hand wheel to

activate 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.

– Lightweight: Suitable for cutting paraffin -embedded mat’l in a continuous ribbon

Types of Microtome

• ROTARY MICROTOME – Heavyweight: motor driven instruments

used with a slow, continuous speed and retracting advance movement to section hard material embedded in synthetic resin.

– Section thickness settings range from 0.5µm to 60µm on most machines.

– Sections of paraffin wax embedded tissues are normally cut within the range 3 to 5µm whilst resin sections are between 0.5 to 1µm.

– Usually used with disposable steel knives

Types of Microtome

• SLIDING MICROTOME

Types of Microtome

• SLIDING MICROTOME– well suited to cutting a wide range

of materials including bone, plastics, resins and wood as well as large area soft tissues

– Thick sections

Types of Microtome

• SLEDGE MICROTOME

Types of Microtome

• SLEDGE MICROTOME – 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.

Types of Microtome

• SLEDGE MICROTOME – very heavy for stability and not

usually subject to vibration– can also be used to cut

materials from various industrial applications (wood, plastics, textile fibers)

– not suitable for cutting very hard resins such as araldite because of the risk of vibration

Types of Microtome

• FREEZING MICROTOME

Types of Microtome

• FREEZING MICROTOME – for cutting thin to semi-thin sections of

fresh, frozen tissue and semi-thin sections from industrial products such as some textiles, paper, leather, soft plastics, rubber, powders, pastes and food products

– 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.

Types of Microtome

• FREEZING MICROTOME – Some cooling systems also allow the

knife to be cooled at the same time. – Note: the knife moves while 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.

Types of Microtome

• ULTRAMICROTOME

Types of Microtome

• ULTRAMICROTOME

Types of Microtome

• ULTRAMICROTOME – used to prepare ultrathin sections

for light and electron microscopy. – Very small samples of tissue or

industrial products are usually embedded in hard resin before cutting.

– It has been reported that sections can be cut as thin as 10 nanometres.

Types of Microtome

• ULTRAMICROTOME – Two forms of advance mechanism

• thermal mechanism relies upon heat induced expansion in a bifurcated metal strip

• mechanical form - a microprocessor coupled to a precise stepping motor controls the advance mechanism. 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.

Types of Microtome

• CRYOSTAT

Types of Microtome

• CRYOSTAT– primarily used for cutting sections

of frozen tissue as well as pastes, powders and some food substances.

– 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.

Types of Microtome

• CRYOSTAT– usually contains a rotary microtome

although some portable units utilise 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.

Types of Microtome

• SAW MICROTOME

Types of Microtome

• SAW MICROTOME– cut sections from very hard material

such as undecalcified bone, glass or ceramics.

– Samples (commonly embedded in resins) are moved extremely slowly against a diamond coated saw rotating at approximately 600 rpm.

– Sections of 20 µm or greater are possible providing the saw blade is in perfect condition.

– Very thin sections are not possible.

Types of Microtome

• VIBRATING MICROTOME

Types of Microtome

• 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.

Types of Microtome

• VIBRATING MICROTOME– 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 while immersed in a fluid which also aids in dissipating heat produced at the vibrating edge of the razor as it cuts.

Types of Microtome

• ROCKING MICROTOME

Types of Microtome

• ROCKING MICROTOME– Comprised of three moving parts– 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.

Types of Microtome

• ROCKING MICROTOME– 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 µm in thickness are possible.

Types of Microtome

• HAND MICROTOME

Types of Microtome

• HAND MICROTOMEThe 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.

Microtome Knives

• STEEL KNIVES– manufactured from high quality carbon

or tool grade steel which is heat treated to harden the edge.

– should be free from impurities, contain anti-corrosives and be rust-resistant.

– The best knives are those that are fully hardened

Microtome Knives

• NON-CORROSIVE KNIVES FOR CRYOSTATS– manufactured from hardened, heat

treated stainless steel free from all impurities and containing 12 to 15% chromium.

Microtome Knives

• DISPOSABLE BLADES– are essentially refined, thickened razor

blades. – produce high quality sections and have

replaced conventional microtome knives in many instances.

– manufactured from high quality stainless steel

– edges can be coated with platinum or chromium to enhance strength and prolong cutting life

– Teflon coated for cryostat

Advantages disposable blades1. Substantial cost reduction2. Consistent section reproducibility & section

quality3. reaches cryostat chamber temperature more

rapidly than a conventional knife minimizing time delay during blade exchanges or temperature adjustments.

4. Support of all common microtome systems 5. For all paraffin embedded samples6. Expensive resharpening no longer required7. No longer dependent on quality of selected

resharpening service8. No downtime while knives are at the

resharpening service

Microtome Knives

• TUNGSTEN CARBIDE– non corrosive– practically non magnetic – 100x harder than hardened tool

steel– excellent resistance to wear – brittle because of their extreme

hardness and should be handled carefully

Microtome Knives

• GLASS KNIVES– 'Ralph knives' – Need Glass knife holders – Glass knives are hard but brittle and

care is required with their handling. – deteriorate with storage due to changes

in the 'flow' or 'strain' of the glass after fracture and from oxidation impurities remaining in the hardened glass after manufacture. Knives should thus be prepared immediately before use.

Microtome Knives

• GLASS KNIVES

Microtome Knives

• DIAMOND KNIVES– manufactured from gem quality

diamonds without flaws.– 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.

– Available in different STYLES for diff. purposes

– Resharpened by manufacturer (unless extremely damaged)

Microtome Knives

• SAPPHIRE KNIVES– manufactured from one piece of solid

sapphire artificially produced from an alumina monocrystal under computer controlled thermal conditions.

– Sapphire is harder than tungsten, carbide or glass which ensures high durability of the cutting edge for all types of material.

– The only restriction when using a sapphire knife is block size as the knife edge is limited to 11 mm. A special knife holder is required.

Profile of Steel Knives

• The cutting edge of all steel knives is produced by grinding a bevel on each side of the knife for profiles A, B and C, or onto the angled surface of a profile D knife. The bevel faces enclose a sharper angle than the main surfaces of the knife

Profile of Steel Knives• Profile A: strongly plane concave/biconcave

– Both knives are extremely sharp and are used for cutting soft, celloidin embedded material or foam compounds.

– not suitable for relatively hard materials, which cause the edge to vibrate and produce the phenomenon known as chattering.

– To obtain the best result the knife should always be oblique to the object when cutting sections.

Profile of Steel Knives

• Profile B: plane concave – similar to a profile A knife but has a

thicker back– used for cutting sections from material

which is too hard to cut with a profile A knife but can also be used for softer materials embedded in paraffin wax.

– also suitable for cutting the softer components (stalks, leaves) of fresh botanical specimens. This knife should be positioned obliquely to the material being sectioned.

Profile of Steel Knives

• Profile C: wedge Shaped / plane wedge– has more rigidity than profile A or B

knives and can therefore be used for cutting harder materials.

– cannot be ground as sharp as profile A or B knives.

– Commonly used for cutting sections from paraffin wax embedded material, frozen sections, cryostat sections and for small, synthetic resin embedded material this knife can also cut soft plastics, rubber, wood and some textile fibres. With this style of knife the cutting plane is transverse to the object.

Profile of Steel Knives

• Profile D: plane Shaped – cut hard and tough material as it

has greater stability than any of the other profile knives.

– least sharp of all of the profiles. – commonly used for cutting

synthetic resin blocks, hard materials embedded in paraffin wax, large wax blocks and various substances used in industry.

Sharpening Steel Knives

• can be achieved manually or by using an automatic knife sharpening machine.

• Automatic machines tend to remove more metal during sharpening so that knives become worn quickly.

• Manual methods on the other hand remove far less metal but require more skill, experience and time to produce a satisfactory edge.

Sharpening Steel Knives