Microtomy

PATHOLOGY -PLANT VIROLOGY

1. Acharya N.G. Ranga Agricultural University S. V. Agricultural College, Tirupati COURSE NO : PL PATH-502 COURSE TITLE : Plant virology TOPIC : Microtomy SUBMITTED BY: M. Lokeswaridevi TAM -2020-030, Dept of Entomology

2. Microtomy The means by which tissue can be sectioned and attached to a surface for further microscopic examination. Derived from the Greek mikros , meaning “small”, and temnein , meaning “to cut”) It is a mechanical device for cutting thin uniform slices of tissue sections.

3. History The earliest form of microtomy was the freehand sectioning of fresh or fixed material using a sharp razor .

4. 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. Invention of the microtome is given to the anatomist Wilhelm His .

5. Based on material of the knife: Steel knives Glass knives Diamond knives Sapphire knives

6. Steel Knives Steel microtome knives are manufactured from high quality carbon or tool grade steel which is heat treated to harden the edge. The steel should be rust resistant, free from impurities and contain anti-corrosives.

7. Non-corrosive knives for cryostat Disposable blades Tungsten carbide knives Based on compositon

8. Tungsten carbide knives Noncorrosive Practically nonmagnetic 100 times harder than hardened tool steel.

9. Non-Corrosive Knives For Cryostats These are manufactured from hardened, heat treated stainless steel free from all impurities. They contain 12 to 15% chromium.

10. Disposable Blades These are essentially refined, thickened razor blades. All disposable blades are manufactured from high quality stainless steel. The edge of disposable blades can be coated with platinum or chromium to enhance strength and prolong cutting life

11. Glass knives

12. Hard but brittle Care is required while handling These knives 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.

13. Diamond knives Manufactured from gem quality diamonds. Very expensive the knives are extremely durable, because of the hardness factor of the diamond, and are used primarily for cutting ultrathin, resin sections.

14. Sapphire knives Manufactured from one piece of solid sapphire artificially produced from an alumina monocrystal under computer controlled thermal conditions.

15.   The knife edge can be classified according to its shape / profile. PROFILE – A: Strongly Plano concave/biconcave PROFILE – B: Plano concave PROFILE – C: Wedge Shaped PROFILE – D: Plane Shaped/Tool edge shaped

16. Profiles of the Knives.

17. Angles of Knives RAKE ANGLE

18. Sharpening of knifes Honning: It removes all irregularities from knife.

19. TYPES OF HONE Belgian black vein Arkansas Aloxite Tam’o Shanter Scotch Carborundum Plate glass

20. Stropping To polish & sharpen the cutting edge .

21. Classification of microtomes

22. Manual microtomes: Rocking microtome Rotary microtome Sledge microtome Freezing microtome Vibrating microtome Ultra microtome Cryostat Sliding microtome Saw microtome Hand microtome

23. Rocking Microtome This instrument is one of the oldest in design, relatively cheap, and is exclusively designed for sectioning paraffin blocks. This microtome comprise of three moving parts, which is extremely reliable and requires minimum maintenance .

24. Mechanism of action Knife is fixed, the block of the tissue moves through an arc to strike the knife. Between strokes the block is moved towards the knife for the required thickness of sections by means of a ratchet operated micrometer thread. Steady backward and forward movement of the handle gives ribbons of good sections.

25. Rotary Microtome Derives its name from the rotary action of the hand-wheel which actuates the cutting movements. Section thickness settings range from 0.5μm to 60μm on most machines. Machines of this sort are general purpose microtomes for cutting semi-thin to thin sections for light microscopy

26. BASE (MICROTOME BODY) KNIFE AND ITS HOLDER BLOCK HOLDER Parts of a Rotary microtome

27. Sledge microtome Become a popular machine for routine use since the world war II Its action is much slower when compared to rocking or rotary type. A large knife is used (24 cm in length) and the knife is usually wedge-shaped which reduces the possibility of vibration and requires less honing.

28. Sliding Microtome In a sliding microtome, the knife is moved horizontally against a fixed block which progresses against it in an inclined plane. The sliding microtome can be used for paraffin-wax embedded sections although it was designed for cutting celloidin- embedded sections

29. Freezing Microtome The machine is clamped to the edge of a bench and is connected to a cylinder of CO 2 by means of a specially strengthened flexible metal tube. Consistent, high quality, thin sections are very difficult to obtain with this type of microtome

30. Vibrating Microtome The vibrating microtome was conceived as a microtome which could produce high quality sections of fresh, unfixed material from animal or botanical sources. The name of the instrument was derived from the high speed vibration produced by a safety razor blade which provided the cutting power.

31. Ultra Microtome Is used to prepare ultra-thin sections for light and electron microscopy . Are usually embedded in hard resin before cutting. That sections can be cut as thin as 10 nanometers. Two forms of advance mechanism have been developed in this style of microtome.

32. Saw Microtome Saw microtome's 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. It is possible to produce sections of 20 μm or greater, provided the saw blade is in perfect condition. The saw microtome is not capable for producing very thin sections.

33. Hand Microtome The hand microtome is limited to sectioning intrinsically rigid botanical material, and it is difficult to obtain thin sections from animal tissues

34. Cryostat Microtome The introduction of fluorescent antibody staining techniques by coons, creech and jones in 1941 led to a need for thin sections (3-5 μm) of fresh frozen tissue free of ice crystal defects . To satisfy these criteria the tissue must be snap frozen at a very low temperature. Linderstrom -lang and mogensen designed the first cryostat in 1938. Coons and his colleagues redesigned it in 1951.

35. microtome of any type but preferably rustproof, which is enclosed and operated within a deep freeze cabinet. The temperature may be regulated between-10ºC to -40ºC. Any crystostat can be used as an alternative to a freezing microtome for rapid sectioning. The microtome may be adjusted to cut sections from 2- 16μm.

36. Laser Microtome Laser microtome is used for precise, non-contact sectioning and was designed to slice samples with high precision. It’s equipped with state- of-the-art femtosecond laser technology. It enables non-contact cutting inside biological tissues and various materials without causing thermal damage. Depending on the material being processed, slice thicknesses of about 5 to 100 μm are feasible.

37. Principle: 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.

38. Computerized Microtome It is equipped with the advanced rapid thermostatic switch, semiconductor freezing, cryo scalped and cryoplate . The computerized microtome can carry out the rapid freezing section or routine paraffin section (dual-purpose).

39. This microtome attains slice thickness in the range of 1- 25μm with least slice adjusting graduation of 1μm and a maximal slice section of 32x32mm. The temperature of cryo scalpel and cryoplate range between 0ºC ~ -18ºC and -10ºC ~ -40ºC respectively

40. Embedding and Sectioning Embedment Light microscopy Tissue infiltrated with molten paraffin wax - which is allowed to cool and harden. Requires dehydration and infiltration with a paraffin solvent - aromatic hydrocarbon (xylene, toluene, benzene ) Provides sufficient support to section to about 3 micrometers minimum with a steel knife. Paraffin can infiltrate deeply into tissue, allowing large blocks and ultimately large sections to be obtained.

41. Precautions to be taken before section cutting Fix Specimens Properly Process Tissue Properly Embed Specimens Carefully Locate Microtome Appropriately Set Blade Clearance Angle Optimally Maximize Blade Life Ensure Blocks are Cold

42. Faults Section too thick Holes from rough trimming Knife lines (vertical striations) Disruption Fine cracks or micro-chatter Coarse chatter

43. SOLUTION Tighten blade and block holders Reduce clearance angle Use softening fluid Rehydrate and surface decalcify Re-embed in fresh paraffin Replace or use new area of blade Clean blade edge to clean excess paraffin

44. Applications Traditional histological technique Cryosectioning Electron microscopy Botanical microtomy

45. REFERENCES MICROTOMES AND MICROTOME KNIVES – A REVIEW AND PROPOSED CLASSIFICATION Ellis RC. The microtome: function and design. Woods and Ellis 2000 Du pont . Sorvall JB-4 Microtome. The light microtome for super thin sections. Newtown, USA; Du Pont Instruments. 1991. http://users.adam.com. au/royellis/microt/microt.ht. Walter F. The microtome manual of the technique of preparation and of section cutting. Germany; Ernst Leitz Wetzlar GMBH. 1980

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