Microtome

MICROTOME

1. MICROTOME

2. Why do we preserve tissue samples ??? For gross display in the Museum . For microscopic evaluation .

3. For Microscopic Study The Major tissue component , Is initially fixed using a suitable fixative . Undergoes adequate change in solutions , at adequate time - PROCESSING . Embedded in paraffin wax as BLOCKS . From the blocks , thin tissue sections are cut using specialized equipment called MICROTOME . Stained & studied under a microscope

4. MICROTOMY The art of section cutting. This is a process of cutting thin sections of tissue embedded in the wax block using a microtome . MICROTOME Basic instrument / Mechanical device for cutting thin uniform slices of tissue .

5. HISTORY In the beginning of microscopy , sections were made with R azor blades. This method failed due to the impossibility of light transmission through thicker tissue . Only with proper clean reproducible cuts of 100 μ m , light can be transmitted , allowing precise observation of tissues to be carried out .

6. With this idea , one of the first devices for such cuts were designed by George Adams in the year 1770 & was further developed by William Cunnings . Named as CUTTING ENGINE . Hand operated machine A cylinder to hold sample & a hand crank to cut .

7. Then the word Microtome was derived from Greek   mikros , meaning "small", &   temnein , meaning " to cut" . It is a tool used to   cut   extremely thin slices of material, known as Sections . Important in science to cut sections for microscopical observation. Microtomes use steel , glass , or diamond blades depending upon the specimen type & desired thickness of the sections to be cut . Sections can be made as thin as , breadth of a human hair , between 50 – 100 μ m .

8. After several modifications , today we use microtomes of KNIFE BLOCK DESIGN. It has a Changeable knife , & Specimen holder. Automatic machines are also available , where the sample moves over the knife with a precision control of tissue thickness.

9. Sectioning using Razor blades .

10. VINTAGE MICROTOMES

11. First clinical use of microtome

12. TYPES OF MICROTOMES Based upon the mechanism , Rotary Rocking Base sledge Freezing Vibrating Saw Sliding Cryostat Ultra-Microtome

13. ROTARY MICROTOME So called because of the Rotary action of hand wheel used for cutting movement. The block holder is mounted on a steel carriage , which moves in grooves , up & down. Most ideal for routine & research work . Excellent for cutting serial sections .

14. PARTS OF THE ROTARY MICROTOMES Block holder Knife clamp screw Knife clamps Block adjustment Thickness gauge Angle of tilt adjustment Operating handle

15. The feed mechanism is activated by turning a wheel on  one side of the machine. The wheel operation may be electrical / manual . The knife is fixed with its edge fixed upwards , while the object moves against the knife . 1 rotation of the operating wheel produces , 1 complete cycle with a downward cutting stroke & an upward return. It is often modified to cut ultrathin sections between 50Å – 200Å.

16. Advantages of the Rotary microtome Heavy & stable. Used for large number of serial sections . Ideal for Paraffin-embedded tissues   . Movable knife holder . The sections cut are flat. It is useful for routine & research papers . Currently most common type of microtome in practice.

17. Base Sledge Microtome L arge & heavy instrument . Very stable & popular for routine use . The feed mechanism has a fixed knife , beneath which , the object mounted on a heavy sliding base moves . Mainly used for cutting sections of cellulose nitrate embedded tissues with an obliquely set knife.

18. Parts of Base-sledge microtome Angular tilt adjustment Knife clamps Block holder Coarse feed adjustment Operating handle Thickness gauge Adjustment locking nut Block adjustment screw Split nut clasp

19. The block holder is mounted on a steel carriage which slides backward & forwards in groups against fixed horizontal knife . The block is raised towards the knife at a predetermined thickness. Advantages of Base-sledge microtome Useful to cut extremely hard blocks & large sections ( eg : brain ). The microtome is heavy & stable. The knife used is   sledge   shaped which requires less honing.

20. CAMBRIDGE ROCKING MICROTOME

21. A simple machine invented by Sir Horace Darwin in 1881 . Developed by Cambridge company . The instrument has an arm that moves in a rocking motion while cutting the sections. Hence named as Cambridge rocking microtome. T he knife is held by means of microtome thread. The rocking microtome was designed primarily for cutting paraffin wax sections . When needed , it can also be used to cut frozen section tissues .

22. PARTS OF THE ROCKING MICROTOME Knife holder Block holder / chuck Upper & lower arms Screw Lever Pawl Ratchet wheel Mill head microtome screw Sleeve Scale

23. It cuts the sections between 1 - 20 microns . The knife is fixed with the edge. The object moves against the knife circularly , producing a sharply curved surface to the block . W ith each stroke the tissue holder automatically moves vertically towards the knife . Cutting stroke is Spring operated & is easy to handle . This microtome must be placed on a solid non-slippery surface to allow a better hold .

24. Advantages of  Cambridge rocking microtomes The cost of knife & microtome is low . Celloidin embedded tissues can be sectioned easily .

25. VIBRATING MICROTOMES Cuts using a vibrating blade . The resultant cut is made with less pressure. Used for difficult samples . Thickness around 30 -500nm for fresh tissues & 10 – 500 nm for fixed tissues .

26. SAW MICROTOME Used for hard tissues ( teeth & bones ). Has a recessed rotating saw which slices through the sample . Minimal cutting thickness is 50 μ m .

27. ULTRA MICROTOMES Designed to cut very thin sections . Parts : Low power Binocular microscope . object stage Has both manual / cutting actions.

28. How does it differ from conventional microtomes ? Fine and precise feedback mechanism . Mechanical / thermally controlled Thickness range 5 – 100 nm Special knives - plate glass / plastic Can cut even small blocks . Can be used even in hard embedding media like araldite

29. Freezing Microtomes Designed for the preparing frozen sections of fluid & non-fluid tissue . Preliminary embedding is not needed . A cylinder of compressed carbon-dioxide for rapid cooling of the tissues is connected with the object stage . Provisions to cool the knife is also present . The knife moves horizontally across the tissue surface . Characterstic features - N on-movable tissue block & cooling system.

30. Parts of freezing type microtome Knife clamps Operating handle Thickness gauge Stage Stage valve Coarse adjustment

31. Advantages of Freezing Microtome U sed to cut sections for Rapid diagnosis . It cuts non-dehydrated fresh tissue in a frozen state . Used for Rapid histopathological diagnosis during surgery . This type of microtome is also used when lipids, enzymes, & neurological structures are to be demonstrated .

32. DRAWBACKS OF FREEZING MICROTOME Inactivation of some enzymes Ribbon sections cannot be prepared using this microtome An inability to prepare multiple sections of uniform thickness . Ultimately led to the invention of CRYOSTAT .

33. CRYOSTAT Pearse 1960 ; Hollands 1962 . Also called as Cold microtome / Deci -slicer in refrigerator . Device by which temperature can be maintained at low level . As described by Linderstorm – Lang and Mogeson , The cryostat consists of a microtome contained within a refrigerated cabinet designed to operate at - 5 to - 30 ° C .

34. PROCEDURE Block tissue - up to 3 cm thick Fresh tissue . Block is mounted in a small quantity of 1% glucose / water on the platform of the chuck standing vertically on a bench . Immediately frozen - liquid carbon dioxide / solid carbon dioxide (dry ice). The chuck with frozen tissue is transferred to microtome maintained at -15 to -20° C in the refrigerated chamber . The knife is moved & sections are cut at 4-5 μ m with anti-roll guide plate in position .

35. ADVANTAGES

36. In earlier models of freezing microtomes , temperature controlled manually In Cryostat , temperature control is automatic .

37. I DON’T NEED TO CHECK THE TEMPERATURE OR TISSUE THICKNESS.. That’s a relief !!

38. DISADVANTAGES Complete defrosting of machine is required from time to time. Slow fixation reaction . Cannot be used for fixed tissues. Not useful if the tissue requies lower temperature than the setting temperature of the machine . Morphological details less accurate. Pigment formation is too prominent. Dermatitis of hand & irritation of nostrils to the microtomist .

39. In simple words , Microtome cuts a tissue of only one millimeter into million pieces …

40. Thank you