Freezing microtome_ cryostat.pptx

Cryostat

1. Freezing microtome/Cryostat

2. Introduction Microtomy Means by which the tissue comes in contact with the cutting tool (knife or blade) such that tissue is sectioned and attached to a surface for microscopic examination.

3. Introduction - 2 MICROTOME It is a mechanical device used for cutting uniform slices of tissue sections

4. Introduction - 2 MICROTOME It is a mechanical device used for cutting uniform slices of tissue sections

5. Types of Microtome There are several types of microtome, each designed for a specific purpose, although many have multifunctional roles.

6. Rotary microtome Rotation of a fine advancing hand-wheel by 360° degrees, moving the specimen vertically past the cutting surface and returning it to the starting position. Most common type

7. Types of rotary microtome Manual rotary microtome Completely manipulated by the operator

8. Types of rotary microtome Semi automated rotary microtome One motor to advance either fine or course hand wheel

9. Types of rotary microtome Fully automated rotary microtome Two motors that drive both the fine and the course advance hand-wheel

10. Types of Microtome Base sledge microtome Specimen is stationary Knife slides across the top of the specimen during sectioning Used for large blocks, hard tissues, or whole mounts, Neuro and ophthalmic pathology.

11. Types of Microtome Rotary Rocking microtome One of the oldest design Knife is fixed, the tissue block moves through an arc and strikes against the knife Disadvantages: Size of the blocks that can be cut is limited Sections are cut in a curved plane Cheap, reliable, simple to use Requires minimal maintenance

12. Types of Microtome Sliding Microtome The knife is stationary Specimen slides under the blade during sectioning. This microtome was developed for use with celloidin-embedded tissue blocks.

13. Types of Microtome Ultra Microtome Exclusively for electron microscopy Ultrathin sections upto 10nm Glass/ diamond/sapphire knives Block is brought close to the knife edge under a microscope As each section is cut, it is floated on a water bath adjacent to the knife

14. Types of Microtome Freezing microtome Consists of a fixed stage over which the knife moves Carbon dioxide gas is used to freeze tissue It absorbs large heat and rapidly freezes the tissue

15. Types of Microtome Vibrating microtome High speed vibration is used Designed to cut tissues which have not been fixed or frozen Sections are thicker Tissue is immersed in water Enzyme histochemistry and ultrastructure histochemistry

16. Types of Microtome Cryostat Refrigerated cabinet with a microtome housed in a deep freeze cabinet - 15 to -30 degrees C Good, thin high quality frozen sections Most of the cryostats use a modified rotary microtome

17. PRINCIPLE of FROZEN SECTION When a tissue is frozen, the water within the tissue turns to ice and in this state the tissue is firm, with the ice acting as the embedding medium.

18. cryostat

19. Access to the chamber is via a sliding window. Working temperature : 0 to -35 degree Celsius Rotary microtome controlled by an external hand wheel. Freezing shelf knife holder

20. HEAT EXTRACTOR

21. HAND WHEEL

22. ANTI ROLL PLATE Prevent rolling or curling of tissue Glass plate supported on an aluminium frame Provides gap between underside of glass and upper surface of knife.

23. Anti roll plate

24. TEMPERATURE SETTINGS Digital display For most tissues: -15 to -23 degree Celsius is used For tissues with more fat: Colder temperature Temperature log maintained Defrosting done daily Rapid freezing to be done to reduce freeze artefacts

25. CHUCKS

26. Thank You

27. 1. To avoid drying, the tissue should be kept in ...................... 2. Tissues can be fixed with ...................... 3. ...................... or ...................... is used as embedding media 4. ...................... gas is most commonly used with freezing microtome