Histopathology - CRYOSTAT

CONTENT
MICROTOME DEFINITION
TYPES OF MICROTOME
USES OF MICROTOME
PROCEDURE
APPLICATIONS

1. Microtome By, S.Shruthi II Bsc.CLT Dr.NGP COLLLEGE

2. TYPES OF MICROTOMES Rotary microtome Rocking microtome Base sledge microtome Sliding microtome Freezing microtome Vibrating microtome Laser microtome Saw microtome Cryostat microtome Ultra microtome

3. Rotary Microtome

4. Rocking Microtome

5. Base Sledge Microtome

6. Sliding Microtome

7. Freezing Microtome

8. Vibrating Microtome

9. Laser Microtome

10. Saw Microtome

11. Cryostat Microtome

12. Ultra Microtome

13. What is cryostat? Cryostat is a device by which temperature can be maintained in a low level. In pathology and histology it is known as a chamber containing a microtome for sectioning frozen tissue.

14. Cryostat is designed for the optimal production of cryo-sections from the tissues to examine. Most frequent use is by pathologist in pathology laboratories , but also many research facilities require a cryostat. This automated cryotome has a UV source which is used for 35 minutes to sterilise the instrument and specimen off-cuts. Another special feature is it facilitates sectioning and especially reduces ice crystal condensation on specimen and knife by using a lower temperature on surrounding parts.

15. The cryostat is essentially an ultrafine “deli-slicer”, called a microtome, placed in a freezer. The temperature can be varied, depending on the tissue being cut - usually from minus 20 to minus 30 degree Celsius. The freezer is either powered by electricity, or by a refrigerant like liquid nitrogen . Small portable cryostats are available and can run off generators or vehicle inverters. To minimize unnecessary warming all necessary mechanical movements of the microtome can be achieved by hand via a wheel mounted outside the chamber.

16. The Newer microtomes have electric push button advancement of the tissue. Tissue are sectioned as thin as 1 micrometre. Once frozen, the specimen on the chuck is mounted on the microtome. The crank is rotated and the specimen advances toward the cutting blade. Once the specimen is cut to a satisfactory quality, it is mounted on a warm (room temperature) clear glass slide, where it will instantaneously melt and adhere. The glass slide and specimen are air dried, and stained. The entire process from mounting to reading the slide takes from 10 to 20 minutes, allowing rapid diagnosis in the operating room, for the surgical excision of cancer. The cryostat section quality is poorer as compared to fixed tissue sections.

17. Although cryosections are physically less stable than paraffin they are generally superior for the preservation of antigenicity and therefore the detection of antigens by microscopy. The preparation of cryosections does not involve the dehydration steps typical of other sectioning methods, and, furthermore, sectioning, labelling, and observation of specimens can usually be carried out in one day. In general, the sample is frozen quickly in either isopentane or liquid nitrogen. Rapid freezing reduces ice crystal formation and minimizes morphological damage. Frozen sections may be used for a variety of procedures, including immunochemistry , enzymatic detection.

18. The equipment contains the following main components: Some of the panels are under computer control ; showing temperature and working conditions . The middle of the unit contains to the low-temperature unit for quick freezing of biopsy tissue and section cutting operations The lower part of the unit holds the refrigeration compressor unit Behind the low-temperature unit holds the mechanical drive and motor drive units .

19. Features : Control System: Automatic temperature is computer-controlled. The LCD displays three temperatures. Shown are The working conditions of all hardware, Compressor A Compressor B Lighting UV Defrost and advance and withdrawal of the motor Freezing of the table and the knife. The instrument has a count-down display when the compressor is started.

20. Four freezing points: the dual-compressor control system has four thermostat control points – the head, knife, table and box. The head can be set to different sectioning temperatures. Freezing speed: there is no need to pre-cool, the instrument can get into freezing mode after only 10 min from start up. It can be switched off after finishing work to save power.

21. Operating temperature: The cryostat sections work with using very low temperatures. This saves energy and time and also reduces ice crystal condensation. A hard matrix achieved by a lower temperature may be required for hard specimens. System protection: The rapid freezing system can be switched off manually at any time. After working 8 hours, the rapid freezing system will switch off automatically.

22. Defrosting system: It has automatic and manual defrosting functions. 1) defrosting time interval; 2) defrosting time; 3) manual defrosting may be set. These settings may be varied according to ambient temperature. The defrosting function can be switched off any time; the instrument will change to freezing mode after the defrosting function is switched off. Mechanical design: The instrument's mechanical construction is superlative . The use of roller bearings makes the instrument very stable in the vertical plane. The mechanism is enclosed and maintenance-free. Mechanical performance is excellent for the production of uniform flat and stable section; a fine anti-curling function is provided.

23. Sterilisation: This cryostat has a UV disinfection function, and the freezing box is designed for easy cleaning of sectioning debris. Tissue endothermic device: It can freeze tissue rapidly using the freezing table. The big freezing table is suitable for hospitals. Steel knives and disposable blade holders are interchangeable. Knife movements control advance, retraction and sectioning speeds.

24. Specifications Section thickness range : 1μm to 80μm Increments of 1μm from 1μm to 20μm Increments of 2μm from 20μm to 40μm Increments of 5μm from 40μm to 80μm Trimming thickness range : 10μm to 400μm adjustable Increments of 5μm from 10μm to 50μm Increments of 10μm from 50μm to 100μm Increments of 50μm from 100μm to 400μm

25. Minimum division value of section: 1μm Vertical movement of head: 60mm Horizontal movement of head: 20mm Rough feeding speed: 0.7mm/s or 0.35mm/s Working time: Rapid freezing system switches off automatically after 8 hours Maximum specimen size: 35 x 35mm Angle adjustment of section knife: 0° - 10°

26. Temperature range: Freezing chamber: -30°C to -10°C Time freezing shelf to reach -30°C: 60 minutes Freezing shelf minimum temperature: -45°C Minimum temperature of heat extractor on freezing shelf: -55°C Heat extractor working time : 15 minutes Electrical: 230VAC 50Hz 650W Dimensions (W x D x H): 660 x 640 x 1130mm Weight: 125 kg Working noise: 65dB (A)

27. Care of the microtome: Dust accumulation must be prevented by putting a cover when not in use. Wipe the moving parts regularly with good neutral oil (e.g. coconut oil) to lubricate and avoid rust. After cutting, clean frequently from accumulated paraffin using a soft brush with xylene. Never adjust the screws too tightly that they may cause binding.

28. METHOD:  Freeze a fresh, unfixed tissue sample, up to 2.0 cm in diameter, in OCT in a suitable tissue mold. Freeze the OCT containing the tissue onto the specialized metal grids that fit onto the cryostat . (OCT is viscous at room temperature and miscible with H 2 O, but freezes into a solid support at −20°C.Certain soft tissues, such as brain, are optimally frozen in M-1 medium at −3°C.) Cut sections 5-15 μm thick in the cryostat at −20°C. If necessary, adjust the temperature of the cutting chamber ±5°C, according to the tissue under study. ( A camel hair brush is useful to help guide the emerging section over the knife blade.)

29. Within 1 min of cutting a tissue section, transfer the section to a room temperature microscope slide by touching the slide to the tissue. (The tissue section will melt onto the slide. This must be accomplished within 1 min of cutting the section to avoid freeze-drying of the tissue.Poly-L-lysine-coated or silanized slides improve the adherence of the section.) To evaluate tissue preservation and orientation, stain the first slide of each set with toluidine blue (1%-2% w/v in H 2 O), haematoxylin, and eosin, or any aqueous stain. Immediately immerse the slide into an appropriate fixative

30. To maximize the adherence of the section to the slide, some researchers allow the section to air-dry onto the slide at room temperature before fixing the sample. The disadvantage of this is that surface tension forces distort the cells, causing loss of high-resolution detail. Air-drying may also cause some changes in immunostaining results. Cover any unused tissue with a layer of OCT to prevent freeze-drying and store the rest of the sample at −70°C. (For long-term storage, a moistened tissue should be added to the container with the block to prevent desiccation)

31. Difference between a Microtome and a Cryostat A Microtome is used to cut very thin sections at room temperature, on the other hand a Cryostat is used to cut frozen sections at sub zero temperatures (generally -30 deg C). A cryostat is used in situations where rapid analysis of tissues is required. The water rich tissue is frozen on a quick freezing shelf inside the cryostat, this makes it very hard and it is then ready to be cut into thin sections in a microtome, also placed inside the cryostat chamber.

32. On the other hand to be cut in a simple microtome, the tissue needs to be first dehydrated and fixed in paraffin before  it can be sectioned. It is a long procedure compared to quick sectioning in a cryostat. The quality of sections cut in a cryostat is inferior compared to those cut in a microtome because dehydrated and paraffin embedded tissues give better sections but when it comes to quick sectioning, Cryostat is the choice. 

33. FILL UP : 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

34. ANSWERS: 10% Formalin. Saline Optimum cooling temperature or 20% sucrose Carbon dioxide, Liquid Nitrogen.