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Leica CM3050 S Sectioning

Leica CM3050 S Research Cryostat

Cryostats

How do I choose the right knife holder and specimen clamp?

There are five different knife holders and four specimen clamps available. Each one has been developed to suit the different types of specimens and to allow you a great variety of applications - whether for use in the histopathology or research lab or for sample preparation in the industrial quality assurance lab. Please refer to the chart by clicking on the below hyperlink. Download the chart

What are the pro's and con's of disposable blades versus conventional knives?

  1. Advantages in the application of disposable blades over conventional knives:
    - Substantial cost reduction
    - Consistent section reproducibility
    - Consistent section quality
    - Support of all common microtome systems
    - For all paraffin embedded samples
    - For all cryosectioning applications
    - Expensive resharpening no longer required
    - No longer dependent on quality of selected resharpening service
    - No downtime while knives are at the resharpening service
  2. Standard re-usable steel knives and disposable blades in comparison with standard microtome blades: In typical histology laboratories, 50-100 blocks on average per day and person will be handled. Depending on the type of specimen and the way of trimming, up to 50 specimens and more can be worked on. Assuming the laboratory is handling all the different types of tissues that are typically processed in a histopathologyl lab, up to 20 blocks can be cut with high quality microtome blades retaining the same sectioning quality.

  3. Conventional steel knife 16 cm c-profile: As a rule, no more than 80 to 100 blocks will be cut by a steel knife with consistent section quality. Thereafter, the steel knife has to be sharpened regularly to regain the required knife performance. Due to the design of microtome knife holders, only 70% to 80% of the total knife edge can be utilized for cutting. The remaining 20% to 30% are not accessible because of the clamping principle commonly used.

What are the reasons for using cryotechniques?

  1. Rapid interoperative consultations and diagnosis.
  2. Preservation of enzyme activity for enzyme histochemistry and antigenicity for immunohistochemistry and in situ hybridization.
  3. Retention of substances that are soluble in routine processing solutions, such as lipids.
  4. Preservation of cell morphology without exposure to chemicals and/or heat.
  5. Can be performed on fixed and unfixed tissue specimens.

How to avoid ridged sections?

  1. The blade edge is damaged; use different part of the cutting edge.
  2. The edge of the anti-roll plate is damaged, replace the anti-roll plate.

How to avoid chatter or Venetian blinds?

  1. The specimen might be improperly frozen onto the specimen disc. Remove the specimen; ensure that the disc is clean and that the grooves are cleared of any build up of cryocompound. Remount the specimen on the disc using a minimum amount of cryocompound.
  2. The specimen disc is not secured properly. Ensure that the disc is properly inserted into the specimen head.
  3. The specimen is very hard, or not homogeneous. Reduce the specimen surface area if necessary; cut at the temperature necessary for the most difficult to freeze component of the tissue; if possible, trim the block into a V shape and orient the block so that the smallest surface will contact the blade first.
  4. Rigorous trimming of the block has caused the specimen to detach from the disc. Re-mount the specimen onto the disc.
  5. The blade is not clamped properly. Check the clamping. It may be overtightened. (Position the locking lever so it is in the same plane as the disposable blade front plate.)
  6. The cutting edge is dull. Use different parts of the cutting edge or replace the blade.
  7. The knife profile is inappropriate for the specimen to be cut. Use knife with different profile that can withstand the cutting force.
  8. The clearance angle is incorrect. Set correct angle.
  9. Cutting speed is too fast and should be slowed down.

How to avoid thick-thin sections?

  1. The temperature is incorrect for the tissue to be cut. Select correct temperature.
  2. The cryocompound and specimen have become detached from disc after freezing. Remove the specimen and ensure that the disc is clean and that the grooves are cleared of any build up of cryocompound. Remount the specimen on the disc using a minimum amount of cryocompound.
  3. The specimen disc is not clamped tightly enough. Check disc clamping.
  4. The specimen arm is fully extended and the knife holder is too far back to be clamped tightly onto the microtome base. Move the specimen arm all the way to the home position and reposition that knife holder squarely over the clamping device.
  5. The knife profile is inappropriate for the specimen to be cut. Use a knife with different profile (c or d) or possibly switch to a disposable blade system.
  6. The knife may have ice or debris built-up on the back side. It should be cleaned.
  7. The knife/blade is clamped using either too much or not enough pressure. Check knife clamping.
  8. You may be using a blunt knife. Use a different area of the knife edge or replace the knife.
  9. The cutting speed is irregular or too fast. Turn the handwheel in a moderate, hesitation-free manner. Automate if possible.
  10. The clearance angle may be incorrect. Set correct angle.
  11. The section thickness may be inappropriate. Select correct section thickness.

Why would the tissue stick or crumble on the anti-roll plate?

  1. The anti-roll plate is too warm or incorrectly positioned. Cool down the anti-roll plate or reposition the plate.
  2. Specimen is too warm. Select lower temperature.
  3. There is adipose tissue, other tissue fragments or debris on the edge of the anti-roll plate or blade edge. Clean with alcohol or acetone.
  4. There is rust on the knife. Remove rust.

Why do sections tear as they are being cut?

  1. The blade/knife edge is blunt or there is dirt, dust, frost or rust on the edge. Remove the cause or replace the blade.
  2. The leading edge of the anti-roll plate is damaged. Replace the plate.
  3. The back of the blade back is dirty. Clean, or replace the blade.
  4. The temperature is too low for the tissue to be cut: increase (raise) the temperature, wait for a few minutes and try sectioning again.

What do I do if I hear a scraping noise during the sectioning and specimen return movement?

  1. The anti-roll plate projects too far beyond the cutting edge and is scraping on the specimen surface. Re-adjust anti-roll plate correctly.

  2. Check knife angle and increase if necessary.
  3. If there is a singing noise, the specimen might be too hard for the type of blade/knife. Select a blade/knife that is appropriate for the specimen.

How does the anti-roll plate get damaged after it is adjusted?

  1. The plate is extended too far past the cutting edge.
  2. The adjustment of the plate was carried out in direction of the cutting edge with the glass on the sharp surface.
  3. If the plate is damaged, it must be replaced or another portion of the plate must be used.

Why should specimens not be stored inside the cryostat?

The cold air inside a cryostat is very dry and will rapidly dehydrate the specimen.
  • Specimens may be stored during the day when the automatic defrost cycle is not activated but they must be covered by a layer of cryocompound or wrapped in aluminum foil to prevent dehydration.
  • Not only is the specimen dehydrated, but also the cryocompound surrounding the specimen. The cryocompound sectioning properties are negatively influenced (feels like sectioning chewing gum).
  • When sectioning of a specimen is completed, remove the remaining sample from the specimen holder while still frozen and wrap in a double layer of aluminum foil, label and store in a sealed container in a -80 °C freezer.