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Leica CM1900 CRYOSTAT - guidelines for use

Who can book the microscope?

The cryostat belongs to Anders Nykjær Lab who has priority in using it. If you are outside AN lab, you must get a permission to use it from Benedicte Vestergaard or Anders Nykjær.

As a general rule, only DANDRITE researchers, including affiliated researchers and team leaders, can be granted permission to use the Leica CM1900 CRYOSTAT in room 374D in building 1116. The access is provided that the user has been properly trained and are in the official online booking system. Any violations of the rules will prohibit the individual/group to use the cryostat.

Booking procedure

  1. Assign a super-user within your group
  2. Introduction and training to the super-user by the contact person
  3. Get trained by your super-user (assigned for each research group)
  4. Get access to the online booking system (contact Benedicte)
  5. Book the cryostat – remember it is NOT ALLOWED to use the cryostat without official training and booking!!!
  6. Bring your own disposable blades for cutting the tissue, OCT, super frost slides.
  7. If you prefer you can bring your own anti-rolling glass (This can be bought here

How to get access to the booking calendar

If you are eligible as user (as defined above) of the CRYOSTAT and want to book user time, then please write an email to Benedicte Vestergaard (benvest@biomed.au.dk), whom will grant you access to the booking calendar.

It is important that all users ensure permission from their Group Leader before usage of the CRYOSTAT, since it is the Group Leader, who will at the end of the year receive invoice for the usage of the CRYOSTAT by her/his group members. To ensure authorization from the respective Group Leader, please include the Group Leader (as cc'ed) in the e-mail correspondence with Benedicte, when asking for access to the booking calendar.

Note that you only need to contact Benedicte to get access to the booking calendar, not to confirm or inform her about your booked slots at the microscope.

User fees for using the CRYOSTAT

There is no fee for using the cryostat however, purchase of knives and other supplies for the usage has to be done by the using groups individually.

Further, prospective costs related to repair/maintenance of the cryostat will be charged on the groups using the microscope proportional to the usage. It is important that all users ensure permission from their Group Leader before usage since it is the Group Leader, who will be charged for prospective costs. However, the total upper limit for maintenance expenses pr. year is set to be: 25,000 DKK.

CRYOSTAT specifications

  • This cryostat has been discontinued by Leica but you can find the user manual here (PDF).
  • Section thickness range: 0 – 60 μm_ _
  • Maximum specimen size: 40 mm
  • Optimal temperature for sectioning (depends on the tissue type): from -15°C to -22°C
  • The knife has a stable position; the specimen is being moved

CRYOSTAT accessories

  • Cryostat chucks (= specimen holders)
  • Absolut or 96% ethanol for cleaning
  • Scalpel to remove excessive OCT
  • Painting brushes for cleaning and collecting the sections
  • Paper tissue for cleaning
  • Light microscope to check the sections
  • Biological waste to trash the samples and the blades
  • Screws for cryostat adjustment
  • Leica CM1900 datasheet
  • If anything is missing, FILL IT UP!

THE MUST RULES when using the CRYOSTAT

  • DO NOT turn off the cryostat. Always check if there is ice accumulation in the chamber. If yes, contact the super-users
  • Allow the cryostat chamber, the blade and the specimen to cool down to the desired temperatures well in advance (~ 30 - 40min)
  • BE GENTLE to the blade holder and the anti-rolling glass. Any scratches will badly affect sections of all users
  • Always keep the glass window closed when not sectioning
  • Always lock the cryostat handle when leaving the cryostat or manipulating in the chamber (safety reasons)
  • Unlock the specimen holder before you turn the knob that adjusts the angles
  • DO NOT leave any stuff not belonging to the cryostat inside.
  • CLEAN UP PROPERLY the whole cryostat and specimen holders when finished
  • Humidity and temperature of the room and thus the cryostat must be as stable as possible because any changes affect the sectioning a lot – do notice any abnormalities and let the super-users know immediately.
  • When working with possibly contaminated or infected material, the general safety guidelines for laboratories must be applied!!!

For good sections:

  • Put the knife, anti-rolling glass and your samples inside of the cryostat to adjust them to the temperature inside of the cryostat (~ 30 - 40min). If they are too warm or too cold it will cause you problems with quality of the sections
  • Make sure all screws are tight - if they are losoe you will get sections of uneven thickness
  • Fix the sample by applying OCT on the cryostat chuck of the right size.
  • Cut away excessive OCT around your tissue
  • Keep the specimen holder temperature around -20°C, some degrees higher for the knife
  • Adjust the height of the anti-rolling glass to the shape of your knife carefully
  • Check your sections under the light microscope and carefully adjust the specimen orientation if needed.
  • Make sure to use clean, sharp, scratches- free knife and anti-rolling glass
  • Use fine sectioning for the actual samples
  • If you have any doubts, check the manual or contact one of the super-users

 

After you are finished with the sectioning:

  • Remove your sample, knife and anti-rolling glass
  • Brush down all the flakes into the tray, take the tray out (make sure the specimen holder and wheel are locked), empty the tray in the biological waste, clean the tray with 96% ethanol and put it back
  • Clean the cryostat chucks and anti-rolling glass properly with 96% ethanol. Make sure there is no OCT left in the cryostat chucks.
  • Set the temperature in cryostat chamber for -11°C.

The Leica CM1900 CRYOSTAT is placed in:
building 1116, room 374D

Leica CM1900 CRYOSTAT coordinator

The contact person is responsible for the cryostat maintenance and super-users training. All groups using the cryostat must assign one “super-user”. After introduction and training the super-user will be responsible for the correct and proper training of all other user from their research group.

For any issues appearing with the cryostat Benedicte must always be contacted.

Technical support

Company Triolab AS (www.triolab.dk)


Jesper Aasted (jesper.aasted@triolab.dk)