Face down cryoembedding technique

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1 )Apply a thin layer of embedding medium to the tip of the slide

2) Place tissue FACE DOWN on the dispensing slide.

3) Look through the underside of the slide while adjusting the tissue so desired margin or other aspect is visible in position.

4) Touch the leading edge of the tissue to the well floor . It will adhere.  Consider the best positioning of tissue in the well. How will it fit best? How do I want the tissue to hit the blade.

5)Pull the dispensing slide out from under the tissue.


 

6)Tissue requiring precise positioning can be flattened or manipulated and guided into desired position by carrying out this step slowly and carefully.

7) Fill the well so that a meniscus of medium is bulging above the brim. 
-Too much medium in not a problem. Too little is a problem.

8) Press the chuck over the well as quickly as possible.

9) Place the over chuck freezing block over the stem.

10) Remove block with a tap to the chuck stem using the over-chuck block

Freezing time

 Approximate freezing times at -24C. using a cold chuck:

18mm well - 20 seconds
24mm well - 35 seconds
30mm well - 60 seconds

Freezing will take longer if the chuck is warm; if the tissue is very thick; if bar is allowed to warm; or at warmer cryostat temperature.

Results

Untrimmed block

Trimmed block

Close-up picture of slide

Micrograph 50x


Various every day uses

Each in a single well

Large piece of kidney

Sample four pieces

Sample nine pieces


Strips

1mm skin strips on edge

Lots of little pieces

The slides


Liquids

This system easily handles liquids and very soft specimens creating three dimensional blocks from specimens that would easily crush using conventional cryostat methods.

Liquid and very bloody or water density specimens such as curettings often shatter when cut because they get icy hard on freezing.

-Keep a household spoon to scrape up these specimens and place them into wells. Stir in a bit of embedding medium in the spoon before  putting tissue into the well

This will decrease the shattering considerably. Scrape the tissue off the spoon with a dispensing slide.


Micro-arrays

These (25) 3-4mm pieces took two minutes to place on the well floor. 50 to 100 pieces or more can be easily arranged in a well where adhesion to the floor makes orderly placement child's play. Even previously frozen cores can be "glued to the well floor with a touch of OCT. These arrays cut like any other blocks. There is no separation of tissue from embedding medium when cutting because there is no coring involved. The sesame seed pinwheel an example of what's possible.


 The Cut off Trick 

A quick way to a very well embedded clean section. Works great with ureter margins which typically have telescoping of the mucosa when trying to cut off the margin section.

 
 

1) Place the end of the tissue in a drop of embedding medium.

2) Touch the margin to the well floor to which it will adhere.

3) Holding the tissue with a forceps cut it off with a scalpel blade at the level of the well bar surface. Make sure your blade is sharp and use a gentle slicing motion with the scalpel.

4) Fill the well and apply the chuck.

This trick can be applied to anything that is difficult to neatly trim a 3 mm. section without disturbing the integrity of the anatomy. The bond of the freezing steel holds the tissue fixed and the well bar surface acts as a guide for the scalpel. Bronchial margins can be cut easily in a large piece and placed margin down on the well floor. Next cut off the excess. Try it with a uterine cervix. The tissue can be oriented on a dispensing slide and applied precisely to the well floor before cutting off the upper portion.


Reducing freezing artifacts

Cold embedding medium

The precision cryoembedding system rapidly begins freezing tissue when the tissue touches the well floor. In my experience there is reduced freezing artifact when compared to conventional cryostat technique. However there will still be some ice crystal formation particularly in very edematous and watery tissues. This can be reduced by keeping a bottle of embedding medium (OCT) in the refrigerator. I routinely use cold embedding medium on my brain biopsies, thyroid lung tissue, kidney and anything that I am concerned with freezing artifact. Bowel submucosa can be very edematous  Cold embedding medium will also further speed the freezing times.

Pre-chilling tissue

There is less freeze artifacts in tissues which are rapidly frozen. The tissues we receive in clinical practice are usually somewhere between body temperature and room temperature.  The amount of heat removed from a piece of tissue to reduce the temperature from 37 degrees C to 0 degrees C is  going to be 37 times more than to go from 1 degree to 0 degrees. Therefore our tissue should freeze much faster if chilled.

I have been keeping a piece of metal and a dispensing slide in my refrigerator. In cases where I am willing to spend the extra effort I put the tissue first in the cold dispensing slide on the metal and use cold embedding medium. Give the tissue 30 seconds or so to cool down and put it in the well. Fill the well with cold embedding medium.

For situations where snap freezing in super cooled liquids in necessary pre-chilling the tissue may improve results.