Placement of tissue in the well
Choose a well that will fit the tissue and leave a rim of embedding medium surrounding the tissue. This rim I call the “handle” This gives us brush users something to grab. It also provides a margin of error before the tissue so if there is curling it may not involve the tissue.
-Consider how the tissue will fall when it’s placed in the well so that you avoid overlapping the tissue on the edge of the well. The dispensing slides make this quite easy. Just look through the slide as it's placed over the well and you will see if and how it fits. I can easily place four 1.0 cm square pieces of tissue into the corners of a 30 mm well.
Filling the well
The well must be filled so that the medium forms a meniscus that bulges above the level of the well. This way the medium engages the channels in the chuck face so that it can adhere. If the well is under filled the chuck will not adhere. If it is overfilled the excess will be extruded and the chuck will adhere well.
The well bar is machined so the base of the well is parallel to the surface. When the chuck is placed flush over the well its surface is parallel to the well base. Therefore the surface of a perfectly prepared block will have its face parallel to the chuck face. When a lot of embedding medium overflows the well there is more likely to be a slight loss of the parallel relationship of the chuck and block face. This apparatus routinely prepares blocks that have faces very close to parallel to the chuck face. This can be maximized by using just enough embedding medium to fill the well and form a meniscus. In this situation there will be minimal extrusion and the chuck can be placed very flush to the surface of the well bar. Using a large chuck on a smaller well will accomplish the same feat. This parallelism is only useful if the chuck holder is stationary or adjusted perfectly parallel to the chuck face. If you get good at filling the wells just right, and have a precisely adjusted cryostat, all of your chucks will approach the blade at the same angle. Trimming will be quite fast and minimal.
A very thin and uniform layer of embedding medium on the dispensing slide will result in tissue in the flattest plane. A thick and irregular coating of medium will end up as an irregular layer on top of the tissue and therefore less flat. For a chunk of fibroid this is of no consequence, but if you want to section four core biopsies and have all four left for permanent sections we need super flat. Using this system you can easily get a flat block to cut, but can you cut it? To get a section of 1mm. biopsy cores the entire face much be reached at the same time within a few hundred microns of trimming. This means the cryostat chuck holder must be perfectly parallel to the face of the chuck. If you lucky enough to have X-Y fine adjustment on your chuck holder you can slowly approach the face of the block as you adjust the plane of the chuck. If you have a stationary chuck holder and have prepared a parallel chuck face as I mentioned above you can achieve this core biopsy magic. The balloons picture on the home page contains 131 sesame seeds cut in a single plane. This is accomplished by carefully approaching the block face a section at a time while adjusting the angle of the chuck holder. My cryostat does not have a precise x-y adjustment. Just a knob that loosens and the chuck holder goes flop! I loosen it very slightly and with minute incremental movements and strong hands until I'm getting the whole face. This is no simple task. Maybe if my system creates a demand for it the cryostat manufacturers will take my advice and put a joystick controller and crossing alignment lasers in their instruments. That will make it easy.
What's a well full?
This depends on several factors including the toughness of the tissue, the state of operation of your cryostat and the rigidity of the microtome blades. In my cryostat, if I fill my largest well with large pieces of scalp it will cut thick and thin because the toughness of this leathery tissue puts too much drag on the system. If a tissue is tough it will stress anything with potential movement. That may be bending of the blade, or slight movements in any aspect of the cryostat such as the chuck holder, blade holder or the mechanisms that hold them. We often don’t appreciate it but to get a uniform 5 micron section there must be less than a micron of movement in any of these parts. Some tissues can be like leather. On the other hand I think I could get a clean section of a piece of liver the size if this book with little effort. The bottom line is, consider what it is you are cutting and always make sure everything is screwed and clamped down tight. Also make sure there is nothing between the chuck and the chuck holder so that the chuck is always flush to the holder.
Liquids.....can you cut ice with a knife?
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 metal or plastic spoon to scrape up these specimens and place them into wells. I always stir in a bit of embedding medium into these in my spoon before I put them in the well. This will decrease the shattering considerably. Scrape the tissue off the spoon with a dispensing slide.
The Tap Out
This is really easier than anyone expects. Use the over-chuck freezing block and give a sharp tap to the chuck stem. If you try to pull the blocks up by hand, although the little one sometimes come up effortlessly, you will be surprised at how the forces of cohesion hold the larger ones. This sharp tap breaks a cleavage plane. Something must be going on at a molecular level having to do with displacing forces creating the adhesion. In any event it works well.
-Tap the block toward you so that the chuck lands on the shelf. You can also tap down like hitting a nail with a hammer. Hold the freezing block by its top and tap the chuck stem with the bottom of the block. And try not to hit your finger!
Imperfections in the block face can be seen in two situations. In face down embedding there may be a small amount of retraction of medium around the tissue. This is most evident when using very little embedding medium on the dispensing slide for "super flat" blocks. The actual depth of this retraction is quite shallow and in most specimens it is reached quickly during superficial trimming and requires no attention. I find it necessary to plaster the defects in very small or thin specimens where you need to take the section early in the first 100 - 200 microns. Defects may be seen around pieces prepared by frozen block cryoembedding. A space or gap in the embedding medium around the tissue can lead to "curling away" of the tissue from the medium. This space is easily filled in a few seconds using the following procedure I call “plastering”.
Plastering "super flat" embedded specimen
1) Close up of a flat embedded block showing slight retraction of medium around the tissue.
2) Place a drop of embedding medium on the chuck face.
3) Press the block face to a flat freezing surface such as cryostat stage or any of the freezing apparatus.
4) Remove the chuck with a tap of the over chuck freezing block.
5) Trimmed block with defects filled.
Plastering frozen block cryoembedding specimen
If the block is already in the chuck holder apply medium and press a freezing block to the face. Medium can be applied with a thin smear from the finger, a drop directly placed on the block or by using a slide or other flat applicator as one would use a putty knife. Use medium sparingly, and work fast, for a very thin coating.
See more everyday used for plastering in the discussion on plastering technique in the frozen section technique section. Frozen section technique III
Why did my chuck come off?
If this ever happens it is because the medium did not freeze into the channels of the chuck.
This is one of three things:
1) The well was under filled and the medium never made it to the channels,
2) The chuck was used warm without a heat extractor and removed too early,
3) The chuck was covered in oil or alcohol.
The fix: Another squirt of embedding medium over the filled well and a new cold chuck