Debbie Knight

A Day in the Life: January 28, 2013

In A Day in the Life on January 28, 2013 at 11:54 am

From time to time, I will give a glimpse into the “glamorous” life of a research associate and talk about what I’m doing in the lab. These entries I will call “A Day in the Life…” 

This week I am making thin slices (called sections) of frozen tissue specimens using an instrument called a “cryostat.”

This is a cryostat, an instrument used to make thin slices (sections) of frozen tissue samples.

This is a cryostat, an instrument used to make thin slices (sections) of frozen tissue samples.

The one I’m using is a little fancier than the one I used ten years ago. This one has a digital display and controls that you can adjust.

controls-sm

This cryostat’s controls are digital, so fine adjustments can be made easily.

The inside, however, looks pretty much the same as the “old” cryostat I used to use.

inside-the-cryostat-sm

Inside the cryostat.

So, the first thing you need is your frozen block of tissue. The “block” is formed in a plastic mold called a “cryomold” which can come in several sizes. Basically, you put your tissue into the mold and then pour this clear gooey (think honey) liquid called “O.C.T embedding medium.”

OCT-medium-sm

The all important O.C.T. embedding medium is a clear and gooey substance that turns white when frozen.

You then quickly freeze (called “snap freeze” or “flash freeze”) the block in a really cold liquid like liquid nitrogen or dry ice-chilled ethanol or isopentane. The clear liquid turns white that’s seen in the photo below.

cryomold-sm

Tissue (red) flash frozen in O.C.T. embedding medium (white) in a plastic mold.

We store our embedded tissue blocks at minus 80 degrees Celsius until we are ready to section them.  The tissue block is pretty brittle at this temperature so we have to “warm” it up to minus 27 degrees Celsius (the temperature inside the cryostat) before we can proceed. You can do this by placing the tissue blocks inside the cryostat and waiting 15 to 30 minutes before proceeding.

Once the tissue block has warmed up a little, you “pop” the tissue block out of the plastic mold. You then add some of the O.C.T. embedding medium to the cryostat adapter (called a “chuck”) and quickly place the tissue block on top of it. The embedding medium acts like a glue to hold the block onto the chuck.

tissue-on-chuck-sm

To attach the frozen tissue block to the cryostat adapter (called a “chuck”), you add a few drops of O.C.T. embedding medium (as shown on right) and quickly place the tissue block over it to essentially glue the block on the chuck (as shown on the right).

Here is the result. I obviously am a little out of practice getting the tissue on the chuck straight, but you get the idea.

chuck-1-sm

Tissue block is mounted on the cryostat chuck. In the background, other tissue blocks are awaiting the same fate.

The next step is to place the chuck on the mount.

chuck-2-sm

Placing the chuck on the mount of he cryostat.

The chuck/tissue block is adjusted so it is as close to parallel with the cutting blade as possible. It is then locked into place to prevent it from shifting while sections are cut.

chuck-3-sm

Adjusting and locking the chuck/tissue block into place.

The cutting surface is a blade that sort of resembles a razor blade. It comes in a dispenser pack as shown in the photo below.

blades-sm

A pack of blades used to cut tissue in the cryostat. They’re kind of like razor blades.  Only one blade is used at a time.

The blade is placed in the holder and locked down. This blade will be used over and over until it has too many nicks or the cutting edge or becomes dull. It is really sharp, so the user has to be careful not to accidentally cut herself on the blade. Some cryostats are used to cut potentially biohazardous materials like human tissues or infectious animal tissues.

blade-on-microtome-sm

The cryostat blade goes here (indicated by pointing finger). It’s really sharp, so care is needed when working with the cryostat.

To cut tissue sections, you use a hand crank on the outside of the cryostat to move the tissue across the blade. It’s nice because you can control the speed of the cut — fast if you’re trying to get to a specific area of tissue or slow if you’re planning on catching and keeping the section.

To make the chuck/tissue block move across the blade, you turn a wheel on the side of the cryostat. You can turn the handle slowly for more precision work or quickly to trim the block to an area of interest.

To make the chuck/tissue block move across the blade, you turn a wheel on the side of the cryostat. You can turn the handle slowly for more precision work or quickly to trim the block to an area of interest.

cutting-tissue-composite-sm

The chuck/tissue block moves across the blade as shown in this sequence of photos.

This particular cryostat is missing a part: a thing called a “roll plate.” The roll plate is a flat piece of plastic that rests against the blade and catches the tissue section as it comes off. It prevents the tissue section from rolling up like a scroll. In lieu of a roll plate, you have to use a paintbrush to catch and unroll the section as it comes off the blade.

catching-the-section-sm

This cryostat is lacking a device called a “roll plate” which helps to catch the tissue section and lay it flat before it rolls up into a tube. Here I am using a camel hair paint brush to catch the tissue section.

The microscope slides used to catch the sections have a special coating on them to help the tissue “stick” better (and stay stuck) to the glass.

slides-sm

The microscope slides we use have a special coating on them which helps the tissue “stick” better to the glass.

Once you have flattened out the tissue section with the paintbrush, you then touch the glass slide (face down) on the tissue. The glass slide is room temperature, the tissue section is cold (at minus 27 degrees Celsius), so the tissue section “melts” onto the slide. You then let the section air dry at room temperature before placing it in a slide box that will go into a freezer for storage.

transfering-the-section-sm

After the tissue section is laid flat using the paintbrush(es), you tap the glass slide (face down) against the tissue section. The section sort of melts onto the slide.

slides-with-sections-sm

The tissue needs to dry a little after it is transferred to the slide.

slides-in-slidebox-sm

The slides are placed in a slide box. The lid is put in place before the slide box goes into the minus 80 degree Celsius freezer for storage.

Not every tissue section that rolls off the blade is usable. Sometimes you just can’t unroll it. Sometimes the sections tears. After a few hours of cutting tissues, you end up with a nice pile of shavings. These have to be cleaned up and disposed of properly.

leftover-sections-sm

Not every section is successfully uncurled. Here are some discarded sections.

So there you have it.

And even though it’s been a good ten years since I last used a cryostat, I think it’s a lot like riding a bike. You might be a little wobbly, but it all comes back to you.

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  1. Oh man, I cut myself once on that blade. They are so sharp you don’t even know you’re cut until the blood is shooting out the side of your finger.

    • I guess it would only take once. Glad you survived your “battle wound” to tell the tale. 🙂
      And, even though I have klutzish tendencies, I’ve never cut myself (knock on wood).

  2. This was an extremely informative post, i’m using the cryostat today! Thanks

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