Debbie Knight

Archive for January, 2013|Monthly archive page

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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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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.

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

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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.

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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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Photo of the Week: Autoclave “Art”

In photo log on January 24, 2013 at 1:10 pm

autoclave art

This is what happens when a plastic bin melts in an autoclave.

I call this “autoclave art.”

Sometimes the “art” is quite interesting. Something you want to keep and hang on the lab wall.

Sometimes, as in this case, I find the “art” a bit pedestrian. However, it might make a cool picture frame. Albeit, its bubbly marshmallow detail might have a limited appeal.

So how did this happen?

Well, normally the autoclave sterilizes glassware, liquids, etc. using a combination of heat (121 degrees Celsius or 250 degrees Fahrenheit) and pressure (18 pounds per square inch). Most bacteria are killed under these conditions.

This particular autoclave malfunctioned and heated the chamber beyond (way beyond) the 250 degrees Fahrenheit. When I heard the autoclave’s alarm sounding, the pressure was 35 pounds per square inch (nearly twice the amount normally used). I’m not sure what the temperature was — the digital readout where the temperature would be displayed was screaming “Error! Error! Error!”

I’d say!

The temperature was high enough to melt the plastic bin the glass bottles and flasks were in.

autoclave art in situ

We had to call the repair guy this morning to fix the overheating issue and to help remove the plastic which had oozed through the wire mesh rack. He will take the rack (and the embedded artwork) back to the shop to melt off the plastic.

So long, autoclave art!

 

For more on autoclaves, see my previous post

 

 

That’s the report I’m filing: the scientific manuscript

In observation on January 16, 2013 at 9:30 am

Submit

A friend of mine, a fourth-year graduate student, just submitted a manuscript to the scientific journal PLOS One.

Her name is listed as the first author on this, her first manuscript submission.

It’s a pretty big deal to write that first first-author manuscript. It’s your research, it’s your baby.

Naturally, she’s very excited about it.

It reminded me of my first first-author manuscript, written so many years ago. I remember it was quite a task to write the beast. It consumed a large part of my life while I gathered and graphed data, plunged into the existing scientific literature.

The writing did not come easily for me.

Every word was written knowing full well that it would be heavily scrutinized by my boss, the reviewers and other scientists.

By the time the manuscript was submitted to a scientific journal for review, my boss had so heavily edited what I wrote I barely recognized it as “mine.” I was really surprised my name was still first.

It was a humbling experience.

But it was a huge learning opportunity.

My next manuscript was a little easier to write.  And a little more of my writing remained untouched.

When that first manuscript was accepted and published in a scientific journal, I sent copies to everyone I knew. It was so cool to see my name in print, to have my research efforts out there in the scientific community.

It’ll be interesting to see how my friend handles it if and when her manuscript is finally published.

Of course the manuscript will have to run the gauntlet of editors and scientific reviewers.

Best case scenario is the manuscript is accepted for publication with no changes required.

But more likely, the reviewers will have some issues that she will have to address either in writing or through more experiments. In this case, she would need to make the necessary changes and resubmit the manuscript for further scrutiny.

Then there’s the worst case scenario. The journal’s editors will reject the manuscript because the work is not appropriate for their journal. In this case, she would have to take stock and retool the manuscript to submit to another perhaps more appropriate scientific journal.

I have my fingers crossed for the best case scenario!

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So what does a scientific manuscript look like?

It is pretty much a technical report. It has a very defined structure, a formula if you will.

First is the introduction which puts the research in context – historically as well as biologically. The last paragraph of the introduction should include what your data shows.

This isn’t storytelling where you hold the punch line until the end. Everything is revealed up front.

I find the introduction one of the hardest things to write.

Quite a bit of digging through the scientific literature happens before the first word is written. And including the appropriate depth of literature review is tricky.

The next section in most scientific manuscripts is the material and methods section.

This is where I usually start the manuscript writing process.

I advise any first-timer to start here!

Why? It is by far the easiest part of the manuscript to write. It involves writing about what reagents you used and how you performed the experiments.

If you kept a very meticulous lab notebook, all this information is at your fingertips. Reagent specifics such as catalog numbers, supplier names and company addresses as well as antibody clone designations, antibody isotypes and associated molecular tags are often needed in this section. Sometimes that means you have to hunt for the data sheets that came with the reagent or dig through a stack of packing slips to find the specific details you need for this section.

You learn quickly to include this information in your lab notebook after you’ve worked on your first manuscript.

The next section is the results section. This is the next easiest section to write — although it’s not nearly as easy or straightforward as the materials and methods section.

This is where you tell your research “story.”

Here you present the data as graphs, tables, figures, photos, etc.. Each figure needs a description, a figure legend, that should be able to stand alone. Sometimes this is the only thing a reader will read in the article.

Generally, the first line of the figure legend summarizes what the figure is showing – kind of a title or a headline.

It is then followed by a brief description of the method used as well as what the figure shows.

If written well, the reader shouldn’t need to even look at the accompanying image.

What’s tricky with the results section is figuring out what data presented in what order will help you tell your story best.

Many researchers tell the story how it unfolded, often starting with the original observation that sent them on their journey.

The researcher will then show how they proved what they thought was happening was actually happening.

Sometimes that story takes a twist – an unexpected outcome of their investigation. I find these most interesting to read, to see how researchers roll with the punches.

Sometimes the story unfolds in a straightforward and logical manner.

Sometimes the researcher offers their interpretation of the data in this section, but more often the significance of their discovery is discussed in the last section, the hardest section to write, the conclusions (or sometimes discussion) section. In this section, the researcher places his data in context with other scientific data. This may require a lengthy explanation why his data flies in the face of other scientist’s data.

Some researchers expound on the subtleties of their findings.

Some researchers will wax philosophical. Some will stand on their soapbox.

There is quite a bit of style variation in the conclusions section.

And, yes, there is a bit of overlap in these various sections.

You need the overlap because different readers start at different places within the research article. Some readers will only look at the conclusions. Some are only interested in a particular method or a particular part of the puzzle.

I know that I can read the same paper at different points in my research project and get very different information out of that paper. At the start of the research project I might only be interested in a particular method. At another stage of the project, I might want to see what their data looked like or how they interpreted their findings.

So there you have it. Scientists do their research, perform the experiments to better understand a phenomenon, and report their findings to the scientific community. (At least that’s how it happens in the university setting.)