What is that? A pipette!

pipetteMost of the time, biologist in the lab aren’t working with things that they can see. You need to look at cell under a microscope, proteins using X-Ray crystallography (for example), and DNA and RNA by various methods like gel electrophoresis or cell staining (see more about visualizing DNA here).  Many experiments require scientists to manipulate DNARNA or proteins in small liquid volumes to do experiments to understand the sequence of these molecules, to discover what other molecules they bind to, or to change them to figure out how they work in cells. To manipulate these small volumes, we use a tool called a pipette (shown at left) – also called a micropipette to be even more precise. It’s similar in concept to the baster you have in your kitchen that sucks up liquid when basting your turkey, except it’s for much smaller volumes and is exquisitely precise.

Pipettes come in different sizes, and the size of the pmulti_size_pipettesipette determines the volume that they can dispense (see picture at right).  They dispense microliter (abbreviated as “ul”) volumes of liquid. To give you an idea of scale, 1 drop of water is about 50ul.  One milliliter (ml) is1000ul, which is the largest micropipette volume you can dispense, and there are about 44ml in a shot of vodka. These are small volumes we are talking about!!

The sizes shown to the right dispense up to 2ul (called a “p2”), 200ul (“p200”), or 1000ul (“p1000”). By spinning the plunger on the pipette, you can change the volume that is dispensed within the range that the pipette allows.  You then put disposable tip to the end of the pipette and suck up that volume of liquid. As an example, check out the photo below of the p1000 dispensing 600ul of water and the p200 dispensing 20ul of water.  The image to the left is the pipette tip filled with that volume and to the right is the water in a tube. This type of tube is used every day in the lab and is called an eppendorf tube. IT can hold 1.5ml (or 1500ul) of water. These small plastic tubes have attached caps that can snap the tube closed to allow you to mix the contents or do whatever is needed for the experiment (like heat it up, cool it down, spin the contents, etc). And if you’re wondering why they are called “eppendorf” tubes, it’s after the name of a German company called Eppendorf that is one manufacturer of this kind of tubes (similar to how the brand Kleenex is now synonymous with tissues)

pipette volumes

Maybe you’re wondering why scientists have to use such small volumes?  One reason is because there isn’t a large amount of DNA or RNA or proteins in cells, so when it’s isolated, it doesn’t take up a large volume. As an example, if you isolate the genomic DNA from  5 million cells, you would isolate 10-30ug  of DNA. That’s MICRO grams of DNA – or 0.00003 grams.  As comparison, a grain of salt weighs more than 1200x more than the amount of DNA that you would isolate from 5 million cells!  And this is all in just 50ul of liquid.  Very small amounts! The good news is that working in large volumes isn’t really needed because most experiments don’t need a large quantity of materials to get the answer. Not to mention that working with larger volumes for experiments would be more expensive.  If you are doing an enzymatic reaction, a larger volume reaction would require more enzyme.  To get an idea of what this would cost,  a very common reaction cuts DNA with restriction enzymes, which are essentially super-specific DNA scissors. These restriction enzymes cost several hundred dollars per tube and you use 0.5-1ul for every 50ul reaction.  If you did a reaction in a large test tube of 10ml, you’d need 200 times more enzyme and would go through hundreds or thousands of dollars if enzyme for each experiment.  Labs are not that rich!

Fortunately scientists have developed techniques and equipment, like the micropipette, that can manipulate, detect and analyze incredibly small amount of DNA or RNA or proteins. For most bench scientists, learning how to use a micropipette is done on day one (I learned how to use one in high school at City Lab) and pretty soon becomes second nature.  In grad school, I pipetted so much that there were days I’d go home with a sore thumb or pipetting  calluses. However all this practice did pay off.  I picked up a pipette for the first time in 8 years a few weeks ago to start doing experiments in my new lab. After all these years, it was just like riding a bike – or in this case just like “using a pipette”.

What is that? A beautiful image of deadly tumor cells

the eye

Thanks to Dr. Roberto Fiorelli of Barrow Neurological Institute for sharing this stunning image

It looks like an eye.  Perhaps a terrifying pink eye, like the Eye of Sauron, coming out of the darkness. It’s not an eye, but it is a bit terrifying. This is an image of a slice of the brain showing tumor cells (in green and red) surrounding a blood vessel.  How does this type of image get made?  How does this type of image help scientists? What does it mean that these tumor cells are near a blood vessel?

This image is created with a microscope – specifically a confocal microscope. I’m going to use a very weird analogy to explain why confocal microscopy is so cool, so stick with me. Imagine that you have a jello mold with an object it and you want to know exactly what the object looks like.  Now imagine a regular microscope is like a flashlight.  When you point the flashlight at the jello mold the whole thing lights up including what’s in front of the object, and if the object is translucent, the jello behind the object lights up too.  This gives you an idea of what the object is, but it may be kind of fuzzy because of all the jello you see in front and behind the object.  Confocal microscopy, on the other hand, is designed to turn that wide flashlight beam into a single pinpoint of light so only one part of the object is illuminated at a time.  So when you move this single pinpoint around (back and forth and up and down) over the object, you can get a clear a crisp image of what is inside the jello.

confocal_microscopy

To bring this analogy back into the science-verse, the jello is a cell or a piece of human tissue with layers of many cells.  The objects inside the jello are certain proteins marked so that they light up in different colors (what we call fluorescence) when excited by the light from the laser (flashlight). When confocal microscopy is used to look at these proteins, you can see clear crisp images of exactly where the proteins are in the cells.  And if you take enough images up and down through the cells and the tissue, you can even create a 3D image of the cell or a piece of tissue. Check out this neat video of a 3D rendering of a piece of the brain called the hippocampus.

Now back to the image above.  This is a piece of tissue taken from a patient with an ependymoma, a tumor derived from brain tissue and is primarily found in younger patients.  The colors you see are:

  1. Blue: a chemical called DAPI (or 4′,6-Diamidino-2-Phenylindole, Dihydrochloride) that binds to DNA. Since DNA is found in the nucleus of every cell, staining cells with DAPI helps you to locate each cell in the image – each blue dot is one cell.
  2. Red: stains a protein called GFAP (Glial Fibrillary Acidic Protein) that is found in different cells of the brain, but is also a marker of particular brain tumors, like ependymomas
  3. Green: stains a protein called vimentin that is also found in different cells of the brain, including cells that make up large blood vessels and brain tumors like ependoymomas

So what are we able to learn from this beautiful picture?  See how there are a lot of red and green cells surrounding an empty round space.  That round space is a blood vessel. Cancer cells need food and oxygen to grow, so the green and red cancer cells are clustering around the blood vessel to get the nutrients they need. Even though this is a beautiful image, it helps scientists to understand how these deadly tumors function within the brain and how they find the resources to grow.

If you want to look at more amazing images taken using confocal microscopy and fluorescently tagged proteins, check out these links

Wellcome Image Awards 2015
The Cell: An Image Library

Thanks to Dr. Roberto Fiorelli of Barrow Neurological Institute for sharing this stunning image from his postdoctoral work in Dr. Nadar Sanai’s Laboratory, the Barrow Brain Tumor Research Center

For more “What is That?”, click here.

What is that? A liquid nitrogen freezer

This is a new feature called “What is that?” where I will show a photo of something from the lab and then discuss what it is, what it does, and why scientists use it.  Every lab that I’ve ever worked in, I gave tons of tours – to my family, to other scientists, to kids and to the public!  Seeing what a real lab looks like and understanding how things work is one of the ways I use to demystify science and scientists. Think of this new series as a virtual lab tour with me as your tour guide!  I hope at some point soon to turn this feature into a short video series…but until then, enjoy learning about what “that” is!

So what are these? These are liquid nitrogen freezers.

liquidnitrogenfreezersLet’s start by talking about freezers in general. What temperature is your freezer at home? Well it needs to be cold enough to freeze things. The freezing temperature of water is 32° Fahrenheit (F) or 0° Celsius (C), so you want your freezer to at least be able to freeze water, but you also want it to preserve your food.  Part of preservation is preventing bacteria from growing since bacteria could potentially cause food poisoning when you thaw and eat the food (especially if it isn’t cooked first).  The colder the temperature, the less likely the bacteria is to be able to grow.  Also, the colder the freezer is, the less likely it is that enzymes (proteins that perform chemical reactions in cells) are active and breaking down the food’s nutrients. So the ideal freezing temperature for food is 0°F or -18°C.

In the lab, we have -20°C freezers that store reagents and look a lot like the upright freezer that my mom has in her basement to store extra food (and chill martini glasses). We also have -80°C freezers, also called ultra low freezers referring to the “ultra low” temperature.  These are better at storing proteins at a temperature that is cold enough to inactivate them. These ultra low freezers are also good for storing RNA.  RNA has a natural enemy, a protein called RNase, which is essentially a high-powered chomping Packman after the Pac-Dots dots that make up the RNA. At really cold temperatures, this RNase Packman is inactive and can’t work, keeping the RNA sample safe.

roses LN2

Creative Commons license. Original photo from Kasi Metcalfe on Flickr

As a bit of a segue, my 4 year old nephew calls me on Facetime a few times a month and always asks me to “show him some science.” One of the first things I showed him was our “Biospecimen Cryostorage” where we store the samples from our biobank, and which is what is pictured above. This room has three Liquid Nitrogen Freezers, and these are even colder that the ultra-low freezers. With liquid nitrogen (abbreviated to LN2) in the base, the whole tank is cooled to below -275°F or -170°C. If you feel like you’ve heard of liquid nitrogen before, you probably have. This is the liquid that is used at Science Centers to freeze roses and then smash them into a million pieces.  If you’ve seen any cooking show in the past 5 years, you’ve probably seen a chef use liquid nitrogen and pour it into a mix of cream and sugar to make a fast ice cream. However, in science we use liquid nitrogen as a way to quickly freeze and preserve living cells and tissue – a process overall known as cryopreservationLN2description

To better understand the photo and to really understand “what this is”, I have labeled areas above.  Each of the two tanks pictured can hold up to 40,000 individual samples.  These samples could be blood samples, tissue samples, samples of cells, etc.  In our case, these samples are stored in 2 ml (milliliter) tubes in plastics boxes in racks inside each freezer. Each box can hold 96 samples and each rack (see photo below) can hold 13 boxes (over 1200 samples). The freezers are automatically filled by tanks filled with liquid nitrogen sitting right next to each freezer.  Each freezer isn’t completely filled with liquid nitrogen for a few reasons. First, it would be really expensive but more importantly, it isn’t necessary. The temperature of liquid nitrogen is -190°C but the vapor keeps the tank colder than -170°C, which is still good enough to keep all of the proteins in these cells completely inactive.

LN2removalSamples are removed from these tanks on a regular basis and distributed to researchers.  You can see an image on the left of a rack of boxes being removed. It is important when selecting individual samples that they are not allowed to warm up and thaw.  This thawing can activate proteins that break down the samples or causes ice crystals when re-thawing that affect the integrity of a tissue sample or the viability of frozen cells, for example.

What is that? Liquid nitrogen freezers and a filler tank
What does it do? Keeps biospecimens or other samples at really low temperatures
Why do scientists use the? To keep samples well preserved and viable so that they work better for their experiments.

For more “What is That?”, click here.