A Foray into Scanning Electron Microscopy

This semester I am enrolled in a very fun seminar called Elementary Scanning Electron Microscopy.  Basically, I am being taught how to use a scanning electron microscope (SEM) and then given free reign to play with it for the rest of the semester.  After a few lectures and labs, the class is given the semester to develop an independent project using the SEM.  In a few months we will all meet again to give a presentation on our results.

For many people the term "scanning electron microscope" means very little.  For others, it may be associated with those characteristic black and white images of teeny tiny little things portrayed in immense detail.  That is pretty much where I was at a few weeks ago, but now, after some lectures and time running the instrument, I have a newfound appreciation for this very cool technology.  As you've probably guessed already, I'm not going to be able to resist giving you at least a brief rundown on how an SEM works.  Cue cartoonish diagram:

Just from looking over this picture you should be able to tell we're dealing with a cool piece of equipment.  I mean the thing at the top is called an "Electron Gun" for crying out loud!  It doesn't get much cooler and more science-y than that.  Let's break this thing down a bit so we can gain a better understanding of what exactly it does.  As you can see, the machine is structured in a vertical column with the electron gun at the top and the specimen at the bottom.  Electrons won't travel through air, so the whole thing is sealed off and the air is sucked out with a pump, leaving a vacuum inside the instrument.  Now the electron gun can fire a beam of electrons down the column, onto the specimen at the bottom.  All those round components inside the column serve to control the size and shape of the beam, as well as determine the number of electrons moving down the column at any given moment.

When the beam of electrons strikes the specimen, it causes a number of things to happen.  Many of the electrons simply bounce back and are aptly called "backscatter electrons."  At the same time, the stimulation of the atoms within the sample by the electron beam produces x-rays and the emission of low-energy "secondary electrons."  While each of these can tell you interesting things about your specimen, we are primarily interested in these secondary electrons for imaging purposes.  They provide detailed information about the topography of the specimen and are used to generate the incredible, close-up images that have made the SEM famous.

One more neat little detail about the SEM is that it works best on specimens that are conductive (i.e. metal).  Many things we are interested in looking at are not at all conductive.  To remedy this, the specimens are coated in a very conductive, malleable metal.  Which one you ask?  Well...gold.  Yes, that's right, if the process weren't cool enough already, it is made all the cooler in that all of the samples we look at are gold coated (even if only a few atoms thick).  Hooray science!

Now, as a reward for making it through all the technical stuff, here are some neat pictures I've taken so far:

This is the tip of a mosquito proboscis.  That's right, that horrible serrated spear is what stabs through your skin so they can drink your blood.  Who knew they were more than just pointy tubes?

Fly face at 37x magnification.  Wait a minute Fly, what's that in your eye?

Let's take a look at 600x magnification. Oh, it looks like dust.  Or pollen?

Still not sure what it is, but here is the presumed pollen at 2600x magnification.

Gelatinization, or Science Makes Me Stay Up All Night

For the second time since I began my lab work, I have arrived at the dreaded gelatinization stage of sample processing.  I say dreaded because this is easily the most time consuming step and requires the samples be regularly checked at odd hours (e.g. 11:30 pm and 2 am last night).

Explaining gelatinization requires a little background about what exactly I do with all of the little
pieces of walrus bone I have gathered over the last semester.  The end goal of the entire process is to analyze the carbon and nitrogen stable isotopes of the collagen in the bones.  Collagen is a structural protein and is found in abundance in connective tissues and in bones.  Unfortunately, all that pesky calcium carbonate (the hard part of the bone) makes it difficult to get to the collagen.  So, after cleaning the bones in a sonic bath and removing any fats/oils using a chloroform-methanol solution, I get rid of the calcium carbonate.

This is a relatively easy process, if a little slow, and involves our old friend hydrochloric acid (or HCl).  Everyone probably knows about HCl as it is fairly ubiquitous, found everywhere from the high school chemistry class to the inside of your stomach.  The acid dissolves the calcium carbonate in the bone, creating calcium chloride, carbon dioxide, and water.  The reaction looks something like this:

This process can be a little slow, taking anywhere from a couple of weeks to a month or more.  As the calcium carbonate dissolves, little bubbles of C02 appear, indicating that the process is working.  I continue adding acid little by little, until all of the hard structure is gone and the bone is soft and rubbery.  Once the demineralized bones have been rinsed back to neutral pH, they are ready for gelatinization.

This step makes use of a machine called a vortex evaporator, which is designed for an entirely different purpose but is very useful for this process.  This machine continuously vibrates and heats the bone samples (colloquially known as the "shake and bake" step), causing the collagen to unravel from its complex and very stable structure, forming gelatin (hence the name "gelatinization").

During this process, the solid pieces of bone dissolve into the solution until they are entirely gone and only a few little tiny white pieces can be seen.  If they stay on the machine too long, however, the collagen will continue to degrade until it is useless and the consequences can be disastrous.  This is why the samples must be checked.... and checked... and checked... and... well you get the idea.  As a result, for the 2 or 3 days (or sadly, sometimes more) that this process is ongoing, I make the 20 minute drive to the lab many times at ungodly hours to ensure the process goes smoothly.

All complaints aside, the process really isn't all that bad.  Once a samples has gelatinized, I pour it into a syringe and squirt it through a very fine filter into a vial then place it in the freezer.  Once all samples have been filtered and frozen, they are put on the freeze drier for 24 hours.  Magically, the clearish liquid turns into a lacy, white, cotton candy-like substance, which is the final product of the process.  I then weigh out a tiny (~0.2 mg) piece, submit it for analysis on the mass spectrometer and VOILA! Science!

For those of you that made it this far and looked at all the boring black and white images, here is a disgruntled looking underwater walrus:

Photo credit: Pete Barrett, National Geographic

New Year, New Opportunities

Hello everyone,

We did it.  We made it through another year and now here we are at the start of a fresh, new one.  The days are getting longer (in the northern hemisphere at least) and everything seems just a little bit brighter.  I am not one to make grand resolutions on the New Year, but it is certainly a period of new beginnings and unfolding opportunities.  So it is with that spirit in mind that I will share with you some of the things that I am looking forward to in 2015:

1. The Alaska Marine Science Symposium

Each year in mid-January, the North Pacific Research Board puts on a conference in Anchorage and nearly everyone involved in marine science in Alaska gets together to share their research, talk, collaborate, and generally have a good time.  I am looking forward to presenting a poster on some of the initial results of my PhD research.

2. Traveling to Barrow!

Sometime this summer, I will have the opportunity to spend around a month in Barrow, helping with the North Slope Borough's marine mammal stranding program and ideally gathering a few new samples for my dissertation work.  I am still very unclear on what exactly this will entail, which makes it all the more exciting.  For those of you who are unfamiliar with Barrow, look on a map for the tippy top bit at the farthest north part of Alaska.  That's where I will be!

3. Publishing my M.S.

This one is a little more tentative, of course, as it requires a journal to accept my manuscript, but I am finally nearing a point where it is ready to submit.  It feels like it has been a long time coming.  I was second author on a note that came out in December in Marine Mammal Science (See it here), so this won't be my first publication.  It will be my first, first-author publication and is much nearer to my heart, as it is the product of 4 years of hard work.  Fingers crossed the reviewers are gentle.


In the end I'm sure the most exciting stuff will be the things I don't even know about yet, but those are a few examples of things I am looking forward to in 2015.  I hope everyone has a great year ahead with many things to look forward to!