Eggshells? AFM? What? why?
Yes, admittedly completely odd and abstract. Let me explain why it makes sense and why it was so much fun, especially if you want to teach serendipity and nanobiology to undergraduate students. Hands-on with an affordable AFM.
The eggshell is a fascinating biomaterial that we all are familiar with. So familiar that we don’t think twice about it. When you do take a closer look, it’s a magnificent design of nature, essentially a layered packaging material consisting of the eggshell and the eggshell membrane. But more on that later.
So why AFM? Atomic force microscopes operate like a sophisticated phonograph by scanning a needle over the surface of a flat material. If you use a nanoscale needle and nanoscale positioning you can image nanomaterials (a 3D map of the surface = topography). More details here. The AFMWorkshop AFMs we use ourselves and distribute are the most affordable and accessible tool to image nanomaterials. A ready-to-use AFM including installation and training starts at ~$30,000 and has been used successfully by many undergraduate students and researchers.
The “experiment” presented here started as pure serendipity and is written with 20/20 hindsight. I hope to illustrate what becomes possible if you have access to one of our AFMs. In the earlier days of BioCubic our HR-AFM was set up in my garage (yep) and I didn’t have any research-grade nanobiology samples. Fortunately, you can’t make an omelet without breaking a few eggs!
Before we dive into the experiment, a quick reminder of the size of things in biology. A hair (~100 um thin or 1/10 of a mm) – a cell (~10-50 um wide) – a bacterium (~1 um) – a virus (~30-400 nm) – a protein (~4-10 nm) – DNA (2.4 nm) – glucose molecule (~1 nm). Our AFMs get you well below 1 nm (see imaging biomolecules)!
Breaking eggs while thinking science
I was thinking science while peeling a hardboiled egg and struggling. This thin eggshell membrane is incredibly tough even after cooking. “That’s funny…” I’m looking for biological samples, why not put the eggshell in the AFM? It’s a biomaterial, it’s flat … could be interesting! I had no idea what to expect, I only remembered that the shell is related to research in biomineralization (bone formation, osteoporosis) and the eggshell membrane reminded me of biomaterials used in grafting procedures (tendon, skin, wound healing). The outside surface of the eggshell was interesting, a uniform surface with microscale topography. Interesting!
Topography of the outside surface of an eggshell, 3D rendered and shadowed. 20 x 20 µm scan, 2.5 µm maximum topography. Interesting, but not exceptional…
Topography of the inside of a hardboiled egg, showing denatured proteins of the eggshell membrane. Huh? There might be something there!
But seeing the inside of the eggshell (the eggshell membrane) really got me excited, the hints of fibers got me reading. The lines could be degraded collagen fibers, the dots aggregated egg white proteins? It turns out that eggshell membranes are mostly collagen I. We use so many eggs in food production that the membrane is separated from the shell at an industrial scale and sold in many forms. Who knew? But this egg was boiled and the structures clearly suffered. What would a fresh membrane look like? I cracked another egg, rinsed out the eggshell, let it dry and mounted it for the AFM. Within minutes I was scanning obvious collagen fibers!
Fresh eggshell membrane washed and dried. This topography image shows the collagen network that gives the membrane its strength. But the features appear slightly blurred?
The images looked a little blurry? I guessed that the sample may not be completely dry, but scanning the same sample the next day gave the same result. Odd! Why would the surface of a dry material appear consistently blurred?
Then it clicked. Maybe because I’m scanning something on top of the collagen? The membrane is in direct contact with the egg white and my gentle rinse didn’t remove all of it? How can we confirm that? We’re using an AFM! So scan a tiny area at high magnification and we see lots of squiggly stuff. Proteins?
The egg white proteins that cover the entire interior surface of the eggshell membrane are visible at high resolution.
A quick literature search informs me that ovalbumin is the most abundant egg white protein (52%) and measures 7.0 × 3.6 × 3.0 nm in size, which nicely agrees with my measurements. Wow, we just went all the way from a banal eggshell to measuring single proteins molecules!
But back to the collagen fibers! How would we get a nice image of collagen? What if we mount the area of the eggshell membrane that faces the air space where there is no egg white?
Exterior surface of the eggshell membrane where it faces the air space in the egg. This topography image shows a denser collagen network that is needed to withstand the additional strain on the membrane in this area.
That worked! And it makes sense, the air space in the egg causes the inner shell membrane away from the supporting shell. To withstand the added strain, the collagen fibers need to be thicker and densely overlapped. And since there is no egg white covering that side of the membrane the collagen fibers appear sharp. Q.E.D.!
what did we learn?
Serendipity is fun, a trivial material can quickly lead you down a nanoscale rabbit hole. Leveraging curiosity-driven discovery with students and giving them ownership of a project through their own hands and mind is the most engaging way to learn about any subject. And even a PhD-level microscopy expert with 25 years of experience can be baffled by something as trivial as an eggshell!
We have the tool, the knowledge, and endless other examples of nanobiology in front of our noses (some on our noses). Why don’t we teach this in school? We absolutely could! And if that’s not enough you can even take a course on how to build one. Help us spread the word!
