Why AFM?

Atomic force microscopes (AFM) are so versatile that it is a challenge to cover all their applications. Nanotechnology can now be found everywhere in our daily lives but only very few people have access to the tools to work with it. Today, AFMs are the simplest, most affordable, versatile tool to discover, understand, and control materials at the nanoscale. Let me illustrate this with an example that starts out as trivial play and ends up being relevant for education and research, from the microstructure all the way to biomolecules: The eggshell membrane. Everyone has seen and touched an eggshell membrane, the layer between the egg white and the eggshell. It is thin and flexible, surprisingly tough, but we don’t give it much thought when we crack an egg. Yet it shares many characteristics of advanced biomaterials in cutting-edge biomedical applications and can serve as a highly relevant test sample for AFM. Below are my first attempts at imaging this material and why it got me thinking about serendipity.

First attempt

“That’s funny…”1. I was peeling a hardboiled egg and noticed how incredibly tough this thin membrane is even after cooking. Why not put the eggshell in the AFM? It’s a biomaterial, it’s flat … should be interesting! I had no idea what to expect, except 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).

Topography of the outside surface of an eggshell, 3D rendered and shadowed. 20 x 20 µm scan, 2.5 µm maximum topography.

Topography of the inside of a hardboiled egg, showing the denatured proteins of the eggshell membrane. The lines are degraded collagen fibers, the dots most likely aggregated egg white proteins.

Seeing the denatured fibers of the eggshell membrane got me reading. It turns out that eggshell membranes are mostly collagen I 2. We use so many eggs in food production that the membrane is separated from the shell and sold as dietary supplement. But this one was boiled and the structures clearly suffered. What would a fresh membrane look like?

Second Attempt

I cracked an egg, rinsed out the eggshell, let it dry completely and mounted it on an AFM disc. Within minutes the AFM was scanning collagen fibers 3, but the images were a little blurry. My immediate suspicion was that the sample is not completely dry, but scanning the same sample days later gave the same result. Odd!

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?

Why would the surface of a dry material appear consistently blurred? Maybe because it is in direct contact with the egg white and the gentle rinse didn’t remove all of it. How do we know? Because scanning a small area at high resolution reveals a sea of egg white proteins! Ovalbumin is the most abundant egg white protein (52%) and measures 7.0 × 3.6 × 3.0 nm in size 4, 5, which nicely agrees with the measurements. It also illustrates the convolution of small features with the AFM tip (in this case scanning a ~3 nm features with a 10 nm tip radius).

The egg white proteins that cover the entire interior surface of the membrane are visible at higher resolution. The height measurement is accurate but in XY the image is a convolution of the 10 nm tip radius of the probe with the proteins, making the proteins appear wider.

So how would we get a nice image of collagen fibers? What if we mount the area of the eggshell membrane that faces the air space where there is no egg white? It makes sense that the collagen fibers need to be thicker and densely spaced since that area is not supported by the eggshell. And since there is no egg white covering that side of the membrane the collagen fibers appear sharp.

Exterior surface of the eggshell membrane where it faces the air space. This topography image shows a denser collagen network that is needed to withstand the additional strain on the membrane in this area. The air space is formed between the inner and outer membrane at the round end of the egg (no structural support from the eggshell).

Why it matters

The eggshell membrane is a fascinating biomaterial and a great test sample for AFM. It is easy to collect, mount on an AFM disc, and contains interesting biological features at multiple size scales. At the microscale, collagens are abundant protein fibers of varying thickness and highly relevant in biology education and human diseases. At the nanoscale, the high protein content and concentration of ovalbumin allows direct visualization of protein molecules. This innocuous material can be used to show students how macroscopic properties are achieved by micro and nanoscale features.

So why don’t we use AFMs to teach about biomolecules in school? These microscopes are robust, easy to use, and the most affordable tools to get us to the nanoscale. The principle of an AFM teaches physics and engineering, the sample teaches molecular biology, hands-on to students as early as high school. But most importantly it teaches discovery and serendipity, with so many examples of “That’s funny…” all around us if we just took a moment to pay attention. Imagine a nanotechnology class that teaches students about learning through discovery.

Ironically, the same applies to research. Why don’t we use AFM more often? Here is a tool that can image biomaterials and measure forces at the micro and nanoscale! It’s easy to do these measurements on dry samples, and we can work in liquids and measure the elasticity of individual fibers.

Stay tuned for more experiments and contact us with other ideas and examples of “That’s funny…”!