FridayAFM - secrets of the egg shell

Héctor here, your AFM expert at Nanosurf calling out for people to share their Friday afternoon experiments. Today I explore chicken eggs, after the egg-cellent suggestion from my colleague Christian.

But why chicken eggs? Because the chicken egg industry is a massive giant, and using egg by-products in bio research is an emerging field (Ref 1). So, let's see if this, easy to access sample, is suitable for AFM imaging, and what is the minimum preparation needed.

Beforehand, some egg-facts to blow your mind about eggs:

• According to the European Commission, there are almost 59 million laying hens in Denmark, 57 million in France, and 47 in Spain. Which is very curious when you consider that only 6 million people live in Denmark, 68 in France, and 48 in Spain.

• The United Egg producers (US), estimate that a laying hen produces on average 300 eggs per year. This means that 17700 million eggs per year are produced in Denmark alone.

• In EU, according to the Egg commision, on average each person consumes about 210 eggs per year . 

If you combine all this information, it means that each year Denmark has to export 16440 millions of eggs, France 2820 millions, and Spain 4020 millions.

This is no small task!

Specially if you consider that they should be inspected, classified, handled, and preserved.

So, without further ado, let's get to business and prepare some samples.  I purchased some brown and white chicken eggs to try seeing some differences. I started by cracking them open, and putting the contents aside (we will look only at the egg-shell). My first approach was to use blue-Tac to secure the shell and the membrane, but it turns out that the pressure needed to secure the shell breaks it completely. However, double-sided sticky tape does the job, and the same for the egg membrane (which I peeled of with tweezers to avoid contaminating it).

I used a glass slide because It was convenient, but it doesn't play any special role, I could have used a metal plate or a ceramic disc for instance. Next step was to place it under the AFM, and secure it to reduce drift and vibrations.

As you can see from the diagram, the egg-shell is composed by a hard part (cuticle, crystal layer and mammilla), and the soft, fragile, membrane. When the egg is open, the membrane peels off easily. However, a fine layer of fibers might stick to the §bottom part of the mamilla. Because of this, I only imaged the cuticle and the membrane, and ignored the rest. 

1 Egg structure and sample preparation.

I started with the brown egg, using Dynamic mode and a Multi 75 probe. I found out that the surface of the cuticle is composed of spherical calcite elements. With a broad distribution of sizes.

Using some previous research to guide me (Ref 2), I identified pores. These are easy to spot in the phase contrast channel, as the AFM probe cannot hit the bottom of the pore and this produces a big phase contrast on that position. As you can see, pores tend to have irregular shapes.

1 Brown egg, calcite nanoparticles in the cuticle, pore, and fibers in the egg-shell membrane. Images taken in Dynamic mode with a Multi 75-E probe.

The membrane part of the brown egg, is composed of fibers (which are a mixture of proteins, up to 50000 different types, see Ref 1), and the difficulty when imaging this part is that it tends to retain liquid, which hides the structures completely, but also, is full of deep trenches where probes can break easily if the scan speed is too fast or the feedback is inappropriate. However, as you can see from the image, imaging is possible, and even the full range of the scanner is accesible without encountering big steps in height. By the way, the nodules, or particles along the fibers are likely to be calcite deposits (Ref 1).

The white egg, (figure below), also presented similar structures in both cuticle and membrane and I cannot identify anything distinguishing the brown from the white eggs, however, because this experiment only included a few images, I don't rule completely that there might be statistical differences between the two once more data is compared.

1 White egg, nanoparticles in the cuticle, pore, and fibers on the egg-shell membrane. Imaged in Dynamic mode with a PPP XYNCHR probe.

So, conclusions, message to take home and future experiments?

• Imaging chicken egg-membranes (and egg shell) in air is feasible, double-sided tape can be used to secure these type of samples, and Dynamic mode can be used to image these samples.

• The samples obtained this way seem simple enough to consider using them in further experiments. They are also clean and homogeneous over large areas.

• In terms of future experiments, I think it will be nice trying to use the membrane as scaffold for tissue growth, or bacteria attachment, or even to capture contaminants (i.e. as a filter).

I hope you like the results shown, and they are useful to you as a guide when working with similar materials.

References:

1 Pillai, M.M., Saha, R. & Tayalia, P. Avian eggshell membrane as a material for tissue engineering: A review. J Mater Sci 58, 6865–6886 (2023). https://doi.org/10.1007/s10853-023-08434-2

2 I. Arzate-Vázquez, J.V. Méndez-Méndez, E.A. Flores-Johnson, J. Nicolás-Bermúdez, J.J. Chanona-Pérez, E. Santiago-Cortés,
Study of the porosity of calcified chicken eggshell using atomic force microscopy and image processing, Micron, Volume 118, (2019). https://doi.org/10.1016/j.micron.2018.12.008.