Study of the porosity of calcified chicken eggshell using atomic force microscopy and image processing

•Porosity of calcified chicken eggshell was examined by AFM and image processing.•The palisade layer exhibits the highest degree of porosity and largest thickness.•Bubble pores in all layers have mainly circular shapes and similar sizes.•A gradient of porosity along the cross-section of calcified eggshell was observed. In this work, the porosity of the layers of calcified chicken eggshell (vertical crystal layer VCL, palisade layer PL and mammillary layer ML) was evaluated using atomic force microscopy (AFM) and image processing (IP). AFM topographic images were obtained from different locations for each layer and along the cross-section of calcified eggshell. Roughness parameters, surface area values, pore size and shape, surface porosity, area occupied by pores and pore density were obtained from AFM and IP. It was observed that the thickest layer (PL) exhibited the highest degree of porosity (surface porosity = 2.75 ± 1.68%, pore density = 162 ± 60 pores/μm2) when compared to the other two layers. In general, the pores located in all layers (“bubble pores”) had circular shape and similar sizes. Measurements revealed a porosity gradient along the cross-section which varied with position, i.e., increasing surface porosity from the VCL towards the region of the PL closer to the ML, and decreasing surface porosity from this location towards the ML innermost surface. This suggests that the calcified eggshell has a sandwich-like structure where porosity may influence gas exchange and mechanical properties. The combination of AFM with IP presented here provides a simple and precise method to study porosity in calcified chicken eggshell, and this methodology could be used to examine other types of porous biological materials.