Quantification of eggshell microstructure using X-ray micro computed tomography

Figures & data

Figure 1. Two-dimensional cross section through the X–Z axis of an eggshell sample at a resolution of 1.5 µm, generated by X-ray mCT. Labels show the thickness of the palisade matrix (distance 1) and the thickness of the outer shell membrane (distance 2) below the mammillary nucleation sites.

Figure 2. Reconstruction showing orientation of 3D axis used. Scale bar = 400 µm.

Figure 3. Negative three-dimensional images showing the quantity, shape and arrangement of pores in the (A) sharp, (B) equator and (C) blunt regions of chicken eggshell. The images are foreshortened, with the horizontal focal point of each image placed midway between the outside of the shell (top) and the mammillary nucleation surface (bottom) but with the horizontal curvature of the shell left intact. Note that pores vary in the size, shape and the completeness with which they traverse the whole shell. Scale bar = 400 µm.

Figure 4. Two-dimensional image (1.5 µm resolution) in the X–Y plane of the junction between the outer shell membrane and the mammillary layer of a chicken eggshell fragment. Because of the curvature of the shell, the centre of the image shows only the collagen fibres of the outer shell membrane and the mammillary knobs (mammillary body nucleation sites) become visible towards the periphery (appearing to increase in frequency and size but then coalescing).

Figure 5. Representative mammillary body profile showing the count of individual mammillary bodies viewed in a flat sampling plane (X–Y) as the curved shell is traversed vertically (Z-axis) from the nucleation sites, through the main palisade matrix to the external surface. The contrast is set such that X-ray dense material (calcite) appears white while other material or empty space appears black. Images A–E represent the visual appearance of the scan, showing the increase from few dense bodies in A and B, to the maximum number in C, before the calcite columns fuse in D and E. The mammillary body density was estimated as the maximum count divided by the unit area.

Figure 6. (A) Three-dimensional reconstruction of an equatorial region of eggshell between the outer surface and the mammillary layer, showing the high-density material of the palisade matrix (calcite) as white and the intrinsic porosity (vesicles) in blue (colour version available online at http://dx.doi.org/10.1080/00071668.2014.924093). (B) The same reconstructed image with the dense material removed to reveal the negative space of the vesicles. Scale bar = 400 µm, resolution = 1.5 µm.

Table. Eggshell microstructural parameters determined using three-dimensional X-ray mCT, showing mean values ± SEM in three regions of the eggshell (n = 10). P-value indicates significance level using one-way analysis of variance (ANOVA); where a significant effect of shell region was observed, values in the row without a common superscript are significantly different (P < 0.05) using Fisher’s least significant difference post hoc test. Definitions of variables are given in the text

Variable	Sharp end	Blunt end	Equator	Significance P
Shell thickness, µm	371 ± 8	367 ± 8	361 ± 10	NS
Mammillary density, N/mm^2	310.5 ± 17.1^a	252.6 ± 8.76^b	286.6 ± 18.9^a,b	<0.05
Pore frequency, N/mm^2	0.82 ± 0.08^a	1.38 ± 0.11^b	1.64 ± 0.06^c	<0.001
Average pore size, µm^2	94.95 ± 8.69^a	129.43 ± 7.25^b	131.18 ± 6.73^b	<0.01
Sponginess, %	0.29 ± 0.05	0.37 ± 0.08	0.33 ± 0.04	NS

Figure 7. Two-dimensional slice-by-slice (traversing the X–Z axis) images of an equatorial region of shell, showing a pore which traverses the palisade matrix. Images are at 6 µm intervals and are at a resolution of 1.5 µm. Scale bar = 400 µm.

Figure 8. Three-dimensional reconstruction of an equatorial region of eggshell between the outer surface and the mammillary layer, generated using X-ray mCT. The reconstruction is positioned (A) to cut through vertically a pore along one face of the image with high-density material (calcite) shown as white and other material/space in grey. In (B), the dense material is shown transparent with the pore false coloured in shaded yellow to show its full shape and orientation through the shell matrix (colour version available online at http://dx.doi.org/10.1080/00071668.2014.924093). Scale bar = 400 µm, resolution = 1.5 µm.

Figure 9. Relationship between mammillary body density and pore frequency across the entire eggshell (r² = 0.1464, P = 0.037). N = 30.

Figure 10. Relationship between pore frequency and average pore size across the entire eggshell (r² = 0.2076, P = 0.011). N = 30.