# Structural Pattern Analysis in _Patella vulgata_ Shells Using Raman Imaging
## 3 Results and Discussion
### Pigment Distribution and Variations  
The presence of pigments within the shell matrix provides insights into biomineralization dynamics and potential environmental influences on shell formation. To examine their spatial organization, Raman intensity maps were generated by analyzing the two predominant bands associated with carotenoids, 1130 cm\({}^{-1}\) and 1521 cm\({}^{-1}\). Figure 8 shows the spatial distribution of carotenoids in _P. vulgaris_ shells. These maps were obtained by summing the Raman intensities of the 1130 cm\({}^{-1}\) and 1521 cm\({}^{-1}\) bands to show their distribution across the shell layers. The Raman intensity maps in Figure 8 reveal a heterogeneous distribution of carotenoids within the shells. Blue-colored regions, indicating lower intensities at the 1130 cm\({}^{-1}\) and 1521 cm\({}^{-1}\) bands, corresponding to areas with reduced carotenoid content. These regions allow the differentiation of growth lines, suggesting that carotenoid incorporation varies during shell formation. This relationship between carotenoid presence and growth lines may be influenced by changes in the organic matrix, metabolic processes, or seasonal factors. Furthermore, carotenoids are most concentrated in the outermost shell layers, particularly near the periostracum, while their presence is noticeably reduced in the inner boundary regions. This pattern is consistent with other mollusks, where pigments are primarily restricted to external layers [36]. However, distribution varies among shells; LAN109 (Figure 8a), LAN112 (Figure 8b), LAN229 (Figure 8d), and LAN258 (Figure 8f) show strong carotenoid signals in the outer shell, while LAN183 (Figure 8c) and LAN255 (Figure 8e) exhibit a more diffuse presence. These differences may reflect individual biological or ecological factors influencing pigment deposition. In most of the shells analyzed, high carotenoid concentrations led to spectral saturation, requiring the filtering and exclusion of affected measurements. This effect is caused by the strong resonance Raman response of carotenoid vibrational modes combined with  
Figure 8: Raman intensity maps of pigments in transversal shell sections of _P. vulgaris_. Maps were obtained by summing the Raman intensities of the 1130 cm\({}^{-1}\) and 1521 cm\({}^{-1}\) bands, associated with carotenoid bonds. The color scale represents intensity variations in arbitrary units (a.u.). These values correspond to detected signal counts after preprocessing, ranging from the lowest values in blue to the highest values in red. Subfigures correspond to the following shells: (**a**) LAN109, (**b**) LAN112, (**c**) LAN183, (**d**) LAN229, (**e**) LAN255, and (**f**) LAN258. Each subfigure includes a scale bar that represents 1 mm. increased fluorescence, which exceeds the detector's range and results in information loss due to saturation. This issue is particularly evident in LAN112 (Figure 8b), where missing spectral points correspond to the outermost shell margin, a region known to exhibit strong vibrational signals associated with shell coloration [17]. The comparison between Figures 7 and 8 suggests that carotenoid incorporation is not influenced by aragonite or calcite layers, indicating that their distribution occurs independently of mineralogical phases. However, our analysis primarily detects carotenoids rather than the full organic composition of the shell, as most organic molecules in the shell matrix do not exhibit a resonance Raman signal. This limitation makes it uncertain how other organic components are distributed within the shell. Figure 9 shows the spectral shifts of the 1521 cm\({}^{-1}\) Raman band to explore possible variations in the chemical structure of carotenoids within the shell matrix. The spectral shifts observed in Figure 9 align with the alternation of aragonite and calcite layers identified in Figure 7, suggesting that the mineral phase influences the structural properties of carotenoids.