# Structural Pattern Analysis in _Patella vulgata_ Shells Using Raman Imaging
## 4 Conclusions  
In this work, Raman imaging was applied to analyze the structural and compositional heterogeneity in six _P. vulgaris_ shells collected from northern Spain. The results demonstrate that Raman spectroscopy is a powerful, non-destructive, and highly precise tool for characterizing the spatial distribution of calcium carbonate polymorphs, organic pigments, and carbonate ion substitutions in the CaCO\({}_{3}\) lattice within _P. vulgaris_ shells. The analysis revealed clear spatial variations in mineralogical composition, with distinct layers of aragonite and calcite that align with the shell's structural organization. These findings reinforce the consistency of biomineralization strategies among limpets and underline the shell's potential as an environmental archive. Carotenoids, responsible for shell pigmentation, exhibited heterogeneous intensity patterns across the shell, which were independent of the underlying mineralogical composition but closely aligned with growth dynamics. Furthermore, spectral shifts in the carotenoid Raman band at 1521 cm\({}^{-1}\), corresponding to the vibrational mode of conjugated C=C bonds, suggest structural modifications likely associated with changes in the surrounding mineral phase during shell formation. In parallel, shifts observed in the symmetric stretching mode \(\
u_{1}\) of the carbonate group (1085 cm\({}^{-1}\)) suggest substitution processes within the carbonate lattice, most likely dominated by magnesium incorporation. The spatial correlation between these shifts and carotenoid distribution points to a coordinated interplay between mineral chemistry and organic compound deposition during biomineralization. Although this study examined a limited number of samples, it allowed the identification of repetitive and consistent internal structural and compositional patterns using Raman imaging. Nevertheless, individual differences were also observed, reflecting the biological differences inherent to living organisms. These differences, however, did not obscure the main spatial patterns. Inter-individual variability in physiological status and genetic background may influence shell geochemistry. Metabolic responses to stress [59] and genetic variability in biomineralization-related gene expression [60] have been reported to modulate elemental incorporation during shell formation. Although not explicitly addressed in the present study, these sources of intra-specimen variability are relevant and will be considered in future research. In addition to individual variability, environmental differences across geographic regions--such as variations in temperature, salinity, or pH--can also affect shell structure and composition. Future research could explore _P. vulgaris_ shells from diverse environmental settings to better understand how external factors influence biomineralization patterns. Other studies have investigated the relationship between shell geochemistry and environmental variables, providing useful references for future approaches [61; 62; 63]. Comparative studies across species, locations, and environmental conditions, as well as analyses at different growth stages, will provide a broader framework for interpreting structural and compositional trends. In this context, Raman imaging could be complemented with techniques such as scanning transmission electron microscopy (STEM) and atom probe tomography (APT) to correlate internal shell structure with elemental distribution. Likewise, analyzing shells at different growth stages could offer insights into the temporal evolution of mineral composition. Finally, the use of portable Raman systems in the field could enable non-invasive, real-time analyses directly on archaeological or ecological samples, expanding the practical applications of this technique. Conceptualization, M.G.F.-M., B.G.G., C.G.-G., J.M., J.F.A., L.R.-C. and A.C.; methodology, M.G.F.-M., B.G.G., C.G.-G., L.R.-C. and A.C.; software, M.G.F.-M., J.M.