## 4 Discussion  
According to a top-down/bottom-up procedure, the multianalytical approach followed in this work provided valuable information for the discussion of all the gathered results, opening new readings on the artistic execution technique of this rare artwork. Over the Ag leaf, two different organic layers were found. An oil-based topcoat, probably blended with a natural resin, might have been used as the \"mecca\" layer to make the silver ground brighter and like gold, thus recalling gilding techniques and procedures mentioned in many historical treaties [10]. However, although analytically challenging, no signals relative to natural dyes (curcuma, saffron, and aloe) added to imitate gold were found neither using DRIFT nor micro-ATR-FTIR spectroscopies. Beneath this layer, a thin organic layer applied originally over the silver leaf to protect it from starnishing was found. The latter was composed of egg yolk with wax, in part or completely transformed over time into a calcium oxalate film. These outcomes were further strengthened by UVIVF microfluorimetry. In contrast to the topcoat, the broad, low-intensity and remarkably red-shifted fluorescence emission of the thin layer over the Ag leaf reflects very well the composition of an aged and altered organic layer. More importantly, the latter figured out also a \"wavy\" profile when observed in the transverse section under UVIVF microscopy, indicating either that it was still fresh when the uppermost layer was applied or that the latter was later added causing softening and partial dissolution of the underlying layer. These two assumptions shed new light on the execution technique of this painting, opening also new possible ways of interpretation. Technically, it has more sense that the uppermost layer was later added as past conservation treatment rather than applied when the underlying layer was still fresh. The lack of the top oil coat in the sampled fragment on the frame went in this direction. Contrarily, what would be the function of this thick oil coat later added? If intended to impart a golden hue to the silver leaf as sort of a \"mecca\" layer it should have been applied over the metal leaf. In this scenario, the second hypothesis becomes more reliable as well as the function of the old thin layer covering the Ag leaf. The latter could have been thought either as a final application for protecting the leaf and for increasing or reducing its shine, thus for an aesthetic reason. It is worth reporting that the use of egg yolk and wax could not be casual and reflects to some extent the cold-encaustic technique, notoriously called \"Punic wax\". In this technique, the wax before to be added to protein compounds (casein, egg, gum, and glue) was made water-soluble following an alkali treatment with lime. The addition of wax leads to improve adhesion, spreading, and transparency of the medium as well as the resistance to moisture [32]. In this painting, \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Compounds** & **Raman Frequency (cm\({}^{-1}\))** & **Assignments** & **Comments** \\ \hline \multirow{6}{*}{CaSO\({}_{4}\) (2H\({}_{2}\)O)} & 415 & \(\
u_{2}\)(SO\({}_{4}\)) & Dihydrate \\ \cline{2-4}  & 496 & \(\
u_{2}\)(SO\({}_{4}\)) & Dihydrate \\ \cline{2-4}  & 607 & \(\
u_{4}\)(SO\({}_{4}\)) & Anhydrate \\ \cline{2-4}  & 627 & \(\
u_{2}\)(SO\({}_{4}\)) & Anhydrate \\ \cline{2-4}  & 675 & \(\
u_{2}\)(SO\({}_{4}\)) & Dihydrate \\ \cline{2-4}  & 1007 & \(\
u_{1}\)(SO\({}_{4}\)) & Dih./Anh. \\ \cline{2-4}  & 1015 & \(\
u_{3}\)(SO\({}_{4}\)) & Anhydrate \\ \hline \multirow{2}{*}{Ag\({}_{2}\)S} & 158 & Ag lattice & \\ \cline{2-4}  & 198 & \(\
u\)(Ag-S) & \\ \cline{2-4}  & 281 & \(\
u\)(Ag-S) & \\ \hline \multirow{2}{*}{Ag\({}_{2}\)O} & 236 & \multirow{2}{*}{\(\
u\)(Ag-O)} & \\ \cline{2-4}  & 425 & \(\delta\)(Ag-O) & \\ \cline{2-4}  & 491 & & \\ \cline{2-4}  & 563 & & \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Raman vibrational modes assignments of CaSO\({}_{4}\), Ag\({}_{2}\)S, and Ag\({}_{2}\)O compounds.