## 2 Materials and Methods  
Four samples were taken from the painting in those areas where silver was applied as background in order to study the stratigraphy and gather information on the technical execution and the chemical composition of the artistic materials. Figure 1 shows a view of the entire painting along with the sampling points. Sampling was carried out on original layers avoiding areas affected by most recent interventions of restoration.  
The fragments were cast in polyester resin (Mecaprex 2S from Presi, Grenoble, France), dried and finely polished for cross-sections preparation. The stratigraphies were then observed with a UV-VIS Nikon Eclipse E400 epi-fluorescence microscope (Tokyo, Japan) provided with 5\(\times\), 10\(\times\), 20\(\times\), and 100\(\times\) objectives and with a Nikon D80 DSLR digital CCD camera. Fluorescence was excited with a mercury lamp and was observed using a V2A Nikon filter cube (Exc.:380-420 nm, DM:420 nm, Bar.:450 nm).  
Scanning Electron Microscopy-Energy Dispersive X-Ray Spectrometry (SEM-EDS) measurements were carried out by using a UHR-SEM Gaia 3 FIB/SEM by Tescan (Brno, Czech Republic) placed at the Center of Electronic Microscopies \"[PERSON]\" (Ce.M.E-CNR).  
UV-Induced VIS Fluorescence (UVIVF) microspectroscopy was performed with a high-sensitivity Avaspec (Avantes, Apeldoorn, The Netherlands) CCD spectrophotometer (200-1100 nm, grating 300 lines/mm) coupled through an optical fiber to a Nikon Eclipse 400 epi-fluorescence microscope. An HBO mercury short-arc lamp (emission above 295 nm) was used as an excitation source, whereas a CFI PlanFluor 100\(\times\)/1.3 OIL objective lens and the V2A Nikon filter cube to collect fluorescence emission from the varnish layers of the prepared cross-sections.  
Fourier Transformed-Infrared (FT-IR) spectra of paint samples in embedded and non-embedded conditions were collected using Diffuse Reflectance Infrared Fourier Transform (DRIFT) and Microattenuated Total Reflection Fourier Transformed-Infrared (\(\mu\)-ATR FT-IR) techniques. An Agilent Cary 630 FTIR portable spectrometer (Agilent Technologies, Santa Clara, CA, USA) fitted with a diffuse reflectance accessory for non-contact measures was exploited for Diffuse Reflectance Infrared Fourier Transform (DRIFT) measurements. Pseudoabsorbance spectra (log(1/R); R = reflectance) were acquired in the 4000-650 cm\({}^{-1}\) range, at 8 cm\({}^{-1}\) resolution and by averaging 64 scans per sample accumulated. The beam spot size was estimated to be approximately 1 mm. No correction algorithms were applied to DRIFT spectra.  
Microattenuated Total Reflection Fourier Transformed-Infrared (\(\mu\)-ATR FT-IR) spectra were instead collected using an Agilent Cary 660 FT-IR spectrometer coupled with the Cary 620 Microscope and equipped with a MCT detector. The spectra were acquired in ATR mode with Germanium crystal, collecting 64 scans, with a resolution of 4 cm\({}^{-1}\) in the 4000-400 cm\({}^{-1}\) range. Spectra were processed using Agilent Resolutions Pro software (Agilent Technologies, Santa Clara, CA, USA ).  
Raman measurements were performed under an XPlora Horiba micro-Raman instrumentation (Chelmsford, Essex, UK) using a 785 nm laser wavelength, a 100\(\times\) objective, and a diffraction grating of 1200 g/mm. Raman spectra were collected using an integration time of 10 s at 4 mW laser power and were averaged over \(N=15\) mapping points.