2.1.4. Confocal Raman Spectroscopy Analyses

Raman spectroscopy was performed at German Aerospace Center in Berlin, using a 532 nm excitation laser, with a Confocal Raman microscope (WITec alpha300), at room temperature, under ambient atmospheric conditions. The spectral resolution of the spectrometer is 4–5 cm<sup>−</sup>1. Before the analyses, the spectrometer was calibrated with pure silicon and paracetamol test samples. A 10× Nikon objective, with a 0.25 numerical aperture, was used to focus the laser on a 1.5 μm spot. For single spectra, all measurements were performed at 0.1 mW laser power with 10 s integration and 50 accumulations. Image scans were done at 0.7 mW with 1 s and 1 accumulation (to avoid signal saturation or damaging effects) on three distinct areas up to 100 μm × 100 μm and up to 500 image points, thus collecting a minimum of 1000 measurements per sample.

All data analyses were performed with the WITec Project FIVE software. Parameters used for analysis were the value of signal coverage on random zones on the samples, to show the presence/absence of the signal defined as spectra having Signal to Noise Ratio (SNR) values superior to 5 and the value of SNR. To analyze the presence of the signal, the region of interest between 200 and 2000 cm−<sup>1</sup> was cropped, then a fifth-order polynomial function was applied for background subtraction; and finally, SNR masks were applied. As previously described in [28] for carotenoid pigments, the SNR was defined as the height of the 1600 cm−<sup>1</sup> peak divided by the noise represented by the standard deviation of a

spectral region without features (1750 to 1950 cm−1). The position of the 1600 cm−<sup>1</sup> peak was also derived from masks available in the WITec Project FIVE software.
