## 4 Discussion  
In this work, we investigated the effectiveness of the combination of time-resolved fluorescence and micro-Raman spectroscopy, taking the advantages of the different sensitivity of the two methods. Raman spectroscopy produces a spectral fingerprint that enables molecular characterization of the sample, but at the cost of relatively time-consuming measurements that are often limited by the inherent weakness of the Raman scattering phenomenon, which can be hampered by other competitive processes such as fluorescence. Implementation of the method to the fiber-based imaging mode is not straightforward, although the possibility of combining Raman-FL in a fiber probe for imaging has already been demonstrated [33]. On the other hand, the analysis of UV-induced fluorescence allows for a limited chemical specificity, especially when organic compounds are considered--such as binders and varnishes, which are composed of a variety of fluorophores that give rise to a broadband signal. Furthermore, FL phasor analysis has proven effective in discriminating different binders in real time with an easy-to-use fiber-based imaging modality. Hence, the combined use of the two techniques can provide both molecular fingerprinting and fast analysis by targeting both components of pigment-binder mixtures.  
In pigmented mixtures, the influence of the pigments on the overall fluorescence signal is widely documented in the literature, and may depend on a number of competing factors, including optical scattering and absorption of fluorescence or luminescence emissions from the pigment, as well as chemical interactions (e.g., quenching) between the binder and the pigment [14; 15; 17]. The change in FL observed in this study shows a possible further correlation with the viscosity of the binder in which the pigment is dispersed. In the cases reported here, the fluorescence lifetime of the pigments seems to have been strongly affected by the high-viscosity medium--beeswax--while it remained almost unchanged  
Figure 5: FL (**a**) and Raman (**b**) results for pigmented and non-pigmented mixtures laid on glass and metal substrates. Raman spectra are plotted after baseline subtraction (default function in TWiRE software).  
when mixed with linseed oil, which is characterized by a lower viscosity. It is known that the non-radiative decay of the excited state of a fluorophore is affected by the medium viscosity, which is one of the main parameters determining the diffusion of fluorescent species [48]. In low-viscosity media, the non-radiative decay via intramolecular charge transfer from the excited fluorophore to absorbing molecules in the surrounding environment is greater than in high-viscosity media, resulting in a decrease in the fluorescence lifetime [49]. Further measurements will allow for verification of this hypothesis. For now, the preliminary results presented in this work demonstrate that FL phasor analysis allows for discrimination between materials, and reveals very subtle differences in the decay times of pure or mixed substances.  
The observed influence on the fluorescence lifetime by the bronze substrate is likely related to the presence of copper in the metal alloy. Indeed, copper(II) ions are well-known quenchers of fluorescence, not only producing evident changes in the fluorescence spectra of protein-based binding media [19], but also significantly reducing the fluorescence lifetime via a Forster resonance energy transfer (FRET) mechanism [50]. As with pigments dispersed in the binder, for mixtures laid on a bronze substrate, the reduction in lifetime can be also related to the concomitant release of non-radiative energy to the molecular environment.