Multi-photon excitation fluorescence and third-harmonic generation microscopy measurements combined with confocal Raman microscopy for the analysis of layered samples of varnished oil films

The non-destructive determination of layer structures in works of art remains a significant challenge. Non-linear microscopy and confocal Raman microscopy (CRM) were employed for characterisation of varnish-media layers in model samples, providing important information regarding the thickness of materials and the identification of different media in depth. Commonly found triterpenoid varnishes mastic and dammar were applied over a single layer of films of linseed oil. Non-linear microscopy of samples was carried out using a 1028-nm femtosecond laser source; both third-harmonic generation signals (THG) and three-photon fluorescence signals (3PEF) of samples were collected in an effort to measure the thickness of mono- and bi-layers; in parallel scans of larger areas were undertaken to assess heterogeneities in samples with spatial resolution of ∼2 μm. Complementary spectroscopic information from CRM collected with both a 514.5-nm argon-ion and a 785-nm diode lasers coupled with a 100X objective and a motorised stage was carried out. Comparison of C–H stretching regions of Raman spectra allowed the differentiation between different molecular materials and the fingerprint region was employed for the depth profiling of the samples.