Correlative nonlinear optical microscopy and infrared nanoscopy reveals collagen degradation in altered parchments

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Non‐invasive investigation techniques are strongly needed to avoid sampling during the examination of heritage artefacts. This study aims at developing specific and non‐destructive mapping of the degradation of parchment, which is mainly composed of dermal fibrillar collagen. The main issue is to characterize gelatinization, its ultimate and irreversible alteration corresponding to collagen denaturation to gelatin, both from the morphological and chemical point of view. To that end, we implement correlative imaging of parchment using nonlinear optical (NLO) microscopy and nanoscale infrared spectroscopy (nanoIR). NLO microscopy, also called multiphoton microscopy, advantageously provides non‐invasive three‐dimensional (3D) multimodal imaging of scattering samples with micrometer‐scale resolution [1]. Among the collected signals, SHG signals are specific for dense non‐centrosymmetric materials. These signals therefore provide a unique structural probe of fibrillar collagen at the micrometer scale since the small signals from the collagen triple helices at molecular scale are amplified by constructive interferences at macromolecular scale due to the tight alignment of the collagen molecules to form collagen fibrils [2]. Nanoscale infrared spectroscopy is carried out thanks to an Atomic Force Microscope (AFM) coupled with an IR pulsed tunable laser [3]. This IR nanoscopy allows acquiring chemical mapping and local IR spectra to characterize and image samples at nanoscale. Using this multiscale approach, key information about collagen and gelatin signatures is obtained in parchments and assessed by characterizing the denaturation of pure collagen reference samples. A new absorbing band is observed near the amide I band in the IR spectra, colocalized with the onset of fluorescence signals in NLO images. Meanwhile, a strong decrease is observed in Second Harmonic signals (see figure 1). NLO microscopy therefore appears as a powerful tool to reveal collagen degradation in a non‐invasive way. It should provide a relevant method to assess or monitor the condition of collagen‐based materials in museum and archival collections and opens avenues for a broad range of applications regarding this widespread biological material.