Abstract

Nonlinear optical (NLO) microscopy has revolutionized three-dimensional (3D) imaging of biological tissues by offering new modes of contrast even in unstained tissues. Notably, two-photon excited fluorescence (2PEF) and second harmonic generation (SHG) signals can be recorded simultaneously in two detection channels to probe different tissue components. Since few years, some studies have shown the potential of this technique for the study of cultural heritage artefacts. 2PEF signals are emitted by a wide range of materials (fluorophores) in historical artefacts with specific absorption and emission fluorescence spectra [1]. SHG signals are specific for dense and well aligned structures and thus enable the visualization of fibrillar collagen without any labelling and with unequalled sensitivity and specificity [2]. In contrast, SHG signals vanish for centrosymmetric materials such a gelatin, which is obtained upon degradation (denaturation and hydrolysis) of collagen. Accordingly, SHG microscopy provides structural information about the 3D organization of the fibrillar collagen within parchments and other skin-based artefacts [3].

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