Résumé
Non-linear optical microscopy (NLOM) is an effective method to follow pathological processes involving collagen fibrosis or collagen destruction. In particular, the intensity of the second harmonic generation (SHG) signal depends on the structure and orientation of the collagen fibers. In this work, we have used two-photon fluorescence (2PEF) and SHG to investigate the dental caries process. The objective of this study is the characterization of the collagen network in sound and carious dentin using NLOM to study non decalcified teeth. Materials and methods: Longitudinal slices with thickness up to 0.5mm were prepared from freshly extracted teeth: 4 from a sound tooth without caries and 4 from a carious tooth. The samples were polished to 0.25 µm and cleaned up in an ultrasound alcohol bath for 5 min. Fluorescence microscopy, at a wavelength of 480nm, was performed on sound and carious dentin structures. Dentin collagen network images were provided by means of 2PEF and SHG, using an incident wavelength of 800nm on areas previously defined by fluorescence microscopy. Results and conclusion: We have visualized groups of collagen fibres that are constituents of the healthy dentin extracellular matrix and succeeded to differentiate between healthy and carious tooth dentin structure. In a carious lesion, the SHG signal is low and the collagen network is difficult to observe. The obtained 2PEF and SHG three-dimensional images of dentine reveal the tubule network. The visualization of the three dimensional structure of the samples and the optical sectioning capacity inherent to the NLOM technique is a powerful approach for non-invasive investigation of dentine structure and caries.