Hard Tissue Characterization by Non-Destructive 3D CLSM/Micro-Raman

Objectives: So far ultra-structural and micro-chemical studies on biological hard tissues require tedious techniques of sample pre-treatments bearing the risk of manifold artefacts. Methods: Innovative fibre optic coupling of a prototype confocal laser scanning microscope (Leica TCS SP2-X1, air objectives) with a micro-Raman spectrometer (Jobin Yvon: stigmatic Raman spectrograph, optimised for CCD Camera, grid 1200 lines/mm) for simultaneously recording CLSM-images and micro-Raman spectra. Specimens: sectioned (otherwise untreated) naturally wet human enamel and dentin. Results: Specimen confocal illumination scans as well as excitation of molecular vibrations (Raman effect) were achieved by a single 632 nm He/Ne laser source, allowing truly three-dimensional confocal hard tissue imaging (reflection mode) together with simultaneous micro-Raman spectra uptake. These conditions compromise between high lateral CLSM image resolution (about 300 nm) and appropriate (natural hard tissue) fluorescence suppression, a prerequisite for Raman spectra uptake. The present technical realization allows the definition of microscopic subsurface Raman regions of interest (less than 1 cubic micrometer) directly on the computer monitor recording the confocal images. Thus non-destructive micro-Raman spectra can be assigned to specific microscopic subsurface compartments of dental hard tissues, enabling ultra-structural and micro-chemical characterization of e.g. prismatic versus interprismatic enamel, tubular versus intertubular dentin, etc. Most sensitive for the characterization of dental hard tissues are the Raman modes attributed to carbonated apatite: the various u₁ - u₄ PO₄ (mainly 960 cm^-1), the CO₃ (1070 cm^-1) as well as the non-apatitic HPO₄ (1003 cm^-1) vibrations. The fine structure of the individual Raman bands allows further interpreting apatitic crystal orientation. Organic components in particular from dentin contribute amide (1245 cm^-1) and C-H (1453 cm^-1) stretch and bend vibrations to the Raman spectra. Conclusions: The present prototype fibre optic CLSM/Raman coupling eliminates problems so far excluding the more widespread adoption of the technique. Prototype experiments demonstrated the feasibility of ultra-structural and micro-chemical characterization of dental hard tissues.