1. Evgenije Novta, Medicinski fakultet Univerziteta u Novom Sadu, Serbia
2. Tijana Lainović, Medicinski fakultet Univerziteta u Novom Sadu, Serbia
3. Dejan Pantelić, Institut za fiziku, Zemun, Serbia
4. Mihailo Rabasović, Institut za fiziku, Zemun, Serbia
5. Aleksandar Krmpot, Institut za fiziku, Zemun, Serbia
6. Danica Pavlović, Institut za fiziku, Zemun, Serbia
7. Larisa Blažić, Medicinski fakultet Univerziteta u Novom Sadu, Serbia
Nonlinear microscopy (NLM) is one of the latest microscopy techniques, which still hasn’t found its wider use in exploration of dental materials and tissues. Noninvasive optical methods, such as nonlinear optical microscopy, allow nondestructive imaging of tissue (without histological fixation, fluorescent or imunological labelling). The aim of this study was to use NLM for examination of hard dental tissues, their interfaces, as well as the tooth-resin interface. Teeth specimens were observed by a homemade nonlinear microscopy setup at the Institute of Physics in Belgrade. Titanium-sapphire laser, adjusted at 730 nm wavelenght, was used as an excitation source for two-photon excitation fluorescence microscopy (TPEF), while Yb:KGW laser set at 1040 nm was used for second harmonic generation (SHG). Using nonlinear optical effects produced by ultrashort laser pulses (in femto-seconds), this microscopy technique allows excitation of the material in the infrared part of the spectrum and detection of its response in the visible. In this way, penetration depth of the laser beam is increased and laser damage effects are reduced. Specific morphological structures of hard dental tissues and their interfaces which possess intrinsic autofluorescence, were clearly presented using TPEF microscopy. The SHG signal was detected in dentine due to presence of collagen type I (triple helical molecule organized in non-centrosymmetric fibrils), while this signal was completely absent for enamel. These techniques also demonstrated its suitability for investigation of tooth-resin interface. Results showed the increase of autofluorescence in this area after placement of dentin-bonding agents, most likely as a result of exposition of collagen fibrils and activation of dentinal proteases. Nonlinear optical microscopy enables precise insight into the morphological structure of dental tissues, and can also serve for acquiring information about the quality of the hybrid layer, which exists on the tooth-resin interface. These results could have a practical value in clinical diagnosis and treatment, and stimulate development of new enhanced dental materials and equipment in collaboration with the industry. Acknowledgments. Research was supported by the Ministry of Education, Science and Technological Development of the Republic of Serbia (Projects: III45016, III 46010, ON 171038 and TR 035020).
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