3D Nanostructuring and Nanoanalysis of Human Enamel and Dentin Using Focused Ion Beams S. Sadighikia*, M. Sezen** and T. Uusimaki*** *Faculty of Natural Sciences and Engineering, Sabanci University, Istanbul, Turkey, ssadighikia@sabanciuniv.edu **Sabanci University Nanotechnology Research and Application Center (SUNUM), Istanbul, Turkey, meltemsezen@sabanciuniv.edu *** ETH Zurich, EAWAG, Switzerland, tonimikael.uusimaeki@eawag.ch ABSTRACT This study particularly focuses on three-dimensional nanoanalysis of human dentin and enamel samples, using FIB nanostructuring methods such as FIB tomography and preparation of 3D TEM samples. Features within the human dentin and enamel with micro and nano sized were revealed with high definition and accuracy. While the micro sized features reconstructed in three dimentions by means of stacking 2D SE images which are aquired by slice and view application of FIB/SEM platform, a novel pin-like TEM sample was prepared with FIB to investigate the nano sized features within the dentin and enamel. The complimentary analysis helped to reveal the microstructure and morphology of human dentin and enamel in three dimensions in detail. Keywords: FIB/SEM, TEM, tomography, dentin, enamel, 3D analysis, dentistry 1 INTRODUCTION Dental hard tissue comprises a combination of enamel and dentin, both of which have different compositions and structures. Such materials have both micro- and nanometer sized channels, pores and features within the structure. One of the well-known ways of revealing those features is X-ray tomography which is not sufficient due to resolution limits. For having a precise and reliable characterization of small features, 3D electron microscopy is a convenient application [1]. Dentin is a hydrated hard tissue that covers the majority of human teeth by both weight and volume [2] The tissue serves as an elastic foundation for the hard, outermost enamel, and as a protective enclosure for the central pulp. Dentin consists of microscopic channels, called dentinal tubules, which radiate outward through the dentin from the pulp to the exterior cementum or enamel border. These tubules contain fluid and cellular structures [3]. As a result, dentin has a degree of permeability, which can increase the sensation of pain and the rate of tooth decay. Dentin is traversed by a network of tubules that are oriented radially outward from the central pulp towards the dentin–enamel junction [2]. On the other hand, type-I collagen forms a fibrous three dimensional network structure which build up the dentin matrix. Compared to bone, the collagen matrix in dentin is more interwoven with numerous crossing of fibrils. The layers of a human tooth are demonstrated in Fig. 1. Figure 1: The scheme of a human tooth showing the individual layers. Enamel is the hardest biological substance in the human body and is a composite material consisting of both a mineral and an organic phase. The mineral phase predominates (95–96 wt.%) and consists primarily of calcium phosphate salts in the form of large hexagonal hydroxyapatite crystals that are both carbonated and defective. Sets of similarly orientated crystals form rod-like structures called enamel prisms, 3–6μm in cross-sectional diameter. Prisms are separated from each other by a thin organic prism sheath and by interprismatic enamel. The protein/organic matrix comprises approximately 1 wt.% of the enamel, and the remaining approximately 3 wt.% is contributed by water. As the demand for providing information at nano and subnano scale especially in the field of “life sciences” increases, the scientists are in search of employing cutting- edge analysis technologies and developing novel