Comparative Sample Preparation Using Focused Ion Beam and Ultramicrotomy of Human Dental Enamel and Dentine for Multimicroscopic Imaging at Micro- and Nanoscale
Katharina Witzke,
Marcus Frank,
Olaf Specht,
Ute Schulz,
Claudia Oehlschläger,
Detlef Behrend,
Peter Ottl,
Mareike Warkentin
Affiliations
Katharina Witzke
Department of Oral and Maxillofacial Surgery/Plastic Surgery, Greifswald University Medicine, Ferdinand-Sauerbruch-Straße DZ7, 17475 Greifswald, Germany
Marcus Frank
Electron Microscopy Centre, Rostock University Medical Center, Strempelstraße 14, 18057 Rostock, Germany
Olaf Specht
Department of Material Science and Medical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119 Rostock, Germany
Ute Schulz
Electron Microscopy Centre, Rostock University Medical Center, Strempelstraße 14, 18057 Rostock, Germany
Claudia Oehlschläger
Department of Material Science and Medical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119 Rostock, Germany
Detlef Behrend
Department of Life, Light and Matter, University of Rostock, Albert-Einstein-Straße 25, 18059 Rostock, Germany
Peter Ottl
Department of Life, Light and Matter, University of Rostock, Albert-Einstein-Straße 25, 18059 Rostock, Germany
Mareike Warkentin
Department of Material Science and Medical Engineering, University of Rostock, Friedrich-Barnewitz-Straße 4, 18119 Rostock, Germany
(1) Background: The aim of this study was to systematically compare TEM sections of mineralized human enamel and dentine prepared by focused ion beam (in situ lift-out) technique and ultramicrotomy through a combination of microscopic examination methods (scanning electron microscopy and transmission electron microscopy). In contrast with published studies, we compared the TEM preparation methods using the same specimen blocks as those for the ultramicrotomy and FIB technique. (2) Methods: A further evaluation of TEM sample preparation was obtained by confocal laser scanning microscopy and atomic force microscopy. In addition, ultramicrotome- and focused ion beam-induced artefacts are illustrated. (3) Results: The FIB technique exposed a major difference between non-decalcified enamel and dentine concerning the ultrastructural morphology compared to ultramicrotome-prepared sections. We found that ultramicrotomy was useful for cutting mineralized dentine, with the possibility of mechanical artefacts, but offers limited options for the preparation of mineralized enamel. FIB preparation produced high-quality TEM sections, showing the anisotropic ultrastructural morphology in detail, with minor structural artefacts. Our results show that the solution of artificial saliva and glutardialdehyde (2.5% by volume) is a very suitable fixative for human mineralized tissue. (4) Conclusions: The protocol that we developed has strong potential for the preparation of mineralized biomaterials for TEM imaging and analysis.
