Comparative Sample Preparation Using Focused Ion Beam and Ultramicrotomy of Human Dental Enamel and Dentine for Multimicroscopic Imaging at Micro- and Nanoscale

Abstract

(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.

Keywords: TEM; artefacts; dentine; enamel; focused ion beam; ultramicrotomy.

Figure 1

The structural hierarchy of mature human enamel and dentine from the macroscale to nanoscale. (a) Stereo-zoom microscopy of a tooth slice from a premolar; thickness, 1 mm; view from mesial, (E) enamel and (D) dentine. (b) SEM micrograph of enamel. The alternating orientation of the prism rods (level 6) reveals the Hunter–Schreger bands (level 7). (c) SEM micrograph of enamel showing interprisms and prisms (level 5) formed by hydroxyapatite crystallite nanofibrils (level 2–4). (d) TEM micrograph of enamel showing hydroxyapatite crystallites (level 1). (e) SEM micrograph showing numerous dentinal tubules, peritubular dentine and intertubular dentine. (f) SEM micrograph illustrating dentinal tubules and odontoblast processes. (g) TEM micrograph of the nanostructure of intertubular dentine.

Figure 2

Experimental workflow of human mineralized enamel and dentine preparation using ultramicrotomy and in situ FIB lift-out technique for TEM imaging. For inspection and quality control in tissue preparation, multimicroscopic imaging was used (Stereozoom, AFM, CLSM, SEM).

Figure 3

Comparison of ultramicrotome and in situ FIB lift-out-prepared human mineralized enamel. (a) Ultramicrotome-prepared enamel section depicting the increasing size gradient of enamel hydroxyapatite crystals towards the outer enamel surface (ES). Delamination of the hydroxyapatite crystals and the organic matrix, indicated by arrow heads. (b) In situ FIB lift-out-prepared enamel section at the interface (white broken line) between an enamel prism (PS) and the interprismatic substance (IPS), showing the different orientation of hydroxyapatite crystals in PS and IPS. (c) Ultramicrotome-prepared enamel section showing hydroxyapatite crystals forming nanofibrils with an aligned course (arrows). (d) In situ FIB lift-out-prepared enamel section showing hydroxyapatite crystals (arrows) inside the interprismatic substance. (e) High magnification of an ultramicrotome-prepared enamel section reveals single hydroxyapatite crystals (arrows) that are covered with nanoparticles between 10 and 30 nm in size. (f) High magnification of an in situ FIB lift-out-prepared section showing the enamel nanostructure in detail, with no nanoparticles on the surface of the hydroxyapatite crystals (arrows).

Figure 4

Comparison of ultramicrotome and in situ FIB lift-out-prepared human mineralized dentine. (a) Ultramicrotome-prepared dentine section showing the peritubular (PTD) and intertubular dentine (ID) interface; (DT) dentinal tubule (b) In situ FIB lift-out-prepared dentine section showing the highly mineralized peritubular dentine (PTD) and the less mineralized intertubular dentine (ID); (DT) dentinal tubule. (c) Ultramicrotome-prepared intertubular dentine. The inset in c is a magnified TEM image of a hydroxyapatite crystal bundle. (d) In situ FIB lift-out-prepared intertubular dentine section. Arrows indicate hydroxyapatite crystal bundles.

Figure 5

Comparison of enamel block surface morphology after mechanical polishing and ultramicrotomy. (a) Reflected-light microscopy (differential interference contrast mode) of mechanically polished enamel showing polishing marks on the enamel surface with enamel (E) and resin (R). (b) Confocal laser scanning microscopy of mechanically polished enamel showing scratches and polishing marks. The microstructure of enamel (prisms and interprismatic substance) is not obvious with enamel (E) and resin (R). (c) AFM micrograph of mechanically polished enamel depicting scratches and polishing marks. (d) Reflected-light microscopy of the enamel block surface after ultramicrotomy, showing the microstructure of enamel (prisms and interprismatic substance) with enamel (E) and resin (R). (e) Confocal laser scanning microscopy of the enamel surface after ultramicrotome preparation, depicting numerous prisms and the interprismatic substance with enamel (E) and resin (R). (f) AFM micrograph of the enamel surface after ultramicrotome preparation showing numerous prisms (dark red area) and the interprismatic substance (yellow area). (g) Horizontal height profile of the mechanically polished enamel surface measured by AFM, showing the surface roughness at the microscale in detail. (h) Horizontal height profile of the same enamel sample after ultramicrotome preparation, measured by AFM.

Figure 6

Comparison of dentine block surface morphology after mechanical polishing and ultramicrotomy. (a) Reflected-light microscopy (differential interference contrast mode) of mechanically polished dentine showing polishing marks on the dentine surface. (b) Confocal laser scanning microscopy of mechanically polished dentine showing scratches and polishing marks. The microstructure of dentine (dentinal tubules and intertubular substance) is obvious. (c) AFM micrograph of mechanically polished dentine depicting scratches and polishing marks. (d) Reflected-light microscopy of the dentine block surface after ultramicrotomy, showing the microstructure of dentine (dentinal tubules and intertubular substance) with dentine (D) and resin (R). (e) Confocal laser scanning microscopy of the dentine surface after ultramicrotome preparation depicting numerous dentinal tubules and the intertubular substance with dentine (D) and resin (R). (f) AFM micrograph of the dentine surface after ultramicrotome preparation showing numerous dentinal tubules and the intertubular substance.

Figure 7

Dentine and enamel ultrathin sections showing ultramicrotome-induced artefacts. (a) SEM image of ultramicrotome-prepared enamel. TEM examination is only possible near the enamel–epoxy–resin interface (white arrow heads). Enamel in the center of the ultrathin section became lost during the preparation process using ultramicrotomy. (b) SEM image of ultramicrotome-prepared dentine showing local folding (white arrow heads) and cutting marks (white arrow). (c) TEM image of ultramicrotome-prepared enamel. The X–Y bending indicates chatter marks. (d) TEM image of ultramicrotome-prepared dentine showing local cracks at the peritubular dentine (pD), with intertubular dentine (itD).

Figure 8

Enamel and dentine sections depicting FIB and electron-beam-induced artefacts. (a) TEM image of an FIB-prepared enamel lamella. (b) SEM micrograph of an FIB-prepared dentine section depicting FIB/TEM–lamella bending. (c) SEM micrograph of FIB-prepared dentine showing enlarged FIB/TEM lamella bending. (d) TEM image of FIB-prepared enamel. The arrow heads indicate a short focus depth at the lateral image margin due to the FIB/TEM lamella bending. (e) TEM image of FIB-prepared enamel depicting the numerous voids introduced during FIB preparation in enamel hydroxyapatite crystals. These voids are more clearly observed at the margin of the electron-transparent area (arrow heads). (f) TEM image of FIB-prepared enamel showing FIB/TEM lamella destruction induced by TEM inspection. Small initial voids induced during FIB milling create beam-sensitive areas that conflate into holes and destroy the hard tissue’s ultrastructure during TEM analysis.