Smf 1000

Dentin exposed at four additional molars were wet sanded using #600 SiC paper, upon which TP and the composite cement were applied following the manufacturer's instructions. The cement surface was covered with a plastic strip (3M Oral Care, St. Paul, MN, USA) prior to being light-cured for 20s. Epoxy resin-embedded tooth specimens were observed by FIB-SEM (SMF-1000, HITACHI High-Tech Science, Tokyo, Japan) (Yoshihara et al., 2020b) and TEM (HR-TEM, 200 kV; JEM-2100, JEOL).

Eight slabs embedded in epoxy resin with the cross-sectioned adhesive-dentin interface exposed at the surface were mechanically polished using diamond lapping films (3M, St.Paul, MN, USA) and argon-ion polishing (SM-090101 Cross-Section Polisher, JEOL, Tokyo, Japan). Orthogonal FIB-SEM (SMF-1000, HITACHI, Tokyo, Japan) imaging was conducted for 3D-ultrastructural characterization. The FIB and SEM ion/electron beams were orthogonally aligned, instead of being set at the standard angle of 60°, to obtain high spatial resolution and high-contrast SEM photomicrographs (Wirth, 2009) . Lucis Pro (Microtechnics, Granite Bay, CA, USA) and Stacker and Visualizer-Kai (System In Frontier, Tokyo, Japan) software packages were utilized for 3D reconstruction. SEM was performed at 0.5 kV. Images were obtained using an annular in-lens secondary electron detector and an annular in-lens energy-selected backscattered electron detector. The observed area was 10x10 µm with 10 nm/pixels each. Serial sectioning observations were carried out with a slice pitch of 10 nm for 990 sheets. The obtained images were rendered by removing resin and pseudo-colored according contrast.