Sm 090101 cross section polisher

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.

Similar to the FIB-SEM specimen-preparation procedure, eight embedded specimens were mechanically polished using diamond lapping films (3M) and argon-ion polished (SM-090101 Cross-Section Polisher, JEOL). The SPM system employed was a Bruker MultiMode 8 SPM equipped with a Nanoscope V controller and Nanoscope analysis software (Bruker Nano Surface Business, Santa Barbara, CA, USA). Cross-sectioned surfaces were quantitatively characterized using the PeakForce QNM (Quantitative NanoMechanics) SPM module under ambient conditions. Elastic modulus (EM) was calculated by Nanoscope analysis software using the Derjaguin-Muller-Toropov (DMT) model (Derjaguin et al., 1975; Wang et al., 2013) . Samples were scanned using a probe (RTESPA-525, Bruker) with a nominal radius of 8 nm and a nominal spring constant of 200 Nm -1 with 0.501 Hz scan rates, a 250-mV amplitude set point and a 150-nm peak force amplitude. After SPM, the samples were observed by SEM (JSM-6701F, JEOL).