Scanning confocal electron microscopy (SCEM) is a new imaging technique that is capable of depth sectioning with nanometer-scale depth resolution. However, the depth resolution in the optical axis direction (Z) is worse than might be expected on the basis of the vertical electron probe size calculated with the existence of spherical aberration. To investigate the origin of the degradation, the effects of electron energy loss and chromatic aberration on the depth resolution of annular dark-field SCEM were studied through both experiments and computational simulations. The simulation results obtained by taking these two factors into consideration coincided well with those obtained by experiments, which proved that electron energy loss and chromatic aberration cause blurs at the overfocus sides of the Z-direction intensity profiles rather than degrade the depth resolution much. In addition, a deconvolution method using a simulated point spread function, which combined two Gaussian functions, was adopted to process the XZ-slice images obtained both from experiments and simulations. As a result, the blurs induced by energy loss and chromatic aberration were successfully removed, and there was also about 30% improvement in the depth resolution in deconvoluting the experimental XZ-slice image.

The mineralized structure of aligned collagen fibrils in a tilapia fish scale was investigated using transmission electron microscopy (TEM) techniques after a thin sample was prepared using aqueous techniques. Electron diffraction and electron energy loss spectroscopy data indicated that a mineralized internal layer consisting of aligned collagen fibrils contains hydroxyapatite crystals. Bright-field imaging, dark-field imaging, and energy-filtered TEM showed that the hydroxyapatite was mainly distributed in the hole zones of the aligned collagen fibrils structure, while needle-like materials composed of calcium compounds including hydroxyapatite existed in the mineralized internal layer. Dark-field imaging and three-dimensional observation using electron tomography revealed that hydroxyapatite and needle-like materials were mainly found in the matrix between the collagen fibrils. It was observed that hydroxyapatite and needle-like materials were preferentially distributed on the surface of the hole zones in the aligned collagen fibrils structure and in the matrix between the collagen fibrils in the mineralized internal layer of the scale.

We evaluated the depth resolution of annular dark-field (ADF) scanning confocal electron microscopy (SCEM) with a stage-scanning system by observation of nanoparticles. ADF-SCEM is a three-dimensional (3D) imaging technique that we recently proposed. An ADF-SCEM instrument involves a pinhole aperture before a detector for rejecting electrons from the out-of-focal plane in a specimen and an annular aperture under the specimen for collecting only scattered electrons. The stage-scanning system enables us to directly obtain optical slice images perpendicular and parallel to an optical axis at a desired position. In particular, the parallel slices visualize the elongation of nanoparticles along the optical axis, which depends on the depth resolution. ADF-SCEM effectively reduced the elongation length of the nanoparticles sufficiently to demonstrate depth sectioning, in comparison with scanning transmission electron microscopy and bright-field SCEM. The experimentally obtained length was nearly equal to the theoretically estimated one from the probe size considering the experimental conditions. Furthermore, we applied this ADF-SCEM technique to analysis of the 3D position of catalytic nanoparticles on carbon nanostructures.