STEM tomography offers several advantages even for biological specimens. The most important advantage is the ability to generate clear images for thick specimens. STEM is free from chromatic aberration, which is main obstructer to generate clear images for TEM. The second advantage is ‘dynamic focus’. Since TEM imaging uses a fixed focus, only a very narrow area can be optimally focused on for obtaining images of highly tilted specimens. Therefore, the introduction of a sufficiently large defocus is required in TEM tomography. However, the scanning beam can be focused flexibly in STEM. Therefore, the entire image area can be optimally focused on even in highly tilted specimens, and defocusing is not necessary. The third advantage is the ability to change the imaging mode. In STEM tomography, it is easy to switch between the bright field, dark field and HAADF modes; in contrast, it is very difficult to obtain stable dark-field images during data collection in TEM tomography. The fourth advantage is the linear contrasts of the images. It is not necessary to consider the contrast transfer function (CTF) of electron lenses in STEM imaging.
Until now, Scanning transmission electron microscopy (STEM) tomography has not yet become popular in the biological research field. It is widely considered that the beam damage of STEM would be very severe, since a convergent electron beam is required for the imaging. On the other hand, STEM allows us taking tilt series of images from thick biological specimens (up to 1μm) without using ultra-high voltage electron microscopy for finding out 3-D reconstruction of the specimens. To demonstrate a benefit of STEM tomography for the analysis of biological specimens, the shrinkage of the specimens was measured for the quantification of the beam irradiation-induced damage. The difference of the size of intracellular organelles, such as mitochondria was determined, before and after taking the tilt series of images for tomography. In the result, it was appeared that the beam irradiation-induce damage of STEM tomography was considerably less than that of TEM tomography.
Dark field imaging of scanning transmission electron microscopy (STEM) was applied to electron tomography (ET) method of biological specimens. Theoretical resolution of ET is reduced by chromatic aberration and unfocused information from the thick section. STEM imaging has effective advantages for these problems. We observed the thick biological specimens such as rat absorptive enterocyte using STEM. The reconstruction data from tilted image series (±70º) of the 250nm and 400nm thick plastic embedded specimens acquired by using 300kV STEM showed that the resolution reached to about 10nm in Z-axis. The slices from the reconstructions showed a clathrin hexagonal lattice structure on the coated pit or hexagonal array of actin filaments within microvilli parallel actin bundle even in the ZX plane. These values are close to the theoretical Z-axis resolutions that are estimated to 10nm or 15nm. It should be noted that these resolutions were obtained even in the peripheral region of the reconstructions. Therefore, we concluded that dark field STEM imaging improves the electron tomography resolution throughout the whole reconstructions not just the center of tomogram especially in thick sections.
The rapidly-frozen and deeply-etched EM image allows us to find that the cytoplasmic surface of the plasma membrane has a large undulation covered with the networks of caveolae, clathrin-coated pits, and actin-based membrane skeletons. In this paper, we compared the three-dimensional reconstruction of the cellular freeze-replica containing of various focal point distances, between the conventional TEM tomography and the variable convergence-angle STEM tomography, by using of the electron microscope equipped with the three condenser-lens system containing Cs correctors. It was clearly found that the small convergence-angular (0.5mrad) HAADF-STEM tomography could make the best 3D reconstruction with little computational artifacts through the missing wedge during EM observation. This proposed freeze-etch tomography could be expected as a novel tool for the plasma membrane research.
We have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area. In this system, cells or organelles are observed by the fluorescence microscopy from the top, and by the high resolution scanning microscopy from the bottom. Cells cultured on the open dish can be externally manipulated under an optical microscopy, fixed, and observed using scanning electron microscopy.
Surface plasmon (SP) is converted to photon when propagating on surface nano-structures such as steps and periodic structures with sub-wavelength intervals. We newly developed an angle resolved light detection system for TEM-CL, and measured surface plasmon induced light emission. Dispersion curve of SPP on an Ag surface was derived from the angle resolved spectrum images. Standing wave patterns of SPP on plasmonic crystals were visualized in the spectrum image taken at a proper emission angle. This technique can be applied to light emission phenomena induced by SP, and provides a powerful tool to characterize SP on nano-plasmonic structures in high spatial resolution.
Although so-called ‘fusion’ morphologically divides into two different types, ‘ostensible fusion’ and ‘actual fusion’, very little is known about the heterogeneity of fusion in maxillo-facial morphogenesis. Moreover, the controversy concerning the mechanism of epithelial disappearance continues even to the fusion of palatal shelves, which has been most often researched as a model of ‘actual fusion’.In this paper, I explained the morphological difference between ‘ostensible fusion’ and ‘actual fusion’ in so-called ‘fusion’. In addition, I showed a case of epithelial disappearance by means of cell migration, epithelial –mesenchymal transformation, and apoptosis, in a single palatal shelf culture method of fetal mice. Such cell differentiation suggests how epithelial cells disappear during ‘actual fusion’.
Convergent-beam electron diffraction (CBED) patterns are obtained by converging a conical electron beam on a nanometer-size specimen area. The CBED method enables us to determine specimen thicknesses, crystal symmetries, lattice parameters and strains, lattice defects and crystal structural parameters. Basics of the CBED method and tips for CBED experiments are described. Some applications of CBED are briefly outlined.
A novel fixative solution which I recently developed enabled to prepare paraffin-embedded lens specimens without artefacts. The growth region and tissue-type stem cells in the lens epithelium were located near the equatorial region of the lens. Under culture conditions, every lens epithelial cells (LEC) from any different areas of the lens exhibited prompt initiation of cell growth.
A high energy-resolution energy dispersive spectrometer (EDS) utilizing a transition-edge-sensor (TES) microcalorimeter is developed for a transmission electron microscope (TEM). As the first step, we have attached a single-pixel TES system cooled by the cryogen-free cooling system to the TEM and obtained an energy resolution of 7.6eV at silicon Kα line without degrading the spatial resolution of the TEM.