In electron tomography the ‘missing wedge’ problem due to limited specimen tilt angles is crucial. In addition, reduction of the number of projection images is highly desired. In this article, a novel reconstruction method using an image gray-level quantization unit (QU) is described, which was devised to resolve the issues. A digital image consists of QUs which are stacked in each pixel for making a brightness. Therefore, it is thinkable that a tomography image is reconstructed by arranging discrete QUs in three-dimensionally, where the 3 axes are two for the image plane and one for the gray-level. Here, the total number of QUs can be approximately determined from the projection theory. Then, a solution which minimize an error calculated from a set of given projection data gives a unique one. As a result of computer model-based simulations and an experiment with a complex nano-particle, successful reconstruction images unaffected by missing wedge problem were obtained. Furthermore, even though a relatively small number of projection images were given to the method, almost the same images output.
Electron tomography is now widely applied to 3-dimensional (3D) structure characterization of various matters in such a high spatial resolution as in nanometer scale. According to significant improvement in the image resolution due to aberration-corrected electron microscopy, several advanced algorisms for reconstruction have been recently proposed to achieve the corresponding spatial resolution in 3D. The present brief review is focused on recent progress in 3D structure characterization of metallic nanoparticles by electron tomography. The first topics demonstrates the advantages of DART algorism established by Batenburg et al., to overcome the serious missing wedge problem involved in electron tomography as well as to reduce the total dose of electron illumination for acquisition of projection images. The second one treats a trial to reveal the 3D atom configuration in a polycrystalline binary alloy particle by reconstruction with EST algorism, which was proposed by Miao et al., from a tilt series of atomic resolution HAADF-STEM images.
We developed a novel straining holder for soft materials that is capable of tilt specimens to a high-angle for electron tomography under stretching. A sample cartridge is mounted on the tensile holder, on which a variety of soft material samples such as microtomed thin sections of bulk specimens and solvent-cast thin films. Fine, stable control of the deformation process with nanoscale magnification was achieved. The holder allows large tensile deformation and a high tilt angle (up to 75°) during in situ observations. With the large tensile deformation, the strain on the specimen can be as large as 50. The dynamic mechanical deformation and fracture processes of soft materials in micro- and meso-scopic scales will be observed with by combining the holder with a transmission electron microscope. We used this technique to study the deformation process in a silica nanoparticle-filled isoprene rubber.
The cryo-electron microscopy is one of the most powerful techniques to elucidate biological molecular structure under nearly physiological conditions. Furthermore, we can also visualise in situ molecular behaviours in cells and tissues at nano-meter resolutions by electron tomography. We here clarified the architecture of filopodia, which is an essential device for cell motility and sensing. We observed the hexagonal actin bundles and their sets, where the actin numbers in the bundles are consistent with those estimated by mechanical balance. The cross-linked structures between actin filaments were averaged and compared with the atomic model resolved by X-ray crystallography; we showed a binding manner of fascin to actin filaments. Besides, we browsed in reconstructed filopodia and so found cooperative bindings of fascin and short actins in the periphery of the actin bundles. Thus we proposed a novel filopodia formation mechanism. We challenged the elucidation of the elongation mechanism by observing the tips of filopodia. Hereafter, cryo-electron tomography will useful for materials in water as well as biological samples.
There are various structures such as micro-sized bone cell and nano-sized collagen fibrils in the bone tissue. The bone tissue is maintained and bone metabolism is regulated by interaction of these structures. Since various diseases are caused without maintenance of the bone tissue and regulation of the bone metabolism properly, it is very important to analyze the microstructures inside of the bone tissue three-dimensionally. We are able to study biological significance of these microstructures more deeply by three-dimensional morphometry. In this paper, we describe three-dimensional analysis of the osteocyte network by the confocal laser microscopy, and the collagen fibrils of the bone tissue by the orthogonally arranged FIB-SEM, which is used our laboratory.
In scanning transmission electron microscopy (STEM), the formula for optimum beam half angle and attainable resolution of STEM proposed by Crewe and Salzman in 1982 are well known. This optimum beam angle is derived from a condition where the statistical mean information content of an optical image is maximum. This paper describes processes that derives statistical mean information content of optical image from information theory of Shannon and that derives the optimum beam half angle of optical system suffering from spherical aberration. A calculation method for information passing capacity and spatial resolution of an optical system for SEM and/or STEM with consideration for spherical aberration, chromatic aberration, and brightness of electron source is also explained.
Limit of detection (LOD) for As dopant in Si wafer was explored using a transmission electrom microscope (TEM) equipped with two large-sized silicon drift detectors. The LODs of As were estimated to be 30 ppm (1.5 x 1018 atoms/cc) with As(Kα) line and 70 ppm(3.5 x 1018 atoms/cc) with As(Lα) line based on the results of secondary ion mass spectroscopy. These concentrations come into a range of practical dopant concentration in a semiconductor device. In case where there are two lines such as K and L of As in an X-ray spectrum, LOD is lower for a line with lower background than a line with higher background. To avoid increasing background of spectra due to contamination during acquisition of X-ray, it is effective to apply plasma or ion cleaner to specimen before installing into the TEM. We measured LODs at 200 and 100 kV to be 30 ppm and 20 ppm respectively. These values tell that LOD is improved at lower accelerating voltage as well as longer acquisition time, larger beam current, since the X-ray generation probability is higher at lower acceleration voltage, though the background is higher for a line at lower accelerating voltage.
Atomic force microscopy (AFM) allows us to visualize atomic-scale surface structures at solid-liquid interfaces. However, the speed of the atomic-scale imaging by conventional AFM is limited to ~1 min/frame and hence imaging of dynamic events on the timescale of seconds has remained challenging. Recently, we have improved the imaging speed of AFM to ~1 s/frame without deteriorating the atomic-scale spatial resolution. In this article, I would like to introduce fundamental techniques that enabled this technical innovation and its application example.