Applications of a heliun ion microscopy (HIM) technology have been eagerly studied at more than 20 organizations in the world, and several unique applications are developed in here. LSI Cu / low-k interconnect structures have been imaged using generated secondary electrons. In comparison with electron, helium ion has larger cross section, and it realized HIM observation of samples with less current because of higher efficiency of secondary electron generation for imaging, which results in less power implant (less themal damage input) into the samples. At the same time helium ions penetrate deeper than electrons at optimal observation conditions. Utilizing these features, a low-k matetrial patten with less deformation (thermal damage) and a Cu metal line underneath a 130 nm dielectric are imaged. A few - 40 nm diameter tungusten pillars are deposited under the helium ion beam irradiation in W(CO)₆ gas atmosphere with high spacial resolution accuracy, which realized precicse electron tomography and re-construction. The helium ion irradiation onto a graphene film could introduce defect controrablly in the film which tune conduction properties of the graphene. Possibility of ion beam luminescence is shown.

Along with the development of nanostructured materials and devices, novel microscopic methods which can characterize the fine surface structures with a nanoscale resolution as close as the real ones are highly demanded. Scanning helium ion microscope (SHIM) is one of such microscopic instruments. SHIM has advantages such as the high spatial resolution, large depth-of-focus, and high material contrast by using a helium ion beam instead of the electron beam while its operability is similar to general scanning electron microscope (SEM). Equipped with electron gun to neutralize electric charging, SHIM enables us to observe insulators without the coating of conductive thin films. In NIMS we have developed a device for the in situ sample heating and have succeeded in the high resolution SHIM observation of various materials at the elevated temperatures. On the other hand, it is possible for the helium ion beam to process a nanoscale direct lithography. Moreover, by installing the devices which may introduce a wide range of gases, using the ion beam induced gas decomposition and deposition, SHIM can create nanoscale structures made of metallic and semiconductor materials. This equipment is one of the shared facilities of NIMS microstructural characterization platform and offers the technical support for domestic and international research.

TOF-SIMS (Time-of-Flight Secondary Ion Mass Spectrometry) is widely used as a highly sensitive surface analysis technique to obtain the information of elements and molecular species, and is the only technique to identify the molecular distribution at sub-micron scale. However, it was impossible to analyze the molecular distribution underneath the surface, because of the damage of the molecular structures during sputtering of the surface layers. Recently, C₆₀ and Ar-gas cluster ions have been proved to cause minimum damage to the molecular structure during sputtering, and their ion guns have been developed for commercial use. They provide us the three dimensional information of molecular species beneath the surface layer. In this article, two-dimensional (2D) molecular imaging using TOF-SIMS will be discussed, and then the applications of three-dimensional (3D) molecular imaging of polymer film will be presented.

The scanning ion conductance microscope (SICM), introduced by Hansma et al., in 1989, uses an electrolyte-filled micropipette electrode as a sensitive probe and detects the ion current between the probe and the bath electrode in an electrolyte solution. When the probing tip approaches the sample surface in the solution, the ion current decreases because the space through which ions can flow is decreased. By monitoring the change in ion current, SICM can obtain contact-free images of the sample topography. This technique is expected to be useful for studying the surface structure of soft biological samples under liquid conditions. In this paper, we explained the principle of the SICM and showed some results of the application of SICM to biosciences. We showed SICM images of collagen fibrils and cultivated cells in liquid. Sequential time-lapsed SICM images of live cells were also shown for investigating the movement of cellular processes on the time scale of minutes. We then applied SICM to the study on the surface topography of tissue blocks (e.g., trachea, etc.). The advantages of SICM imaging in biosciences were discussed by comparison with imaging by scanning electron microscopy.

Vortices in superconductors (quantized magnetic flux-line) play an important role not only in physical properties but also in practical applications of superconductivity. The small size and weak magnetic flux of vortices, however, have been obstacles to their observation for a long time. Lorentz microscopy with a field emission transmission electron microscope and with a magnetic field application system is indispensable in order to observe individual vortices, and to reveal their structure, configurations and behaviors. In this paper, the Lorentz microscopy and a “direction-free magnetic field application system” dedicatedly developed for a 1-MV field-emission electron microscope are described. This paper also introduces a few observation results in the high-T_c superconductors, Bi₂Sr₂CaCu₂O_8+δ and YBa₂Cu₃O_7-δ.

To understand mechanisms of neural networks, we explore structures of the networks by tracing morphologies of single neurons which are involved in the networks and further investigate correlation between the characteristics of the network structure and properties of neural activity. Here, I show three examples of experiments in our strategy. One: axon collaterals which had been derived from corticospinal neurons were visualized by GFP expression after infection with a recombinant Sindbis virus. We traced the labeled axons one by one and found that the striatum, zona incerta and pontine nuclei received the axon collaterals frequently. Two: dendrites of neurons in the neostriatum were visualized using the recombinant virus and were analyzed with respect to appositions of immunolabeled excitatory axon terminals under a confocal laser scanning microscope (CF-LSM) and scanning electron microscope equipped with focused ion beam (FIB-SEM). Synapses between thalamostriatal terminals and GFP-labeled dendrites were observed in the 3D ultrastructure data which was obtained under the FIB-SEM. Three: in the rat whisker system, correlations between distribution of thalamocortical axons and size of receptive fields were revealed by juxtacellular labeling method. I bereave that these bottom-up experiments contribute to building theoretical models which explain mechanisms of neural circuits.

We have been developing an electron diffractive imaging method by using “low-voltage” electron beam to analyze the atomic structures of light-element materials. Light elements, which are considered to be the key materials in the fields of sustainable energy and environmental engineering, are cumbersome materials for conventional electron microscopy because they are damaged easily by “high-voltage” electron beam irradiation. The electron diffractive imaging method, which reconstructs the structures of specimens from electron diffraction pattern via iterative phase retrieval, can obtain high-resolution images avoiding lens aberrations and also obtain phase images. By using a prototype microscope, characteristic features (outer and inner diameters, number of walls) of multiwall carbon nanotube were reconstructed from a diffraction pattern recorded at 20 kV. To solve difficulties that arose in the prototype, we developed an electron diffraction microscope based on a conventional scanning electron microscope. Using this microscope, atomic arrangement of a single wall carbon nanotube (SWCNT) was reconstructed from a 30-kV diffraction pattern. Before the iterative phase retrieval, image processing to reduce noises in the diffraction pattern was executed. In the reconstructed SWCNT image, intensity difference between single isolated atoms and two overlapped atoms could be divided.

Optical and electron microscopes are widely used for morphological observation of microbes and cultured cells. However, the small size of microbes (flagella and pili) and cells (filopodia) limits optical microscope observation. An electron microscope has a high resolution. However, lengthy pretreatments such as dehydration or metal evaporation are basically necessary. This may obscure delicate structures in SEM images. For the observation of natural structures in microbes and cells, we have developed an in-liquid gold labeling technique using an atmospheric SEM (ASEM), which observes a sample in liquid in open atmosphere. Sample pretreatment required only simple tasks including fixation, gold labeling, and medium exchange, taking less than one hour in total. ASEM imaging of the bacteria in aqueous solution revealed pili and delicate spiral flagella in natural form. The gold labeling also allowed filopodia as thin as 100 nm in diameter to be clearly visualized.

The detailed atomic and electronic structures of the interfaces between ZnO precipitate and palladium were examined employing high-resolution transmission electron microscopy (HRTEM), electron energy-loss spectroscopy (EELS), and ab-initio calculation. It was indicated that (1–11)_Pd//{0002}_ZnO polar planes tend to be parallel to the interface. A ZnO precipitate was terminated by oxygen on (000-2) surface at one end of the precipitate and by zinc on (0002) surface at the other end of the precipitate. The presence of both zinc- and oxygen-terminated interfaces was also indicated by the EEL spectra acquired from both the interfaces. At the oxygen-terminated interface, a shoulder was detected in O-K energy-loss near-edge structure (ELNES) below the first peak in the O-K ELNES. From the ab-initio calculation, it was suggested that the origin of the shoulder could be attributed to the strong chemical bonding and hybridization of Pd-d and O-p orbital at the oxygen-terminated polar interface.

PEEM, which gives the information about the spatiotemporal distribution under the reaction conditions, has been improved to possess chemical sensitivities by analyzing emitted photoelectron kinetic energy. This new type of PEEM is called as EXPEEM(Energy filtered PEEM). We adopted a Wien filter as a linear energy analyzer of photoelectron. We achieved high senitivity EXPEEM system by adopting the mulitpole Wien filter. We have developed a user friendly EXPEEM using the local network in Hokkaido.

A dual-axis 360° rotation holder for a pillar-shaped specimen was developed in order to improve the three-dimensional reconstruction of a magnetic field.  Two magnetic field components are reconstructed by using the dual rotation series, and the other component is calculated from the two reconstructed components using the Maxwell's equation, but the reconstructed components were disturbed by the projection angle limitation incurred when using a conventional holder for a thin specimen.  However, reconstructed components without the projection angle limitation effect were obtained when using our new holder and the calculated component was improved.  We show this improvement by using a magnetic field distribution known specimen.

The budding yeast Saccharomyces cerevisiae is an excellent model organism with which to study intracellular trafficking pathways, such as the secretory and endocytic pathways. While “forward genetics”, which involves the identification of a gene responsible for a specific phenotype, is still useful in studies of yeast, recent advances in fluorescence microscopy have facilitated a “reverse genetics” approach for revealing the functions of genes by analyzing the phenotypes resulting from mutation. Here we introduce a technique for live-cell imaging of yeast endocytosis and analysis of the molecular mechanisms involved.

Fluorescent protein revolutionized our understanding of biological processes. However the requirement for external illumination definitely precludes its universal application to certain biological processes. On the other hand, bioluminescent proteins such as luciferase don’t require the external illumination, thereby bioluminescence can be imaged without phototoxicity and auto-fluorescence from the specimen, allowing us to detect signals with high signal-to-noise ratio. These properties make bioluminescent proteins potentially superior to fluorescent proteins as a bioimaging tool. However, existing bioluminescent proteins are too dim to be measured in real time, requiring longer exposure than fluorescence imaging that takes less than 1 second. To overcome this drawback, we conducted random mutagenesis on Renilla reniformis luciferase (Rluc) to improve the intensity. Then, the luminescence intensity was further increased by fusion of the improved Rluc to a yellow fluorescent protein with high FRET efficiency. The chimeric protein named ‘Nano-lantern’ showed much brighter luminescence than the commercially available Rluc, enabling not only real-time imaging of intracellular structures in living cells with spatial resolution equivalent to fluorescence but also sensitive tumor detection in freely moving unshaved mouse which has never been possible before. This super-duper luminescent protein will revolutionize conventional bioimaging by allowing visualization of biological phenomena not seen before.

Single and clustering dislocations introduced into the deformed steel have been investigated using the back scattering electron signals in a scanning electron microscope. The development of the electron channeling contrast imaging technique has been simply reviewed, in which the inelastic scattering contrast has been discussed to understand the dislocation contrast observed by SEM, which is treated by a similar manner to that observed by TEM.  With improvement of the control of incident electron beam direction, the ECCI technique will be widely applied to the field of study in materials.