We develop a compact scanning probe microscope (SPM), named a pencil-type SPM, which can be installed into a commercial-available superhigh-resolution in-lens cold-cathode field-emission scanning electron microscope (SEM). Here we demonstrate their combined performance utilizing their outstanding features. While observing SEM image, we prepare a Ge-deposited Pt-It tip, and bring it in touch with a W filament heated at about 1400°C, resulting in a bridge of melting Ge between the tip and the W filament; the Ge bridge gets wet well with the filament. We draw them apart, and observe the solidification and crystallization of the Ge on the tip side at lower temperature. Further separation of them breaks the Ge bridge, leading to formation of a protrusion with a radius of about 50 nm at the end of the tip. Energy dispersive X-ray spectroscopy (EDX) equipped with the SEM reveals the precipitation of Pt in the inside of protrusion. This protrusion is mostly covered with Ge, ex-situ revealed by scanning Auger electron spectroscopy/microscopy (SAM). This indicates that the combined microscope has potential to analyze the alloying, precipitation and formation of a contact on a micro- to nano-scale, and to fabricate nanostructures.
With the size reduction in structures, the difference in the electronic properties, for example, caused by the structural nonuniformity in each element, has an ever more crucial influence on macroscopic functions of materials and current devices. Thus, for further advances of nanoscale science and technology, the direct observation of the local quantum properties, which provides us with the basis for the characteriscics of macroscopic functions, is of great importance. We have developed a new microscopy technique that enables the visualization of femtosecond carrier dynamics in nanometer-scale structures. The ultimate temporal and spatial resolutions were simultaneously realized by combining an advanced quantum optical technology with scanning tunneling microscopy. The optical pulses used for pump-probe method are selectively transmitted using a pulse picker, and the delay time is controlled digitally, which, for the first time, has allowed the probing of carrier dynamics in nanometer-scale organized structures over a wide range of time scales. Furthermore, this method reduces the measurement time and hence enables the spatial mapping of time-resolved signals, which has been desired for a long time.
Microscope having atomic resolution with chemical sensitivity is one of the ultimate microscopes for the material science. Scanning tunneling microscope (STM) assisted by the core-level excitation using synchrotron radiation (SR) may be a possible candidate of such an ultimate microscope. We have demonstrated that we can observe element specific images of surfaces in the spatial resolution of several tens of nanometer by detecting the secondary electrons produced by the electron-hole recombination after the core-level excitation with an STM tip and taking the photon-induced current images. Recently, we have modified the system in order not only to improve its performance but also to accommodate a focused beam of a newly renovated beamline. Here, we briefly report on the modification and future prospects of this method.
The surface potential distribution and the surface charge distribution are very important properties of materials, which influence various physical and chemical processes. In this paper, we describe the measurement principle of Kelvin Probe Force Microscopy (KPFM), which detects the electrostatic force between a tip and a sample surface and can measure the contact potential difference (CPD) between the work function of a tip and that of a sample surface quantitatively. The operation of KPFM is based on the dynamic mode atomic force microscopy (AFM), which uses the enhancement of the force sensitivity by oscillating the cantilever at resonance. We also describe the frequency-modulation (FM) method to measure the very weak tip-sample interaction. Finally, as a practical application of AFM/KPFM, we introduce the experimental result of the discrimination between the CaF1 interface layer and the CaF2 bulk layer on thin film epitaxiallly grown on Si(111) surface with high spatial resolution.
Frequency modulation atomic force microscopy (FM-AFM) is a powerful method not only for imaging surfaces at the atomic scale but also for investigating surface properties. However, the high-resolution FM-AFM observations have been limited in vacuum environments where the Q-factor of the cantilever resonance usually exceeds 10,000. It is heavily reduced in air or liquids and hence the effective force sensitivity is decreased. We have developed a low noise cantilever deflection sensor by optimizing the optical design of the sensor and by modulating the laser power with a high frequency signal. Using this sensor, we have developed a high-resolution FM-AFM working in both air and liquids.
Secondary-electron (SE) image information in a scanning ion microscope (SIM) using gallium (Ga) and helium (He) ion species has been described comparing with a scanning electron microscope (SEM). Using the Monte Carlo (MC) simulation, the trajectories of all the collision partners (i.e., primary ions, recoiled target atoms, and excited electrons (electron cascade) have been simulated to excite SEs under the ion impacts. For Ga ion impact, the SE yields show a gradual decrease with increasing Z2, being superimposed with a periodic change. This general Z2-dependency is opposite to that for electron impact. The Z2-dependency for He ion impact is characterized to the middle position of Ga ion and electron impacts and is weak. The characteristics of SIM images have been compared with SEM images in material contrast, information depth, incident-angle dependency of SE yields dominating topography contrast, and energy distribution of SEs influencing voltage contrast.
In vivo, the integration of the complex cellular interactions takes place most efficiently within the organized architecture of secondary lymphoid organs. This review outlines both structures and molecules of rat secondary lymphoid organs that support trafficking of immune cells by a multicolor immunoenzyme staining technique using a panel of antibodies to rat cell and tissue markers. Cellular interactions during immune responses including the cluster formation, proliferative response are visualized in situ on tissue sections. This approach has a potential to discover truth in disease processes and host responses because it can directly observe the immune response in situ.
Osteoclasts are specialized in cells that play a central role in bone remodeling. Polarized osteoclasts are equipped with specialized structures called podosomes, dynamic actin rich adhesions, which distribute over the entire ventral membrane. To explore their cytoplasmic and membrane cytoskeletal organizations, we used a three-dimensional (3D) ultrastructural approach to visualize employed a method of “unroofing”. Podosomal cytoskeletons are incorporated into a dense network of actin cytoskeletal organization in the lamellipodium. On the ventral adherent membranes, an extensive flat clathrin lattice has been shown. Although clathrin-dependent endocytosis is the major pathway for the uptake of nutrients in eukaryotic cells, recent evidence shows that some flat clathrin plaques are apparently restricted to the adherent membrane which may reflect cell adhesion to the substrate. We will review the potential role of podosomes as an adhesion structure, summarize current understanding about functional roles of podosomes and discuss further research on these unique structures.
Cryo-electron microscopy of vitreous sections (CEMOVIS) enables us to observe hydrated biological materials that are cut into thin sections following physical fixation by high-pressure freezing. Thin sectioning conventionally embedded specimens causes certain artifacts, including a lack of molecules that are unfixed and deformation of molecules through chemical fixation, dehydration, and heavy metal staining. On the other hand, by avoiding the abovementioned processes, vitrification yields specimens that are closer to the native state. However, some artifacts caused by direct sectioning of vitrified ice have been verified since this technique first appeared. Here, we introduce the pros and cons of CEMOVIS, the methods for utilization of CEMOVIS, and the protocol and materials necessary to implement it.
Transition metal oxides in strongly correlated electron systems exhibit a variety of physical properties sensitively related to their crystal structures and constituent elements. Since these structurally complex crystals often have some nonequivalent atomic sites in a unit cell, the local electronic structure is different even for the same element. To further understand such materials it is important to examine the local electronic structure at high spatial resolution. Here, we show the results of the local electronic structure analysis for transition metal oxides with layered structure by using site-resolved EELS measured by the STEM equipped with a spherical aberration corrector for the illumination lens system.
Epigenetic modification such as DNA methylation is implicated in the induction of cell differentiation and its maintenace in eukaryotes. In spite of potential importance of the analysis of methylation states of sequence specific DNA in indivisual cells, it was impossible. Here we presented a new histochemical method, HELMET, which permits us to analyze methylated levels of specific sites of DNA using an isoshizomeric set of restriction enzymes. When the method was applied to examine the methylation states of CCGG sequences in mouse testis, we effectively found differetiation stage-dependent changes in the ratio of the number of nonmethylated CCGG sites to that of methylated ones during spermatogenesis.
We have developed an “adaptive SEM” technology; a newly developed nano-particle probe set for simultaneous labeling of different target molecules was combined with backscattered electron (BE) observation mode of field emission scanning electron microscopy (FE-SEM) to carry out single-molecular expression analysis. Polystyrene spheres prepared with precise size control were used as a template for production of nano-particle probe sets; various elements were thermally deposited on these template spheres. After removal of the template, cup-shaped metal nano-particle probes were obtained. By varying the size and element, more than 500 kinds of probes with different characteristics were obtained. Identification of nano-particle probes was accomplished by comparing BE intensities reflecting differences in the atomic number in FE-SEM with a pre-established BE image library of metal nano-particles. By using the “adaptive” SEM technology (i.e., nano-particle identification is “adaptive” under various sample conditions), we could simultaneously identify six different elements with hardly any increase in measurement time and spatial resolution in comparison to secondary electron observation. This wide-reaching technology based on nano-particle labeling and BE analysis could allow simultaneous detection of a large number of target biomolecules at a single-cell level with a nanometer spatial resolution, holding great promise for the post-genome/proteome era.