Yuji Konyuba

The Atmospheric Scanning Electron Microscope with open sample space observes dynamic phenomena in liquid or gas.

Although conventional electron microscopy (EM) requires samples to be in vacuum, most chemical and physical reactions occur in liquid or gas. The Atmospheric Scanning Electron Microscope (ASEM) can observe dynamic phenomena in liquid or gas under atmospheric pressure in real time. An electron-permeable window made of pressure-resistant 100 nm-thick silicon nitride (SiN) film, set into the bottom of the open ASEM sample dish, allows an electron beam to be projected from underneath the sample. A detector positioned below captures backscattered electrons. Using the ASEM, we observed the radiation-induced self-organization process of particles, as well as phenomena accompanying volume change, including evaporation-induced crystallization. Using the electrochemical ASEM dish, we observed tree-like electrochemical depositions on the cathode. In silver nitrate solution, we observed silver depositions near the cathode forming incidental internal voids. The heated ASEM dish allowed observation of patterns of contrast in melting and solidifying solder. Finally, to demonstrate its applicability for monitoring and control of industrial processes, silver paste and solder paste were examined at high throughput. High resolution, imaging speed, flexibility, adaptability, and ease of use facilitate the observation of previously difficult-to-image phenomena, and make the ASEM applicable to various fields.

Atmospheric scanning electron microscope system with an open sample chamber: configuration and applications.

An atmospheric scanning electron microscope (ASEM) with an open sample chamber and optical microscope (OM) is described and recent developments are reported. In this ClairScope system, the base of the open sample dish is sealed to the top of the inverted SEM column, allowing the liquid-immersed sample to be observed by OM from above and by SEM from below. The optical axes of the two microscopes are aligned, ensuring that the same sample areas are imaged to realize quasi-simultaneous correlative microscopy in solution. For example, the cathodoluminescence of ZnO particles was directly demonstrated. The improved system has (i) a fully motorized sample stage, (ii) a column protection system in the case of accidental window breakage, and (iii) an OM/SEM operation system controlled by a graphical user interface. The open sample chamber allows the external administration of reagents during sample observation. We monitored the influence of added NaCl on the random motion of silica particles in liquid. Further, using fluorescence as a transfection marker, the effect of small interfering RNA-mediated knockdown of endogenous Varp on Tyrp1 trafficking in melanocytes was examined. A temperature-regulated titanium ASEM dish allowed the dynamic observation of colloidal silver nanoparticles as they were heated to 240°C and sintered.

Direct observation of protein microcrystals in crystallization buffer by atmospheric scanning electron microscopy.

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called “crystallization bottleneck”. Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few μm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.

Computer simulations analysis for determining the polarity of charge generated by high energy electron irradiation of a thin film

Detailed simulations are necessary to correctly interpret the charge polarity of electron beam irradiated thin film patch. Relying on systematic simulations we provide guidelines and movies to interpret experimentally the polarity of the charged area, to be understood as the sign of the electrostatic potential developed under the beam with reference to a ground electrode. We discuss the two methods most frequently used to assess charge polarity: Fresnel imaging of the irradiated area and Thon rings analysis. We also briefly discuss parameter optimization for hole free phase plate (HFPP) imaging. Our results are particularly relevant to understanding contrast of hole-free phase plate imaging and Berriman effect.

Development of Amorphous Carbon Thin Film Phase Plate

However, thin film Zernike phase plate had some problems, those are, their reliability, lifetime (due to charging and aging) and cost (due to craft production including hole forming by a focused ion beam). To solve these problems, we have been challenging to fabricate several kinds of the thin film phase plates with various materials and structures by a high throughput fabrication method utilizing a micro electro mechanical systems (MEMS) technology. As a first trial, we have fabricated titanium (Ti) / silicon nitride (SiN) / Ti sandwich type thin film Zernike phase plates [3] and we improved the manufacturing yield. However, we could not achieve sufficient stability, due to charging of the Ti/SiN/Ti thin film Zernike phase plate.

Contrast Enhancement of Long-Range Periodic Structures using Hole-Free Phase Plate

Phase contrast transmission electron microscopy is a powerful tool to enhance the image contrast of transparent materials such as ice-embedded biological specimens and polymer materials. In this method, a phase plate, which is placed at the back-focal plane of the objective lens, gives a phase shift for scattered electron waves, resulting in a significant enhancement contrast of specimens in images. Zernike phase plate (ZPP), consisting of a thin carbon film with a small central hole, is first tested practical phase plate [1]. However, ZPP has disadvantages for image quality. Phase contrast of specimens in low spatial frequency does not improve, since the scattered electron passing through a central hole of ZPP does not change their phases. The threshold frequency at the center hole edge is called cut-on frequency. The additional disadvantageous effect of the abrupt cut-on frequency reveals that strong fringes appear.

Hole Free Phase Plate Tomography for Materials Sciences Samples

We report, for the first time, the three dimensional reconstruction (3D) of a transistor from a microprocessor chip and roughness of molecular electronic junction obtained by electron tomography with Hole Free Phase Plate (HFPP) imaging. The HFPP appears to enhance contrast between inorganic materials and also increase the visibility of interfaces between different materials. We demonstrate that the degree of enhancement varies depending on material and thickness of the samples using experimental and simulation data.

Fabrication and characterization of sample-supporting film made of silicon nitride for large-area observation in transmission electron microscopy

In transmission electron microscopy (TEM), silicon nitride (SiN) films are widely used as sample-supporting films owing to their robustness. We fabricated large-scale SiN films deposited by low-pressure chemical vapor deposition (LPCVD). This preparation method is advantageous for large window areas, since it yields films with control over properties such as tension and thickness. We fabricated large SiN windows for mounting large ultrathin sections and for acquiring large-area TEM images. Thus, sample sections sliced by conventional sample preparation techniques were successfully mounted on these sample-supporting films. We successfully obtained a 680 × 250 μm2 TEM montage image of a whole Drosophila embryo.

High Throughput Fabrication Process of a Zernike Phase Plate

In transmission electron microscopy (TEM) for biological and polymer samples, it is difficult to image with high contrast, since they are mostly composed of light elements and have similar density. One solution to enhance the contrast is utilization of phase contrast microscopy, which is realized in optical microscopy. Accordingly, many types of phase plates for electron microscopy have been proposed. Zernike phase contrast TEM (ZPC-TEM) with a Zernike phase plate (ZPP) provides higher contrast at Af (defocus) = 0 with respect to one in conventional TEM [1]. ZPC-TEM attracts much attention in Cryo-TEM applications such as cryo-electron tomography and single-particle analysis [2], because their specimens are easy to be damaged with electrons and they need high contrast with minimum dose on the specimen.

The New Atmospheric Scanning Electron Microscope allows in situ observation of dynamic phenomena under atmospheric pressure

Most chemical and physical reactions occur in liquid or gas. The new Atmospheric Scanning Electron Microscope (ASEM) is able to observe samples directly in liquid or gas under atmospheric pressure [1, 2]. In this system, an electron-transparent window made of pressure-resistant 100nm-thick silicon nitride (SiN) film, set into the bottom of the ASEM dish, allows an electron beam to be projected from underneath the sample (Fig. 1 a). Electrons backscattered from the sample are captured by a detector positioned below. Above the dish, an optical microscope (OM) realizes quasi-simultaneous observation. This combined system is called ClairScope (JASM-6200). We have also developed ASEM dishes with different functionalities to observe various phenomena in controlled environments.