Sukehiro Ito

A novel approach to scanning electron microscopy at ambient atmospheric pressure

Scanning electron microscopy (SEM) for observing samples at ambient atmospheric pressure is introduced in this study. An additional specimen chamber with a small window is inserted in the main specimen chamber, and the window is separated with a thin membrane or diaphragm allowing electron beam propagation. Close proximity of the sample to the membrane enables the detection of back-scattered electrons sufficient for imaging. In addition to the empirical imaging data, a probability analysis of the un-scattered fraction of the incident electron beam further supports the feasibility of atmospheric SEM imaging over a controlled membrane-sample distance.

A novel transmission electron imaging technique for observation of biological samples on a plate

We introduce a novel transmission electron imaging method to clarify the internal structure of single whole cells by scanning electron microscopy (SEM). In this method, whole cells are cultivated on a transparent flat plate in advance, which is made of a scintillator that emits photons by irradiating an electron beam. The detector developed here can obtain both the secondary electron (SE) surface images and transmission electron (TE) images. Observations of whole mount cells by this technique clearly show that the cellular internal structure can be observed as transmission images which are produced by photons emitted from the scintillator plate by electron beam irradiation.

A novel approach for scanning electron microscopic observation in atmospheric pressure

Atmospheric scanning electron microscopy (ASEM) for observing samples at ambient atmospheric pressure is introduced in this study. An additional specimen chamber with a thin membrane allowing electron beam propagation is inserted in the main specimen chamber. Close proximity of the sample to the membrane enables the detection of backscattered electrons (BSEs) sufficient for imaging. A probability analysis of the un-scattered fraction of the incident electron beam and the beam profile further supports the feasibility of atmospheric SEM imaging over a controlled membrane-sample distance. An image enhancement method based on the analysis is introduced for the ASEM.

Electron tomography of whole cultured cells using novel transmission electron imaging technique

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research.

Reduction of Electron Scattering Image Blur for Atmospheric Scanning Electron Microscopy

Recently, methods for observing samples under atmospheric pressure in a scanning electron microscope (SEM) have been reported by some investigators. We proposed a novel atmospheric SEM (ASEM) technique for observing samples which are present in ambient air conditions but are separated from the membrane [1]. In our system, the environment around the sample can be kept in ambient air conditions (Fig. 1(a)). While wet materials is clearly observed without direct sample membrane contact at an optimized distance, typical atmospheric SEM image taken in atmosphere is more blurred compared to conventional SEM image taken in vacuum condition. The reason why ASEM images looks like “blurred” is because electron beam is scattered by electron scattering region shown in Fig. 1(b). In order to reduce the electron scattering effect, some methods utilizing light element gas [2] or additional vacuum pump to reduce pressure [1] (10 4 ~10 5 Pa) have been developed. A typical atmospheric SEM image is shown in Fig. 1(c). Brightness of point B is brighter than that of point A, although the edge of number “9” is clear. The image gives us a consideration that the profile of electron beam arriving at sample is estimated as sum of scattered and un-scattered electrons beam. As a result, the image in Fig. 1(c) seems to be blurred. Based on the consideration, we develop an image enhancement algorism for ASEM (electron scattering corrector: ES-Corrector). By using this algorism, blurring created by scattered electrons in ASEM image can be improved after detection of SEM image. Figure 2 shows SEM images of Cu mesh (Fig. 2(a)(b)) taken in atmospheric pressure. Figure 2(c) and (d) are restored images using ES-Corrector. The images show great improvements in clarity and edge sharpness than the observed images. The microstructures on Cu mesh observed in Fig. 2(c) and (d) are compatible to those in SEM images taken in vacuum Fig. 2(e) and (f). Figure 3 shows SEM images of a filter paper (Fig. 3(a)), renal glomerulus without metal staining (Fig. 2(b)), a leaf surface of the Japanese radish(Fig. 3(c)), and blood cells fixed with 1% glutaraldehyde and immune-stained with gold particles (Fig. 3(d)) taken in atmospheric pressure at room temperature. Figure 3(a)-(h) is the original and restored images. The images show great improvements in clarity and edge sharpness than the observed images. It has been shown that the ES-Corrector algorism to reduce effect of scattered electrons from ASEM image can improve image quality.

Observation of Wet Samples Using a Novel Atmospheric Scanning Electron Microscope

The scanning electron microscope (SEM) has been used as a powerful tool for providing surface information of micro and nanostructures. In recent years, SEM methods for observing wet samples under atmospheric pressure have been reported by some investigators [1-2]. With these methods, the sample space is separated by a thin transparent membrane from vacuum environment where electron beam is propagated, and samples attaching to the membrane are observed by SEM.