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 (HFPP) is one of other types of phase plate, consisting only of thin carbon film. In HFPP, the charging on a carbon film due to high-density electron beam (cross-over) acts like a small central hole of ZPP. The size of cross-over on HFPP is smaller than typical size of a central hole of ZPP, which is typically 1 m in diameter. Thus, the cut-on frequency of HFPP is lower than ZPP and phase contrast of larger structures is enhanced by HFPP. Also with HFPP, The fringing effects, which is observed in ZPP, reduces because the width of the fringes is increasing to be wider than sample size with the very small cut-on frequency.In this study, we applied the HFPP to enhance the phase contrast for unstained polymer materials. A 200kV field-emission electron microscope (JEM-2200FS) equipped with Schottky electron source was used to obtain a small cross-over on a HFPP. A HFPP, which is made of 15nm-thick amorphous carbon, is set on the back focal plane of the objective lens of focal length 2.8mm. Figure 1 compares conventional TEM and HFPP-TEM images for 50 nm-thick unstained polymer section (Polystyrene-b-isoprene, Polymer Source Inc.). The conventional TEM images have low contrast, even with the large defocus (Figure 1 (a), (b)). In the HFPP-TEM image, lamellar structures of the polymer are clearly observed (Figure 1 (c)). Figure 2 shows the diffractogram of HFPP-TEM image in Figure 1. Peaks in the diffractogram are corresponding to 0.02 nm -1 in reciprocal space, indicating the lamellar period of specimen is 50 nm (Figure 2). A reason for that such a long periodic structure can be observed by the HFPP-TEM is that the cross-over on a HFPP, which is supposed to be a phase modulation region, is small enough to enhance the low-spatial frequency of the lamellar structures. Figure 3 shows an intensity profile of a cross-over spot, which is formed on a back focal plane and observed in diffraction mode. The measured size of a cross-over on a HFPP in real space (scale in lower 60

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.

Recent years, in transmission electron microscopy (TEM), the phase plate enhances phase contrast of a specimen image. Naturally, we expect that TEM with the phase plate enables us to obtain high contrast images of the samples, which are composed of light elements such as biological samples and polymer samples.So far, many types of phase plates for electron microscopy have been proposed. The most productive type of the phase plate is the thin film. Above all thin film, Zernike phase plate has been producing promising results [1-2].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.Next, we tried to fabricate the amorphous carbon thin film Zernike phase plate. The fabrication method for this phase plate is similar to one for the Ti/SiN/Ti thin film phase plate [4]. To improve the characteristics for the thin film phase plate: crystallization of the amorphous film, electro resistibility, thickness controllability and others, we adjusted the deposition condition for amorphous carbon thin film Zernike phase plates. Finally, we have succeeded to fabricate an amorphous carbon thin film phase plate having sufficient characteristics.Figures 1(a) and 1(b) show SEM images of top and bottom surfaces of amorphous carbon thin film Zernike phase plate. As shown in the figures, the amorphous carbon thin films are clean. The thickness of the amorphous carbon film was fabricated to be approximately 30 nm that gave ʌ/2 phase shift for 200 kV electrons. In the center of each square window, there is a small hole where the unscattered electrons pass through. The diameter of the hole was fabricated to be approximately 0.7 ȝm as shown in Figure. 2. The stability of thus-fabricated phase plate in TEM imaging was good enough for sample observation. It is worthy to say that a hole-free phase plate [5][6] fabricated with the same method was also confirmed to show good performance.

Recently, we have developed a large solid angle EDS (Energy Dispersive Spectrometer) detector for transmission electron microscope, whose maximum solid angle reaches ~1 sr [1][2][3]. The detector is a silicon drift type and can provide highly sensitive X-ray acquisition even at high counting rate. Therefore, it is expected that the EDS detector enables us to analyze electron beam sensitive samples such as biological and polymer materials, which are easily damaged or broken under electron beam irradiation. However, it is difficult to determine the chemical composition of those samples even with the highly sensitive detector, because their chemical composition may change during the analysis. To evaluate the change, we have developed a function to record the time change of the chemical composition in frame-by-frame. The function, enables us to perform the time-resolved X-ray analyses. The software for the function has three main functions: 1) recording scanning electron and X-ray images during the X-ray acquisition, 2) calculating the gross X-ray intensity of interested elements, and its change can be plotted after the X-ray acquisition, 3) extracting and displaying the integrated X-ray images in the arbitrary frames after the X-ray acquisition. Figures 1 and 2 show the time-resolved results for a halite (NaCl) sample on a carbon support film. The X-ray images with a size of 128 x 128 pixels were obtained by using a field emission electron microscope (JEM-2800), equipped with the silicon drift detector with sensor area of 100 mm 2 (solid angle is ~0.95 sr), operated at the accelerating voltage of 200 kV. The probe size, the probe current, the dwell time for each pixel and the irradiated area were 1 nm , 1 nA, 100 sec, and 920 x 920 nm 2 respectively, resulting in the estimated dose rate of 1.2 x 10 4 e/nm 2 /frame. Figure 1 shows the change of the X-ray intensities of C, O, Na, and Cl from NaCl particles with frame. Figure 2 shows three sets of the time-resolved X-ray images of the Na and the Cl and the DF-STEM images, which were integrated in 20 frames. They were sampled from early frames (1 st -20 th frames), middle frames (171 st -180 th ), and late frames (376 th -395 th ).The plots showing the change of the X-ray intensities of C, O, Na, and Cl with frame, shown in Fig. 1, reveals that the intensities of the Na and the Cl gradually decreased with the increase of frames. The intensity of the Cl decreased more rapidly than that of the Na (Fig. 1). The Cl preferentially decreased at the periphery of the NaCl particles and disappeared in the irradiated region, while the Na diffused out to the carbon support film around the particles (Fig. 2). This result suggests that loss of the Na and the Cl under the electron beam irradiation can be evaluated by this method. Although the detail of the loss mechanism has not been confirmed, we may consider that the Cl evaporated as vapor and dispersed into the vacuum as gas, while the Na deposited as solid and diffused to the surroundings. As described above, it is expected that ...

Modern electron microscopes feature fully digital components and are operated by personal computers running standard operating systems. A benefit of this operation is that these instruments can be controlled by a computer sitting next to the instrument, or by one connected to the instrument via a local network (Intranet) or by the Internet. This remote operation is identical to local operation and lends itself to a number of applications: remote operation for enhanced performance, instrument sharing, experiment collaboration, remote diagnostics, remote servicing, and remote teaching and training. 1As microscopes have become more complex, they have become more specialized. Due to the expense of these instruments, not every laboratory that has a need for a specific feature on a microscope can afford such a machine. This has led to an increase in collaborative funding for instrumentation, with the instrument residing in one centralized facility. This means that teaching of electron microscope fundamentals as well as training for the instruments is not necessarily limited to a single facility. Through remote operation of the microscope, users and students at remote facilities can get the education and training they need without the added expense of travel.Classroom teaching of electron microscopy has typically been the teaching of the theory and operation of these instruments without the benefit of actually operating one during class. The electron microscope often resides in an environmentally controlled laboratory that is not conducive to a large number of students being near by for education and training. Remote operation eliminates this problem by instead bringing the instrument to the students.Full remote control of TEMs has been a reality for nearly a decade. 2 One system for remotely operating these instruments is Sirius. Sirius is a client / server platform that interfaces directly with the control software of the TEM. The remote software terminal is designed to work with a trackball and knob set so that remote microscope operation is completely identical to local operation ( Figure 1). Sirius operates using the TCP/IP protocol, with the remote client interfacing directly to a server. This makes the connection secure, and ensures that only one client (and thus one user) can be connected to the microscope at any given time. All operation and data collection is integrated by combining Sirius with a remote connection to a PC operating the Gatan Microscopy Suite (Figure 2). The system sends small compressed data communications to conserve network bandwidth, resulting in live time operation.