Low-aberration ExB deflector optics for scanning electron microscopy.

To suppress aberrations in the signal electron optics of a scanning electron microscope, we propose ExB deflector (deflector with superimposed electric and magnetic fields) optics that cancel the aberrations generated during large-angle deflection. This improves the resolution of the angle or position of the signal electrons on the sample surface, allowing them to be discriminately detected. The proposed optics consist of two ExB deflectors and a transfer system with two 4-f systems, or systems that have four times the focal length, placed between them. This configuration maintains the symmetry of the electron beam trajectory throughout the transfer system such that aberrations generated by the first ExB deflector are negated by the second. The effect of the proposed optics was confirmed using a ray-tracing simulation of the electron beam, and the aberration was reduced to at most one-tenth of that in the case with only one ExB deflector. Furthermore, as an example, we examined the implementation of the proposed ExB deflector optics to resolve the signal electron angle and found that the sample emission angle range of 80° can be resolved with an angular resolution of 1°. Therefore, the proposed ExB deflector optics can be applied to the signal electron optics of a scanning electron microscope to improve the resolution of the signal electrons.

Spherical aberration correction with an in-lens N-fold symmetric line currents model.

In our previous works, we have proposed N-SYLC (N-fold symmetric line currents) models for aberration correction. In this paper, we propose "in-lens N-SYLC" model, where N-SYLC overlaps rotationally symmetric lens. Such overlap is possible because N-SYLC is free of magnetic materials. We analytically prove that, if certain parameters of the model are optimized, an in-lens 3-SYLC (N = 3) doublet can correct 3rd order spherical aberration. By computer simulation, we show that the required excitation current for correction is less than 0.25 AT for beam energy 5 keV, and the beam size after correction is smaller than 1 nm at the corrector image plane for initial slope less than 4 mrad.

Automatic system for electron tomography data collection in the ultra-high voltage electron microscope.

In this study, we report an automatic system for collection of tilt series for electron tomography based on the ultra-HVEM in Osaka University. By remotely controlling the microscope and reading the observation image, the system can track the field of view and do focus in each tilt angle. The automatic tracking is carried out with an image matching technique based on normalized correlation coefficient. Auto focus is realized by the optimization of image sharpness. A toolkit that can expand the field of view with technique of image stitching is also developed. The system can automatically collect the tilt series with much smaller time consumption.

Axial geometrical aberration correction up to 5th order with N-SYLC.

We present N-SYLC (N-fold symmetric line currents) models to correct 5th order axial geometrical aberrations in electron microscopes. In our previous paper, we showed that 3rd order spherical aberration can be corrected by 3-SYLC doublet. After that, mainly the 5th order aberrations remain to limit the resolution. In this paper, we extend the doublet to quadruplet models also including octupole and dodecapole fields for correcting these higher order aberrations, without introducing any new unwanted ones. We prove the validity of our models by analytical calculations. Also by computer simulations, we show that for beam energy of 5keV and initial angle 10mrad at the corrector object plane, beam size of less than 0.5nm is achieved at the corrector image plane.

The influence of structure depth on image blurring of micrometres-thick specimens in MeV transmission electron imaging.

This study investigates the influence of structure depth on image blurring of micrometres-thick films by experiment and simulation with a conventional transmission electron microscope (TEM). First, ultra-high-voltage electron microscope (ultra-HVEM) images of nanometer gold particles embedded in thick epoxy-resin films were acquired in the experiment and compared with simulated images. Then, variations of image blurring of gold particles at different depths were evaluated by calculating the particle diameter. The results showed that with a decrease in depth, image blurring increased. This depth-related property was more apparent for thicker specimens. Fortunately, larger particle depth involves less image blurring, even for a 10-µm-thick epoxy-resin film. The quality dependence on depth of a 3D reconstruction of particle structures in thick specimens was revealed by electron tomography. The evolution of image blurring with structure depth is determined mainly by multiple elastic scattering effects. Thick specimens of heavier materials produced more blurring due to a larger lateral spread of electrons after scattering from the structure. Nevertheless, increasing electron energy to 2MeV can reduce blurring and produce an acceptable image quality for thick specimens in the TEM.

Spherical aberration correction with threefold symmetric line currents.

It has been shown that N-fold symmetric line current (henceforth denoted as N-SYLC) produces 2N-pole magnetic fields. In this paper, a threefold symmetric line current (N3-SYLC in short) is proposed for correcting 3rd order spherical aberration of round lenses. N3-SYLC can be realized without using magnetic materials, which makes it free of the problems of hysteresis, inhomogeneity and saturation. We investigate theoretically the basic properties of an N3-SYLC configuration which can in principle be realized by simple wires. By optimizing the parameters of a system with beam energy of 5.5keV, the required excitation current for correcting 3rd order spherical aberration coefficient of 400 mm is less than 1AT, and the residual higher order aberrations can be kept sufficiently small to obtain beam size of less than 1 nm for initial slopes up to 5 mrad.

Double-twist cylinders in liquid crystalline cholesteric blue phases observed by transmission electron microscopy.

Cholesteric blue phases are liquid crystalline phases in which the constituent rod-like molecules spontaneously form three-dimensional, helical structures. Despite theoretical predictions that they are composed of cylindrical substructures within which the liquid crystal molecules are doubly twisted, real space observation of the arrangement of such structures had not been performed. Through transmission electron microscopy of photopolymerized blue phases with controlled lattice plane orientations, we report real space observation and comparison of the lattice structures of blue phases I and II. The two systems show distinctly different contrasts, reflecting the theoretically predicted, body centred and simple cubic arrangement of the double-twist cylinders. Transmission electron microscopy also reveals different tendencies of the two blue phases to align on unidirectionally rubbed surfaces. We thus show that TEM observation of alignment-controlled, photopolymerized liquid crystals can be a powerful tool to investigate complex liquid crystalline order.

Fast auto-acquisition tomography tilt series by using HD video camera in ultra-high voltage electron microscope.

The ultra-high voltage electron microscope (UHVEM) H-3000 with the world highest acceleration voltage of 3 MV can observe remarkable three dimensional microstructures of microns-thick samples[1]. Acquiring a tilt series of electron tomography is laborious work and thus an automatic technique is highly desired. We proposed the Auto-Focus system using image Sharpness (AFS)[2,3] for UHVEM tomography tilt series acquisition. In the method, five images with different defocus values are firstly acquired and the image sharpness are calculated. The sharpness are then fitted to a quasi-Gaussian function to decide the best focus value[3]. Defocused images acquired by the slow scan CCD (SS-CCD) camera (Hitachi F486BK) are of high quality but one minute is taken for acquisition of five defocused images.In this study, we introduce a high-definition video camera (HD video camera; Hamamatsu Photonics K. K. C9721S) for fast acquisition of images[4]. It is an analog camera but the camera image is captured by a PC and the effective image resolution is 1280×1023 pixels. This resolution is lower than that of the SS-CCD camera of 4096×4096 pixels. However, the HD video camera captures one image for only 1/30 second. In exchange for the faster acquisition the S/N of images are low. To improve the S/N, 22 captured frames are integrated so that each image sharpness is enough to become lower fitting error. As countermeasure against low resolution, we selected a large defocus step, which is typically five times of the manual defocus step, to discriminate different defocused images.By using HD video camera for autofocus process, the time consumption for each autofocus procedure was reduced to about six seconds. It took one second for correction of an image position and the total correction time was seven seconds, which was shorter by one order than that using SS-CCD camera. When we used SS-CCD camera for final image capture, it took 30 seconds to record one tilt image. We can obtain a tilt series of 61 images within 30 minutes. Accuracy and repeatability were good enough to practical use (Figure 1). We successfully reduced the total acquisition time of a tomography tilt series in half than before.jmicro;63/suppl_1/i25/DFU066F1F1DFU066F1Fig. 1.Objective lens current change with a tilt angle during acquisition of tomography series (Sample: a rat hepatocyte, thickness: 2 m, magnification: 4k, acc. voltage: 2 MV). Tilt angle range is ±60 degree with 2 degree step angle. Two series were acquired in the same area. Both data were almost same and the deviation was smaller than the minimum step by manual, so auto-focus worked well. We also developed a computer-aided three dimensional (3D) visualization and analysis software for electron tomography "HawkC" which can sectionalize the 3D data semi-automatically[5,6]. If this auto-acquisition system is used with IMOD reconstruction software[7] and HawkC software, we will be able to do on-line UHVEM tomography. The system would help pathology examination in the future.This work was supported by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, under a Grant-in-Aid for Scientific Research (Grant No. 23560024, 23560786), and SENTAN, Japan Science and Technology Agency, Japan.

Lorentzian-like image blur of gold nanoparticles on thick amorphous silicon films in ultra-high-voltage transmission electron microscopy.

We quantitatively analyzed the contrast degradation and blur of 20-nm gold nanoparticles adsorbed on the top of amorphous silicon films of thicknesses of 0.54, 1.09, 1.63 and 2.2 µm in bright-field transmission electron microscope (TEM) images taken at accelerating voltages of 0.5, 1, 2 and 3 MeV. The thickness dependence of the transmission was well explained and consistent with our calculations. The blur function, derived by assuming that the TEM image of a thick specimen can be reproduced by convolving the TEM image of a very thin specimen with it, was found to be expressed by a two-dimensional Lorentzian function. Considering the two characteristics of the Lorentzian function, a sharp peak around the center and a long tail, we concluded that, for TEM observations of thick specimens, the image contrast is degraded predominantly by inelastic scattering and the image is blurred predominantly by multiple elastic scattering.

Structural modifications of dentinal microcracks with human aging.

Several studies have reported the mechanism of crack propagation with aging. Although structural modifications of dentinal microcracks with aging have been evaluated by observing the cracked surface using scanning electron microscope (SEM), very few attempts have been made at sectional observation of the microcracks inside dentine using transmission electron microscopy (TEM). The objectives of this study were to: (1) examine the process of dentinal microcrack formation using TEM and (2) to morphologically evaluate the relation between dentinal microcrack propagation and human aging. Molars from 'young' (16-28 years) and 'aged' (62-76 years) subjects were evaluated. Dentine blocks were cracked with an indenter and sectioned using a diamond knife and ultramicrotome after embedding in epoxy resin. Microcracks were observed by TEM and ultra-high-voltage electron microscope tomography to determine the characteristics of crack propagation in the young and aged teeth. The results show that, in young teeth, crack propagation tended to pass through the dentinal tubules, while in aged teeth, it tended to deflect to the outer side of peritubular dentine (PTD), especially in coronal dentine. The advantage of this method is that it allows visualization and evaluation of the ultrastructural propagation of microcracks in dentine. The differences in crack propagation between young and aged dentine could be explained by differences in PTD thickness.

Electron tomographic resolution of microns-thick specimens in the ultrahigh voltage electron microscope.

In this study, we determine the electron tomography (ET) resolution for microns-thick specimens by experiment in the ultra-high voltage electron microscope. A tilt series of projection images of a tilted 8µm thick epoxy-resin film are first acquired. Tomographic reconstructions are then calculated and the resolution is evaluated with the Fourier shell correlation method. The ET resolution of 32nm is achieved under the condition of 2MV accelerating voltage. We also demonstrate that some high tilt angle projections may be little useful for improving the final ET resolution because of the corresponding poor image qualities. These results are helpful to understand the possibility and limitation of ET applications in microns-thick specimens.

Note: direct measurement of the point-to-point resolution for microns-thick specimens in the ultrahigh-voltage electron microscope.

We report on a direct measurement method and results of the point-to-point resolution for microns-thick amorphous specimens in the ultrahigh-voltage electron microscope (ultra-HVEM). We first obtain the ultra-HVEM images of nanometer gold particles with different sizes on the top surfaces of the thick epoxy-resin specimens. Based on the Rayleigh criterion, the point-to-point resolution is then determined as the minimum distance between centers of two resolvable tangent gold particles. Some values of resolution are accordingly acquired for the specimens with different thicknesses at the accelerating voltage of 2 MV, for example, 18.5 nm and 28.4 nm for the 5 µm and 8 µm thick epoxy-resin specimens, respectively. The presented method and results provide a reliable and useful approach to quantifying and comparing the achievable spatial resolution for the thick specimens imaged in the mode of transmission electron including the scanning transmission electron microscope.

Image blurring of thick specimens due to MeV transmission electron scattering: a Monte Carlo study.

Image blurring of MeV transmission electrons for gold nanoparticles on the top surface of micrometer-thick specimens has been investigated using the Monte Carlo simulation. Both the simulated line density profile and therefore image blurring were in good agreement with the experimental ones in the ultrahigh voltage electron microscope. Quantitative effects of specimen thickness and electron energy on image blurring were presented, in which the specimen thickness had a greater influence. Image blurring was demonstrated to be caused mainly by multiple elastic scattering, but it could be reduced to several nanometers for a 5 µm thick epoxy-resin specimen at the electron energy of 2 MeV.

Use of an external real-time image viewer coupled with prespecified actions enhanced the complete examinations for capsule endoscopy.

BACKGROUND AND AIM: The clinical utility of capsule endoscopy (CE) is often limited by incomplete small-bowel transit. The aim was to determine whether the use of an external real-time viewer could reduce delays caused by delayed gastric emptying of the capsule or delayed intestinal transit and also improve the rate of positive findings. METHODS: We compared the proportion of completed exams and positive results among a group of patients studied before introduction of real-time viewer and a group in which capsule transit through the esophagus, stomach, and small bowel was regularly monitored and actions (e.g. administration of water or intravenous metoclopramide) were taken if it was delayed. RESULTS: One hundred procedures in the viewer group and 100 control procedures in the age-matched controls were analyzed. In the viewer group, additional water intake (22 cases) and/or administration of metoclopramide (26 cases) were required. Endoscopic-assisted duodenal placement of the capsule was required in three cases. Overall one-third (n=33) of cases required viewer-prompted interventions. The completion rate (86% vs 66%, P=0.002) and the rate of positive findings (80% vs 67%, P=0.04) were significantly higher in the viewer group compared to the no viewer group. CONCLUSIONS: Checking the progress of the capsule with the external real-time viewer improved the diagnostic yield and completion rate of CE.

Renin-angiotensin system associated with risk of upper GI mucosal injury induced by low dose aspirin: renin angiotensin system genes' polymorphism.

BACKGROUND: We have previously shown that co-treatment of angiotensin type 1 receptor (AT1R) blocker (ARB) or angiotensin converting enzyme (ACE) inhibitor seem to reduce peptic ulcer among patients taking low dose aspirin. It is reported that a series of renin-angiotensin system (RAS) gene polymorphisms significantly influence the rate of the gene transcription. AIM: The aim of this study was to examine the genotypes of RAS genes related to the risk of peptic ulcer and ulcer bleeding among patients taking low dose aspirin. METHODS: Patients taking 100 mg of aspirin who were planning to undergo endoscopy for surveillance or who had history of recent upper GI ulcer bleeding were included. ACE (Ins/Del), angiotensinogen (AGT; G-217A, A-20C, A-6G, T174 M, M235T), and AT1R (T-713G, C-521T, A1166C) genotypes were determined by PCR or PCR-RFLP. RESULTS: Four hundred twenty-five patients were enrolled including 68 patients with peptic ulcer and 20 patients with ulcer bleeding. Co-treatment of ARB was significantly associated with peptic ulcer and ulcer bleeding. AGT-20 CC (adjusted OR 4.94, 95% CI 1.21-20.2) was significantly associated with ulcer bleeding. The CC genotype of AT1R-521 was significantly associated with peptic ulcer only in the subgroup taking neither ACE inhibitor nor ARB. CONCLUSIONS: Co-treatment of ARB reduces peptic ulcer and bleeding among patients taking low dose aspirin. RAS may play an important role in the development of upper GI mucosal injury induced by low dose aspirin.

An automatic method of detecting and tracking fiducial markers for alignment in electron tomography.

We presented an automatic method for detecting and tracking colloidal gold fiducial markers for alignment in electron tomography (ET). The second-order derivative of direction was used to detect a fiducial marker accurately. The detection was optimized to be selective to the size of fiducial markers. A preliminary tracking result from the normalized correlation coefficient was refined using the detector. A constraint model considering the relationship among the fiducial markers on different images was developed for removing outlier. The three-dimensional positions of the detected fiducial markers and the projection parameters of tilt images were calculated for post process. The accuracy of detection and tracking results was evaluated from the residues by the software IMOD. Application on transmission electron microscopic images also indicated that the presented method could provide a useful approach to automatic alignment in ET.

Aspirin-induced peptic ulcer and genetic polymorphisms.

There are a few studies of the association between genetic polymorphisms and the risks of acetylsalicylic acid (aspirin)-induced ulcer or its complications. Two single nucleotide polymorphisms (SNP) of cyclooxygenase-1 (COX-1), A-842G and C50T, exhibited increased sensitivity to aspirin and had lower prostaglandin synthesis capacity, lacking statistical significance in the association with bleeding peptic ulcer. A recent Japanese study indicated that the number of COX-1-1676T alleles was a significant risk factor for peptic ulcer in users of non-steroidal anti-inflammatory drugs (NSAIDs). There are some genetic polymorphisms for aspirin resistance, such as platelet membrane glycoproteins, thromboxane A2 (TXA2) receptor, platelet activating factor acetylhydrolase and coagulation factor XIII; however, data on the frequency of gastrointestinal (GI) events in these variants are lacking. Carrying the CYP2C9 variants is reported a significantly increased risk of non-aspirin NSAID-related GI bleeding. The polymorphisms of interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) have been associated with development of peptic ulcer or gastric cancer. In a recent investigation, carriage of the IL-1beta-511 T allele was significantly associated with peptic ulcer among low-dose aspirin users. Hypoacidity in corpus gastritis related to polymorphisms of pro-inflammatory cytokines seems to reduce NSAIDs or aspirin-related injury. Data on which polymorphisms are significant risk factors for GI events in aspirin users are still lacking and further large-scale clinical studies are required.

Determination of the linear attenuation range of electron transmission through film specimens.

We have investigated the linear attenuation range of electron transmission through film specimens and its dependence on the electron energy, the acceptance half-angle of a detector or an objective aperture, and specimen properties, in the scanning transmission electron microscope (STEM) and the conventional transmission electron microscope (TEM). Electron transmission in the bright-field mode was calculated by the Monte Carlo simulation of electron scattering, and its range of the linear attenuation in film thickness was then determined by a linear least squares fit. The corresponding linear thickness range was shown to increase with the electron energy and the acceptance half-angle, although it decreased with the increase in the atomic number of specimen materials. Under the condition of a 300kV STEM or a 3MV ultra-high voltage electron microscope (ultra-HVEM), the linear attenuation range could extend to several microns for light specimen materials, and this was validated by experimental data in the ultra-HVEM. The presented results can be helpful for accurately measuring the specimen thickness or mass from electron transmission, and estimating the deviation of electron transmission from linearity when tilting a specimen in electron tomography.

Image quality of microns-thick specimens in the ultra-high voltage electron microscope.

Image quality of MeV transmission electrons is an important factor for both observation and electron tomography of microns-thick specimens with the high voltage electron microscope (HVEM) and the ultra-HVEM. In this work, we have investigated image quality of a tilted thick specimen by experiment and analysis. In a 3 MV ultra-HVEM, we obtained transmission electron images in amplitude contrast of 100 nm gold particles on the top surface of a tilted 5 microm thick amorphous epoxy-resin film. From line profiles of the images, we then measured and evaluated image blurring, contrast, and the signal-to-noise ratio (SNR) under different effective thicknesses of the tilted specimen and accelerating voltages of electrons. The variation of imaging blurring was consistent with the analysis based on multiple elastic scattering. When the effective thickness almost tripled, image blurring increased from approximately 3 to approximately 20 nm at the accelerating voltage of 3 MV. For the increase of accelerating voltage from 1 to 3 MV in the condition of the 14.6 microm effective thickness, due to the reduction of multiple scattering effects, image blurring decreased from approximately 54 to approximately 20 nm, and image contrast and SNR were both obviously enhanced by a factor of approximately 3 to preferable values. The specimen thickness was shown to influence image quality more than the accelerating voltage. Moreover, improvement on image quality of thick specimens due to increasing the accelerating voltage would become less when it was further increased from 2 to 3 MV in this work.

Helicobacter pylori eradication prevents extension of intestinalization even in the high-risk group for gastric cancer.

BACKGROUND/AIMS: CDX2 is associated with the intestinal phenotype in the gastrointestinal tracts and is expressed in the intestinal type of gastric cancer. Helicobacter pylori-associated atrophic gastritis is characterized by aberrant expression of CDX2. The aim was to investigate the effects of eradication to the expression of genes related to the gastric and intestinal phenotype including CDX2. We compared the effect of eradication between the patients at high risk for gastric cancer and controls. METHODS: 20 patients with endoscopic resection for early gastric cancer and 12 sex- and age-matched controls were studied. CDX2 and mucin mRNA expressions were examined using whole biopsy specimens and microdissected gastric glands taken from corpus lesser and greater curves before and 1 year after eradication. RESULTS: CDX2 and MUC2 expressions in the cancer group were significantly higher than in the controls and were significantly decreased after eradication. MUC5AC (p = 0.01) and MUC6 (p = 0.02) expression significantly increased in the control group; the difference between the two groups became significant after eradication. CDX2 expression in the glands without goblet cells was detectable and disappeared after eradication. CONCLUSION: H. pylori eradication can reverse gastric phenotype and diminish aberrant CDX2 expression in the early stage of intestinalization.