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.

Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs.

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.

Microscale fluid flow analysis in a human osteocyte canaliculus using a realistic high-resolution image-based three-dimensional model.

Osteocytes play a pivotal role in the regulation of skeletal mass. Osteocyte processes are thought to sense the flow of interstitial fluid that is driven through the osteocyte canaliculi by mechanical stimuli placed upon bone, but how this flow elicits a cellular response is virtually unknown. Modern theoretical models assume that osteocyte canaliculi contain ultrastructural features that amplify the fluid flow-derived mechanical signal. Unfortunately the calcified bone matrix has considerably hampered studies on the osteocyte process within its canaliculus. Using one of the few ultra high voltage electron microscopes (UHVEM) available worldwide, we applied UHVEM tomography at 2 MeV to reconstruct unique three-dimensional images of osteocyte canaliculi in 1 µm sections of human bone. A realistic three-dimensional image-based model of a single canaliculus was constructed, and the fluid dynamics of a Newtonian fluid flow within the canaliculus was analyzed. We created virtual 2.2 nm thick sections through a canaliculus and found that traditional TEM techniques create a false impression that osteocyte processes are directly attached to the canalicular wall. The canalicular wall had a highly irregular surface and contained protruding axisymmetric structures similar in size and shape to collagen fibrils. We also found that the microscopic surface roughness of the canalicular wall strongly influenced the fluid flow profiles, whereby highly inhomogeneous flow patterns emerged. These inhomogeneous flow patterns may induce deformation of cytoskeletal elements in the osteocyte process, thereby amplifying mechanical signals. Based on these observations, new and realistic models can be developed that will significantly enhance our understanding of the process of mechanotransduction in bone.

Chromosome observation by scanning electron microscopy using ionic liquid.

Electron microscopy has been used to visualize chromosome since it has high resolution and magnification. However, biological samples need to be dehydrated and coated with metal or carbon before observation. Ionic liquid is a class of ionic solvent that possesses advantageous properties of current interest in a variety of interdisciplinary areas of science. By using ionic liquid, biological samples need not be dehydrated or metal-coated, because ionic liquid behaves as the electronically conducting material for electron microscopy. The authors have investigated chromosome using ionic liquid in conjunction with electron microscopy and evaluated the factors that affect chromosome visualization. Experimental conditions used in the previous studies were further optimized. As a result, prewarmed, well-mixed, and low concentration (0.5∼1.0%) ionic liquid provides well-contrasted images, especially when the more hydrophilic and the higher purity ionic liquid is used. Image contrast and resolution are enhanced by the combination of ionic liquid and platinum blue staining, the use of an indium tin oxide membrane, osmium tetroxide-coated coverslip, or aluminum foil as substrate, and the adjustment of electron acceleration voltage. The authors conclude that the ionic-liquid method is useful for the visualization of chromosome by scanning electron microscopy without dehydration or metal coating.

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.

Controlled structure and properties of silicate nanoparticle networks for incorporation of biosystem components.

Inorganic nanoparticles are of technological interest in many fields. We created silicate nanoparticle hydrogels that effectively incorporated biomolecules that are unstable and involved in complicated reactions. The size of the silicate nanoparticles strongly affected both the physical characteristics of the resulting hydrogel and the activity of biomolecules incorporated within the hydrogel. We used high-resolution transmission electron microscopy (TEM) to analyze in detail the hydrogel network patterns formed by the silicate nanoparticles. We obtained clear nanostructured images of biomolecule-nanoparticle composite hydrogels. The TEM images also showed that larger silicate nanoparticles (22 nm) formed more loosely associated silicate networks than did smaller silicate nanoparticles (7 nm). The loosely associated networks formed from larger silicate nanoparticles might facilitate substrate diffusion through the network, thus promoting the observed increased activity of the entrapped biomolecules. This doubled the activity of the incorporated biosystems compared with that of biosystems prepared by our own previously reported method. We propose a reaction scheme to explain the formation of the silicate nanoparticle networks. The successful incorporation of biomolecules into the nanoparticle hydrogels, along with the high level of activity exhibited by the biomolecules required for complicated reaction within the gels, demonstrates the nanocomposites' potential for use in medical applications.

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.

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.

Multiple scattering effects of MeV electrons in very thick amorphous specimens.

Multiple scattering has an important influence on the analysis of microns-thick specimens with MeV electrons. In this paper, we report on effects of multiple scattering of MeV electrons on electron transmission and imaging of tilted and thick amorphous film specimens by experiment and theoretical analysis. Electron transmission for microns-thick epoxy-resin and SiO(2) specimens calculated by the multiple elastic-scattering theory is in good agreement with measurements in the ultrahigh voltage electron microscope (ultra-HVEM) at Osaka University. Electron transmission and electron energy are then presented in an approximate power law. The bright-field ultra-HVEM images of gold particles on the top or bottom surfaces of 5 and 15mum thick specimens further illustrate the effect of multiple scattering on image quality. The observed top-bottom effect for the very thick specimens appears to be mainly caused by multiple elastic scattering. With increase in the accelerating voltage from 1 to 2MV, image blurring, contrast, the signal-to-noise ratio, and the top-bottom effect are improved because of reduction in the influence of multiple scattering. However, the effect of specimen thickness on image blurring is shown to be stronger than that of accelerating voltage. At the 2MV accelerating voltage, the 100nm gold particle can be imaged with less blurring of approximately 4nm when located at the bottom surface of a 15mum thick epoxy-resin specimen.

A method for observing silver-stained osteocytes in situ in 3-microm sections using ultra-high voltage electron microscopy tomography.

Osteocytes are surrounded by hard bone matrix, and it has not been possible previously to directly observe the in situ architecture of osteocyte morphology in bone. Electron microscope tomography, however, is a technique that has the unique potential to provide three-dimensional (3D) visualization of cellular ultrastructure. This approach is based on reconstruction of 3D volumes from a tilt series of electron micrographs of cells, and resolution at the nanometer level has been achieved. We applied electron microscope tomography to thick sections of silver-stained osteocytes in bone using a Hitachi H-3000 ultra-high voltage electron microscope equipped with a 360 degrees tilt specimen holder, at an accelerating voltage of 2 MeV. Osteocytes with numerous processes and branches were clearly seen in the serial tilt series acquired from 3-microm-thick sections. Reconstruction of young osteocytes showed the 3D topographic morphology of the cell body and processes at high resolution. This morphological data on osteocytes should provide useful information to those who study osteocyte physiology and the several models used to explain their mechanosensory properties.

High sintering resistance of platinum nanoparticles embedded in a microporous hollow carbon shell fabricated through a photocatalytic reaction.

Platinum (Pt) nanoparticles encapsulated in microporous carbon with a hollow structure (nPt@hC) were fabricated on the basis of a titanium(IV) oxide (TiO2) photocatalytic reaction. From the tomogram of a sample studied by using a transmission electron microscope (TEM), the Pt nanoparticles were found to be embedded in the carbon shell and were physically separated from each other by the carbon matrix. Owing to this unique structure, the Pt particles showed high resistance to sintering when subjected to thermal treatment at temperatures up to 800 degrees C. As a result, hydrogenation reactions using various heat-treated nPt@hCs as catalysts indicated that loss of catalytic activity was minimized. Thus, the present system will be a promising system for optimizing catalyst nanostructures utilized in processes requiring rigorous conditions.

Microscopic tomography with ultra-HVEM and applications.

The ultra-HVEM with an accelerating voltage of 3 MV at Osaka University is capable of achieving excellent penetration and resolution for thick specimens. We obtained images of 5-microm-thick slices tilted at angles of up to 70 degrees for biological samples and observed stick-shaped samples of Si devices free from missing zone. These features make the ultra-HVEM an invaluable extension of 3D observation by electron tomography. In this paper, we introduce aspects of ultra-HVEM tomography; specifically, the magnification, the amount of image blurring for thick samples and the electron staining method. Finally, we give some typical applications in the fields of cell biology, pathology and electrical engineering.

Measurement of electron transmission through tilted thick specimens with an ultrahigh voltage electron microscope.

Electron transmission through tilted epoxy-resin film specimens with thicknesses of 1 and 5 microm has been measured under different accelerating voltages and diameters of the objective aperture in a 3 MV ultrahigh voltage electron microscope. Measurements show that the linearity between the logarithmic electron transmission and effective thickness of the tilted specimens holds valid only for some smaller effective thicknesses. However, the higher accelerating voltage leads to a wider linear attenuation range. The linear range can reach 7 microm for the 3 MeV incident electrons. Moreover, the measurements were analyzed with the electron-scattering theory. Actually, the high-energy electrons are capable of penetrating much thicker specimens, but many electrons elastically scattered at large angles cannot be collected as useful signals because of being blocked by the objective aperture in the mode of amplitude contrast. The need for further investigations was suggested to determine theoretically the electron transmission through a thick low-Z specimen and its exponential attenuation range.

Three-dimensional, computer-tomographic analysis of membrane proteins (TrkA, caveolin, clathrin) in PC12 cells.

Signaling of nerve growth factor (NGF) and its receptor (TrkA) promotes neuronal differentiation, synapse formation and survival. It has been known that the complex of NGF and TrkA is internalized into the cytoplasm and transported for further signal transduction, but the ultrastructural information of this process is virtually unknown. In order to clarify the relationship between the internalization of TrkA and the membrane-associated proteins (caveolin and clathrin), the localization and three-dimensional structures of those proteins were examined with computer tomography of high voltage electron microscopy in PC12 cells. TrkA immunoreactivity was found only at definite areas in the plasma membrane, as ring and cluster structures. Its 3D image indicated that those cluster structures contained small pits, which did not appear to be typical caveolae in size and shape. 3D images of clathrin and caveolin-1 immunoreactivities indicated that the formation of those small pits was associated with clathrin, but not with caveolin-1. Caveolin-1 immunoreactivity was found as a mesh-like structure just beneath the plasma membrane. These results suggest that clathrin rather than caveolin is mainly involved in the process of TrkA internalization, at least in differentiated PC12 cells.

Tomography experiment of an integrated circuit specimen using 3 MeV electrons in the transmission electron microscope.

The possibility of utilizing high-energy electron tomography to characterize the micron-scale three dimensional (3D) structures of integrated circuits has been demonstrated experimentally. First, electron transmission through a tilted SiO(2) film was measured with an ultrahigh-voltage electron microscope (ultra-HVEM) and analyzed from the point of view of elastic scattering of electrons, showing that linear attenuation of the logarithmic electron transmission still holds valid for effective specimen thicknesses up to 5 microm under 2 MV accelerating voltages. Electron tomography of a micron-order thick integrated circuit specimen including the Cu/via interconnect was then tried with 3 MeV electrons in the ultra-HVEM. Serial projection images of the specimen tilted at different angles over the range of +/-90 degrees were acquired, and 3D reconstruction was performed with the images by means of the IMOD software package. Consequently, the 3D structures of the Cu lines, via and void, were revealed by cross sections and surface rendering.

Actin dynamics in papilla cells of Brassica rapa during self- and cross-pollination.

The self-incompatibility system of the plant species Brassica is controlled by the S-locus, which contains S-RECEPTOR KINASE (SRK) and S-LOCUS PROTEIN11 (SP11). SP11 binding to SRK induces SRK autophosphorylation and initiates a signaling cascade leading to the rejection of self pollen. However, the mechanism controlling hydration and germination arrest during self-pollination is unclear. In this study, we examined the role of actin, a key cytoskeletal component regulating the transport system for hydration and germination in the papilla cell during pollination. Using rhodamine-phalloidin staining, we showed that cross-pollination induced actin polymerization, whereas self-pollination induced actin reorganization and likely depolymerization. By monitoring transiently expressed green fluorescent protein fused to the actin-binding domain of mouse talin, we observed the concentration of actin bundles at the cross-pollen attachment site and actin reorganization and likely depolymerization at the self-pollen attachment site; the results correspond to those obtained by rhodamine-phalloidin staining. We further showed that the coat of self pollen is sufficient to mediate this response. The actin-depolymerizing drug cytochalasin D significantly inhibited pollen hydration and germination during cross-pollination, further emphasizing a role for actin in these processes. Additionally, three-dimensional electron microscopic tomography revealed the close association of the actin cytoskeleton with an apical vacuole network. Self-pollination disrupted the vacuole network, whereas cross-pollination led to vacuolar rearrangements toward the site of pollen attachment. Taken together, our data suggest that self- and cross-pollination differentially affect the dynamics of the actin cytoskeleton, leading to changes in vacuolar structure associated with hydration and germination.

Observations of unstained biological specimens using a low-energy, high-resolution STEM.

Low-energy, high-resolution scanning transmission electron microscopy (STEM) is introduced as a convenient method for observing unstained biological specimens. By reducing the electron energy, the cross section for light elements becomes comparable to that of conventional electron microscopy observations. The STEM mode exhibited the advantage that the induced energy loss and charge build-up in the sample affected the image to a lesser extent than in the TEM or SEM mode. Furthermore, the efficiency of an STEM detector is high, and the total radiation damage can be reduced if thermal damage due to localized heating at a slow scan operation can be overcome. We applied this method for observations of biological samples that were in the form of thin slices, fine fibers and small particles. When the supporting film for samples is absent, the resolution and the contrast of STEM images can be maintained similar to SEM and TEM images, respectively.