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Anomaly detection is a very effective method to extract useful information from abundant data. Most existing anomaly detection methods are based on normal region or some specific algorithms, which ignore the fact that many actual datasets are mainly imbalanced, resulting in not function properly or effectively in practical, especially in the medical field. On the other hand, imbalanced dataset is also a frequently encountered problem in the learning of neural network because the lack of data in a minority class may lead to uneven classification accuracy. In this paper, inspired by these observations, a novel anomaly detection approach by using classical echo state network (ESN), a brain-inspired neural computing model, is presented. The entire dataset of the proposed method obeys an extremely imbalanced distribution, that is, anomalies are much rarer than normal data. And the training dataset has only the normal data. When the ESN is well trained, the parameters in ESN are the memory of normal data. If the normal data are added into the well-trained network, the error between the input data and the corresponding output is smaller compared with the error between abnormal input data and its corresponding output. Then anomaly behavior is detected if the error between the input data and the corresponding predictive value exceeds a certain threshold. Different from setting an invariable threshold arbitrarily for all of the data, the threshold value used in the proposed method is determined from the analysis of information theory and can be adjust adaptively according to different datasets. Experiments on abnormal heart rate detection are conducted to demonstrate and verify the effectiveness of the proposed detection algorithm and theory.

SummaryStain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue, and liver tissue.

UNLABELLED ESSENTIALS: How platelets organize their α-granule cargo and use their canalicular system remains controversial. Past structural studies were limited due to small sampling volumes or decreased resolution. Our analyses revealed homogeneous granules and a closed canalicular system that opened on activation. Understanding how platelets alter their membranes during activation and secretion elucidates hemostasis. SUMMARY BACKGROUND Platelets survey the vasculature for damage and, in response, activate and release a wide range of proteins from their α-granules. Alpha-granules may be biochemically and structurally heterogeneous; however, other studies suggest that they may be more homogeneous with the observed variation reflecting granule dynamics rather than fundamental differences. OBJECTIVES Our aim was to address how the structural organization of α-granules supports their dynamics. METHODS To preserve the native state, we prepared platelets by high-pressure freezing and freeze-substitution; and to image nearly entire cells, we recorded tomographic data in the scanning transmission electron microscope (STEM). RESULTS AND CONCLUSIONS In resting platelets, we observed a morphologically homogeneous α-granule population that displayed little variation in overall matrix electron density in freeze-substituted preparations (i.e., macro-homogeneity). In resting platelets, the incidence of tubular granule extensions was low, ~4%, but this increased by > 10-fold during early steps in platelet secretion. Using STEM, we observed that the initially decondensing α-granules and the canalicular system remained as separate membrane domains. Decondensing α-granules were found to fuse heterotypically with the plasma membrane via long, tubular connections or homotypically with each other. The frequency of canalicular system fusion with the plasma membrane also increased by about three-fold. Our results validate the utility of freeze-substitution and STEM tomography for characterizing platelet granule secretion and suggest a model in which fusion of platelet α-granules with the plasma membrane occurs via long tubular connections that may provide a spatially limited access route for the timed release of α-granule proteins.

Microscopies based on focused electron probes allow the cell biologist to image the 3D ultrastructure of eukaryotic cells and tissues extending over large volumes, thus providing new insight into the relationship between cellular architecture and function of organelles. Here we compare two such techniques: electron tomography in conjunction with axial bright-field scanning transmission electron microscopy (BF-STEM), and serial block face scanning electron microscopy (SBF-SEM). The advantages and limitations of each technique are illustrated by their application to determining the 3D ultrastructure of human blood platelets, by considering specimen geometry, specimen preparation, beam damage and image processing methods. Many features of the complex membranes composing the platelet organelles can be determined from both approaches, although STEM tomography offers a higher ∼3 nm isotropic pixel size, compared with ∼5 nm for SBF-SEM in the plane of the block face and ∼30 nm in the perpendicular direction. In this regard, we demonstrate that STEM tomography is advantageous for visualizing the platelet canalicular system, which consists of an interconnected network of narrow (∼50–100 nm) membranous cisternae. In contrast, SBF-SEM enables visualization of complete platelets, each of which extends ∼2 µm in minimum dimension, whereas BF-STEM tomography can typically only visualize approximately half of the platelet volume due to a rapid non-linear loss of signal in specimens of thickness greater than ∼1.5 µm. We also show that the limitations of each approach can be ameliorated by combining 3D and 2D measurements using a stereological approach.

The nanoscale structure of the ionomer film located in the catalyst layer of polymer exchange membrane fuel cells (PEMFCs) is of vital importance to proton transport and catalyst utilization. Classical molecular dynamic simulations are conducted to explore the molecular-level structure as well as the structure–property relationships in the ionomer film. Twenty-four systems are simulated to investigate the effect of (i) hydration, (ii) ionomer film thickness, (iii) oxidation of the carbon support surface, and (iv) the presence of catalyst nanoparticles on film adhesion and morphology. The ionomer does not form a continuous film on the carbon surface; rather, the ionomer forms irregular patches through which proton transport from the catalyst to the membrane must occur. These ionomer films are not able to retain water to the same extent as bulk ionomer membranes. However, thicker films retain proportionally more water than thinner films, allowing for a larger and better connected aqueous domain required for...

Carbon supported platinum (Pt/C) catalyst remains among the most preferable catalyst materials for Proton Exchange Membrane (PEM) fuel cells. However, platinum (Pt) particles suffer from poor durability and encounter electrochemical surface area (ESA) loss under operation with the accompany of Pt nanoparticle coarsening. Several proposed mechanisms have involved the Pt detachment from its carbonate support as an initial step for the deactivation of Pt nanoparticles. In this study, we investigated the detachment mechanism from the nano-adhesion point of view. Classic molecular dynamics simulations are performed on systems contain Pt nanoparticles of different sizes and shapes. A thin Nafion film (1 nm) at different hydration levels is also included in the system to study the environmental effect on nanoparticle adhesion. We found that the adhesion force strengthens as the Pt size goes up. Pt nanoparticles of tetrahedral shape exhibit relatively stronger connection with the carbon substrate due to its unique ‘anchor-like’ structure. Adhesion is enhanced with the introduction of a Nafion. The humidity level in the Nafion film has a rather complicated effect on the strength of nanoparticle adhesion. The binding energies and maximum adhesive forces are reported for all systems studied.

Summary Electron microscopy is an essential tool for the evaluation of microstructure and properties of the catalyst layer (CL) of proton exchange membrane fuel cells (PEMFCs). However, electron microscopy has one unavoidable drawback, which is radiation damage. Samples suffer temporary or permanent change of the surface or bulk structure under radiation damage, which can cause ambiguity in the characterization of the sample. To better understand the mechanism of radiation damage of CL samples and to be able to separate the morphological features intrinsic to the material from the consequences of electron radiation damage, a series of experiments based on high-angle annular dark-field–scanning transmission scanning microscope (HAADF-STEM), energy filtering transmission scanning microscope (EFTEM), and electron energy loss spectrum (EELS) are conducted. It is observed that for thin samples (0.3–1 times λ), increasing the incident beam energy can mitigate the radiation damage. Platinum nanoparticles in the CL sample facilitate the radiation damage. The radiation damage of the catalyst sample starts from the interface of Pt/C or defective thin edge and primarily occurs in the form of mass loss accompanied by atomic displacement and edge curl. These results provide important insights on the mechanism of CL radiation damage. Possible strategies of mitigating the radiation damage are provided. SCANNING 36:338–346, 2014. © 2013 Wiley Periodicals, Inc.

Platinum catalysts play a critical role in fuel cell technology. Current optimization efforts focus on reducing the amount of Pt in the system and optimizing the utilization of that which remains. The effect of the presence of Pt nanoparticles on the local structure and morphology of the polymer electrolyte membrane, water, and hydronium ions has been studied at molecular level in this work. Classical molecular dynamics simulation has been used to examine a system containing a 4 nm fcc cubic ({100} face) platinum nanoparticle at the center surrounded by Nafion polymer, water molecules, and hydronium ions at λ = 3, 6, 9, 15, and 22. The changes in density and orientation distribution of sulfonic acid groups in the side-chains, water, and hydronium as a function of distance from platinum surface are analyzed in this study. Sulfonic acid groups and hydronium ions show a very high increase in density near the platinum surface, and they approach the bulk value as they move away from the platinum surface. At lo...

Electron tomography in the scanning transmission electron microscope (STEM) can be performed on sections of stained plastic-embedded tissues or cells of 1 to 2 micrometer thickness without effects of chromatic aberration because there are no imaging lenses after the specimen. By using a small STEM probe convergence angle of 1-2 mrad the geometrical broadening of the probe is restricted, which enables a spatial resolution of a few nanometers. Furthermore, by using an axial bright-field detector instead of the standard high-angle annular dark-field detector, image blurring due to multiple elastic scattering can be reduced in the lower part of the specimen. Here, we have applied STEM tomography to elucidate the 3D ultrastructure of human blood platelets, which are small anucleate blood cells that aggregate to seal leaks at sites of vascular injury and are important in the pathology of atherosclerosis and other diseases. Of particular interest are the morphological changes that occur in alpha-granules, which contain important proteins released when platelets are activated. Axial bright-field STEM electron tomographic tilt series were acquired at an accelerating voltage of 300 kV from 1.5-micrometer thick sections of platelets that had been prepared by rapid freezing and freeze-substitution; and the tomograms were reconstructed from dual-axis tilt series. The tomographic reconstructions revealed changes in ultrastructure that occurred on platelet activation including release of alpha granules through channels connecting to the plasma membrane. The research was supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering, and the research in the Storrie laboratory was supported in part by NIH grant R01 HL119393.

Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale 3D ultrastructure of entire cells and tissue volumes. In SBF-SEM, an ultramicrotome built into the SEM specimen stage successively removes thin sections from a plastic-embedded, heavy metal-stained specimen. After each cut, the freshly exposed block face is imaged at a low incident electron energy using a backscattered electron detector to provide 3D ultrastructure with a resolution of approximately 5 nm in the plane of the block face and around 25 nm in the perpendicular z-direction, as limited by the slice thickness. We have explored the feasibility of improving the z-resolution in SBF-SEM by recording images at multiple primary beam energies, thus sampling different depths below the block surface.A linear relationship was found between the depth of test structures, generated by Monte Carlo simulations, and the ratio of backscattered image intensities recorded at primary beam energies between 1.4 keV and 6.8 keV. This enabled us to reconstruct the 3D model within a 25-nm surface layer at a z-resolution of around 5 nm. We used a Zeiss Sigma-VP SEM equipped with a Gatan 3View SBF system to acquire 3D data from a specimen consisting of gold spheres embedded in carbon. Experiments were also performed on embedded blocks of stained biological tissues.Although damage of the block under electron irradiation limits the signal to noise ratio, the use of multiple primary beam energies, coupled with a physics-based Monte Carlo model, provides the possibility of obtaining cellular ultrastructure at nearly isotropic 3D spatial resolution.

Electron microscopy is an essential tool for the evaluation of microstructure and properties of the catalyst layer (CL) of proton exchange membrane fuel cells (PEMFCs). However, electron microscopy has one unavoidable drawback, which is radiation damage. Samples suffer temporary or permanent change of the surface or bulk structure under radiation damage, which can cause ambiguity in the characterization of the sample. To better understand the mechanism of radiation damage of CL samples and to be able to separate the morphological features intrinsic to the material from the consequences of electron radiation damage, a series of experiments based on high-angle annular dark-field-scanning transmission scanning microscope (HAADF-STEM), energy filtering transmission scanning microscope (EFTEM), and electron energy loss spectrum (EELS) are conducted. It is observed that for thin samples (0.3-1 times λ), increasing the incident beam energy can mitigate the radiation damage. Platinum nanoparticles in the CL sample facilitate the radiation damage. The radiation damage of the catalyst sample starts from the interface of Pt/C or defective thin edge and primarily occurs in the form of mass loss accompanied by atomic displacement and edge curl. These results provide important insights on the mechanism of CL radiation damage. Possible strategies of mitigating the radiation damage are provided.

Extended abstract of a paper presented at Microscopy and Microanalysis 2011 in Nashville, Tennessee, USA, August 7–August 11, 2011.

Carbon supported platinum (Pt/C) catalysts are of vital importance to today's fine chemical industry. However, both Pt and its carbon support surface undergo oxidation during operation. There is a lack of information on how surface oxidation affects the durability of Pt/C catalysts. In this study, we report the impact of oxidation of Pt/C on the binding energy and nanoparticle adhesion force. Classic molecular dynamics simulations are performed on systems containing PtO nanoparticles of 4 nm and graphite surfaces oxidized with either epoxy or hydroxyl groups at several oxidation extents (10%, 25% and 50%). Appropriate for service in polymer electrolyte membrane fuel cells (PEMFC), the effect of a thin Nafion film (1 nm thick) at different hydration levels (λ = 3, 6, 9 and 15) is also included in the system to study the impact of oxidation on the interface structure of the electrolyte and the electrode. This study shows that the adhesion of both the Nafion film and the PtO nanoparticle is drastically affected by the type and degree of oxidation on the carbon surface. The oxidation with hydroxyl groups on the graphite surface enhanced the binding energy between the polymer electrolyte and the carbon electrode, while oxidation with the epoxy group beyond a certain amount caused delamination of the film. The adhesion of the PtO nanoparticle was similarly enhanced by the hydroxylated surface and diminished by the epoxidized surface. The binding energies and adhesive forces are reported for all systems studied.

not Available.

Serial block face scanning electron microscopy (SBF-SEM) provides nanoscale 3D ultrastructure of tissue samples up to several hundred micrometers in size. In SBF-SEM, an ultramicrotome built into the SEM specimen stage successively removes thin sections from a plastic-embedded, heavy metal-stained specimen. After each cut, the freshly exposed block face is imaged at a low incident electron energy using the backscattered electron signal, which is sensitive to heavy atoms in the sample. Although the x-y resolution in the plane of the block face is approximately 5 nm, the resolution along the z-axis in SBF-SEM is limited by the minimum slice thickness of around 25 nm. We have explored the feasibility of improving the z-resolution in SBF-SEM by recording images at more than one primary beam energy, thus sampling different depths below the block surface. We used Monte Carlo simulations of SEM images from an epoxy block containing 5-nm diameter carbon spheres stained with 14% osmium positioned at different depths, as a model for small biological structures. A linear relationship was found between the depth of the spheres and the ratio of backscattered signals at primary beam energies of 1.4 keV and 6.8 keV, which allowed us to generate 3D tomograms with a depth resolution of around 5 nm. Experiments are in progress to test this technique using a Zeiss Sigma-VP SEM equipped with a Gatan 3View SBF system. Sub-surface SBF-SEM could potentially match focused ion beam (FIB) SEM in terms of z-resolution, but with the added advantage of providing higher throughput and larger tissue volumes. The research was supported by the intramural program of NIBIB.