Enhanced imaging in low dose electron microscopy using electron counting

We compare the direct electron imaging performance at 120 keV of a monolithic active pixel sensor (MAPS) operated in a conventional integrating mode with the performance obtained when operated in a single event counting mode. For the combination of sensor and incident electron energy used here, we propose a heuristic approach with which to process the single event images in which each event is renormalised to have an integrated weight of unity. Using this approach we find enhancements in the Nyquist frequency modulation transfer function (MTF) and detective quantum efficiency (DQE) over the corresponding integrating mode values by factors of 8 and 3, respectively.     

Detective quantum efficiency of electron area detectors in electron microscopy

Recent progress in detector design has created the need for a careful side-by-side comparison of the modulation transfer function (MTF) and resolution-dependent detective quantum efficiency (DQE) of existing electron detectors with those of detectors based on new technology. We present MTF and DQE measurements for four types of detector: Kodak SO-163 film, TVIPS 224 charge coupled device (CCD) detector, the Medipix2 hybrid pixel detector, and an experimental direct electron monolithic active pixel sensor (MAPS) detector. Film and CCD performance was measured at 120 and 300 keV, while results are presented for the Medipix2 at 120 keV and for the MAPS detector at 300 keV. In the case of film, the effects of electron backscattering from both the holder and the plastic support have been investigated. We also show that part of the response of the emulsion in film comes from light generated in the plastic support. Computer simulations of film and the MAPS detector have been carried out and show good agreement with experiment. The agreement enables us to conclude that the DQE of a backthinned direct electron MAPS detector is likely to be equal to, or better than, that of film at 300 keV.     

New system for secondary electron detection in variable-pressure scanning electron microscopy

A novel secondary electron detection system combining a two‐stage detector head and a differential pumping system is presented. The detector head consisted of a scintillation Everhart‐Thornley detector and a microsphere plate, separating it from the lower vacuum in the intermediate chamber (below 0.1 mbar). The system was arranged asymmetrically, which should contribute to a lower gas leakage through the plate and a longer life span of the plate. The system offered all the advantages of the scintillator detector in a wide range of gas pressures, from high vacuum to those of the order of 10 mbar, typical of high‐pressure scanning electron microscopy.     

透射电镜生物样品冷冻置换技术的方法

本文介绍了制备透射电镜生物样品冷冻置换技术的基本方法及其优点。     

Thermal diffuse scattering in transmission electron microscopy

In conventional transmission electron microscopy, thermal scattering significantly affects the image contrast. It has been suggested that not accounting for this correctly is the main cause of the Stobbs factor, the ubiquitous, large contrast mismatch found between theory and experiment. In the case where a hard aperture is applied, we show that previous conclusions drawn from work using bright field scanning transmission electron microscopy and invoking the principle of reciprocity are reliable in the presence of thermal scattering. In the aperture-free case it has been suggested that even the most sophisticated mathematical models for thermal diffuse scattering lack in their numerical implementation, specifically that there may be issues in sampling, including that of the contrast transfer function of the objective lens. We show that these concerns can be satisfactorily overcome with modest computing resources; thermal scattering can be modelled accurately enough for the purpose of making quantitative comparison between simulation and experiment. Spatial incoherence of the source is also investigated. Neglect or inadequate handling of thermal scattering in simulation can have an appreciable effect on the predicted contrast and can be a significant contribution to the Stobbs factor problem.     

Identification of bacteria by studying one section under light microscopy,scanning, and transmission electron microscopy

A method for bacterial identification has been developed by means of studying the same histological sections through several types of microscopy. With this method, one section was processed and analyzed respectively for light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Sections of gingival biopsies were Gram stained and bacteria tentatively identified by LM. Photographs of the sections were taken and presketched transparent acetate sheets (PTAS) were made from the photos. The same section was later prepared for SEM, areas previously thought to contain bacteria were localized by placing the PTAS onto the SEM monitoring screen. The SEM specimens were subsequently processed for TEM, bacteria were located, and micrographs obtained. The results showed that out of ten diseased gingival biopsies observed under the LM, bacteria were found to be present in all the specimens and were identified as both Gram positive and Gram negative. By transferring the section from LM to SEM, the bacteria could be relocated and their morphotype (cocci, rods, etc.) clearly identified in most of the cases. Since cocci may resemble other biological granular structures under SEM, they require further analysis under TEM for additional positive identification. This study demonstrated that the method described here is a useful tool for assessing the presence and identifying bacteria within the gingival tissues.     

Characterization of an oil-degrading Microcoleus consortium by means of confocal scanning microscopy, scanning electron microscopy and transmission electron microscopy

A consortium of microorganisms with the capacity to degrade crude oil has been characterized by means of confocal laser scanning microscopy (CLSM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The analysis using CLSM shows that Microcoleus chthonoplastes is the dominant organism in the consortium. This cyanobacterium forms long filaments that group together in bundles inside a mucopolysaccharide sheath. Scanning electron microscopy and transmission electron microscopy have allowed us to demonstrate that this cyanobacterium forms a consortium primarily with three morphotypes of the heterotrophic microorganisms found in the Microcoleus chthonoplastes sheath. The optimal growth of Microcoleus consortium was obtained in presence of light and crude oil, and under anaerobic conditions. When grown in agar plate, only one type of colony (green and filamentous) was observed.     

Regenerating titanium ventricular assist device surfaces after gold/palladium coating for scanning electron microscopy

Titanium is one of the most commonly used materials for implantable devices in humans. Scanning electron microscopy (SEM) serves as an important tool for imaging titanium surfaces and analyzing cells and other organic matter adhering to titanium implants. However, high‐vacuum SEM imaging of a nonconductive sample requires a conductive coating on the surface. A gold/palladium coating is commonly used and to date no method has been described to “clean” such gold/palladium covered surfaces for repeated experiments without etching the titanium itself. This constitutes a major problem with titanium‐based implantable devices which are very expensive and thus in short supply. Our objective was to devise a protocol to regenerate titaniumsurfaces after SEM analysis. In a series of experiments, titanium samples from implantable cardiac assist devices were coated with fibronectin, seeded with cells and then coated with gold/palladium for SEM analysis. X‐ray photoelectron spectroscopy spectra were obtained before and after five different cleaning protocols. Treatment with aqua regia (a 1:3 solution of concentrated nitric and hydrochloric acid), with or without ozonolysis, followed by sonication in soap solution and sonication in deionized water, allowed regenerating titanium surfaces to their original state. Atomic force microscopy confirmed that the established protocol did not alter the titanium microstructure. The protocol described herein is applicable to almost all titanium surfaces used in biomedical sciences and because of its short exposure time to aqua regia, will likely work for many titanium alloys as well. Microsc. Res. Tech., 2009. © 2009 Wiley‐Liss, Inc.     

Platinum-shadowed,whole-mounts for transmission electron microscopy of cells cultured on plastic

A simple and convenient method is described whereby cells cultured on polystyrene plastic substrata may be critical point-dried, rotary-shadowed with platinum/carbon and finally released by means of propylene oxide for examination whole in the transmission electron microscope. The technique is particularly useful for the localization of colloidal gold probes, both intra- and extra-cellularly. The problem of premature melting of polystyrene at about 307 K after prolonged exposure to carbon dioxide is solved by limiting the residence time in the critical point-drier to 1 h. Details for cleaning and mounting the released films of cells on specimen grids are given. The technique expands the application of the ‘whole-mount’ approach of Hopkins and co-workers (Hopkins et al., 1981; Hopkins, 1985) to cells cultured directly on polystyrene substrata, that is, in conventional plastic culture vessels.     

利用CMOS图像传感器测试成像镜头MTF的实用方法

介绍了一种用CMOS图像传感器测量镜头MTF的实用方法及其实用实例。该方法通过引入参考空间频率,利用在CMOS图像传感器像面上,对被测空间频率与参考空间频率的像素灰度值的读取,能够便捷并且比较准确地测定镜头的MTF值。由于参考空间频率的引入,大体消除了CMOS图像传感器本身MTF对测量结果的影响,从而使测量结果更接近理论运算结果。     

Segmentation of virus particle candidates in transmission electron microscopy images

In this paper, we present an automatic segmentation method that detects virus particles of various shapes in transmission electron microscopy images. The method is based on a statistical analysis of local neighbourhoods of all the pixels in the image followed by an object width discrimination and finally, for elongated objects, a border refinement step. It requires only one input parameter, the approximate width of the virus particles searched for. The proposed method is evaluated on a large number of viruses. It successfully segments viruses regardless of shape, from polyhedral to highly pleomorphic.     

Atom probe tomography and transmission electron microscopy of a Mg-doped AlGaN/GaN superlattice

The electronic characteristics of semiconductor-based devices are greatly affected by the local dopant atom distribution. In Mg-doped GaN, the clustering of dopants at structural defects has been widely reported, and can significantly affect p-type conductivity. We have studied a Mg-doped AlGaN/GaN superlattice using transmission electron microscopy (TEM) and atom probe tomography (APT). Pyramidal inversion domains were observed in the TEM and the compositional variations of the dopant atoms associated with those defects have been studied using APT. Rarely has APT been used to assess the compositional variations present due to structural defects in semiconductors. Here, TEM and APT are used in a complementary fashion, and the strengths and weaknesses of the two techniques are compared.     

Sectionable microcarrier beads: An improved method for the preparation of cell monolayers for electron microscopy

Cross-linked dextran beads provide an excellent surface for tissue-cultured cell monolayers, and can be processed for transmission (TEM) and scanning (SEM) electron microscopy, as well as light microscopy (LM). Cells are grown to confluency on the surface of the microcarriers, where at any point aliquots can be removed and experimentally treated as desired (e.g. immunocytochemistry) providing a representative sample. Sample preparation for TEM follows standard procedures for any cell monolayer, but infiltration times must be at least doubled to allow penetration of the beads. The polymerized blocks can then be sectioned for TEM or LM with no additional steps required. SEM sample preparation involves attaching the fixed bead/cell suspension to a glass coverslip with poly-1-lysine, dehydration, critical point drying, and coating for conductivity. The fixed and dried sample can also be attached directly to the SEM stub as free beads and subsequently gold coated. These beads provide (1) an increased surface area of cells visible per area of thin section, (2) eliminates the careful orientation required for flat substrate methods of embedding, (3) decreases the amount of sample manipulation in the forms of re-embedding and gluing, and (4) decreases the amount of drying artifact seen as cracking in SEM monolayer preparations.     

High‐quality imaging in environmental scanning electron microscopy – optimizing the pressure limiting system and the secondary electron detection of a commercially available ESEM

In environmental scanning electron microscopy applications in the kPa regime are of increasing interest for the investigation of wet and biological samples, because neither sample preparation nor extensive cooling are necessary. Unfortunately, the applications are limited by poor image quality. In this work the image quality at high pressures of a FEI Quanta 600 (field emission gun) and a FEI Quanta 200 (thermionic gun) is greatly improved by optimizing the pressure limiting system and the secondary electron (SE) detection system. The scattering of the primary electron beam strongly increases with pressure and thus the image quality vanishes. The key to high‐image quality at high pressures is to reduce scattering as far as possible while maintaining ideal operation conditions for the SE‐detector. The amount of scattering is reduced by reducing both the additional stagnation gas thickness (aSGT) and the environmental distance (ED). A new aperture holder is presented that significantly reduces the aSGT while maintaining the same field‐of‐view (FOV) as the original design. With this aperture holder it is also possible to make the aSGT even smaller at the expense of a smaller FOV. A new blade‐shaped SE‐detector is presented yielding better image quality than usual flat SE‐detectors. The electrode of the new SE detector is positioned on the sample table, which allows the SE‐detector to operate at ideal conditions regardless of pressure and ED.     

Scanning electron microscopy and transmission electron microscopy study of hot-deformed γ-TiAl-based alloy microstructure

The aim of this work was to assess the changes in the microstructure of hot‐deformed specimens made of alloys containing 46–50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 °C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.     

High temperature controlled atmosphere experiments for catalysts in a transmission electron microscope

A new transmission electron microscopy (TEM) specimen preparation procedure for high temperature experiments using a controlled atmosphere specimen holder (HTCASH) has been developed. It is designed for studying the microstructure of catalyst specimens before and after treatments in various gases. The procedure involved (1) finding a new formula for the embedding material, (2) devising a new method of making specimen supports, and (3) developing a method for removing the embedding material after the specimen has been microtomed. These techniques were then brought together to produce the ideal specimens for the HTCASH experiments. As an extra benefit, this procedure is also suitable for preparing specimens for ultrahigh resolution imaging experiments. The application of the new procedure in HTCASH experiments is illustrated through a high temperature reduction of a Co/SiO₂-923 catalyst.     

Prospects for lithium imaging using annular bright field scanning transmission electron microscopy: a theoretical study

There is strong interest in lithium imaging, particularly because of its significance in battery materials. However, light atoms only scatter electrons weakly and atomic resolution direct imaging of lithium has proven difficult. This paper explores theoretically the conditions under which lithium columns can be expected to be directly visible using annular bright field scanning transmission electron microscopy. A detailed discussion is given of the controllable parameters and the conditions most favourable for lithium imaging.     

Image simulations of kinked vortices for transmission electron microscopy

We present an improved model of kinked vortices in high-T_c superconductors suitable for the interpretation of Fresnel or holographic observations carried out with a transmission electron microscope. A kinked vortex is composed of two displaced half-vortices, perpendicular to the film plane, connected by a horizontal flux-line in the plane, resembling a connecting Josephson vortex (JV) segment. Such structures may arise when a magnetic field is applied almost in the plane, and the line tension of the fluxon breaks down under its influence. The existence of kinked vortices was hinted in earlier observations of high-T_c superconducting films, where the Fresnel contrast associated with some vortices showed a dumbbell like appearance. Here, we show that under suitable conditions the JV segment may reveal itself in Fresnel imaging or holographic phase mapping in a transmission electron microscope.