Effect of a physical phase plate on contrast transfer in an aberration-corrected transmission electron microscope

In this theoretical study we analyze contrast transfer of weak-phase objects in a transmission electron microscope, which is equipped with an aberration corrector (C(s)-corrector) in the imaging lens system and a physical phase plate in the back focal plane of the objective lens. For a phase shift of pi/2 between scattered and unscattered electrons induced by a physical phase plate, the sine-type phase contrast transfer function is converted into a cosine-type function. Optimal imaging conditions could theoretically be achieved if the phase shifts caused by the objective lens defocus and lens aberrations would be equal to zero. In reality this situation is difficult to realize because of residual aberrations and varying, non-zero local defocus values, which in general result from an uneven sample surface topography. We explore the conditions--i.e. range of C(s)-values and defocus--for most favourable contrast transfer as a function of the information limit, which is only limited by the effect of partial coherence of the electron wave in C(s)-corrected transmission electron microscopes. Under high-resolution operation conditions we find that a physical phase plate improves strongly low- and medium-resolution object contrast, while improving tolerance to defocus and C(s)-variations, compared to a microscope without a phase plate.     

Imaging of radiation-sensitive samples in transmission electron microscopes equipped with Zernike phase plates

We have optimized a bright-field transmission electron microscope for imaging of high-resolution radiation-sensitive materials by calculating the imaging dose n(0) needed to obtain a signal-to-noise ratio (SNR)=5. Installing a Zernike phase plate (ZP) decreases the dose needed to detect single atoms by as much as a factor of two at 300 kV. For imaging larger objects, such as Gaussian objects with full-width at half-maximum larger than 0.15 nm, ZP appears more efficient in reducing the imaging dose than correcting for spherical aberration. The imaging dose n(0) does not decrease with extending of chromatic resolution limit by reducing chromatic aberration, using high accelerating potential (U(0)=300 kV), because the image contrast increases slower than the reciprocal of detection radius. However, reducing chromatic aberration would allow accelerating potential to be reduced leading to imaging doses below 10 e(-)/A(2) for a single iodine atom when a CS-corrector and a ZP are used together. Our simulations indicate that, in addition to microscope hardware, optimization is heavily dependent on the nature of the specimen under investigation.     

Experimental observation of the improvement in MTF from backthinning a CMOS direct electron detector

The advantages of backthinning monolithic active pixel sensors (MAPS) based on complementary metal oxide semiconductor (CMOS) direct electron detectors for electron microscopy have been discussed previously; they include better spatial resolution (modulation transfer function or MTF) and efficiency at all spatial frequencies (detective quantum efficiency or DQE). It was suggested that a ‘thin’ CMOS detector would have the most outstanding properties [1], [2] and [3] because of a reduction in the proportion of backscattered electrons. In this paper we show, theoretically (using Monte Carlo simulations of electron trajectories) and experimentally that this is indeed the case.     

A model of secondary electron imaging in the helium ion scanning microscope

A combination of the ‘semi-empirical’ model for secondary electron production and the TRIM routines which describe ion stopping power, scattering, and transport, has been used to construct a Monte Carlo simulation (IONiSE) that can quantitatively interpret the generation of secondary electrons (SE) from materials by fast helium ions. This approach requires that the parameters of the semi-empirical model be determined by fitting to experimental yield data but has the merit that, unlike more fundamental models, it can be applied with equal ease to both pure elements and complex compounds. The application of the model to predict the topographic yield variation of helium generated SE as a function of energy and material, and to investigate the ratio between SE generated by incident and backscattered ions, is demonstrated.     

Imaging thin and thick sections of biological tissue with the secondary electron detector in a field-emission scanning electron microscope

A field-emission scanning electron microscope (FESEM) equipped with the standard secondary electron (SE) detector was used to image thin (70–90 nm) and thick (1–3 μm) sections of biological materials that were chemically fixed, dehydrated, and embedded in resin. The preparation procedures, as well as subsequent staining of the sections, were identical to those commonly used to prepare thin sections of biological material for observation with the transmission electron microscope (TEM). The results suggested that the heavy metals, namely, osmium, uranium, and lead, that were used for postfixation and staining of the tissue provided an adequate SE signal that enabled imaging of the cells and organelles present in the sections. The FESEM was also used to image sections of tissues that were selectively stained using cytochemical and immunocytochemical techniques. Furthermore, thick sections could also be imaged in the SE mode. Stereo pairs of thick sections were easily recorded and provided images that approached those normally associated with high-voltage TEM.     

Depth sectioning in scanning transmission electron microscopy based on core-loss spectroscopy

Recent and ongoing improvements in aberration correction have opened up the possibility of depth sectioning samples using the scanning transmission electron microscope in a fashion similar to the confocal scanning optical microscope. We explore questions of principle relating to image interpretability in the depth sectioning of samples using electron energy loss spectroscopy. We show that provided electron microscope probes are sufficiently fine and detector collection semi-angles are sufficiently large we can expect to locate dopant atoms inside a crystal. Furthermore, unlike high angle annular dark field imaging, electron energy loss spectroscopy can resolve dopants of smaller atomic mass than the supporting crystalline matrix.     

Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM)

We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.     

Atomic imaging using secondary electrons in a scanning transmission electron microscope: experimental observations and possible mechanisms

We report detailed investigation of high-resolution imaging using secondary electrons (SE) with a sub-nanometer probe in an aberration-corrected transmission electron microscope, Hitachi HD2700C. This instrument also allows us to acquire the corresponding annular dark-field (ADF) images both simultaneously and separately. We demonstrate that atomic SE imaging is achievable for a wide range of elements, from uranium to carbon. Using the ADF images as a reference, we studied the SE image intensity and contrast as functions of applied bias, atomic number, crystal tilt, and thickness to shed light on the origin of the unexpected ultrahigh resolution in SE imaging. We have also demonstrated that the SE signal is sensitive to the terminating species at a crystal surface. A possible mechanism for atomic-scale SE imaging is proposed. The ability to image both the surface and bulk of a sample at atomic-scale is unprecedented, and can have important applications in the field of electron microscopy and materials characterization.     

Scanning reflection electron microscopy of surface topography by diffusely scattered electrons in the scanning electron microscope

In this paper, the technique of scanning reflection electron microscopy (SREM) by diffusely scattered electrons in the scanning electron microscope is described in detail. A qualitative account of the formation of image contrast in SREM is also described. It is assumed that, for grazing geometry, forward-scattered electrons reflect from regions close to the surface, following a few scattering events within the first few atomic layers, and lose very little energy in the process. The penetration depth of the primary electrons is very limited, resulting in strongly peaked envelopes of forward-scattered electrons. It is also assumed that a surface containing topographic features presents a range of tilt angles, resulting in different reflection coefficients. Tilt contrast results because each facet has a different scattering yield, which is dependent upon local surface inclination. Full details of the instrumentation designed for SREM are described, and to illustrate the technique, results recorded from an epitaxial GaAs on GaAs crystal, Pb₂(Zr,Ta)O₆ thin film on silicon, and SiO₂ amorphous film on silicon are presented.     

Compensation of the shadowing error in three-dimensional imaging with a multiple detector scanning electron microscope

A method for three‐dimensional quantitative surface characterization for scanning electron microscopy is presented. The method used a quadruple scintillator detector developed by us. A surface reconstruction algorithm was performed by special software, with new algorithms for error compensation. Among these errors, detector shadowing was of particular importance. This was due to the disturbance in integration continuity when one or more detectors was screened from the flow of electrons. Several methods for the reduction of this error have been proposed and tested by us. The methods were based on software processing of complementary information, such as unshadowed detector signals, shadow depth and modified integration schemes.     

Effect of shot noise and secondary emission noise in scanning electron microscope images

The effect of shot noise and emission noise due to materials that have different emission properties was simulated. Local variations in emission properties affect the overall signal‐to‐noise ratio (SNR) value of the scanning electron microscope image. In the case in which emission noise is assumed to be absent, the image SNRs for silicon and gold on a black background are identical. This is because only shot noise in the primary beam affects the SNRs, irrespective of the assumed noiseless secondary electron emission or backscattered electron emission processes. The addition of secondary emission noise degrades the SNR. Materials with higher secondary electron yield and backscattering electron yield give rise to higher SNR. For images formed from two types of material, the contrast of the image is lower. The reduction in image signal reduces the overall image SNR. As expected, large differences in δ or η give rise to higher SNR images.     

Advantages of stereo imaging of metallic surfaces with low voltage backscattered electrons in a field emission scanning electron microscope

High emission current backscattered electron (HC-BSE) stereo imaging at low accelerating voltages (≤ 5 keV) using a field emission scanning electron microscope was used to display surface structure detail. Samples of titanium with high degrees of surface roughness, for potential medical implant applications, were imaged using the HC-BSE technique at two stage tilts of + 3° and − 3° out of the initial position. A digital stereo image was produced and qualitative height, depth and orientation information on the surface structures was observed. HC-BSE and secondary electron (SE) images were collected over a range of accelerating voltages. The low voltage SE and HC-BSE stereo images exhibited enhanced surface detail and contrast in comparison to high voltage (> 10 keV) BSE or SE stereo images. The low voltage HC-BSE stereo images displayed similar surface detail to the low voltage SE images, although they showed more contrast and directional sensitivity on surface structures. At or below 5 keV, only structures a very short distance into the metallic surface were observed. At higher accelerating voltages a greater appearance of depth could be seen but there was less information on the fine surface detail and its angular orientation. The combined technique of HC-BSE imaging and stereo imaging should be useful for detailed studies on material surfaces and for biological samples with greater contrast and directional sensitivity than can be obtained with current SE or BSE detection modes.     

Measurements of stretch lengths of gold mono-atomic wires covered with 1,6-hexanedithiol in 0.1 M NaClO4 with an electrochemical scanning tunneling microscope

Stretch lengths of pure gold mono-atomic wires have been studied recently with an electrochemical scanning tunneling microscope (STM). Here, we will report a study of stretch lengths of gold mono-atomic wires with and without 1,6-hexanedithiol (HDT) using the STM break-junction method. First, the stretch length was measured as a function of electrode potentials of a bare Au(111) substrate and a gold STM tip in a 0.1M NaClO(4) aqueous solution. Second, a self-assembled monolayer (SAM) was fabricated on an Au(111) substrate by dipping the substrate into a 1mM HDT ethanol solution. At last, we measured the stretch length of gold mono-atomic wires on a substrate covered with the SAM in place of the bare Au(111) substrate. We compared the electrode potential dependence of the stretch lengths of gold mono-atomic wires covered with and without HDT. We will discuss the effect of the electrode potential on the stretch lengths by taking account of electrocapillarity of gold mono-atomic wires.     

Monte Carlo simulation of secondary electron and backscattered electron images in scanning electron microscopy for specimen with complex geometric structure

A new Monte Carlo technique for the simulation of secondary electron (SE) and backscattered electron (BSE) of scanning electron microscopy (SEM) images for an inhomogeneous specimen with a complex geometric structure has been developed. The simulation is based on structure construction modeling with simple geometric structures, as well as on the ray-tracing technique for correction of electron flight-step-length sampling when an electron trajectory crosses the interface of the inhomogeneous structures. This correction is important for the simulation of nanoscale structures of a size comparable with or even less than the electron scattering mean free paths. The physical model for electron transport in solids combines the use of the Mott cross section for electron elastic scattering and a dielectric function approach for electron inelastic scattering, and the cascade SE production is also included.     

气相色谱法测定土壤中阿特拉津、百菌清、溴氰菊酯和环氧七氯的微量残留

目的:研究采用加速溶剂萃取法提取土壤中的阿特拉津、百菌清、溴氰菊酯和环氧七氯微量残留,气相色谱-电子捕获检测器法定量检测的分析方法。方法:采集某地土壤样品,风干后,打成细粉,取粉碎后的土壤样品约12 g,置萃取池中,用环己烷进行萃取,萃取液氮吹至近干,加1mL乙腈溶解,涡旋1 min后滤过,上气相色谱仪定量检测。结果:阿特拉津、百菌清、溴氰菊酯和环氧七氯浓度范围0.01~8.0μg/mL内线性关系良好,相关系数均不低于0.995;检出限在0.006~0.010 mg/kg范围内;加标回收率在84.1%~92.3%之间;重复性RSD分别为2.85%、3.19%、2.77%和3.47%。结论:结果表明,方法具有操作简单、检测准确等特点,可以用于土壤中微量农药残留的检测。     

Comparative study by computed radiography,histology, and scanning electron microscopy of the articular cartilage of normal goats and in chronic infection with caprine arthritis‐encephalitis virus

In the northeast of Brazil, caprine arthritis‐encephalitis (CAE) is one of the key reasons for herd productivity decreasing that result in considerable economic losses. A comparative study was carried out using computed radiography (CR), histological analysis (HA), and scanning electronic microscopy (SEM) of the joints of CAE infected and normal goats. Humerus head surface of positive animals presented reduced joint space, increased bone density, and signs of degenerative joint disease (DJD). The carpal joint presented no morphological alterations in CR in any of the animals studied. Tarsus joint was the most affected, characterized by severe DJD, absence of joint space, increased periarticular soft tissue density, edema, and bone sclerosis. Histological analysis showed chronic tissue lesions, complete loss of the surface zone, absence of proteoglycans in the transition and radial zones and destruction of the cartilage surface in the CAE positive animals. Analysis by SEM showed ulcerated lesions with irregular and folded patterns on the joint surface that distinguished the limits between areas of normal and affected cartilage. The morphological study of the joints of normal and CAE positive goats deepened understanding of the alteration in the tissue bioarchitecture of the most affected joints. The SEM finding sustained previous histological reports, similar to those found for rheumatoid arthritis, suggesting that the goat infected with CAE can be considered as a potential model for research in this area. Microsc. Res. Tech. 77:11–16, 2014. © 2013 Wiley Periodicals, Inc.     

Intermediate aggregates resulting in the interaction of bile salt with liposomes studied by transmission electron microscopy and light scattering techniques

The interaction of sodium cholate (SC) with phosphatidylcholine liposomes was studied by means of transmission electron microscopy (TEM), changes in the mean particle size (quasielastic light scattering, QELS) and in the static light scattering (SLS) of the system during liposome solubilization. A good correlation was found between the TEM diameter of particles and the mean hydrodynamic diameter (HD) determined by QELS. The intermediate aggregates resulting in this interaction were dependent on the SC concentration in the system. Thus, an initial vesicle growth occurred when the SC concentration in the system was 13.79 mol%. Additional SC amounts (41.17 mol% SC) led to the formation of the largest vesicles (HD 410 nm). Increasing SC amounts led to a slight fall in the vesicle diameter and in the SLS of the system. Thus, for 47.08 mol% SC, TEM images still showed the presence of vesicles albeit with traces of smaller structures and signs of vesicle fusion. When SC concentration exceeded 48 mol% an abrupt decrease in SLS occurred, the size curve starting to show a bimodal distribution. Thus, for 50 mol% SC a sharp distribution curve appeared at 52 nm indicating the formation of small particles and TEM images showed clear signs of vesicle disintegration with formation of tubular structures. The subsequent self organization of these tubular structures (54 mol% SC) led to the formation of open multilayered structures in coexistence with small particles. A gradual increase in the number of these small particles (mixed micelles) led to the complete solubilization of liposomes.