Advantages of indium–tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.     

Sample preparation and microscopical investigation techniques for metal and ceramics containing graphite and graphene‐like layered particles

Scanning electron microscopy (SEM) techniques are widely used in microstructural investigations of materials since it can provide surface morphology, topography, and chemical information. However, it is important to use correct imaging and sample preparation techniques to reveal the microstructures of materials composed of components with different polishing characteristics such as grey cast iron, graphene platelets (GPLs)‐added SiAlON composite, SiC and B₄C ceramics containing graphite or graphene‐like layered particles. In this study, all microstructural details of gray cast iron were successfully revealed by using argon ion beam milling as an alternative to the standard sample preparation method for cast irons, that is, mechanical polishing followed by chemical etching. The in‐lens secondary electron (I‐L‐SE) image was clearly displayed on the surface details of the graphites that could not be revealed by backscattered electron (BSE) and Everhart–Thornley secondary electron (E‐T SE) images. Mechanical polishing leads to pull‐out of GPLs from SiAlON surface, whereas argon ion beam milling preserved the GPLs and resulted in smooth surface. Grain and grain boundaries of polycrystalline SiC and B₄C were easily revealed by using I‐L SE image in the SEM after only mechanical polishing without any etching process. While the BSE and E‐T SE images did not clearly show the residual graphites in the microstructure, their distribution in the B₄C matrix was fully revealed in the I‐L SE image.     

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.     

A double detector system for BSE and SE imaging

The double detector system described here is a simple device suitable for any SEM. It permits efficient imaging of specimen surfaces in either the secondary electron (SE) or backscattered electron (BSE) mode. The BSE detector is an annular single-crystal scintillator made of yttrium aluminium garnet (YAG) and the SE detector has a scintillator of the same material. Both detectors have their own light guides which are connected to a single photomultiplier. The choice of signal is made with a mechanical diaphragm mounted on a flange between the light guide and the photomultiplier. The SE detector may be replaced by a second BSE detector to allow the detection of “low” take-off angle BSEs to provide information which differs from that given by the annular BSE detector which operates to detect BSEs with a “high” take-off angle. In this way it is possible to image either material or topographic contrast with high resolution and to take advantage of the choice of detected electrons.     

Use of backscattered electron detector arrays for forming backscattered electron images in the scanning electron microscope

The backscattered electron (BSE) signal in the scanning electron microscope (SEM) can be used in two different ways. The first is to give a BSE image from an area that is defined by the scanning of the electron beam (EB) over the surface of the specimen. The second is to use an array of small BSE detectors to give an electron backscattering pattern (EBSP) with crystallographic information from a single point. It is also possible to utilize the EBSP detector and computer-control system to give an image from an area on the specimen--for example, to show the orientations of the grains in a polycrystalline sample ("grain orientation imaging"). Some further possibilities based on some other ways for analyzing the output from an EBSP detector array, are described.     

Fundamental problems of imaging subsurface structures in the backscattered electron mode in scanning electron microscopy

In‐depth imaging of subsurface structures in scanning electron microscopy (SEM) is usually obtained by detecting backscattered electrons (BSE). For a layer‐by‐layer imaging in BSE microtomography, it is preferable to use an energy filtering of BSE. A simple approach is used to estimate the contrast by using backscattering coefficients of bulk materials and the maximum escape depths of the BSE. The contrast obtained by BSE energy filtering is about twice that of the standard BSE method by varying the acceleration voltage. The contrast decreases with increasing information depth. The information depth is about four times smaller than the electron range. The transmission of the spectrometer influences the minimum current of the order of 10^?8 A that is needed to get a contrast of 1%, for example.     

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.     

用同位素质谱技术发现山西古风化壳型稀土金属矿床

对山西石炭纪铝土矿中钐 (Sm )和钕 (Nd)元素含量的化学制备和同位素质谱稀释分析方法进行了详细探讨 ,采用固体同位素质谱技术发现了山西古风化壳型稀土金属矿床。可能总稀土的强富集是在本区形成风化壳吸附型轻稀土金属矿床的主要成因。研究结果表明 :在山西沁源大峪和平陆曹川的稀土含量达到风化壳吸附型轻稀土金属和稀土铝土矿矿床工业指标。山西古风化壳型稀土金属矿床是在华北地区首次发现的稀土金属矿床。     

Topographic and material contrast in low-voltage scanning electron microscopy

Two scintillation backscattered electron (BSE) detectors with a high voltage applied to scintillators were built and tested in a field emission scanning electron microscope (SEM) at low primary beam energies. One detector collects BSE emitted at low take-off angles, the second at high takeoff angles. The low take-off detector gives good topographic tilt contrast, stronger than in the case of the secondary electron (SE) detection and less sensitive to the presence of contamination layers on the surface. The high take-off detector is less sensitive to the topography and can be used for detection of material contrast, but the contrast becomes equivocal at the beam energy of 1 keV or lower.     

Influence of topography and specimen preparation on backscattered electron images of bone

Backscattered electron (BSE) images of bone exhibit graylevel contrast between adjacent lamellae. Mathematical models suggest that interlamellar contrast in BSE images is an artifact due to topographic irregularities. However, little experimental evidence has been published to support these models, and it is not clear whether submicron topographical features will alter BSE graylevels. The goal of this study was to determine the effects of topography on BSE image mean graylevels and graylevel histogram widths using conventional specimen preparation techniques. White-light interferometry and quantitative BSE imaging were used to investigate the relationship between the BSE signal and specimen roughness. Backscattered electron image graylevel histogram widths correlated highly with surface roughness in rough preparations of homogeneous materials. The relationship between BSE histogram width and surface roughness was specimen dependent. Specimen topography coincided with the lamellar patterns within the bone tissue. Diamond micromilling reduced average surface roughness when compared with manual polishing techniques but did not significantly affect BSE graylevel histogram width. The study suggests that topography is a confounding factor in quantitative BSE analysis of bone. However, there is little quantitative difference between low-to-moderate magnification BSE images of bone specimens prepared by conventional polishing or diamond micromilling.     

Automated in‐chamber specimen coating for serial block‐face electron microscopy

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally (‘sample charging’). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block‐face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1–2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal‐to‐noise ratio (SNR) caused by the film is smaller than that caused by the widely used low‐vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non‐conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.     

Errors in quantitative backscattered electron analysis of bone standardized by energy-dispersive x-ray spectrometry

Backscattered electron (BSE) imaging has proven to be a useful method for analyzing the mineral distribution in microscopic regions of bone. However, an accepted method of standardization has not been developed, limiting the utility of BSE imaging for truly quantitative analysis. Previous work has suggested that BSE images can be standardized by energy-dispersive x-ray spectrometry (EDX). Unfortunately, EDX-standardized BSE images tend to underestimate the mineral content of bone when compared with traditional ash measurements. The goal of this study is to investigate the nature of the deficit between EDX-standardized BSE images and ash measurements. A series of analytical standards, ashed bone specimens, and unembedded bone specimens were investigated to determine the source of the deficit previously reported. The primary source of error was found to be inaccurate ZAF corrections to account for the organic phase of the bone matrix. Conductive coatings, methyl-methacrylate embedding media, and minor elemental constituents in bone mineral introduced negligible errors. It is suggested that the errors would remain constant and an empirical correction could be used to account for the deficit. However, extensive preliminary testing of the analysis equipment is essential.     

Correlative scanning electron and confocal microscopy imaging of labeled cells coated by indium‐tin‐oxide

Confocal microscopy imaging of cells allows to visualize the presence of specific antigens by using fluorescent tags or fluorescent proteins, with resolution of few hundreds of nanometers, providing their localization in a large field‐of‐view and the understanding of their cellular function. Conversely, in scanning electron microscopy (SEM), the surface morphology of cells is imaged down to nanometer scale using secondary electrons. Combining both imaging techniques have brought to the correlative light and electron microscopy, contributing to investigate the existing relationships between biological surface structures and functions. Furthermore, in SEM, backscattered electrons (BSE) can image local compositional differences, like those due to nanosized gold particles labeling cellular surface antigens. To perform SEM imaging of cells, they could be grown on conducting substrates, but obtaining images of limited quality. Alternatively, they could be rendered electrically conductive, coating them with a thin metal layer. However, when BSE are collected to detect gold‐labeled surface antigens, heavy metals cannot be used as coating material, as they would mask the BSE signal produced by the markers. Cell surface could be then coated with a thin layer of chromium, but this results in a loss of conductivity due to the fast chromium oxidation, if the samples come in contact with air. In order to overcome these major limitations, a thin layer of indium‐tin‐oxide was deposited by ion‐sputtering on gold‐decorated HeLa cells and neurons. Indium‐tin‐oxide was able to provide stable electrical conductivity and preservation of the BSE signal coming from the gold‐conjugated markers. Microsc. Res. Tech. 78:433–443, 2015. © 2015 Wiley Periodicals, Inc.     

High-resolution backscatter electron imaging of colloidal gold in LVSEM

High‐resolution backscatter electron (BSE) imaging of colloidal gold can be accomplished at low voltage using in‐lens or below‐the‐lens FESEMs equipped with either Autrata‐modified yttrium aluminium garnet (YAG) scintillators doped with cerium, or with BSE to secondary electron (SE) conversion plates. The threshold for BSE detection of colloidal gold was 1.8 keV for the YAG detector, and the BSE/SE conversion was sensitive down to 1 keV. Gold particles (6, 12 and 18 nm) have an atomic number of 79 and were clearly distinguished at 500 000× by materials contrast and easily discriminated from cell surfaces coated with platinum with an atomic number of 78. BSE imaging was relatively insensitive to charging, and build up of carbon contamination on the specimen was transparent to the higher energy BSE.     

About the role of the various types of secondary electrons (SE1; SE2; SE3) on the performance of LVSEM

The relative weight, δ_Β, of the yield of secondary electrons, SE₂, induced by the backscattered electrons, BSE, with respect to that, δ_P, of secondary electrons, SE₁, induced by the primary electrons, PE, is deduced from simple theoretical considerations. At primary energies E₀ larger than E_M (where the total SE yield δ = δ_P + δ_B is maximum), the dominant role of the backscattering events is established. It is illustrated in SEM by a direct comparison of the contrast between SE images and BSE images obtained at E₀ ～ 5 keV and E₀ ～ 15 keV on a stratified specimen. At energies E₀ less than E_M, the dominant role of SE₁ electrons with respect to SE₂ (and SE₃) is established. It is illustrated by the better practical resolution of diamond images obtained with an in‐lens detection in low voltage SEM E₀ ～ 0.2–1 keV range compared with that obtained with a lateral detector. The present contribution illustrates the improved performance of LVSEM in terms of contrast and of practical resolution as well as the importance of variable voltage methods for subsurface imaging. The common opinion that the practical lateral resolution is given by the incident spot diameter is also reconsidered in LVSEM.     

Backscattered electron imaging and electron backscattered diffraction in the study of bacterial attachment to titanium alloy structure

The application of secondary electron (SE) imaging, backscattered electron imaging (BSE) and electron backscattered diffraction (EBSD) was investigated in this work to study the bacterial adhesion and proliferation on a commercially pure titanium (cp Ti) and a Ti6Al4V alloy (Ti 64) with respect to substrate microstructure and chemical composition. Adherence of Gram‐positive Staphylococcus epidermidis 11047 and Streptococcus sanguinis GW2, and Gram‐negative Serratia sp. NCIMB 40259 and Escherichia coli 10418 was compared on cp Ti, Ti 64, pure aluminium (Al) and vanadium (V). The substrate microstructure and the bacterial distribution on these metals were characterised using SE, BSE and EBSD imaging. It was observed that titanium alloy‐phase structure, grain boundaries and grain orientation did not influence bacterial adherence or proliferation at microscale. Adherence of all four strains was similar on cp Ti and Ti 64 surfaces whilst inhibited on pure Al. This work establishes a nondestructive and straight‐forward statistical method to analyse the relationship between microbial distribution and metal alloy structure.     

Image restoration for TV‐scan moving images acquired through a semiconductor backscattered electron detector

A semiconductor backscattered electron (BSE) detector has become popular in scanning electron microscopy session. However, detectors of semiconductor type have a serious disadvantage on the frequency characteristics. As a result, fast scan (e.g. TV‐scan) BSE image should be blurred remarkably. It is the purpose of this study to restore this degradation by using digital image processing technology. In order to improve it practically, we have to settle several problems, such as noise, undesirable processing artifacts, and ease of use. Image processing techniques in an impromptu manner like a conventional mask processing are unhelpful for this study, because a complicated degradation of output signal affects severely the phase response as well as the amplitude response of our SEM system. Hence, based on the characteristics of an SEM signal obtained from the semiconductor BSE detector, a proper inverse filter in Fourier domain is designed successfully. Finally, the inverse filter is converted to a special convolution mask, which is skillfully designed, and applied for TV‐scan moving BSE images. The improved BSE image is very effective in the work for finding important objects. SCANNING 31: 229–235, 2009. © 2010 Wiley Periodicals, Inc.     

The morphology and composition of cholesterol,protein, and bilirubin deposits in dried human bile: Cathodoluminescence and backscattered electron imaging

This work is the first to deal with the application of color cathodoluminescence scanning electron microscopy (CCL SEM) and a novel version of combined imaging with backscattered electrons (CCL+BSE SEM) for the study of the composition of bile and its precipitation mechanisms. The present study demonstrates cholesterol, protein, and bilirubin distribution in deposits of normal and abnormal humanbile after solution evaporation to full dryness. Qualitative CCL SEM analysis showed that dried bile remnants include different proportions of the above components. Three types of deposits were observed: Arborescent crystals, typical cholesterol crystals, and amorphous bilirubin particles. The selection of crystalline or amorphous precipitate phases is determined by the dehydration/concentration process. The findings may explain key features in lithogenesis.     

A simple backscattered electrons and specimen current detection device for the scanning electron microscope

A simple, low-investment device has been developed that allows the collection of backscattered electrons (BSEs) and specimen current (SC) signals for imaging purposes and current measurement. Originally, this system was designed for detection, measurement, and display of specimen current, with a video signal output whose level was modulated by this current. Eventually, a BSE detector was developed, using a graphite disk (about 8 cm in diameter) to collect the BSEs. The disk was mounted on a Philips SEM 5O5, attached and concentrically to the final lens aperture. This configuration gives a large solid angle of collection. The collected charge is further processed by the same electronics used in the aforementioned SC detection system. Electron channeling, topographic contrast with BSE, and material contrast with BSE and SC images can be obtained with reasonably good edge definition.