Backscattered electron imaging of the undersurface of resin-embedded cells by field-emission scanning electron microscopy

In this study backscattered electron (BSE) imaging was used to display cellular structures stained with heavy metals within an unstained resin by atomic number contrast in successively deeper layers. Balb/c 3T3 fibroblasts were cultured on either 13-mm discs of plastic Thermanox, commercially pure titanium or steel. The cells were fixed, stained and embedded in resin and the disc removed. The resin block containing the cells was sputter coated and examined in a field-emission scanning electron microscope. The technique allowed for the direct visualization of the cell undersurface and immediately overlying areas of cytoplasm through the surrounding embedding resin, with good resolution and contrast to a significant depth of about 2 μm, without the requirement for cutting sections. The fixation protocol was optimized in order to increase heavy metal staining for maximal backscattered electron production. The operation of the microscope was optimized to maximize the number of backscattered electrons produced and to minimize the spot size. BSE images were collected over a wide range of accelerating voltages (keV), from low values to high values to give ‘sections' of information from increasing depths within the sample. At 3–4 keV only structures a very short distance into the material were observed, essentially the areas of cell attachment to the removed substrate. At higher accelerating voltages information on cell morphology, including in particular stress fibres and cell nuclei, where heavy metals were intensely bound became more evident. The technique allowed stepwise ‘sectional’ information to be acquired. The technique should be useful for studies on cell morphology, cycle and adhesion with greater resolution than can be obtained with any light-microscope-based system.     

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.     

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.     

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.     

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.     

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.     

Choosing the optimum accelerating voltage (EO) to visualize submicron precipitates with a field emission scanning electron microscope

With the advent of field emission scanning electron microscopes (FESEM), the observation of small phases in the 5 to 50 nm range seems to be possible at low accelerating voltage using backscattered electron imaging mode. In this context, it is important to understand the contrast of multiphased materials at such low energy. A Monte Carlo program to simulate electron trajectories of multiphased materials (CASINO) was used to compute electron backscattering images. Simulations of images for various compositions of spherical precipitates embedded in a homogeneous matrix as a function of precipitate size and accelerating voltage are presented. These simulations show the concept of an optimum accelerating voltage to maximize the contrast of electron backscattering images. The results presented in this paper show that the contrast of backscattering images of multiphased images in the scanning electron microscope is not only a function of the atomic number difference, but that it is also strongly related to the geometry and the size of the phases.     

Measurement of the electric field distribution and potentials on the object surface in an emission electron microscope without restriction of the electron beams

An emission electron microscope without restriction of the electron beams was used to visualize and measure the distribution of electric fields and potentials on the surface under study. Investigations of this kind can be performed in an emission electron microscope without any aperture diaphragm. The potentialities of this method have been demonstrated using measurements with a silicon p–n junction to which a voltage has been applied in the reverse direction. The quantitative analysis becomes more complicated if the specimen is characterized by a heterogeneous intensity distribution of the electron emission from different areas of its surface. In the latter case two images obtained at different accelerating voltages (i.e. different voltages of the microscope extractor) provide the information necessary for an analysis of electric field and potential distributions.     

Imaging of immunolabeled membrane receptors in uncoated sem specimens

Epidermal growth factor receptors (EGFR) were labeled with 10 nm immunogold and examined on uncoated specimens of A431 human epidermoid carcinoma cells. A field emission gun and a high-sensitivity YAG ring detector were used to demonstrate the affinity labeling simultaneously in the secondary-electron (SE) and backscattered-electron (BSE) modes with a low accelerating voltage (V_o). At V_o=2kV, the SE and BSE signals were too weak to identify all markers, while at V_o=3–7 kV labeling was observed unambiguously in both the SE and BSE modes with smaller and higher working distances. Increasing the V_o to above 7 kV sometimes provokes instability of the specimens. A V_o of ? 10 kV produces charging artifacts in the SE image, but permits a BSE image of the gold markers providing additional topographic information. In conclusion, immunogold labeling can be used with good results for uncoated specimens.     

The imaging mechanism of single-walled carbon nanotubes on Si/SiO₂ wafer in scanning electron microscopy

Scanning electron microscopy imaging of both suspended single‐walled carbon nanotubes (SWNTs) and contacted SWNTs with Si/SiO₂ substrate has been studied in this paper. The voltage contrast has been investigated by supplying external electric field around the samples. The results show that the image contrast of SWNTs attributes to both voltage contrast from the area surrounding SWNTs (tens of nanometres in both sides of the SWNTs) and electron beam induced emission from SWNTs themselves under low primary beam energy. Under high primary beam energy, however, EBIE dominates the image contrast due to the fact that the voltage contrast caused by implanted charges of the SiO₂ layer is weakened. Imaging under the primary beam energy lower than 1 keV offers widened diameter of SWNTs, which promises that the SWNTs are observable at very low magnification (lower than 100×). At a larger magnification, however, imaging under the primary beam energy higher than 10 keV can display more realistic images of the SWNTs. In addition, an appropriate external electric field can improve the images.     

SEM electron channelling patterns as a technique for the characterization of ion-implantation damage

Electron channelling patterns (ECPs) formed in back-scattered images in the scanning electron microscope (SEM) have been used occasionally to confirm surface amorphization during ion implantation. In order to place such observations on a more quantitative basis, the study reported here has explored the variation of ECP appearance with both specimen damage levels (and thus subsurface structures) and SEM accelerating voltage (i.e. sampled depth). Polished and annealed (0001) single crystal sapphire discs were implanted to various damage levels up to both subsurface and full surface amorphization. Damage levels were measured independently by Rutherford back-scattering (RBS). Selected-area ECPs were obtained in a Jeol-840 electron microscope operating over the range 5–40 kV in 5-kV steps. Progressive ECP degradation—in terms of high-order line disappearance—was observed with increasing dose, culminating in total pattern loss when full surface amorphization occurred. However, ECP information could still be obtained from the damaged near-surface material even when a subsurface amorphous layer was present, thus demonstrating the shallow retrieval depth of information from the ECP technique. Indeed, because the spatial distribution of damage from ion implantation is both calculable and measurable, these experiments have also allowed us, for the first time, to explore and demonstrate the shallow sample depths from which the majority of ECP contrast originates (< 150 nm in sapphire at an accelerating voltage of 35 kV), even when the beam penetration is considerable by comparison (～ 5 μm). Furthermore, the way in which this sampled depth varies with SEM accelerating voltage is both demonstrated and shown to be a powerful diagnostic technique for studying the distribution of near-surface structural damage.     

Comparison in spatial spreads of secondary electron information between scanning ion and scanning electron microscopy

Monte Carlo simulations have been carried out to compare the spatial spreads of secondary electron (SE) information in scanning ion microscopy (SIM) with scanning electron microscopy (SEM). Under Ga ion impacts, the SEs are excited by three kinds of collision-partners, that is, projectile ion, recoiled target atom, and target electron. The latter two partners dominantly contribute to the total SE yield gamma for the materials of low atomic number Z2. For the materials of high Z2, on the other hand, the projectile ions dominantly contribute to gamma. These Z2 dependencies generally cause the gamma yield to decrease with an increasing Z2, in contrast with the SE yield delta under electron impacts. Most of the SEs are produced in the surface layer of about 5lambda in depth (lambda: the mean free path of SEs), as they are independent of the incident probe. Under 30 keV Ga ion impacts, the spatial spread of SE information is roughly as small as 10 nm, decreasing with an increasing Z2. Under 10 keV electron impacts, the SEI excited by the primary electrons has a small spatial spread of about 5lambda, but the SEII excited by the backscattered electrons has a large one of several 10 to several 100 nanometers, decreasing with an increasing Z2. The main cause of a small spread of SE information at ion impact is the short ranges of the projectile ions returning to the surface to escape as backscattered ions, the recoiled target atoms, and the target electrons in collision cascade. The 30 keV Ga-SIM imaging is better than the 10 keV SEM imaging in spatial resolution for the structure/material measurements. Here, zero-size probes are assumed.     

Relativistic calculations of intensity distributions in elemental maps using contrast transfer functions

The interpretation of highly resolved elemental maps is not straightforward: one has to consider the quantum mechanical nature of the scattering process as well as the influence of the microscope. Existing calculations of the contrast in elemental maps are based on a non-relativistic approach, while in most of the currently installed electron microscopes, the electrons penetrate the specimen with relativistic energies ≥ 200 keV. Therefore, we have recalculated the intensity distribution in elemental maps based on a fully relativistic theory. Using the concept of contrast transfer functions, the simulations account for lens aberrations as well as the defocus. Surprisingly, the results exhibit considerable deviations between the relativistic and non-relativistic calculations even in the region of low acceleration voltages such as 100 kV. These differences increase with increasing acceleration voltage and are strongly dependent on aperture and energy loss. Quantitative simulations and evaluations of highly resolved elemental maps should therefore make use of a fully relativistic theory.     

Electron-specimen interactions in low-voltage scanning electron microscopy

Measurements of the electron range R, and the backscattering coefficient η and the secondary electron yield δ at normal and tilted incidence for different elements show characteristic differences for electron energies in the range of 0.5 to 5 keV, compared with energies larger than 5 keV. The backscattering coefficient does not increase monotonically with increasing atomic number; for example, the secondary electron yield shows a lesser increase with increasing tilt angle. This can be confirmed in back-scattered electron (BSE) and secondary electron (SE) micrographs of test specimens. The results are in rather good agreement with Monte Carlo simulations using elastic Mott cross-sections and a continuous-slowing-down model with a Rao Sahib-Wittry approach for the stopping power at low electron energies. Therefore, this method can be used to calculate quantities of BSE and SE emission, which need a larger experimental effort. Calculations of the angular distribution of BSEs show an increasing intensity with increasing atomic number at high takeoff angles than expected from a cosine law that describes the angular characteristics at high electron energies. When simulating the energy distribution of BSEs, the continuous-slowing-down model should be substituted by using an electron energy-loss spectrum (EELS) that considers plasmon losses and inner-shell ionizations individually (single-scattering-function model). The EELS can be approached via the theory for aluminium or from EELS spectra recorded in a transmission electron microscope for other elements. Measurements of electron range Rα Eⁿ of 1 to 10 keV electrons are obtained from transmission experiments with thin films of known mass thickness. In agreement with other authors the exponent n is lower than at higher electron energies.     

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.     

Effects of hot electron emission on a low-conductivity tetracyanoethylene polymer layer including studies of the corrugation of the film surface

The effect of strong field electron emission (FEE) on a tetracyanoethylene (TCNE) polymer layer was studied by Field Ion Microscopy (FIM) using TCNE and Ne as the imaging gases. The TCNE polymer was formed on each tungsten tip by radical polymerisation before FEE. The FIM images show field emission spots all over the surface of the tip. The FEM images show a random distribution of several field emission areas at the onset of FEE. After sometime at a current of about microA, there is a transition to higher currents at the same voltage, in which the electron emission pattern changes to have only one emitting area. After this transition, two different types of FIM images were observed, depending on the imaging gas that was used. Neon FIM images at low tip voltages show spots in the areas where the electron emission current was greatest, and at much higher voltages these images show emission from other areas with lower surface corrugation. However, the FIM images with TCNE as the imaging gas do not show any differences between the areas with and without electron emission. The FIM images remain as before FEE, which can be explained by the formation of a new polymer by the reaction of the surface layer with the imaging gas. It is assumed that chemically reactive fragments at the polymer/vacuum interface, which are needed for the polymerisation reaction, are formed by pyrolysis and sputtering processes during FEE.     

Mineral content changes in bone associated with damage induced by the electron beam

Energy-dispersive x-ray (EDX) spectroscopy and backscattered electron (BSE) imaging are finding increased use for determining mineral content in microscopic regions of bone. Electron beam bombardment, however, can damage the tissue, leading to erroneous interpretations of mineral content. We performed elemental (EDX) and mineral content (BSE) analyses on bone tissue in order to quantify observable deleterious effects in the context of (1) prolonged scanning time, (2) scan versus point (spot) mode, (3) low versus high magnification, and (4) embedding in poly-methylmethacrylate (PMMA). Undemineralized cortical bone specimens from adult human femora were examined in three groups: 200x embedded, 200x unembedded, and 1000x embedded. Coupled BSE/EDX analyses were conducted five consecutive times, with no location analyzed more than five times. Variation in the relative proportions of calcium (Ca), phosphorous (P), and carbon (C) were measured using EDX spectroscopy, and mineral content variations were inferred from changes in mean gray levels ("atomic number contrast") in BSE images captured at 20 keV. In point mode at 200x, the embedded specimens exhibited a significant increase in Ca by the second measurement (7.2%, p < 0.05); in scan mode, a small and statistically nonsignificant increase (1.0%) was seen by the second measurement. Changes in P were similar, although the increases were less. The apparent increases in Ca and P likely result from decreases in C: -3.2% (p < 0.05) in point mode and -0.3% in scan mode by the second measurement. Analysis of unembedded specimens showed similar results. In contrast to embedded specimens at 200x, 1000x data showed significantly larger variations in the proportions of Ca, P, and C by the second or third measurement in scan and point mode. At both magnifications, BSE image gray level values increased (suggesting increased mineral content) by the second measurement, with increases up to 23% in point mode. These results show that mineral content measurements can be reliable when using coupled BSE/EDX analyses in PMMA-embedded bone if lower magnifications are used in scan mode and if prolonged exposure to the electron beam is avoided. When point mode is used to analyze minute regions, adjustments in accelerating voltages and probe current may be required to minimize damage.     

Application of ion-beam etching techniques to the fine structure of biological specimens as examined with a field emission SEM at low voltage

The term “etching,” in electron microscopy, refers to the removal of specimen surface layers and includes chemical, electrolytic, and ion-beam methods. The ion-beam etching process is used to remove layers of a target material by bombarding it with ionized gas molecules. Recently, the method has been applied to the field of biological specimens; however, the practical procedures for such organic materials have not been developed. In the present study, we used an apparatus in which a beam of argon ions is collimated and focused by electrostatic lenses onto an appropriate target. We demonstrated the optimum conditions to observe biological specimens that were treated with osmium tetroxide and tannic acid. The specimens were examined uncoated at low accelerating voltage using a field emission scanning electron microscope. According to our experiments, when a biological specimen was observed under high-resolution conditions at over 50,000x magnification, the optimum condition of ion-beam etching consisted of an accelerating voltage of E = 1 keV and an ion-beam dose of It = 360 ～ 400 μA. min, depending on parts of the specimens. In order to decrease overetching, we had to choose factors such as E = 1 ～ 2 keV and It = 500 μA. min.     

Charge contrast imaging of material growth and defects in environmental scanning electron miscroscopy--linking electron emission and cathodoluminescence

An electron-based technique for the imaging of crystal defect distribution such as material growth histories in non- and poorly conductive materials has been identified in the variable pressure or environmental scanning electron microscope. Variations in lattice coherence at the meso-scale can be imaged in suitable materials. Termed charge contrast imaging (CCI), the technique provides images that correlate exactly with emitted light or cathodoluminescence in suitable materials. This correlation links cathodoluminescence and an electron emission. The specific operating conditions for observation of these images reflect a complex interaction between the electron beam, the positive ions generated by electron-gas interactions in the chamber, a biased detector, and the sample. The net result appears to be the suppression of all but very near surface electron emission from the sample, probably from of the order of a few nanometres. Consequently, CCI are also sensitive to very low levels of surface contaminants. Successful imaging of internal structures in a diverse range of materials indicate that the technique will become an important research tool.     

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.