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.     

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.     

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.     

The correlation of light and electron microscope observations

A technique is described to allow electron microscopic investigation of a specific feature of a section on a glass slide. A section on a glass slide (previously treated with a silicone release agent) is processed as required for light microscopy. The section is then impregnated with Araldite and cured with an epoxy resin block on top of it. The section and block are removed from the slide and viewed with a light microscope. The selected area for ultrastructural study remains under continuous observation while the block is trimmed. Semi-thin (1 μm) sections retain the original staining for light microscopy and ultra-thin sections are stained with heavy metals in the normal manner. We show how an inflammatory lesion in a large area of muscle in a case of polymyositis may be quickly located and studied at the ultrastructural level.     

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.     

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.     

A new method of compositional image formation by a four-element backscattered electron detector in an SEM

A special mixing procedure for signals from a four element backscattered electron (BSE) detector is proposed for compositional image formation when a sample with a rough surface is examined by a scanning electron microscope (SEM). The new method allows appreciable suppression of the influence of the sample surface topography in a compositional mode for take-off angles less than about 30°, relative to the microscope axis. The theoretical approach based on the analysis of BSE angular distribution is compared with the experiment. The mixing procedure uses a dimensionless parameter, which depends mainly on take-off angle. Photographs of the Ge-Zn structure with its rough surface were taken in conventional and proposed compositional modes for take-off angle 11° and electron energy 20 keV and show a considerable suppression of the topographic effect when the new method is used.     

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.     

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.     

Use of reciprocal lattice layer spacing in electron backscatter diffraction pattern analysis

In the scanning electron microscope using electron backscattered diffraction, it is possible to measure the spacing of the layers in the reciprocal lattice. These values are of great use in confirming the identification of phases. The technique derives the layer spacing from the higher-order Laue zone rings which appear in patterns from many materials. The method adapts results from convergent-beam electron diffraction in the transmission electron microscope. For many materials the measured layer spacing compares well with the calculated layer spacing. A noted exception is for higher atomic number materials. In these cases an extrapolation procedure is described that requires layer spacing measurements at a range of accelerating voltages. This procedure is shown to improve the accuracy of the technique significantly. The application of layer spacing measurements in EBSD is shown to be of use for the analysis of two polytypes of SiC.     

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.     

Backscattered electron SEM imaging of cells and determination of the information depth

Backscattered electron imaging of HT29 colon carcinoma cells in a scanning electron microscope was studied. Thin cell sections were placed on indium‐tin‐oxide‐coated glass slides, which is a promising substrate material for correlative light and electron microscopy. The ultrastructure of HT29 colon carcinoma cells was imaged without poststaining by exploiting the high chemical sensitivity of backscattered electrons. Optimum primary electron energies for backscattered electron imaging were determined which depend on the section thickness. Charging effects in the vicinity of the SiO₂ nanoparticles contained in cell sections could be clarified by placing cell sections on different substrates. Moreover, a method is presented for information depth determination of backscattered electrons which is based on the imaging of subsurface nanoparticles embedded by the cells.     

Application of channel electron multipliers in an electron detector for low‐voltage scanning electron microscopy

An electron detector containing channel electron multipliers was built and tested in the range of low‐voltage scanning electron microscopy as a detector of topographic contrast. The detector can detect backscattered electrons or the sum of backscattered electrons and secondary electrons, with different amount of secondary electrons. As a backscattered electron detector it collects backscattered electrons emitted in a specific range of take‐off angles and in a large range of azimuth angles enabling to obtain large solid collection angle and high collection efficiency. Two arrangements with different channel electron multipliers were studied theoretically with the use of the Monte Carlo method and one of them was built and tested experimentally. To shorten breaks in operation, a vacuum box preventing channel electron multipliers from an exposure to air during specimen exchanges was built and placed in the microscope chamber. The box is opened during microscope observations and is moved to the side of the scanning electron microscope chamber and closed during air admission and evacuation cycles enabling storing channel electron multipliers under vacuum for the whole time. Experimental tests of the detector included assessment of the type of detected electrons (secondary or backscattered), checking the tilt contrast, imaging the spatial collection efficiency, measuring the noise coefficient and recording images of different specimens.     

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.     

Simultaneously identifying S-phase labelled cells and immunogold-labelling of vinculin in focal adhesions

A new combination of autoradiography and immunolabelling techniques is presented that allows the simultaneous identification of both S‐phase cells and their focal adhesions using scanning electron microscopy. The technique allows both labels to be discerned visually by their unique shapes and location within and on the cell. S‐phase cells were radio‐labelled with a pulse of tritiated thymidine, selectively incorporated into synthesizing DNA. The cells were then immunogold‐labelled for the focal adhesion protein, vinculin, prepared for autoradiography, and embedded in resin. The resin was then polymerized before removing the substrate, to expose the embedded cell undersurface. Electron‐energy ‘sectioning’ of the sample by varying the accelerating voltage of the electron beam allowed separate S‐phase cell identification in one electron‐energy ‘section’ and visualization of immunogold label in another ‘section’, within the same cell. As a result of applying this technique it was possible to positively identify S‐phase cells and immunogold‐labelled focal adhesions on the same cell simultaneously, which could be used to quantify focal adhesion sites on different substrates.     

Influence of the angular distribution of backscattered electrons on signals at different take-off angles in low-voltage scanning electron microscopy (LVSEM)

It is well known that the differential Mott cross section for large-angle elastic scattering shows maxima and minima at angles depending on material and electron energy. For electron energies of 10–30 keV, the averaging by frequent elastic scattering processes results in approximate Lambert angular distributions of backscattered electrons (BSE). However, the present Monte Carlo calculations for electron energies E = 1–5 keV and different angles of incidence show strong deviations from a Lambert distribution which increases with decreasing energy. The signals of the BSE detector with five annular segments for different take-off directions show good agreement with the calculations for normal electron incidence.     

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.     

Detectors for low voltage scanning electron microscopy

Two simple electron detectors (low and high take-off angles) for low-voltage scanning electron microscopy were built and tested. They contain large area scintillators with an applied high voltage and are able to detect backscattered electrons with high efficiency. These detectors also can record the sum of backscattered and secondary electrons. The primary beam of the microscope is screened from the scintillator high voltage by grids, which also permit switching from the BSE to the (SE + BSE) mode. The circular symmetry of the grids minimizes the influence of applied potentials on the primary beam. The use of the low and high take-off detectors permits the detection of back-scattered electrons emitted from the specimen surface into different ranges of take-off angles.     

High-voltage electron microscopy of inorganic particles in intact blood cells

Preliminary results obtained by examining intact fixed human red and white blood cells containing inorganic particles under the high-voltage transmission electron microscope are described. Iron filings and ferritin ingested in vitro by granulocytes were observed as were inorganic particles in red cells from a case of lead poisoning. The nature of the particles in red cells is discussed. A faint intracellular network was seen in normal red cells and in cells from a case of lead poisoning. It was found possible to focus the electron microscope at different planes within the cell. The optimum accelerating voltage for red cells appeared to be around 750 kV, whereas a clear image of granulocytes was obtained at voltages between 750 and 1000 kV. The first results indicate that it is possible to examine intact blood cells under the high-voltage electron microscope. Further work is in progress to determine if more information can be acquired by this technique.