Diffraction effects and inelastic electron transport in angle‐resolved microscopic imaging applications

We analyse the signal formation process for scanning electron microscopic imaging applications on crystalline specimens. In accordance with previous investigations, we find nontrivial effects of incident beam diffraction on the backscattered electron distribution in energy and momentum. Specifically, incident beam diffraction causes angular changes of the backscattered electron distribution which we identify as the dominant mechanism underlying pseudocolour orientation imaging using multiple, angle‐resolving detectors. Consequently, diffraction effects of the incident beam and their impact on the subsequent coherent and incoherent electron transport need to be taken into account for an in‐depth theoretical modelling of the energy‐ and momentum distribution of electrons backscattered from crystalline sample regions. Our findings have implications for the level of theoretical detail that can be necessary for the interpretation of complex imaging modalities such as electron channelling contrast imaging (ECCI) of defects in crystals. If the solid angle of detection is limited to specific regions of the backscattered electron momentum distribution, the image contrast that is observed in ECCI and similar applications can be strongly affected by incident beam diffraction and topographic effects from the sample surface. As an application, we demonstrate characteristic changes in the resulting images if different properties of the backscattered electron distribution are used for the analysis of a GaN thin film sample containing dislocations.     

Quantitative secondary ion mass spectrometry imaging of self-assembled monolayer films for electron beam dose mapping in the environmental scanning electron microscope

Fluorinated alkanethiol self-assembled monolayers (SAM) films immobilized on gold substrates have been used as electron-sensitive resists to map quantitatively the spatial distribution of the primary electronbeam scattering in an environmental scanning electron microscope (ESEM). In this procedure, a series of electron dose standards are prepared by exposing a SAM film to electron bombardment in well-defined regions at different levels of electron dose. Microbeam secondary ion mass spectrometry (SIMS) using Cs⁺ bombardment is then used to image the F^- secondary ion signal from these areas. From the reduction in F^- intensity as a function of increasing electron dose, a calibration curve is generated that allows conversion of secondary ion signal to electron dose on a pixel-by-pixel basis. Using this calibration, electron dose images can be prepared that quantitatively map the electron scattering distribution in the ESEM with micrometer spatial resolution. The SIMS imaging technique may also be used to explore other aspects of electron-surface interactions in the ESEM.     

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.     

Multiple scattering effects of MeV electrons in very thick amorphous specimens

Multiple scattering has an important influence on the analysis of microns-thick specimens with MeV electrons. In this paper, we report on effects of multiple scattering of MeV electrons on electron transmission and imaging of tilted and thick amorphous film specimens by experiment and theoretical analysis. Electron transmission for microns-thick epoxy-resin and SiO₂ specimens calculated by the multiple elastic-scattering theory is in good agreement with measurements in the ultrahigh voltage electron microscope (ultra-HVEM) at Osaka University. Electron transmission and electron energy are then presented in an approximate power law. The bright-field ultra-HVEM images of gold particles on the top or bottom surfaces of 5 and 15 μm thick specimens further illustrate the effect of multiple scattering on image quality. The observed top‐bottom effect for the very thick specimens appears to be mainly caused by multiple elastic scattering. With increase in the accelerating voltage from 1 to 2 MV, image blurring, contrast, the signal-to-noise ratio, and the top‐bottom effect are improved because of reduction in the influence of multiple scattering. However, the effect of specimen thickness on image blurring is shown to be stronger than that of accelerating voltage. At the 2 MV accelerating voltage, the 100 nm gold particle can be imaged with less blurring of ∼4 nm when located at the bottom surface of a 15 μm thick epoxy-resin specimen.     

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) of biological specimens: Preliminary results with a novel beam separating system

Low-energy electron microscopy (LEEM) and mirror electron microscopy (MEM) utilize a parallel beam of slow-moving electrons backscattered from the specimen surface to form an image. If the electrons strike the surface an LEEM image is produced and if they are turned back just before reaching the surface an MEM image results. The applications thus far have been in surface physics. In the present study, applications of LEEM and MEM in the biological sciences are discussed. The preliminary results demonstrate the feasibility of forming images of uncoated cultured cells and cellular components using electrons in the threshold region (i.e. 0–10 V). The results also constitute a successful test of a novel beam-separating system for LEEM and MEM.     

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.     

Application of the low-loss scanning electron microscope image to integrated circuit technology. Part 1--Applications to accurate dimension measurements

Scanning electron microscopes (SEMs) are the most extensively used tools for dimensional metrology and defect inspection for integrated circuit technologies with 180 nm and smaller features. Currently, almost all SEMs are designed to collect as many secondary and backscattered electrons as possible. These signals are mainly secondary electrons (SE1, SE2, and SE3) detected with various detection schemes. To facilitate the electron collection, very strong electric and magnetic fields are applied not just in the path of the primary electron beam but to the emerging electrons as well. These new systems provide strong signals, thus better signal-to-noise ratio, and thus resulting in higher throughput than older ones. On the other hand, the use of secondary electrons means that measurement results are much more prone to the detrimental effects of electron beam interactions, sample charging, and sample contamination than measurements with higher-energy backscattered electrons. The use of backscattered electrons, especially low-loss electrons (LLE), can provide better surface sensitivity, edge accuracy, and repeatability, possibly at the expense of measurement speed. This two-part study investigates the benefits and drawbacks of low-loss electron imaging to edge characterization for dimensional metrology and enhancement of fine surface features done through filtration or separation of the generated LLE signal and the use of energy-dependent signals. Part 1 reviews and illustrates the potential for accurate dimensional measurements at low accelerating voltage by LLE, and Part 2 will concentrate on the enhancement of surface features in chemical-mechanically planarized specimens with the use of a novel LLE detector.     

Imaging of thermal and elastic surface properties by scanning electron acoustic microscopy

Scanning electron acoustic microscopy is a new technique for imaging the thermal and elastic properties of surfaces and detecting subsurface flaws. It can be carried out in a modified scanning electron microscope. The effects of electron beam energy and phase angle on scanning electron acoustic images of the thermal and elastic properties of surfaces were studied with an alumina fiber/aluminum matrix composite for fiber directions both transverse and coaxial to the surface. Images produced with 10- and 30-keV electrons at beam modulation frequencies of 80–1200 kHz appeared to be identical, with the exception of a lower signal-to-noise ratio for the lower electron energy. This observation suggests that the energy input from the beam can be considered to occur at the surface for electron energies below 30 keV and frequencies below 1200 kHz. Images recorded at 0° phase angle mapped regions of different thermal and elastic properties. Images recorded at 90° phase angle highlighted the boundaries between such regions. Scanning electron acoustic microscopy can image features of different thermal and elastic properties at greater depth than traditional imaging with backscattered electrons. The practical application of the technique to the study of surfaces is illustrated by the imaging of grain structure and subsurface particles for an extruder barrel.     

Simulations of scanning electron microscopy imaging and charging of insulating structures

We present a three‐dimensional simulation of scanning electron microscope (SEM) images and surface charging. First, the field above the sample is calculated using Laplace's equation with the proper boundary conditions; then, the simulation algorithm starts following the electron trajectory outside the sample by using electron ray tracing. When the electron collides with the specimen, the algorithm keeps track of the electron inside the sample by simulating the electron scattering history with a Monte Carlo code. During this phase, secondary and backscattered electrons are emitted to form an image and primary electrons are absorbed; therefore, a charge density is formed in the material. This charge density is used to recalculate the field above and inside the sample by solving the Poisson equation with the proper boundary conditions. Field equation, Monte Carlo scattering simulation, and electron ray tracing are therefore integrated in a self‐consistent fashion to form an algorithm capable of simulating charging and imaging of insulating structures. To maintain generality, this algorithm has been implemented in three dimensions. We shall apply the so‐defined simulation to calculate both the global surface voltage and local microfields induced by the scanning beam. Furthermore, we shall show how charging affects resolution and image formation in general and how its characteristics change when imaging parameters are changed. We shall address magnification, scanning strategy, and applied field. The results, compared with experiments, clearly indicate that charging and the proper boundary conditions must be included in order to simulate images of insulating features. Furthermore, we shall show that a three‐dimensional implementation is mandatory for understanding local field formation.     

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.     

Recent developments and new strategies in scanning electron microscopy*

In addition to improvements in lateral resolution in scanning electron microscopy, recent developments of interest here concern extension of the incident beam energy, E₀, over two decades, from ≈ 20 keV to ≈ 0.1–0.5 keV and the possibility of changing the take-off emission, α, of detected secondary electrons. These two degrees of freedom for image acquisition permit a series of images of the same field of view of a specimen to be obtained, each image of the series differing from the others in some aspect. The origins of these differences are explored in detail and they are tentatively interpreted in terms of the change in the secondary electron emission yield δ vs. E₀, δ = f(E₀), and also of the change in δ vs. α, ∂δ/∂α. Various origins for the chemical contrast and topographic contrast have been identified. Illustrated by correlating a secondary electron image and a backscattered electron image, use of the scatter diagram technique facilitates image comparison. The difference between the lateral resolution and the size of the minimum detectable detail is outlined to avoid possible errors in nanometrology. Some aspects related to charging are also considered and possible causes of contrast reversal are suggested. Finally, the suggested strategy consists of the acquisition of various images of a given specimen by changing one parameter: primary beam energy and take-off angle for conductive specimens; working distance or beam intensity for high-resolution experiments; scanning frequency for insulating specimens.     

Simulation of ion imaging: sputtering, contrast, noise

Scanning ion microscopy has received a boost in the last decade, thanks to the development of novel ion sources employing light ions, like He⁺, or ions from inert gases, like Ne⁺ and Ar⁺. Scanning ion images, however, might not be as easy to interpret as SEM micrographs. The contrast mechanisms are different, and there is always a certain degree of sample sputtering. The latter effect, on the one hand, prevents assessing the resolution on the basis of a single image, and, on the other hand, limits the probing time and thus the signal-to-noise ratio that can be obtained. In order to fully simulate what happens when energetic ions impact on a sample, a Monte Carlo approach is often used. In this paper, a different approach is proposed. The contrast is simulated using curves of secondary electron yields versus the incidence angle of the beam, while the surface modification prediction is based on similar curves for the sputtering yield. Finally, Poisson noise from primary ions and secondary electrons is added to the image. It is shown that the evaluation of an ion imaging tool cannot be condensed in a single number, like the spot size or the edge steepness, but must be based on a more complex analysis taking into account at least three parameters: sputtering, contrast and signal-to-noise ratio. It is also pointed out that noise contributions from the detector cannot be neglected for they can actually be the limiting factor in imaging with focused ion beams. While providing already good agreement with experimental data in some imaging aspects, the proposed approach is highly modular. Further effects, like edge enhancement and detection, can be added separately.     

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.     

Specimen‐thickness effects on transmission Kikuchi patterns in the scanning electron microscope

We report the effects of varying specimen thickness on the generation of transmission Kikuchi patterns in the scanning electron microscope. Diffraction patterns sufficient for automated indexing were observed from films spanning nearly three orders of magnitude in thickness in several materials, from 5 nm of hafnium dioxide to 3 μm of aluminum, corresponding to a mass‐thickness range of ～5 to 810 μg cm^–2. The scattering events that are most likely to be detected in transmission are shown to be very near the exit surface of the films. The energies, spatial distribution and trajectories of the electrons that are transmitted through the film and are collected by the detector are predicted using Monte Carlo simulations.     

A high signal-to-noise ratio toroidal electron spectrometer for the SEM

This paper presents a high signal-to-noise ratio electron energy spectrometer attachment for the scanning electron microscope (SEM), designed to measure changes in specimen surface potential from secondary electrons and extract specimen atomic number information from backscattered electrons. Experimental results are presented, which demonstrate that the spectrometer can in principle detect specimen voltage changes well into the sub-mV range, and distinguish close atomic numbers by a signal-to-noise ratio of better than 20. The spectrometer has applications for quantitatively mapping specimen surface voltage and atomic number variations on the nano-scale.     

About the role of boundary conditions on compositional imaging with a scanning electron microscope

We consider the effects of different boundaries on the visibility of a specimen detail providing a compositional contrast in scanning electron microscopy, operating with backscattered electrons or secondary electrons. An object characterized by a gradual variation in composition, an As-doped region in Si, is investigated. The different boundaries in the cross-sectioned specimen correspond to the absence or presence of a poly-Si layer on top of the implanted region, deposited after the annealing treatment. It is shown that the interpretation model used for image formation is of paramount relevance for understanding the experimental results, indicating that the boundaries of the doped region are important in hindering or enhancing its visibility. The relevance of experimental parameters such as electron energy and probe dimension is also reported.     

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.     

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.     

Optimal strategies for imaging thick biological specimens: exit wavefront reconstruction and energy-filtered imaging

In transmission electron microscopy (TEM) of thick biological specimens, the relationship between the recorded image intensities and the projected specimen mass density is distorted by incoherent electron–specimen interactions and aberrations of the objective lens. It is highly desirable to develop a strategy for maximizing and extracting the coherent image component, thereby allowing the projected specimen mass density to be directly related to image intensities. For this purpose, we previously used exit wavefront reconstruction to understand the nature of image formation for thick biological specimens in conventional TEM. Because electron energy-loss filtered imaging allows the contributions of inelastically scattered electrons to be removed, it is potentially advantageous for imaging thick, biological samples. In this paper, exit wavefront reconstruction is used to quantitatively analyse the imaging properties of an energy-filtered microscope and to assess its utility for thick-section microscopy. We found that for imaging thick biological specimens (> 0.5 μm) at 200 keV, only elastically scattered electrons contribute to the coherent image component. Surprisingly little coherent transfer was seen when using energy-filtering at the most probable energy loss (in this case at the first plasmon energy-loss peak). Furthermore, the use of zero-loss filtering in combination with exit wavefront reconstruction is considerably more effective at removing the effects of multiple elastic and inelastic scattering and microscope objective lens aberrations than either technique by itself. Optimization of the zero-loss signal requires operation at intermediate to high primary voltages (> 200 keV). These results have important implications for the accurate recording of images of thick biological specimens as, for instance, in electron microscope tomography.