Surface roughness of preparations for backscattered electron-scanning electron microscopy: the image differences and their Monte Carlo simulation

Patterns and levels of mineralisation in the biological hard tissues have been studied using the backscattered electron (BSE) mode in the scanning electron microscope (SEM). To prevent gross topographic detail overwhelming changes in signal from composition, samples are embedded in polymethylmethacrylate (PMMA) and a flat block surface produced by polishing or micromilling. This study was undertaken to establish the degree of residual topography achieved in these finishing processes. A sample of human rib was embedded in PMMA and prepared, as for examination in the SEM, by polishing on graded abrasives and pre- and, finally, ultramilling. After each preparation step, the block face was imaged using a confocal reflection microscope surface mapping facility. The recorded topographies were used in a Monte Carlo simulation to model the surface interface and thus, for each of the sample preparation techniques, to calculate predicted variations in BSE signal. The latter were compared with experimental data derived under standard operating conditions in the SEM. Micromilling produced block faces with typical peak-trough relief of 80 nm, while hand polishing left occasional scratches 1.5 microns deep with a general undulation of 150-250 nm. Monte Carlo simulations of a rough surface of bone using these data predicted that additional contrast levels of 5% could be expected from micromilled surfaces and > 10% for hand polished samples of bone. Thus, micromilling is the best preparation method for bone, since this tissue develops a collagen orientation-related relief on polishing, which may be largely responsible for the (incorrect) supposition that lamellation in bone is related to changes in net degree of mineralisation.     

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.     

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.     

An image-based methodology to establish correlations between porosity and cutting force in micromilling of porous titanium foams

Porous titanium foam is now a standard material for various dental and orthopedic applications due to its light weight, high strength, and full biocompatibility properties. In practical biomedical applications, outer surface geometry and porosity topology significantly influence the adherence between implant and neighboring bone. New microfabrication technologies, such as micromilling and laser micromachining opened new technological possibilities for shape generation of this class of products. Besides typical geometric alterations, these manufacturing techniques enable a better control of the surface roughness that in turn affects to a large extent the friction between implant and surrounding bone tissue. This paper proposes an image analysis approach for optical investigation of the porosity that is tailored to the specifics of micromilling process, with emphasis on cutting force monitoring. According to this method, the area of porous material removed during micromilling operation is estimated from optical images of the micromachined surface, and then the percentage of solid material cut is calculated for each tool revolution. The employment of the aforementioned methodology in micromilling of the porous titanium foams revealed reasonable statistical correlations between porosity and cutting forces, especially when they were characterized by low-frequency variations. The developed procedure unlocks new opportunities in optimization of the implant surface micro-geometry, to be characterized by an increased roughness with minimal porosity closures in an attempt to maximize implant fixation through an appropriate level of bone ingrowth.     

Thickness determination of ultra-thin films using backscattered electron spectra of a new toroidal electrostatic spectrometer

Backscattered electron spectroscopy offers detailed information for multilayer and subsurface-layer materials with distinct Z contrast: It can be used for the validation of Monte Carlo calculations, to obtain depth selective electron microtomographic images, and to determine the thickness of ultra thin films on bulk substrates. In this paper we describe a new energy-dispersive method for thickness determination of thin films on bulk aluminum using backscattered electron (BSE) spectra obtained by a polar, toroidal, electrostatic spectrometer. After a brief recapitulation of the spectrometer's geometry, the techniques for its energy calibration and the preparation of thin double-layer films are introduced. Backscattered electron measurements and thickness calibrations for Au and Cu films with thicknesses of 0–200 nm on bulk aluminum will be presented for various primary electron energies.     

Imaging individual atoms inside crystals with ADF-STEM

The quantitative imaging of individual impurity atoms in annular dark-field scanning transmission electron microscopy (ADF-STEM) requires a clear theoretical understanding of ADF-STEM lattice imaging, nearly ideal thin samples, and careful attention to image processing. We explore the theory using plane-wave multislice simulations that show the image intensity of substitutional impurities is depth-dependent due to probe channeling, but the intensity of interstitial impurities need not be. The images are only directly interpretable in thin samples. For this reason, we describe a wedge mechanical polishing technique to produce samples less than <50 A thick, with low surface roughness and no amorphous surface oxide. This allows us to image individual dopants as they exist within a bulk-like silicon environment. We also discuss the image analysis techniques used to extract maximum quantitative information from the images. Based on this information, we conclude that the primary nanocluster defect responsible for the electrical inactivity of Sb in Si at high concentration consists of only two atoms.     

Collection deficiencies of scanning electron microscopy signal contrasts measured and corrected by differential hysteresis image processing

Limitations of scanning electron microscopy (SEM) image resolution and quality were measured in digital image data and their effect on image contrasts was analyzed and corrected by differential hysteresis (DH) processing. DH processing is a mathematical procedure that utilizes hysteresis properties of intensity variations in the image for a segmentation of differential contrast patterns. These patterns display contrast properties of the data as coherent full-frame images. The contrast segmentation is revertible so that the original image can be restored from the sum of the sequentially extracted DH contrast patterns. DH imaging enhances weak contrast components so that they are more easily recognizable and displays SEM image data free of signal collection efficiency contrasts. Example image data include environmental SEM (ESEM) and SEM images of low and mediumhigh magnifications where collection deficiencies included charging of the specimen surface, obstructions from specimen topography, and uneven signal collection properties of the detector. ESEM low-vacuum image data, which appear to be of high quality, contained local areas of reduced contrasts due to residual surface charging. In such areas, signal contrasts were reduced up to 80%, which suppressed most of the weak short-range contrasts. In low-magnification SEM images, up to 93% of the local high precision contrast was lost from the various adverse effects which diminished the pixel-related contrast resolution of the microscope and resulted in images with low detail. Also, at medium magnification, surface charging effects dramatically reduced the image quality because contrasts resulting from local electron beam/specimen interactions were reduced by as much as 71%. DH imaging restored the local contrast losses by elimination of the collected distorted fraction of signal contrasts and reconstitution of the collected maintained fraction. Restored DH images are of superior quality and enhance the imaging capability of the conventional SEM. DH contrast segmentation provides an improved basis for the measurement of various signal contrast components and detector performances. The DH analysis will ultimately facilitate a precise deduction of specimen properties from extracted contrast patterns.     

Automated compensation of light attenuation in confocal microscopy by exact histogram specification

Confocal laser scanning microscopy (CLSM) enables us to capture images representing optical sections on the volume of a specimen. The images acquired from different layers have a different contrast: the images obtained from the deeper layers of the specimen will have a lower contrast with respect to the images obtained from the topmost layers. The main reasons responsible for the effects described above are light absorption and scattering by the atoms and molecules contained in the volume through which the light passes. Also light attenuation can be caused by the inclination of the observed surface. In the case of the surfaces that have a steep inclination, the reflected light will have a different direction than the one of the detector. We propose a technique of digital image processing that can be used to compensate the effects of light attenuation based on histogram operations. We process the image series obtained by CLSM by exact histogram specification and equalization. In this case, a strict ordering among pixels must be induced in order to achieve the exact histogram modeling. The processed images will end up having exactly the specified histogram and not a histogram with a shape that just resembles to the specified one, as in the case of classical histogram specification algorithms. Experimental results and theoretical aspects of the induced ordering are discussed, as well as a comparison between several histogram modeling techniques with respect to the processing of image series obtained by confocal microscopy. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

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.     

Analysis on formation mechanism of ultra-smooth surfaces in atmospheric pressure plasma polishing

Atmospheric pressure plasma polishing (APPP) is developed for the final finishing of high quality ultra-smooth surfaces. To improve surface quality, formation mechanism of ultra-smooth surfaces in APPP is studied. Quantum chemistry simulation is used to investigate the interaction between atoms. Simulation of single Si-F bonding process indicates 0.2 eV difference of binding energy between convex and concave models, which reflects the reaction probability of convex structure is higher than that of concave structure. By comparing the spatial atomic configuration and species diffusion path, it is also demonstrated convex topography should be removed faster than concave topography. So roughness of optical surfaces can be reduced further to form ultrasmooth surfaces. And experimental results accord well with theoretical analysis. Detected by atomic force microscopy every 40 s, the average maximum height of surface is testified to decrease faster than the maximum depth obviously, which makes the whole surface going toward a new equilibrium status with lower roughness. Another experiment proves the average surface roughness decreases from Ra 4.529 to 0.926 nm after 100 s continuous machining. And the stereo images also indicate obvious improvement of surface topography. Moreover, free outmost electron is proved to be helpful to promote chemical reaction by simulation, so fresh surfaces may be more favorable for APPP which makes sample preparation more purposeful.     

Effects of micromilled NiP mold surface topography on the optical characteristics of injection molded prismatic retroreflectors

Automotive lighting applications require mold micro-features with increasingly better surface finishing, especially for optical lenses and retroreflectors. In this work innovative micromilling experiments were conducted on electroless-plated nickel phosphorous (NiP) surfaces to fabricate a mold for prismatic retroreflectors. Cutting parameters effects on surface topography of mold micro-features were experimentally characterized and optimized, monitoring moreover the cutting forces generated by NiP micromilling. Furthermore, the mold surface topography was replicated on polycarbonate injection molded retroreflectors and its effects on their optical performance was characterized by means of a dedicated spectrophotometric technique. The obtained results show that micromilling of electroless-plated NiP can substitute polishing in the fabrication of high surface finish mold micro-features.     

Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

A method using an on-line digital computer for the improvement of resolution of backscattered electron images

In principle, the resolution of backscattered electron (BSE) images can be little improved, even though an infinitely small beam size is achieved by various improvements in the intrinsic instrument. In order to circumvent this problem, a method is proposed which utilizes an on-line digital computer for the image recording and processing. The major image-processing tools are reduction, expansion, super-imposition with matching of the images, and high-emphasis filtering in the Fourier domain. By using various combinations of these techniques, the resolution of BSE images has been significantly improved. The validity of these improved images has been confirmed. In the case of a BSE image with too wide a dynamic range, both the present method and digital homomorphic filtering provide successful results.     

Theoretical explanation of the relationship between backscattered electron and x-ray linear attenuation coefficients in calcified tissues

X-ray absorption and backscattered electron (BSE) microscopies are two commonly used techniques for estimating mineral contents in calcified tissues. The resolution in BSE images is usually higher than in x-ray images, but due to the previous lack of good standards to quantify the grey levels in BSE images of bones and teeth, x-ray microtomog-raphy (XMT) images of the same specimens have been used for calibration. However, the physics of these two techniques is different: for a specimen with a given composition, the x-ray linear attenuation coefficient is proportional to density, but there is no such relation with the BSE coefficient. To understand the reason that this calibration appears to be valid, the behaviour of simulated bone samples was investigated. In this, the bone samples were modelled as having three phases: hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂), protein, and void (either empty or completely filled with polymethylmethacrylate (PMMA), a resin which is usually used for embedding bones and teeth in microscopic studies). The x-ray linear attenuation coefficients (calculated using published data) and the BSE coefficients (calculated using Monte Carlo simulation) were compared for samples of various phase proportions. It was found that the BSE coefficient correlated only with the x-ray attenuation coefficient for samples with PMMA infiltration. This was attributed to the properties of PMMA (density and mean atomic number) being very similar to those of the protein; therefore, the sample behaves like a two-phase system which allows the establishment of a monotonic relation between density and BSE coefficient. With the newly developed standards (brominated and iodinated dimethacrylate esters) for BSE microscopy of bone, grey levels can be converted to absolute BSE coefficients by linear interpolation, from which equivalent densities can be determined.     

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.     

Beam interactions,contrast and resolution in the SEM

Backscattered and secondary electrons are both used in the SEM for imaging purposes. The backscattered signal is the result of high angle elastic scattering events, while the secondary signal is the result of knock-on inelastic collisions. The characteristic differences between images in the two modes arise from the details of the relevant interactions in the two cases. In order to examine this in a quantitative manner Monte Carlo electron trajectory simulation techniques have been used. Calculations of the ultimate resolution and depth of information of the secondary and backscattered images are presented, together with simulations of the edge brightness effect in high resolution secondary images and an analysis of the microanalytical application of atom number contrast in the backscattered mode.     

Electron backscattered diffraction characterization technique for analysis of a Ti2AlNb intermetallic alloy

This investigation was conducted to ascertain the benefits of electropolishing after mechanical polishing for electron backscattered diffraction of a Ti₂AlNb intermetallic Ti−21Al−29Nb (at.%) alloy containing the orthorhombic (O) and body-centered-cubic (BCC) phases. Electropolishing was performed at −40 °C in 6% H₂SO₄ methanol solution. Atomic force microscopy was used to measure the surface topography in attempt to correlate nano-scale surface roughness with electron backscatter diffraction pattern quality. The results suggest that mechanically polishing with colloidal silica (SiO₂) or alumina followed by electropolishing is a sufficient surface preparatory technique for producing quality electron backscattered diffraction patterns for O + BCC microstructures. However, poor pattern quality results after mechanically polishing without electropolishing. High-quality orientation maps for O-dominated O + BCC microstructures were only possible through mechanical polishing followed by electropolishing. The data also suggest that surface roughness, on the order of 50 nm, has less effect on pattern quality than subsurface deformation. Overall, removing the near-surface damage was more critical than reduction of topography.     

Surface roughness and texture analysis in microscale

The capacity of various instruments in roughness measurements and analysis is compared. Review of various models of roughness is made and the models of contact mechanics are presented, when taking account the nanometer scale roughness and relating phenomena of adhesion and surface forces. The concept of multi-level models of roughness and contact area is presented. Analysis of surface topography as a spatial pattern is given, when using the approaches of image recognition theory operating with the 3D digital images processing. Qualitatively the spatial structure is often characterized in terms of texture features such as random, linear, wavy etc., and some national standards introduce spatial structure of machined surfaces. However, texture characteristics are not adequately investigated. AFM images of different surfaces were used as initial data and multi-dimensional scaling technique was used for the data analysis. The study has shown that there are at least four types surface textures on nanoscale level. The correlation was found between texture types and reasons of their formation.     

Gradient based intensity normalization

Intensity normalization is important in quantitative image analysis, especially when extracting features based on intensity. In automated microscopy, particularly in large cellular screening experiments, each image contains objects of similar type (e.g. cells) but the object density (number and size of the objects) may vary markedly from image to image. Standard intensity normalization methods, such as matching the grey-value histogram of an image to a target histogram from, i.e. a reference image, only work well if both object type and object density are similar in the images to be matched. This is typically not the case in cellular screening and many other types of images where object type varies little from image to image, but object density may vary dramatically. In this paper, we propose an improved form of intensity normalization which uses grey-value as well as gradient information. This method is very robust to differences in object density. We compare and contrast our method with standard histogram normalization across a range of image types, and show that the modified procedure performs much better when object density varies between images.