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.     

A simple method for AFM tip characterization by polystyrene spheres

An AFM image would not be the true topography of a surface because of the limitation of a finite size of the tip. The true topography of the surface can be deduced if we can know the tip shape. In this paper a simple method has been established to determine the profile of an AFM tip. A geometrical model for the tip and a spherical object has been proposed to show the procedure for deducing the tip shape from AFM images. Isolated spheres and closely packed spheres with different diameters have been observed to confirm the tip shape by this method. It is a non-destructive method to determine the tip shape and the results can be used for future reconstruction of an AFM image.     

An expanded approach to noise reduction from high-resolution STEM images based on the maximum entropy method

An expanded use of the maximum entropy method (MEM) is suggested to reduce noise from an experimental high-angle annular dark-field (HAADF) scanning transmission electron microscope (STEM) image. The MEM is combined with an estimate of the standard deviation of noise from an experimental HAADF STEM image and low-pass filtering using the information limit for an incoherent STEM image. Consequently, the present method has just one parameter of a Lagrange multiplier. It is demonstrated that the present method can reduce noise efficiently in high-resolution HAADF STEM images.     

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.     

Reconstruction of a scanned topographic image distorted by the creep effect of a Z scanner in atomic force microscopy

We analyzed the illusory slopes of scanned images caused by the creep of a Z scanner in an atomic force microscope (AFM) operated in constant-force mode. A method to reconstruct a real topographic image using two scanned images was also developed. In atomic force microscopy, scanned images are distorted by undesirable effects such as creep, hysteresis of the Z scanner, and sample tilt. In contrast to other undesirable effects, the illusory slope that appears in the slow scanning direction of an AFM scan is highly related to the creep effect of the Z scanner. In the controller for a Z scanner, a position-sensitive detector is utilized to maintain a user-defined set-point or force between a tip and a sample surface. This serves to eliminate undesirable effects. The position-sensitive detector that detects the deflection of the cantilever is used to precisely measure the topography of a sample. In the conventional constant-force mode of an atomic force microscope, the amplitude of a control signal is used to construct a scanned image. However, the control signal contains not only the topography data of the sample, but also undesirable effects. Consequently, the scanned image includes the illusory slope due to the creep effect of the Z scanner. In an automatic scanning process, which requires fast scanning and high repeatability, an atomic force microscope must scan the sample surface immediately after a fast approach operation has been completed. As such, the scanned image is badly distorted by a rapid change in the early stages of the creep effect. In this paper, a new method to obtain the tilt angle of a sample and the creep factor of the Z scanner using only two scanned images with no special tools is proposed. The two scanned images can be obtained by scanning the same area of a sample in two different slow scanning directions. We can then reconstruct a real topographic image based on the scanned image, in which both the creep effect of the Z scanner and the slope effect of the sample have been eliminated. The slope effect of the sample should be eliminated so as to avoid further distortion after removal of the creep effect. The creep effect can be removed from the scanned image using the proposed method, and a real topographic image can subsequently be efficiently reconstructed.     

Evaluation of image based Abbott–Firestone curve parameters using machine vision for the characterization of cylinder liner surface topography

In this paper, a method is proposed for the evaluation of image based Abbott–Firestone curve parameters aiming to characterize the cylinder bore surface topography using machine vision. Plateau honing experiments are performed to generate sixteen cylinder liners with different surface topographies and the 2-D and 3-D Abbott–Firestone parameters are measured using a stylus instrument and Coherence Scanning Interferometer (CSI), respectively. The images are captured from the corresponding portions of the cylinder liner surfaces using a Charge Coupled Device (CCD) camera connected with different microscopic attachments. The captured images are filtered using a Butterworth high pass filter followed by the adaptation of the double step Gaussian filtering procedure specified by the ISO 13565-1. An Abbott–Firestone curve is constructed by finding the cumulative of the intensity histogram of the filtered images. Five image based parameters are evaluated from the constructed Abbott curve by adapting the procedures presented in ISO 13565-2. The computed image based Abbott–Firestone curve parameters are observed to bear a statistically significant correlation with the measured 2-D and 3-D Abbott–Firestone curve parameters. An artificial neural network (ANN) is trained and tested to arrive at the actual values of the Abbott–Firestone curve parameters using the computed image based feature parameters. The results indicate that the multiple surface topography parameters of the cylinder bore surface could be estimated/predicted with a reasonable accuracy using machine vision technique coupled with ANN.     

Surface profiling of combustion chamber deposits using aperture correlation confocal microscopy

The depth discrimination capability of a confocal microscope can be used to generate height-coded maps of surface topography from reflective surfaces. However, this surface profiling ability is severely limited when black surfaces are examined. In this paper we describe how a new form of confocal microscopy, known as self-correlating aperture microscopy, can be used to obtain surface topographies from the black carbonaceous deposits found in the combustion chambers of internal combustion engines. The technique is nondestructive and requires no sample preparation. The stereo pair images presented show the range of different morphologies found in combustion deposits generated by different fuel chemistries.     

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.     

Reconstruction of the images of reflectors from ultrasonic echo signals using the maximum-entropy method

It is proposed to use the maximum-entropy method (MEM) for processing ultrasonic echo signals for reconstructing images of reflectors with a high signal-to-noise ratio and a low level of “side lobes” of the point-scattering function. When processing echo signals, the pulse-propagation paths can be considered taking reflections from irregular boundaries of a tested object with the wave-type transformation into account. In model experiments, images of reflectors were obtained taking the refractions of rays at the rough surface into account, when echo signals were recorded both using an ordinary single-element transducer in the transceiver mode and an antenna array that recorded echo signals in the double- and triple-scanning modes. The reconstructed images have a resolution that exceeds the resolution according to the Rayleigh criterion. The MEM makes it possible to obtain images of flaws with low-level side lobes, when less than 10% of the complete set of echo signals are used.     

Polystyrene spheres on mica substrates: AFM calibration, tip parameters and scan artefacts

Atomic force microscopy (AFM), in various versions, has had major impact as a surface structural and spectroscopic tool since its invention in 1986. At its present state of development, however, the interpretation of AFM images is limited by the current state of methodologies for calibration over the wide dynamic range of magnification. Also, the parameters of individual tips, as well as the generic characteristics of different kinds of tips, affect both the quality of the images and their interpretation. Finally, the very nature of the tip-to-surface interaction will generate artefacts, in addition to those associated with tip shape, which need to be fully understood by the practitioners of force microscopy. This project seeks to address and shed light on some of these issues. Polystyrene beads deposited on mica substrates form hexagonal close-packed layers. The unit cell parameters are suitable for calibration of the AFM in the lateral plane, while the perpendicular spacing of the layers is appropriate for calibration along the vertical axis. Using different size fractions, it is straightforward to determine the extents of linearity, orthogonality, thermal and instrumental drifts over distances from 100 nm to tens of micrometres. The present results show that the methodologies for contact mode operation can be adapted to noncontact modes. It is known that an AFM image arises from a convolution of surface topography and tip shape, and is mediated by the interaction. In principle it is possible to carry out a deconvolution, if we have complete knowledge about two of the three elements (i.e. tip, surface and interaction). In practice we rarely have the requisite information. Prominent artefacts will occur when the characteristic parameters of the tip are comparable to those of the surface topography, and/or if there is a variable strength, or extent of localization, of the interaction. The present results demonstrate artefacts due to effects of geometry as well as interaction.     

High resolution images of a reconstructed surface structure on (111) gold platelets: Interpretation and comparison with theoretical models

Reconstruction of the (111) gold surface has been previously observed, and it has been shown that the surface net is compressed in the <110> direction, as compared with the bulk lattice. We present here some high resolution studies made on this surface reconstruction: the problem was to obtain a structure image corresponding mainly to the uppermost plane which forms the surface. For this, we have formed high resolution images by interferences between the so-called forbidden diffracted beams $\text{1}\text{3}$ (4?22): bulk gold, when observed along the <111> direction, is better described as a stacking of hexagonal two-dimensional layers, and the $\text{1}\text{3}$ (4?22) beams occur as diffracted beams from 1 (or2) hexagonal layers; they nearly disappear every 3 slices. If the top layer is reconstructed, the diffracted beams from the first slice will be different and will not cancel with the diffracted beams from the bulk layers. Hence the image formed with these beams will give some information on the reconstructed top layer. Computed images have been obtained for different models of the reconstructed surface. They show that, indeed, images obtained under these conditions are linked with the topography of the surface. They also show the importance of different experimental parameters, in particular the beam divergence.     

Depth of information in photoelectron microscopy

The depth of information is defined as the distance below the surface of a specimen from which information is contributed at a specified resolution. A simplified model of photoemission is used to explore the relationship between electron escape depths and depth of information in photoelectron microscopy (PEM or photoemission electron microscopy). The depth of information is equal to the escape depth when the escape depth is small relative to the instrument resolution. When the escape depth is large compared to the instrument resolution or when information is carried for example by reflected light, the image consists of well resolved surface detail at the instrument resolution and dimmer, more diffuse, images of detail below the surface. Thus the same sample can exhibit different depths of information depending on the image details of interest. Other mechanisms of transmitting information to the surface, for example induced topography, are discussed, and experimental examples are given.     

Three-dimensional surface topography segmentation through clustering

In digital image analysis, segmentation is the partitioning of the image into multiple regions according to a given criterion. This work investigates the adaptation of segmentation techniques originally developed for digital images to the partitioning of the three-dimensional micro and nano topography of engineered surfaces. In particular, a segmentation technique is introduced for partitioning a surface into regions characterized by homogeneous local texture properties. Local surface texture properties are captured through roughness parameters evaluated over surface patches centered about each surface point. Roughness parameters are chosen depending on texture properties to be highlighted, and collected into feature vectors that are then subjected to clustering. Several issues are addressed, ranging from the choice of appropriate clustering techniques to the design of proper feature vectors and similarity metrics for capturing relevant aspects of three-dimensional surface topography and achieving a meaningful partitioning. The advantages in terms of improved morphologic, structural and tribologic analysis capabilities are highlighted and discussed through the application of the proposed technique to example real-life industrial applications, mainly concerned with the discrimination of localized surface modifications either generated on purpose and in controlled conditions (such as indentations and scratches produced during surface testing) or generated by random interaction of the surface with the environment (scratches, bumps and other types of marking due to accidental damage or use-related wear phenomena).     

Three-channel false colour AFM images for improved interpretation of complex surfaces: A study of filamentous cyanobacteria

Imaging signals derived from the atomic force microscope (AFM) are typically presented as separate adjacent images with greyscale or pseudo-colour palettes. We propose that information-rich false-colour composites are a useful means of presenting three-channel AFM image data. This method can aid the interpretation of complex surfaces and facilitate the perception of information that is convoluted across data channels. We illustrate this approach with images of filamentous cyanobacteria imaged in air and under aqueous buffer, using both deflection-modulation (contact) mode and amplitude-modulation (tapping) mode. Topography-dependent contrast in the error and tertiary signals aids the interpretation of the topography signal by contributing additional data, resulting in a more detailed image, and by showing variations in the probe-surface interaction. Moreover, topography-independent contrast and topography-dependent contrast in the tertiary data image (phase or friction) can be distinguished more easily as a consequence of the three dimensional colour-space.     

Improving immunoassays based on the analysis of scanning force microscopy images

Scanning force microscopy has been demonstrated to be an effective binding event detection step for immunoassays. In its simplest form--analysing small area images--the detection limit of the scanning force microscopic immunoassay (SFMIA) has been shown to be comparable to existing techniques. In the present work, we have examined how the performance of image analysis-based SFMIA can be improved. Firstly, we have used a surface analysis parameter that increases linearly with the concentration of binding events. This parameter--the surface area ratio--is the ratio of the surface area after antigen binding to the surface area of the original biospecific surface. With this parameter, SFMIA images can be rapidly analysed and converted into assay units. Secondly, we have demonstrated that by using silicon wafer supports that carry fiducial marks we can relocate to very high accuracy onto the biospecific surfaces and identify the changes due to antigen binding. By relocating in this manner the signal to noise ratio of the technique is enhanced. Thirdly, from simulations we have determined the SFM tip size and image area that optimizes the immunoassay sensitivity.     

An improved surface roughness measurement method for micro-heterogeneous texture in deep hole based on gray-level co-occurrence matrix and support vector machine

Microscopic vision system has been employed to measure the surface roughness of micro-heterogeneous texture in deep hole, by virtue of frequency domain features of microscopic image and back-propagation artificial neural network optimized by genetic algorithm. However, the measurement accuracy needs to be improved for engineering application. In this paper, we propose an improved method based on microscopic vision to detect the surface roughness of R-surface in the valve. Firstly, the measurement system for the roughness of R-surface in deep hole is described. Thereafter, the surface topography images of R-surface are analyzed by the gray-level co-occurrence matrix (GLCM) method, and several features of microscopic image, which are nearly monotonic with the surface roughness, are extracted to fabricate the prediction model of the roughness of R-surface accurately. Moreover, a support vector machine (SVM) model is presented to describe the relationship of GLCM features and the actual surface roughness. Finally, experiments on measuring the surface roughness are conducted, and the experimental results indicate that the GLCM-SVM model exhibits higher accuracy and generalization ability for evaluating the microcosmic surface roughness of micro-heterogeneous texture in deep hole.     

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.     

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.