The method of increasing COMPO contrast by linearization of backscattering characteristic η =f(Z)

The backscattered electron signal (BSE) in the scanning electron microscope (SEM) has been used for investigation of a specimen surface composition (COMPO mode). Creation of a material composition map is difficult because the dependence of backscattering coefficient η on the atomic number Z for Z > 40 is nonlinear. The method of increase in SEM resolution for the BSE signal by use of digital image processing has been proposed. This method is called the linearization of the η =f(Z) characteristic. The function approximating the experimental η =f (Z) dependence was determined by numerical methods. After characteristics linearization, the digital image in COMPO mode allows to distinguish between two elements with high atomic numbers if their atomic numbers differ by ΔZ = 1.     

Image restoration for TV‐scan moving images acquired through a semiconductor backscattered electron detector

A semiconductor backscattered electron (BSE) detector has become popular in scanning electron microscopy session. However, detectors of semiconductor type have a serious disadvantage on the frequency characteristics. As a result, fast scan (e.g. TV‐scan) BSE image should be blurred remarkably. It is the purpose of this study to restore this degradation by using digital image processing technology. In order to improve it practically, we have to settle several problems, such as noise, undesirable processing artifacts, and ease of use. Image processing techniques in an impromptu manner like a conventional mask processing are unhelpful for this study, because a complicated degradation of output signal affects severely the phase response as well as the amplitude response of our SEM system. Hence, based on the characteristics of an SEM signal obtained from the semiconductor BSE detector, a proper inverse filter in Fourier domain is designed successfully. Finally, the inverse filter is converted to a special convolution mask, which is skillfully designed, and applied for TV‐scan moving BSE images. The improved BSE image is very effective in the work for finding important objects. SCANNING 31: 229–235, 2009. © 2010 Wiley Periodicals, Inc.     

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.     

A double detector system for BSE and SE imaging

The double detector system described here is a simple device suitable for any SEM. It permits efficient imaging of specimen surfaces in either the secondary electron (SE) or backscattered electron (BSE) mode. The BSE detector is an annular single-crystal scintillator made of yttrium aluminium garnet (YAG) and the SE detector has a scintillator of the same material. Both detectors have their own light guides which are connected to a single photomultiplier. The choice of signal is made with a mechanical diaphragm mounted on a flange between the light guide and the photomultiplier. The SE detector may be replaced by a second BSE detector to allow the detection of “low” take-off angle BSEs to provide information which differs from that given by the annular BSE detector which operates to detect BSEs with a “high” take-off angle. In this way it is possible to image either material or topographic contrast with high resolution and to take advantage of the choice of detected electrons.     

Crystals with fivefold symmetry

A digital processing system has been applied to the signals of a multiple detector system for secondary (SE) and backscattered electrons (BSE) in a SEM. The system provides the usual contrast enhancement procedures, Fourier transform and correlation and, in addition, the summation, subtraction and division of images from different detectors. The difference signal of two SE detectors can be used to reconstruct the local surface tilt and the surface profile, and a subtraction of a BSE image from a SE image allows one to extract the pure surface information. Methods for correcting image shifts of sequentially recorded micrographs have been applied by making use of a Fourier transform or a cross-correlation.     

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.     

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.     

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.     

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.     

High-resolution backscatter electron imaging of colloidal gold in LVSEM

High‐resolution backscatter electron (BSE) imaging of colloidal gold can be accomplished at low voltage using in‐lens or below‐the‐lens FESEMs equipped with either Autrata‐modified yttrium aluminium garnet (YAG) scintillators doped with cerium, or with BSE to secondary electron (SE) conversion plates. The threshold for BSE detection of colloidal gold was 1.8 keV for the YAG detector, and the BSE/SE conversion was sensitive down to 1 keV. Gold particles (6, 12 and 18 nm) have an atomic number of 79 and were clearly distinguished at 500 000× by materials contrast and easily discriminated from cell surfaces coated with platinum with an atomic number of 78. BSE imaging was relatively insensitive to charging, and build up of carbon contamination on the specimen was transparent to the higher energy BSE.     

Extended algorithm for simulation of light transport in single crystal scintillation detectors for S(T)EM

The new extended Monte Carlo (MC) simulation method for photon transport in S(T)EM back scattered electron (BSE) scintillation detection systems of various shapes is presented in this paper. The method makes use of the random generation of photon emission from a scintillator luminescent centre and describes the trajectory of photons and the efficiency of their transport toward the photocathode of the photomultiplier tube. The paper explains a new algorithm for determining the position of interaction of the photon with the surface of the single crystal scintillator or of the light guide with nearly arbitrary shapes. Some examples of the utilization of the simulation method are also included, and conclusions for very simple edge-guided signal (EGS) scintillation detection systems made. The computer optimized design of the BSE scintillation detector for the S 4000 Hitachi SEM was chosen to demonstrate the capability of this MC simulation method.     

Use of a multichannel pulse height analyser for the analysis of back-scattered SEM images

The use of a simple electronic switch to convert the video signal from a scanning electron microscope to a series of pulses is described. A multichannel pulse height analyser may then be used to perform quantitative image analysis. Examples are given showing how composition analysis, area fraction measurement and image contouring may be performed using the atomic number contrast signal from a back-scattered electron detector. Other detector systems such as scintillators or specimen current imaging could also be used.     

Mechanical scanning of the specimen in the scanning electron microscope

A scanning electron microscope (SEM) system equipped with a motor drive specimen stage fully controlled with a personal computer (PC) has been utilized for obtaining ultralow magnification SEM images. This modem motor drive stage works as a mechanical scanning device. To produce ultra-low magnification SEM images, we use a successful combination of the mechanical scanning, electronic scanning, and digital image processing techniques. This new method is extremely labor and time saving for ultra-low magnification and wide-area observation. The option of ultra-low magnification observation (while maintaining the original SEM functions and performance) is important during a scanning electron microscopy session.     

A nondestructive method for three-dimensional reconstruction of luminescence materials: Principles,data acquisition,image processing

In the study presented here we have tried to state the principles and calculate and visualize models of three-dimensional (3-D)-cathodoluminescence reconstruction of luminescence structures by scanning electron microscopy (SEM). The new technique does not destroy the specimen and uses the variable energy of the electron beam to penetrate to different depths in the specimen volume. The SEM in color cathodoluminescence mode (CCL-SEM) detects integrated panchromatic CL-images for different energies of the electron beam. The use of electron scattering theory in solids and theories of cathodoluminescence and color allow the production of problem-oriented software for the routine processing of primary images. Processed images represent the CCL-SEM displays of separated layers (without CL information from other layers) up to the maximum depth penetrated by the beam. The 3-D reconstruction is carried out through algorithms developed using a personal computer, software, and a set of processed two-dimensional (2-D) images. The first experimental work was accomplished using a multilayer SiC mesastructure. The final reconstructed image of SiC material demonstrates separated epitaxial layers of different SiC polytypes and Z sections (YOZ and XOZ sections). The 3-D image represents the space distribution of CL-spectral data in color CL interpretation.     

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.     

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.     

A method for preparing colored scanning electron micrographs using SE and BSE images

Biological specimens impregnated with heavy metal are observed in an SEM equipped with a sensitive BSE detector. The SE image and the BSE image are successively recorded under the same conditions. Both pictures are displayed coincidentally on a screen, using two projectors of the same type, but with different color filters. The resulting color picture is photographed with color film. Specific regions of the specimen which were impregnated with heavy metal are clearly demonstrated.     

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.     

A simple backscattered electrons and specimen current detection device for the scanning electron microscope

A simple, low-investment device has been developed that allows the collection of backscattered electrons (BSEs) and specimen current (SC) signals for imaging purposes and current measurement. Originally, this system was designed for detection, measurement, and display of specimen current, with a video signal output whose level was modulated by this current. Eventually, a BSE detector was developed, using a graphite disk (about 8 cm in diameter) to collect the BSEs. The disk was mounted on a Philips SEM 5O5, attached and concentrically to the final lens aperture. This configuration gives a large solid angle of collection. The collected charge is further processed by the same electronics used in the aforementioned SC detection system. Electron channeling, topographic contrast with BSE, and material contrast with BSE and SC images can be obtained with reasonably good edge definition.     

Quality improvement of environmental secondary electron detector signal using helium gas in variable pressure scanning electron microscopy

The quality of the image signal obtained from the environmental secondary electron detector (ESED) employed in a variable pressure (VP) SEM can be dramatically improved by using helium gas. The signal-to-noise ratio (SNR) increases gradually in the range of the pressures that can be used in our modified SEM. This method is especially useful in low-voltage VP SEM as well as in a variety of SEM operating conditions, because helium gas can more or less maintain the amount of unscattered primary electrons. In order to measure the SNR precisely, a digital scan generator system for obtaining two images with identical views is employed as a precondition.