Interpretation of secondary electron images obtained using a low vacuum SEM

Charging of insulators in a variable pressure environment was investigated in the context of secondary electron (SE) image formation. Sample charging and ionized gas molecules present in a low vacuum specimen chamber can give rise to SE image contrast. "Charge-induced" SE contrast reflects lateral variations in the charge state of a sample caused by electron irradiation during and prior to image acquisition. This contrast corresponds to SE emission current alterations produced by sub-surface charge deposited by the electron beam. "Ion-induced" contrast results from spatial inhomogeneities in the extent of SE signal inhibition caused by ions in the gaseous environment of a low vacuum scanning electron microscope (SEM). The inhomogeneities are caused by ion focusing onto regions of a sample that correspond to local minima in the magnitude of the surface potential (generated by sub-surface trapped charge), or topographic asperities. The two types of contrast exhibit characteristic dependencies on microscope operating parameters such as scan speed, beam current, gas pressure, detector bias and working distance. These dependencies, explained in terms of the behavior of the gaseous environment and sample charging, can serve as a basis for a correct interpretation of SE images obtained using a low vacuum SEM.     

A comparison of conventional Everhart-Thornley style and in-lens secondary electron detectors: a further variable in scanning electron microscopy

The secondary electron (SE) imaging of several samples across a range of scanning electron microscopes (SEM) and SE detectors under matched operating conditions has generated a highly variable image data set. Using microanalytical conditions (10-15?kV), images from in-column SE detectors reveal the presence of surface films and contaminants that are invisible to conventional Everhart-Thornley SE detectors under the same conditions. Data from studying the effects of working distance, the image resolution derived through contrast transfer function analysis and electrostatic mirror imaging of the SE detectors in operation combine with other studies to suggest that the classically defined SE1 component can be separated from other SE components. SE images obtained by tailored mechanical design and energy-filtering will provide SE images with probe-sized resolution and dominated by surface detail currently only seen in low-voltage SEM, potentially even from thermionic-sourced columns.     

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.     

Objective comparison of scanning ion and scanning electron microscope images

Common and different aspects of scanning electron microscope (SEM) and scanning ion microscope (SIM) images are discussed from a viewpoint of interaction between ion or electron beams and specimens. The SIM images [mostly using 30 keV Ga focused ion beam (FIB)] are sensitive to the sample surface as well as to low-voltage SEM images. Reasons for the SIM images as follows: (1) no backscattered-electron excitation; (2) low yields of backscattered ions; and (3) short ion ranges of 20–40nm, being of the same order of escape depth of secondary electrons (SE) [=(3–5) times the SE mean free path]. Beam charging, channeling, contamination, and surface sputtering are also commented upon.     

Electron and ion imaging of gland cells using the FIB/SEM system

The FIB/SEM system was satisfactorily used for scanning ion (SIM) and scanning electron microscopy (SEM) of gland epithelial cells of a terrestrial isopod Porcellio scaber (Isopoda, Crustacea). The interior of cells was exposed by site-specific in situ focused ion beam (FIB) milling. Scanning ion (SI) imaging was an adequate substitution for scanning electron (SE) imaging when charging rendered SE imaging impossible. No significant differences in resolution between the SI and SE images were observed. The contrast on both the SI and SE images is a topographic. The consequences of SI imaging are, among others, introduction of Ga⁺ ions on/into the samples and destruction of the imaged surface. These two characteristics of SI imaging can be used advantageously. Introduction of Ga⁺ ions onto the specimen neutralizes the charge effect in the subsequent SE imaging. In addition, the destructive nature of SI imaging can be used as a tool for the gradual removal of the exposed layer of the imaged surface, uncovering the structures lying beneath. Alternative SEM and SIM in combination with site-specific in situ FIB sample sectioning made it possible to image the submicrometre structures of gland epithelium cells with reproducibility, repeatability and in the same range of magnifications as in transmission electron microscopy (TEM). At the present state of technology, ultrastructural elements imaged by the FIB/SEM system cannot be directly identified by comparison with TEM images.     

Application of the low-loss scanning electron microscope image to integrated circuit technology part II--chemically-mechanically planarized samples.

Chemical-mechanical planarization (CMP) is a process that gives a flat surface on a silicon wafer by removing material from above a chosen level. This flat surface must then be reviewed (typically using a laser) and inspected for scratches and other topographic defects. This inspection has been done using both the atomic force microscope (AFM) and the scanning electron microscope (SEM), each of which has its own advantages and disadvantages. In this study, the low-loss electron (LLE) method in the SEM was applied to CMP samples at close to a right angle to the beam. The LLEs show shallower topographic defects more clearly than it is possible with the secondary electron (SE) imaging method. These images were then calibrated and compared with those obtained using the AFM, showing the value of both methods. It is believed that the next step is to examine such samples at a right angle to the beam in the SEM using the magnetically filtered LLE imaging method.     

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.     

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.     

Monte Carlo simulation of secondary electron and backscattered electron images in scanning electron microscopy for specimen with complex geometric structure

A new Monte Carlo technique for the simulation of secondary electron (SE) and backscattered electron (BSE) of scanning electron microscopy (SEM) images for an inhomogeneous specimen with a complex geometric structure has been developed. The simulation is based on structure construction modeling with simple geometric structures, as well as on the ray-tracing technique for correction of electron flight-step-length sampling when an electron trajectory crosses the interface of the inhomogeneous structures. This correction is important for the simulation of nanoscale structures of a size comparable with or even less than the electron scattering mean free paths. The physical model for electron transport in solids combines the use of the Mott cross section for electron elastic scattering and a dielectric function approach for electron inelastic scattering, and the cascade SE production is also included.     

Imaging colloidal gold labels in LVSEM

On biological samples, the topographic imaging capabilities of the new generation of scanning electron microscopes (SEM) (those having both field-emission guns and low aberration lenses) rival those of the replica techniques. In addition, they permit the localization of specific molecules on the sample surface using one of several labeling techniques utilizing heavy metal colloids. Normally, colloidal gold can be detected in the SEM both by the secondary electron signal (shape) and by the backscattered electron signal (BSE, Z-contrast). The new instruments seem to produce their best topographic images using low-beam voltage (1–5 kV) where topographic contrast is higher and the required thickness of the metal coating is less (Haggis and Pawley 1988, Ris and Pawley 1988). Although the detection of backscattered electrons is more difficult at low-beam voltage, we are able to show here that the secondary electron (SE) signal produced with a 2–5-kV beam permits the unambiguous detection of gold particles as small as 5 nm on carbon-coated specimens while a 1-kV beam produces a high-quality topographic image of the same sample.     

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.     

Monte Carlo Simulation of CD‐SEM Images for Linewidth and Critical Dimension Metrology

In semiconductor industry, strict critical dimension control by using a critical dimension scanning electron microscope (CD‐SEM) is an extremely urgent task in near‐term years. A Monte Carlo simulation model for study of CD‐SEM image has been established, which is based on using Mott's cross section for electron elastic scattering and the full Penn dielectric function formalism for electron inelastic scattering and the associated secondary electron (SE) production. In this work, a systematic calculation of CD‐SEM line‐scan profiles and 2D images of trapezoidal Si lines has been performed by taking into account different experimental factors including electron beam condition (primary energy, probe size), line geometry (width, height, foot/corner rounding, sidewall angle, and roughness), material properties, and SE signal detection. The influences of these factors to the critical dimension metrology are investigated, leading to build a future comprehensive model‐based library. SCANNING 35: 127‐139, 2013. © 2012 Wiley Periodicals, Inc.     

Measurement technique for the incident electron current in secondary electron detectors and its application in scanning electron microscopes.

A measurement technique for incident electron current in secondary electron (SE) detectors, especially the Everhart-Thornley (ET) detector, based on signal-to-noise ratio (SNR), which uses the histogram of a digital scanning electron microscope (SEM) image, is described. In this technique, primary electrons are directly incident on the ET detector. This technique for measuring the correlation between incident electron current and SNR is applicable to the other SE detectors. This correlation was applied to estimate the efficiency of the ET detector itself, to evaluate SEM image quality, and to measure the geometric SE collection efficiency and the SE yield. It was found that the geometric SE collection efficiency at each of the upper and lower detectors of a Hitachi S-4500 SEM was greater than 0.78 at all working distances.     

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.     

Image contrast enhancement of Ni/YSZ anode during the slice‐and‐view process in FIB‐SEM

Focused ion beam‐scanning electron microscopy (FIB‐SEM) is a widely used and easily operational equipment for three‐dimensional reconstruction with flexible analysis volume. It has been using successfully and increasingly in the field of solid oxide fuel cell. However, the phase contrast of the SEM images is indistinct in many cases, which will bring difficulties to the image processing. Herein, the phase contrast of a conventional Ni/yttria stabilized zirconia anode is tuned in an FIB‐SEM with In‐Lens secondary electron (SE) and backscattered electron detectors. Two accessories, tungsten probe and carbon nozzle, are inserted during the observation. The former has no influence on the contrast. When the carbon nozzle is inserted, best and distinct contrast can be obtained by In‐Lens SE detector. This method is novel for contrast enhancement. Phase segmentation of the image can be automatically performed. The related mechanism for different images is discussed.     

Observation of GaAs—AlxGa1 — x as heterostructures and quantum-well-wire structures using backscattered electron image

As a microscale tool for observing GaAs-Al_x Ga_1–xAs heterostructures, backscattered electron (BE) images in the scanning electron microscope (SEM) were compared with conventional secondary electron (SE) images. BE images were found to be more sensitive to compositional differences between GaAs and Al_xGa_1–xAs and less sensitive to surface roughness. BE images have a spatial resolution of 10 nm or better. This method enables the nondestructive observation of ultrafine lateral periodic structures, such as quantum-well-wire (QWW) structures, fabricated by compositional disordering technology using focused Ga ion-beam (Ga-FIB) implantation into GaAs-Al_xGa_1–xAs material.     

Transient analysis of gaseous electron-ion recombination in the environmental scanning electron microscope

Most of the work carried out in relation to contrast mechanisms and signal formation in an environmental scanning electron microscope has yet to consider the time dependent aspects of image generation at a quantitative level. This paper quantitatively describes gaseous electron‐ion recombination (also known as ‘signal scavenging’) in an environmental scanning electron microscope at a transient level by utilizing the dark shadows/streaks seen in gaseous secondary electron detector images of alumina (Al₂O₃) immediately after a region of enhanced secondary electron emission is encountered by a scanning electron beam. The investigation firstly derives a theoretical model of gaseous electron‐ion recombination that takes into consideration transients caused by the time constant of the gaseous secondary electron detector electronics and external circuitry used to generate images. Experimental data of pixel intensity versus time of the streaks are then simulated using the model enabling the relative magnitudes of (i) ionization and recombination rates, (ii) recombination coefficients and (iii) electron drift velocities, as well as absolute values of the total time constant of the gaseous secondary electron detection system and external circuitry, to be determined as a function of microscope operating parameters such as gaseous secondary electron detector bias, sample‐electrode separation, imaging gas pressure, and scan speed. The results revealed, for the first time, the exact dependence that the effects of secondary electron‐ion recombination on signal formation has on reduced electric field and time in an environmental scanning electron microscope. Furthermore, the model implicitly demonstrated that signal loss as a consequence of field retardation due to ion space charges, although obviously present, is not the foremost phenomenon causing streaking in images, as previously thought.     

Chitosan utilized for bacterial preparation for scanning electron microscopy

Bacterial sample preparation is crucial for its observation by scanning electron microscopy (SEM). However, the current polylysine (PLL) method leads to bacterial morphological changes. To overcome this problem, we employed chitosan (CS) to coat coverslips to prepare bacteria for SEM and compared it with the PLL method. Coverslips coated with 0.025% (w/v) CS showed satisfactory bacterial binding ability. Within 30 min of binding time, the number of bacteria on CS-coated and PLL-coated coverslips exhibited no differences. Four bacteria strains were employed to compare the differences in SEM images between the two methods. Most of the bacteria showed irregular surface or sticky substances after settling on PLL-coated coverslips, while bacteria with clear surface texture were observed on CS-coated coverslips. Transmission electron microscopy (TEM) images showed deformed bacterial envelope on PLL-coated coverslips; meanwhile, similar intact envelope was observed from the bacteria on CS-coated coverslips and the bacteria without any treatment. The TEM results verified the morphological differences of SEM between the two methods. Except for morphology, the length of the rod-shaped bacteria was longer on CS-coated coverslips than that on PLL-coated coverslips, less shrinkage of the sample was observed, and CS could preserve the length of the rod-shaped bacteria better than PLL in its preparation for SEM. It is demonstrated that the low-cost CS could be utilized in bacterial preparation for SEM to acquire preferable images. Bacterial suspension with optical density at 600 nm of about 0.5, deposited on 0.025% CS-coated coverslips for 30 min, and followed by routine fixation, dehydration, and drying are optimal parameters.     

Scanning microscopies of indented MgO crystals

The combination of scanning electron microscopy (SEM) and scanning optical microscopy (SOM), including a computer-controlled signal detection system, is promising in the study of a variety of materials, especially such alkaline-earth oxides with a rock salt structure, such as MgO. Among the SEM modes of this technique used to investigate deformed zones in indented MgO single crystals are: secondary electrons (SE), cathodoluminescence (CL) (total, pointal, color), electron beam-induced current (EBIC), electron beam-induced voltage (EBIV), as well as both polarized and transmitted light modes in SOM. The present experiments were designed to clarify the correlation between the optical, luminescent, electrical, and plastic properties of deformed MgO. An attempt has been made to explain the results in terms of dislocations created during deformation.     

Advantages of indium–tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium–tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.