Improved depth of field in the scanning electron microscope derived from through-focus image stacks

The depth of field limit in the scanning electron microscope (SEM) can be overcome by recording stacks of through-focus images (as in conventional and confocal optical microscopy) which are postprocessed to generate an all-in-focus image. Images are recorded under constant electron optical conditions by mechanical Z-axis movement of the sample. This gives rise to a change in magnification through the stack due to the perspective projection of the SEM image. Calculation of the necessary scaling as well as the derivation of best focus information at every patch in the image--and a contour map function derived from the selected patch depths--are incorporated in a new software package (Auto-Montage Pro). The utility of these procedures is demonstrated with examples from the study of human osteoporotic bone, where results show uncoupling of resorption and formation. The procedure can be combined with pseudo-colour coding for the direction of apparent illumination when using backscattered electron (BSE) detectors in contrasting positions.     

Reproducibility and accuracy of spatial measurements from digitally captured images in the environmental scanning electron microscope

Accurate spatial measurements in a scanning electron microscope (SEM) require calibration of the magnification as a function of working distance and microscope operating conditions. This work presents the results of the calibration of an environmental SEM for the accurate spatial measurement of dimensions and areas in experiments, both for the measurement of strain in steel specimens under applied loads and the measurement of dimensional changes in timber with changes in relative humidity.     

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.     

Practical method for high-resolution imaging of polymers by low-voltage scanning electron microscopy

Morphologic characterization of polymers by scanning electron microscopy (SEM) is often made difficult by their sensitivity to electron beam damage. We describe here a specimen preparation method for the imaging of polymer blends by low-voltage SEM (LV-SEM) that improves their stability in the electron beam and hence facilitates focusing and recording of high magnification images. Its application to nanosized core-shell latexes embedded in a polymethylmethacrylate matrix and semi-crystalline polypropylene/ethylene-propylene rubber blends is discussed.     

Natural color scanning electron microscopy based on the frequency characteristics of the human visual system

The principles of image formation in natural color scanning electron microscopy (NC-SEM) are discussed in detail. This method is based on the frequency characteristic of the human visual system. It is shown that the Mach effect and masking effect are important in the characteristics. The former, which can enhance structural details, is visually similar to the edge effect in secondary electron (SE) images, and the latter is required for proper representation of very degraded color information obtained from a light microscope. When using these effects suitably, an NC-SEM image with the resolution equivalent to that of an SEM image can be acquired, though it is composed of an SEM image and a special video microscopy (VM) image with a resolution much lower than the SEM image of the identical view. The NC-SEM is more effective than the SEM in observation. interpretation, and analysis of various samples with important color information.     

A simple cryo-holder facilitates specimen observation under a conventional scanning electron microscope

A pre-cryogenic holder (cryo-holder) facilitating cryo-specimen observation under a conventional scanning electron microscope (SEM) is described. This cryo-holder includes a specimen-holding unit (the stub) and a cryogenic energy-storing unit (a composite of three cylinders assembled with a screw). After cooling, the cryo-holder can continue supplying cryogenic energy to extend the observation time for the specimen in a conventional SEM. Moreover, the cryogenic energy-storing unit could retain appropriate liquid nitrogen that can evaporate to prevent frost deposition on the surface of the specimen. This device is proved feasible for various tissues and cells, and can be applied to the fields of both biology and material science. We have employed this novel cryo-holder for observation of yeast cells, trichome, and epidermal cells in the leaf of Arabidopsis thaliana, compound eyes of insects, red blood cells, filiform papillae on the surface of rat tongue, agar medium, water molecules, penicillium, etc. All results suggested that the newly designed cryo-holder is applicable for cryo-specimen observation under a conventional SEM without cooling system. Most importantly, the design of this cryo-holder is simple and easy to operate and could adapt a conventional SEM to a plain type cryo-SEM affordable for most laboratories.     

An approach to the reduction of hydrocarbon contamination in the scanning electron microscope

The deposition of electron beam-induced specimen contamination in both the transmission (TEM) and scanning electron microscopes (SEM) has remained a problem since the beginning of these forms of microscopy. Generally, sources of SEM contamination can be attributed to one or a combination of three major contributors: (1) the pumping system; (2) outgassing of other internal SEM component parts (i.e., specimen stage, stage lubricants, O-rings, etc.), or (3) the sample (including its preparation and handling). Generally, because of the nature of SEM, specimen contamination can be minimized but is difficult to avoid fully. This work outlines three approaches taken with instruments at NIST to reduce the deposition of contamination in high-resolution cold-field emission SEMs. With some modification these techniques could be applied to any SEM. These approaches have been in successful operation for several years, resulting in a reduction in electron beam-induced hydrocarbon contamination.     

Time-lapse scanning electron microscopy for measurement of contamination rate and stage drift

Measurements done with scanning electron microscopes (SEMs) may lose their validity due to contamination and charging caused by the primary electron beam. The sample stage and the electron beam also slightly drift during the course of the measurements. Consequently, it is essential to find out the time limit of valid measurements, that is, the maximum time before the sample or its position changes too much. This paper describes digital time-lapse SEM, a useful tool for investigating the extent and effects of contamination and stage drift. It works with hardware and software that create a stack of sequential images. Later these images can be viewed as a short movie; it is also possible to apply all the image processing and analysis procedures that are otherwise applied to separate, individual images. This method gives a reliable way of measuring the rate of contaminant deposition and of stage and electron beam drift of SEMs, and it could be helpful in controlling these problems.     

Nanotip electron gun for the scanning electron microscope

Experimental nanotips have shown significant improvement in the resolution performance of a cold field emission scanning electron microscope (SEM). Nanotip electron sources are very sharp electron emitter tips used as a replacement for the conventional tungsten field emission (FE) electron sources. Nanotips offer higher brightness and smaller electron source size. An electron microscope equipped with a nanotip electron gun can provide images with higher spatial resolution and with better signal-to-noise ratio. This could present a considerable advantage over the current SEM electron gun technology if the tips are sufficiently long-lasting and stable for practical use. In this study, an older field-emission critical dimension (CD) SEM was used as an experimental test platform. Substitution of tungsten nanotips for the regular cathodes required modification of the electron gun circuitry and preparation of nanotips that properly fit the electron gun assembly. In addition, this work contains the results of the modeling and theoretical calculation of the electron gun performance for regular and nanotips, the preparation of the SEM including the design and assembly of a measuring system for essential instrument parameters, design and modification of the electron gun control electronics, development of a procedure for tip exchange, and tests of regular emitter, sharp emitter and nanotips. Nanotip fabrication and characterization procedures were also developed. Using a "sharp" tip as an intermediate to the nanotip clearly demonstrated an improvement in the performance of the test SEM. This and the results of the theoretical assessment gave support for the installation of the nanotips as the next step and pointed to potentially even better performance. Images taken with experimental nanotips showed a minimum two-fold improvement in resolution performance than the specification of the test SEM. The stability of the nanotip electron gun was excellent; the tip stayed useful for high-resolution imaging for several hours during many days of tests. The tip lifetime was found to be several months in light use. This paper summarizes the current state of the work and points to future possibilities that will open when electron guns can be designed to take full advantage of the nanotip electron emitters.     

Focused ion beam scanning electron microscopy in biology

Since the end of the last millennium, the focused ion beam scanning electron microscopy (FIB‐SEM) has progressively found use in biological research. This instrument is a scanning electron microscope (SEM) with an attached gallium ion column and the 2 beams, electrons and ions (FIB) are focused on one coincident point. The main application is the acquisition of three‐dimensional data, FIB‐SEM tomography. With the ion beam, some nanometres of the surface are removed and the remaining block‐face is imaged with the electron beam in a repetitive manner. The instrument can also be used to cut open biological structures to get access to internal structures or to prepare thin lamella for imaging by (cryo‐) transmission electron microscopy. Here, we will present an overview of the development of FIB‐SEM and discuss a few points about sample preparation and imaging.     

Optimisation of the environmental scanning electron microscope for observation of drying of matt water-based lacquers

Environmental scanning electron microscopy (ESEM) modifies conventional SEM through the use of a partial gas pressure in the microscope specimen chamber. Like conventional SEM, it has the resolution to image structure on the submicron lengthscale, but can also tolerate hydrated specimens if water vapour is used in the specimen chamber. This ability to image aqueous specimens leaves ESEM uniquely placed to study in situ drying in polymer latexes. However, there are two key practical difficulties associated with in situ drying. First, the size of the latex particles: larger latex particles are typically around 500 nm in diameter. Although ESEM can resolve structure on this lengthscale without difficulty, the magnification required results in radiation damage of the specimen due to the electron beam. This means that a given region can be imaged only once during film formation, so the evolution of particular features cannot be followed. Second, the change from ambient temperature and pressure to the ESEM conditions of 7 degrees C and 7.5 torr (100 Pa) can subject the specimen to a very high evaporation rate, which can disrupt film formation. The inclusion of a drop of water in the specimen chamber is shown largely to alleviate this, enabling successful imaging of film formation in the lacquer. Instead of the polymer latex itself, this work concentrates on a matting lacquer with silica inclusions. The silica matting agent particles are 1-10 microm in size, allowing for a lower magnification to be used, massively reducing specimen damage. Furthermore, the contrast during drying is much enhanced in the presence of silica. The images reveal the silica as bright regions against a darker background of polymer and water. Film formation shows the transition from a uniform, featureless aqueous solution to a polymer film with silica particles present on the surface. The appearance of individual silica particles can be followed. The particles are generally revealed quite early, after a few minutes of drying time. As film formation progresses, these same particles appear larger and more distinct. Few new particles are revealed at longer film formation times.     

An automated image alignment system for the scanning electron microscope

We have developed an automated image alignment system for the scanning electron microscope (SEM). This system enables specific locations on a sample to be located and automatically aligned with submicron accuracy. The system comprises a sample stage motorization and control unit together with dedicated imaging electronics and image processing software. The standard SEM sample stage is motorized in the X and Y axes with stepping motors which are fitted with rotary optical encoders. The imaging electronics are interfaced to beam deflection electronics of the SEM and provide the image data for the image processing software. The system initially moves the motorized sample stage to the area of interest and acquires an image. This image is compared with a reference image to determine the required adjustments to the stage position or beam deflection. This procedure is repeated until the area imaged by the SEM matches the reference image. A hierarchical image correlation technique is used to achieve submicron alignment accuracy in a few seconds. The ability to control the SEM beam deflection enables the images to be aligned with an accuracy far exceeding the positioning ability of the SEM stage. The alignment system may be used on a variety of samples without the need for registration or alignment marks since the features in the SEM image are used for alignment. This system has been used for the automatic inspection of devices on semiconductor wafers, and has also enabled the SEM to be used for direct write self-aligned electron beam lithography.     

Imaging of specimens at optimized low and very low energies in scanning electron microscopes.

The modern trend towards low electron energies in scanning electron microscopy (SEM), characterised by lowering the acceleration voltages in low-voltage SEM (LVSEM) or by utilising a retarding-field optical element in low-energy SEM (LESEM), makes the energy range where new contrasts appear accessible. This range is further extended by a scanning low-energy electron microscope (SLEEM) fitted with a cathode lens that achieves nearly constant spatial resolution throughout the energy scale. This enables one to optimise freely the electron beam energy according to the given task. At low energies, there exist classes of image contrast that make particular specimen data visible most effectively or even exclusively within certain energy intervals or at certain energy values. Some contrasts are well understood and can presently be utilised for practical surface examinations, but others have not yet been reliably explained and therefore supplementary experiments are needed.     

Software acceleration techniques for the simulation of scanning electron microscope images

A scanning electron microscope (SEM) simulator was developed based on the models used in the MONSEL software. This simulator extends earlier work by introducing an object-oriented framework and adding optimization methods based on precomputation of electron trajectories. Several optimizations enable speedup by factors of 5-100 on a single processor over unoptimized simulations without introducing additional approximations. The speedup for a particular surface depends on the self-similarity of the surface at the scale of the electron penetration depth. We further accelerate by parallelizing the calculations for a total speedup of about 100-2000 on 30 processors. The goal of this work was to create a system capable of simulating a quantitatively accurate SEM image of a relatively unconstrained surface. Results of this work include simulation software, optimization algorithms, performance measurements with various optimizations, and examples of simulated images.     

Image sharpness measurement in the scanning electron-microscope--part III.

Fully automated or semi-automated scanning electron microscopes (SEM) are now commonly used in semiconductor production and other forms of manufacturing. Testing and proving that the instrument is performing at a satisfactory level of sharpness is an important aspect of quality control. The application of Fourier analysis techniques to the analysis of SEM images is a useful methodology for sharpness measurement. In this paper, a statistical measure known as the multivariate kurtosis is proposed as an additional useful measure of the sharpness of SEM images. Kurtosis is designed to be a measure of the degree of departure of a probability distribution. For selected SEM images, the two-dimensional spatial Fourier transforms were computed. Then the bivariate kurtosis of this Fourier transform was calculated as though it were a probability distribution. Kurtosis has the distinct advantage that it is a parametric (i.e., a dimensionless) measure and is sensitive to the presence of the high spatial frequencies necessary for acceptable levels of image sharpness. The applications of this method to SEM metrology will be discussed.     

Correction of image drift and distortion in a scanning electron microscopy

Continuous research on small‐scale mechanical structures and systems has attracted strong demand for ultrafine deformation and strain measurements. Conventional optical microscope cannot meet such requirements owing to its lower spatial resolution. Therefore, high‐resolution scanning electron microscope has become the preferred system for high spatial resolution imaging and measurements. However, scanning electron microscope usually is contaminated by distortion and drift aberrations which cause serious errors to precise imaging and measurements of tiny structures. This paper develops a new method to correct drift and distortion aberrations of scanning electron microscope images, and evaluates the effect of correction by comparing corrected images with scanning electron microscope image of a standard sample. The drift correction is based on the interpolation scheme, where a series of images are captured at one location of the sample and perform image correlation between the first image and the consequent images to interpolate the drift–time relationship of scanning electron microscope images. The distortion correction employs the axial symmetry model of charged particle imaging theory to two images sharing with the same location of one object under different imaging fields of view. The difference apart from rigid displacement between the mentioned two images will give distortion parameters. Three‐order precision is considered in the model and experiment shows that one pixel maximum correction is obtained for the employed high‐resolution electron microscopic system.     

Performance of signal‐to‐noise ratio estimation for scanning electron microscope using autocorrelation Levinson–Durbin recursion model

A new technique to quantify signal‐to‐noise ratio (SNR) value of the scanning electron microscope (SEM) images is proposed. This technique is known as autocorrelation Levinson–Durbin recursion (ACLDR) model. To test the performance of this technique, the SEM image is corrupted with noise. The autocorrelation function of the original image and the noisy image are formed. The signal spectrum based on the autocorrelation function of image is formed. ACLDR is then used as an SNR estimator to quantify the signal spectrum of noisy image. The SNR values of the original image and the quantified image are calculated. The ACLDR is then compared with the three existing techniques, which are nearest neighbourhood, first‐order linear interpolation and nearest neighbourhood combined with first‐order linear interpolation. It is shown that ACLDR model is able to achieve higher accuracy in SNR estimation.     

An improved scanning method based on characteristics of the human visual system for scanning electron microscopy

An improved scanning method for the scanning electron microscope (SEM) is proposed. Here, quincuncial scanning (sampling) instead of a conventional (raster) scanning is used. This scanning method is very effective for quality improvement of an SEM image obtained under undersampling conditions (rough sampling). The present study focuses on characteristics of the human visual system, specifically the low response of eyes in diagonal directions. When using this method coupled with a high-precision interpolation, the number of pixels necessarily doubles. It is not surprising that it is advantageous for printing. A more important advantage is the fact that SEM images can be acquired with a shorter recording time. Hence, this type of scanning will be helpful for quick and frequent recordings in a "snapshot" mode, which up to now has not been achieved successfully by SEM.     

Reduction of charging effects using vector scanning in the scanning electron microscope.

We describe a vector scanning system to reduce charging effects during scanning electron microscope (SEM) imaging. The vector scan technique exploits the intrinsic charge decay mechanism of the specimen to improve imaging conditions. We compare SEM images obtained by conventional raster scanning versus vector scanning to demonstrate that vector scanning successfully reduces specimen-charging artifacts.