Reduction in acquisition time of scanning electron microscopy image using complex hysteresis smoothing

Complex hysteresis smoothing (CHS), which was developed for noise removal of scanning electron microscopy (SEM) images some years ago, is utilized in acquisition of an SEM image. When using CHS together, recording time can be reduced without problems by about one-third under the condition of SEM signal with a comparatively high signal-to-noise ratio (SNR). We do not recognize artificiality in a CHS-filtered image, because it has some advantages, that is, no degradation of resolution, only one easily chosen processing parameter (this parameter can be fixed and used in this study), and no processing artifacts. This originates in the fact that its criterion for distinguishing noise depends simply on the amplitude of the SEM signal. The automation of reduction in acquisition time is not difficult, because CHS successfully works for almost all varieties of SEM images with a fairly high SNR.     

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.     

A statistical model of signal-noise in scanning electron microscopy

A statistical model describing signal-noise generation and development along the signal formation process in a standard scanning electron microscope (SEM) using an Everhart-Thornley secondary electron detector is derived. Noise in the detector signal is modeled to originate from a cascade of five signal conversion stages. Based on the derived model, general conclusions are drawn concerning the total signal-to-noise ratio (SNR) at each stage, and the influence of each stage on the total SNR of the detector signal. The model is furthermore applied to a real-world SEM, and verified by experimental data.     

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.     

Image signal-to-noise ratio estimation using the autoregressive model

In the last two decades, a variety of techniques for signal-to-noise ratio (SNR) estimation in scanning electron microscope (SEM) images have been proposed. However, these techniques can be divided into two groups: first, SNR estimators of good accuracy, but based on impractical assumptions; second, estimators based on realistic assumptions but of poor accuracy. In this paper we propose the implementation of autoregressive (AR)-model interpolation as a solution to the problem. Unlike others, the proposed technique is based on a single SEM image and offers the required accuracy and robustness in estimating SNR values.     

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.     

Nonlinear least squares regression for single image scanning electron microscope signal‐to‐noise ratio estimation

A new method based on nonlinear least squares regression (NLLSR) is formulated to estimate signal‐to‐noise ratio (SNR) of scanning electron microscope (SEM) images. The estimation of SNR value based on NLLSR method is compared with the three existing methods of nearest neighbourhood, first‐order interpolation and the combination of both nearest neighbourhood and first‐order interpolation. Samples of SEM images with different textures, contrasts and edges were used to test the performance of NLLSR method in estimating the SNR values of the SEM images. It is shown that the NLLSR method is able to produce better estimation accuracy as compared to the other three existing methods. According to the SNR results obtained from the experiment, the NLLSR method is able to produce approximately less than 1% of SNR error difference as compared to the other three existing methods.     

Highly accurate SNR measurement using the covariance of two SEM images with the identical view

Quality of an SEM image is strongly influenced by the extent of noise. As a well-known method in the field of SEM, the covariance is applied to measure the signal-to-noise ratio (SNR). This method has potential ability for highly accurate measurement of the SNR, which is hardly known until now. If the precautions discussed in this article are adopted, that method can demonstrate its real ability. These precautions are strongly related to "proper acquisition of two images with the identical view," "alignment of an aperture diaphragm," "reduction of charging phenomena," "elimination of particular noises," and "accurate focusing," As necessary, characteristics in SEM signal and noise are investigated from a few standpoints. When using the maximum performance of this measurement, SNR of many SEM images obtained in a variety of the SEM operating conditions and specimens can be measured accurately.     

The use of helium gas to reduce beam scattering in high vapour pressure scanning electron microscopy applications

Both image quality and the accuracy of x-ray analysis invariable pressure scanning electron microscopes (VPSEMs) are often limited by the spread of the primary electronbeam due to scattering by the introduced gas. The degree of electron scattering depends partly on the atomic number Z of the gas, and the use of a low Z gas such as helium should reduce beam scattering and enhance image quality. Using anuncoated test sample of copper iron sulphide inclusions in calcium fluorite, we show that the reduction in beam scatter produced by helium is more than sufficient to compensate for its reduced efficiency of charge neutralisation. The relative insensitivity to pressure of x-ray measurements in a helium atmosphere compared with air, and the consequent ability to work over a wider range of working distances, pressures, and voltages, make helium potentially the gas of choice for many routine VPSEM applications.     

The reduction of the beam gas interactions in the variable pressure scanning electron microscope with the use of helium gas

This paper presents experimental observations on electron scattering by gases (helium and air) in the specimen chamber of a variable pressure scanning electron microscope. It shows an important reduction of the beam scattering with the use of helium gas, and the consequences for the x-ray microanalysis are discussed.     

Image noise cross-correlation for signal-to-noise ratio estimation in scanning electron microscope images

A new and robust parameter estimation technique, named image noise cross-correlation, is proposed to predict the signal-to-noise ratio (SNR) of scanning electron microscope images. The results of SNR and variance estimation values are tested and compared with nearest neighborhood and first-order interpolation. Overall, the proposed method is best as its estimations for the noise-free peak and SNR are most consistent and accurate to within a certain acceptable degree, compared with the others.     

Rapid freezing and electron microscopy for the arrest of physiological processes

An apparatus for the rapid freezing of tissue is described, which can be used for the electron microscopy of arrested physiological processes. The material is frozen by bringing it in contact with a silver surface cooled to liquid nitrogen temperature at reduced pressure. The freezing surface is protected from condensation of moisture and gases from the air by a flow of helium gas. The cooling of the specimen during its descent through the cold helium is not large enough to interfere with physiological processes. Freezing occurs very rapidly in the surface but is retarded to about 8 msec at a depth of 10 μm. The apparatus was used to freeze frog muscle during contraction.     

Performance of a mixed Lagrange time delay estimation autoregressive (MLTDEAR) model for single-image signal- to-noise ratio estimation in scanning electron microscopy

A novel technique based on the statistical autoregressive (AR) model has recently been developed as a solution to estimate the signal-to-noise ratio (SNR) in scanning electron microscope (SEM) images. In another research study, the authors also developed an algorithm by cascading the AR model with the Lagrange time delay (LTD) estimator. This technique is named the mixed Lagrange time delay estimation autoregressive (MLTDEAR) model. In this paper, the fundamental performance limits for the problem of single-image SNR estimation as derived from the Cramer–Rao inequality is presented. We compared the experimental performances of several existing methods – the simple method, the first-order linear interpolator, the AR-based estimator as well as the MLTDEAR method – with respect to this performance bound. In a few test cases involving different images, the efficiency of the MLTDEAR single-image estimation technique proved to be significantly better than that of the other three methods. Study of the effect of different SEM setting conditions that affect the autocorrelation function curve is also discussed.     

Metrics for focusing in extremely noisy scanning electron microscopy condition

Finding a best focused image in very noisy condition is an extremely difficult task for the SEM user. If a performance, which is much higher than that of an expert in focusing, can be achieved in a computer-controlled scanning electron microscope (SEM), it will be very helpful for our field due to the many possible applications. To accomplish this work, we employ a powerful metric-the covariance obtained by a special scanning method. It can select the best focused image from a series of SEM images acquired by altering the focus of the objective lens under an extremely noisy SEM image condition. The noise immunity of the present method is quantitatively evaluated, and it is further improved based on the obtained evaluation result.     

A retractable electron emitter for the creation of unperturbed pure electron plasmas

A retractable electron emitter has been constructed for the creation of unperturbed pure electron plasmas on magnetic surfaces in the Columbia Non-neutral Torus stellarator. The previous method of electron emission using emitters mounted on stationary rods limited the confinement time to 20 ms. A pneumatically driven system that can retract from the magnetic axis to the last closed flux surface in less than 20 ms while filling the surfaces with electrons was designed. The motion of the retractable emitter was modeled with a system of dynamical equations. The measured position versus time of the emitter agrees well with the model and the fastest axis-to-edge retraction was measured to be 20 ms with 40 psig helium gas driving the pneumatic piston.     

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.     

Automatic 300-4 K temperature cycling apparatus

The apparatus described here automatically cycles small samples between 300 and 4 K by alternately raising and lowering the sample through the neck of a commercial liquid helium storage Dewar. A bellows, which is pressurized by the helium boil-off gas, provides all of the required mechanical motion. By utilizing the cooling available from the boil-off gas, liquid helium helium consumption is limited to 0.03 l/cyc for a 12-g sample. Cycle times can be as short as 5 min.     

A spectroscopic scanning electron microscope design

This paper describes a proposal to improve the design of scanning electron microscopes (SEMs). The design is based upon using an SEM column similar to the conventional one, magnetic sector plates and a mixed field immersion objective lens. The optical axis of the SEM column lies in the horizontal direction and the primary beam is turned through 90 degrees before it reaches the specimen. This arrangement allows for the efficient collection, detection and spectral analysis of the scattered electrons on a hemispherical surface that is located well away from the rest of the SEM column. The proposed SEM design can also be easily extended to incorporate time multiplexed columns and multi-column arrays.     

Electron irradiation-induced destruction of carbon nanotubes in electron microscopes

Observations of carbon nanotubes under exposure to electron beam irradiation in standard transmission electron microscope (TEM) and scanning electron microscope (SEM) systems show that such treatment in some cases can cause severe damage of the nanotube structure, even at electron energies far below the approximate 100 keV threshold for knock-on damage displacing carbon atoms in the graphene structure. We find that the damage we observe in one TEM can be avoided by use of a cold finger. This and the morphology of the damage imply that water vapour, which is present as a background gas in many vacuum chambers, can damage the nanotube structure through electron beam-induced chemical reactions. Though, the dependence on the background gas makes these observations specific for the presently used systems, the results demonstrate the importance of careful assessment of the level of subtle structural damage that the individual electron microscope system can do to nanostructures during standard use.     

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.