Differential contrast imaging of secondary electron signals in cryo-field-emission scanning electron microscopy

Low-temperature scanning electron microscopy (LTSEM) is limited in resolution and image quality by charging of frozen hydrated samples and collection deficiencies of secondary electron signal contrasts. We measured and corrected both effects using differential hysteresis processing (DHP) of LTSEM images, scanned at 15-bit from 5×4 inch Polaroid negatives. Bulk charging produced a major contrast component equal to 44–87% of the intensity range of the image. The strong charging contrast reduced the local high-resolution signal contrasts to an unrecognizable level. Segmentation and imaging of the unaffected surface contrasts produced high-quality images of high contrast from metal-coated samples as well as from uncoated samples. The differential contrast imaging can be used for control of the sequential etching of ice from the non metal-coated sample as well as improved LTSEM imaging of the finally coated sample.     

The role of induced contrast in images obtained using the environmental scanning electron microscope

Generation of contrast in images obtained using the environmental scanning electron microscope (ESEM) is explained by interpretation of images acquired using the gaseous secondary electron detector (GSED), ion current, and the Everhart-Thornley detector. We present a previously unreported contrast component in GSED and ion current images attributed to signal induction by changes in the concentration of positive ions in the ESEM chamber during image acquisition. Changes in positive ion concentration are caused by changes in electron emission from the sample during image acquisition and by a discrepancy between the drift velocities of negative and positive charge carriers in the imaging gas. The proposed signal generation mechanism is used to explain contrast reversal in images produced using the GSED and ion current signals and accounts for discrepancies in contrast observed, under some conditions, in these types of images. Combined with existing models of signal generation in the ESEM, the proposed model provides a basis for correct interpretation of ESEM images.     

High‐performance serial block‐face SEM of nonconductive biological samples enabled by focal gas injection‐based charge compensation

A longstanding limitation of imaging with serial block‐face scanning electron microscopy is specimen surface charging. This charging is largely due to the difficulties in making biological specimens and the resins in which they are embedded sufficiently conductive. Local accumulation of charge on the specimen surface can result in poor image quality and distortions. Even minor charging can lead to misalignments between sequential images of the block‐face due to image jitter. Typically, variable‐pressure SEM is used to reduce specimen charging, but this results in a significant reduction to spatial resolution, signal‐to‐noise ratio and overall image quality. Here we show the development and application of a simple system that effectively mitigates specimen charging by using focal gas injection of nitrogen over the sample block‐face during imaging. A standard gas injection valve is paired with a precisely positioned but retractable application nozzle, which is mechanically coupled to the reciprocating action of the serial block‐face ultramicrotome. This system enables the application of nitrogen gas precisely over the block‐face during imaging while allowing the specimen chamber to be maintained under high vacuum to maximise achievable SEM image resolution. The action of the ultramicrotome drives the nozzle retraction, automatically moving it away from the specimen area during the cutting cycle of the knife. The device described was added to a Gatan 3View system with minimal modifications, allowing high‐resolution block‐face imaging of even the most charge prone of epoxy‐embedded biological samples.     

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.     

Imaging of semiconducting polymer blend systems using environmental scanning electron microscopy and environmental scanning transmission electron microscopy

This study has investigated the potential of environmental electron microscopy techniques for studying the structure of polymer‐based electronic devices. Polymer blend systems composed of F8BT and PFB were examined. Excellent contrast, both topographical and compositional, can be achieved using both conventional environmental scanning electron microscopy (ESEM) and a transmission detector giving an environmental scanning transmission electron microscope (ESTEM) configuration. Controllable charging effects present in the ESEM were observed, giving rise to a novel voltage contrast. This shows the potential of such contrast to provide excellent images of phase structure and charge distributions.     

Image quality evaluation for FIB-SEM images

Focused ion beam scanning electron microscopy (FIB-SEM) tomography is a serial sectioning technique where an FIB mills off slices from the material sample that is being analysed. After every slicing, an SEM image is taken showing the newly exposed layer of the sample. By combining all slices in a stack, a 3D image of the material is generated. However, specific artefacts caused by the imaging technique distort the images, hampering the morphological analysis of the structure. Typical quality problems in microscopy imaging are noise and lack of contrast or focus. Moreover, specific artefacts are caused by the FIB milling, namely, curtaining and charging artefacts. We propose quality indices for the evaluation of the quality of FIB-SEM data sets. The indices are validated on real and experimental data of different structures and materials.     

Parameters and mechanisms governing image contrast in scanning electron microscopy of single-walled carbon nanotubes

Image formation of single-walled carbon nanotubes (SWNTs) in the scanning electron microscope (SEM) is peculiarly sensitive to primary electron landing energy, imaging history, sample/substrate geometry, electrical conductivity, sample contamination, and substrate charging. This sensitivity is probably due to the extremely small interaction volume of the SWNTs' monolayered, nanoscale structures with the electron beam. Traditional electron beam/bulk specimen interaction models appear unable to explain the contrast behavior when directly applied to SWNTs. We present one systematic case study of SWNT SEM imaging with special attention to the above parameters and propose some physical explanations for the effect of each. We also demonstrate that it is possible to employ voltage biasing to counteract this extrinsic behavior, gain better control of the image contrast, and facilitate the interpretation of SWNT images in the SEM.     

Scanning electron microscope charging effect model for chromium/quartz photolithography masks

We propose a new method for fitting a model of specimen charging to scanning electron microscope (SEM) images. Charging effects cause errors when one attempts to infer the size or shape of a specimen from an image. The goal of our method is to enable image analysis algorithms for measurement, segmentation, and three-dimensional (3-D) reconstruction that would otherwise fail on images containing charging effects. Our model is applied to images of chromium/quartz photolithography masks and may also work in the more general case of isolated metal islands on a flat insulating substrate. Unlike methods based on Monte Carlo simulation, our simulation method does not handle more general topographies or specimens composed entirely of an insulator; it is a crude approximation to the physical charging process described in more detail in Cazaux (1986) and Melchinger and Hofmann (1985), but can be fit with quantitative accuracy to real SEM images. We only consider changes in intensity and do not model charging-induced distortion of image coordinates. Our approach has the advantage over existing methods of enabling fast prediction of charging effects so it may be more practical for image analysis applications.     

Reducing charging effects in scanning electron microscope images by Rayleigh contrast stretching method (RCS)

To reduce undesirable charging effects in scanning electron microscope images, Rayleigh contrast stretching is developed and employed. First, re-scaling is performed on the input image histograms with Rayleigh algorithm. Then, contrast stretching or contrast adjustment is implemented to improve the images while reducing the contrast charging artifacts. This technique has been compared to some existing histogram equalization (HE) extension techniques: recursive sub-image HE, contrast stretching dynamic HE, multipeak HE and recursive mean separate HE. Other post processing methods, such as wavelet approach, spatial filtering, and exponential contrast stretching, are compared as well. Overall, the proposed method produces better image compensation in reducing charging artifacts.     

Images of paraffin monolayer crystals with perfect contrast: minimization of beam-induced specimen motion

Quantitative analysis of electron microscope images of organic and biological two-dimensional crystals has previously shown that the absolute contrast reached only a fraction of that expected theoretically from the electron diffraction amplitudes. The accepted explanation for this is that irradiation of the specimen causes beam-induced charging or movement, which in turn causes blurring of the image due to image or specimen movement. In this paper, we used three different approaches to try to overcome this image-blurring problem in monolayer crystals of paraffin. Our first approach was to use an extreme form of spotscan imaging, in which a single image was assembled on film by the successive illumination of up to 50,000 spots, each of a diameter of around 7 nm. The second approach was to use the Medipix II detector with its zero-noise readout to assemble a time-sliced series of images of the same area in which each frame from a movie with up to 400 frames had an exposure of only 500 electrons. In the third approach, we simply used a much thicker carbon support film to increase the physical strength and conductivity of the support. Surprisingly, the first two methods involving dose fractionation in space or time produced only partial improvements in contrast whereas the third approach produced many virtually perfect images, where the absolute contrast predicted from the electron diffraction amplitudes was observed in the images. We conclude that it is possible to obtain consistently almost perfect images of beam-sensitive specimens if they are attached to an appropriately strong and conductive support; however great care is needed in practice and the problem remains of how to best image ice-embedded biological structures in the absence of a strong, conductive support film.     

Derivation of Optimal Global Equalization Function with Variable Size Block Based Local Contrast Enhancement

A conventional global contrast enhancement is difficult to apply in various images because image quality and contrast enhancement are depndent on image characteristics largely.And a local contrast enhancement not only causes a washed-out effect,but also blocks.To solve these drawbacks,this paper derives an optimal global equalization function with variable size block based local contrast enhancement.The optimal equalization function makes it possible to get a good quality image through the global contrast enhancement.The variable size block segmentation is firstly executed using intensity differences as a measure of similarity.In the second step,the optimal global equalization function is obtained from the enhanced contrast image having variable size blocks.Conformed experiments have showed that the proposed algorithm produces a visually comfortable result image.     

High-pressure scanning electron microscopy of insulating materials: A new approach

A new SEM technique for imaging uncoated non-conducting specimens at high beam voltages is described which employs a high-pressure environment and an electric field to achieve charge neutralization. During imaging, the specimen surface is kept at a stable low voltage, near earth potential, by directing a flow of positive gas ions at the specimen surface under the action of an electric bias field at a pressure of about 200 Pa. In this way charge neutrality is continuously maintained to obtain micrographs free of charging artefacts. Images are formed by specimen current detection containing both secondary electron and backscattered electron signal information. Micrographs of geological, ceramic, and semiconductor materials obtained with this method are presented. The technique is also useful for the SEM examination of histological sections of biological specimens without any further preparation. A simple theory for the charge neutralization process is described. It is based on the interaction of the primary and emissive signal components with the surrounding gas medium and the resulting neutralizing currents. Further micrographs are presented to illustrate the pressure dependence of the charge neutralization process in two glass specimens which show clearly identifiable charging artefacts in conventional microscopy.     

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.     

Variable bright-darkfield-contrast, a new illumination technique for improved visualizations of complex structured transparent specimens

Variable bright-darkfield contrast (VBDC) is a new technique in light microscopy which promises significant improvements in imaging of transparent colorless specimens especially when characterized by a high regional thickness and a complex three-dimensional architecture. By a particular light pathway, two brightfield- and darkfield-like partial images are simultaneously superimposed so that the brightfield-like absorption image based on the principal zeroth order maximum interferes with the darkfield-like reflection image which is based on the secondary maxima. The background brightness and character of the resulting image can be continuously modulated from a brightfield-dominated to a darkfield-dominated appearance. When the weighting of the dark- and brightfield components is balanced, medium background brightness will result showing the specimen in a phase- or interference contrast-like manner. Specimens can either be illuminated axially/concentrically or obliquely/eccentrically. In oblique illumination, the angle of incidence and grade of eccentricity can be continuously changed. The condenser aperture diaphragm can be used for improvements of the image quality in the same manner as usual in standard brightfield illumination. By this means, the illumination can be optimally adjusted to the specific properties of the specimen. In VBDC, the image contrast is higher than in normal brightfield illumination, blooming and scattering are lower than in standard darkfield examinations, and any haloing is significantly reduced or absent. Although axial resolution and depth of field are higher than in concurrent standard techniques, the lateral resolution is not visibly reduced. Three dimensional structures, reliefs and fine textures can be perceived in superior clarity.     

The use of charging effects in Al/Al2O3 metal-matrix composites as a contrast mechanism in the SEM

A technique is described to image two phases (alumina and spinel) within a metal-matrix composite which takes advantage of charging effects that occur during examination in an SEM. Microscope and specimen parameters which affect the amount of contrast generated via charging are discussed, and imaging strategies are introduced to optimize the effect. “Model” metal-matrix composite specimens were developed to verify the degree of charging in each phase.     

Three‐dimensional imaging of whole mouse models: comparing nondestructive X‐ray phase‐contrast micro‐CT with cryotome‐based planar epi‐illumination imaging

In this study, we compare two evolving techniques for obtaining high‐resolution 3D anatomical data of a mouse specimen. On the one hand, we investigate cryotome‐based planar epi‐illumination imaging (cryo‐imaging). On the other hand, we examine X‐ray phase‐contrast micro‐computed tomography (micro‐CT) using synchrotron radiation. Cryo‐imaging is a technique in which an electron multiplying charge coupled camera takes images of a cryo‐frozen specimen during the sectioning process. Subsequent image alignment and virtual stacking result in volumetric data. X‐ray phase‐contrast imaging is based on the minute refraction of X‐rays inside the specimen and features higher soft‐tissue contrast than conventional, attenuation‐based micro‐CT. To explore the potential of both techniques for studying whole mouse disease models, one mouse specimen was imaged using both techniques. Obtained data are compared visually and quantitatively, specifically with regard to the visibility of fine anatomical details. Internal structure of the mouse specimen is visible in great detail with both techniques and the study shows in particular that soft‐tissue contrast is strongly enhanced in the X‐ray phase images compared to the attenuation‐based images. This identifies phase‐contrast micro‐CT as a powerful tool for the study of small animal disease models.     

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.     

Charging control using pulsed scanning electron microscopy

This paper describes a novel method to observe highly charging specimens at high-beam voltages without specimen preparation. It is found that the technique greatly reduces charging artifacts such as image shift, astigmatism, and defocussing without sacrificing image quality. Images obtained of uncoated specimens are found to be comparable to gold-coated specimens and without exhibiting charging effects. The technique also allows the study of charge distribution effects in specimen charging of which very little understanding exists, particularly as far as the spatial and time-dependent properties of charging are concerned.     

Use of spot-scan procedure for recording low-dose micrographs of beam-sensitive specimens

Electron micrographs of monolayer crystals of paraffin have been recorded with a spot-scan mode of imaging which uses a small 50 nm diameter moving beam. In comparison with normal stationary beam images using 5 μm illuminating beams, the spot-scan micrographs show a higher and more consistent contrast from the 3.8–4.2 Å hydrocarbon chain spacings. On average the improvement in contrast is twofold, but this still leaves scope for further improvement: the best spot-scan images still do not quite reach the level of contrast calculated theoretically from electron diffraction. We believe that the cause of the low contrast in paraffin images must be specimen motion caused by radiation damage rather than a charging effect, for two reasons. First, it does not occur in control images of vermiculite, a non-beam-sensitive mineral, when treated identically. Secondly, although charging might still be a problem with paraffin, when images are taken with the objective aperture in the column, a procedure which is normally expected to reduce charging, no improvement in contrast is found. Thus we think that the use of the small beam minimizes the effect of specimen motion on image contrast by minimizing the specimen area exposed at each instant and therefore the resultant image blurring. Further improvements with even smaller illumination beam diameters might be expected.