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.     

Specimen biasing to enhance or suppress secondary electron emission from charging specimens at low accelerating voltages,

Biasing of the specimen is shown to produce improved images in the scanning electron microscope at low beam energies (0.8–2.5 keV) when charging effects (induced by the primary electron beam), topographic effects, or detector shadowing effects would otherwise be present. Examples of such improvement are given for gallium arsenide field-effect transistors (positive charging), patterned photoresist layers on silicon wafers (negative charging and shadowing in contact holes), fractured polymethylmethacrylate (negative charging), polyethylene wrapper material (positive charging), and polished diamond tools (positive charging). It is concluded that specimen biasing may be a simpler and more convenient way to achieve some of the advantages of the converted backscattered secondary electron (CBSE) technique for imaging, but without some of the fundamental disadvantages of that technique. Characterization of this backscattered electron-derived image bears further investigation for possible metrological applications.     

Simulations of scanning electron microscopy imaging and charging of insulating structures

We present a three‐dimensional simulation of scanning electron microscope (SEM) images and surface charging. First, the field above the sample is calculated using Laplace's equation with the proper boundary conditions; then, the simulation algorithm starts following the electron trajectory outside the sample by using electron ray tracing. When the electron collides with the specimen, the algorithm keeps track of the electron inside the sample by simulating the electron scattering history with a Monte Carlo code. During this phase, secondary and backscattered electrons are emitted to form an image and primary electrons are absorbed; therefore, a charge density is formed in the material. This charge density is used to recalculate the field above and inside the sample by solving the Poisson equation with the proper boundary conditions. Field equation, Monte Carlo scattering simulation, and electron ray tracing are therefore integrated in a self‐consistent fashion to form an algorithm capable of simulating charging and imaging of insulating structures. To maintain generality, this algorithm has been implemented in three dimensions. We shall apply the so‐defined simulation to calculate both the global surface voltage and local microfields induced by the scanning beam. Furthermore, we shall show how charging affects resolution and image formation in general and how its characteristics change when imaging parameters are changed. We shall address magnification, scanning strategy, and applied field. The results, compared with experiments, clearly indicate that charging and the proper boundary conditions must be included in order to simulate images of insulating features. Furthermore, we shall show that a three‐dimensional implementation is mandatory for understanding local field formation.     

Attenuation correction in confocal laser microscopes: a novel two-view approach

Confocal microscopy is a three‐dimensional (3D) imaging modality, but the specimen thickness that can be imaged is limited by depth‐dependent signal attenuation. Both software and hardware methods have been used to correct the attenuation in reconstructed images, but previous methods do not increase the image signal‐to‐noise ratio (SNR) using conventional specimen preparation and imaging. We present a practical two‐view method that increases the overall imaging depth, corrects signal attenuation and improves the SNR. This is achieved by a combination of slightly modified but conventional specimen preparation, image registration, montage synthesis and signal reconstruction methods. The specimen is mounted in a symmetrical manner between a pair of cover slips, rather than between a slide and a cover slip. It is imaged sequentially from both sides to generate two 3D image stacks from perspectives separated by approximately 180° with respect to the optical axis. An automated image registration algorithm performs a precise 3D alignment, and a model‐based minimum mean squared algorithm synthesizes a montage, combining the content of both the 3D views. Experiments with images of individual neurones contrasted with a space‐filling fluorescent dye in thick brain tissue slices produced precise 3D montages that are corrected for depth‐dependent signal attenuation. The SNR of the reconstructed image is maximized by the method, and it is significantly higher than in the single views after applying our attenuation model. We also compare our method with simpler two‐view reconstruction methods and quantify the SNR improvement. The reconstructed images are a more faithful qualitative visualization of the specimen's structure and are quantitatively more accurate, providing a more rigorous basis for automated image analysis.     

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.     

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.     

A Study on Central Moments of the Histograms from Scanning Electron Microscope Charging Images

This paper presents a study on using the statistical parameter, central moment, to describe the properties of the histograms of scanning electron microscope (SEM) images. Various charging effects of SEM images and the corresponding histogram profiles are analyzed. The central moment distributions are used to describe the overview of the histograms of an image. The results show that central moments can be used to quantify and characterize the charging images. In particular, the second moment (variance) and third moment (skewness) can be used to distinguish the differences between charging and noncharging images. SCANNING 29: 211‐218, 2007. © 2007 Wiley Periodicals, Inc.     

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.     

Stroboscopic image capture: reducing the dose per frame by a factor of 30 does not prevent beam-induced specimen movement in paraffin

Beam-induced specimen movement may be the major factor that limits the quality of high-resolution images of organic specimens. One of the possible measures to improve the situation that was proposed by Henderson and Glaeser [Ultramicroscopy 16 (1985) 139-150], which we refer to here as "stroboscopic image capture", is to divide the normal exposure into many successive frames, thus reducing the amount of electron exposure--and possibly the amount of beam-induced movement--per frame. The frames would then be aligned and summed. We have performed preliminary experiments on stroboscopic imaging using a 200-kV electron microscope that was equipped with a high dynamic range Charge-coupled device (CCD) camera for image recording and a liquid N2-cooled cryoholder. Single-layer paraffin crystals on carbon film were used as a test specimen. The ratio F(g)/F(0) of paraffin reflections, calculated from the images, serves as our criterion for the image quality. In the series that were evaluated, no significant improvement of the F(image)(g)/F(image)(0) ratio was found, even though the electron exposure per frame was reduced by a factor of 30. A frame-to-frame analysis of image distortions showed that considerable beam-induced movement had still occurred during each frame. In addition, the paraffin crystal lattice was observed to move relative to the supporting carbon film, a fact that cannot be explained as being an electron-optical effect caused by specimen charging. We conclude that a significant further reduction of the dose per frame (than was possible with this CCD detector) will be needed in order to test whether the frame-to-frame changes ultimately become small enough for stroboscopic image capture to show its potential.     

The electron microscopic studies of pore space of solids by a method of conjugate surfaces

This paper presents a new method of studying pore space of solids with the help of SEM images. The method in question consists of obtaining true images of the specimen microstructure and its further processing by the Image Analyser. The above-mentioned image appears as the result of superimposing exactly corresponding SEM images which can be observed on both (conjugate) surfaces of the fractured specimen. Therewith, the dark spots on the total image unambiguously represent pores, whilst bright spots represent grains and particles. The following estimates of the pore space are made by automatic Image Analyser of the total image: the determination of total, open and effective porosity; permeability; distribution pores according to its size; the channel tortuosity.     

Methods for compensation of the light attenuation with depth of images captured by a confocal microscope

A confocal laser scanning microscope (CLSM) enables us to capture images from a biological specimen in different depths and obtain a series of precisely registered fluorescent images. However, images captured from deep layers of the specimen may be darker than images from the topmost layers because of light loss distortions. This effect causes difficulties in subsequent analysis of biological objects. We propose a solution using two approaches: either an online method working already during image acquisition or an offline method assisting as a postprocessing step. In the online method, the gain value of a photomultiplier tube of a CLSM is controlled according to the difference of mean image intensities between the reference and currently acquired image. The offline method consists of two stages. In the first stage, a standard histogram maintaining relative frequencies of gray levels and improving brightness and contrast is created from all images in the series. In the second stage, individual image histograms are warped according to this standard histogram. The methods were tested on real confocal image data captured from human placenta and rat skeletal muscle specimens. It was shown that both approaches diminish the light attenuation in images captured from deep layers of the specimen.     

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.     

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.     

Computer-simulated high-resolution transmission electron microscopy (HRTEM) in tourmaline

The contrast distributions observed in high-resolution transmission electron microscopy (HRTEM) images of tourmaline depend on the types and magnitudes of the exchange components present and on the degree of atom overlap along the direction of observation. Furthermore, the fractional atomic coordinates in tourmalines are valid only for the specific specimen refined. These properties make the interpretation of experimental HRTEM images of tourmaline using image simulation if not impossible at least extremely difficult. A correct interpretation of experimental HRTEM images of tourmaline is possible provided the structural refinement data on the same crystal are available. Nevertheless, it is possible to interpret the experimental HRTEM images of tourmaline if the composition of the structural model chosen during image simulations approximates the composition of the specimen studied by electron microscopy. A good control of the composition of the specimen studied and an appropriate choice of a structural model for image simulation are therefore as important as properly controlling specimen thickness, specimen tilt, beam tilt and objective lens defocus.     

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.     

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.     

On some contrast reversals in SEM: application to metal/insulator systems

Contrast changes of SEM images with experimental conditions (beam energy, angle of detection, etc.) are analyzed by combining physical arguments based on secondary electron emission (SEE) to instrumental arguments involving detection. Possible occurrences of contrast reversals are explored to illustrate these changes in a striking manner. Deduced from SEE yield data, simulated SEM images show a material contrast reversal for a Pt/quartz specimen, a result partly supported by real images of a Cr/quartz integrated circuit. A shift of reversal energy with the detector's position is deduced from a difference in secondary electrons (SE) angular distributions between metals and insulators. Similarly, changes of topographic contrast with detection conditions, specimen composition and angle of tilt are investigated and a possible contrast reversal is again indicated. Finally, it is shown how charging contrast deduced from the expected evolution of SEE yield during irradiation is amplified by in-lens detection: a point illustrated by a contrast reversal of images of SiC particles. The main application concerns a proper interpretation of SEM images that is essential in the investigation of devices obtained from lithographic processes. The discussion on material contrast outlines the difficulty in generalizing the present analysis based on published data and experimental strategies based on implementing specific attachments in the SEM or on biasing the specimen holder are suggested.     

Color cathodoluminescence display in the scanning electron microscope of deep relief surfaces

A scanning electron microscope (SEM) is a multifunctional instrument for the measurement of topographic relief on the surface of bulk specimen images. This instrument is also available to detect the physical effects induced by an electron beam into subsurface layers. Space distribution of the physical properties of measured effects in the relative microrelief is a very important problem in the SEM. We describe a method of displaying specimen information in the SEM using the color cathodoluminescence (CCL-SEM) technique nondistorted by relief influence and CCL-SEM images with composite (color and black / white ) contrast using CCL+BSEmode.