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.     

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.     

A flexible instrument control and image acquisition system for a scanning electron microscope

We have developed an instrument control and image acquisition system for use with scanning electron microscopes. By making the system flexible over a wide range of operating voltages, scan generation and image acquisition modes can be easily accommodated to a wide range of instruments. We show the implementation of this system for use with a custom‐built low‐voltage scanning electron microscope. We then explore the simple modifications that are required for control of two instruments intended for use as free electron lasers.     

Low-cost image-capture system for a scanning electron microscope

We describe a PC-based active-capture system for recording digital images from a scanning electron microscope. The system is based on a National Instruments data-acquisition board and a Pentium computer, controlled by software that we have written in Visual Basic.     

Application of channel electron multipliers in an electron detector for low‐voltage scanning electron microscopy

An electron detector containing channel electron multipliers was built and tested in the range of low‐voltage scanning electron microscopy as a detector of topographic contrast. The detector can detect backscattered electrons or the sum of backscattered electrons and secondary electrons, with different amount of secondary electrons. As a backscattered electron detector it collects backscattered electrons emitted in a specific range of take‐off angles and in a large range of azimuth angles enabling to obtain large solid collection angle and high collection efficiency. Two arrangements with different channel electron multipliers were studied theoretically with the use of the Monte Carlo method and one of them was built and tested experimentally. To shorten breaks in operation, a vacuum box preventing channel electron multipliers from an exposure to air during specimen exchanges was built and placed in the microscope chamber. The box is opened during microscope observations and is moved to the side of the scanning electron microscope chamber and closed during air admission and evacuation cycles enabling storing channel electron multipliers under vacuum for the whole time. Experimental tests of the detector included assessment of the type of detected electrons (secondary or backscattered), checking the tilt contrast, imaging the spatial collection efficiency, measuring the noise coefficient and recording images of different specimens.     

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells.     

Resolution in low voltage scanning electron microscopy

As the energy of an electron beam is reduced, the range falls and the secondary electron yield rises. A low voltage scanning electron microscope can therefore, in principle, examine without damage or charging samples such as insulators, dielectrics or beam sensitive materials. This paper investigates the way in which the choice of beam energy affects the spatial resolution of a secondary electron image. It is shown that for samples which are thin compared to the electron range, the edge resolution and contrast in the image improve with increasing beam energy. In samples that are thicker than the electron range, the resolution can be optimized at either high or low energies, but low energy operation will produce images of higher contrast. At an energy of 2 keV or less beam interaction limited resolutions of the order of 3 nm should be possible.     

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.     

Mechanical scanning of the specimen in the scanning electron microscope

A scanning electron microscope (SEM) system equipped with a motor drive specimen stage fully controlled with a personal computer (PC) has been utilized for obtaining ultralow magnification SEM images. This modem motor drive stage works as a mechanical scanning device. To produce ultra-low magnification SEM images, we use a successful combination of the mechanical scanning, electronic scanning, and digital image processing techniques. This new method is extremely labor and time saving for ultra-low magnification and wide-area observation. The option of ultra-low magnification observation (while maintaining the original SEM functions and performance) is important during a scanning electron microscopy session.     

A systematic investigation of the charging effect in scanning electron microscopy for metal nanostructures on insulating substrates

Scanning electron microscopy is perhaps the most important method for investigating and characterizing nanostructures. A well‐known challenge in scanning electron microscopy is the investigation of insulating materials. As insulating materials do not provide a path to ground they accumulate charge, evident as image drift and image distortions. In previous work, we have seen that sample charging in arrays of metal nanoparticles on glass substrates leads to a shrinkage effect, resulting in a measurement error in the nanoparticle dimension of up to 15% at 10 kV and a probe current of 80 ± 10 pA. In order to investigate this effect in detail, we have fabricated metal nanostructures on insulating borosilicate glass using electron beam lithography. Electron beam lithography allows us to tailor the design of our metal nanostructures and the area coverage. The measurements are carried out using two commonly available secondary electron detectors in scanning electron microscopes, namely, an InLens‐ and an Everhart–Thornley detector. We identify and discriminate several contributions to the effect by varying microscope settings, including the size of the aperture, the beam current, the working distance and the acceleration voltage. We image metal nanostructures of various sizes and geometries, investigating the influence of scan‐direction of the electron beam and secondary electron detector used for imaging. The relative measurement error, which we measure as high as 20% for some settings, is found to depend on the acceleration voltage and the type of secondary electron detector used for imaging. In particular, the Everhart–Thornley detectors lower sensitivity to SE₁ electrons increase the magnitude of the shrinkage of up to 10% relative to the InLens measurements. Finally, a method for estimating charge balance in insulating samples is presented.     

Reversed scan direction reduces electron beam damage in EBSD maps

The deleterious effects of electron beam damage on high‐resolution electron backscatter diffraction (EBSD) maps of undeformed quartz are significantly reduced by scanning in the direction opposite to that dictated by widely used EBSD acquisition software. Higher quality electron backscatter patterns are produced when the electron beam moves progressively down the sample (the apparent ‘up’ direction in the resulting maps) for all step sizes where beam damage affects EBSD map quality (≤ ～0.4 μm in this study). The relative improvement associated with downward scanning increases as step size is reduced. A comparison of high‐resolution maps made in experimentally deformed quartz demonstrates that downward scanning reduces by a factor of ～2 the lower limit in step size relative to maps scanned in the conventional direction. The electron beam damages quartz at its point of entry, forming ～0.1‐μm diameter bumps visible in Scanning electron microscope (SEM) images. Downward scanning produces better results because it minimizes the flux of electrons through these loci of damaged crystal.     

Use of backscattered electron detector arrays for forming backscattered electron images in the scanning electron microscope

The backscattered electron (BSE) signal in the scanning electron microscope (SEM) can be used in two different ways. The first is to give a BSE image from an area that is defined by the scanning of the electron beam (EB) over the surface of the specimen. The second is to use an array of small BSE detectors to give an electron backscattering pattern (EBSP) with crystallographic information from a single point. It is also possible to utilize the EBSP detector and computer-control system to give an image from an area on the specimen--for example, to show the orientations of the grains in a polycrystalline sample ("grain orientation imaging"). Some further possibilities based on some other ways for analyzing the output from an EBSP detector array, are described.     

Real-time image quality assessment with mixed Lagrange time delay estimation autoregressive (MLTDEAR) model

A proposal to assess the quality of scanning electron microscope images using mixed Lagrange time delay estimation technique is presented. With optimal scanning electron microscope scan rate information, online images can be quantified and improved. The online quality assessment technique is embedded onto a scanning electron microscope frame grabber card for real‐time image processing. Different images are captured using scanning electron microscope and a database is built to optimally choose filter parameters. An optimum choice of filter parameters is obtained. With the optimum choice of scan rate, noise can be removed from real‐time scanning electron microscope images without causing any sample contamination or increasing scanning time.     

Personal-computer-based system for electron beam X-ray microanalysis of biological samples

A system based on a personal computer has been developed which provides a relatively inexpensive way to equip an electron microscopy laboratory for quantitative elemental analyses of cryosectioned biological samples. This system demonstrates the feasibility of making an X-ray analyser from a personal computer, together with commercially available hardware and software components. Hardware and software have been assembled to drive the beam in a scanning electron microscope, collect and analyse X-ray spectra, and save, retrieve, and analyse data. Our software provides a menu-controlled user interface to direct spectra acquisition and analysis. Spot analyses, video images, and quantitative elemental images may be obtained and results transferred in ASCII format to other computers. Wet weight, as well as dry weight, concentrations are calculated, if measurements were made of areas of the hydrated sample before it was freeze-dried. Grey-level copies of video and quantitative elemental images may be made on a laser printer.     

Automated in‐chamber specimen coating for serial block‐face electron microscopy

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally (‘sample charging’). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block‐face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1–2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal‐to‐noise ratio (SNR) caused by the film is smaller than that caused by the widely used low‐vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non‐conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast.     

Elimination of scanning electron microscopy image periodic distortions with digital signal-processing methods

Electromagnetic interference is one of the main distortion sources in scanning electron microscopy. Electromagnetic interference‐generated scanning electron microscopy image distortions are usually visible as edge blur (at low scan rates) or vibration (at high scan rates). Hardware solutions to this problem, e.g. electrostatic and magnetic shielding, are expensive and, in some cases, difficult to implement. The current investigations led to a significant decrease in the periodic distortions by a novel adaptation of software‐based digital signal processing to scanning electron microscopy problems, without any hardware modification.     

Redesign of the scanning electron microscope for parallel energy spectral acquisition

This paper presents a scanning electron microscope (SEM) design that is compatible with parallel electron energy spectrum acquisition. The SEM should in principle be capable of capturing the energy spectrum of all scattered electrons simultaneously, from low energy secondary electrons to elastic backscattered electrons. Preliminary simulation results predict that the beam separator spectrometer will have a relatively high transmission-energy resolution performance, comparable or better than the cylindrical mirror analyzer (CMA), while at the same time being able to capture the entire energy range of scattered electrons.     

‘Collapsing rings’ on Schottky electron emitters

The electron beam in systems that use a Schottky emitter as the electron source can display periodic fluctuations when the emitter is operated at an extraction voltage that gives a relatively low field strength at the tip. In the past, these fluctuations have been associated with the so-called “collapsing rings” without much further information. In this paper, the tip’s geometry changes associated with these beam instabilities are investigated in more detail by recording the evolution of the emission pattern of a Schottky emitter showing ‘collapsing rings’ for different operating conditions. Scanning electron microscope (SEM) images of different Schottky emitters have been used to support the interpretation.     

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.     

Measurement of electron probe beam diameter by digital image processing

The present report illustrates a computerized method for precise measurement of the diameter of an electron beam. The value of this measurement extends beyond simply providing an accurate estimate of resolution. Other salient areas which will benefit include quantitative X-ray microanalysis, energy loss spectroscopy, diffraction studies, and electron beam lithography. The biological sciences as well as the material sciences will gain enormously from improved accuracy in measurement (control) of beam diameter. It is anticipated that most or all of the mathematical manipulations outlined in this paper will be incorporated into digital electronic packages which will perform the functions automatically for setting the electron beam diameter to the scientist's choice. The purpose of the present report is to indicate some of the principles involved so that as electron microscopy becomes more computerized and automated, the user will have some understanding of what the electronics are doing rather than simply depressing a button or two and ignoring the power of what resides within the walls of the instrument. The performance of a scanning electron microscope (SEM) and a scanning transmission electron microscope (STEM) is roughly determined by the incident electron probe beam size (diameter) involving a sufficient electron current. In the present paper, the diameter of an ultrafine electron beam is measured indirectly from the information given by the blurring of an edge in a STEM or a SEM image of a crystalline specimen with fine, sharp edges. The obtained data were processed by digital image processing methods which give an accurate value of the beam diameter. For confirming the validity of this method, a suitable simulation based on the convolution theorem was performed. By using this measurement, we could measure the diameter of an ultrafine electron beam down to 2 nm, which could not be measured easily by previous techniques.