New developments in fast image processing and data acquisition for STM

We have developed new easy-to-use but powerful software in order to obtain the best scan rates in data acquisition and image processing for a PC-AT microcomputer. The programs have been completely written in the C-language, which made it possible to combine low-level characteristics (quick access to memory, interrupt control, etc.) without losing the advantages of a high-level language. The data acquisition software was intended to be sufficiently flexible to acquire either topographical or spectroscopic data with no configuration changes. In topographical (z=f(x,y)) and spectroscopic (I=f(V, z)) measurements, we can obtain up to 15,000 samples/s rates, with 12-bit resolution in a ±10V range. Spectroscopic measurements are done by acquiring I(V) with the feedback off at several z values (up to 256) until I reaches a preset value; afterwards, z is reset automatically to the initial voltage to avoid tip-sample contact. The image processing was designed to get the best performances in time and quality with a simple system such as a PC-AT equipped with an IBM Professional Graphics Display (640times480 pixels and 256 simultaneous colours). It was structured in three parts: plane subtraction, image display (3-D shaded surfaces, 3-D surfaces with colour scales or 2-D contour maps), and filtering (smoothing filters or FFT convolution-deconvolution techniques).     

A method for personal expertise-independent evaluation of image resolution in scanning electron microscopy

The two conventional methods currently employed for the evaluation of image resolution in scanning electron microscopy are the gap method and a fast Fourier transform (FFT) method. These can be highly dependent on personal expertise on the distinction between signal information and noise contained in a micrograph. Hence, the present paper proposes an alternative method (referred to as a contrast-to-gradient (CG) method) that can determine the image resolution of a micrograph without requiring personal expertise on the judgment of noise. The image resolution in the CG method is defined as a weighted harmonic mean of the local resolution, which is proportional to the quotient of the threshold contrast divided by the local gradient. The local gradient is calculated from the quadratic function that best fits the local pixel intensities over 5 x 5 pixels. It has been shown that the CG method, compared with the FFT method, has a broader range of applications for various types of images, such as low-contrast, noise-containing, filter-processed, highly directional, and quasi-periodic feature images.     

Study of the direct Fourier method for the three-dimensional reconstruction of objects in the case of a missing angular data range

Some practical and theoretical considerations on the three-dimensional reconstruction of an object from its two-dimensional projections by the direct Fourier method have been obtained using computer generated images. The procedure applied has been designed to study the specific problems derived from the collection of images in transmission electron microscopes (TEM), making special emphasis on images of biological objects. The study of the calculated point spread function (PSF) of the experimental system and the mean and variance profiles of the reconstructions have provided useful criteria to assess the quality and the interpretability of three-dimensional reconstructions of real data.     

An electron beam lithography and digital image acquisition system for scanning electron microscopes

A low‐cost microcontroller based control and data acquisition unit for digital image recording of scanning electron microscope (SEM) images and scanning electron microscope based electron beam lithography (EBL) is described. The developed microcontroller low‐level embedded software incorporates major time critical functions for image acquisition and electron beam lithography and makes the unit an intelligent module which communicates via USB with the main computer. The system allows recording of images with up to 4096 × 4096 pixel size, different scan modes, controllable dwell time, synchronization with main power frequency, and other user controllable functions. The electron beam can be arbitrary positioned with 12‐bit precision in both dimensions and this is used to extend the scanning electron microscope capabilities for electron beam lithography. Hardware and software details of the system are given to allow its easy duplication. Performance of the system is discussed and exemplary results are presented.     

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.     

A guide to analysis and reconstruction of serial block face scanning electron microscopy data

Serial block face scanning electron microscopy (SBF‐SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers – how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D. However, the software programmes can appear overwhelming, time‐consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore, the aim of this paper is to provide a detailed step‐by‐step protocol, accompanied by tutorial videos, for several types of analysis programmes that can be used on raw SBF‐SEM data, although there are more options available than can be covered here. To showcase the programmes, datasets of skeletal muscle from foetal and adult guinea pigs are initially used with procedures subsequently applied to guinea pig cardiac tissue and locust brain. The tissue is processed using the heavy metal protocol developed specifically for SBF‐SEM. Trimmed resin blocks are placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks are analysed in three different programmes, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a particular type of structure. For example, larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast‐based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximize informative output from valuable tissue.     

New generation scanning electron microscopy technology based on the concept of active image processing

A new generation of scanning electron microscopy (SEM) technology is proposed based on the concept of “active image processing.” In order to collect sufficient data for a purpose which is defined in the utilization of active image processing, we may need more devices from among a variety of useful hardware, for example, a digital scan generator with meaningful parameters and an analog-to-digital converter for ultrahigh density recording. After the data acquisition, the application of some digital image processing techniques is certainly effective, because the method in question is specially designed so that the property of obtained data will be suitable for the application of these techniques. The present technology should produce a variety of attractive options in the field of SEM.     

Determination of a homogeneity factor for composite materials by a microstructural image analysis method

The physical properties of particle‐reinforced composite materials are highly affected by the distribution of particles within a matrix material. In this study, a microstructural image analysis method with a new distribution index for quantifying the degree of distribution in composite materials was developed. The free‐path spacing between particles was measured to calculate the distribution (D) index based on the coefficient of variation. The proposed method was applied to six digitally created reference patterns as representative binary composite microstructures and three actual ceramic‐matrix composites, respectively. It is found that the D index increased from 0.00 to 0.67 depending on the degree of distribution or homogeneity level based on the reference patterns. The homogeneity levels for the binary composites are then classified from a perfect (maximum) to very low level (minimum) based on increasing D index values, where a high D index presents a poorer distribution. The results obtained for reference patterns and metal silicide‐refractory oxide composite microstructures indicate that the proposed method is a useful tool to quantify the degree of distribution with high accuracy, and can be efficiently used for different types of composite microstructures.     

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.     

Porosity determination of ceramic materials by digital image analysis--a critical evaluation

Measuring the porosity of materials by digital image analysis of micrographs is a well-established and convenient method for the testing of metallic samples. However, when applied to ceramic materials, this method has been shown to be much less reliable and poorly reproducible. The purpose of this present work is to clarify the reason for this deficiency, involving many porosity measurements, performed on plasma-sprayed zirconia, under systematically varied microscopic imaging conditions, and the porosities being calculated using various evaluation methods. Comparison between of the results has shown that the present state of the image analysis method is not satisfactory for absolute porosity measurements on ceramic materials. It can be useful as a convenient tool for comparative measurements, however, if the imaging conditions maintained in the microscope and the evaluation method are held to be exactly identical.     

A method for processing polymeric beads for critical point drying

Polymeric beads are increasingly being used for surface studies. Processing of these beads for critical point drying (CPD) poses a problem since these beads must be handled in suspension. For this processing, a method has been described here which relies on a capsule made by an ordinary micropipette tip covered on the end by parafilm and on the other by a filter paper which permits the exchange of fluid without the loss of beads.     

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.     

Computer modeling of three-dimensional reconstruction algorithm of cathodoluminescence material properties,analysis of errors,and optimization of variable parameters

Three-dimensional (3-D) reconstruction of cathodoluminescence (CL) properties of materials (Saparin et al. 1997) is a new nondestructive technique that measures quantum yield information of subsurface layers as a set of two-dimensional image sections. This technique is able to synthesize a 3-D image without destroying the sample. The basis for this technique is the fact that the electrons in scanning electron microscopy penetrate the sample at different depths with a variation of accelerating voltage. The detected CL emission integrates the information from the full volume occupied by the charge carriers. Estimating the errors that occur during the reconstruction process is complex since these errors are caused by necessary approximations, noise, and the imperfection of the 3-D reconstruction algorithm that is employed. An analysis of the factors that affect the accuracy of the technique has been made. The estimation of systematic errors and the optimization of variable parameters were calculated by computer modeling.     

Some aspects of optimizing contrasts for the investigation of joint materials in the environmental scanning electron microscope

In the environmental scanning electron microscope, material joints of different atomic mass and different electrical conducting properties can easily be observed simultaneously without coating the specimen. For such heterogeneous materials, the quality of the image can be optimized with respect to contrast and resolution if the contrast types as well as their significance to the composition of the image are known.     

Colour display of video information in scanning electron microscopy: Principles and applications to physics,geology, soil science,biology, and medicine

From a colourimetric point of view, colour has two independent aspects: brightness and chromaticity. In black and white images, all elements are of the same chromaticity and can be distinguished only by brightness contrast. In the colour image, elements of the same brightness can be discriminated by chromaticity (colour) contrast. Generally, colour image elements can be discriminated both by brightness and by chromaticity. As the human eye can distinguish a number of hues two orders of magnitude larger than the number of grey levels, it is safe to say that the colour image is much more informative than the black and white image. There are some peculiarities of the colour image and methods of its formation in SEM. Two principles of image formation are used. The first consists of the formation of a real colour image in the cathodoluminescence mode. In this case the colour of an image element is determined by the spectrum of the luminescence emission excited in the corresponding point of an object by the electron beam. The second principle is that of colour coding (quasicolour, pseudocolour), when a video signal in colour (either digital or analog) corresponds to a video signal (amplitude, frequency, phase, etc.) produced by any mode in the scanning electron microscope. We present a review of the methods of colour display of video information in scanning electron microscopy and their applications to physics, geology, soil science, biology, and medicine.     

Comparison of restorative materials and surface alterations after prebiotic and probiotic beverages: A nanoindentation and SEM study

The purpose was to investigate the surface characteristics of various resin-based materials by immersing in probiotic beverages. A total of 420 disc-shaped samples (5 mm × 2 mm) were prepared from resin-based composites. Samples were divided into four groups and immersed for 10 min/day for 1 month in either a probiotic sachet, kefir, kombucha, or artificial saliva (control). Surface roughness was measured at baseline and 1 month. One sample of each of the tested materials was examined under nanoindentation to evaluate the reduced elasticity modulus and nanohardness scores. Scanning electron microscopy (SEM) was used to compare surface differences. Data were analyzed statistically using one-way ANOVA test and the significance was set at p < .05. The lowest roughness scores were observed in Z250, Estelite Bulk Fill, and HRi ENA in most of the test groups. Among conventional composites, Z250 group had the highest nanohardness and elasticity modulus scores. Among bulk-fill composites, Estelite Bulk Fill Flow had the lowest surface roughness after immersion in probiotic beverages and the highest nanohardness values. Reveal HD, as a bulk-fill group showed higher surface roughness and considerably lower nanohardness and elasticity modulus scores. Maximum height levels of samples were recorded. SEM images revealed voids and microcracks on the surfaces of test materials. Dentists may prefer Z250 as microhybrid and Estelite Bulk Fill Flow as bulk-fill composites for the restorations of patients who consume gut-friendly drinks regularly. When there are various types of materials, nanoindentation is a useful method for evaluating surface alterations and sensible comparisons.     

Quantitative and reliable in vitro method combining scanning electron microscopy and image analysis for the screening of osteotropic modulators

The increased generation and up-regulated activity of bone resorbing cells (osteoclasts) play a part in the impairment of bone remodeling in many bone diseases. Numerous drugs (bisphosphonates, calcitonin, selective estrogen receptor modulators) have been proposed to inhibit this increased osteoclastic activity. In this report, we describe a pit resorption assay quantified by scanning electron microscopy coupled with image analysis. Total rabbit bone cells with large numbers of osteoclasts were cultured on dentin slices. The whole surface of the dentin slice was scanned and both the number of resorption pits and the total resorbed surface area were measured. Resorption pits appeared at 48 h and increased gradually up to 96 h. Despite the observation of a strong correlation between the total resorption area and the number of pits, we suggest that area measurement is the most relevant marker for osteoclastic activity. Osteotropic factors stimulating or inhibiting osteoclastic activity were used to test the variations in resorption activity as measured with our method. This reproducible and sensitive quantitative method is a valuable tool for screening for osteoclastic inhibitors and, more generally, for investigating bone modulators.     

Quantitative fractography by digital image processing: NIH Image macro tools for stereo pair analysis and 3-D reconstruction

A set of NIH Image macro programs was developed to make qualitative and quantitative analyses from digital stereo pictures produced by scanning electron microscopes. These tools were designed for image alignment, anaglyph representation, animation, reconstruction of true elevation surfaces, reconstruction of elevation profiles, true-scale elevation mapping and, for the quantitative approach, surface area and roughness calculations. Limitations on time processing, scanning techniques and programming concepts are also discussed.