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.     

Atomic force microscope tip cleaning by using gratings as micro‐washboards

The sharpness of atomic force microscope (AFM) tips is essential for acquiring high quality AFM images. However, AFM tips would easily get contaminated during scanning and storage at ambient condition, which influences image resolution and causes image distortion. Replacing the probe frequently is a solution, but uneconomical. To solve this problem, several tip cleaning methods have been proposed but there is space for further improvement. Therefore, this article developed a method of tip cleaning by using a one‐dimensional grating (600 lines/mm) as a micro‐washboard to “wash” contaminated tips. We demonstrate that the contaminants can be scrubbed away by rapidly scanning such micro‐washboard against the tip in the aids of Z‐dithering (10–20 Hz) exerted on the washboard. This method is highly efficient and proved to be superior to traditional ones. Experiments show that AFM images acquired with “washed” tips have higher resolution and less distortion compared with images acquired using contaminated tips, even comparable to those scanned by new ones. Microsc. Res. Tech. 76:1131–1134, 2013. © 2013 Wiley Periodicals, Inc.     

A Model‐based approach for microvasculature structure distortion correction in two‐photon fluorescence microscopy images

This paper investigates a postprocessing approach to correct spatial distortion in two‐photon fluorescence microscopy images for vascular network reconstruction. It is aimed at in vivo imaging of large field‐of‐view, deep‐tissue studies of vascular structures. Based on simple geometric modelling of the object‐of‐interest, a distortion function is directly estimated from the image volume by deconvolution analysis. Such distortion function is then applied to subvolumes of the image stack to adaptively adjust for spatially varying distortion and reduce the image blurring through blind deconvolution. The proposed technique was first evaluated in phantom imaging of fluorescent microspheres that are comparable in size to the underlying capillary vascular structures. The effectiveness of restoring three‐dimensional (3D) spherical geometry of the microspheres using the estimated distortion function was compared with empirically measured point‐spread function. Next, the proposed approach was applied to in vivo vascular imaging of mouse skeletal muscle to reduce the image distortion of the capillary structures. We show that the proposed method effectively improve the image quality and reduce spatially varying distortion that occurs in large field‐of‐view deep‐tissue vascular dataset. The proposed method will help in qualitative interpretation and quantitative analysis of vascular structures from fluorescence microscopy images.     

Piezoelectric field around threading dislocation in GaN determined on the basis of high-resolution transmission electron microscopy image

A new method of determining the piezoelectric field around dislocations from high‐resolution transmission electron microscopy images is presented. In order to determine the electrical potential distribution near a dislocation core, we used the distortion field, obtained using the geometrical phase method and the non‐linear finite element method. The electrical field distribution was determined taking into account the inhomogeneous strain distribution, finite geometry of the sample and the full couplings between elastic and electrical fields. The results of the calculation for a transmission electron microscopy thin sample are presented.     

Pipeline for illumination correction of images for high‐throughput microscopy

The presence of systematic noise in images in high‐throughput microscopy experiments can significantly impact the accuracy of downstream results. Among the most common sources of systematic noise is non‐homogeneous illumination across the image field. This often adds an unacceptable level of noise, obscures true quantitative differences and precludes biological experiments that rely on accurate fluorescence intensity measurements. In this paper, we seek to quantify the improvement in the quality of high‐content screen readouts due to software‐based illumination correction. We present a straightforward illumination correction pipeline that has been used by our group across many experiments. We test the pipeline on real‐world high‐throughput image sets and evaluate the performance of the pipeline at two levels: (a) Z′‐factor to evaluate the effect of the image correction on a univariate readout, representative of a typical high‐content screen, and (b) classification accuracy on phenotypic signatures derived from the images, representative of an experiment involving more complex data mining. We find that applying the proposed post‐hoc correction method improves performance in both experiments, even when illumination correction has already been applied using software associated with the instrument. To facilitate the ready application and future development of illumination correction methods, we have made our complete test data sets as well as open‐source image analysis pipelines publicly available. This software‐based solution has the potential to improve outcomes for a wide‐variety of image‐based HTS experiments.     

Strain mapping accuracy improvement using super‐resolution techniques

Super‐resolution (SR) software‐based techniques aim at generating a final image by combining several noisy frames with lower resolution from the same scene. A comparative study on high‐resolution high‐angle annular dark field images of InAs/GaAs QDs has been carried out in order to evaluate the performance of the SR technique. The obtained SR images present enhanced resolution and higher signal‐to‐noise (SNR) ratio and sharpness regarding the experimental images. In addition, SR is also applied in the field of strain analysis using digital image processing applications such as geometrical phase analysis and peak pairs analysis. The precision of the strain mappings can be improved when SR methodologies are applied to experimental images.     

The influence of low-strain thermo-mechanical processing on grain boundary network characteristics in type 304 austenitic stainless steel

Grain boundary engineering of austenitic stainless steel, through the introduction of plastic strain and thermal annealing, can be used to develop microstructures with improved resistance to inter‐granular degradation. The influence of low‐strain thermo‐mechanical processing on grain boundary network development, with systematic variations of annealing treatments, has been investigated. Three stages of the microstructure development during grain boundary engineering in low‐strain processing conditions are identified, and correlated with changes in grain boundary character and deviation distributions. Low‐energy connected length segments at triple junctions, which have been proposed to be responsible for crack bridging during inter‐granular stress corrosion cracking, can be influenced by the choice of the annealing treatment parameters. The development of individual grain boundary length segments of different character showed consistent trends with increasing grain size. Crack length predictions are consistent with the beneficial effect of designing microstructures with high fractions of twin grain boundaries and smaller grain size.     

High resolution electron microscopy of interfaces in chlorinated phthalocyanine molecular crystals

Molecular images of chlorinated copper phthalocyanine have shown the arrangement of molecules at the interface between two crystals. Mismatch does not cause distortion or recrystallization for low index junctions but for more irregular interfaces strain causes alteration of the molecular tilt angle and accommodation of the mismatch by the formation of edge dislocations whose Burger's vectors are parallel to the interface.     

遥感图像条带噪声的多尺度变分模型去除

多片CCD拼接遥感成像系统由于存在非均匀性问题,导致遥感图像中常存在条带噪声,本文在分析条带噪声的主要来源和模型的基础上,提出了多尺度变分模型的条带噪声去除方法。首先,分析了条带噪声的特点并建立了图像退化模型。其次,结合条带噪声的单向性特点与多尺度分层分解方法构造能量泛函。然后,利用不动点Gauss-Seidel迭代法多尺度分级极小化能量泛函,将条带噪声和图像有用信息分离。最后,对各尺度结构分量和细节分量进行累加,得到去噪图像。实验结果表明:对于周期条带噪声,图像畸变量为2‰,图像辐射质量提升到11.715dB;对于随机条带噪声,图像畸变量为3.3‰,图像辐射质量提升到11.092 5dB。与典型条带噪声去除方法相比,不管是周期条带噪声还是随机条带噪声,本文方法均能够在保证畸变量很小的情况下,将其完全去除,满足遥感图像低畸变量的预处理要求。     

Influence of scanning rate on quality of AFM image: Study of surface statistical metrics

The purpose of this work is to study the dependence of AFM‐data reliability on scanning rate. The three‐dimensional (3D) surface topography of the samples with different micro‐motifs is investigated. The analysis of surface metrics for estimation of artifacts from inappropriate scanning rate is presented. Fractal analysis was done by cube counting method and evaluation of statistical metrics was carrying out on the basis of AFM‐data. Combination of quantitate parameters is also presented in graphs for every measurement. The results indicate that the sensitivity to scanning rate growths with fractal dimension of the sample. This approach allows describing the distortion of the images against scanning rate and could be applied for dependences on the other measurement parameters. The article explains the relevance and comparison of fractal and statistical surface parameters for characterization of data distortion caused by inappropriate choice of scanning rate.     

Correction of distortion due to thermal drift in scanning probe microscopy

A common source of distortion in scanning probe microscope (SPM) images is “thermal drift,” the slow thermal expansion of different materials in the sample and microscope due to small changes in temperature over the course of a scan. We describe here a method for correcting this distortion by immediately following each image scan with a rescan of a small, narrow portion of the same area with the slow and fast scan axes reversed. The original, full image is corrected using a low-order polynomial mapping function, with coefficients determined by a pixel-wise comparison between the original full and rescanned partial images. We demonstrate here that this method can correctly remove distortion from a wide range of images with a precision of better than one pixel, and is also robust to common imaging artifacts. We also address some of the programming considerations that have gone into implementing this computationally intensive technique, which can now be performed using standard desktop hardware in times that range between a few seconds and a few minutes.     

MALDI TOF MS profiling of bacteria at the strain level: A review

Since the advent of the use of matrix‐assisted laser desorption/ionization (MALDI) time‐of‐flight mass spectrometry (TOF MS) as a tool for microbial characterization, efforts to increase the taxonomic resolution of the approach have been made. The rapidity and efficacy of the approach have suggested applications in counter‐bioterrorism, prevention of food contamination, and monitoring the spread of antibiotic‐resistant bacteria. Strain‐level resolution has been reported with diverse bacteria, using library‐based and bioinformatics‐enabled approaches. Three types of characterization at the strain level have been reported: strain categorization, strain differentiation, and strain identification. Efforts to enhance the library‐based approach have involved sample pre‐treatment and data reduction strategies. Bioinformatics approaches have leveraged the ever‐increasing amount of publicly available genomic and proteomic data to attain strain‐level characterization. Bioinformatics‐enabled strategies have facilitated strain characterization via intact biomarker identification, bottom‐up, and top‐down approaches. Rigorous quantitative and advanced statistical analyses have fostered success at the strain level with both approaches. Library‐based approaches can be limited by effects of sample preparation and culture conditions on reproducibility, whereas bioinformatics‐enabled approaches are typically limited to bacteria, for which genetic and/or proteomic data are available. Biological molecules other than proteins produced in strain‐specific manners, including lipids and lipopeptides, might represent other avenues by which strain‐level resolution might be attained. Immunological and lectin‐based chemistries have shown promise to enhance sensitivity and specificity. Whereas the limits of the taxonomic resolution of MALDI TOF MS profiling of bacteria appears bacterium‐specific, recent data suggest that these limits might not yet have been reached. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:188–217, 2013     

The effect of surface strain relaxation on HAADF imaging

In this work the effects of strain on high-angle annular dark field (HAADF) images taken in zone axis conditions have been quantitatively studied. In particular, the presence of dark contrast zones in experimental HAADF images of InGaAs–GaAs interfaces is here interpreted in terms of strain relaxation at the surface. The consistence of this assumption is demonstrated by means of experiments and simulations performed for different In compositions, specimen tilt and thickness conditions. It is shown how the HAADF contrast mechanism is related to the bending of the lattice planes in the first surface region. Finally, a generalization of the 1s approximation that is able to qualitatively describe the effect of strain on HAADF images is presented.     

Monte Carlo Simulation of CD‐SEM Images for Linewidth and Critical Dimension Metrology

In semiconductor industry, strict critical dimension control by using a critical dimension scanning electron microscope (CD‐SEM) is an extremely urgent task in near‐term years. A Monte Carlo simulation model for study of CD‐SEM image has been established, which is based on using Mott's cross section for electron elastic scattering and the full Penn dielectric function formalism for electron inelastic scattering and the associated secondary electron (SE) production. In this work, a systematic calculation of CD‐SEM line‐scan profiles and 2D images of trapezoidal Si lines has been performed by taking into account different experimental factors including electron beam condition (primary energy, probe size), line geometry (width, height, foot/corner rounding, sidewall angle, and roughness), material properties, and SE signal detection. The influences of these factors to the critical dimension metrology are investigated, leading to build a future comprehensive model‐based library. SCANNING 35: 127‐139, 2013. © 2012 Wiley Periodicals, Inc.     

Intensity correction of fluorescent confocal laser scanning microscope images by mean-weight filtering

This paper addresses the problem of intensity correction of fluorescent confocal laser scanning microscope images. Confocal laser scanning microscope images are frequently used in medicine for obtaining 3D information about specimen structures by imaging a set of 2D cross sections and performing 3D volume reconstruction afterwards. However, 2D images acquired from fluorescent confocal laser scanning microscope images demonstrate significant intensity heterogeneity, for example, due to photo‐bleaching and fluorescent attenuation in depth. We developed an intensity heterogeneity correction technique that (a) adjusts the intensity heterogeneity of 2D images, (b) preserves fine structural details and (c) enhances image contrast, by performing spatially adaptive mean‐weight filtering. Our solution is obtained by formulating an optimization problem, followed by filter design and automated selection of filtering parameters. The proposed filtering method is experimentally compared with several existing techniques by using four quality metrics: contrast, intensity heterogeneity (entropy) in a low frequency domain, intensity distortion in a high frequency domain and saturation. Based on our experiments and the four quality metrics, the developed mean‐weight filtering outperforms other intensity correction methods by at least a factor of 1.5 when applied to fluorescent confocal laser scanning microscope images.     

航空变焦距斜视成像几何畸变的自动校正

针对变焦距航空摄像机斜视成像产生的几何变形,提出一种同时校正斜视梯形失真和变焦距镜头非线性畸变的自动校正方法。根据直线透视投影不变性原理,利用单参数除式模型通过变步长优化搜索方法得到不同焦距对应的镜头畸变系数和畸变中心坐标;研究了焦距变化对畸变的影响规律,校正了镜头畸变使其满足针孔成像模型;引入飞机位置、姿态和摄像机视轴指向方位等因素,将航空图像重投影到地图坐标系中,对坐标变换后的像素亮度值进行重采样得到校正斜视变形和镜头畸变后的正射投影图像。对不同焦距和位置姿态下拍摄的地面靶标畸变图像和实际航空变焦距斜视图像进行了校正。结果表明,该方法能够有效准确地校正图像的几何变形,当飞行高度为2 500m时,在文中给定的位置姿态精度下的图像几何校正均方误差约为2m,较好地满足了后续图像拼接需求。该方法效率高,便于自动化实现,对提高图像拼接精度和实现目标精确定位与实时稳定跟踪具有重要意义。     

An automated feedback system with the hybrid model of scoring and classification for solving over-segmentation problems in RNAi high content screening

Background: High content screening (HCS) via automated fluorescence microscopy is a powerful technology for generating cellular images that are rich in phenotypic information. RNA interference is a revolutionary approach for silencing gene expression and has become an important method for studying genes through RNA interference‐induced cellular phenotype analysis. The convergence of the two technologies has led to large‐scale, image‐based studies of cellular phenotypes under systematic perturbations of RNA interference. However, existing high content screening image analysis tools are inadequate to extract content regarding cell morphology from the complex images, thus they limit the potential of genome‐wide RNA interference high content screening screening for simple marker readouts. In particular, over‐segmentation is one of the persistent problems of cell segmentation; this paper describes a new method to alleviate this problem. Methods: To solve the issue of over‐segmentation, we propose a novel feedback system with a hybrid model for automated cell segmentation of images from high content screening. A Hybrid learning model is developed based on three scoring models to capture specific characteristics of over‐segmented cells. Dead nuclei are also removed through a statistical model. Results: Experimental validation showed that the proposed method had 93.7% sensitivity and 94.23% specificity. When applied to a set of images of F‐actin‐stained Drosophila cells, 91.3% of over‐segmented cells were detected and only 2.8% were under‐segmented. Conclusions: The proposed feedback system significantly reduces over‐segmentation of cell bodies caused by over‐segmented nuclei, dead nuclei, and dividing cells. This system can be used in the automated analysis system of high content screening images.     

Defect formation in self-assembling quantum dots of InGaAs on GaAs: a case study of direct measurements of local strain from HREM

The growth and defect structures in free-standing self-assembled In_0.6Ga_0.4As quantum dots (QDs) grown on (001) GaAs by solid source molecular beam epitaxy has been investigated. The QDs are elongated along [1¯ 1 0]. At a nominal thickness of eight monolayers defect complexes, associated with intrinsic stacking faults, have been generally observed on both sides of a QD in (1¯ 1 0) cross-section. The total defect vector of such defect complexes is a /3 <111>. Local strain components on {111} slip planes in the QDs without defects have been measured directly from digitized high-resolution electron microscopy images. The distortion on the two sets of {111} planes of a (1¯ 1 0) cross-section is different owing to elastic relaxation. The results of strain measurements suggest that a 60° dislocation nucleates first on the set of {111} planes of higher contractive shear strain, i.e. (111) planes on the right side of the QDs, and (1¯ 1¯ 1) planes on the left side. A 30° partial dislocation forms subsequently on the other set of {111} planes, i.e. (1¯ 1¯ 1) planes on the right side of the QDs and (111) planes on the left side, when the 60° dislocation glides down towards the In_0.6Ga_0.4As/GaAs interface, as a result of the additional strain field of the 60° dislocation. The efficiency of the defect complexes in strain relaxation of the QDs has been shown by strain measurements in QDs with the presence of defects.     

Effects of damping and stiffness of AFM cantilever on the imaging of fine surfaces

In this paper, by applying the differential quadrature (DQ) method, a semi analytical model has been developed for atomic force microscope cantilever, and then by using the interfacial forces between the cantilever tip and imaged surfaces, a 2D model has been extracted for imaging nano‐sized fine samples. By employing the present model, several simple and standard samples have been imaged, and finally the effects of the microcantilever's structural damping and its stiffness on the imaging results have been investigated. It has been observed that, through the control of damping, the quality of the acquired images is considerably improved. It has also been shown that the self‐softening and self‐hardening properties of cantilever have serious effects on the obtained images. The present model can be used to study the effects of different parameters on the process of imaging small‐scale samples. Also, as one of its most important applications, this model can be used in common multiscale models for simulating and predicting the effects of large and small fields on each other.     

Improved correction of axial geometrical distortion in index-mismatched fluorescent confocal microscopic images using high-aperture objective lenses

Extracting quantitative data from microscopic volume images is straightforward when the refractive indices of the immersion medium and the mounting medium are equal. The readings of the position of the specimen stage can be directly used to measure depth and width. Imperfectly matched immersion and mounting media result in axial geometrical distortion. Linear correction of the axial distortion using the paraxial estimate of the axial scaling factor yields results that may differ as much as 4% from the actual values. From calculations based on a theoretical expression of the 3‐D point‐spread function in the focal region of a high‐aperture microscope focussing into a mismatched mounting medium, we derived axial scaling factors that result in quantitative results accurate to better than 1%. From a non‐linear correction procedure, an improved formula for the paraxial estimate of the axial scaling factor is derived.