A simple method to 'point count' silt using scanning electron microscopy aided by image analysis

This study presents a simple method to ‘point count’ silt‐sized grains using backscattered scanning electron microscopy together with image analysis. The work materialized out of the need to determine the heavy mineral abundance within silt obtained from coastal dunes to aid in the interpretation of dune weathering. This technique allows two broad mineral groups to be quantified according to their modal abundance. The groups are characterized by their dominant atomic elements present; atomic numbers > 20 are classified as ‘high’ (metal oxides, zircon, monazite, carbonates, pyroxenes and amphiboles) and those < 20 as ‘low’ (quartz, feldspars and organics). As a check on this technique, X‐ray fluorescence was used. This showed a strong positive correlation (r² = 0.85) with the developed point counting technique.     

Evaluation of scanning electron microscopy images of a model dressing using image feature extraction techniques and principal component analysis

Twelve dressing systems made by varying protein type, oil level, CaCl₂, NaCl, and sucrose, were examined using scanning electron microscopy. Images from the 12 systems were quantitatively analysed using methods of feature extraction. These methods were based on vectorisations of the images followed by principal component analysis on the extracted vectors. These techniques were used to examine the reproducibility of the acquired images as well as to relate the images to rheologic and sensory texture parameters. Two feature extraction methods were used: the angle measure technique (AMT) and the absolute difference method (ABDF). The ABDF method used fewer principal components to extract information from images relevant to the complex modulus/sensory viscosity of the system, but the information seemed equally well preserved by the two-feature extraction methods. The AMT was more efficient in classifying the images with respect to protein type. A fair correlation between images and complex modulus was obtained (R=0.73). It is suggested that a better correlation might be obtained by adding more systems, increasing the number of areas imaged for each system as well as avoiding systems of low viscosity.     

Correcting for 3D distortion when using backscattered electron detectors in a scanning electron microscope

A variable pressure scanning electron microscope (VPSEM) can produce a topographic surface relief of a physical object under examination, in addition to its two‐dimensional (2D) image. This topographic surface relief is especially helpful when dealing with porous rock because it may elucidate the pore‐space structure as well as grain shape and size. Whether the image accurately reproduces the physical object depends on the management of the hardware, acquisition, and postprocessing. Two problems become apparent during testing: (a) a topographic surface relief of a precision ball bearing is distorted and does not correspond to the physical dimensions of the actual sphere and (b) an image of a topographic surface relief of a Berea sandstone is geometrically tilted and topographically distorted even after standard corrections are applied. The procedure presented here is to ensure the veracity of the image, and includes: (a) adjusting the brightness and contrast levels originally provided by the manufacturer and (b) tuning the amplifiers of the backscatter detector plates to be equal to each other, and producing zero voltage when VPSEM is idle. This procedure is tested and verified on the said two physical samples. SCANNING 31: 59–64, 2009. © 2009 Wiley Periodicals, Inc.     

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.     

Assessment and correction of geometric distortions in low-magnification scanning electron microscopy images

A method is introduced to assess and correct the geometric distortions which frequently occur in low-magnification scanning electron microscopy (SEM) images. Such images typically exhibit a complex pattern of varying deviations from orthogonality which cannot be adequately corrected by simple geometric transformations such as shifting, scaling, rotation, or shearing. A suitable approach to rectify low-magnification SEM images is polynomial warping, a correction procedure which also accomplishes rubber sheet transformation. To demonstrate the approach, a reference grid for low magnifications has been scanned at 40- and 55-fold magnifications by means of a microanalyzer. Calculated geometric distortions range from 1.5 to 3.5% of the image dimensions; applying polynomial warping, distortions could be reduced to approximately 0.1% of the image dimensions. Because of its easy application and the widespread availability in image processing packages, polynomial warping can be recommended as a routine procedure for rectifying low-magnification SEM images.     

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.     

Proper acquisition and handling of scanning electron microscopy images using a high-performance personal computer

The modern high-performance personal computer (PC) has very recently expanded the range of utilization of digital scanning electron microscopy (SEM) images, and the PC will be used increasingly with SEMs. However, the image quality of digital SEM images may be considerably influenced by scanning and digitization conditions. In particular, the effects of the aliasing error peculiar to digital data are often serious in the low-magnification acquisition (undersampling) of SEM images, and moreover even a high-magnification image (oversampling) is disturbed by the undersampled noise (a sort of aliasing error). Furthermore, the signal-to-noise ratio of a digitized SEM image is closely related to the performance of the analog-to-digital converter. To prevent a flood of low-quality digital images with artifacts by the aliasing and additional noise, we propose a method using very high-density sampling (scanning). In addition, we will discuss how to handle digital SEM images from the point of view of the sampling and quantization.     

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.     

Comparison of three‐dimensional analysis and stereological techniques for quantifying lithium‐ion battery electrode microstructures

Lithium‐ion battery performance is intrinsically linked to electrode microstructure. Quantitative measurement of key structural parameters of lithium‐ion battery electrode microstructures will enable optimization as well as motivate systematic numerical studies for the improvement of battery performance. With the rapid development of 3‐D imaging techniques, quantitative assessment of 3‐D microstructures from 2‐D image sections by stereological methods appears outmoded; however, in spite of the proliferation of tomographic imaging techniques, it remains significantly easier to obtain two‐dimensional (2‐D) data sets. In this study, stereological prediction and three‐dimensional (3‐D) analysis techniques for quantitative assessment of key geometric parameters for characterizing battery electrode microstructures are examined and compared. Lithium‐ion battery electrodes were imaged using synchrotron‐based X‐ray tomographic microscopy. For each electrode sample investigated, stereological analysis was performed on reconstructed 2‐D image sections generated from tomographic imaging, whereas direct 3‐D analysis was performed on reconstructed image volumes. The analysis showed that geometric parameter estimation using 2‐D image sections is bound to be associated with ambiguity and that volume‐based 3‐D characterization of nonconvex, irregular and interconnected particles can be used to more accurately quantify spatially‐dependent parameters, such as tortuosity and pore‐phase connectivity.     

Quantitative analysis of keratin filament networks in scanning electron microscopy images of cancer cells

The keratin filament network is an important part of the cytoskeleton. It is involved in the regulation of shape and viscoelasticity of epithelial cells. The morphology of keratin networks depends on post-translational modifications of keratin monomers. In-vitro studies indicated that network characteristics, such as filament crosslink density, determines the biophysical properties of the filament network. This report presents a quantitative method for the morphological analysis of keratin filament networks. Visualization of filaments was based on prefixation extraction of epithelial cells and scanning electron microscopy (SEM). SEM images were processed by a skeletonization algorithm to obtain a graph structure that represents individual filaments as well as their connections. This method was applied to investigate the effects of transforming growth factor α (TGFα) on the morphology of keratin networks in pancreatic cancer cells. TGFα contributes to pancreatic cancer progression and activates signalling pathways phosphorylating keratin monomers. Using this new method, a significant alteration to the keratin network morphology could be detected in response to TGFα.     

Algorithms for accurate 3D registration of neuronal images acquired by confocal scanning laser microscopy

This paper presents automated and accurate algorithms based on high‐order transformation models for registering three‐dimensional (3D) confocal images of dye‐injected neurons. The algorithms improve upon prior methods in several ways, and meet the more stringent image registration needs of applications such as two‐view attenuation correction recently developed by us. First, they achieve high accuracy (≈ 1.2 voxels, equivalent to 0.4 µm) by using landmarks, rather than intensity correlations, and by using a high‐dimensional affine and quadratic transformation model that accounts for 3D translation, rotation, non‐isotropic scaling, modest curvature of field, distortions and mechanical inconsistencies introduced by the imaging system. Second, they use a hierarchy of models and iterative algorithms to eliminate potential instabilities. Third, they incorporate robust statistical methods to achieve accurate registration in the face of inaccurate and missing landmarks. Fourth, they are fully automated, even estimating the initial registration from the extracted landmarks. Finally, they are computationally efficient, taking less than a minute on a 900‐MHz Pentium III computer for registering two images roughly 70 MB in size. The registration errors represent a combination of modelling, estimation, discretization and neuron tracing errors. Accurate 3D montaging is described; the algorithms have broader applicability to images of vasculature, and other structures with distinctive point, line and surface landmarks.     

Three-dimensional analysis of high voltage electron microscope tilt images: Methods and problems

A quantification program for obtaining three-dimensional image information from high voltage electron microscope stereo pair pictures was developed for a Luzex 5000 image analyzer. A zero tilt image was used as a reference. A real-time image processor system was introduced to improve the image quality and to reduce the beam damage of the specimens. Some modifications of the optical system and the specimen stage were made to improve the accuracy and efficiency of stereoscopy. The accuracy of the measurement was considerably improved by these modifications. Some basic problems and limitations of the method are discussed.     

Argon broad ion beam tomography in a cryogenic scanning electron microscope: a novel tool for the investigation of representative microstructures in sedimentary rocks containing pore fluid

The contribution describes the implementation of a broad ion beam (BIB) polisher into a scanning electron microscope (SEM) functioning at cryogenic temperature (cryo). The whole system (BIB‐cryo‐SEM) provides a first generation of a novel multibeam electron microscope that combines broad ion beam with cryogenic facilities in a conventional SEM to produce large, high‐quality cross‐sections (up to 2 mm²) at cryogenic temperature to be imaged at the state‐of‐the‐art SEM resolution. Cryogenic method allows detecting fluids in their natural environment and preserves samples against desiccation and dehydration, which may damage natural microstructures. The investigation of microstructures in the third dimension is enabled by serial cross‐sectioning, providing broad ion beam tomography with slices down to 350 nm thick. The functionalities of the BIB‐cryo‐SEM are demonstrated by the investigation of rock salts (synthetic coarse‐grained sodium chloride synthesized from halite‐brine mush cold pressed at 150 MPa and 4.5 GPa, and natural rock salt mylonite from a salt glacier at Qom Kuh, central Iran). In addition, results from BIB‐cryo‐SEM on a gas shale and Boom Clay are also presented to show that the instrument is suitable for a large range of sedimentary rocks. For the first time, pore and grain fabrics of preserved host and reservoir rocks can be investigated at nm‐scale range over a representative elementary area. In comparison with the complementary and overlapping performances of the BIB‐SEM method with focused ion beam‐SEM and X‐ray tomography methods, the BIB cross‐sectioning enables detailed insights about morphologies of pores at greater resolution than X‐ray tomography and allows the production of large representative surfaces suitable for FIB‐SEM investigations of a specific representative site within the BIB cross‐section.     

Correlative light and backscattered electron microscopy of bone--part II: automated image analysis

Detailed studies of biological phenomena often involve multiple microscopy and imaging modes and media. For bone biology, various forms of light and electron microscopy are used to study the microscopic structure of bone. Integrating information from the different sources is necessary to understand how different aspects of the bone structure interact. To accomplish this, methods were developed to prepare and image thin sections for correlative light microscopy (LM) and backscattered electron imaging in the scanning electron microscope (BSE-SEM). Images of the same fields of view may then be analyzed for degrees of relationships between specimen features not observed by LM or SEM alone. These methods are applied here to study possible associations between the degree of bone mineralization and pattern of collagen fiber orientation in the mid-shaft of the human femur. The "relational images" obtained allow us to examine the relationship between these two variables, both objectively and quantitatively.     

Extraction of yarn positional information from three-dimensional CT image of textile fabric using a yarn model for its structure analysis

This paper proposes a novel method for analyzing a textile fabric structure to extract positional information regarding each yarn using three-dimensional X-ray computed tomography (3D CT) image. Positional relationship among the yarns can be reconstructed using the extracted yarn positional information. In this paper, a sequence of points on the center line of each yarn of the sample is defined as the yarn positional information, since the sequence can be regarded as the representative position of the yarn. The sequence is extracted by tracing the yarn. The yarn is traced by estimating the yarn center and direction and correlating the yarn part of the 3D CT image with a 3D yarn model, which is moved along the estimated yarn direction. The trajectory of the center of the yarn model corresponds to the positional information of the yarn. The application of the proposed method is shown by experimentally applying the proposed method to a 3D CT image of a double-layered woven fabric. Furthermore, the experimental results for a plain knitted fabric show that this method can be applied to even knitted fabrics.     

Computerized morphometry of the cirrhotic liver: comparative analysis in primary biliary cirrhosis, alcoholic cirrhosis, and posthepatitic cirrhosis

Fibrosis and nodular regeneration are the hallmarks of liver cirrhosis. To assess the degree of fibrosis and the severity of the structural changes affecting parenchymal and extraparenchymal components in liver cirrhosis, a computerized morphometric model has been applied to liver specimens from patients undergoing liver transplantation for primary biliary cirrhosis, posthepatitic and alcoholic cirrhosis. Fifty-eight hepatectomy specimens from patients undergoing liver transplantation for cirrhosis were analyzed: 17 alcoholic, 28 posthepatitic (HBV-related and HCV-related cirrhosis), and 13 primary biliary cirrhoses. Liver specimens were fixed in 10% neutral-buffered formalin and embedded in paraffin. Sections were stained with chromotrope-aniline blue method and monoclonal antibodies against cytokeratin 7 and CD31. Volume fractions of parenchymal compartment and fibrosis were stereologically determined on the specimens stained with chromotrope-aniline blue method. Volume fractions of portal bile ducts, proliferated bile ductules, and hepatocytes with biliary metaplasia were measured on cytokeratin 7 stains, while volume fractions of capillary units have been evaluated on CD31 staining. Volume fraction of fibrosis was higher in primary biliary cirrhosis than in the other disease-induced cirrhosis. The main differences were related to immunohistochemical staining. Volume fraction of hepatocytes with biliary metaplasia was higher in HCV-related cirrhosis, whereas volume fractions of biliary structures were more prominent in HBV-related cirrhosis. Primary biliary cirrhosis was characterized by a reduced number of bile ducts and by a wider expression of cytokeratin 7 into periportal hepatocytes. Capillary units were more prominent in primary biliary cirrhosis than alcoholic and posthepatitic cirrhosis. Our computerized morphometric model well describes and quantifies the morphological alterations of the liver and it could represent an adjunctive tool to evaluate the degree of dysplastic phenomena involving parenchymal and extraparenchymal compartments.