3D Scanning Electron Microscopy Research Articles

Bayesian deconvolution of scanning electron microscopy images using point-spread function estimation and non-local regularization.

Microscopy is one of the most essential imaging techniques in life sciences. High-quality images are required in order to solve (potentially life-saving) biomedical research problems. Many microscopy techniques do not achieve sufficient resolution for these purposes, being limited by physical diffraction and hardware deficiencies. Electron microscopy addresses optical diffraction by measuring emitted or transmitted electrons instead of photons, yielding nanometer resolution. Despite pushing back the diffraction limit, blur should still be taken into account because of practical hardware imperfections and remaining electron diffraction. Deconvolution algorithms can remove some of the blur in post-processing but they depend on knowledge of the point-spread function (PSF) and should accurately regularize noise. Any errors in the estimated PSF or noise model will reduce their effectiveness. This paper proposes a new procedure to estimate the lateral component of the point spread function of a 3D scanning electron microscope more accurately. We also propose a Bayesian maximum a posteriori deconvolution algorithm with a non-local image prior which employs this PSF estimate and previously developed noise statistics. We demonstrate visual quality improvements and show that applying our method improves the quality of subsequent segmentation steps.

Tribological study in microscale using 3D SEM surface reconstruction

Tribology as a branch of materials and mechanical engineering sciences incorporates concepts of friction, wear, and lubrication to discover knowledge and facts about different surfaces. The scanning electron microscope (SEM) and the optical microscope (OM) are two common imaging equipment that have been used in tribological research to visualize and characterize worn surfaces. SEMs are more practical than the OMs since: (1) they are able to generate higher resolution and increased magnification, and (2) they can also provide a greater depth of field. While SEM micrographs still remain two-dimensional (2D), tribological studies truly require information about three-dimensional (3D) surface structures. 3D surface reconstruction of SEM images is very useful in the literature since it helps to analyze the surface roughness of worn surfaces which can imply the wear and friction behavior of materials. In this contribution, copper pins were tested by pin-on-disk tribometer on three different counterfaces (aluminum as softer material, copper with the same hardness, and stainless steel as harder material) and worn surfaces further characterized by using an optimized multi-view 3D SEM surface reconstruction framework. The surface roughness of pin is also calculated by using the 3D SEM surface reconstruction algorithm. In addition, wear mechanisms were studied in further details. Results show that the COF and wear of copper pin against stainless steel are less than COF and wear of copper pin against aluminum and copper due to the formation of in situ iron rich layer on the surface.

Biomedical Applications of Stereoscopy for Three-Dimensional Surface Reconstruction in Scanning Electron Microscopes

The scanning electron microscope (SEM) offers two-dimensional (2D) micrographs of three-dimensional (3D) objects due to its inherent operating mechanisms. To overcome this limitation, other devices have been used for quantitative morphological analysis. Many efforts have been made on the applications of software-based approaches to 3D reconstruction and measurements by SEM. Based on the acquisition of two stereo images, a multi-view technique consists of two parts: (i) geometric calibration and (ii) image matching. Quantitative morphological parameters such as height and depth could be nondestructively measured by SEM combined with special software programs. It is also possible to obtain conventional surface parameters such as roughness and volume of biomedical specimens through 3D SEM surface reconstruction. There is growing evidence that conventional 2D SEM without special electron detectors can be transformed to 3D SEM for quantitative measurements in biomedical research.

3DSEM++: Adaptive and intelligent 3D SEM surface reconstruction

Structural analysis of microscopic objects is a longstanding topic in several scientific disciplines, such as biological, mechanical, and materials sciences. The scanning electron microscope (SEM), as a promising imaging equipment has been around for decades to determine the surface properties (e.g., compositions or geometries) of specimens by achieving increased magnification, contrast, and resolution greater than one nanometer. Whereas SEM micrographs still remain two-dimensional (2D), many research and educational questions truly require knowledge and facts about their three-dimensional (3D) structures. 3D surface reconstruction from SEM images leads to remarkable understanding of microscopic surfaces, allowing informative and qualitative visualization of the samples being investigated. In this contribution, we integrate several computational technologies including machine learning, contrario methodology, and epipolar geometry to design and develop a novel and efficient method called 3DSEM++ for multi-view 3D SEM surface reconstruction in an adaptive and intelligent fashion. The experiments which have been performed on real and synthetic data assert the approach is able to reach a significant precision to both SEM extrinsic calibration and its 3D surface modeling.

Quantitative characterization of cleavage and hydrogen-assisted quasi-cleavage fracture surfaces with the use of confocal laser scanning microscopy

“True” cleavage (TC) and quasi-cleavage (QC) fracture surfaces of low-carbon steel specimens tested in liquid nitrogen and after hydrogen charging respectively were investigated by quantitative confocal laser scanning microscopy (CLSM) and conventional scanning electron microscopy (SEM) with electron-backscattered diffraction (EBSD). Topological and crystallographic features of the TC fracture surface are found in good agreement with the generally accepted cleavage mechanism: TC facets diameters correspond to those of grains; the crack path strictly follows the crystallographic orientation of grains and the most of the cleavage cracks are parallel to {100} planes. On the 2D SEM images, the QC facets appeared resembling the TC ones in terms of river line patterns, shapes and sizes. However, the substantial differences between the topography of these two kinds of fracture surfaces were revealed by 3D CLSM: the average misorientation angle between QC facets and the roughness of the QC fracture surface were much lower than those measured for TC. It is demonstrated that all these features are attributed to the specific fracture mechanism operating during hydrogen-assisted cracking.

A comparative study on the application of SIFT, SURF, BRIEF and ORB for 3D surface reconstruction of electron microscopy images

Image feature detector and descriptor algorithms have made a big advance in almost every area of computer vision applications including object localisation, object tracking, mobile robot mapping, watermarking, panorama stitching and 3D surface reconstruction by assisting the detection and description of feature points in a set of given images. In this paper, we evaluate the performance of four robust feature detection algorithms namely SIFT, SURF, BRIEF and ORB on multi-view 3D surface reconstruction of microscopic samples obtained by a scanning electron microscope (SEM), a widely used equipment in biological and materials sciences for determining the surface attributes of micro objects. To this end, we first develop an optimised multi-view framework for SEM extrinsic calibration and its 3D surface reconstruction. We design a Differential Evolutionary-based algorithm to solve the problem in a global optimisation platform. Several qualitative and quantitative comparisons such as reliability on SEM extrinsic calibration and validity on 3D visualisation performed on real microscopic objects as well as a synthetic model. The present evaluation is expected to provide better insights and consideration to determine which algorithm is well deserved for multi-view 3D SEM surface reconstruction.

3D scanning electron microscopy applied to surface characterization of fluorosed dental enamel

The enamel surfaces of fluorotic teeth were studied by scanning electron stereomicroscopy. Different whitening treatments were applied to 25 pieces to remove stains caused by fluorosis and their surfaces were characterized by stereomicroscopy in order to obtain functional and amplitude parameters. The topographic features resulting for each treatment were determined through these parameters. The results obtained show that the 3D reconstruction achieved from the SEM stereo pairs is a valuable potential alternative for the surface characterization of this kind of samples.

3DSEM: A 3D microscopy dataset.

The Scanning Electron Microscope (SEM) as a 2D imaging instrument has been widely used in many scientific disciplines including biological, mechanical, and materials sciences to determine the surface attributes of microscopic objects. However the SEM micrographs still remain 2D images. To effectively measure and visualize the surface properties, we need to truly restore the 3D shape model from 2D SEM images. Having 3D surfaces would provide anatomic shape of micro-samples which allows for quantitative measurements and informative visualization of the specimens being investigated. The 3DSEM is a dataset for 3D microscopy vision which is freely available at [1] for any academic, educational, and research purposes. The dataset includes both 2D images and 3D reconstructed surfaces of several real microscopic samples.

Al2O3/Al Cermets by Plasma Spraying: Optical Response of Experimental and Numerically Represented Materials

Optical properties of plasma-sprayed coatings and numerically represented samples were studied at wavelengths ranging from visible to mid-infrared. The paper focuses on Al2O3 and Al2O3/Al cermet coatings with different metal concentrations. Microstructure and composition of the samples were characterized in order to explain their optical response that is highly dependent on volume and/or surface scattering as a function of the wavelength range. 2D scanning electron microscopy and 3D x-ray microtomography images were exploited to get statistical data in order to numerically represent simplified samples from the complex microstructure of plasma-sprayed coatings. A Monte Carlo ray-tracing model, based on geometrical optical laws, was then applied to reproduce experimental trends of the acquired optical spectra. Good agreement with the experimental data was obtained.

Micro-crack enhanced permeability in tight rocks: An experimental and microstructural study

The elastic and hydraulic response of a rock and its stress sensitivity are strongly affected by the presence of micro-cracks. Therefore, a full characterization and quantification of cracks at the micro-scale is essential for understanding the physical and transport properties of rocks under stress. As yet, there is no uniquely accepted method to precisely quantify the density and geometrical characteristics of such microstructural features. In this contribution, we present results of quantitative analyses of 2D scanning electron microscopy (SEM) images and 3D X-ray microtomograms acquired on three samples of Carrara Marble artificially cracked by thermal shock. New semi-automatic workflows have been developed to perform these 2D and 3D analyses. The main outcome is the quantification of average length, aspect ratio, and density per unit surface (2D) or volume (3D) of micro-cracks observed. The thermal treatment only opens grain boundaries and does not result in the creation of new intragranular cracks. The results are consistent with the degree of thermal cracking artificially induced on the rock sample prior to the imaging/analysis procedure, i.e., more and wider micro-cracks are measured on samples heated to higher temperatures. The results of these quantitative microstructural analyses are also consistent with nuclear magnetic resonance (NMR) data independently acquired on the same samples saturated with water.

3D fibre deposition and stereolithography techniques for the design of multifunctional nanocomposite magnetic scaffolds.

Magnetic nanocomposite scaffolds based on poly(ε-caprolactone) and poly(ethylene glycol) were fabricated by 3D fibre deposition modelling (FDM) and stereolithography techniques. In addition, hybrid coaxial and bilayer magnetic scaffolds were produced by combining such techniques. The aim of the current research was to analyse some structural and functional features of 3D magnetic scaffolds obtained by the 3D fibre deposition technique and by stereolithography as well as features of multimaterial scaffolds in the form of coaxial and bilayer structures obtained by the proper integration of such methods. The compressive mechanical behaviour of these scaffolds was investigated in a wet environment at 37°C, and the morphological features were analysed through scanning electron microscopy (SEM) and X-ray micro-computed tomography. The capability of a magnetic scaffold to absorb magnetic nanoparticles (MNPs) in water solution was also assessed. confocal laser scanning microscopy was used to assess the in vitro biological behaviour of human mesenchymal stem cells (hMSCs) seeded on 3D structures. Results showed that a wide range of mechanical properties, covering those spanning hard and soft tissues, can be obtained by 3D FDM and stereolithography techniques. 3D virtual reconstruction and SEM showed the precision with which the scaffolds were fabricated, and a good-quality interface between poly(ε-caprolactone) and poly(ethylene glycol) based scaffolds was observed for bilayer and coaxial scaffolds. Magnetised scaffolds are capable of absorbing water solution of MNPs, and a preliminary information on cell adhesion and spreading of hMSCs was obtained without the application of an external magnetic field.

Deposition of Ag 2~6 mol%-Doped ZnO Photocatalyst Thin Films by Thermal Spray Coating Method for E.coli Bacteria Degradation

ZnO doped with 2~6 mol% Silver (Ag) photocatalyst thin films has been deposited on glass substrate by thermal spray coating with temperature deposition of 250°C. A gel of ZnO:Ag precursor has been synthesized by sol-gel route from aqueous/alcoholic solution of zinc acetate dehydrate and silver nitrate mixture at room temperature. The morphology of ZnO:Ag films were investigated scanning electron microscopy (SEM). 3D SEM images of ZnO:Ag thin films show the rough morphology with roughness mean square (rms) of 150 to 195 nm. The grain size of ZnO:Ag films were found in the range 76,5 to 304,8 nm. The photoactivity examination of ZnO:Ag photocatalyst films show the E. Coli bacteria degraded up to 99.99% under sunlight irradiation for 4 hours.

Recent advances in 3D SEM surface reconstruction

The scanning electron microscope (SEM), as one of the most commonly used instruments in biology and material sciences, employs electrons instead of light to determine the surface properties of specimens. However, the SEM micrographs still remain 2D images. To effectively measure and visualize the surface attributes, we need to restore the 3D shape model from the SEM images. 3D surface reconstruction is a longstanding topic in microscopy vision as it offers quantitative and visual information for a variety of applications consisting medicine, pharmacology, chemistry, and mechanics. In this paper, we attempt to explain the expanding body of the work in this area, including a discussion of recent techniques and algorithms. With the present work, we also enhance the reliability, accuracy, and speed of 3D SEM surface reconstruction by designing and developing an optimized multi-view framework. We then consider several real-world experiments as well as synthetic data to examine the qualitative and quantitative attributes of our proposed framework. Furthermore, we present a taxonomy of 3D SEM surface reconstruction approaches and address several challenging issues as part of our future work.

The ultrastructure of mono- and holocentric plant centromeres: an immunological investigation by structured illumination microscopy and scanning electron microscopy.

The spatial distribution of the three centromere-associated proteins α-tubulin, CENH3, and phosphorylated histone H2A (at threonine 120, H2AThr120ph) was analysed by indirect immunodetection at monocentric cereal chromosomes and at the holocentric chromosomes of Luzula elegans by super-resolution light microscopy and scanning electron microscopy (SEM). Using structured illumination microscopy (SIM) as the super-resolution technique on squashed specimens and SEM on uncoated isolated specimens, the three-dimensional (3D) distribution of the proteins was visualized at the centromeres. Technical aspects of 3D SEM are explained in detail. We show that CENH3 forms curved "pads" mainly around the lateral centromeric region in the primary constriction of metacentric chromosomes. H2AThr120ph is present in both the primary constriction and in the pericentromere. α-tubulin-labeled microtubule bundles attach to CENH3-containing chromatin structures, either in single bundles with a V-shaped attachment to the centromere or in split bundles to bordering pericentromeric flanks. In holocentric L. elegans chromosomes, H2AThr120ph is located predominantly in the centromeric groove of each chromatid as proven by subsequent FIB/FESEM ablation and 3D reconstruction. α-tubulin localizes to the edges of the groove. In both holocentric and monocentric chromosomes, no additional intermediate structures between microtubules and the centromere were observed. We established models of the distribution of CENH3, H2AThr120ph and the attachment sites of microtubules for metacentric and holocentric plant chromosomes.

Study on torsional fretting wear behavior of a ball-on-socket contact configuration simulating an artificial cervical disk.

A ball-on-socket contact configuration was designed to simulate an artificial cervical disk in structure. UHMWPE (ultra high molecular weight polyethylene) hot pressed by powders and Ti6Al4V alloy were selected as the material combination of ball and socket. The socket surface was coated by a ~500 nm C-DLC (carbon ion implantation-diamond like carbon) mixed layer to improve its surface nano hardness and wear resistance. The torsional fretting wear behavior of the ball-on-socket model was tested at different angular displacements under 25% bovine serum lubrication with an axial force of 100 N to obtain more realistic results with that in vivo. The fretting running regimes and wear damage characteristics as well as wear mechanisms for both ball and socket were studied based on 2D (two dimension) optical microscope, SEM (scanning electron microscope) and 3D (three dimension) profiles. With the increase of angular displacement amplitude from 1° to 7°, three types of T-θ (Torsional torque-angular displacement amplitude) curves (i.e., linear, elliptical and parallelogram loops) corresponding to running regimes of PSR (partial slip regime), MR (mixed regime) and SR (slip regime) were observed and analyzed. Both the central region and the edge zone of the ball and socket were damaged. The worn surfaces were characterized by wear scratches and wear debris. In addition, more severe wear damage and more wear debris appeared on the central region of the socket at higher angular displacement amplitude. The dominant damage mechanism was a mix of surface scratch, adhesive wear and abrasive wear for the UHMWPE ball while that for the coated socket was abrasive wear by PE particles and some polishing and rolling process on the raised overgrown DLC grains. The frictional kinetic behavior, wear type, damage region and damage mechanism for the ball-on-socket model revealed significant differences with those of a ball-on-flat contact while showing better consistency with that of in vitro cervical prosthesis simulations according to the literature.

Dynamic studies of human corneal fibroblasts.

Keratocytes ordinarily exist in a quiescent state. However, following injury or after surgery they become activated and fibroblastic and drive the repair of the corneal matrix, a process that is highly dynamic and occurs in three dimensions (3D). Previously, MironMendoza and associates used time-lapse microscopy to study the migration of human corneal fibroblasts out of compressed extracellular matrix constructs into a surrounding uncompressed 3D matrix of either collagen or fibrin. This showed that cells migrated individually into collagen matrices (where they subsequently formed connections), but as small groups into fibrin matrices. In their current study, Miron-Mendoza et al. further demonstrate that cells migrate separately into uncompressed 3D collagen matrices even in the presence of thrombin, which they had found to induce cell clustering in 2D on compliant, but not rigid, collagen matrices. The authors also discovered that cells migrating into fibrin moved collectively and formed an interconnected network, even when the cytoskeleton was perturbed by the Rho kinase inhibitor Y-27632 and/or the myosin II inhibitor blebbistatin. The results elegantly demonstrate how time-lapse microscopy helps elucidate the integrated, dynamic behavior of corneal fibroblasts in 3D, and the videos of this make compelling viewing. As the authors point out, the interplay between cells, and their interaction with their environment, are important for corneal wound healing, tissue engineering, and development. Keratocytes do not exist in isolation but form highly extended networks, and the work of Miron-Mendoza and colleagues has provided extra insights into the dynamic nature of cell–cell connectivity. Previously, Nishida and colleagues beautifully documented an integrated system of long, thin cytoplasmic cell processes in the corneas of young adult rats seen by scanning electron microscopy (SEM). More recently, Young and coworkers used a serial block face ablative 3D SEM approach and found that long, thin cell protrusions of presumptive keratocytes in developing cornea extended large distances into the extracellular space, connecting with each other and mirroring (perhaps dictating) the alignment of collagen bundles and emergent lamellae. These studies, however, are merely ‘‘snapshots in time,’’ and the work of Miron-Mendoza and associates significantly adds to the field in the quest for a more comprehensive appreciation of corneal cell–cell and cell–matrix interactions.

Developing 3D SEM in a broad biological context

When electron microscopy (EM) was introduced in the 1930s it gave scientists their first look into the nanoworld of cells. Over the last 80 years EM has vastly increased our understanding of the complex cellular structures that underlie the diverse functions that cells need to maintain life. One drawback that has been difficult to overcome was the inherent lack of volume information, mainly due to the limit on the thickness of sections that could be viewed in a transmission electron microscope (TEM). For many years scientists struggled to achieve three-dimensional (3D) EM using serial section reconstructions, TEM tomography, and scanning EM (SEM) techniques such as freeze-fracture. Although each technique yielded some special information, they required a significant amount of time and specialist expertise to obtain even a very small 3D EM dataset. Almost 20 years ago scientists began to exploit SEMs to image blocks of embedded tissues and perform serial sectioning of these tissues inside the SEM chamber. Using first focused ion beams (FIB) and subsequently robotic ultramicrotomes (serial block-face, SBF-SEM) microscopists were able to collect large volumes of 3D EM information at resolutions that could address many important biological questions, and do so in an efficient manner. We present here some examples of 3D EM taken from the many diverse specimens that have been imaged in our core facility. We propose that the next major step forward will be to efficiently correlate functional information obtained using light microscopy (LM) with 3D EM datasets to more completely investigate the important links between cell structures and their functions.Lay DescriptionLife happens in three dimensions. For many years, first light, and then EM struggled to image the smallest parts of cells in 3D. With recent advances in technology and corresponding improvements in computing, scientists can now see the 3D world of the cell at the nanoscale. In this paper we present the results of high resolution 3D imaging in a number of diverse cells and tissues from multiple species. 3D reconstructions of cell structures often revealed them to be significantly more complex when compared to extrapolations made from 2D studies. Correlating functional 3D LM studies with 3D EM results opens up the possibility of making new strides in our understanding of how cell structure is connected to cell function.

Pore scale imaging and modelling of coal properties

A key parameter in determining the productivity and commercial success of coal seam gas wells is the permeability of individual seams. Laboratory testing of core plugs is commonly used as an indicator of potential seam permeability. The highly heterogeneous and stress-dependent nature of coal makes laboratory measurements difficult to perform and the results difficult to interpret. Consequently, permeability in coal is poorly understood. The permeability of coal at the core scale depends on the geometry, topology, connectivity, mineralisation and spatial distribution of the cleat system, and a better understanding of coal permeability, that and the factors that control this depends on having a better understanding and detailed characterisation of the system. The authors used high resolution micro-focus X-ray computed tomography and 2D-3D image registration techniques to overlay tomograms of the same core plug, with and without X-ray attenuating fluids present in the pore space, with 2D scanning electron microscope images to produce detailed 3D visualisations of the geometry and topology of the cleat systems in the coal plugs. Novel filtering algorithms were used to produce segmentations that preserve cleat aperture dimensions and together with large-scale fluid flow simulations, they performed directly on the images and were used to compute porosities and permeabilities. Image resolution and segmentation sensitivity studies are also described, which show that the core scale permeability is controlled by a small number of well-connected percolating cleats. The results of this study demonstrate the potential of simple image-based analysis techniques to provide rapid estimates of core plug permeabilities.

Further Closing the Resolution Gap: Integrating Cryo-Soft X-Ray and Light Microscopies

Further Closing the Resolution Gap: Integrating Cryo-Soft X-Ray and Light Microscopies

A novel surface quality evaluation method in ultra-precision raster milling using cutting chips

Although online surface quality evaluation is important in ultra-precision machining technologies, very little research has been reported on the topic. In this paper, cutting chips were employed to evaluate machined surface quality online under the occurrence of tool wear in ultra-precision raster milling. Cutting chips were collected and examined by a 3D scanning electron microscope. The inspected cross-sectional shape of ‘ridges’ imprinted by the tool fracture was approximated into two geometric elements so that it could be determined by a mathematical model. The geometric elements for every ridge were assembled into a virtual cutting edge so as to rebuild the machined surface. A mathematical model was established to realize the rebuilt machined surface and calculate the surface roughness under the consideration of tool fractures effects. The theoretical and experimental results show that the ridges imprinted on the machined surface can be predicted by examining a section of the collected cutting chips. It is interesting to note that the quality of the machined surface is reflected in the location and the height of ridges. This demonstrates that the online surface quality evaluation using cutting chips is a novel method as compared to conventional methods, since it can evaluate the machined surface and simulate the machined surface topography without the need to stop the cutting process.