Backscattered Electron Signal Research Articles

Microstructure of confectionery masses revealed by cryo-planing

Here we present a novel, combined cryo-scanning electron microscopy (cryo-SEM) and confocal laser scanning microscopy (CLSM) method for imaging of chocolate confectionery product microstructures of several millimeters down to about 100 nm. The SEM part of the method is based on cryo-fixation, cryo-polishing, and scanning electron microscopy, at low vacuum and low temperature using the backscattered electron signal. Starting with cryo-fixation of the chocolate sample in a desired state (cooled, ambient, or melted), the sample is cryo-planed in a cryo-ultramicrotome. Once a polished cut is obtained, the sample is analyzed using a cryo-SEM technique, with the unusual combination of low temperature and low vacuum settings, without heavy metal coating. Imaging is done based on material density contrast. Elemental composition of particles is recorded by energy dispersive x-ray spectroscopy (EDS). The combination of imaging contrast and EDS allows identification and measurement of the four main constituents of chocolate (cocoa solids, fat, sugar, and milk solids). Finally, the same cryo-polished sample sections of solid chocolate products are analyzed using a CLSM imaging technique to reveal complementary microstructural details. An obvious application of the method could be the visualization and quantitative analysis of the size, shape, and composition of chocolate confectionery products.

Extracting ductile cast iron microstructure parameters from fracture surfaces: A deep learning based instance segmentation approach

This study investigates the deep-learning based microstructural analysis from SEM images of ductile cast iron fracture surfaces. A Mask R-CNN model was trained, achieving 70% precision and 75% recall in graphite particle detection. Combined with a fracture surface reconstruction using the.4-quadrant backscattered electron signal, key parameters, including the particle size, shape and distance were extracted accurately. Compared to micrograph analysis, following probabilistic simulations showed the impact of the higher microstructural variance for the fracture surfaces on crack initiation, leading to higher scatter and elevated crack resistance curves. This highlights the potential of deep-learning based analysis for comprehensive microstructural characterization.

KMnO4/Pb staining allows uranium free imaging of tissue architectures in low vacuum scanning electron microscopy

Scanning electron microscopy under low-vacuum conditions allows high-resolution imaging of complex cell/tissue architectures in nonconductive specimens. However, the conventional methods for metal staining of biological specimens require harmful uranium compounds, which hampers the applications of electron microscopy. Here, we introduce a uranium-free KMnO4/Pb metal staining protocol that allows multiscale imaging of extensive cell/tissue architectures to intensive subcellular ultrastructures. The obtained image contrast was equivalent to that of Ur/Pb staining and sufficient for ultrastructural observation, showing the fine processes of podocytes in the glomerulus, which were invisible by light microscopy. The stainability in the elastic tissue indicated that the distinct histochemical properties of KMnO4 oxidation led to Pb deposition and BSE signal enhancement superior to Ur staining. Elemental analysis clarified that the determinant of the backscattered electron signal intensity was the amount of Pb deposition enhanced by KMnO4 oxidation. This user-friendly method is anticipated to create a new approach for biomedical electron microscopy.

Imaging and Segmenting Grains and Subgrains Using Backscattered Electron Techniques.

We present two new methods of processing data from backscattered electron signals in a scanning electron microscope to image grains and subgrains. The first combines data from multiple backscattered electron images acquired at different specimen geometries to (1) better reveal grain boundaries in recrystallized microstructures and (2) distinguish between recrystallized and unrecrystallized regions in partially recrystallized microstructures. The second utilizes spherical harmonic transform indexing of electron backscatter diffraction patterns to produce high angular resolution orientation data that enable the characterization of subgrains. Subgrains are produced during high-temperature plastic deformation and have boundary misorientation angles ranging from a few degrees down to a few hundredths of a degree. We also present an algorithm to automatically segment grains from combined backscattered electron image data or grains and subgrains from high angular resolution electron backscatter diffraction data. Together, these new techniques enable rapid measurements of individual grains and subgrains from large populations.

Standardization and quantification of backscattered electron imaging in scanning electron microscopy

Backscattered electron (BSE) imaging based on scanning electron microscopy (SEM) has been widely used in scientific and industrial disciplines. However, achieving consistent standards and precise quantification in BSE images has proven to be a long-standing challenge. Previous methods incorporating dedicated calibration processes and Monte Carlo simulations have still posed practical limitations for widespread adoption. Here we introduce a bolometer platform that directly measures the absorbed thermal energy of the sample and demonstrates that it can help to analyze the atomic number (Z) of the investigated samples. The technique, named Atomic Number Electron Microscopy (ZEM), employs the conservation of energy as the foundation of standardization and can serve as a nearly ideal BSE detector. Our approach combines the strengths of both BSE and ZEM detectors, simplifying quantitative analysis for samples of various shapes and sizes. The complementary relation between the ZEM and BSE signals also makes the detection of light elements or compounds more accessible than existing microanalysis techniques.

A novel method for measuring the charging kinetics of dielectrics under electron irradiation in SEM

A novel method is proposed to measure the charging potential of dielectric targets under medium energy electron irradiation in a scanning electron microscope. The method is based on the measurement of backscattered electron signals by standard semiconductor or scintillation detectors. The signal is pre-calibrated by grey scale levels on the SEM screen. The detector signal is made up with backscattered and secondary electrons which are accelerated in the electric field created above the dielectric surface under irradiation.

Influence of Curing Agent Amount on Properties of Dynamic Vulcanized Phenyl Silicone Rubber-SEBS-SBS System.

In this paper, we prepared a new type of thermoplastic vulcanizate (TPV) by melt blending methyl vinyl phenyl silicone rubber (PSR), styrene butylene copolymer (SBS), and hydrogenated SBS (SEBS) and then dynamically vulcanizing it. At the same time, we studied the influence of the content of the vulcanizing agent on the properties. The corresponding backscattered electron images were obtained by a scanning electron microscope (SEM) test of each group of samples, as well as the distribution of the PSR phase and the SEBS-SBS phase, and the vulcanization process of the samples with a vulcanizing agent content of 1 phr were characterized. According to the imaging principle of the backscattered electron signal, we found that the atomic number contrast can be clearly reflected in the backscattered image. From the obtained images, we found that PSR is a dispersed phase, while SEBS and SBS are continuous phases, that is, they had a "Sea-Island" structure. In the first 30 s of the vulcanization reaction, the "Sea-Island" structure is formed, and then the vulcanization reaction rate gradually slows down. We then printed the images and analyzed them using a colorimeter and found that it was feasible to quantitatively characterize the size of the compatible layer between the continuous and dispersed phases. According to the quantitative characterization results, we found that the silane coupling agent KH-172 can increase the thickness of the compatible layer by nearly 35%. In addition, we also tested the mechanical properties and low-temperature elastic properties of the material. Finally, we found that when the content of the vulcanizing agent was 1 phr, the elastic properties and tensile properties were the best, and when the content of the vulcanizing agent was more than 1 phr, the tensile and elastic properties of the material decreased significantly. At the same time, we also found that the addition of the silane coupling agent KH-172 can also significantly improve the tensile properties and elastic properties of TPV, which we believe is related to the increase in the thickness of the compatible layer. The test results of dynamic mechanics show that PSR has good compatibility with SEBS-SBS. When the vulcanizing agent content is less than or equal to 1 phr, the material exhibits good low-temperature resistance. In addition, through the test of the melt index of each group, it was also found that the addition of the vulcanizing agent will affect the fluidity of the melt to a certain extent. When the content of the vulcanizing agent is greater than 1 phr, the melt fluidity decreases more obviously.

A Morphological Study of Dynamically Vulcanized Styrene-Ethylene-Butylene-Styrene/Styrene-Butylene-Styrene/MethylVinylSilicon Rubber Thermoplastic Elastomer.

In this work, we prepared thermoplastic silicone rubber (TPSiV) by dynamically vulcanizing different relative proportions of methyl vinyl silicone rubber (MVSR), styrene ethylene butene styrene block copolymer (SEBS), and styrene butadiene styrene block copolymer (SBS). The compatibility and distribution of the MVSR phase and SEBS/SBS phase were qualitatively characterized by Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM) tests on TPSiV. Subsequently, the backscattered electron signal image was analyzed using a colorimeter, and it was found that the size of the interface layer between the MVSR phase and the SEBS-SBS phase could be quantitatively characterized. This method overcomes the defect of the etching method, which cannot quantitatively analyze the size of the compatible layer between the two polymers. The final experiment proved that the two phases in TPSiV exhibited a “sea-island” structure, in which the MVSR phase acted as a dispersed phase in the SEBS-SBS phase. In addition, the addition of the silane coupling agent KH-907 (γ-isocyanatopropyltriethoxysilane) improved the mechanical properties of TPSiV, increasing the tensile strength by about 40% and the elongation at break by 30%. The permanent tensile deformation increase rate was about 15%. Through the quantitative measurement of the compatible layer, it was found that KH-907 could increase the thickness of the interface layer between the MVSR phase and the SEBS-SBS phase by more than 30%, which explained why the silane coupling agent KH-907 improved the mechanical properties of TPSiV at the micro level.

Applications of Scanning Electron Microscopy Using Secondary and Backscattered Electron Signals in Neural Structure.

Scanning electron microscopy (SEM) has contributed to elucidating the ultrastructure of bio-specimens in three dimensions. SEM imagery detects several kinds of signals, of which secondary electrons (SEs) and backscattered electrons (BSEs) are the main electrons used in biological and biomedical research. SE and BSE signals provide a three-dimensional (3D) surface topography and information on the composition of specimens, respectively. Among the various sample preparation techniques for SE-mode SEM, the osmium maceration method is the only approach for examining the subcellular structure that does not require any reconstruction processes. The 3D ultrastructure of organelles, such as the Golgi apparatus, mitochondria, and endoplasmic reticulum has been uncovered using high-resolution SEM of osmium-macerated tissues. Recent instrumental advances in scanning electron microscopes have broadened the applications of SEM for examining bio-specimens and enabled imaging of resin-embedded tissue blocks and sections using BSE-mode SEM under low-accelerating voltages; such techniques are fundamental to the 3D-SEM methods that are now known as focused ion-beam SEM, serial block-face SEM, and array tomography (i.e., serial section SEM). This technical breakthrough has allowed us to establish an innovative BSE imaging technique called section-face imaging to acquire ultrathin information from resin-embedded tissue sections. In contrast, serial section SEM is a modern 3D imaging technique for creating 3D surface rendering models of cells and organelles from tomographic BSE images of consecutive ultrathin sections embedded in resin. In this article, we introduce our related SEM techniques that use SE and BSE signals, such as the osmium maceration method, semithin section SEM (section-face imaging of resin-embedded semithin sections), section-face imaging for correlative light and SEM, and serial section SEM, to summarize their applications to neural structure and discuss the future possibilities and directions for these methods.

Three-dimensional reconstruction of the Golgi apparatus in osteoclasts by a combination of NADPase cytochemistry and serial section scanning electron microscopy.

The three-dimensional morphology of the Golgi apparatus in osteoclasts was investigated by computer-aided reconstruction. Rat femora were treated for nicotinamide adenine dinucleotide phosphatase (NADPase) cytochemistry, and light microscopy was used to select several osteoclasts in serial semi-thin sections to investigate the Golgi apparatus by backscattered electron-mode scanning electron microscopy. Lace-like structures with strong backscattered electron signals were observed around the nuclei. These structures, observed within the Golgi apparatus, were attributed to the reaction products (i.e., lead precipitates) of NADPase cytochemistry. Features on the images corresponding to the Golgi apparatus, nuclei, and ruffled border were manually traced and three-dimensionally reconstructed using ImageJ/Fiji (an open-source image processing package). In the reconstructed model, the Golgi apparatus formed an almost-continuous structure with a basket-like configuration, which surrounded all the nuclei and also partitioned them. This peculiar three-dimensional morphology of the Golgi apparatus was discovered for the first time in this study. On the basis of the location of the cis- and trans-sides of the Golgi apparatus and the reported results of previous studies, we postulated that the nuclear membrane synthesized specific proteins in the osteoclasts and, accordingly, the Golgi apparatus accumulated around the nuclei as a receptacle.

Tracing method based on heterodyne interferometer for scanning electron microscopes

In this study, a tracing method for a scanning electron microscope was developed based on a heterodyne interferometer, which can directly trace the measured values to the laser wavelength and then to the internationally defined meter length reference. In this method, a sample scanning motion is introduced, and the measuring mirror of the interferometer is fixed on the sample stage. The movement and displacement of the sample stage is recorded by the laser interferometer displacement measurement system, which synchronously collects the secondary electrons or backscattered electron signals excited on the sample surface. The feature size of the sample is measured, and the value can be traced directly to the international unit of length using the frequency-stabilized laser reference, which proves that the proposed method is an absolute measurement method. This study analyzed the specific technology implementation methods and experimental results, including positioning control of the displacement system and its measurement and control during the measurement process. Measurement uncertainty budgets are also discussed. Experimental measurements were carried out and their accuracy was verified. Results showed that this method could effectively obtain the performance parameters of the feature size intuitively and significantly improve the measurement accuracy.

Inelastic Scattering in Electron Backscatter Diffraction and Electron Channeling Contrast Imaging.

Electron backscatter diffraction (EBSD) and electron channeling contrast imaging (ECCI) are used to extract crystallographic information from bulk samples, such as their crystal structure and orientation as well as the presence of any dislocation and grain boundary defects. These techniques rely on the backscattered electron signal, which has a large distribution in electron energy. Here, the influence of plasmon excitations on EBSD patterns and ECCI dislocation images is uncovered by multislice simulations including inelastic scattering. It is shown that the Kikuchi band contrast in an EBSD pattern for silicon is maximum at small energy loss (i.e., few plasmon scattering events following backscattering), consistent with previous energy-filtered EBSD measurements. On the other hand, plasmon excitation has very little effect on the ECCI image of a dislocation. These results are explained by examining the role of the characteristic plasmon scattering angle on the intrinsic contrast mechanisms in EBSD and ECCI.

An optimized approach using cryofixation for high-resolution 3D analysis by FIB-SEM

Compared with conventional two-dimensional transmission electron microscopy (TEM), focused ion beam scanning electron microscopy (FIB-SEM) can provide more comprehensive 3D information on cell substructures at the nanometer scale. Biological samples prepared by cryofixation using high-pressure freezing demonstrate optimal preservation of the morphology of cellular structures, as these are arrested instantly in their near-native states. However, samples from cryofixation often show a weak back-scatter electron signal and bad image contrast in FIB-SEM imaging. In addition, it is impossible to do large amounts of heavy metal staining. This is commonly achieved via established osmium impregnation (OTO) en bloc staining protocols. Here, we compared the FIB-SEM image quality of brain tissues prepared using several common freeze-substitution media, and we developed an approach that overcomes these limitations through a combination of osmium tetroxide, uranyl acetate, tannic acid, and potassium permanganate at proper concentrations, respectively. Using this optimized sample preparation protocol for high-pressure freezing and freeze-substitution, perfect smooth membrane morphology, even of the lipid bilayers of the cell membrane, was readily obtained using FIB-SEM. In addition, our protocol is broadly applicable and we demonstrated successful application to brain tissues, plant tissues, Caenorhabditis elegans, Candida albicans, and chlorella. This approach combines the potential of cryofixation for 3D large volume analysis of subcellular structures with the high-resolution capabilities of FIB-SEM.

Enhancement of image contrast for carbon nanotube and polymer composite film in scanning electron microscope.

Image contrast between carbon nanotubes (CNTs) and polytetrafluoroethylene (PTFE) in a CNT/PTFE composite film, which is difficult to obtain by conventional backscattered electron (BSE) imaging, was optimized to better elucidate the distribution of CNT in the film. Ultra-low landing energy condition (0.3keV in this study) was used to prevent specimen damage due to electron beam irradiation. Signal acceptance maps, which represent the distributions of energy and take-off angle, were calculated to evaluate the features of the signal detection system used in this study. SEM images of this composite film were taken under several sets of conditions and analyzed using these acceptance maps. CNT and PTFE in the composite film can be clearly distinguished with material and topographic contrasts using the BSE signal under optimized energy and take-off angle ranges, even at ultra-low landing energy conditions.

Observation of the microstructure of the metastable Ti15Mo alloy after ageing

Omega phase particles can be observed by conventional SEM. Metastable Ti15Mo alloy was annealed at 500 °C for 16 hours. During annealing, ω phase particles grew and got chemically stabilized by expelling Mo atoms. As the result, these ellipsoidal particles, approx. 100 nm long and 50 nm wide can be observed using back-scattered electrons signal in conventional SEM due to chemical contrast. TEM study proved that these particles indeed belong to ω phase. Co-existence of β, ω and α phases was observed. Thin α lamellae were observed along with distorted ω phase particles by TEM.

Depth-correlated backscattered electron signal intensity for 3D-profile measurement of high aspect ratio holes.

In-line metrology for measuring 3D features of the high aspect ratio (HAR) holes is becoming more challenging due to the progressing semiconductor technology, particularly in memory devices. Measurements of the bottom critical dimension (CD), taper angles and 3D profiles of the HAR holes require new imaging capabilities. In this work, we explored the characteristics of high-energy backscattered electron (BSE) signals and demonstrated their promising application to 3D metrology. From Monte Carlo simulation results, it is worth noting that BSE signal intensity emitted from an irradiated location in the depth of the hole decreases exponentially with the increase of the depth from the top surface (perpendicular depth) of the hole. Furthermore, the influences of various factors including the electron energy, the depth and the sidewall angle (SWA) of the hole on the attenuation of the BSE signal intensity were investigated. The simulation results show that the attenuation of the BSE signal intensity depends on the electron energy, the depth and the density of the hole but is independent of the SWA and the incident angle of the primary electron beam. Based on the characteristics of the BSE signal intensity, an algorithm was proposed for the 3D metrology of the HAR holes. Finally, the differences in CDs between the measured value and the target value of HAR holes with various geometries were examined. A maximum measurement bias within ±2.0nm for various holes with different depths, densities and SWA values shows great potential of depth-correlated BSE signals in 3D metrology.

SUN-017 LOW-VACUUM SCANNING ELECTRON MICROSCOPY ANALYSIS FOR HUMAN BIOPSY SAMPLES

One of the major goal of biological microscopy is to elucidate the structural evidence relating functional activity or pathophysiological condition. High resolution imaging by electron microscopy can provide detailed tissue architectures, but the specific and skillful technics are required and it takes long time to the sample preparation. Low-vacuum scanning electron microscopy (LV-SEM) developed recently is a simple and fast technic to observe higher resolution of histological paraffin sections prepared for conventional light microscopy. Periodic acid methenamine silver (PAM) stain is a routine staining for basement membrane in renal biopsy, and contains silver, a heavy metal, which enhances the backscattered electron signal. To evaluate the benefit of LV-SEM, we observed PAM stained glomerular basement membrane (GBM) in human renal biopsy samples. The study was performed under the declaration of Helsinki principles. The study protocol was approved by the Institutional Review Board of the Tohoku Medical and Pharmaceutical University. All participants provided written informed consent after a full explanation of the purpose of the study and the potential risk involved. The human kidneys were obtained from patients with membranous nephropathy, IgA nephropathy, Alport syndrome, or thin basement membrane disease by renal biopsy and embedded in paraffin. Four-μm sections were deparaffinized with xylene, hydrated with ethanol, and stained with periodic acid methenamine silver (PAM). Miniscope TM4000 (Hitachi High-Technologies) was used to observe the GBM by detecting backscattered electron signal. After evacuation of the specimen chamber a few minutes, the sections were observed under the electron beam accelerating voltage of 10 kV for higher magnification (x2,500 to x20,000) or 15 kV for lower magnification (x40 to x2,000) with 30 Pa. The observations at higher magnification were easily performed under LV-SEM by zooming up from x40 to x20,000. The PAM-positive GBMs were bright under LV-SEM. The GBMs showed dark electron dense deposits in membranous nephropathy and IgA nephropathy, and characteristic coarse meshwork appearances in Alport syndrome. The GBMs in thin basement membrane disease seemed relatively thinner than the others. Direct observation of conventional renal biopsy paraffin sections by LV-SEM is a simple and beneficial method for the rapid and detailed analysis of the diseases. It will likely be useful for the observation of GBM morphological change.

Detecting Gunshot Residue from Sellier & Bellot Nontox Heavy Metal-free Primer by in situ Cathodoluminescence.

Gunshot residue (GSR) from the discharge of ammunition can provide crucial information in reconstructing criminal cases. Traditional primers create particles of heavy metals such as lead, barium, and antimony. In forensic laboratories, automatic inorganic particle detection is performed by scanning electron microscopy (SEM), using the backscattered electron signal to search for bright residues among the many darker environmental particles, due to higher electron density of the former. Some innovative primers, indicated as heavy metal-free (HMF), produce a residue of elements with atomic numbers below 21, urgently demanding new detecting solutions. For the first time, residues from Sellier & Bellot Nontox HMF primer are demonstrated to emit visible light under electron beam stimulation in a SEM. Cathodoluminescence is then proposed as a promising tool to both detect and characterize residues in forensic cases involving HMF primers, with minor changes to traditional analytical apparatus used for inorganic GSR analysis.

Monte Carlo simulation and theoretical calculation of SEM image intensity and its application in thickness measurement

The intensity profiles of backscattered and secondary electrons from a pure Mg sample have shown a variation with sample thickness and acceleration voltage in the range of 5–30 kV, depending on the specimen holder used. The intensities of backscattered electron (BSE) and secondary electron (SE) signals increases with the sample thickness until saturation when using a scanning transmission electron microscopy (STEM) holder with a closed tube below the sample. However the SE signal increases to the maximum and then decreases with the sample thickness when using a transmission Kikuchi diffraction (TKD) holder with no shielding below the sample whereas the BSE signal again increases until saturation. The influence of the holder on the SE signals is caused by the fact that secondary electrons emitted from the bottom surface could be detected only when using the TKD holder but not the STEM holder. The experimental results obtained are consistent with the Monte Carlo simulation results. Application of the magnitude of the SE and BSE signals to measurement of sample thickness has been considered and the BSE image profile shows a reasonably good accuracy.

The Use of Low Vacuum Scanning Electron Microscopy (LVSEM) to Analyze Peripheral Nerve Samples.

The use of electron microscopy allows analysis of the microarchitecture of peripheral nerves both during development and in the processes of nerve regeneration and repair.We describe a novel method for the rapid analysis and quantification of myelin in peripheral nerve using a low vacuum scanning electron microscopy protocol. For this methodology, excised nerves are prepared for traditional transmission electron microscopy (TEM) imaging, but at the stage where semi-thin sections would be taken, the resin block is instead imaged at low vacuum in the scanning electron microscope (SEM) using the backscattered electron signal. Any features in the tissue which have incorporated high concentrations of osmium from the fixation process (e.g., myelin) appear as bright regions in the image.Myelin therefore is easily identifiable in the images, and since there is a high contrast difference between it and the surrounding tissue, automated measurements for myelin thickness (e.g., G ratio) using standard and freely available image analysis software (e.g., ImageJ) are easily achievable, consistent, and repeatable. This method therefore greatly speeds up the analysis of nerve samples and, in many cases, will obviate the need for ultrathin sections and TEM for analyzing nerve morphology.Low vacuum (LV) SEM imaging has benefits compared with light microscopy; magnification is continuous, resolution is higher, and contrast and brightness are controllable. Since the resin block does not need a metal coating for imaging in LV mode, once the images have been collected, the block is still ready to section for both light microscopy (LM) and TEM.