Specimens For Electron Microscopy Research Articles

Fast Processing of Electron Microscopic Specimen Preserved Ultrastructure of Glomeruli and Electron-Dense Deposits in Diagnostic Renal Biopsies: A Prospective and Retrospective Comparative Study

Optimization of electron microscopy (EM) tissue processing protocols is essential to handle the global increase in the number of renal biopsies requiring EM for accurate diagnosis. The conventional EM processing method (CEM) is the standard method used by >95% of laboratories worldwide and it takes at least 48 to 52 hours for completion. In contrast, a fast-processing EM (FEM) method using microwave irradiation can be completed in 8 hours, allowing EM findings to be reported within 24 hours for most cases. There is widespread concern about the suboptimal quality of the FEM that may compromise its diagnostic roles; however, qualitative and quantitative data supporting the noninferiority of FEM compared with CEM has not been reported. We performed both prospective and retrospective studies. The prospective analysis compares FEM and CEM images from the same biopsy samples. For each case, the tissue was divided into 2 pieces: 1 piece for FEM processing and the second for CEM processing. The retrospective study compares the EM images of renal cases with electron-dense deposits from our archives that were processed either by FEM or CEM. The prospective analysis included 4 cases: lupus membranous nephropathy, IgA nephropathy, immune complex–mediated glomerulonephritis, and acute tubular injury. Both FEM and CEM methods obtained high-resolution images with comparable quality. A quantitative morphometric analysis of the glomerular basement membrane (GBM) in the IgA nephropathy case showed similar GBM thickness when processed by the FEM and the CEM, suggesting that FEM did not affect GBM thickness. The retrospective study of 42 cases with electron-dense deposits showed that the ultrastructural features of electron-dense deposits were indistinguishable between the FEM and the CEM. This included microtubular substructures in immunotactoid glomerulonephritis, the "fingerprint" deposits in cryoglobulinemic glomerulonephritis, fibril deposits in the light chain amyloidosis as well as fibrillary glomerulonephritis, with comparable morphometric measurements of the deposits. The FEM is efficient, consistent, reproducible, and delivers comparable high-quality sections and images for diagnostic assessment of renal biopsies, compared with those attained by the CEM while decreasing turnaround time significantly, making it possible to provide faster and accurate diagnostic results.

Investigation of static mechanical characteristics and numerical simulation of fractal gangue cemented backfill materials

This study investigated the static mechanical responses of gangue cemented backfill materials (GCBM) with aggregate particle size distribution (APSD) satisfied fractal grading theory. The recycling of gangue in GCBM alleviates gangue accumulation pollution and improves mining production efficiency. Macroscopically, uniaxial compression experiments regarding various loading strain rates (ε̇) on gangue cemented backfill specimens (GCBS) were conducted. Acoustic emission monitoring and digital image correlation technique were employed to reveal crack activities and strain field evolution in real time. Microscopically, scanning electron microscopy and numerical specimens considering APSD were utilized to analyze the microstructure and damage process. The deterioration mechanisms and quantified number of cracks were explored at the micro level. The conclusions are as follows: (1) The axial stress (σ) of GCBM increased with fractal dimension (D) of APSD and ε̇. For the same σ, cumulative AE counts decreased with increasing ε̇ and D. (2) The main failure mode of the GCBS under static loading was tensile failure, exhibiting tensile cracks initiating at the bonding–aggregate interface. (3) The increase in the proportion of fine aggregate contributed to the optimization of the microstructures of the GCBS (4) An increased proportion of fine aggregate in the GCBS improved the synergistic load-bearing capacity between the cementing and aggregate mediums, leading to an enhancement in the σ.

Natural History and Clinicopathological Associations of TRPC6-Associated Podocytopathy.

Understanding the genetic basis of human diseases has become integral to drug development and precision medicine. Recent advancements have enabled the identification of molecular pathways driving diseases, leading to targeted treatment strategies. The increasing investment in rare diseases by the biotech industry underscores the importance of genetic evidence in drug discovery and approval processes. Here we studied a monogenic Mendelian kidney disease, TRPC6-associated podocytopathy (TRPC6-AP), to present its natural history, genetic spectrum, and clinicopathological associations in a large cohort of patients with causal variants in TRPC6, in order to help define the specific features of disease and further facilitate drug development and clinical trials design. the study involved 64 individuals from 39 families with TRPC6 causal missense variants. Clinical data, including age of onset, laboratory results, response to treatment, kidney biopsy findings, and genetic information, were collected from multiple centers nationally and internationally. Exome or targeted sequencing was performed and variant classification was based on strict criteria. Structural and functional analyses of TRPC6 variants were conducted to understand their impact on protein function. In depth re-analysis of light and electron microscopy specimens for 9 available kidney biopsies was conducted to identify pathological features and correlates of TRPC6-AP. Large-scale sequencing data did not support causality for TRPC6 protein-truncating variants. We identified 21 unique TRPC6 missense variants, clustering in three distinct regions of the protein, and with different effects on TRPC6 3D protein structure. Kidney biopsy analysis revealed FSGS patterns of injury in most cases, along with distinctive podocyte features including diffuse foot process effacement and swollen cell bodies. The majority of patients presented in adolescence or early adulthood but with ample variation (average 22, SD ± 14 years), with frequent progression to kidney failure but with variability in time between presentation and ESKD. This study provides insights into the genetic spectrum, clinicopathological associations, and natural history of TRPC6-AP.

Calibrating cryogenic temperature of TEM specimens using EELS

Cryogenic Scanning/Transmission Electron Microscopy has been established as a leading method to image sensitive biological samples and is now becoming a powerful tool to understand materials' behavior at low temperatures. However, achieving precise local temperature calibration at low temperatures remains a challenge, which is especially crucial for studying phase transitions and emergent physical properties in quantum materials. In this study, we employ electron energy loss spectroscopy (EELS) to measure local cryogenic specimen temperatures. We use the temperature-dependent characteristics of aluminum's bulk plasmon peak in EEL spectra, which shifts due to changes in electron density caused by thermal expansion and contraction. We successfully demonstrate the versatility of this method by calibrating different liquid nitrogen cooling holders in various microscopes, regardless of whether a monochromated or non-monochromated electron beam is used. Temperature discrepancies between the actual temperature and the setpoint temperatures are identified across a range from room temperature to 100 K. This work demonstrates the importance of temperature calibrations at intermediate temperatures and presents a straightforward, robust method for calibrating local temperatures of cryogenically-cooled specimens in electron microscopes.

Fs-laser preparation of half grid specimens for atom probe tomography and transmission electron microscopy

For atom probe tomography (APT) and transmission electron microscopy (TEM) half grids are commonly applied as specimen carriers, serving as mount for focused ion beam (FIB) processed lift-outs. This technical note presents an alternative way to fabricate half grids directly from the material to be investigated using femtosecond (fs)-laser ablation. After cutting ∼200 µm thin in-plane and cross-plane slices from a TiN coated cemented carbide substrate using a precision cutting saw, two different half grid geometries, one suitable for APT and the other for TEM, were cut via fs-laser processing. Freestanding posts for APT were further sharpened and the areas intended for TEM were thinned top-down using the fs-laser system followed by final annular FIB milling of the APT and thinning of the TEM specimens. For proof of concept, the TiN APT half grid specimens in in- and cross-plane direction were successfully measured in voltage and laser-assisted mode, utilizing a local electrode atom probe, while electron transparency of the in- and cross-plane TEM half grid specimens was proven using a scanning electron microscope equipped with a scanning transmission electron microscopy (STEM) detector. The herein presented method is simple, fast and omits the necessity of a lift-out, reducing the complexity of FIB operations. Additionally, a frequently considered weak point of APT lift-out specimens, the Pt weld between specimen and half grid or microtip coupon is avoided, whilst a wide range of materials can be processed and consumables consumption is reduced. Thus, this work clearly demonstrates the great potential of fs-laser processing for APT and TEM specimen preparation.

Creating Efficient Workflows for Electron Microscopy Laboratories with Automated Specimen Preparation

Abstract Electron microscopy (EM) is essential to the biological and biomedical sciences and clinical diagnostic pathology. Electron microscopy specimen preparation is laborious and time-consuming, with transmission EM (TEM) chemical preparation typically requiring 1–3 days, while volume electron microscopy (vEM) takes 3–5 days of tedious manual reagent exchanges every few minutes or hours. This places a considerable burden on laboratory scientists with the ongoing demand for TEM, and the rapidly growing demand for vEM due to its potential to revolutionize structural biology, connectomics, and related fields. This burden is exacerbated by a shortage of trained electron microscopy scientists as current staff retire, and few enter the workforce. This report provides four case studies to illustrate how automated and faster specimen preparation workflows using mPrep™ Automated Specimen Processors (ASP-1000™ and ASP-2000™, Microscopy Innovations, LLC) free electron microscopy staff in academic and pre-clinical research labs and a clinical pathology laboratory.

Nanoscale liquid Al phase formation through beam heating of MgAl2O4 in TEM.

In this study, electron-beam irradiation of a MgAl2O4 single-crystalline thin-film specimen in a transmission electron microscope reveals an unexpected formation of nanoscale liquid Al droplets. Despite the comparable melting temperatures of Mg and Al, the resulting liquid phase is predominantly composed of Al. This predominant presence of Al in the liquid phase is attributed to the selective evaporation of Mg, driven by its higher vapor pressure at elevated temperatures. Our observations suggest a correlation between electron-beam irradiation and a subsequent rise in specimen temperature. In particular, the observed melting of Al defies explanation by the widely accepted mechanism that attributes specimen heating to electron-energy loss, given the negligible energy deposited as determined by the collision stopping power. Instead, we suggest that the significant specimen heating is due to Auger excitation, a process known to deposit substantial energy. This contention is supported by a quantitative heat-transfer finite element analysis.

Optimized procedure for conventional TEM sample preparation using birefringence

Specimens for quality transmission electron microscopy (TEM) analyses must fulfil a range of requirements, which demand high precision during the prior preparation process. In this work, an optimized procedure for conventional TEM specimen preparation is presented that exploits the thickness-dependence of interference colors occurring in birefringent materials. It facilitates the correct estimation of specimen thickness to avoid damage or breaking during mechanical thinning and reduces ion-milling times below 30minutes. The benefits of the approach are shown on sapphire and silicon carbide cross-section samples. The presented method is equally suitable for assessing specimen thickness during dimpling and wedge-polishing, and is particularly useful at thicknesses below 20 μm, where the accuracy of mechanical techniques is insufficient. It is precise enough to be employed for a visual thickness estimation during the thinning process, but can be additionally optimized by analyzing the RGB spectrum of the occurring interference colors.

In Situ Transmission Electron Microscopy Study of Bubble Behavior Near the Surface of Ice Crystals by Using a Liquid Cell With a Peltier Cooling Holder.

Liquid cell transmission electron microscopy (LC-TEM) is a unique technique that permits in situ observations of various phenomena in liquids with high spatial and temporal resolutions. One difficulty with this technique is the control of the environmental conditions in the observation area. Control of the temperature ranging from room temperature to minus several tens of degrees Celsius, is desirable for controlling the supersaturation in various materials and for observing crystallization more easily. We have developed a cooling transmission electron microscopy specimen holder that uses Peltier devices, and we have combined it with a liquid cell to realize accurate temperature control in LC-TEM. We evaluated this system by using water as a specimen. Motionless bubbles, shown to be voids containing pressurized gas, formed in the specimen sometime after the temperature had reached -12°C. An electron diffraction pattern showed that the specimen turned into ice Ih after the formation of these bubbles, confirming that our system works properly and can induce crystallization. In addition, we analyzed the behavior of bubbles formed in the ice Ih, and we discussed the formation of these bubbles and their internal pressure.

Experimental and modelling evidence for hydrogen trapping at a β-Nb second phase particle and Nb-rich nanoclusters in neutron-irradiated low Sn ZIRLO

Zirconium-based alloys used for fuel cladding in nuclear fission reactors are susceptible to hydrogen embrittlement during operation, but we currently lack the necessary mechanistic understanding of how hydrogen behaves in the materials during service to properly address this issue. Imaging the distribution of hydrogen within material microstructures is key to creating or validating models that predict the behaviour and influence of hydrogen on material properties, but is experimentally difficult. Studying hydrogen in zirconium-alloys is further complicated by the fact that the most common routes for preparing specimens for Transmission Electron Microscopy and Atom Probe Tomography (APT) analysis, electropolishing and focused ion beam (FIB) milling, are known to induce hydride formation. This introduces uncertainty as to whether the hydrogen distribution in the analysed specimen is actually representative of the entire sample a priori. Recent work has shown that this effect can be mitigated by performing the final specimen thinning stages at cryogenic temperatures. In this paper we use cryo-FIB to prepare APT specimens of neutron-irradiated low Sn ZIRLO, showing that hydrogen is trapped within a β-Nb SPP and at Nb-rich nanoclusters formed by exposure to neutron irradiation. We then use density functional theory calculations to explain these experimental observations. These results highlight the importance of including niobium-rich features in models used to predict hydrogen pick-up in zirconium alloys during service and delayed hydride cracking during storage.

High-quality TEM specimen preparation for lithium-ion conducting solid electrolytes by low-energy ion milling

Focused ion beam (FIB) is a widely used method to prepare transmission electron microscopy (TEM) specimen from bulk materials. However, the surface amorphous layer induced by ion beam is an obstacle to obtain high-quality atomic-scale images for quantitative analysis, especially for the analysis of light elements such as lithium in lithium-ion conducting solid electrolytes. Here, taking lithium-ion conducting solid electrolyte materials as an example, the advantages and disadvantages of applying low-energy Ar+ ion for fine milling after FIB are investigated. Combining Monte-Carlo simulations with ion milling experiments, the milling parameters are evaluated and discussed in detail. With optimized parameters, TEM specimens with less beam damage and thinner amorphous layer were prepared, enabling the acquisition of high-quality atomic-scale images. Furthermore, low-energy Ar+ ion milling is also able to remove hydrocarbon contamination formed during the electron beam illumination inside the microscope, making the contaminated TEM specimens reusable.

A Method for Epicuticular Wax and Contaminant Removal from Herbarium Specimens for Scanning Electron Microscopy

Abstract We describe a method for cleaning leaf epicuticular wax and contaminants from herbarium specimens in preparation for scanning electron microscopy. Specimens are rinsed in chloroform, heated at 50°C for 8 hrs, ultrasonicated in 70% ethanol, and subjected to critical-point drying. This method helps preserve and reveal the surface details of specimens that are heavily covered with epicuticular wax and contaminants, making many morphologically and functionally important details readily visible. We tested our method by using it to clean herbarium specimens and comparing the results with those obtained using three other chemicals: acetone, xylene, and 1:1 acetone:xylene, confirming that chloroform treatment was the most effective.

Alternative Forward Models for Imaging Thick Specimens in Transmission Electron Microscopy.

I have investigated two different forward models for image formation in transmission electron microscopy of thick specimens, the 3DCtf model, which introduces a defocus gradient in the linear approximation, and the multislice model. An important result is that the 3DCtf model does not seem to be compatible with the multislice image formation model. A second very useful finding is that the exit wave in the multislice model has an imaginary part, which, in first-order approximation, is a pure projection of the specimen and is not affected by the defocus gradient. The defocus gradient only comes into play in real valued and higher-order imaginary terms. If the multislice model is closer to reality than the 3DCtf-model, then the best way to retrieve the specimen projection for thicker specimens should be a procedure for retrieving the exit wave's imaginary term, for example using images recorded at different defocus values.

Grain Boundary Plane Measurement Using Transmission Electron Microscopy Automated Crystallographic Orientation Mapping for Atom Probe Tomography Specimens.

Grain boundaries are critical in determining the properties of materials, including mechanical stability, conductivity, and corrosion resistance. The specific properties of materials depend not only on the misorientation of the crystals, the three most commonly characterized parameters, but also on the angle of the grain boundary plane between the two crystals, the final two parameters in the five-parameter macroscopic description of the grain boundary. The method presented here allows for the direct measurement of all five parameters of the grain boundary in a transmission electron microscopy specimen of various morphologies. This is especially applicable to atom probe specimens, where only a single-tilt axis is generally available, allowing the crystallographic description to be matched to the detailed chemical data available in the atom probe tomography. This method provides a platform for efficient grain boundary analysis in unique samples, saving operator time and allowing for ease of acquisition and interpretation in comparison with traditional electron diffraction methods.

Observation of the same asbestos body by both phase contrast microscope and analytical transmission electron microscope.

The amount of asbestos body (AB) in the human lungs is used as an index to assess asbestos lung cancer (ALC). This study reports a new method to observe the same AB previously observed by analytical transmission electron microscope (ATEM) by phase contrast microscope (PCM) or the contrary order. Four kinds of specimens were prepared from the lung tissue of an asbestos related worker: ordinary PCM specimen (A); PCM specimen (B) of which the cover glass was stripped off and ashed at a low temperature; transmission electron microscope (TEM) specimen (C); and PCM specimen (D) covered a TEM specimen (C) with immersion liquid and cover glass. These specimens were all observed by PCM, and the specimen (C) by analytical TEM (ATEM). The results showed that the TEM specimen (C) is transparent in visible light and we can also see the particles by PCM. The image by PCM of the TEM specimen (C) showed very similar features to that of PCM specimens (A) and (B). Accordingly, we could observe various same particles by both ATEM and PCM. In conclusion, the method observing the same AB by both PCM and ATEM will contribute to standardize the recognition of AB for PCM analysts.

Transition to body-centered cubic structure in Au thin films under electron-beam irradiation

Gold (Au) is well known to transition to a body-centered cubic (bcc) structure at high temperature and pressure. However, this study shows that such conditions may not be necessary. In this study, Au thin film specimens for transmission electron microscopy (TEM) undergo a transition from face-centered cubic (fcc) to bcc structure under electron-beam irradiation in TEM. Some regions in the Au thin film specimens are selectively thinned during electron-beam irradiation. The thin regions are considered to be subjected to the localization of energy deposition by electronic excitations by incident electrons due to their finite size, which causes the regions to be heated. Of the excitations, Auger decays are a possible source of Au heating. The temperature rise due to Auger decays is successfully simulated to be accompanied by stress development in the thin regions. Sometimes the thin regions transition from face-centered cubic (fcc)-to-bcc phases. The fcc matrix and the bcc phase have orientation relationships similar to those found in the fcc-to-bcc phase transformation in steels, which indicate that the transition to the bcc structure occurs through a displacive mechanism. It is suggested that the phase transition is facilitated for thin film morphology and that crystal defects caused by electron-beam irradiation and stress development stabilize the more open bcc structure.

Influence of thermal ageing on tensile-plastic flow and work hardening parameters of Indian reduced activated Ferritic Martensitic steel

The present study investigates the influence of thermal ageing (873K/5000h) on the microstructure, tensile-plastic flow behavior, and work hardening parameters of normalized and tempered (N&T) 1.4W-0.06Ta Indian Reduced Activated Ferritic Martensitic (INRAFM) steel. To comprehensively understand the tensile-plastic flow response of the INRAFM steel across a broad temperature spectrum of 300-873 K, the Hollomon, Ludwigson and Voce equations were employed. The results reveal an augmentation in the strain hardening exponent following the ageing process, attributed to the heightened work hardening capability of the aged steel, while the strain hardening coefficient exhibited a decline post ageing. To elucidate the dislocation debris structure?s formation and movement within both the N&T and thermally aged specimens, transmission electron microscopy (TEM) specimens were procured in close proximity to the tensile tested specimens at varying temperatures of 300, 573 and 873 K. The tensile plastic flow behavior at diverse temperatures was aptly described by Hollomon, Ludwigson, and Voce equations. The fitting accuracy of these equations was determined using the goodness of fit, as indicated by the lowest ?2 values. The constitutive Voce equation successfully captured the yield strength (YS) and ultimate tensile strength (UTS) with the initial stress and saturation stress acting as fitting parameters. Distinct patterns of initial stress and saturation stress variations were observed concerning both N&T and aged steel in relation to temperature. Furthermore, absolute value of the Voce strain component (nv) demonstrated a reduction consequent to ageing, manifesting a two-stage behavior corresponding to temperature elevation. Notably, the deceleration of the recovery process during high temperature conditions was more pronounced in the thermally aged steel when contrasted with the N&T steel. Conclusively, the Voce relation proved efficacious in predicting the yield stress (YS) and ultimate tensile strength (UTS) of both the thermally aged and N&T INRAFM steel at varying temperatures.

An apparatus for plasma cleaning and storage of transmission electron microscopy specimens and specimen holders.

The transmission electron microscopy (TEM) specimen with thickness in nanometer scale is susceptible to hydrocarbon contamination and oxidation, and the specimen holder is also susceptible to contaminants, which would deteriorate the quality of TEM imaging and degrade the efficiency of TEM experiments. Conventional pretreatment devices often have limited functions and low practicability, which may cause problems for TEM specimens and holders. In this work, a multifunctional apparatus for plasma cleaning and storage of TEM specimens and specimen holders is developed based on the specific design of the vacuum joints. The apparatus includes a plasma cleaning system, holder storage station, and specimen storage station, which share the same vacuum system. The cleaning of hydrocarbon contaminants on the specimen and storage of the specimens and holders can be achieved simultaneously in this apparatus. TEM imaging and energy-dispersive X-ray spectroscopy (EDS) analyses of two treated specimens using the apparatus demonstrated that it could effectively remove hydrocarbon contaminants on the specimen. The holder storage station, used to preserve TEM holders in vacuum conditions, can also be modified as a specimen storage station by an appropriate design of the specimen storage platform, in which specimens are protected from water and contaminations. The designed apparatus not only robustly avoids damage to the ultrathin specimen and holders but also improves the working efficiency and reduces costs. These advantages could make our apparatus more appealing for the complement to the present commercial plasma cleaning and storage devices. HIGHLIGHTS: An apparatus for the pretreatment of transmission electron microscopy (TEM) specimens and specimen holders with three functions-plasma cleaning, holder storage, and specimen storage-was designed and fabricated. Using this single apparatus, the cleaning of hydrocarbon contaminants on the specimen and storage of the specimens and holders can be achieved simultaneously. The designed apparatus can not only robustly avoid damage to the ultrathin specimen and holders but also improve the working efficiency and reduce costs by adopting a single vacuum system. These advantages could make our apparatus more appealing for the complement to the present commercial plasma cleaning and storage devices.

Caries-inhibiting Effect of Microencapsulated Active Components in Pit and Fissure Sealants.

The aim of the present in vitro study was to examine the caries-inhibiting effect of a pit and fissure sealant (PFS) containing ion-releasing microcapsules under cariogenic conditions in a biofilm artificial mouth. Forty-eight human third molars were divided into four groups (n=12 per group). Fissures were extended with burs and sealed with experimental PFS. The four groups of specimens were treated as follows: 1) EPFS 1: EPFS (Premier Dental) of increasing viscosity, containing microcapsules loaded with remineralizing agents (calcium, phosphate, and fluoride ions); 2) US: fluoride-releasing PFS (UltraSeal XT plus, UltraDent Products, South Jordan, UT, USA); 3) EPFS 2: experimental PFS of constant viscosity containing microcapsules loaded with calcium, phosphate, and fluoride ions; and 4) FT: glass ionomer cement (GIC) (GC Fuji Triage CAPSULE WHITE glass ionomer cement, GC Europe NV, Leuven, Belgium). FT and US were used as control groups. EPFS 1 and EPFS 2 were the experimental groups. Specimens were stored in distilled water for 14 days at 37°C, subjected to 10,000 thermocycles (5°C and 55°C) and finally exposed to microbiological cycling in a Streptococcus mutans-based artificial mouth for 10 days. Replicas were made for scanning electron microscopic (SEM) evaluation and specimens were cut for fluorescence microscopy. Overall demineralization depths at the margin of Fuji Triage were significantly shallower than in the other groups (p<0.05). Overall demineralization depths adjacent to the experimental pit and fissure sealant EPFS 2 (59±15 μm) were comparable to the values of the resin-based pit and fissure sealant UltraSeal XT plus (58±10 μm, p≥0.05). SEM revealed surface roughness of the GIC-based PFS. The experimental PFS with microcapsules containing active components for remineralization did not show a caries-inhibiting effect compared to a fluoride-releasing resin-based PFS. Lower demineralization depths adjacent to GIC sealants indicate an anticariogenic effect through fluoride ion release.

Atomistic observation of in situ fractured surfaces at a V-doped WC Co interface

• Crack propagation dynamic across a V-doped WC Co interface is studied in TEM. • Aberration-corrected STEM is used to examine the as-fractured surface. • The fracture propagates between the V-containing trilayers and the abutting cobalt. • A loss of interfacial coherency accounts for the selected propagation path. An in situ mechanical test was performed on a V-doped WC Co specimen in a transmission electron microscope (TEM) to understand the crack propagation dynamics. The fracture occurs along a WC Co interface. Aberration-corrected high-angle annular dark-field (HAADF) imaging and energy-dispersive X-ray spectroscopy (EDS) mapping are combined to investigate the as-fractured surface. The interfacial cleavage plane is atomically flat, taking place between the V-containing trilayers and the abutting cobalt grain. No noticeable structural or compositional change is detected for the trilayer superstructure coherent with the underneath WC grain. Density functional theory (DFT) calculations reveal that the interlayer bonding within the coherent interfacial trilayer superstructure is strong while that between the superstructure and cobalt is substantially weaker due to incoherency. This study provides insights into the interfacial fracture behavior in hard metals and suggests the importance of interfacial coherency to fracture resistance.