Contrast Transmission Electron Microscopy Research Articles

SOX4 promotes the growth and metastasis of breast cancer

PurposeIncreasing evidence has shown that the transcription factor SOX4 is closely associated with the development and progression of many malignant tumors. However, the effect of SOX4 on breast cancer is unclear. In this study, we purposed to investigate the role of SOX4 in the growth and metastasis in breast cancer and the underlying mechanism. Moreover, the effect of SOX4 on cancer cell resistance to chemotherapeutic agents was also evaluated in vitro and in vivo.MethodsWe used lentivirus technique to ectopically express SOX4 in MDA-MB-231 and SUM149 cells or knockdown SOX4 in BT474 cells, and examined the effect of these changes on various cellular functions. MTT assay was used to determine the cell viability as well as resistance to chemotherapeutic agents. The regulation of SOX4 on epithelial-mesenchymal transition (EMT)-related genes was analyzed using qRT-PCR. The binding of SOX4 to the CXCR7 gene was demonstrated using chromatin immunoprecipitation assay and dual-luciferase reporter activity assay. The effect of SOX4/CXCR7 axis on metastasis was examined using Transwell migration and Matrigel invasion assays. The expression of SOX4/CXCR7 in primary tumors and metastatic foci in lymph nodes was assessed using immunohistochemistry. Cellular morphology was investigated under phase contrast microscope and transmission electron microscopy. Moreover, the effect of SOX4 on tumor growth, metastasis, and resistance to chemotherapy was also studied in vivo by using bioluminescent imaging.ResultsSOX4 increased breast cancer cell viability, migration, and invasion in vitro and enhanced tumor growth and metastasis in vivo. It regulated EMT-related genes and bound to CXCR7 promoter to upregulate CXCR7 transcription. Both SOX4 and CXCR7 were highly expressed in human primary tumors and metastatic foci in lymph nodes. Treatment of breast cancer cells with the CXCR7 inhibitor CCX771 reversed the SOX4 effect on cell migration and invasion. Ectopic expression of SOX4 increased the susceptibility of cells to paclitaxel.ConclusionsSOX4 plays an important role in the growth and metastasis of breast cancer. SOX4/CXCR7 may serve as potential therapeutic targets for the treatment. Paclitaxel may be a good therapeutic option if the expression level of SOX4 is high.

Strengthening the FeCoCrNiMo0.15 high entropy alloy by a gradient structure

In present work, a gradient structured FeCoCrNiMo0.15 high entropy alloy (HEA) is processed by shot peening treatment. Microstructures of the gradient structured HEA are characterized by electron backscatter diffraction (EBSD), electron channeling contrast imaging (ECCI) and transmission electron microscopy (TEM). Complex microstructures consisting of nanograins, nanotwins, with high densities of dislocations are formed on the surface of the shot peening treated HEA. With the increase of depth from surface to center, a planar dislocation slip occurs while no deformation twin is observed. Un-deformed grains were observed in the regions of ∼100 μm–150 μm depth from the surface. Such gradient structured HEA shows an increase of yield strength from 418 MPa to 486 MPa when compared to the one with homogeneous structure, with a slight decrease in tensile elongation from 50.9% to 46.8%. During tensile deformation, strong deformation twinning including the formation of hierarchical nanotwins occurs in the gradient structured HEA. These unique deformation microstructures are associated with high strain hardening and show increased ductility, which creates excellent combination of strength and tensile elongation. Our work offers valuable insight into achieving high-performance HEAs.

Metabolic labeling of Escherichia coli genomic DNA with erythrosine-11-dUTP for functional imaging via correlative microscopy.

The fluorescent metabolic labeling of microorganisms genome is an advanced imaging technique to observe and study the native shapes, structural changes, functions, and tracking of nucleic acids in single cells or tissues. We have attempted to visualize the newly synthesized DNA within the intact nucleoid of ice-embedded proliferating cells of Escherichia coli K-12 (thymidine-requiring mutant, strain N4316) via correlative light-electron microscopy. For that purpose, erythrosine-11-dUTP was synthesized and used as a modified analog of the exogenous thymidine substrate for metabolic incorporation into the bacterial chromosome. The formed fluorescent genomic DNA during in cellulo polymerase reaction caused a minimal cellular arrest and cytotoxicity of E. coli at certain controlled conditions. The stained cells were visualized in typical red emission color via an epifluorescence microscope. They were further ice-embedded and examined with a Hilbert differential contrast transmission electron microscopy. At high-resolution, the ultrastructure of tagged nucleoid appeared with significantly higher electron dense in comparison to the unlabeled one. The enhanced contrast areas in the chromosome were ascribed to the presence of iodine contents from erythrosine dye. The presented labeling approach might be a powerful strategy to reveal the structural and dynamic changes in natural DNA replication including the relationship between newly synthesized in vivo nucleic acid and the physiological state of the cell.

Characterization of Tobacco Mosaic Virus Virions and Repolymerized Coat Protein Aggregates in Solution by Small-Angle X-Ray Scattering.

The structure of tobacco mosaic virus (TMV) virions and stacked disk aggregates of TMV coat protein (CP) in solution was analyzed by synchrotron-based small-angle X-ray scattering (SAXS) and negative contrast transmission electron microscopy (TEM). TMV CP aggregates had a unique stability but did not have helical symmetry. According to the TEM data, they were stacked disks associated into transversely striated rod-shaped structures 300 to 800 Å long. According to modeling based on the crystallographic model of the 4-layer TMV CP aggregate (PDB: 1EI7), the stacked disks represented hollow cylinders. The calculated SAXS pattern for the disks was compared to the experimental one over the entire measured range. The best correlation with the SAXS data was found for the model with the repeating central pair of discs; the SAXS curves for the stacked disks were virtually identical irrespectively of the protein isolation method. The positions of maxima on the scatter curves could be used as characteristic features of the studied samples; some of the peaks were assigned to the existing elements of the quaternary structure (periodicity of aggregate structure, virion helix pitch). Low-resolution structural data for the repolymerized TMV CP aggregates in solution under conditions similar to natural were produced for the first time. Analysis of such nano-size objects is essential for their application in biomedicine and biotechnology.

How Nanoscale Dislocation Reactions Govern Low- Temperature and High-Stress Creep of Ni-Base Single Crystal Superalloys

The present work investigates γ-channel dislocation reactions, which govern low-temperature (T = 750 °C) and high-stress (resolved shear stress: 300 MPa) creep of Ni-base single crystal superalloys (SX). It is well known that two dislocation families with different b-vectors are required to form planar faults, which can shear the ordered γ’-phase. However, so far, no direct mechanical and microstructural evidence has been presented which clearly proves the importance of these reactions. In the mechanical part of the present work, we perform shear creep tests and we compare the deformation behavior of two macroscopic crystallographic shear systems [ 01 1 ¯ ] ( 111 ) and [ 11 2 ¯ ] ( 111 ) . These two shear systems share the same glide plane but differ in loading direction. The [ 11 2 ¯ ] ( 111 ) shear system, where the two dislocation families required to form a planar fault ribbon experience the same resolved shear stresses, deforms significantly faster than the [ 01 1 ¯ ] ( 111 ) shear system, where only one of the two required dislocation families is strongly promoted. Diffraction contrast transmission electron microscopy (TEM) analysis identifies the dislocation reactions, which rationalize this macroscopic behavior.

Phase Separation Studies of Complexes of Intrinsically Disordered Protein Tau and Anionic Liposomes

Tau is a highly soluble intrinsically disordered protein (IDP) characterized classically as a microtubule-associated protein (MAP) in axons of mature neurons. The most well established primary function of tau is to suppress microtubule (MT) dynamic instability providing a stable foundation for organelle trafficking and cell communication. However, the dynamic and polyampholytic nature of tau's peptide composition implies a capacity to interact with charged biological substrates in the cytosol for other distinct functions. There is emerging evidence suggesting that tau associations with charged, specifically anionic, cytosolic molecules may prime pathological structural states seen in neurodegenerative diseases. However, the mechanisms of these secondary interactions are still poorly understood. Therefore, our goal is to understand the structural implications of tau associations with anionic molecules, specifically biological lipid membranes. Here we probe phase transitions of tau-anionic liposomes interactions at different anionic liposome formulations using differential interference contrast microscopy (DIC), fluorescence microscopy, and transmission electron microscopy (TEM).

The challenges of applying an Activity-Based Sampling methodology to estimate the cancer risk associated with asbestos contaminated landfilled zones

Inhabitants of Sibaté (Colombia) report that between approximately 1975 and 1985 asbestos-containing materials (ACM) were dumped at different locations in the urban area of the municipality. Starting in around 1986, the dumping of materials resulted in landfilled zones, on top of which different facilities were then constructed. In a previous study, an underground friable asbestos layer was discovered in these landfilled zones. However, potential exposure to asbestos on the surface of landfilled zones in Sibaté has not been determined. In the current study, the U.S. EPA Activity-Based Sampling (ABS) methodology was adapted and applied in three scenarios located on potential landfilled areas in Sibaté, to estimate the current risk of exposure to asbestos through inhalation, and the resulting excess lifetime cancer risk (ELCR). For this purpose, generic ABS in a football stadium, and specific ABS in both a public playground and a school courtyard were conducted. Personal, area and blank samples were collected and analyzed using phase contrast microscopy (PCM) and transmission electron microscopy (TEM) following NIOSH 7400 and ISO 13794 methods, respectively. Exposure point concentrations were determined and compared against the action level for asbestos in air (ALAA), and were also used to calculate the ELCR of each scenario.A total of 25 airborne asbestos samples were collected, and 22 of these (12 personal samples, 7 area samples and 3 blank samples) were analyzed using PCM. Eighteen of these samples (12 personal, 3 area samples and 3 blank samples) were analyzed using TEM. The total asbestos structures concentration of personal samples ranged from non-detected to 0.326 S/cc (i.e., total asbestos structures counts ranged from 0 to 12). All samples had PCM-Equivalent asbestos structures concentrations below analytical sensitivity. Of the 22 samples analyzed, 18 were overloaded with particles.Although chrysotile and actinolite were identified in some personal samples, suggesting a potential risk of asbestos exposure, the ELCR was at U.S. EPA acceptable risk levels. Since the ABS methodology was applied in a limited number of scenarios and a small number of samples were collected, these results should be interpreted with caution and additional sampling campaigns are required to fully understand the risk of asbestos exposure in Sibaté. Methodological and analytical challenges encountered in the current study are discussed in detail, which could inform future ABS studies, not only in Sibaté, but also in other areas with asbestos-contaminated soils.

Optical and Structural Characterization of CdTe Nanoparticles Synthesized Using Chemical Bath Deposition Technique

The purpose of this research is the elaboration of cadmium telluride nanoparticles by using the chemical bath deposition technique. The formulation procedure consists of preparing tellurium ions (Te−2) and combination of tellurium ion solution with deionized water, cadmium chloride, and polyethyleneimine (PEI). The nanoparticles were synthesized at three different concentrations denoted C1, C2, and C3. Furthermore, for each CdTe, nanoparticles were obtained with absorption edges at 404.02 nm, 428 nm and 452.66 nm related to C1, C2, and C3, respectively. Also, the direct energy gaps of CdTe nanoparticle semiconductors were: Eg(C1) = 3.07 eV, Eg(C2) = 2.9 eV, and Eg(C3) = 2.74 eV. Transmission electron microscopy (TEM) indicated three different average diameter sizes of a single nanoparticle: D(C1) = 3.23 nm, D(C2) = 6.46 nm, and D(C3) = 7.73 nm with average sizes of 6.11 nm, 5.29 nm, and 9.76 nm, respectively. The fast Fourier transform of TEM contrast micrographs corresponded with a hexagonal structure. The presence of Cd, Te, and TeO2 was identified by x-ray photoelectron spectroscopy. Raman spectroscopy was used to identify three peaks at 122 cm−1, 140 cm−1 and 165 cm−1 that proved the formation of CdTe nanoparticles.

A multiscale study on the morphology and evolution of slip bands in a nickel-based superalloy during low cycle fatigue

Plastic deformation during low cycle fatigue in fcc materials with low stacking fault energy is accumulated in slip bands, which become preferential sites for crack initiation. Whilst these dislocation structures have been studied before, little has been done to assess the effect and evolution of the individual slip lines within them. In this study, samples of a γ′ precipitate strengthened nickel-based superalloy are fatigued at room temperature and 700 ∘C for 1, 40 and 500 cycles. The resulting dislocation structures are characterised via Electron Channeling Contrast Imaging and Transmission Electron Microscopy. We introduce a new methodology to measure slip band parameters such as the slip line spacing and shear step length by analysing the holes left by sheared precipitates in γ′-etched secondary electron micrographs. Statistics of these parameters are obtained and compared for different conditions. Advantages of this technique include resolution at the scale of individual planes, acquisition of true three-dimensional data and applicability in the bulk of the material. The combination of these techniques provides a unique mechanistic and quantitative insight into the slip band and precipitate morphology evolution.

3D characterization of nanowire devices with STEM based modes

Advanced semiconductor devices have 3D morphologies with nanometer sized features. Therefore, their structural and chemical characterization requires analysis techniques with high spatial 3D resolution. Through focal integrated differential phase contrast and high angle annular dark field scanning transmission electron microscopy (STEM) depth sectioning are among the techniques that can potentially fulfill these needs. In this work they are applied to Si and Ge nanowire structures with gate all around replacement metal gates. It is shown that with both imaging modes slicing with 2D lattice resolution through the polycrystalline gate stack and the monocrystalline wires is possible, while a resolution in the viewing direction on the order of 5 nm is obtained. Based on fast Fourier transformation analysis the crystal distribution in the gate stack and morphology of the nanowires are analyzed. Similar structures are investigated with combined STEM-energy dispersive spectroscopy 180° tomography on pillar shaped TEM specimens. Comparison of the 3D imaging modes and standard TEM/STEM imaging is discussed.

Martensite Reversion Duality Behavior in a Cold-Rolled High Mn Transformation-Induced Plasticity Steel

The martensite reversion behavior of a cold-rolled high Mn transformation-induced plasticity (TRIP) steel was investigated through an elaborate continuous annealing procedure. The dilatometry analysis was employed to ensure a precise heating rate (50 °C/s) control during annealing along with acquiring the critical temperature range of the martensite reversion. The magnetic measurement was used for $$ \alpha^{\prime} $$-martensite quantification through annealing, and electron channeling contrast imaging (ECCI) and transmission electron microscopy (TEM) analyses were also performed to characterize the microstructure. The dilatometry results revealed the dual temperature ranges for the $$ \alpha^{\prime} $$-martensite reversion in the experimental TRIP steel; the reversion occurred in two separate temperature regions of 540 °C to 730 °C and 730 °C to 850 °C. Although the inhomogeneity in the morphology of $$ \alpha^{\prime} $$-martensite (lath type and dislocation cell type), which was detected in the ECCI image of the cold-rolled structure, might be responsible for the reversion temperature range broadening, the discontinuity of the reversion was mainly attributed to the partitioning of Mn from $$ \alpha^{\prime} $$-martensite to austenite through annealing. The microstructural characterizations indicated that the duality would also exist in the case of the martensite reversion mechanism. Accordingly, the reversion of lath-type martensite appeared to occur merely through an athermal mechanism, while the dislocation cell-type martensite might be reversed to austenite via both shear and diffusional mechanisms.

Nitrogen deprivation elicits dimorphism, capsule biosynthesis and autophagy in Papiliotrema laurentii strain RY1

Stress response due to the lack of essential nutrient(s) for an organism has been a focal point of several scientific investigations. The present study investigates the cellular adaptations behind the ability of Papiliotrema laurentii strain RY1 to perpetuate without added nitrogen and propagate robustly in growth- limiting amount of nitrogen. We executed phenotypic (using scanning electron microscopy, differential interference contrast microscopy and transmission electron microscopy), microbiological and computational analyses to show multiple responses of dimorphism, capsule formation and autophagy as a survival strategy by the yeast upon nitrogen starvation. The roles of phosphomannose isomerase, phosphomannomutase and several autophagy-related transcripts aiding in such a response have been discussed.

Advanced laboratory techniques characterising solids, fluids and pores in shales

Shales are of significant interest as reservoirs, seals and overburden in petroleum exploration and production, geological storage of CO2 and in the nuclear waste industry. Their properties and microstructure are difficult to measure and image and as such, there are still considerable gaps in our understanding of shale behaviour. The advent of more advanced and quantitative imaging methods along with novel sample preparation techniques including synchrotron imaging and K-edge subtraction with a contrast fluid, scanning (SEM) and transmission electron microscopy (TEM), have combined to allow the resolution of pores in the matrix and the organic matter in gas shales down to the nanometre scale. Textural quantification methods include neutron diffraction to quantify clay fabric alignment in siliciclastic shales as well as stress-dependent calcite c-axis orientation in carbonate-rich gas shales. The contents of the pores are also critical to the assessment of the preservation state of shales as well as the degree of water/gas saturation in resource shales. Combinations of dielectric analysis and 2 and 23 MHz nuclear magnetic resonance (NMR) measurements in siliciclastic and carbonate-rich gas shales can resolve not only the location of water, but also the interaction between water and mineral/organic surfaces in addition to the water and oil content. The organic content of shales is of particular interest in unconventional resources and this has driven application of Fourier transform infra-red (FTIR), to distinguish both mineral and organic maceral components in gas shales and Raman spectroscopy, which is increasingly used for organic matter characterisation in high maturity shales. Ultimately, shales are extremely complex nano-composite materials which require the application of multiple techniques to help determine their bulk physical and flow properties as well as factors controlling fabric and pore orientations and structure.

Iron-Sequestering Nanocompartments as Multiplexed Electron Microscopy Gene Reporters.

Multicolored gene reporters for light microscopy are indispensable for biomedical research, but equivalent genetic tools for electron microscopy (EM) are still rare despite the increasing importance of nanometer resolution for reverse engineering of molecular machinery and reliable mapping of cellular circuits. We here introduce the fully genetic encapsulin/cargo system of Quasibacillus thermotolerans (Qt), which in combination with the recently characterized encapsulin system from Myxococcus xanthus (Mx) enables multiplexed gene reporter imaging via conventional transmission electron microscopy (TEM) in mammalian cells. Cryo-electron reconstructions revealed that the Qt encapsulin shell self-assembles to nanospheres with T = 4 icosahedral symmetry and a diameter of ∼43 nm harboring two putative pore regions at the 5-fold and 3-fold axes. We also found that upon heterologous expression in mammalian cells, the native cargo is autotargeted to the inner surface of the shell and exhibits ferroxidase activity leading to efficient intraluminal iron biomineralization, which enhances cellular TEM contrast. We furthermore demonstrate that the two differently sized encapsulins of Qt and Mx do not intermix and can be robustly differentiated by conventional TEM via a deep learning classifier to enable automated multiplexed EM gene reporter imaging.

Visualization of unstained DNA nanostructures with advanced in-focus phase contrast TEM techniques

Over the last few years, tremendous progress has been made in visualizing biologically important macromolecules using transmission electron microscopy (TEM) and understanding their structure-function relation. Yet, despite the importance of DNA in all forms of life, TEM visualization of individual DNA molecules in its native unlabeled form has remained extremely challenging. Here, we present high-contrast images of unstained single-layer DNA nanostructures that were obtained using advanced in-focus phase contrast TEM techniques. These include sub-Ångstrom low voltage electron microscopy (SALVE), the use of a volta-potential phase plate (VPP), and dark-field (DF) microscopy. We discuss the advantages and drawbacks of these techniques for broad applications in structural biology and materials science.

Synergies of accelerating differentiation of bone marrow mesenchymal stem cells induced by low intensity pulsed ultrasound, osteogenic and endothelial inductive agent

In terms to investigate the effect of low-intensity pulsed ultrasound (LIPUS) for differentiation of bone marrow mesenchymal stem cells (BMSCs) and the feasibility of simultaneously inducing into osteoblasts and vascular endothelial cells within the cell culture medium in which two inductive agents are added at the same time with or without LIPUS. Cells were divided into a non-induced group, an osteoblast-induced group, a vascular endothelial-induced group, and a bidirectional differentiation-induced group. Each group was further subdivided into LIPUS and non-LIPUS groups. The cell proliferation in each group was measured by MTT assay. Cell morphological and ultrastructural changes were observed by inverted phase contrast microscopy and transmission electron microscopy. The differentiation of BMSCs was detected by confocal microscopy, flow cytometry and quantitative RT-PCR. Results demonstrated that both osteoblast and vascular endothelial cell differentiation markers were expressed in the bidirectional differentiation induction group and early osteogenesis and angiogenesis appeared. The cell proliferation, differentiation rate and expression of osteocalcin and vWF in the LIPUS groups were all significantly higher than those in the corresponding non-LIPUS group (p < .05), suggesting LIPUS treatment can promote the differentiation efficiency and rate of BMSCs, especially in the bidirectional differentiation induction group. This study suggests the combination of LIPUS and dual-inducing agents could induce and accelerate simultaneous differentiation of BMSCs to osteoblasts and vascular endothelial cells. These findings indicate the method could be applied to research on generating vascularized bone tissue with a shape and function that mimics natural bone to accelerate early osteogenesis and angiogenesis.

Integrative taxonomy of a new Redudasys species (Gastrotricha: Macrodasyida) sheds light on the invasion of fresh water habitats by macrodasyids

The order Macrodasyida (Gastrotricha) includes over 350 marine species, and only 3 freshwater species (Marinellina flagellata, Redudasys fornerise, R. neotemperatus). Herein we describe a new freshwater species of Macrodasyida, Redudasys brasiliensis sp. nov., from Brazil through an integrative taxonomic approach. The external morphology and internal anatomy were investigated using differential interference contrast microscopy, confocal microscopy, scanning and transmission electron microscopy. The systematization of the new taxon was inferred by nuclear (18S and 28S) and mitochondrial (COI) genes, and its intra-order relationships were assessed using data from most of available macrodasyids. Phylogenetic analyses yielded congruent trees, in which the new taxon is nested within the family Redudasyidae, but it was genetically distinct from the other species of the genus Redudasys. The new species shares the gross morphology and reproductive traits with other Redudasyidae and the presence of only 1 anterior adhesive tube per side with Redudasys neotemperatus, but it has a specific pattern of ventral ciliation and muscle organization. Results support the hypothesis that dispersion into fresh water habitats by Macrodasyida and Chaetonotida taxa occurred independently and that within Macrodasyida a single lineage invaded the freshwater environment only once. Furthermore, the Neotropical region seems to be peculiar for the evolution of the freshwater macrodasyid clade.

Empirical determination of transmission attenuation curves in mass–thickness contrast TEM imaging

The thickness dependence of electron transmission, observed as mass–thickness contrast in transmission electron microscopy (TEM) images, was precisely measured for polystyrene and amorphous carbon. In the early stages of transmission attenuation, a slight increase in the attenuation coefficient was observed, although a constant value is generally expected according to Beer's law. In contrast, as generally known as nonlinear behavior due to multiple scattering, the coefficient decreased during the intermediate stages. In the later stages, an asymptotic behavior in which the transmission approached a constant value was observed. Based on these results, a function containing three parameters was proposed to express the nonlinear transmission attenuation with increasing thickness. Results obtained using this new model and other previously proposed models were compared with experimental data measured over a wide range of conditions: acceleration voltage of 200–3000 kV, objective aperture radius of 0.85–30 nm−1, and thickness of 0.25–10 μm. It was confirmed that our model can well reproduce all of the measurements with a high degree of precision, while the other models were valid only under limited imaging conditions and/or for limited thickness ranges. Thus, a quantitative description of transmission attenuation under normal TEM observation conditions, that is, over a thickness range without physical absorption and a scattering angular range of a few degrees, is finally obtained after more than a century since early studies on β-rays. Based on a simplified model of multiple scattering, the characteristic behavior of the attenuation curves is intuitively explained.

High piezoelectricity of BiScO3-PbTiO3 ceramics prepared by two step sintering

(1-x)BiScO3-xPbTiO3 (BSPT) ferroelectric ceramics were prepared by two-step sintering. Dielectric measurements in combination with diffraction contrast transmission electron microscopy (TEM) were used to confirm the phase diagram of BSPT system. From these results, the low temperature phase diagram for BSPT was achieved. In present study, high piezoelectric properties with piezoelectric coefficients (d33 ≈ 545 pC/N, d33* ≈ 920 pm/V) and planar electromechanical coupling factor (kp ≈ 57%) are achieved in the ceramics at the composition x = 0.64, indicating a future high-temperature piezoelectric application.

Combining ECCI and FIB milling techniques to prepare site-specific TEM samples for crystal defect analysis of deformed minerals at high pressure

Dislocation microstructures in experimentally deformed single-crystal pyrope-rich garnet, (Mg,Fe)3(Al,Cr)3Si3O12, and polycrystalline forsterite, Mg2SiO4, were investigated by using electron channeling contrast imaging (ECCI) and transmission electron microscopy (TEM) combined with a focused ion beam (FIB)-microsampling. In the orientation-optimized ECCI method, we successfully observed individual dislocations across subgrain boundaries in a low-atomic-number mineral, pyrope-rich garnet (averaged Z-numbers, AZs∼10). Dislocations in a deformed forsterite (iron-free olivine) were also visible in the ECCI. In the ECCI on the single-crystal garnet, deformation bands consisting of dislocations, unusual contrasts in stripes and inhomogeneous distributions of sub-micrometer-sized pores were found. Further site-specific TEM observation on the deformation band revealed a high density of partial dislocations and stacking fault ribbons. The site-specific characterizations from ECCI to TEM, with assistance of FIB, can provide a new approach to investigate dislocation microstructures of deformed materials at high pressure and high temperature.