Focused Ion Beam Scanning Electron Microscopy Research Articles

Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) for Analysis of Surface and Interface Chemistry of Porous Transport Layers

As the United States energy infrastructure moves towards the integration of hydrogen energy, the reliable production of hydrogen through water electrolyzers is imperative. In proton exchange membrane water electrolyzes (PEMWE’s), the porous transport layer (PTL) plays an important role. When choosing PTL material, one should consider corrosion resistance, electrical conductivity, and ability to support the structure of the electrochemical cell. Due to these requirements, titanium is the current state-of-the-art anode PTL material. However, titanium quickly forms a layer of titanium oxide which significantly decreases conductivity of the PTL and respectively decreases the overall efficiency of the PEMWE system. Coatings are commonly applied to the PTL to combat this complication, but the use of noble metals leads to cost concerns. Additionally, the degradation of coated PTLs is not yet well known.Advanced physicochemical characterization of PTLs at various stages of fabrication and testing is vital to understand their properties and the impact on electrochemical performance in order to improve durability and meet cost targets. Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) is currently used to cross section areas of the PTL for subsequent analysis with Scanning Transmission Electron MicroscopyEnergy-dispersive X-ray Spectroscopy (STEM-EDS) analysis to visualize the elemental information of the PTL materials and PTL coatings, specifically looking at detrimental oxide layer formation. Unfortunately, this approach is time-consuming and difficult, motivating the development of alternative approaches that allow the characterization of wide sample sets more efficiently. Additionally, STEM-EDS analysis only provides elemental information, so if several oxide layers preside, it can be difficult to differentiate them. Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) depth profiling is a very powerful technique that has been commonly used to characterize thin films and buried interfaces. Unlike the FIB-SEM and STEM-EDS combination, ToF-SIMS can be performed relatively quickly in several areas of the PTL, provides chemical information, and is sensitive to trace elements. An added advantage of ToF-SIMS is ability to do 2D and 3D chemical analysis on different scales. This enables visualization of elemental and chemical distribution on both larger scale (70x70 um) areas, and smaller areas (20x10 um) which provides more detailed surface and interface information. This allows for detailed tracking of all major constituents of the PTL and coating. It also allows to follow changes in oxide layers upon fabrication and after electrochemical testing, as well as track homogeneity of the surface and oxide layer interfaces throughout different parts of the PTL. This presentation will demonstrate capabilities of this technique for characterization of PTLs, along with progress and potential challenges. ToF-SIMs results will be compared to TEM analysis of cross-sections obtained with FIB-SEM. This study will highlight similarities and differences between the techniques, technique optimization for these morphologically challenging samples, and paths for future investigation moving forward.

Combining State of the Art Open Source and Proprietary Machine Learning Technologies to Build a Data Analysis Pipeline for Gasoline Particulate Filters using X-Ray Microscopy, Focused Ion Beam-Scanning Electron Microscopy and Transmission Electron Microscopy

The performance of a particulate filter is determined by properties that span the macro, meso and atomic scales. Traditionally, the primary role of a gasoline particulate filter (GPF) is to reduce solid particles and liquid droplets. At the macro scale, transport of gas through a filter’s channels and interconnecting pores act as main transport arteries for catalytically active sites. At the meso scale, the micropore structure is important for ensuring that enough active sites are accessible for the gas to reach the catalyst nanoparticles. At the atomic scale, the structure of the catalyst material determines the performance and selectivity within the filter. Understanding all length scales requires a correlative approach but this is often quite difficult to achieve due to the number of software packages a scientist has to deal with. We demonstrate how current state-of-the-art approaches in the field can be combined into a streamlined pipeline to characterise particulate filters by digitally reconstructing the sample, analysing it at high throughput, and eventually use the result as an input for gas flow simulations and better product design.

Pyrite Characteristics and Its Environmental Significance in Marine Shale: A Case Study from the Upper Ordovician Wufeng–Lower Silurian Longmaxi Formation in the Southeast Sichuan Basin, SW China

Pyrite, as a characteristic mineral in organic-rich marine shale, is a significant index for the interpretation of paleoredox conditions. In this study, based on drilling cores and focused ion beam-scanning electron microscopy (FIB-SEM), the occurrence, diameter and particle size distribution of pyrites from 32 samples obtained from the Wufeng–Longmaxi Formation in the southeast Sichuan Basin were analyzed. The results show that pyrite displays various occurrences at the macro-scale and micro-scale. At the macro-scale (mm–cm), pyrite laminations, nodular pyrites and lenticular pyrites can be found from drilling cores. At the micro-scale (nm–µm), the common occurrences of pyrite are pyrite framboids, euhedral pyrites and infilled pyrite framboids. According to the formation mechanism of pyrites, pyrites can be divided into syngenetic pyrites and diagenetic pyrites. The infilled pyrite framboids are categorized as diagenetic pyrites. The mean pyrite framboid diameters (Mean, D) range from 2.94 µm to 5.33 µm (average of 4.26 µm), with most samples showing pyrite framboid diameters from 3.5 μm to 4.8 μm. Most of the diameters of the framboid microcrystals (Mean, d) are less than 0.4 µm. Therefore, according to the (Mean, D) and the (Mean, d), the pyrite framboids can be divided into three sizes: syngenetic framboids (SF, D < 4.8 µm, d ≤ 0.4 µm), early diagenetic framboids (EDF, D > 4.8 µm, d > 0.4 µm) and late diagenetic framboids (LDF, D < 4.8 µm, d > 0.4 µm). Additionally, box-and-whisker charts of the diameter, standard deviation/skewness value of the mean diameter of pyrite framboids (Mean, D) and the ratio of trace elements indicate that the sedimentary water body was a euxinic–dysoxic environment. Euxinic conditions dominated the Wufeng Formation to the lower part of the Long11-3 section, which is beneficial for the preservation of organic matter. However, the middle-upper part of the Long13-Long12 sub-member is a dysoxic sedimentary environment.

Silver nanoparticles penetration in dentin: Implications for long-term caries arrestment

The effectiveness of silver nanoparticles (AgNPs) as a caries arrestment agent was demonstrated in clinical trials some years ago; however, the mechanisms underlying their action remain unclear. It was hypothesized that the AgNPs could penetrate the dentinal tubules, contributing to their enduring cariostatic activity. The experiments were carried out with teeth samples with the incisal coronal third removed with a rotating diamond disk, exposing the dentin beneath the enamel to simulate shallow occlusal caries lesions. The dentin surfaces were treated with two different AgNP colloids: (i) spherical nanoparticles (7.5 nm) and (ii) a mixture of spherical (24.3 nm), triangular (33.8 nm), and rod-shaped (40.1/18.8 nm) nanoparticles. The samples were analyzed by scanning electron microscopy (SEM) and focused-ion beam SEM (FIB-SEM), allowing for the observation of the inner areas of the dentin. It was observed that AgNPs are able to penetrate dentinal tubules and adhere to their walls. The depth depends on the exposure time of the dentin surface to the colloid and the average nanoparticle size. The smaller the nanoparticles and the longer the exposure time, the deeper their penetration. At maximum, the AgNPs were detected up to 362.1 µm from the surface. The total silver amount in the sub-surface intra-tubular dentin was estimated to be within the concentration range expected to inhibit bacterial growth. The maximum penetration depth detected is shallow enough to avoid AgNP contact with the dental pulp.

Cerium metal oxidation studied by IR reflection-absorption and Raman scattering spectroscopies

Oxidation of cerium metal is a complex process which is strongly affected by the presence of water vapor in the oxidative atmosphere. Here, we explore, by means of infrared reflection-absorption spectroscopy (IRRAS) and Raman scattering spectroscopies, thin oxide films, formed on cerium metal during oxidation, under dry vs ambient (humid) air conditions (∼0.2% and ∼50% relative humidities, respectively) and compare them with a thin film of CeO2 deposited on a Si substrate. Complementary analysis of the thin films using x-ray diffraction and focused ion beam-scanning electron microscopy enables the correlation between their structure and spectroscopic characterizations. The initial oxidation of cerium metal results in the formation of highly sub-stoichiometric CeO2−x . Under dry air conditions, a major fraction of that oxide reacts with oxygen to form CeO∼2, which is spectroscopically detected by Raman scattering F 2g symmetry mode and by IRAAS F 1u symmetry mode, splitted into doubly-degenerate transverse optic and mono-degenerate longitudinally optic (LO) modes. In contrast, under ambient (humid) conditions, the oxide formed is more heterogenous, as the reaction of CeO2−x diverges towards the dominant formation of Ce(OH)3. Prior to the spectral emergence of Ce(OH)3, hydrogen ions incorporate into the highly sub-stoichiometric oxide, as manifested by Ce–H local vibrational mode detected in the Raman spectrum. The spectroscopic response of the thin oxide layer thus formed is more complex; particularly noted is the absence of the LO mode. It is attributed to the high density of microstructural and compositional defects in the oxide layer, which results in a heterogenous dielectric nature of the thin film, far from being representable by a single phase of CeO∼2.

Biofilm Development on Carbon Steel by Iron Reducing Bacterium Shewanella putrefaciens and Their Role in Corrosion

Microscopic, electrochemical and surface characterization techniques were used to investigate the effects of iron reducing bacteria (IRB) biofilm on carbon steel corrosion for 72 and 168 h under batch conditions. The organic nutrient availability for the bacteria was varied to evaluate biofilms formed under nutritionally rich, as compared to nutritionally deficient, conditions. Focused ion beam-scanning electron microscopy (FIB-SEM) was used to investigate the effect of subsurface biofilm structures on the corrosion characteristics of carbon steel. Hydrated biofilms produced by IRB were observed under environmental scanning electron microscope (ESEM) with minimal surface preparation, and the elemental composition of the biofilms was investigated using energy dispersive spectroscopy (EDX). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) was used to provide information on the organic and inorganic chemical makeup of the biofilms. Electrochemical techniques employed for assessing corrosion, by open circuit potential, linear polarization and potentiodynamic polarization tests indicated no significant difference in the corrosion resistance for carbon steel in IRB-inoculated, compared to the abiotic solutions of common Postgate C after 72 and 168 h. However, the steel was found to be more susceptible to corrosion when the yeast extract was removed from the biotic environment for the 168 h test. In the absence of yeast nutrient, it is postulated that IRB received energy by transforming the protective film of Fe3+ into more soluble Fe2+ products.

Hierarchical 2D to 3D micro/nano-histology of human dental caries lesions using light, X-ray and electron microscopy

Dental caries is a widespread disease that proceeds by damaging superficial tooth enamel by heterogeneous dissolution. Conventional histology identifies different zones within carious lesions by their optical appearance, but fails to quantify the underlying nanoscale structural changes as a function of specific location, impeding better understanding of the demineralisation process. We employ detailed collocative analysis using different imaging modalities, resolutions and fields of view. Focused ion beam-scanning electron microscopy (FIB-SEM) reveals subsurface 3D nanostructure within milled micro-sized volumes, whilst X-ray tomography allows minimally destructive 3D imaging over large volumes. Correlative combination of these techniques reveals fine detail of enamel rods, inter-rod substance, sheaths, crystallites and voids as a function of location. The degree of enamel demineralisation within the body of the lesion, near its front, and at the surface is visualized and quantified in 3D. We thus establish the paradigm of dental 3D nano-histology as an advanced platform for quantitative evaluation of caries-induced structural modification.

Evolution of Interfacial Phenomena Induced by Electrolyte Formulation and Hot Cycling of Anode-Free Li-Metal Batteries

Even though the anode-free lithium metal battery (AFLMB) is the next promising energy-storage device because of its high energy density, the irreversible interfacial phenomena at the Cu/electrolyte interface are the key failure mechanisms of the AFLMBs. To the best of our knowledge, the interfacial phenomena associated with the capacity fading of AFLMBs are not reported yet. Herein, hot cycling combined with electrolyte formulation using 1% tris(trimethylsilyl) phosphite (TMSP) additive has been used as a protocol to systematically investigate the Cu/electrolyte interfacial phenomena of Li nucleation, compactness of deposited Li, inactive Li (“dead Li” and Li for solid–electrolyte interphase (SEI) formation “SEI-Li”), electrolyte decomposition, and SEI layer formation using a scanning electron microscope, scanning electron microscope-focused ion beam, titration gas chromatography, in situ gas chromatography–mass spectroscopy, and X-ray photoelectron spectroscopy, respectively. The synergy of hot cycling and the TMSP additive lowers the nucleation barrier in Li/Cu from 76.2 to 21.7 mV and increases the compactness of deposited Li from 32.3 to 43.8% and decreases “dead Li” from 45.18 to 10.86% and SEI-Li from 31.04 to 12.49% of anode-free Cu/NMC111. Because of the synergy effects, the Cu/NMC111 cell provides over 100 cycles with an average coulombic efficiency (avg. CE) of 98.6% for the first 60 cycles and a capacity retention (CR) of 60.4% at the end of the 60th cycle at 0.2/0.5 mA cm–2 charger/discharge current density, while the cell with 1 M LiPF6 EC/DEC at 25 °C offers only an avg. CE of 92.8% for the first 15 cycles with a CR of 40.4% at the 15th cycle. The results suggest that the synergy of hot cycling and electrolyte formulation decreases the surface area of deposited Li and stabilizes the interface that suppresses inventory Li loss. This work probes the Cu/electrolyte interfacial phenomena, attributed to the failure mechanisms of AFLMBs. It also sheds light on suitable strategies to stabilize the Cu interface for stable cycling of AFLMBs.

Exploring critical parameters of electrode fabrication in polymer electrolyte membrane fuel cells

Microstructure and electrochemical properties of the cathode catalyst layers (CCL) of a polymer electrolyte membrane fuel cells (PEMFC) have great impact on the performance and durability of the cell. The aim of this work is to establish a link between CCL structure and fuel cell performance. To obtain CCLs with unique structures six types of electrodes were prepared, each with a different coating technique but with the same Pt loading. The coating techniques are airbrush, screen printing, inkjet printing, dry spraying, doctor blade and drop casting. Moreover, intrinsic properties of PEMFC electrodes such as porosity, permeability, diffusivity as well as ionomer distribution are determined by Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and Atomic Force Microscopy (AFM). Overall, 12 parameters have been evaluated. Generally, CCLs with low fractions of uncovered Pt/C show higher performance at low current densities. In this case the more homogeneous ionomer distribution leads to a higher catalyst utilization. At high current densities transport properties of the CCL have to be considered in addition to the catalyst utilization to explain their performance. The CCL prepared by screen printing shows a low fraction of uncovered Pt/C in combination with good transport properties, leading to the best performance at high currents.

Effect Investigation of Ultraviolet Ageing on the Rheological Properties, Micro-Structure, and Chemical Composition of Asphalt Binder Modified by Modifying Polymer

In order to investigate the effect of nano polymer materials on the UV aged performance of asphalt binder, a nano PA modifying agent was selected to prepare modified asphalt. Under the effect of UV radiation, the modifying effect of nano PA on the rheological properties of asphalt was studied. With the extension of UV ageing time, the microstructure variation of PA modified asphalt was characterized by using a focused ion beam scanning electron microscope (FIB-SEM). The thermophysical properties of PA modified asphalt were investigated by thermogravimetric analysis (TG). Furthermore, the functional group compositions of PA modified asphalt were investigated by dynamic FTIR. The results showed that with the extension of UV ageing time, the PA modifier could mitigate the ageing effect of UV radiation on the asphalt binder. PA modifier could achieve an increasing effect on the viscoelasticity of asphalt during the UV ageing process. PA modifier suppressed the generation of microcracks in the surface microzone of UV aged asphalt samples. PA modifier could promote the thermal stability improvement of asphalt binder when subjected to UV radiation. The generation of carbonyl and sulfoxide groups in asphalt was inhibited during the UV ageing period, which indicated that the UV induced reaction of asphalt could be postponed by the PA modifying agent.

Investigating the Role of Spermidine in a Model System of Alzheimer's Disease Using Correlative Microscopy and Super-resolution Techniques.

Background: Spermidine has recently received major attention for its potential therapeutic benefits in the context of neurodegeneration, cancer, and aging. However, it is unclear whether concentration dependencies of spermidine exist, to differentially enhance autophagic flux. Moreover, the relationship between low or high autophagy activity relative to basal neuronal autophagy flux and subsequent protein clearance as well as cellular toxicity has remained largely unclear. Methods: Here, we used high-resolution imaging and biochemical techniques to investigate the effects of a low and of a high concentration of spermidine on autophagic flux, neuronal toxicity, and protein clearance in in vitro models of paraquat (PQ) induced neuronal toxicity and amyloid precursor protein (APP) overexpression, as well as in an in vivo model of PQ-induced rodent brain injury. Results: Our results reveal that spermidine induces autophagic flux in a concentration-dependent manner, however the detectable change in the autophagy response critically depends on the specificity and sensitivity of the method employed. By using correlative imaging techniques through Super-Resolution Structured Illumination Microscopy (SR-SIM) and Focused Ion Beam Scanning Electron Microscopy (FIB-SEM), we demonstrate that spermidine at a low concentration induces autophagosome formation capable of large volume clearance. In addition, we provide evidence of distinct, context-dependent protective roles of spermidine in models of Alzheimer’s disease. In an in vitro environment, a low concentration of spermidine protected against PQ-induced toxicity, while both low and high concentrations provided protection against cytotoxicity induced by APP overexpression. In the in vivo scenario, we demonstrate brain region-specific susceptibility to PQ-induced neuronal toxicity, with the hippocampus being highly susceptible compared to the cortex. Regardless of this, spermidine administered at both low and high dosages protected against paraquat-induced toxicity. Conclusions: Taken together, our results demonstrate that firstly, administration of spermidine may present a favourable therapeutic strategy for the treatment of Alzheimer’s disease and secondly, that concentration and dosage-dependent precision autophagy flux screening may be more critical for optimal autophagy and cell death control than previously thought.

レーザ粉末床溶融結合法によるAl-10%Si-0.4%Mg合金積層造形体の特異的な熱伝導率のイメージベース有限要素解析

Laser powder bed fused Al-10Si-0.4Mg (mass%) alloy exhibits low and anisotropic thermal conductivity. To elucidate dominant microstructural features generating the characteristic thermal conductivity, thermal conduction analysis was performed by an image-based finite element method (FEM) using scanning electron microscope (SEM) images. The serial sectioning images of as-fabricated and 300ºC/2 h heat treated Al-10Si-0.4Mg alloys were obtained by focused ion beam SEM (FIB-SEM) and were used for constructing three-dimensional models. The homogenization method was applied to calculate average thermal conductivity of these models. When the thermal conductivity of α-Al phase was set at the thermal conductivity of pure aluminum, calculated thermal conductivity of as-fabricated model was much higher than experimentally measured thermal conductivity of as-fabricated Al-10Si-0.4Mg alloy, indicating that α-Al phase in the as-fabricated Al-10Si-0.4Mg alloy had much lower thermal conductivity than pure aluminum. In addition, the anisotropy of thermal conductivity appeared only in the as-fabricated model in which the α-Al/Si interfacial thermal resistance was taken into account. These results were used for discussing dominant contributors of low and anisotropic thermal conductivity of laser additive manufactured Al-10Si-0.4Mg alloy.

Poisoning Effects of Cerium Oxide (CeO2) on the Performance of Proton Exchange Membrane Fuel Cells (PEMFCs)

In this study, the poisoning effects of cerium oxide (CeO2) as the contaminant on the performance of proton exchange membrane fuel cells (PEMFCs) are evaluated. An experimental setup was developed to analyze the performance characteristic (I-V) curves in contaminated and non-contaminated conditions. Focused ion-beam scanning electron microscopy (FIB-SEM) cross-section images were obtained as an input for the energy dispersive X-ray (EDX) analysis. The results of the EDX analysis verified the presence of CeO2 in the contaminated membrane electrode assembly (MEA), in addition to fluorine and sulfur. EDX analysis also revealed that as a result of CeO2 contamination, sulfur and fluorine would be distributed all around the MEA, instead of being only in the membrane. The results illustrate that hydrofluoric acid (HF), sulfuric acid (H2SO4), and fluorinated polymer fragments are released, which enhance the crossover of the reactant gases through the membrane, hence reducing the cell’s performance. The I-V characteristic curves proved that the non-contaminated PEMFC setup had double the performance of the contaminated PEMFC.

Volumetric 3D reconstruction of plant leaf cells using SEM, ion milling, TEM, and serial sectioning.

Focused ion beam scanning electron microscopy is well suited for volumetric extractions and 3D reconstructions of plant cells and its organelles. The three-dimensional (3D) reconstruction of individual plant cells is an important tool to extract volumetric data of organelles and is necessary to fully understand ultrastructural changes and adaptations of plants to their environment. Methods such as the 3D reconstruction of cells based on light microscopical images often lack the resolution necessary to clearly reconstruct all cell compartments within a cell. The 3D reconstruction of cells through serial sectioning transmission electron microscopy (ssTEM) and focused ion beam scanning electron microscopy (FIB-SEM) are powerful alternatives but not widely used in plant sciences. Here, we present a method for the 3D reconstruction and volumetric extraction of plant cells based on FIB milling and compare the results with 3D reconstructions obtained with ssTEM. When compared to 3D reconstruction based on ssTEM, FIB-SEM delivered similar results. The data extracted in this study demonstrated that tobacco cells were larger (31410 µm3) than pumpkin cells (20697 µm3) and contained more chloroplasts (175 vs. 124), mitochondria (1317 vs. 291) and peroxisomes (745 vs. 79). While individual chloroplasts, mitochondria, peroxisomes were larger in pumpkin plants (25, 53, and 50%, respectively) they covered more total volume in tobacco plants (5390, 395, 374 µm3, respectively) due to their higher number per cell when compared to pumpkin plants (4762, 134, 59 µm3, respectively). While image acquisition with FIB-SEM was automated, software controlled, and less difficult than ssTEM, FIB milling was slower and sections couldnot be revised or re-imaged as they were destroyed by the ion beam. Nevertheless, the results in this study demonstrated that both, FIB-SEM and ssTEM, are powerful tools for the 3D reconstruction of and volumetric extraction from plant cells and that there were large differences in size, number, and organelle composition between pumpkin and tobacco cells.

Optical flow driven interpolation for isotropic FIB-SEM reconstructions

Background and Objective: Focused Ion Beam - Scanning Electron Microscopy (FIB-SEM) allows three-dimensional ultrastructural analysis of cells and tissues at the nanoscale. The technique iteratively removes a section of the sample with a FIB and takes an SEM image from the exposed surface. The section thickness is usually higher than the image pixel size to reduce acquisition time, thus resulting in anisotropic resolution. In this work, we explore novel interpolation methods along the sectioning direction to produce isotropic resolution and facilitate proper interpretation of the FIB-SEM 3D volumes.Methods: Classical interpolation methods are usually applied in this context under the assumption that the changes through successive images are relatively smooth. However, the actual 3D arrangement of the structures in the sample may cause significant changes in the biological features between consecutive images of the FIB-SEM stacks. We have developed a novel interpolation strategy that accounts for this variation by using the Optical Flow (OF) to estimate it. As an intermediate stage, OF-compensated images are produced by aligning the spatial regions of the biological structures. Interpolated images are then generated from these OF-compensated images. The final isotropic stack is assembled by interleaving the interpolated images with the original images of the anisotropic stack.Results: OF-driven and classical interpolation methods were compared using an objective assessment based on Pearson Correlation Coefficient (PCC) and a qualitative evaluation based on visual results, using public datasets and representative anisotropy conditions. The objective assessment demonstrated that the OF-driven interpolation always yields higher PCC values, with interpolated images closer to the ground truth. The qualitative evaluation corroborated those results and confirmed that classical interpolation may blur areas with substantial changes between consecutive images whereas OF-driven interpolation provides sharpness.Conclusions: We have developed an OF-driven interpolation approach to generating FIB-SEM stacks with isotropic resolution from experimental anisotropic data. It adapts to the rapid variation of the biological structures observed through the images of the FIB-SEM stack. Our approach outperforms classical interpolation and manages to produce sharp interpolated views in cases where there are significant changes between consecutive experimental images.

Recent advances in electron microscopy for the diagnosis and research of glomerular diseases.

Recent technical advances in the detection of backscattered electrons during scanning electron microscopy (SEM) have improved resolution and have provided several new technologies for research and clinical practice in kidney disease. The advances include three-dimensional (3D) electron microscopy (3D-EM), correlative light and electron microscopy (CLEM), low-vacuum SEM (LVSEM), and scanning transmission electron microscopy (STEM). 3D-EM analysis used to be laborious, but recently three different technologies, serial block-face SEM, focused ion beam SEM, and array tomography, have made 3D-EM easier by automating sectioning and the subsequent image acquisition in an SEM. CLEM is a method to correlate light microscopic images, especially immunofluorescent and electron microscopy images, providing detailed ultrastructure of the area of interest where the immunofluorescent marker is located. LVSEM enables the use of SEM on materials with poor electron conductivity. For example, LVSEM makes it possible for high resolution, 3D observation of paraffin sections. Finally, STEM is a method to observe ultrathin sections with improved resolution by using the focused electron beam scanning used in SEM and not the broad electron beam used in transmission electron microscopy. These technical advances in electron microscopy are promising to provide plenty of novel insights for understanding the pathogenesis and diagnosis of various glomerular diseases.

3D FIB-SEM structural insights into the architecture of sub-pellicular microtubules of Trypanosoma cruzi epimastigotes.

Trypanosomatidae, which includes eukaryotic species agents of diseases like leishmaniasis, sleeping sickness, and Chagas disease, have special structures and organelles not found in mammalian cells. They present a layer of microtubules, known as subpellicular microtubules (SPMT), located underneath the plasma membrane and responsible for preserving cell morphology, cell polarity, the position of single copy organelles, and morphological changes that occur throughout the protozoan life cycle. Even though a lot of knowledge about the SPMT is available, we still do not know exactly how each microtubule in the system is organized in three dimensions. Here, we use focused ion beam scanning electron microscopy (FIB-SEM) to analyze the tridimensional organization of epimastigotes SPMT. The high-resolution 3D analyses revealed that certain microtubules of the SPMT end more prematurely than the neighboring ones. These microtubules could (1) be shorter or (2) have the same length as the neighboring ones, assuming that those end up earlier at their other end, might be treadmilling/catastrophe events that have not yet been described in trypanosomatids.

ATG9 Vesicles Are Incorporated Into Nascent Autophagosome Membranes

The autophagosome is a double‐membrane organelle that traps cytoplasmic cargo and traffics it to the lysosome for degradation. How the autophagosome forms is uncertain, but a prevailing model suggests lipids are moved from the ER through the lipid transporter ATG2 to ATG9 vesicles, which then expand to comprise the growing autophagosomal membrane. However, evidence that ATG9 is ever resident within the autophagosome is scant; detection of this putative autophagosome‐resident protein is made challenging both because most ATG9 vesicles in the cell are not involved in the biogenesis at any given time and because the dilution of one or a few vesicle membranes by potentially millions of transported lipids would result in a very low density of ATG9 on the mature autophagosome. Here we develop approaches to address each of these limitations. First, we show that in genetic knockouts of ATG2, ATG9 vesicles accumulate to very high numbers at sites of aborted autophagosome formation. Focused‐ion beam scanning electron microscopy reveals that without ATG2, these putative autophagosome seed vesicles do not expand, but instead accumulate within a large vesicle cluster surrounded by ER. By fluorescence microscopy, we also detect downstream modifiers of the autophagosome membrane at these sites, including the lipid‐anchored form of the LC3 proteins, which suggests that a biochemically competent seed membrane is present. To establish whether ATG9A is found on the same membrane as LC3B, we use styrene maleic acid (SMA) copolymer nanodiscs, to isolate very small intact sections of autophagosome membrane away from all other potential contaminants. Through rigorous combinations of isolation and purification, we reveal that ATG9A and LC3B are co‐resident within the same 10 nm diameter membrane segment and likely engage in a protein‐protein complex. We then apply the same SMA‐based approach to show that even in wildtype autophagosomes, ATG9 and LC3 are co‐resident. Thus, we assert that ATG9 vesicles are the seed membrane for the autophagosome.

Developmental Regulation of Sarcomere Branching in Cardiomyocytes

Cardiac muscles undergo constant contractions to continuously supply oxygenated blood to both central and peripheral organs. To sustain such large demand, cardiac muscles must convert biological fuel to functional force via the contraction of their sarcomeres. Using nanoscale 3D imaging analysis, our group demonstrated that sarcomeres within the cardiac muscle of mature mice (≥ 2 months) are frequently branched to form a unified myofibrillar matrix across the entire cell, thereby providing the architecture needed for both longitudinal and lateral transmission of active force. How these contractile networks are formed during postnatal development remains unclear. Here we used 3D focused‐ion beam scanning electron microscopy (FIB‐SEM) to evaluate myofibrillar ultrastructure within cardiac muscle cells in mice at postnatal (P) days 1, 7, 14, and 42, respectively. 3D reconstruction of myofibrillar structures showed highly connected networks at all time points. Quantitative assessment of sarcomere branch‐points revealed that branching frequency is varied by developmental process. The percentages of sarcomeres with at least one branch‐point are as follows: 57 ± 2.4% (mean ± SE, 14 cells, 129 myofibrils, 802 sarcomeres) in P1, 64 ± 2.1% (17 cells, 161 myofibrils, 795 sarcomeres) in P7, 49 ± 3.9% (4 cells, 50 myofibrils, 250 sarcomeres) in P14, and 27 ± 1.1 % (7 cells, 236 myofibrils, 2320 sarcomeres) in P42. Data was further separated based on whether a single sarcomere is branched into two (single‐branch, Fig 1E, F) or more (multi‐branch, Fig 1G, H) structures to delineate the nature and overall magnitude of branching within cardiomyocytes (Fig 2B‐ D). Across the postnatal development (P1 to P42), there was a 0.4‐fold change in the magnitude of branching within ten sarcomeres (Fig 2D; 7.9±0.41, 11±0.45, 6.5±0.58, and 3.3±0.16 for P1, P7, P14, and P42, respectively). All changes were statistically significant at P<0.05. In conclusion, the frequency of sarcomere branching was highest at P7 and progressively declined until maturation. Findings indicate that while the architecture needed for active force transmission across the length and width of the cell is already formed at birth, the connectivity of cardiac contractile networks is dynamic throughout postnatal development. These results suggest that sarcomere branching dynamics may provide a target for modulating the contractility of cardiac muscle cells.

Volume electron microscopy analysis reveals a new type of membrane junction required for mixing of parental genomes after fertilization

Imaging cells by light or fluorescence microscopy often provides great insights into mechanisms and dynamics of cellular processes. However, to gain information regarding the cellular structures that underlie these processes, nm‐scale resolution is often required. This type of resolution can be obtain using electron microscopy (EM). Traditionally, investigators imaged slices of chemically fixed cells or tissue in order to glimpse at relevant structures in 2‐dimensions. While powerful, this approach can potentially miss important information due to the somewhat random location of the planes analyzed, especially if a small number of images is a acquired. More recently, methodologies of volume EM have been developed that allow for sampling of a continuous volume at high resolution. Here we will describe how one such methodology, Focused Ion Beam‐ Scanning Electron Microscopy (FIB‐SEM), has been used to understand the process by which parental genomes mix after fertilization in C. elegans embryos. One‐cell embryos were collected during different stages of interphase and mitosis, fixed using high‐pressure freezing followed by freeze substitution, and stained to maximize membrane contrast. Images were acquired at 9 nm lateral (x,y) and axial (z) resolution, for volumes of approximately 10 X 10 X 25 µm3. These analyses revealed that for parental chromosomes to mix, the membranes of the two pronuclei must fused via a membrane structure that we termed “3‐way sheet junction”. The steps leading to the formation of 3‐way sheet junctions, and the more recent discovery of a novel membrane structure around centrosomes using these datasets, will be discussed.