Focused Ion Beam Scanning Electron Microscopy Research Articles

Characterization of human trabecular bone across multiple length scales using a correlative approach combining X-ray tomography with LaserFIB and plasma FIB-SEM

Three-dimensional correlative multimodal and multiscale imaging is an emerging method for investigating the complex hierarchical structure of biological materials such as bone. This approach synthesizes images acquired across multiple length scales, for the same region of interest, to provide a comprehensive view of the material structure of a sample. Here, we develop a workflow for the structural analysis of human trabecular bone using a femtosecond laser to produce a precise grid to facilitate correlation between imaging modalities and identification of structures of interest, in this case, a single trabecula within a volume of trabecular bone. Through such image registration, high resolution X-ray microscopy imaging revealed fine architectural details, including the cement sheath and bone cell lacunae of the selected bone trabecula. The selected bone volume was exposed with a combination of manual polishing and site-specific femtosecond laser ablation and then examined with plasma focused ion beam-scanning electron microscopy. This reliable and versatile correlation approach has the potential to be applied to a variety of biological tissues and traditional engineered materials. The proposed workflow has the enhanced capability for generating highly resolved and broadly contextualized structural data for a better understanding of the architectural features of a material spanning its macroscopic to nanoscopic levels.

Video frame interpolation neural network for 3D tomography across different length scales

Three-dimensional (3D) tomography is a powerful investigative tool for many scientific domains, going from materials science, to engineering, to medicine. Many factors may limit the 3D resolution, often spatially anisotropic, compromising the precision of the information retrievable. A neural network, designed for video-frame interpolation, is employed to enhance tomographic images, achieving cubic-voxel resolution. The method is applied to distinct domains: the investigation of the morphology of printed graphene nanosheets networks, obtained via focused ion beam-scanning electron microscope (FIB-SEM), magnetic resonance imaging of the human brain, and X-ray computed tomography scans of the abdomen. The accuracy of the 3D tomographic maps can be quantified through computer-vision metrics, but most importantly with the precision on the physical quantities retrievable from the reconstructions, in the case of FIB-SEM the porosity, tortuosity, and effective diffusivity. This work showcases a versatile image-augmentation strategy for optimizing 3D tomography acquisition conditions, while preserving the information content.

Advanced multi-scale characterization of loess microstructure: Integrating μXCT and FIB-SEM for detailed fabric analysis and geotechnical implications

Loess, a Quaternary wind-blown deposit, is a problem soil that gives rise to frequent geohazards such as landslides and water-induced subsidence. The behaviour of loess is controlled by its microstructure, consisting of silt-sized skeleton particles and complex bonding structures formed by clay-sized particles. Achieving a deep understanding and precise modelling of loess behaviour necessitates comprehensive knowledge of the realistic 3D microstructure. In this paper, a correlative investigation of the 3D loess microstructure is performed using X-ray micro-computed tomography (μXCT) and focused ion beam scanning electron microscope (FIB-SEM). Details of clay structures in loess, such as clay coatings, clay bridges and clay buttresses, are visualized and characterized in 3D based on FIB-SEM images with a voxel size of 10 × 10 × 10 nm3. The clay structures exhibit a diverse degree of complexity and their impact on the mechanical properties of loess is highlighted. Statistical analysis of the skeleton particles, including size, shape and orientation, are derived from μXCT images with a voxel size of 0.7 × 0.7 × 0.7 μm3. The findings provide insights into the collapse mechanism and particle-scale modelling of loess. The combination of μXCT and FIB-SEM proves to be a powerful approach for characterizing the intricate micro-structures of loess, as well as other geomaterials.

Tuning α Precipitation via Post-Heat Treatments in Direct Energy Deposited Metastable β Ti-5Al-5Mo-5V-3Cr Alloy and Its Impact on Mechanical Properties

In this study, the strength and ductility of a direct energy deposited (DEDed) metastable β Ti-5Al-5Mo-5V-3Cr (wt.%, Ti-5553) alloy was explored by tuning the formation of various α microstructures through post-heat treatments. The microstructure of DEDed Ti-5553 with and without post-heat treatments was systematically studied using scanning electron microscopy, three-dimensional (3D) focused ion beam-scanning electron microscopy tomography, transmission electron microscopy, scanning transmission electron microscopy, and atom probe tomography. Nanoscale ω phase and O′ phase particles were observed in the as-built DEDed Ti-5553 without post-heat treatment for the first time. By altering the aging temperatures and heating rates during post-heat treatments, various microstructural evolution pathways were activated, leading to α microstructures with different sizescales, morphologies, and number densities. By fast heating and isothermal aging at 600°C for 2hours, refined α microstructure was formed through the pseudospinodal decomposition mechanism. While slow heating (at the heating rates of 5°C/min or 0.5°C/min) to 600°C and isothermal aging for 2hours, super-refined α microstructures were produced respectively, primarily via ω-assisted and O′-assisted α precipitation mechanisms. By fast heating to and isothermal aging at 700°C, fine-scaled intragranular α microstructure was formed, dramatically different in morphology and sizescale from the coarse α microstructure formed in the casted Ti-5553, which is suspected to be related to the indirect influence of nanoscale isothermal ω phase particles formed during the DED process and the excess oxygen introduced during the DED process. With fast heating to and isothermal aging at 800°C, coarse α microstructure forms through the classical nucleation and growth mechanism. Various α microstructures led to a drastic change of mechanical properties with improvements up to a 55% increase in hardness and 92% increase in tensile yield strength, compared with as-built DEDed Ti-5553. Our work indicates the feasibility of achieving tailored mechanical properties in DEDed metastable β-Ti alloys by tuning α microstructures through selectively activating various phase transformation mechanisms via post-heat treatments.

Chronic undernutrition impairs renal mitochondrial respiration accompanied by intense ultrastructural damage in juvenile rats

This study investigated whether chronic undernutrition alters the mitochondrial structure and function in renal proximal tubule cells, thus impairing fluid transport and homeostasis. We previously showed that chronic undernutrition downregulates the renal proximal tubules (Na++K+)ATPase, the main molecular machine responsible for fluid transport and ATP consumption. Male rats received a multifactorial deficient diet, the so-called Regional Basic Diet (RBD), mimicking those used in impoverished regions worldwide, from weaning to a juvenile age (3 months). The diet has a low content (8 %) of poor-quality proteins, low lipids, and no vitamins compared to control (CTR). We investigated citrate synthase activity, mitochondrial respiration (oxygraphy) in phosphorylating and non-phosphorylating conditions with different substrates/inhibitors, potential across the internal membrane (Δψ), and anion superoxide/H2O2 formation. The data were correlated with ultrastructural alterations evaluated using transmission electron microscopy (TEM) and focused ion beam scanning electron microscopy (FIB-SEM). Citrate synthase activity decreased (∼50 %) in RBD rats, accompanied by a similar reduction in respiration in non-phosphorylating conditions, maximum respiratory capacity, and ATP synthesis. The Δψ generation and its dissipation after carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone remained unmodified in the survival mitochondria. H2O2 production increased (∼100 %) after Complex II energization. TEM demonstrated intense matrix vacuolization and disruption of cristae junctions in a subpopulation of RBD mitochondria, which was also demonstrated in the 3D analysis of FIB-SEM tomography. In conclusion, chronic undernutrition impairs mitochondrial functions in renal proximal tubules, with profound alterations in the matrix and internal membrane ultrastructure that culminate with the compromise of ATP supply for transport processes.

Correlative Microscopy Analysis for Battery Materials

Lithium batteries (LB) are in high demand for a diverse range of applications, such as automotive industry, consumer electronics, wearable and medical devices, grid storage, etc. Each application demands a specific set of performance advantages, such as high capacity and/or energy density, long cycle life, superior electrochemical stability. Therefore, there is no one-size-fits-all for a LB chemistry and its form factor. Battery performance advantages rely on optimization of the battery components, including active materials, electrolyte, separator, binder system, and current collectors. For meaningful device optimization, electrochemical battery testing needs to be combined with a deep understanding of the material and device microstructure. Physical and chemical characterization on the material- and electrode-level is needed to establish root-cause relationships with battery performance. Properties of the interfaces between layers of the battery components often define the device characteristics. Critical information is usually buried beneath the surface of a material specimen or encapsulated in a closed-form device. Microstructural defect identification in the battery needs to be followed by a targeted defect study. Different battery form factors and chemistries make it hard to streamline root-cause analysis.Recently, a commercial focused-ion beam scanning electron microscope was developed with an integrated fs-laser mill attached to the load lock of the microscope, opening the door to new and more powerful analysis approaches in energy materials research. Applications include rapid access to deeply buried structures for high resolution imaging and analytics, large area cross-section preparation, and massive material ablation for sample preparation of structures; e.g. FIB-SEM tomography, TEM, APT, or X-ray nanoCT. Additionally, each of these methods may be correlatively guided by prior imaging, with techniques like 3D X-ray microscopy (XRM), to enable targeted analysis and preparation. The non-destructive nature of 3D XRM makes it an ideal tool for microstructural defect identification in LB. Laser mill allows researchers to combine non-destructive X-ray defect identification with electron microscopy methods for targeted defect characterization. This approach can be used for various battery types and form factors.By combining the laser mill with 3D X-ray microscopy, this platform enables comprehensive analysis of energy materials at the nanoscale. We illustrate these concepts with application examples, and use cases demonstrating the utility of the approach.

Mass Transport Regimes in a CO2R Gas Diffusion Electrode Pore with Coupled Microkinetics Model

Gas Diffusion Electrodes (GDEs) have arisen as a popular CO2 electrolyser configuration due to the improved mass transfer properties and higher current densities possible in comparison to a liquid-fed system. In this work, we aim to understand the mass transport regimes in a single liquid-filled pore of a GDE, i.e., an idealized model of the porous catalyst layer. In previous work, Butler-Volmer (BV) expressions have been used to model the CO evolution reaction and the Hydrogen Evolution Reaction (HER). In this study, we extend that in a few ways: (1) a MicroKinetic Model (MKM) of Au at the surface of the electrode is used instead of the BV expressions, (2) volume/steric effects are incorporated through the Generalized Modified Poisson-Nerst-Planck (GMPNP) equation, and (3) more reactions are considered in the electrolyte. By analyzing pores of varying sizes, following the size distribution measured by Focused Ion Beam Scanning Electron Microscopy (FIB/SEM) in a GDE from the literature, we assess conditions where concentration profiles are no longer effectively one-dimensional in the pore axis. We discuss how these three-dimensional mass transfer effects impact CO2 conversion efficiency. In such regimes, we describe why special treatment is required when calling BV expressions in macroscale pore-averaged models.This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344. LLNL release number: LLNL-ABS-858384.

Three-dimensional structural analyses of the oxygen electrodes in solid oxide electrolysis cells

Solid oxide electrolysis cells (SOECs) have garnered interest as efficient devices for production of clean fuel such as hydrogen gas. However, the industrialization of SOEC technology has encountered significant challenges, such as unsatisfactory performance and insufficient durability. In this study, we aimed to improve the electrochemical performance and stability of the SOEC air electrode by modifying it at various annealing temperatures (900, 1000, and 1100 °C). Electrochemical testing revealed that the SOEC cell with the electrode annealed at 1000 °C for 1 h exhibited the highest current density of 0.88 A/cm2 at 750 °C and 1.3 V, which is 1.76-times higher than that of the cell with the unoptimized electrode annealed at 900 °C. Three-dimensional microstructural analyses using Focused Ion Beam-Scanning Electron Microscope (FIB-SEM) confirmed that the optimized cell is the most porous with sufficient pore surface area, thereby facilitating electrochemical reactions and gas transport, and preventing delamination of the air electrode resulting from lower oxygen partial pressure difference between the barrier layer and air/electrode interface. Side reactions at the interface, including the generation of resistant SrZrO3, are concurrently avoided. These findings demonstrate the potential of the modified SOEC air electrode for improving electrochemical performance and stability.

Combining ternary, ionic liquid-based, polymer electrolytes with a single-ion conducting polymer-based interlayer for lithium metal batteries

Among the many approaches to improve the performance of lithium-metal batteries, ternary polyethylene oxide/ionic liquid/lithium salt electrolytes offer several advantages such as low flammability, high conductivity (vs. polyethylene oxide/lithium salt electrolytes) and, to a large extent, limiting the growth of dendrites at moderate currents. However, they suffer from relatively low mechanical strength for lithium metal confinement. Besides, the lithium transport numbers are very low, which is conducive to lithium depletion during plating at high current densities at the lithium/electrolyte interface. Thus, we show here that the combination of a ternary solid polymer electrolyte with a single-ion polymer-based conducting interlayer allows for a significant improvement of the cyclability of the lithium metal anode. This results in a strong improvement of the electrochemical performance of lithium-metal batteries using solid polymer electrolytes at 80 °C, with an 85% capacity retention after 350 cycles (vs. 60% after 62 cycles for the uncoated anode). This is attributed, via focused ion beam-scanning electron microscopy and X-ray photoelectron spectroscopy, to a denser lithium deposit, better contact with the electrolyte and a reduced reactivity of electrolyte species with the interlayer.

Gamma‐ray‐induced modification of poly(ethylene terephthalate) and polypropylene solid materials with halogen‐containing olefins for X‐ray CT image contrast enhancement

AbstractIt is known that X‐ray CT of organic polymer materials usually provides low‐contrast images because of the low X‐ray absorption of these materials. In this paper, we describe a new method to enhance the contrast of X‐ray CT images of organic polymer materials. We demonstrate that gamma‐ray irradiation of poly (ethylene terephthalate) (PET) fibers in dichloromethane containing 2‐bromoethyl methacrylate, 2‐chloroethyl methacrylate, or 4‐bromostyrene results in the grafting of halogen‐containing oligomeric chains onto the PET fibers. Focused ion beam scanning electron microscopy (FIB‐SEM) and time‐of‐flight secondary ion mass spectrometry (TOF‐SIMS) analyses revealed a homogeneous distribution of bromine atoms within the 2‐bromoethyl methacrylate‐modified PET fibers, not only on the surface. This uniform incorporation translated to a significant improvement in X‐ray CT image contrast compared with pristine fibers. Similar treatment of polypropylene pellets also enhanced the contrast of their X‐ray CT images. This is a new simple and effective method to enhance the X‐ray CT image contrast and potentially applicable to various polymer materials including polymer composites and porous polymer materials, readily allowing the observation of their 3D microstructures finely.

Porosity and Organic Content in the Tuscaloosa Marine Shale: Insights from Focused Ion Beam-Scanning Electron Microscopy Characterization

Porosity and Organic Content in the Tuscaloosa Marine Shale: Insights from Focused Ion Beam-Scanning Electron Microscopy Characterization

Multifaceted dinoflagellates and the marine model Prorocentrum cordatum.

Dinoflagellates are a monophyletic group within the taxon Alveolata, which comprises unicellular eukaryotes. Dinoflagellates have long been studied for their organismic and morphologic diversity as well as striking cellular features. They have a main size range of 10‒100 µm, a complex "cell covering", exceptionally large genomes (~1‒250 Gbp with a mean of 50,000 protein-encoding genes) spread over a variable number of highly condensed chromosomes, and perform a closed mitosis with extranuclear spindles (dinomitosis). Photosynthetic, marine, and free-living Prorocentrum cordatum is a ubiquitously occurring, bloom-forming dinoflagellate and an emerging model system, particularly with respect to systems biology. Focused ion beam/scanning electron microscopy (FIB/SEM) analysis of P. cordatum recently revealed (i) a flattened nucleus with unusual structural features and a total of 62 tightly packed chromosomes, (ii) a single, barrel-shaped chloroplast devoid of grana and harboring multiple starch granules, (iii) a single, highly reticular mitochondrion, and (iv) multiple phosphate and lipid storage bodies. Comprehensive proteomics of subcellular fractions suggested (i) major basic nuclear proteins to participate in chromosome condensation, (ii) composition of nuclear pores to differ from standard knowledge, (iii) photosystems I and II, chloroplast complex I, and chlorophyll a-b binding light harvesting complex to form a large megacomplex (>1.5 MDa), and (iv) an extraordinary richness in pigment-binding proteins. Systems biology-level investigation of heat stress response demonstrated a concerted down-regulation of CO2-concentrating mechanisms, CO2-fixation, central metabolism, and monomer biosynthesis, which agrees with reduced growth yields. FIB/SEM analysis revealed new insights into the remarkable subcellular architecture of P. cordatum, complemented by proteogenomic unravelling of novel nuclear structures and a photosynthetic megacomplex. These recent findings are put in the wider context of current understanding of dinoflagellates.

High-Resolution Focused-Ion Beam Scanning Electron Microscopy Reveals Differentially Organized F-actin Compartments in Cochlear Hair Cell Stereocilia

High-Resolution Focused-Ion Beam Scanning Electron Microscopy Reveals Differentially Organized F-actin Compartments in Cochlear Hair Cell Stereocilia

Postembedding Iodine Staining for Contrast‐Enhanced 3D Imaging of Bone Tissue Using Focused Ion Beam‐Scanning Electron Microscopy

For a better understanding of living tissues and materials, it is essential to study the intricate spatial relationship between cells and their surrounding tissue on the nanoscale, with a need for 3D, high‐resolution imaging techniques. In the case of bone, focused ion beam‐scanning electron microscopy (FIB‐SEM) operated in the backscattered electron (BSE) mode proves to be a suitable method to image mineralized areas with a nominal resolution of 5 nm. However, as clinically relevant samples are often resin‐embedded, the lack of atomic number (Z) contrast makes it difficult to distinguish the embedding material from unmineralized parts of the tissue, such as osteoid, in BSE images. Staining embedded samples with iodine vapor has been shown to be effective in revealing osteoid microstructure by 2D BSE imaging. Based on this idea, an iodine (Z = 53) staining protocol is developed for 3D imaging with FIB‐SEM, investigating how the amount of iodine and exposure time influences the imaging outcome. Bone samples stained with this protocol also remain compatible with confocal laser scanning microscopy to visualize the lacunocanalicular network. The proposed protocol can be applied for 3D imaging of tissues exhibiting mineralized and nonmineralized regions to study physiological and pathological biomineralization.

Characterizing PEM fuel cell catalyst layer properties from high resolution three-dimensional digital images, part I: A numerical procedure for the ionomer distribution reconstruction

In this work, a numerical approach is presented to reconstruct the ionomer three-dimensional distribution in the microstructure of the cathode catalyst layer (CCL) of a polymer electrolyte membrane fuel cell (PEMFC) from a 3D segmented image of the CCL obtained by focused ion beam-scanning electron microscopy (FIB-SEM). Three forms of ionomer are distinguished: the ionomer filaments, the coating ionomer and the inter-carbon grains ionomer. The ionomer filaments are identified by applying a filtering procedure to the size distribution in the segmented solid phase obtained via a maximum sphere inscription algorithm. The coating ionomer is reconstructed via a filtering procedure of the curvature distribution computed over the interface between the two segmented phases from the consideration that concave regions are preferential ionomer accumulation zones during the drying step of the CCL fabrication procedure. This procedure of coating controlled by the local curvature is applied until a desired value of coverage is reached. In a last step, catalyst nanoparticles, also not visible in the FIB-SEM image, are reconstructed in the image.

Characterizing PEM fuel cell catalyst layer properties from high resolution three-dimensional digital images, Part II: Oxygen effective diffusion and proton effective conductivity tensors

The oxygen effective diffusion and proton effective conductivity tensors of a cathode catalyst layer (CCL) are computed using a two-scale approach on CCL microstructure three-dimensional images obtained by focused ion beam-scanning electron microscopy (FIB-SEM) after reconstruction of the ionomer distribution. The results show that the obtained tensors are not isotropic. It is also found that some off-diagonal components are not negligible in the case of the proton effective conductivity tensor. The simulations confirm that Knudsen diffusion has a significant impact on oxygen diffusion. The results are comparable to those obtained in previous works on synthetic images as regards the oxygen effective diffusion through-plane component but differ as regards the proton conductivity through-plane component. Contrary to previous works using synthetic images which tend to underestimate the proton conductivity, they are globally in better agreement with experimental data. The results also show that the effective proton conductivity is heterogeneous at the scale of the computational domains typically considered in several previous works due to the variability in the ionomer distribution at this scale. The impact of liquid water in the primary pores on the proton transport is explored and found to have a significant impact.

Lipid droplet dynamics are essential for the development of the malaria parasite Plasmodium falciparum.

Lipid droplets (LDs) are organelles that are central to lipid and energy homeostasis across all eukaryotes. In the malaria-causing parasite Plasmodium falciparum the roles of LDs in lipid acquisition from its host cells and their metabolism are poorly understood, despite the high demand for lipids in parasite membrane synthesis. We systematically characterised LD size, composition and dynamics across the disease-causing blood infection. Applying split fluorescence emission analysis and three-dimensional (3D) focused ion beam-scanning electron microscopy (FIB-SEM), we observed a decrease in LD size in late schizont stages. LD contraction likely signifies a switch from lipid accumulation to lipid utilisation in preparation for parasite egress from host red blood cells. We demonstrate connections between LDs and several parasite organelles, pointing to potential functional interactions. Chemical inhibition of triacylglyerol (TAG) synthesis or breakdown revealed essential LD functions for schizogony and in counteracting lipid toxicity. The dynamics of lipid synthesis, storage and utilisation in P. falciparum LDs might provide a target for new anti-malarial intervention strategies.

Mesoscale characterization of osseointegration around an additively manufactured genistein-coated implant

Given the hierarchical nature of bone and bone interfaces, osseointegration, namely the formation of a direct bone-implant contact, is best evaluated using a multiscale approach. However, a trade-off exists between field of view and spatial resolution, making it challenging to image large volumes with high resolution. In this study, we combine established electron microscopy techniques to probe bone-implant interfaces at the microscale and nanoscale with plasma focused ion beam-scanning electron microscopy (PFIB-SEM) tomography to evaluate osseointegration at the mesoscale. This characterization workflow is demonstrated for bone response to an additively manufactured Ti-6Al-4V implant which combines engineered porosity to facilitate bone ingrowth and surface functionalization via genistein, a phytoestrogen, to counteract bone loss in osteoporosis. SEM demonstrated new bone formation at the implant site, including in the internal implant pores. At the nanoscale, scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy confirmed the gradual nature of the bone-implant interface. By leveraging mesoscale analysis with PFIB-SEM tomography that captures large volumes of bone-implant interface with nearly nanoscale resolution, the presence of mineral ellipsoids varying in size and orientation was revealed. In addition, a well-developed lacuno-canalicular network and mineralization fronts directed both towards the implant and away from it were highlighted.

Quantitative atlas of collagen hydrogels reveals mesenchymal cancer cell traction adaptation to the matrix nanoarchitecture

Collagen-based hydrogels are commonly used in mechanobiology to mimic the extracellular matrix. A quantitative analysis of the influence of collagen concentration and properties on the structure and mechanics of the hydrogels is essential for tailored design adjustments for specific in vitro conditions. We combined focused ion beam scanning electron microscopy and rheology to provide a detailed quantitative atlas of the mechanical and nanoscale three-dimensional structural alterations that occur when manipulating different hydrogel's physicochemistry. Moreover, we study the effects of such alterations on the phenotype of breast cancer cells and their mechanical interactions with the extracellular matrix. Regardless of the microenvironment's pore size, porosity or mechanical properties, cancer cells are able to reach a stable mesenchymal-like morphology. Additionally, employing 3D traction force microscopy, a positive correlation between cellular tractions and ECM mechanics is observed up to a critical threshold, beyond which tractions plateau. This suggests that cancer cells in a stable mesenchymal state calibrate their mechanical interactions with the ECM to keep their migration and invasiveness capacities unaltered. Statement of significanceThe paper presents a thorough study on the mechanical microenvironment in breast cancer cells during their interaction with collagen based hydrogels of different compositions. The hydrogels’ microstructure were obtained using state-of-the-art 3D microscopy, namely focused ion beam-scanning electron microscope (FIB-SEM). FIB-SEM was originally applied in this work to reconstruct complex fibered collagen microstructures within the nanometer range, to obtain key microarchitectural parameters. The mechanical microenvironment of cells was recovered using Traction Force Microscopy (TFM). The obtained results suggest that cells calibrate tractions such that they depend on mechanical, microstructural and physicochemical characteristics of the hydrogels, hence revealing a steric hindrance. We hypothesize that cancer cells studied in this paper tune their mechanical state to keep their migration and invasiveness capacities unaltered.

Quantitative 3D electron microscopy characterization of mitochondrial structure, mitophagy, and organelle interactions in murine atrial fibrillation

Atrial fibrillation (AF) is the most common clinical arrhythmia, however there is limited understanding of its pathophysiology including the cellular and ultrastructural changes rendered by the irregular rhythm, which limits pharmacological therapy development. Prior work has demonstrated the importance of reactive oxygen species (ROS) and mitochondrial dysfunction in the development of AF. Mitochondrial structure, interactions with other organelles such as sarcoplasmic reticulum (SR) and T-tubules (TT), and degradation of dysfunctional mitochondria via mitophagy are important processes to understand ultrastructural changes due to AF. However, most analysis of mitochondrial structure and interactome in AF has been limited to two-dimensional (2D) modalities such as transmission electron microscopy (EM), which does not fully visualize the morphological evolution of the mitochondria during mitophagy. Herein, we utilize focused ion beam-scanning electron microscopy (FIB-SEM) and perform reconstruction of three-dimensional (3D) EM from murine left atrial samples and measure the interactions of mitochondria with SR and TT. We developed a novel 3D quantitative analysis of FIB-SEM in a murine model of AF to quantify mitophagy stage, mitophagosome size in cardiomyocytes, and mitochondrial structural remodeling when compared with control mice. We show that in our murine model of spontaneous and continuous AF due to persistent late sodium current, left atrial cardiomyocytes have heterogenous mitochondria, with a significant number which are enlarged with increased elongation and structural complexity. Mitophagosomes in AF cardiomyocytes are located at Z-lines where they neighbor large, elongated mitochondria. Mitochondria in AF cardiomyocytes show increased organelle interaction, with 5X greater contact area with SR and are 4X as likely to interact with TT when compared to control. We show that mitophagy in AF cardiomyocytes involves 2.5X larger mitophagosomes that carry increased organelle contents. In conclusion, when oxidative stress overcomes compensatory mechanisms, mitophagy in AF faces a challenge of degrading bulky complex mitochondria, which may result in increased SR and TT contacts, perhaps allowing for mitochondrial Ca2+ maintenance and antioxidant production.