Focused Ion Beam Scanning Electron Microscopy Research Articles

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

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.

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.

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 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.

Infection of Alfalfa Cotyledons by an Incompatible but Not a Compatible Species of Colletotrichum Induces Formation of Paramural Bodies and Secretion of EVs.

Hemibiotrophic fungi in the genus Colletotrichum employ a biotrophic phase to invade host epidermal cells followed by a necrotrophic phase to spread through neighboring mesophyll and epidermal cells. We used serial block face-scanning electron microscopy (SBF-SEM) to compare subcellular changes that occur in Medicago sativa (alfalfa) cotyledons during infection by Colletotrichum destructivum (compatible on M. sativa) and C. higginsianum (incompatible on M. sativa). Three-dimensional reconstruction of serial images revealed that alfalfa epidermal cells infected with C. destructivum undergo massive cytological changes during the first 60 h following inoculation to accommodate extensive intracellular hyphal growth. Conversely, inoculation with the incompatible species C. higginsianum resulted in no successful penetration events and frequent formation of papilla-like structures and cytoplasmic aggregates beneath attempted fungal penetration sites. Further analysis of the incompatible interaction using focused ion beam-scanning electron microscopy (FIB-SEM) revealed the formation of large multivesicular body-like structures that appeared spherical and were not visible in compatible interactions. These structures often fused with the host plasma membrane, giving rise to paramural bodies that appeared to be releasing extracellular vesicles (EVs). Isolation of EVs from the apoplastic space of alfalfa leaves at 60 h postinoculation showed significantly more vesicles secreted from alfalfa infected with incompatible fungus compared with compatible fungus, which in turn was more than produced by noninfected plants. Thus, the increased frequency of paramural bodies during incompatible interactions correlated with an increase in EV quantity in apoplastic wash fluids. Together, these results suggest that EVs and paramural bodies contribute to immunity during pathogen attack in alfalfa. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Anomalous temperature dependence mechanism of the electrochemical dissolution efficiency of iron-based materials

In tests of typical iron-based materials, it has been found that the dissolution efficiency at low temperature is greater than that at ambient temperature. By utilizing sputtering X-ray photoelectron spectroscopy (sputtering XPS) and focused ion beam scanning electron microscopy (FIB-SEM), it was found that this abnormal phenomenon is rooted in a thinner passive film and lower valence oxides formed on the surface at low temperature. Transmission electron microscopy (TEM) analysis supported these conclusions at the microscopic level. First-principles calculations further revealed that lower valence oxides of iron are more conductive than higher valence oxides.

Physicochemical investigation on the hard carbon interface in ionic liquid electrolyte

An investigation of the solid electrolyte interphase (SEI) on hard carbon (HC) anodes in aprotic sodium batteries was carried out by post-mortem analyses. Electrodes cycled in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide (EMIFSI), and N-trimethyl-N-butylammonium bis(fluorosulfonyl)imide (N1114FSI) ionic liquid electrolytes have been collected and studied: benchmark electrodes cycled in commercial organic solutions were examined for comparison purpose. The SEI composition and morphology of post-mortem HC electrodes were analyzed by X-ray Photoelectron Spectroscopy (XPS) and focused ion beam scanning electron microscopy (FIB-SEM) analysis. Overall, the HC electrodes cycled in carbonate-based electrolyte have shown thicker SEI mainly composed of organic compounds, whereas in ILs electrolytes they have shown thinner layer richer in inorganic species, such as NaF, Na2CO3, N-containing species, and “small” sulfide-based compounds, these improving the SEI interfacial properties towards the Na+ migration kinetics. Focusing on ILs based cell formulations, anodes cycled in the EMIFSI-based electrolyte shows several cracks on its surface.

Identification of solid phase speciation of trace elements in dust dry deposition using focused ion beam and selected area electron diffraction

BackgroundFew studies have been conducted to identify solid phase speciation of trace elements within the interior of airborne particles, although the adverse effects of ambient particles are closely related to the speciation of toxic elements and therefore the identification of chemical binding sites and solid phase associations is essential for assessing the impacts of trace elements in ambient air on environmental quality and human health. ObjectiveA combined use of focused ion beam scanning electron microscopy (FIB-SEM) and selected area electron diffraction (SAED) patterns from transmission electron microscopy (TEM) was applied with an energy dispersive X-ray spectroscopy (EDS) to investigate the solid phase speciation of trace elements within the interior of dust dry deposition obtained from an urban area. ResultsThe study results using the high-resolution microscopic techniques for the characterization of nano- and micron-sized particles revealed lead chromate for Pb and Cr6+, barite for Ba, rutile for Ti, and calcite, gypsum, and quicklime for Ca within the carbon matrix, implying their traffic-related sources. Fe oxides were observed for Fe, Mn, Ni, and Cr3+, with cerianite and Fe-La alloys for rare earth elements (REEs). ConclusionsThree types of anthropogenic sources were inferred based on the morphology and texture of the solid phases, as well as their primary usage: traffic road marking paints for Pb, Cr6+, Ba, and Ti, cement materials for Ca, and industrial activities for Fe, Mn, Ni, Cr3+, and REEs. The presence of submicron-sized toxic metal-bearing particles in dust dry deposition indicated that the toxic metals (i.e., Pb, Cr6+) can penetrate into the respiratory system along with the nanoparticles. The study results demonstrate that the solid phase speciation of trace elements detected using FIB-SEM and TEM-SAED can be effectively utilized for the identification of sources and the assessment of chemical toxicity of atmospheric dust.

Editors’ Choice—Visualizing the Impact of the Composite Cathode Microstructure and Porosity on Solid-State Battery Performance

The kinetics of composite cathodes for solid-state batteries (SSBs) relies heavily on their microstructure. Spatial distribution of the different phases, porosity, interface areas, and tortuosity factors are important descriptors that need accurate quantification for models to predict the electrochemistry and mechanics of SSBs. In this study, high-resolution focused ion beam-scanning electron microscopy tomography was used to investigate the microstructure of cathodes composed of a nickel-rich cathode active material (NCM) and a thiophosphate-based inorganic solid electrolyte (ISE). The influence of the ISE particle size on the microstructure of the cathode was visualized by 3D reconstruction and charge transport simulation. By comparison of experimentally determined and simulated conductivities of composite cathodes with different ISE particle sizes, the electrode charge transport kinetics is evaluated. Porosity is shown to have a major influence on the cell kinetics and the evaluation of the active mass of electrochemically active particles reveals a higher fraction of connected NCM particles in electrode composites utilizing smaller ISE particles. The results highlight the importance of homogeneous and optimized microstructures for high performance SSBs, securing fast ion and electron transport.

Quantifying Microstructure Features for High-Performance Solid Oxide Cells.

The drive for sustainable energy solutions has spurred interest in solid oxide fuel cells (SOFCs). This study investigates the impact of sintering temperature on SOFC anode microstructures using advanced 3D focused ion beam-scanning electron microscopy (FIB-SEM). The anode's ceramic-metal composition significantly influences electrochemical performance, making optimization crucial. Comparing cells sintered at different temperatures reveals that a lower sintering temperature enhances yttria-stabilized zirconia (YSZ) and nickel distribution, volume, and particle size, along with the triple-phase boundary (TPB) interface. Three-dimensional reconstructions illustrate that the cell sintered at a lower temperature exhibits a well-defined pore network, leading to increased TPB density. Hydrogen flow simulations demonstrate comparable permeability for both cells. Electrochemical characterization confirms the superior performance of the cell sintered at the lower temperature, displaying higher power density and lower total cell resistance. This FIB-SEM methodology provides precise insights into the microstructure-performance relationship, eliminating the need for hypothetical structures and enhancing our understanding of SOFC behavior under different fabrication conditions.

The physical and cellular mechanism of structural color change in zebrafish

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20° tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

Comparative investigation on the tribocorrosion resistance of Ti6Al4V and 60NiTi alloys inimulated seawater environment

The Ti6Al4V alloy is extensively utilized in critical marine equipment components due to its low density, high specific strength, and exceptional corrosion resistance in marine environments. However, its low hardness and inadequate wear resistance pose challenges in meeting the urgent demand for prolonged service life of relative motion friction pairs under complex wear and corrosion conditions. As a newly developed lightweight friction pair material, hardened 60NiTi is considered highly desirable owing to its combination of high hardness, high compressive strength, and excellent corrosion resistance. Nevertheless, the tribocorrosion properties of 60NiTi compared to those of Ti6Al4V are not yet fully comprehended. The present study conducted a series of sliding wear tests using a ball-on-plate configuration in artificial seawater environment to compare the response of 60NiTi with Ti6Al4V. In order to gain a comprehensive understanding of the factors contributing to the divergence in tribocorrosion response between both materials, further analysis was performed on the wear track subsurface and transfer film using focused ion beam-scanning electron microscopy, Raman spectroscopy, and x-ray photoelectron spectroscopy respectively. The 60NiTi demonstrates significantly enhanced wear resistance in artificial seawater compared to Ti6Al4V, attributed to its combination of high hardness and excellent corrosion resistance. However, Ti6Al4V exhibits a lower friction coefficient (∼0.30) than 60NiTi (∼0.45) due to the formation of a stable transfer film primarily composed of SiO2, Al(OH)3, and Al2O3.

Ultrastructural 3D Microscopy for Biomedicine: Principles, Applications, and Perspectives.

Modern biomedical research often requires a three-dimensional microscopic analysis of the ultrastructure of biological objects and materials. Conceptual technical and methodological solutions for three-dimensional structure reconstruction are needed to improve the conventional optical, electron, and probe microscopy methods, which to begin with allow one to obtain two-dimensional images and data. This review discusses the principles and potential applications of such techniques as serial section transmission electron microscopy; techniques based on scanning electron microscopy (SEM) (array tomography, focused ion beam SEM, and serial block-face SEM). 3D analysis techniques based on modern super-resolution optical microscopy methods are described (stochastic optical reconstruction microscopy and stimulated emission depletion microscopy), as well as ultrastructural 3D microscopy methods based on scanning probe microscopy and the feasibility of combining them with optical techniques. A comparative analysis of the advantages and shortcomings of the discussed approaches is performed.

Volume microscopic analysis of membrane contact sites in mouse kidney renal proximal tubule epithelial cells

Subcellular organelles communicate with one another and with the plasma membrane (PM). A wide variety of membrane contact sites (MCS) tether subcellular structures to one another and mediate inter-organelle communication and exchange. The nature, distribution and function of membrane contact sites within the segment-specific architecture of renal epithelial cells has only just begun to be elucidated. We have used advanced imaging techniques, including Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) and machine learning-based segmentation, to create a comprehensive, high resolution, 3D and quantifiable volume microscopic atlas of the ultrastructure of proximal tubule epithelial cells of the mouse kidney. We applied this data set to identify and quantify MCS between the endoplasmic reticulum and mitochondria and the PM in these cells. FIB-SEM was performed on a 50 μm by 50 μm by 44 μm block of tissue prepared from the renal cortex of a perfusion fixed 12-week-old male mouse. OsO4-stained tissue was embedded in Durcupan and a 3D X-ray tomogram of the block was used to select a volume of interest that included extended lengths of multiple proximal tubule (PT). The volume was imaged by a customized enhanced FIB-SEM system. The X-Y scanning resolution was set to 4-nm, and 4-nm FIB milling was used to expose surfaces for successive imaging. Generation of the complete image stack required ~11,000 cycles of scanning/FIB ablation over ~4 weeks of uninterrupted imaging. Segmentation of the resultant image set was performed with Zeiss Arivis Pro technology. The boundary box for reconstruction had the dimensions of 29.2 μm x 24.8 μm x 8 μm with a volume of 5793.280 μm3 comprising of 2000 consecutive images at an isotropic voxel size of 4 nm. Deep learning segmentation teased apart 10 cells with distinct boundaries in the PT structure. The volume of the cells in our clipped volume ranged from 8.79 μm3 to 1438.53 μm3 with a median of 45.20 μm3. In one representative proximal tubule cell the volume of mitochondria makes up 84.91 μm3 (31.62%) of the cell volume and the volume of Endoplasmic Reticulum (ER) makes up 23.92 μm3 (8.91%) of the cell volume. The 3D reconstruction and quantitative analysis revealed that there is one dominant ER object accounting for most of the ER volume in the whole cell: i.e., the largest ER object takes up 22.37 μm3 or 93.5% of the ER volume in the cell. A post deep-learning processing algorithm used 3D object modifications to identify 11,109 3D MCS between mitochondria and ER in the 10 cells. The Mito-ER MCS ranged from 512 nm3 to 354,304 nm3 with a median volume of 1792 nm3. Visually we can see Mito-ER and Mito-PM MCS extending across a region of up to ~45 nm between the two organelles. Taken together, these data show that the basal lateral surfaces of PT cell PMs are generously invested with fenestrated smooth ER, as are mitochondria and peroxisomes. Extensive regions of direct contact between mitochondria and PM are also observed. The biochemical identities of these contacts and their physiological functions are being actively pursued. NIDDK DK072612 NIDDK DK120534. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.