Unraveling the Impact of Boron Nitride and Silicon Nitride Nanoparticles on Thermoplastic Polyurethane Fibers and Mats for Advanced Heat Management.

The urgent challenges posed by the energy crisis, alongside the heat dissipation of advanced electronics, have embarked on a rising demand for the development of highly thermally conductive polymer composites. Electrospun composite mats, known for their flexibility, permeability, high concentration and orientational degree of conductive fillers, stand out as one of the prime candidates for addressing this need. This study explores the efficacy of boron nitride (BN) and its potential alternative, silicon nitride (SiN) nanoparticles, in enhancing the thermal performance of the electrospun composite thermoplastic polyurethane (TPU) fibers and mats. The 3D reconstructed models obtained from FIB-SEM imaging provided valuable insights into the morphology of the composite fibers, aiding the interpretation of the measured thermal performance through scanning thermal microscopy for the individual composite fibers and infrared thermography for the composite mats. Notably, we found that TPU-SiN fibers exhibit superior heat conduction compared to TPU-BN fibers, with up to a 6 °C higher surface temperature observed in mats coated on copper pipes. Our results underscore the crucial role of arrangement of nanoparticles and fiber morphology in improving heat conduction in the electrospun composites. Moreover, SiN nanoparticles are introduced as a more suitable filler for heat conduction enhancement of electrospun TPU fibers and mats, suggesting immense potential for smart textiles and thermal management applications.

Ingenious Architecture and Coloration Generation in Enamel of Rodent Teeth.

Teeth exemplify architectures comprising an interplay of inorganic and organic constituents, resulting in sophisticated natural composites. Rodents (Rodentia) showcase extraordinary adaptations, with their continuously growing incisors surpassing human teeth in functional and structural optimizations. In this study, employing state-of-the-art direct atomic-scale imaging and nanoscale spectroscopies, we present compelling evidence that the release of material from ameloblasts and the subsequent formation of iron-rich enamel and surface layers in the constantly growing incisors of rodents are complex orchestrated processes, intricately regulated and independent of environmental factors. The synergistic fusion of three-dimensional tomography and imaging techniques of etched rodent́s enamel unveils a direct correlation between the presence of pockets infused with ferrihydrite-like material and the acid resistant properties exhibited by the iron-rich enamel, fortifying it as an efficient protective shield. Moreover, observations using optical microscopy shed light on the role of iron-rich enamel as a microstructural element that acts as a path for color transmission, although the native color remains indistinguishable from that of regular enamel, challenging the prevailing paradigms. The redefinition of "pigmented enamel" to encompass ferrihydrite-like infusion in rodent incisors reshapes our perception of incisor microstructure and color generation. The functional significance of acid-resistant iron-rich enamel and the understanding of the underlying coloration mechanism in rodent incisors have far-reaching implications for human health, development of potentially groundbreaking dental materials, and restorative dentistry. These findings enable the creation of an entirely different class of dental biomaterials with enhanced properties, inspired by the ingenious designs found in nature.

The Potential of Subsampling and Inpainting for Fast Low-Dose Cryo FIB-SEM Imaging.

Traditional image acquisition for cryo focused ion-beam scanning electron microscopy (FIB-SEM) tomography often sees thousands of images being captured over a period of many hours, with immense data sets being produced. When imaging beam sensitive materials, these images are often compromised by additional constraints related to beam damage and the devitrification of the material during imaging, which renders data acquisition both costly and unreliable. Subsampling and inpainting are proposed as solutions for both of these aspects, allowing fast and low-dose imaging to take place in the Focused ion-beam scanning electron microscopy FIB-SEM without an appreciable loss in image quality. In this work, experimental data are presented which validate subsampling and inpainting as a useful tool for convenient and reliable data acquisition in a FIB-SEM, with new methods of handling three-dimensional data being employed in the context of dictionary learning and inpainting algorithms using a newly developed microscope control software and data recovery algorithm.

Advances in Focused Ion Beam Tomography for Three-Dimensional Characterization in Materials Science.

Over the years, FIB-SEM tomography has become an extremely important technique for the three-dimensional reconstruction of microscopic structures with nanometric resolution. This paper describes in detail the steps required to perform this analysis, from the experimental setup to the data analysis and final reconstruction. To demonstrate the versatility of the technique, a comprehensive list of applications is also summarized, ranging from batteries to shale rocks and even some types of soft materials. Moreover, the continuous technological development, such as the introduction of the latest models of plasma and cryo-FIB, can open the way towards the analysis with this technique of a large class of soft materials, while the introduction of new machine learning and deep learning systems will not only improve the resolution and the quality of the final data, but also expand the degree of automation and efficiency in the dataset handling. These future developments, combined with a technique that is already reliable and widely used in various fields of research, are certain to become a routine tool in electron microscopy and material characterization.

The Impact of Intergrain Phases on the Ionic Conductivity of the LAGP Solid Electrolyte Material Prepared by Spark Plasma Sintering.

Li1.5Al0.5Ge1.5(PO4)3 (LAGP) is a promising oxide solid electrolyte for all-solid-state batteries due to its excellent air stability, acceptable electrochemical stability window, and cost-effective precursor materials. However, further improvement in the ionic conductivity performance of oxide solid-state electrolytes is hindered by the presence of grain boundaries and their associated morphologies and composition. These key factors thus represent a major obstacle to the improved design of modern oxide based solid-state electrolytes. This study establishes a correlation between the influence of the grain boundary phases, their 3D morphology, and compositions formed under different sintering conditions on the overall LAGP ionic conductivity. Spark plasma sintering has been employed to sinter oxide solid electrolyte material at different temperatures with high compacity values, whereas a combined potentiostatic electrochemical impedance spectroscopy, 3D FIB-SEM tomography, XRD, and solid-state NMR/materials modeling approach provides an in-depth analysis of the influence of the morphology, structure, and composition of the grain boundary phases that impact the total ionic conductivity. This work establishes the first 3D FIB-SEM tomography analysis of the LAGP morphology and the secondary phases formed in the grain boundaries at the nanoscale level, whereas the associated 31P and 27Al MAS NMR study coupled with materials modeling reveals that the grain boundary material is composed of Li4P2O7 and disordered Li9Al3(P2O7)3(PO4)2 phases. Quantitative 31P MAS NMR measurements demonstrate that optimal ionic conductivity for the LAGP system is achieved for the 680 °C SPS preparation when the disordered Li9Al3(P2O7)3(PO4)2 phase dominates the grain boundary composition with reduced contributions from the highly ordered Li4P2O7 phases, whereas the 27Al MAS NMR data reveal that minimal structural change is experienced by each phase throughout this suite of sintering temperatures.

Quantitative FIB/SEM tomogram analysis of closed and open porosity of spheroidized graphite anode materials for LiBs applications.

The electrochemical behaviour of rounded graphite particles as anode material in a lithium-ion battery strongly depends on the particle properties. The spheroidization process directly affects these properties, including the open porosity that determines the extent of direct contact between liquid electrolyte and carbon surface. Therefore, the quantification of the proportion between open and closed pores is of great interest. Here, we quantify the open and closed porosity of spheroidized porous graphite particles from FIB-SEM tomograms. Quantification is achieved based on two developments: (1) a new sample preparation strategy and (2) a newly developed image evaluation scheme based on neural networks. The sample preparation strategy involves embedding of many graphite powder particles in indium enabling the investigation of several graphite particles in one FIB/SEM tomogram with high stability and with high contrast between the conductive embedding material and porous graphite. A quantitative evaluation of closed and open porosity is achieved by machine learning in form of convolutional neural networks. The convolutional neural network is used to detect the bulk graphite and by further morphological operations, closed and open pores are identified. An error is determined by comparing automatically created quantifications with manual reference values. Our porosity data for two differently spheroidized graphite samples agree qualitatively well with corresponding results from nitrogen physisorption measurements. This approach may allow quantitative data evaluation from porous powders and support understanding of the correlation to the electrochemical behaviour in the lithium-ion battery.

Structure-preserving Gaussian denoising of FIB-SEM volumes.

FIB-SEM (Focused Ion Beam-Scanning Electron Microscopy) is an imaging technique that allows 3D ultrastructural analysis of cells and tissues at the nanoscale. The acquired FIB-SEM data are highly noisy, which makes denoising an essential step prior to volume interpretation. Gaussian filtering is a standard method in the field because it is fast and straightforward. However, it tends to blur the biological features due to its linear nature that ignores the rapid changes of the structures throughout the volume. To address this issue, we have developed a new approach to structure-preserving noise reduction for FIB-SEM. It has abilities to locally adapt the filtering to the biological structures while taking advantage of the simplicity of Gaussian filtering. It uses the Optical Flow (OF) to estimate the variations of the structural features across the volume, so that they are compensated before the subsequent filtering with a Gaussian function. As demonstrated qualitatively and objectively with datasets from different samples and acquired under different conditions, our denoising approach outperforms the standard Gaussian filtering and is competitive with state-of-the-art methods in terms of noise reduction and preservation of the sharpness of the structures.

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.

Changes in Envelope Structure and Cell-Cell Communication during Akinete Differentiation and Germination in Filamentous Cyanobacterium Trichormus variabilis ATCC 29413.

Planktonic freshwater filamentous cyanobacterium Trichormus variabilis ATCC 29413 (previously known as Anabaena variabilis) can differentiate heterocysts and akinetes to survive under different stress conditions. Whilst heterocysts enable diazotrophic growth, akinetes are spore-like resting cells that make the survival of the species possible under adverse growth conditions. Under suitable environmental conditions, they germinate to produce new vegetative filaments. Several morphological and physiological changes occur during akinete formation and germination. Here, using scanning electron microscopy (SEM), we found that the mature akinetes had a wrinkled envelope, and the surface of the envelope smoothened as the cell size increased during germination. Thereupon, the akinete envelope ruptured to release the short emerging filament. Focused ion beam-scanning electron microscopy (FIB/SEM) tomography of immature akinetes revealed the presence of cytoplasmic granules, presumably consisting of cyanophycin or glycogen. In addition, the akinete envelope architecture of different layers, the exopolysaccharide and glycolipid layers, could be visualized. We found that this multilayered envelope helped to withstand osmotic stress and to maintain the structural integrity. Furthermore, by fluorescence recovery after photobleaching (FRAP) measurements, using the fluorescent tracer calcein, we found that intercellular communication decreased during akinete formation as compared with the vegetative cells. In contrast, freshly germinating filaments restored cell communication.

Three-dimensional architecture of Cyrilia lignieresi gametocyte-stage development inside red blood cells.

The Haemogregarinidae family (Apicomplexa: Adeleina) comprises hemoprotozoa that infect mammals, birds, amphibians, fish, and reptiles. Some morphological characteristics of the Cyrilia lignieresi have been described previously, but the parasite-erythrocyte relationship is still poorly understood. In order to understand the structural architecture of C. lignieresi-infected red blood cells, electron microscopy-based three-dimensional reconstruction was carried out using TEM as well as FIB-SEM tomography. Results showed that development of the macrogametocyte-stage inside the red blood cell is related to an increase in cleft-like structures in the host cell cytoplasm. Furthermore, other aspects related to parasite intraerythrocytic development were explored by 3D visualization techniques. We observed the invagination of a large extension of the Inner Membrane Complex (IMC) on the parasite body, which results from or induces a folding of the posterior end of the parasite. Small tubular structures were seen associated with areas related to IMC folding. Taken together, results provide new information on the remodeling of erythrocytes induced by the protozoan C. lignieresi.

Mineral tessellation in bone and the stenciling principle for extracellular matrix mineralization.

We review here the Stenciling Principle for extracellular matrix mineralization that describes a double-negative process (inhibition of inhibitors) that promotes mineralization in bone and other mineralized tissues, whereas the default condition of inhibition alone prevents mineralization elsewhere in soft connective tissues. The stenciling principle acts across multiple levels from the macroscale (skeleton/dentition vs soft connective tissues), to the microscale (for example, entheses, and the tooth attachment complex where the soft periodontal ligament is situated between mineralized tooth cementum and mineralized alveolar bone), and to the mesoscale (mineral tessellation). It relates to both small-molecule (e.g. pyrophosphate) and protein (e.g. osteopontin) inhibitors of mineralization, and promoters (enzymes, e.g. TNAP, PHEX) that degrade the inhibitors to permit and regulate mineralization. In this process, an organizational motif for bone mineral arises that we call crossfibrillar mineral tessellation where mineral formations - called tesselles - geometrically approximate prolate ellipsoids and traverse multiple collagen fibrils (laterally). Tesselle growth is directed by the structural anisotropy of collagen, being spatially restrained in the shorter transverse tesselle dimensions (averaging 1.6 × 0.8 × 0.8 µm, aspect ratio 2, length range 1.5-2.5 µm). Temporo-spatially, the tesselles abut in 3D (close ellipsoid packing) to fill the volume of lamellar bone extracellular matrix. Poorly mineralized interfacial gaps between adjacent tesselles remain discernable even in mature lamellar bone. Tessellation of a same, small basic unit to form larger structural assemblies results in numerous 3D interfaces, allows dissipation of critical stresses, and enables fail-safe cyclic deformations. Incomplete tessellation in osteomalacia/odontomalacia may explain why soft osteomalacic bones buckle and deform under loading.

Multiscale Characterization of an Oxide Scale Formed on the Creep-Resistant ATI 718Plus Superalloy during High-Temperature Oxidation.

The ATI 718Plus® is a creep-resistant nickel-based superalloy exhibiting high strength and excellent oxidation resistance in high temperatures. The present study is focused on multiscale 2D and 3D characterization (morphological and chemical) of the scale and the layer beneath formed on the ATI 718Plus superalloy during oxidation at 850 °C up to 4000 h in dry and wet air. The oxidized samples were characterized using various microscopic methods (SEM, TEM and STEM), energy-dispersive X-ray spectroscopy and electron diffraction. The 3D visualization of the microstructural features was achieved by means of FIB-SEM tomography. When oxidized in dry air, the ATI 718Plus develops a protective, dense Cr2O3 scale with a dual-layered structure. The outer Cr2O3 layer is composed of coarser grains with a columnar shape, while the inner one features fine, equiaxed grains. The Cr2O3 scale formed in wet air is single-layered and features very fine grains. The article discusses the difference between the structure, chemistry and three-dimensional phase distribution of the oxide scales and near-surface areas developed in the two environments. Electron microscopy/spectroscopy findings combined with the three-dimensional reconstruction of the microstructure provide original insight into the role of the oxidation environment on the structure of the ATI 718Plus at the nanoscale.

Highly Selective Cu Staining of Sulfur-Containing Polymers Facilitates 3D Nanomorphology Reconstruction of Polymer:Fullerene Blends in Organic Solar Cells by FIB-SEM Tomography.

The distinction of different organic materials in phase mixtures is hampered in electron microscopy because electron scattering does not strongly differ in carbon-based materials that mainly consist of light elements. A successful strategy for contrast enhancement is selective staining where one phase of a material mixture is labeled by heavier elements, but suitable staining agents are not available for all organic materials. This is also the case for bulk-heterojunction (BHJ) absorber layers of organic solar cells, which consist of interpenetrating networks of donor and acceptor domains. The domain structure strongly influences the power conversion efficiency, and nanomorphology optimization often requires real-space information on the sizes and interconnectivity of domains with nanometer resolution. In this work, we have developed an efficient approach to selectively stain sulfur-containing polymers by homogeneous Cu infiltration, which generates strong material contrast in scanning (transmission) electron microscopy (S(T)EM) images of polymer:fullerene BHJ layers. Cross-section lamellae of BHJ layers are prepared for STEM by focused-ion-beam milling and are attached to a Cu lift-out grid as a copper source. After thermal treatment at 200 °C for 3 h in air, sulfur-containing polymers are homogeneously infiltrated by Cu, while the fullerenes are not affected. Selective Cu staining is applied to map the phase distribution in PTB7:PC71BM BHJ layers fabricated with different processing additives to tailor the nanomorphology. The strong contrast between polymer and fullerene domains is the prerequisite for the three-dimensional reconstruction of the domain structure by focused-ion-beam/scanning-electron-microscopy tomography.

Comparison of Structural Properties Between Postnatal and Adult Tendon Insertion with FIB/SEM Tomography in Rat.

OBJECTIVE: The repaired tendon-bone interface after rotator cuff (RC) repair has been identified as a mechanical weak point, which may contribute to re-tearing. Analyzing the postnatal development of a normal tendon insertion in detail may be useful in helping to promote the regeneration of a normal tendon insertion. We verified the morphological differences between postnatal and adult tendon insertions in terms of the cellular structural properties using FIB/SEM tomography. MATERIALS AND METHOD: SPostnatal and adult Sprague-Dawley rats were used as a model of tendon insertion. The morphological structure of the insertion was evaluated using hematoxylin and eosin (HE) staining, and the 3D ultrastructure of the cells in the insertion was evaluated using FIB/SEM tomography. Additionally, the volume of the cell bodies, nuclei, and cytoplasm were measured and compared in a quantitative analysis. RESULTS: On conventional histology, the boundary line between the fibrocartilage and mineralized cartilage was flat in the adult insertions; however, the boundary line between the mineralized cartilage and bone formed deep interdigitations. The morphology of the cells among the collagen bundles in the adult insertions was completely different from those in the postnatal insertions at the 3D ultrastructural level. The cellular structural properties were statistically different between the postnatal and adult insertions. CONCLUSIONS: In the present study, the morphological differences between postnatal and adult tendon insertion in terms of the ultrastructural cellular properties were clarified. These findings may aid in determining how to regenerate a clinically stable tendon insertion at the tendon-bone interface after RC repair.

Accurate post-mortem alignment for Focused Ion Beam and Scanning Electron Microscopy (FIB-SEM) tomography.

Drifts in the three directions (X, Y, Z) during the FIB-SEM slice-and-view tomography is an important issue in 3D-FIB experiments which may induce significant inaccuracies in the subsequent volume reconstruction and further quantification of morphological volume parameters of the sample microstructure. Cross-correlation is frequently applied directly to the cross-section image series for aligning FIB sliced images. This solution is hazardous and can be flawed as it has been easily demonstrated by a dedicated test experiment. As a result, a novel aligning procedure based on the quantification of the topography of the sample surface has been developed. This new approach will be compared to the common cross-correlation methods, as well as another approach consisting in using an artificial reference marker fabricated during the FIB procedure. All these methods will then be discussed in terms of accuracy and liability.

3D Interrelationship between Osteocyte Network and Forming Mineral during Human Bone Remodeling.

During bone remodeling, osteoblasts are known to deposit unmineralized collagenous tissue (osteoid), which mineralizes after some time lag. Some of the osteoblasts differentiate into osteocytes, forming a cell network within the lacunocanalicular network (LCN) of bone. To get more insight into the potential role of osteocytes in the mineralization process of osteoid, sites of bone formation are three-dimensionally imaged in nine forming human osteons using focused ion beam-scanning electron microscopy (FIB-SEM). In agreement with previous observations, the mineral concentration is found to gradually increase from the central Haversian canal toward pre-existing mineralized bone. Most interestingly, a similar feature is discovered on a length scale more than 100-times smaller, whereby mineral concentration increases from the LCN, leaving around the canaliculi a zone virtually free of mineral, the size of which decreases with progressing mineralization. This suggests that the LCN controls mineral formation but not just by diffusion of mineralization precursors, which would lead to a continuous decrease of mineral concentration from the LCN. The observation is, however, compatible with the codiffusion and reaction of precursors and inhibitors from the LCN into the bone matrix.

Three-Dimensional Architecture of Glomerular Endothelial Cells Revealed by FIB-SEM Tomography.

Focused-ion beam-scanning electron microscopic (FIB-SEM) tomography enables easier acquisition of a series of ultrastructural, sectional images directly from resin-embedded biological samples. In this study, to clarify the three-dimensional (3D) architecture of glomerular endothelial cells (GEnCs) in adult rats, we manually extracted GEnCs from serial FIB-SEM images and reconstructed them on an Amira reconstruction software. The luminal and basal surface structures were clearly visualized in the reconstructed GEnCs, although only the luminal surface structures could be observed by conventional SEM. The luminal surface visualized via the reconstructed GEnCs was quite similar to that observed through conventional SEM, indicating that 3D reconstruction could be performed with high accuracy. Thus, we successfully described the 3D architecture of normal GEnCs in adult rats more clearly and precisely than ever before. The GEnCs were found to consist of three major subcellular compartments, namely, the cell body, cytoplasmic ridges, and sieve plates, in addition to two associated subcellular compartments, namely, the globular protrusions and reticular porous structures. Furthermore, most individual GEnCs made up a "seamless" tubular shape, and some of them formed an autocellular junction to make up a tubular shape. FIB-SEM tomography with reconstruction is a powerful approach to better understand the 3D architecture of GEnCs. Moreover, the morphological information revealed in this study will be valuable for the 3D pathologic evaluation of GEnCs in animal and human glomerular diseases and the structural analysis of developmental processes in the glomerular capillary system.

Advanced cryo-tomography workflow developments - correlative microscopy, milling automation and cryo-lift-out.

Cryo-electron tomography (cryo-ET) is a groundbreaking technology for 3D visualisation and analysis of biomolecules in the context of cellular structures. It allows structural investigations of single proteins as well as their spatial arrangements within the cell. Cryo-tomograms provide a snapshot of the complex, heterogeneous and transient subcellular environment. Due to the excellent structure preservation in amorphous ice, it is possible to study interactions and spatial relationships of proteins in their native state without interference caused by chemical fixatives or contrasting agents. With the introduction of focused ion beam (FIB) technology, the preparation of cellular samples for electron tomography has become much easier and faster. The latest generation of integrated FIB and scanning electron microscopy (SEM) instruments (dual beam microscopes), specifically designed for cryo-applications, provides advances in automation, imaging and the preparation of high-pressure frozen bulk samples using cryo-lift-out technology. In addition, correlative cryo-fluorescence microscopy provides cellular targeting information through integrated software and hardware interfaces. The rapid advances, based on the combination of correlative cryo-microscopy, cryo-FIB and cryo-ET, have already led to a wealth of new insights into cellular processes and provided new 3D image data of the cell. Here we introduce our recent developments within the cryo-tomography workflow, and we discuss the challenges that lie ahead. LAY DESCRIPTION: This article describes our recent developments for the cryo-electron tomography (cryo-ET) workflow. Cryo-ET offers superior structural preservation and provides 3D snapshots of the interior of vitrified cells at molecular resolution. Before a cellular sample can be imaged by cryo-ET, it must be made accessible for transmission electron microscopy. This is achieved by preparing a 200-300 nm thin cryo-lamella from the cellular sample using a cryo-focused ion beam (cryo-FIB) microscope. Cryo-correlative light and electron microscopy (cryo-CLEM) is used within the workflow to guide the cryo-lamella preparation to the cellular areas of interest. We cover a basic introduction of the cryo-ET workflow and show new developments for cryo-CLEM, which facilitate the connection between the cryo-light microscope and the cryo-FIB. Next, we present our progress in cryo-FIB software automation to streamline cryo-lamella preparation. In the final section we demonstrate how the cryo-FIB can be used for 3D imaging and how bulk-frozen cellular samples (obtained by high-pressure freezing) can be processed using the newly developed cryo-lift-out technology.

FIB-SEM-based analysis of Borrelia intracellular processing by human macrophages.

Borrelia burgdorferi is the causative agent of Lyme disease, a multisystemic disorder affecting primarily skin, joints and nervous system. Successful internalization and intracellular processing of borreliae by immune cells, like macrophages, is decisive for the outcome of a respective infection. Here, we use, for the first time, focused ion beam scanning electron microscopy tomography (FIB-SEM tomography) to visualize the interaction of borreliae with primary human macrophages with high resolution. We report that interaction between macrophages and the elongated and highly motile borreliae can lead to formation of membrane tunnels that extend deeper into the host cytoplasm than the actual phagosome, most probably as a result of partial extrication of captured borreliae. We also show that membrane tubulation at borreliae-containing phagosomes, a process suggested earlier as a mechanism leading to phagosome compaction but hard to visualize in live-cell imaging, is apparently a frequent phenomenon. Finally, we demonstrate that the endoplasmic reticulum (ER) forms multiple STIM1-positive contact sites with both membrane tunnels and phagosome tubulations, confirming the important role of the ER during uptake and intracellular processing of borreliae.

3D microstructural study of selachimorph enameloid evolution.

Enameloid, the hyper-mineralized tissue covering shark teeth is a complex structure resulting from both ameloblast and odontoblast activity. The way these two types of cells interact to set up this tissue is not fully understood and results in the formation of subunits in the enameloid: the Single Crystallite Enameloid (SCE) and the Bundled Crystallite Enameloid (BCE). Using the Focused Ion Beam Nanotomography (FIB-nt), 3D images were produced to assess the relationship between the SCE and BCE of one fossil and one recent neoselachian shark teeth. 3D analysis of crystallite bundles reveals a strong connection between the crystallites forming the SCE and those forming the bundles of the Radial Bundle Enameloid (RBE), a component of the BCE, although it has been suggested that SCE and BCE have a different origin: epithelial for the SCE and mesenchymal for the BCE. Another significant result of the use of FIB-nt is the visualization of frequent branching among the radial bundles forming the RBE, including horizontal link between adjacent bundles. FIB-nt demonstrates therefore a strong potential to decipher the complex evolution of hyper-mineralised tissue in shark teeth, and, therefore, to better understand the evolution of tooth structure among basal Gnathostomes.