3D Analysis of Ordered Porous Polymeric Particles using Complementary Electron Microscopy Methods

Mechanical serial block face scanning electron microscopy (SBFSEM) has emerged as a means of obtaining three dimensional (3D) electron images over volumes much larger than possible by focused ion beam (FIB) serial sectioning and at higher spatial resolution than achievable with conventional X-ray computed tomography (CT). Such high resolution 3D electron images can be employed for precisely determining the shape, volume fraction, distribution and connectivity of important microstructural features. While soft (fixed or frozen) biological samples are particularly well suited for nanoscale sectioning using an ultramicrotome, the technique can also produce excellent 3D images at electron microscope resolution in a time and resource-efficient manner for engineering materials. Currently, a lack of appreciation of the capabilities of ultramicrotomy and the operational challenges associated with minimising artefacts for different materials is limiting its wider application to engineering materials. Consequently, this paper outlines the current state of the art for SBFSEM examining in detail how damage is introduced during slicing and highlighting strategies for minimising such damage. A particular focus of the study is the acquisition of 3D images for a variety of metallic and coated systems.

Extract Extended abstract of a paper presented at Microscopy and Microanalysis 2007 in Ft. Lauderdale, Florida, USA, August 5 – August 9, 2007

Microglia are non-neuronal cells of the myeloid lineage that invade and take up long-term residence in the brain during development (Ginhoux et al. 2010) and are increasingly implicated in neuronal maturation, homeostasis, and pathology (Bessis et al. 2007; Paolicelli et al. 2011; Li et al. 2012; Aguzzi et al. 2013, Cunningham 2013, Cunningham et al. 2013). Since the early twentieth century several methods for staining and visualizing microglia have been developed. Scientists in Ramon y Cajal's group (Achucarro 1913, Rio-Hortega 1919) pioneered these methods and their work led to the christening of microglia as the third element of the nervous system, distinct from astrocytes and neurons. More recently, a combination of imaging, genetic, and immunological tools has been used to visualize microglia in living brain (Davalos et al. 2005; Nimmerjahn et al. 2005). It was found that microglia are highly motile under resting conditions and rapidly respond to injuries (Kettenmann et al. 2011) suggesting a role for microglia in both brain homeostasis and pathology. Transmission Electron microscopy (TEM) has provided crucial complementary information on microglia morphology and physiology but until recently EM analyses have been limited to single or limited serial section studies (Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012; Tremblay et al. 2012 ; Sipe et al. 2016). TEM studies were successful in defining a set of morphological criteria for microglia: a polygonal nucleus with peripheral condensed chromatin, a relatively small cytoplasm with abundant presence of rough endoplasmic reticulum (RER), and a large volume of lysosomes and inclusions in the perikaryon. Recent advances in volumetric electron microscopy techniques allow for 3D reconstruction of large samples at nanometer-resolution, thus opening up new avenues for the understanding of cell biology and architecture in intact tissues. At the same time, correlative light and electron microscopy (CLEM) techniques have been extended to 3D brain samples to help navigate and identify critical molecular landmarks within large EM volumes (Briggman and Denk 2006; Maco et al. 2013; Blazquez-Llorca et al. 2015, Bosch et al. 2015). Here we present the first volumetric ultrastructural reconstruction of an entire mouse hippocampal microglia using serial block face scanning electron microscopy (SBEM). Using CLEM we have ensured the inclusion of both large, small, and filopodial microglia processes. Segmentation of the dataset allowed us to carry out a comprehensive inventory of microglia cell structures, including vesicles, organelles, membrane protrusions, and processes. This study provides a reference that can serve as a data mining resource for investigating microglia cell biology.

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.

Serial block-face electron microscopy (SBEM) provides nanoscale 3D ultrastructure of embedded and stained cells and tissues in volumes of up to 107 µm3. In SBEM, electrons with 1–3 keV energies are incident on a specimen block, from which backscattered electron (BSE) images are collected with x, y resolution of 5–10 nm in the block-face plane, and successive layers are removed by an in situ ultramicrotome. Spatial resolution along the z-direction, however, is limited to around 25 nm by the minimum cutting thickness. To improve the z-resolution, we have extracted depth information from BSE images acquired at dual primary beam energies, using Monte Carlo simulations of electron scattering. The relationship between depth of stain and ratio of dual-energy BSE intensities enables us to determine 3D structure with a ×2 improvement in z-resolution. We demonstrate the technique by sub-slice imaging of hepatocyte membranes in liver tissue.

Molecular MicrobiologyVolume 102, Issue 3 p. i-i Front CoverFree Access The parasitic protozoan Trypanosoma brucei contains a non canonical protein translocase in the outer membrane of its mitochondrion. A 3D reconstruction of the insect stage form of the parasite (obtained by serial block face scanning electron microscopy) illustrates the network-like mitochondrion (green), basal body (white), flagellum (purple) and DNA (blue). Image courtesy: Jiří Týč and Sue Vaughan (Oxford Brookes University, Oxford, UK). For details see article by Desy et al. on pp. 520–529 of this issue. First published: 21 October 2016 https://doi.org/10.1111/mmi.13195AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume102, Issue3November 2016Pages i-i RelatedInformation

This paper describes an unsupervised algorithm, which segments the nuclear envelope of HeLa cells imaged by Serial Block Face Scanning Electron Microscopy. The algorithm exploits the variations of pixel intensity in different cellular regions by calculating edges, which are then used to generate superpixels. The superpixels are morphologically processed and those that correspond to the nuclear region are selected through the analysis of size, position, and correspondence with regions detected in neighbouring slices. The nuclear envelope is segmented from the nuclear region. The three-dimensional segmented nuclear envelope is then modelled against a spheroid to create a two-dimensional (2D) surface. The 2D surface summarises the complex 3D shape of the nuclear envelope and allows the extraction of metrics that may be relevant to characterise the nature of cells. The algorithm was developed and validated on a single cell and tested in six separate cells, each with 300 slices of 2000 × 2000 pixels. Ground truth was available for two of these cells, i.e., 600 hand-segmented slices. The accuracy of the algorithm was evaluated with two similarity metrics: Jaccard Similarity Index and Mean Hausdorff distance. Jaccard values of the first/second segmentation were 93%/90% for the whole cell, and 98%/94% between slices 75 and 225, as the central slices of the nucleus are more regular than those on the extremes. Mean Hausdorff distances were 9/17 pixels for the whole cells and 4/13 pixels for central slices. One slice was processed in approximately 8 s and a whole cell in 40 min. The algorithm outperformed active contours in both accuracy and time.

Durability of building materials and components: Edited by: P. J. Sereda and G. G. Litvan ASTM STP 691 (1980) $74.95

The global energy landscape is undergoing a profound transformation, shifting from traditional fossil fuel-based systems to sustainable, renewable energy sources like wind, solar, bioenergy, hydroelectric, and tidal energy. This transition signifies a significant evolution in global energy generation and consumption patterns, necessitating advanced energy storage solutions to manage the intermittent nature of renewables and ensure a stable and reliable energy supply. Advanced energy storage technologies, including batteries, supercapacitors, and other innovative systems, play a pivotal role in this transition by enabling effective load balancing, supporting the electrification of transportation through electric vehicles and charging infrastructure, managing peak demand, and facilitating the seamless integration of renewable energy into the grid. This evolution not only advances sustainability goals but also enhances energy security and independence. [1]Advanced analytical techniques are essential for developing energy storage systems, especially batteries, by providing critical insights into their complex chemical and physical processes. Battery materials' microstructure, composition, and operational dynamics can be explored using analytical techniques like scanning electron microscopy (SEM), focused ion beam (FIB), transmission electron microscopy (TEM), X-ray diffraction (XRD), and spectroscopy. The detailed analysis through these techniques is crucial for optimizing material properties, enhancing energy density, improving safety, and extending battery lifespan. By identifying degradation mechanisms and optimizing electrode and electrolyte materials, these techniques contribute to precise battery engineering. As demand grows for more efficient and higher-capacity batteries, these sophisticated tools are indispensable for advancing energy storage technology. [2]At Shell Technology Center Houston, we have developed air-controlled and cryogenic electron microscopy (EM) workflows for studying advanced energy storage systems in preparation for the energy transition. The air-controlled EM workflow, employing SEM, FIB, TEM, and XRD, plays a pivotal role in battery research by providing a controlled environment that prevents exposure to atmospheric elements that could alter material properties. These techniques are essential for examining the fine structural details and chemical compositions of battery components under near-native conditions. The cryo-EM workflow using SEM, FIB, and TEM is vital in battery research for studying materials and interfaces sensitive to environmental conditions or rapid degradation upon exposure. These cryogenic techniques involve rapid freezing of battery components to preserve their structure in a close-to-native state and prevent deleterious effects of air exposure such as oxidation or moisture interaction. [3]In our study, we present comprehensive results obtained using both air-controlled EM and cryogenic EM workflows to investigate advanced energy storage systems, including lithium-ion batteries (LIBs) and beyond-LIBs such as multivalent rechargeable batteries. The utilization of air-controlled EM techniques such as SEM, FIB, TEM, and XRD allows us to examine battery components under controlled environmental conditions, ensuring accurate analysis of structural details and chemical compositions without the risk of atmospheric contamination. Additionally, the use of cryo-EM techniques, including cryo-SEM, cryo-FIB, and cryo-TEM, enables the study of sensitive battery materials and interfaces in a preserved, near-native state. These cryogenic methods prevent degradation effects such as oxidation and moisture interaction, providing unprecedented insights into the ultrastructural properties and operational dynamics of battery materials at atomic or molecular levels. By leveraging both air-controlled and cryogenic EM workflows, we have achieved a comprehensive understanding of battery mechanics and chemistry, paving the way for improved battery design and enhanced performance in energy storage applications.Reference[1] International Renewable Energy Agent (IRENA) 2021 https://www.irena.org/publications/2021/Aug/Renewable-energy-statistics-2021[2] Tarascon, J.-M., Armand, M., Nature 414, 359–367 (2001). https://doi.org/10.1038/35104644[3] Cheng, D., Lu, B., Raghavendran, G., Zhang, M., and Meng, Y. S., Matter, Volume 5, Issue 1, p26-42, 2022 https://doi.org/10.1016/j.matt.2021.11.019

Comparison of 3-D Cell and Tissue Imaging Techniques Based on Scanning Electron Probes

The Skogsbergia lerneri is a marine ostracod which possesses a carapace that is both protective and transparent. Since development of this carapace and how it is maintained in the adult is not known, the aim of this investigation was to carry out an in-depth ultrastructural study of the ostracod carapace at different developmental stages. Standard transmission electron microscopy and novel serial block face scanning electron microscopy (SBF-SEM) were undertaken to discern carapace ultrastructure in both two and three dimensions. Analysis revealed a carapace consisting of the same basic layer structure as other myodocopid ostracods, namely an epicuticle, exocuticle, endocuticle and membranous layer, but with a thinner adult carapace of mean thickness of 19.2 ± 1.78 µm, n = 5. The carapace layers, except for instar 1 ostracods, had similar relative proportions throughout development. The endocuticle and membranous layer thickened through advancing developmental stages due to an increase in calcified crystalline polyhedrons and a greater number of chitinous lamellae in the membranous layer. Crystalline polyhedron dimensions were significantly smaller near the boundary with the membranous layer. The borders between the carapace layers were indistinct; SBF-SEM revealed an abundance of epicuticle projections into the exocuticle and apparent gradual merging at the boundary of the exocuticle and the endocuticle. Here, we discuss how the S. lerneri carapace layer structure has evolved to serve a specific mechanical function, allowing surface protection and rigidity. In addition, we suggest that the lack of pigment and graduated layer boundaries contribute to the transparency of the carapace.

Platelet aggregation at sites of vascular injury is essential for hemostasis. However, the mechanisms that prevent excessive clot growth are not fully understood. In the prevailing "core and shell" model, based largely on small vessel injury studies, a central core of highly activated platelets is surrounded by a limited signal intensity shell of less activated, minimally degranulated platelets. Recent reports, especially in mouse models of profuse bleeding, suggest thrombus architecture and platelet activation states are more heterogeneous than the binary core and shell model proposes. Here, we performed high-resolution morphometric mapping of individual platelet activation states in mouse jugular vein and femoral artery puncture wound thrombi, using serial block face scanning electron microscopy and wide-area transmission electron microscopy. Manually annotated images were analyzed at multiple time points, revealing initial, 1-minute, near-complete intermixing of platelet activation states with no distinct core of highly activated platelets. At 5 minutes, highly activated, degranulated platelets became concentrated along the interior surfaces of vaulted thrombus structures. At 20 minutes, platelet numbers decreased and distinct clustering of degranulated, highly activated platelets was observed within central portions of the intravascular platelet-rich crown, limiting their access to the circulation. Deletion of the α-granule vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor, vesicle associated membrane protein 8, increased both the frequency and clustering of highly activated platelets. Similar patterns were observed in femoral artery wounds. We conclude that thrombus organization is more complex than previously recognized and provide evidence that progressive structural changes help limit procoagulant surface exposure and thrombus growth during hemostasis after puncture wounding.

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

Electron microscopy (EM) and especially three-dimensional (3D) EM techniques have become established methods in structural virology. Investigation of virus-induced alteration of the cellular ultrastructure, such as Zika virus (ZIKV)-induced replication factories or coronavirus replication organelles, demands 3D imaging. Transmission electron microscopy (TEM) tomography is a widely used method, despite its limitation to samples with a thickness of up to 200 nm. Focused ion beam-scanning electron microscopy (SEM)-tomography can produce 3D data of larger volumes with isotropic, albeit typically lower resolution. Alternative techniques, such as block face-scanning electron microscopy (BF-SEM), SEM array tomography, or TEM imaging of serial sections are used for imaging of larger volumes. However, compared to the previously mentioned techniques, these techniques come at the cost of much lower resolution along the Z-axis of the sample. A technique that provides 3D information of samples up to 1 µm thickness with isotropic resolution of a few nanometers is scanning transmission electron microscopy (STEM) tomography. Here, we present a protocol for the preparation of high-pressure frozen, freeze-substituted, and resin-embedded virological specimens and their analysis using STEM tomography. This protocol benefits from the advantages of room temperature imaging while preserving the biological ultrastructure in a near-native state. We show two representative examples for questions that can be answered using STEM tomography. First, we apply the protocol for studying virion morphogenesis of a recombinant vesicular stomatitis virus (VSV). The STEM tomograms offer information on recombinant VSV budding that is otherwise not accessible by 2D imaging. Second, we show correlative light and electron microscopy (CLEM) using Foerster resonance energy transfer (FRET) imaging and STEM tomography (FRET-3D-CLEM) of EF-C peptide nanofibrils. This recently published combination gives new insights into the uptake and disassembly of infection-enhancing peptide nanofibrils, especially profiting from the large volume that is accessible by STEM tomography.

Focused ion beam-scanning electron microscopy provides novel insights of drug delivery phenomena