Serial Block-face Electron Microscopy Research Articles

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.

Impact of Obesity and Age on Mouse Corneal Innervation at the Epithelial-Stromal Interface.

The corneal epithelium is the most highly innervated structure in the body. Previously, we reported a novel event whereby stromal axons fuse with basal epithelial cells, limiting nerve penetration into the epithelium. Although corneal-epithelial nerves undergo changes in sensitivity and distribution throughout life and in response to an obesogenic diet, it is unknown if neuronal-epithelial cell fusion is altered. Here, we sought to determine if neuronal-epithelial cell fusion frequency correlates with obesogenic diet consumption and age. Corneas were collected from C57BL/6 mice and evaluated for neuronal-epithelial cell fusion frequency using serial block-face scanning electron microscopy. To assess the correlation between diet-induced obesity and fusion frequency, 6-week-old mice were fed either a normal diet or an obesogenic diet for 10 weeks. To assess changes in fusion frequency between young and adult mice under normal dietary conditions, 9- and 24-week-old mice were used. Mice fed a 10-week obesogenic diet showed 87% of central-cornea stromal nerves engaged in fusion compared with only 54% in age-matched controls (16 weeks old). In 9-week-old normal-diet animals, 48% of central-cornea stromal nerves contained fusing axons and increased to 81% at 24 weeks of age. Corneal sensitivity loss correlated with increased body weight and adiposity regardless of age and diet. Neuronal-epithelial cell fusion positively correlates with age and obesogenic diet consumption, and corneal nerve sensitivity loss correlates with increased body weight and adiposity, regardless of age and diet. As such, neuronal-epithelial cell fusion may play a role in corneal nerve density and sensitivity regulation.

Three-Dimensional Ultrastructure of Arabidopsis Cotyledons Infected with Colletotrichum higginsianum.

We used serial block-face scanning electron microscopy (SBF-SEM) to study the host-pathogen interface between Arabidopsis cotyledons and the hemibiotrophic fungus Colletotrichum higginsianum. By combining high-pressure freezing and freeze-substitution with SBF-SEM, followed by segmentation and reconstruction of the imaging volume using the freely accessible software IMOD, we created 3D models of the series of cytological events that occur during the Colletotrichum-Arabidopsis susceptible interaction. We found that the host cell membranes underwent massive expansion to accommodate the rapidly growing intracellular hypha. As the fungal infection proceeded from the biotrophic to the necrotrophic stage, the host cell membranes went through increasing levels of disintegration culminating in host cell death. Intriguingly, we documented autophagosomes in proximity to biotrophic hyphae using transmission electron microscopy (TEM) and a concurrent increase in autophagic flux between early to mid/late biotrophic phase of the infection process. Occasionally, we observed osmiophilic bodies in the vicinity of biotrophic hyphae using TEM only and near necrotrophic hyphae under both TEM and SBF-SEM. Overall, we established a method for obtaining serial SBF-SEM images, each with a lateral (x-y) pixel resolution of 10 nm and an axial (z) resolution of 40 nm, that can be reconstructed into interactive 3D models using the IMOD. Application of this method to the Colletotrichum-Arabidopsis pathosystem allowed us to more fully understand the spatial arrangement and morphological architecture of the fungal hyphae after they penetrate epidermal cells of Arabidopsis cotyledons and the cytological changes the host cell undergoes as the infection progresses toward necrotrophy. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Comparative analysis of traction forces in normal and glaucomatous trabecular meshwork cells within a 3D, active fluid-structure interaction culture environment

This study presents a 3D in vitro cell culture model, meticulously 3D printed to replicate the conventional aqueous outflow pathway anatomical structure, facilitating the study of trabecular meshwork (TM) cellular responses under glaucomatous conditions. Glaucoma affects TM cell functionality, leading to extracellular matrix (ECM) stiffening, enhanced cell-ECM adhesion, and obstructed aqueous humor outflow. Our model, reconstructed from polyacrylamide gel with elastic moduli of 1.5 and 21.7 kPa, is based on serial block-face scanning electron microscopy images of the outflow pathway. It allows for quantifying 3D, depth-dependent, dynamic traction forces exerted by both normal and glaucomatous TM cells within an active fluid-structure interaction (FSI) environment. In our experimental design, we designed two scenarios: a control group with TM cells observed over 20 hours without flow (static setting), focusing on intrinsic cellular contractile forces, and a second scenario incorporating active FSI to evaluate its impact on traction forces (dynamic setting). Our observations revealed that active FSI results in higher traction forces (normal: 1.83-fold and glaucoma: 2.24-fold) and shear strains (normal: 1.81-fold and glaucoma: 2.41-fold), with stiffer substrates amplifying this effect. Glaucomatous cells consistently exhibited larger forces than normal cells. Increasing gel stiffness led to enhanced stress fiber formation in TM cells, particularly in glaucomatous cells. Exposure to active FSI dramatically altered actin organization in both normal and glaucomatous TM cells, particularly affecting cortical actin stress fiber arrangement. This model while preliminary offers a new method in understanding TM cell biomechanics and ECM stiffening in glaucoma, highlighting the importance of FSI in these processes. Statement of significanceThis pioneering project presents an advanced 3D in vitro model, meticulously replicating the human trabecular meshwork's anatomy for glaucoma research. It enables precise quantification of cellular forces in a dynamic fluid-structure interaction, a leap forward from existing 2D models. This advancement promises significant insights into trabecular meshwork cell biomechanics and the stiffening of the extracellular matrix in glaucoma, offering potential pathways for innovative treatments. This research is positioned at the forefront of ocular disease study, with implications that extend to broader biomedical applications.

Regional differences in the ultrastructure of mucosal macrophages in the rat large intestine.

We previously clarified the histological characteristics of macrophages in the rat small intestine using serial block-face scanning electron microscopy (SBF-SEM). However, the regional differences in the characteristics of macrophages throughout the large intestine remain unknown. Here, we performed a pilot study to explore the regional differences in the ultrastructure of mucosal macrophages in the large intestine by using SBF-SEM analysis. SBF-SEM analysis conducted on the luminal side of the cecum and descending colon revealed macrophages as amorphous cells possessing abundant lysosomes and vacuoles. Macrophages in the cecum exhibited a higher abundance of lysosomes and a lower abundance of vacuoles than those in the descending colon. Macrophages with many intraepithelial cellular processes were observed beneath the intestinal superficial epithelium in the descending colon. Moreover, macrophages in contact with nerve fibers were more prevalent in the cecum than in the descending colon, and a subset of them surrounded a nerve bundle only in the cecum. In conclusion, the present pilot study suggested that the quantity of some organelles (lysosomes and vacuoles) in macrophages differed between the cecum and the descending colon and that there were some region-specific subsets of macrophages like nerve-associated macrophages in the cecum.

Zygospore development of Spirogyra (Charophyta) investigated by serial block-face scanning electron microscopy and 3D reconstructions.

Sexual reproduction of Zygnematophyceae by conjugation is a less investigated topic due to the difficulties of the induction of this process and zygospore ripening under laboratory conditions. For this study, we collected field sampled zygospores of Spirogyra mirabilis and three additional Spirogyra strains in Austria and Greece. Serial block-face scanning electron microscopy was performed on high pressure frozen and freeze substituted zygospores and 3D reconstructions were generated, allowing a comprehensive insight into the process of zygospore maturation, involving storage compound and organelle rearrangements. Chloroplasts are drastically changed, while young stages contain both parental chloroplasts, the male chloroplasts are aborted and reorganised as 'secondary vacuoles' which initially contain plastoglobules and remnants of thylakoid membranes. The originally large pyrenoids and the volume of starch granules is significantly reduced during maturation (young: 8 ± 5 µm³, mature: 0.2 ± 0.2 µm³). In contrast, lipid droplets (LDs) increase significantly in number upon zygospore maturation, while simultaneously getting smaller (young: 21 ± 18 µm³, mature: 0.1 ± 0.2 and 0.5 ± 0.9 µm³). Only in S. mirabilis the LD volume increases (34 ± 29 µm³), occupying ~50% of the zygospore volume. Mature zygospores contain barite crystals as confirmed by Raman spectroscopy with a size of 0.02 - 0.05 µm³. The initially thin zygospore cell wall (~0.5 µm endospore, ~0.8 µm exospore) increases in thickness and develops a distinct, electron dense mesospore, which has a reticulate appearance (~1.4 µm) in Spirogyra sp. from Greece. The exo- and endospore show cellulose microfibrils in a helicoidal pattern. In the denser endospore, pitch angles of the microfibril layers were calculated: ~18 ± 3° in S. mirabilis, ~20 ± 3° in Spirogyra sp. from Austria and ~38 ± 8° in Spirogyra sp. from Greece. Overall this study gives new insights into Spirogyra sp. zygospore development, crucial for survival during dry periods and dispersal of this genus.

High-resolution 3D ultrastructural analysis of developing mouse neocortex reveals long slender processes of endothelial cells that enter neural cells.

The development of the neocortex involves an interplay between neural cells and the vasculature. However, little is known about this interplay at the ultrastructural level. To gain a 3D insight into the ultrastructure of the developing neocortex, we have analyzed the embryonic mouse neocortex by serial block-face scanning electron microscopy (SBF-SEM). In this study, we report a first set of findings that focus on the interaction of blood vessels, notably endothelial tip cells (ETCs), and the neural cells in this tissue. A key observation was that the processes of ETCs, located either in the ventricular zone (VZ) or subventricular zone (SVZ)/intermediate zone (IZ), can enter, traverse the cytoplasm, and even exit via deep plasma membrane invaginations of the host cells, including apical progenitors (APs), basal progenitors (BPs), and newborn neurons. More than half of the ETC processes were found to enter the neural cells. Striking examples of this ETC process "invasion" were (i) protrusions of apical progenitors or newborn basal progenitors into the ventricular lumen that contained an ETC process inside and (ii) ETC process-containing protrusions of neurons that penetrated other neurons. Our observations reveal a - so far unknown - complexity of the ETC-neural cell interaction.

The application and development of electron microscopy for three-dimensional reconstruction in life science: a review.

Imaging technologies have played a pivotal role in advancing biological research by enabling visualization of biological structures and processes. While traditional electron microscopy (EM) produces two-dimensional images, emerging techniques now allow high-resolution three-dimensional (3D) characterization of specimens in situ, meeting growing needs in molecular and cellular biology. Combining transmission electron microscopy (TEM) with serial sectioning inaugurated 3D imaging, attracting biologists seeking to explore cell ultrastructure and driving advancement of 3D EM reconstruction. By comprehensively and precisely rendering internal structure and distribution, 3D TEM reconstruction provides unparalleled ultrastructural insights into cells and molecules, holding tremendous value for elucidating structure-function relationships and broadly propelling structural biology. Here, we first introduce the principle of 3D reconstruction of cells and tissues by classical approaches in TEM and then discuss modern technologies utilizing TEM and on new SEM-based as well as cryo-electron microscope (cryo-EM) techniques. 3D reconstruction techniques from serial sections, electron tomography (ET), and the recent single-particle analysis (SPA) are examined; the focused ion beam scanning electron microscopy (FIB-SEM), the serial block-face scanning electron microscopy (SBF-SEM), and automatic tape-collecting lathe ultramicrotome (ATUM-SEM) for 3D reconstruction of large volumes are discussed. Finally, we review the challenges and development prospects of these technologies in life science. It aims to provide an informative reference for biological researchers.

A new look at the architecture and dynamics of the Hydra nerve net

The Hydra nervous system is the paradigm of a ‘simple nerve net’. Nerve cells in Hydra, as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of the Hydra nerve net by immunostaining fixed polyps with a novel antibody that stains all nerve cells in Hydra. Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution TEM (transmission electron microscopy) and serial block face SEM (scanning electron microscopy) show that the nerve nets consist of bundles of parallel overlapping neurites. Results from transgenic lines show that neurite bundles include different neural circuits and hence that neurites in bundles require circuit-specific recognition. Nerve cell-specific innexins indicate that gap junctions can provide this specificity. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells.

HeLa cell segmentation using digital image processing

Computational cell segmentation is a vital area of research, particularly in the analysis of images of cancer cells. The use of cell lines, such as the widely utilized HeLa cell line, is crucial for studying cancer. While deep learning algorithms have been commonly employed for cell segmentation, their resource and data requirements can be impractical for many laboratories. In contrast, image processing algorithms provide a promising alternative due to their effectiveness and minimal resource demands. This article presents the development of an algorithm utilizing digital image processing to segment the nucleus and shape of HeLa cells. The research aims to segment the cell shape in the image center and accurately identify the nucleus. The study uses and processes 300 images obtained from Serial Block-Face Scanning Electron Microscopy (SBF-SEM). For cell segmentation, the morphological operation of erosion was used to separate the cells, and through distance calculation, the cell located at the center of the image was selected. Subsequently, the eroded shape was employed to restore the original cell shape. The nucleus segmentation uses parameters such as distances and sizes, along with the implementation of verification stages to ensure accurate detection. The accuracy of the algorithm is demonstrated by comparing it with another algorithm meeting the same conditions, using four segmentation similarity metrics. The evaluation results rank the proposed algorithm as the superior choice, highlighting significant outcomes. The algorithm developed represents a crucial initial step towards more accurate disease analysis. In addition, it enables the measurement of shapes and the identification of morphological alterations, damages, and changes in organelles within the cell, which can be vital for diagnostic purposes.

Energetics of the microsporidian polar tube invasion machinery

Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3–4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60–140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.

Energetics of the microsporidian polar tube invasion machinery.

Microsporidia are eukaryotic, obligate intracellular parasites that infect a wide range of hosts, leading to health and economic burdens worldwide. Microsporidia use an unusual invasion organelle called the polar tube (PT), which is ejected from a dormant spore at ultra-fast speeds, to infect host cells. The mechanics of PT ejection are impressive. Anncaliia algerae microsporidia spores (3-4 μm in size) shoot out a 100-nm-wide PT at a speed of 300 μm/s, creating a shear rate of 3000 s-1. The infectious cargo, which contains two nuclei, is shot through this narrow tube for a distance of ∼60-140 μm (Jaroenlak et al, 2020) and into the host cell. Considering the large hydraulic resistance in an extremely thin tube and the low-Reynolds-number nature of the process, it is not known how microsporidia can achieve this ultrafast event. In this study, we use Serial Block-Face Scanning Electron Microscopy to capture 3-dimensional snapshots of A. algerae spores in different states of the PT ejection process. Grounded in these data, we propose a theoretical framework starting with a systematic exploration of possible topological connectivity amongst organelles, and assess the energy requirements of the resulting models. We perform PT firing experiments in media of varying viscosity, and use the results to rank our proposed hypotheses based on their predicted energy requirement. We also present a possible mechanism for cargo translocation, and quantitatively compare our predictions to experimental observations. Our study provides a comprehensive biophysical analysis of the energy dissipation of microsporidian infection process and demonstrates the extreme limits of cellular hydraulics.

A presynaptic source drives differing levels of surround suppression in two mouse retinal ganglion cell types

In early sensory systems, cell-type diversity generally increases from the periphery into the brain, resulting in a greater heterogeneity of responses to the same stimuli. Surround suppression is a canonical visual computation that begins within the retina and is found at varying levels across retinal ganglion cell types. Our results show that heterogeneity in the level of surround suppression occurs subcellularly at bipolar cell synapses. Using single-cell electrophysiology and serial block-face scanning electron microscopy, we show that two retinal ganglion cell types exhibit very different levels of surround suppression even though they receive input from the same bipolar cell types. This divergence of the bipolar cell signal occurs through synapse-specific regulation by amacrine cells at the scale of tens of microns. These findings indicate that each synapse of a single bipolar cell can carry a unique visual signal, expanding the number of possible functional channels at the earliest stages of visual processing.

Electron Microscopy Techniques for 3D Plant ER Imaging.

The endoplasmic reticulum (ER) forms an extensive network in plant cells. In leaf cells and vacuolated root cells it is mainly restricted to the cortex, whereas in the root meristem the cortical and cytoplasmic ER takes up a large volume throughout the entire cell. Only 3D electron microscopy provides sufficient resolution to understand the spatial organization of the ER in the root. Here we present two protocols for 3D EM imaging of the ER across a range of scales. For large-scale ER structure analysis, we describe selective ER staining with ZIO that allows for automated or semi-automated ER segmentation. For smaller regions of ER, we describe high-pressure freezing, which enables almost instantaneous fixation of plant tissues but without organelle specific staining. These fixation and staining techniques are suitable for a range of imaging modalities, including serial sections, array tomography, serial block face-scanning electron microscopy (SBF-SEM), or focused ion beam (FIB) SEM.

Volumetric Reconstruction of a Human Retinal Pigment Epithelial Cell Reveals Specialized Membranes and Polarized Distribution of Organelles.

Despite the centrality of the retinal pigment epithelium (RPE) in vision and retinopathy our picture of RPE morphology is incomplete. With a volumetric reconstruction of human RPE ultrastructure, we aim to characterize major membranous features including apical processes and their interactions with photoreceptor outer segments, basolateral infoldings, and the distribution of intracellular organelles. A parafoveal retinal sample was acquired from a 21-year-old male organ donor. With serial block-face scanning electron microscopy, a tissue volume from the inner-outer segment junction to basal RPE was captured. Surface membranes and complete internal ultrastructure of an individual RPE cell were achieved with a combination of manual and automated segmentation methods. In one RPE cell, apical processes constitute 69% of the total cell surface area, through a dense network of over 3000 terminal branches. Single processes contact several photoreceptors. Basolateral infoldings facing the choriocapillaris resemble elongated filopodia and comprise 22% of the cell surface area. Membranous tubules and sacs of endoplasmic reticulum represent 20% of the cell body volume. A dense basal layer of mitochondria extends apically to partly overlap electron-dense pigment granules. Pores in the nuclear envelope form a distinct pattern of rows aligned with chromatin. Specialized membranes at the apical and basal side of the RPE cell body involved in intercellular uptake and transport represent over 90% of the total surface area. Together with the polarized distribution of organelles within the cell body, these findings are relevant for retinal clinical imaging, therapeutic approaches, and disease pathomechanisms.

Ultrastructural analysis of nigrostriatal dopaminergic terminals in a knockin mouse model of DYT1 dystonia.

DYT1 dystonia is associated with decreased striatal dopamine release. In this study, we examined the possibility that ultrastructural changes of nigrostriatal dopamine terminals could contribute to this neurochemical imbalance using a serial block face/scanning electron microscope (SBF/SEM) and three-dimensional reconstruction to analyse striatal tyrosine hydroxylase-immunoreactive (TH-IR) terminals and their synapses in a DYT1(ΔE) knockin (DYT1-KI) mouse model of DYT1 dystonia. Furthermore, to study possible changes in vesicle packaging capacity of dopamine, we used transmission electron microscopy to assess the synaptic vesicle size in striatal dopamine terminals. Quantitative comparative analysis of 80 fully reconstructed TH-IR terminals in the WT and DYT1-KI mice indicate (1) no significant difference in the volume of TH-IR terminals; (2) no major change in the proportion of axo-spinous versus axo-dendritic synapses; (3) no significant change in the post-synaptic density (PSD) area of axo-dendritic synapses, while the PSDs of axo-spinous synapses were significantly smaller in DYT1-KI mice; (4) no significant change in the contact area between TH-IR terminals and dendritic shafts or spines, while the ratio of PSD area/contact area decreased significantly for both axo-dendritic and axo-spinous synapses in DYT1-KI mice; (5) no significant difference in the mitochondria volume; and (6) no significant difference in the synaptic vesicle area between the two groups. Altogether, these findings suggest that abnormal morphometric changes of nigrostriatal dopamine terminals and their post-synaptic targets are unlikely to be a major source of reduced striatal dopamine release in DYT1 dystonia.

Atypical flagella assembly and haploid genome coiling during male gamete formation in Plasmodium

Gametogenesis in Plasmodium spp. occurs within the Anopheles mosquito and is essential for sexual reproduction / differentiation and onwards transmission to mammalian hosts. To better understand the 3D organisation of male gametogenesis, we used serial block face scanning electron microscopy (SBF-SEM) and serial-section cellular electron tomography (ssET) of P. berghei microgametocytes to examine key structures during male gamete formation. Our data reveals an elaborate organisation of axonemes coiling around the nucleus in opposite directions forming a central axonemal band in microgametocytes. Furthermore, we discover the nucleus of microgametes to be tightly coiled around the axoneme in a complex structure whose formation starts before microgamete emergence during exflagellation. Our discoveries of the detailed 3D organisation of the flagellated microgamete and the haploid genome highlight some of the atypical mechanisms of axoneme assembly and haploid genome organisation during male gamete formation in the malaria parasite.

A high-throughput 3D X-ray histology facility for biomedical research and preclinical applications

Background The University of Southampton, in collaboration with the University Hospital Southampton (UHS) NHS Foundation Trust and industrial partners, has been at the forefront of developing three-dimensional (3D) imaging workflows using X-ray microfocus computed tomography (μCT) -based technology. This article presents the outcomes of these endeavours and highlights the distinctive characteristics of a μCT facility tailored explicitly for 3D X-ray Histology, with a primary focus on applications in biomedical research and preclinical and clinical studies. Methods The UHS houses a unique 3D X-ray Histology (XRH) facility, offering a range of services to national and international clients. The facility employs specialised μCT equipment explicitly designed for histology applications, allowing whole-block XRH imaging of formalin-fixed and paraffin-embedded tissue specimens. It also enables correlative imaging by combining μCT imaging with other microscopy techniques, such as immunohistochemistry (IHC) and serial block-face scanning electron microscopy, as well as data visualisation, image quantification, and bespoke analysis. Results Over the past seven years, the XRH facility has successfully completed over 120 projects in collaboration with researchers from 60 affiliations, resulting in numerous published manuscripts and conference proceedings. The facility has streamlined the μCT imaging process, improving productivity and enabling efficient acquisition of 3D datasets. Discussion & Conclusions The 3D X-ray Histology (XRH) facility at UHS is a pioneering platform in the field of histology and biomedical imaging. To the best of our knowledge, it stands out as the world's first dedicated XRH facility, encompassing every aspect of the imaging process, from user support to data generation, analysis, training, archiving, and metadata generation. This article serves as a comprehensive guide for establishing similar XRH facilities, covering key aspects of facility setup and operation. Researchers and institutions interested in developing state-of-the-art histology and imaging facilities can utilise this resource to explore new frontiers in their research and discoveries.

Size and shape analysis of micro- to nano-particles of quartz powders using advanced electron microscopy and laser diffraction methods

The paper deals with particle analysis and the evaluation of the shape and size distribution of micro- to nano-sized quartz particles as a key parameter for the evaluation of quality in many industrial fields, particularly for high-purity applications. Samples are analyzed by laser diffraction, the most commonly used method for determining the size of the particles; for precise measurement with the possibility of high-resolution imaging of particles, scanning electron microscopy was applied. For morphological and size analysis of electrically non-conductive micro- to nano-particles under environmentally compatible conditions of elevated gas pressure and humidity, environmental scanning electron microscopy was selected as the most suitable. Moreover, the high-end methods of focused ion beam scanning electron microscopy and serial block-face scanning electron microscopy enabled the analysis of particles in the epoxy resin volume including spatial imaging. The application of image post-processing as an important part of the electron microscopy workflow is also discussed.

Partial inhibition of complex I restores mitochondrial morphology and mitochondria‐ER communication in hippocampus of APP/PS1 mice

AbstractBackgroundAlzheimer’s Disease (AD) has no cure. We showed that partial inhibition of mitochondrial complex I (MCI) with small molecule CP2 induced adaptive stress response activating multiple neuroprotective mechanisms. Chronic treatment reduced inflammation, improved synaptic and mitochondrial functions, and blocked neurodegeneration in symptomatic APP/PS1 mice, a translational model of AD.MethodsAnalysis of mitochondrial morphology and mitochondria‐endoplasmic reticulum (ER) contacts (MERCS) was done in the hippocampal CA1 tissue from APP/PS1 mice treated with CP2 and compared to APP/PS1 and non‐transgenic mice treated with vehicle using serial block‐face scanning electron microscopy (SBFSEM) and three‐dimensional (3D) EM reconstructions combined with western blot analysis and next generation RNA sequencing.ResultsWe demonstrate that CP2 treatment restores mitochondrial morphology and mitochondria‐ ER communication, reducing ER stress in the APP/PS1 mouse brain. Using 3D EM reconstructions, we found that mitochondria in AD dendrites existed primarily as mitochondria‐on‐a‐string (MOAS), a morphological phenotype that is difficult to detect using conventional 2D EM or fluorescence spectroscopy. Compared to other morphological phenotypes, MOAS are extensively enveloped in the ER membranes forming multiple MERCS known to contribute to abnormal lipid and calcium homeostasis, and ER stress in AD. CP2 treatment specifically reduced MOAS formation, consistent with improved energy homeostasis in the brain, with concomitant reduction in MERCS, ER stress, and improved lipid homeostasis.ConclusionsThese data provide novel information on the role MOAS play in AD. Our data demonstrate that in response to energetic stress, mitochondria in dendrites acquire MOAS phenotype with increased interaction with the ER, which may synergistically contribute to the AD development. Since CP2 treatment alleviated the ER stress and improved mitochondrial dynamics and function, these data further support the development of partial MCI inhibitors as disease modifying strategy for AD.