Serial Block-face Electron Microscopy Research Articles

Melanophages give rise to hyperreflective foci in AMD, a disease-progression marker

Retinal melanosome/melanolipofuscin-containing cells (MCCs), clinically visible as hyperreflective foci (HRF) and a highly predictive imaging biomarker for the progression of age-related macular degeneration (AMD), are widely believed to be migrating retinal pigment epithelial (RPE) cells. Using human donor tissue, we identify the vast majority of MCCs as melanophages, melanosome/melanolipofuscin-laden mononuclear phagocytes (MPs). Using serial block-face scanning electron microscopy, RPE flatmounts, bone marrow transplantation and in vitro experiments, we show how retinal melanophages form by the transfer of melanosomes from the RPE to subretinal MPs when the “don’t eat me” signal CD47 is blocked. These melanophages give rise to hyperreflective foci in Cd47−/−-mice in vivo, and are associated with RPE dysmorphia similar to intermediate AMD. Finally, we show that Cd47 expression in human RPE declines with age and in AMD, which likely participates in melanophage formation and RPE decline. Boosting CD47 expression in AMD might protect RPE cells and delay AMD progression.

3D reconstruction shows mouse skeletal muscle may decline in function across aging due to the MICOS complex

Mitochondria are active organelles, undergoing harmonized cycles of fission and fusion, driven by ‘mitochondrial dynamics,’ to retain their morphology, distribution, and size. Mitochondrial dynamics has emerged as a critical process in maintaining cellular homeostasis. Interestingly, it has been long respected that a decrease in mitochondrial function escorts aging in specific tissues. For example, skeletal muscle gradually loses mass, strength, endurance, and oxidative capacity during aging. This decrease might, in turn, contribute to the observed age-dependent decline in organ function by mutations or alterations in mitochondrial fusion and fission proteins associated with several diseases. However, new players in regulating mitochondrial shape and cristae shape have been recently studied, such as the mitochondrial contact site and cristae organizing system (MICOS) complex. The MICOS complex is a multi-protein interaction hub that helps define cristate and mitochondria architecture. However, the MICOS complex has not been implicated in regulating organelle structure changes during aging. Thus, we hypothesized that loss of the MICOS complex during aging may increase mitochondrial fragmentation, decrease nanotunnels, and alter cristae morphology. To do this, we examined the three-dimensional morphology of mitochondria networks in young (3-month) and aged (2-year) murine gastrocnemius muscle via serial block face-scanning electron microscopy and the Amira program for segmentation, analysis, and quantification. We found differences in mitochondrial network configuration, nanotunneling, size, shape, number, contact sites, MICOS dynamics, and gene expression in skeletal muscle during aging. We also found an association between OPA-1 and the MICOS complex in the gastrocnemius with mitochondrial aging. Furthermore, the loss of the MICOS complex was linked with decreased oxidative capacity and altered mitochondrial metabolism. Potentially, this suggests a novel relationship between the MICOS complex and aging in skeletal muscle.

Myelination generates aberrant ultrastructure that is resolved by microglia.

To enable rapid propagation of action potentials, axons are ensheathed by myelin, a multilayered insulating membrane formed by oligodendrocytes. Most of the myelin is generated early in development, resulting in the generation of long-lasting stable membrane structures. Here, we explored structural and dynamic changes in central nervous system myelin during development. To achieve this, we performed an ultrastructural analysis of mouse optic nerves by serial block face scanning electron microscopy (SBF-SEM) and confocal time-lapse imaging in the zebrafish spinal cord. We found that myelin undergoes extensive ultrastructural changes during early postnatal development. Myelin degeneration profiles were engulfed and phagocytosed by microglia using exposed phosphatidylserine as one "eat me" signal. In contrast, retractions of entire myelin sheaths occurred independently of microglia and involved uptake of myelin by the oligodendrocyte itself. Our findings show that the generation of myelin early in development is an inaccurate process associated with aberrant ultrastructural features that require substantial refinement.

3D Ultrastructural Visualization of Mitosis Fidelity in Human Cells Using Serial Block Face Scanning Electron Microscopy (SBF-SEM).

Errors in chromosome segregation during mitosis lead to chromosome instability, resulting in an unbalanced number of chromosomes in the daughter cells. Light microscopy has been used extensively to study chromosome missegregation by visualizing errors of the mitotic spindle. However, less attention has been paid to understanding spindle function in the broader context of intracellular structures and organelles during mitosis. Here, we outline a protocol to visualize chromosomes and endomembranes in mitosis, combining light microscopy and 3D volume electron microscopy, serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM provides high-resolution imaging of large volumes and subcellular structures, followed by image analysis and 3D reconstruction. This protocol allows scientists to visualize the whole subcellular context of the spindle during mitosis.

A conventional fixation volume electron microscopy protocol for plants.

Volume electron microscopy techniques play an important role in plant research from understanding organelles and unicellular forms to developmental studies, environmental effects and microbial interactions with large plant structures, to name a few. Due to large air voids central vacuole, cell wall and waxy cuticle, many plant tissues pose challenges when trying to achieve high quality morphology, metal staining and adequate conductivity for high-resolution volume EM studies. Here, we applied a robust conventional chemical fixation strategy to address the special challenges of plant samples and suitable for, but not limited to, serial block-face and focused ion beam scanning electron microscopy. The chemistry of this protocol was modified from an approach developed for improved and uniform staining of large brain volumes. Briefly, primary fixation was in paraformaldehyde and glutaraldehyde with malachite green followed by secondary fixation with osmium tetroxide, potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide and finally uranyl acetate and lead aspartate staining. Samples were then dehydrated in acetone with a propylene oxide transition and embedded in a hard formulation Quetol 651 resin. The samples were trimmed and mounted with silver epoxy, metal coated and imaged via serial block-face scanning electron microscopy and focal charge compensation for charge suppression. High-contrast plant tobacco and duckweed leaf cellular structures were readily visible including mitochondria, Golgi, endoplasmic reticulum and nuclear envelope membranes, as well as prominent chloroplast thylakoid membranes and individual lamella in grana stacks. This sample preparation protocol serves as a reliable starting point for routine plant volume electron microscopy.

Mitochondria morphometry in 3D datasets obtained from mouse brains with serial block-face scanning electron microscopy.

The dysfunction of mitochondria is linked with many diseases. In the nervous system, evidence of their implication in neurodegenerative disease is growing. Mitochondria health is assessed by their impact on cellular metabolism but alterations in their morphologies and locations in the cells can also be markers of dysfunctions. Light microscopy techniques allow us to look at mitochondria in vivo in cells or tissue. But in the case of the nervous system, in order to assess the precise location of mitochondria in different cell types and neuronal compartments (cell bodies, dendrites or axons), electron microscopy is required. While the percentage of volume occupied by mitochondria can be assessed on 2D images, alterations in length, branching, and interactions with other organelles require three-dimensional (3D) segmentation of mitochondria in volumes imaged at ultrastructural level. Nowadays three-dimensional volume electron microscopy (vEM) imaging techniques such as serial block face scanning electron microscopy (SBF-SEM) enable us to image 3D volumes of tissue at ultrastructural level and can be done routinely. Segmentation of all the neuropil is also successfully achieved at a large scale in the nervous system. Here, we show a workflow based on open access resources, which allows us to image, segment, and analyze mitochondria in 3D volumes of regions of interest in the mouse brain. Taking advantage of recent developments, e.g., pre-trained models for mitochondria, we speed up the reconstruction and analysis. We also critically assess the impact on the results of the different reconstruction methods chosen and the level of manual corrections invested.

Histological study on regional specificity of the mucosal nerve network in the rat large intestine.

Our previous studies and others have revealed detailed characteristics of the mucosal nerve network in the small intestine, but much remains unknown about the corresponding network in the large intestine. We herein investigated regional differences in the expression of neurochemical markers, the nerve network structure, and the cells in contact with nerve fibers by histological analysis using both immunohistochemistry and serial block-face scanning electron microscopy (SBF-SEM). Immunohistochemistry revealed that immunopositive structures for protein gene product 9.5, vasoactive intestinal peptide (VIP), calretinin and vesicular acetylcholine transporter were more prevalent in the lamina propria of the ascending colon than the cecum and descending colon (DC). There was no significant difference in the frequency of most neurochemical markers between the cecum and DC, but the frequencies of VIP+ structures were higher in the cecum than in the DC. SBF-SEM analysis showed that the nerve network structure was more developed on the luminal side of the DC than the cecum. The cells that nerve fibers abundantly contacted were subepithelial and lamina propria fibroblast-like cells and macrophages. In addition, nerve fibers in the cecum were in more frequent contact with immune cells such as macrophages and plasma cells than nerve fibers in the DC. Thus, the present histological analysis suggested that the mucosal nerve network in the large intestine possessed both regional universality and various specificities, and revealed the intimate relationship between the nerve network and immune cells, especially in the cecum.

Histological study on the reginal difference in the localization of mucosal enteric glial cells and their sheath structure in the rat intestine.

The present study aimed to histologically clarify the regional specificity of the mucosal enteric glial cells (mEGCs) in the rat intestine with serial block-face scanning electron microscopy (SBF-SEM). SBF-SEM analysis with the ileum, the cecum and the descending colon revealed that mEGC nuclei were more abundant in the data stacks from the apical portion of the villus and the lateral portion of the crypt of the ileum. mEGCs exhibited a high rate of coverage over the nerve bundle around the lateral portion of the ileal crypt, but showed an extremely low level of coverage in the luminal portion of the cecum. These findings evidenced regional differences in the localization of mEGCs and in their sheath structure in the rat intestine.

Correlative multiscale microCT-SBF-SEM imaging of resin-embedded tissue.

Three-dimensional biological microscopy presents a trade-off between spatial resolution and field of view. Correlative approaches applying multiple imaging techniques to the same sample can therefore mitigate against these trade-offs. Here, we present a workflow for correlative microscopic X-ray microfocus computed tomography (microCT) and serial block face scanning electron microscopy (SBF-SEM) imaging of resin-embedded tissue, using mammalian placental tissue samples as an example. This correlative X-ray and electron microscopy (CXEM) workflow allows users to image the same sample at multiple resolutions, and target the region of interest (ROI) for SBF-SEM based on microCT. We detail the protocols associated with this workflow and demonstrate its application in multiscale imaging of horse placental villi and ROI selection in the labyrinthine zone of a mouse placenta. These examples demonstrate how the protocol may need to be adapted for tissues with different densities.

Automated large volume sample preparation for vEM.

New developments in electron microscopy technology, improved efficiency of detectors, and artificial intelligence applications for data analysis over the past decade have increased the use of volume electron microscopy (vEM) in the life sciences field. Moreover, sample preparation methods are continuously being modified by investigators to improve final sample quality, increase electron density, combine imaging technologies, and minimize the introduction of artifacts into specimens under study. There are a variety of conventional bench protocols that a researcher can utilize, though most of these protocols require several days. In this work, we describe the utilization of an automated specimen processor, the mPrep™ ASP-2000™, to prepare samples for vEM that are compatible with focused ion beam scanning electron microscopy (FIB-SEM), serial block face scanning electron microscopy (SBF-SEM), and array tomography (AT). The protocols described here aimed for methods that are completed in a much shorter period of time while minimizing the exposure of the operator to hazardous and toxic chemicals and improving the reproducibility of the specimens' heavy metal staining, all without compromising the quality of the data acquired using backscattered electrons during SEM imaging. As a control, we have included a widely used sample bench protocol and have utilized it as a comparator for image quality analysis, both qualitatively and using image quality analysis metrics.

Methods to expose subsurface objects of interest identified from 3D imaging: The intermediate sample preparation stage in the correlative microscopy workflow.

The correlative imaging workflow is a method of combining information and data across modes (e.g. SEM, X-ray CT, FIB-SEM), scales (cm to nm) and dimensions (2D-3D-4D), providing a more holistic interpretation of the research question. Often, subsurface objects of interest (e.g. inclusions, pores, cracks, defects in multilayered samples) are identified from initial exploratory nondestructive 3D tomographic imaging (e.g. X-ray CT, XRM), and those objects need to be studied using additional techniques to obtain, for example, 2D chemical or crystallographic data. Consequently, an intermediate sample preparation step needs to be completed, where a targeted amount of sample surface material is removed, exposing and revealing the object of interest. At present, there is not one singular technique for removing varied thicknesses at high resolution and on a range of scales from cm to nm. Here, we review the manual and automated options currently available for targeted sample material removal, with a focus on those methods which are readily accessible in most laboratories. We summarise the approaches for manual grinding and polishing, automated grinding and polishing, microtome/ultramicrotome, and broad-beam ion milling (BBIM), with further review of other more specialist techniques including serial block face electron microscopy (SBF-SEM), and ion milling and laser approaches such as FIB-SEM, Xe plasma FIB-SEM, and femtosecond laser/LaserFIB. We also address factors which may influence the decision on a particular technique, including the composition, shape and size of the samples, sample mounting limitations, the amount of surface material to be removed, the accuracy and/or resolution of peripheral parts, the accuracy and/or resolution of the technique/instrumentation, and other more general factors such as accessibility to instrumentation, costs, and the time taken for experimentation. It is hoped that this study will provide researchers with a range of options for removal of specific amounts of sample surface material to reach subsurface objects of interest in both correlative and non-correlative workflows.

Volume electron microscopy reveals age-related circuit remodeling in the auditory brainstem.

The medial nucleus of the trapezoid body (MNTB) is an integral component of the auditory brainstem circuitry involved in sound localization. The giant presynaptic nerve terminal with multiple active zones, the calyx of Held (CH), is a hallmark of this nucleus, which mediates fast and synchronized glutamatergic synaptic transmission. To delineate how these synaptic structures adapt to reduced auditory afferents due to aging, we acquired and reconstructed circuitry-level volumes of mouse MNTB at different ages (3 weeks, 6, 18, and 24 months) using serial block-face electron microscopy. We used C57BL/6J, the most widely inbred mouse strain used for transgenic lines, which displays a type of age-related hearing loss. We found that MNTB neurons reduce in density with age. Surprisingly we observed an average of approximately 10% of poly-innervated MNTB neurons along the mouse lifespan, with prevalence in the low frequency region. Moreover, a tonotopy-dependent heterogeneity in CH morphology was observed in young but not in older mice. In conclusion, our data support the notion that age-related hearing impairments can be in part a direct consequence of several structural alterations and circuit remodeling in the brainstem.

Ultrastructural characteristics of oligodendrocyte precursor cells in the early postnatal mouse optic nerve observed by serial block-face scanning electron microscopy.

Oligodendrocyte precursor cells (OPC) arise from restricted regions of the central nervous system (CNS) and differentiate into myelin-forming cells after migration, but their ultrastructural characteristics have not been fully elucidated. This study examined the three-dimensional ultrastructure of OPCs in comparison with other glial cells in the early postnatal optic nerve by serial block-face scanning electron microscopy. We examined 70 putative OPCs (pOPC) that were distinct from other glial cells according to established morphological criteria. The pOPCs were unipolar in shape with relatively few processes, and their Golgi apparatus were localized in the perinuclear region with a single cisterna. Astrocytes abundant in the optic nerve were distinct from pOPCs and had a greater number of processes and more complicated Golgi apparatus morphology. All pOPCs and astrocytes contained a pair of centrioles (basal bodies). Among them, 45% of pOPCs extended a short cilium, and 20% of pOPCs had centrioles accompanied by vesicles, whereas all astrocytes with basal bodies had cilia with invaginated ciliary pockets. These results suggest that the fine structures of pOPCs during the developing and immature stages may account for their distinct behavior. Additionally, the vesicular transport of the centrioles, along with a short cilium length, suggests active ciliogenesis in pOPCs.

A Putative Erythroid Synapse Linking Developing Erythroblasts and Macrophages in Erythroblastic Islands

An estimated 25% of the global population suffer from anemia, a major cause being deficient erythropoiesis. Mammalian erythropoiesis in the adult occurs primarily in the bone marrow (BM), with the spleen being an active site of extramedullary erythropoiesis during anemic stress. Here, we examine regulators of the anemic stress response using an unbiased transcriptomic approach to compare FACS-sorted splenic Ter-119+ erythroid precursors, from untreated mice or phenylhydrazine (PHZ)-treated mice exhibiting chemically-induced anemia. Transcriptomic analyses revealed unexpected expression of a broad range of immune markers in untreated mouse splenic erythroid cells, most which were conspicuously diminished in expression during PHZ-induced anemic stress. These surface antigens, previously unreported on erythroid cells, include markers of B lymphocytes (CD22, CD74), T lymphocytes (CD2, CD3ε) and leukocytes including CD11a/LFA-1, CD45, CD48, CD53, as well as the homing receptors L-selectin and PSGL-1. Immunophenotyping confirmed expression of these immune markers on a subpopulation of steady state BM and splenic erythroid precursors, with a significant reduction in immune marker-expressing erythroid cells seen in the spleen during anemic stress. Erythropoiesis is governed by a close relationship between erythroid precursors and central macrophages in multicellular clusters termed erythroblastic islands (EBIs). Confocal microscopy demonstrated localization of the immune markers CD2, CD3ε, CD11a and PSGL-1 on Ter-119+ mouse erythroid precursors with frequent clustering of markers seen at intercellular contact points where erythroid cells interact with the central EBI macrophage. PHZ-treated mice exhibited a significantly reduced frequency of EBIs in both the BM and spleen compared to control mice. PHZ-treated mice also demonstrated downregulated Interferon Regulatory Factor-8 (Irf8) transcripts on transcriptomic analysis of splenic erythroid precursors. Irf8-deficient mice have been reported to have a profoundly reduced erythroid compartment. There are also reports that Irf8 may be a regulator of macrophage morphology. We therefore studied Irf8-deficient mice to evaluate whether Irf8 has a role in EBI-regulation. BM analysis of Irf8-deficient mice showed a reduced frequency of EBI-like structures compared to wildtype (WT) mice. Scanning electron micrographs revealed that Irf8-/- macrophages are smaller with fewer erythroid precursors attached compared to WT macrophages. Irf8-/- EBIs harbored no identifiable reticulocytes in contrast to WT EBIs. EBI rosette reconstitution assays demonstrated that Irf8-/- erythroid precursors were unable to bind to WT or Irf8-/- macrophages, whereas Irf8-/- macrophages could interact with WT erythroid cells. Immunophenotyping of Irf8-/- erythroid precursors revealed that immune marker expression (CD2, CD38, CD43, CD45, PSGL-1) was significantly lower compared to WT counterparts. These data suggest that Irf8 may regulate both macrophage morphology and erythroid cell surface phenotype influencing erythropoiesis. The newly uncovered immune markers expressed by erythroid cells are also involved in formation of immunological synapses between lymphocytes and macrophages. This led us to question whether an analogous synapse-like structure may exist in EBIs. Serial block face scanning electron microscopy (SBF-SEM) was used to assess EBI morphology at the nanometer scale. SBF-SEM micrographs show erythroblasts extending pseudopods towards the macrophage for attachment. Three-dimensional volumetric reconstruction of SBF-SEM micrographs revealed en face spatial topologies present on the surface of EBI central macrophages similar to those seen in immunological synapses. Our findings suggest that immune markers, possibly mediated by Irf8, may function to form synaptic-like interactions between erythroid cells and macrophages that result in EBIs - an "erythroid synapse". Failure in forming these interactions may result in alterations in EBI formation with potential downstream impacts on erythropoiesis. Improved understanding of how EBIs form and function may aid in the development of therapeutic agents to combat anemia.

No cost but high performance-An alternative open source solution for 3D-visualizations in morphology.

3D-visualization has become common courtesy in science and also found its way into teaching in schools and universities. Nevertheless, the way to high performance 3D-visualization and analyses remains difficult and is often also a matter of budget. Due to the obvious advantages of presenting morphological and anatomical datasets with the help of 3D-figures, all in one software solutions usually come along with high rental and maintenance fees. For that reason, it is more than overdue to establish and use open source software solutions with all their obvious advantages - as other disciplines already do. Here we provide a modular, highly adaptive and freely available software pipeline for high performance 3D-visualizations of (not only) morphological datasets by combining features of ImageJ, MeshLab and Blender, without any additional costs. Exemplarily using serial-block face SEM data as well as serial AZAN-stained histological sections, the herein presented step-by-step protocol allows for a fast and efficient analysis, visualization and animation of large, anatomical datasets. Regardless which type of serial, morphological datasets needs to be analyzed, our open source guide provides an easy to handle and promptly adaptable solution. Therefore, our pipeline for 3D-visualization represents a valuable alternative to conventional, commercial packages. RESEARCH HIGHLIGHTS: We provide a highly modular and easy to learn open source solution for multiple 3D-visualizations. The step-by-step-guide make it easy to start, and advanced users can replace software, add others and such build your own individual software pipeline.

A fibre tracking algorithm for volumetric microstructural data - application to tendons

Tendons are crucial connective tissues made almost entirely of bundles of long, near-parallel collagen fibrils, and are vitally important to skeletal stability and mechanical function. Tendon structure is typically quantified in 2D, whereas, in this work, we have used serial block face-scanning electron microscopy to image tendons in 3D. We present a custom fibre tracking algorithm (FTA), with which we have characterised the 3D microstructure of tendon. Currently available tools for fibre tracing were unsuitable for tracking large numbers of fibrils and handling imaging artefacts associated with EM. We have tracked fibrils through a representative tendon volume and measured their relative length, diameter, orientation, chirality, tortuosity and volume fraction, which are just some of the measurements it is possible to make with the FTA depending on the research question. This algorithm has been developed in a general way and can be applied to a range of biological research questions relating to tendon structure-function relationships, on topics such as ageing, disease, development and injury. The FTA is also applicable to other fibrous biological materials, as well as engineered materials and textiles; it is written using Python and is freely available to download. Statement of significanceWe have created an algorithm for tracking fibres in 3D image stacks and applied it to tendon tissue. Previous studies have examined tendon structure in 2D, whereas we have imaged tendons in 3D using volumetric electron microscopy. Currently available fibre tracing tools could not track the large numbers of fibres or tolerate the artefacts present in biological imaging data. Using our algorithm, we have reconstructed and characterised the geometrical properties of the collagen fibrils (length, width, alignment, area, location). This algorithm could help to answer questions in biology which relate tissue microstructure to function in areas such as ageing, disease, development and injury. It could also be used to study engineered materials and textiles and is available freely to download.

Automated synapse-level reconstruction of neural circuits in the larval zebrafish brain

Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm3. We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system.

Serial block face scanning electron microscopy reveals region-dependent remodelling of transverse tubules post-myocardial infarction.

The highly organized transverse tubule (t-tubule) network facilitates cardiac excitation–contraction coupling and synchronous cardiac myocyte contraction. In cardiac failure secondary to myocardial infarction (MI), changes in the structure and organization of t-tubules result in impaired cardiac contractility. However, there is still little knowledge on the regional variation of t-tubule remodelling in cardiac failure post-MI. Here, we investigate post-MI t-tubule remodelling in infarct border and remote regions, using serial block face scanning electron microscopy (SBF-SEM) applied to a translationally relevant sheep ischaemia reperfusion MI model and matched controls. We performed minimally invasive coronary angioplasty of the left anterior descending artery, followed by reperfusion after 90 min to establish the MI model. Left ventricular tissues obtained from control and MI hearts eight weeks post-MI were imaged using SBF-SEM. Image analysis generated three-dimensional reconstructions of the t-tubular network in control, MI border and remote regions. Quantitative analysis revealed that the MI border region was characterized by t-tubule depletion and fragmentation, dilation of surviving t-tubules and t-tubule elongation. This study highlights region-dependent remodelling of the tubular network post-MI and may provide novel localized therapeutic targets aimed at preservation or restoration of the t-tubules to manage cardiac contractility post-MI.This article is part of the theme issue ‘The cardiomyocyte: new revelations on the interplay between architecture and function in growth, health, and disease’.

Biogenesis and release of endothelial extracellular vesicles: Morphological aspects

BackgroundCell-cell communication through extracellular vesicles (EVs) including exosomes, microvesicles and apoptotic bodies has been shown to be important in physiological homoeostasis as well as pathological processes such as atherosclerosis. However, while the cellular machinery controlling EV formation and composition has been studied during the past decade, less is known about the morphological process of their formation and release. MethodsUsing different electron microscopic approaches including transmission-, scanning-, immun-, and serial block face electron microscopy we studied the morphogenetic events of EV formation and release. We analysed the different steps of EV formation and release in cultured myocardial endothelial (MyEnd) and aortic endothelial (AoEnd) cell lines under serum starvation and under inflammatory conditions. ResultsWe show that in a narrow time frame, the number of active cells and microvesicle (MV) producing cells increased in dependence of time spent in cultivation and additional stimulation by TNF-α. However, MV secretion was a highly heterogeneous process which couldn´t be seen in all cells cultivated under the same conditions. Release of MVs could be observed all over the cells’ surface with no preferred release site. While no single specific microscopic approach applied was sufficient to provide a comprehensive analysis of EV biogenesis, we show that the limitations of one technique could be compensated by the qualities of the respective other applied techniques, thus enabling us to provide a detailed ultrastructural analysis of MV and exosome biogenesis. Surprisingly, exosome release in endothelial cells occurred via a yet undescribed process indicating that MVBs were incorporated into a novel distinct cellular compartment covered by fenestrated endothelium before exosome release. Lastly, we could show that TNF-α stimulation of AoEnd cells leads not only to the upregulation of CD44 in parental cells, but also to incorporation of CD44 into the membranes of generated MVs and exosomes. ConclusionsTaken together, our data contribute to a better understanding of biogenesis and release of EVs. We conclude that under inflammatory conditions, EVs can mediate the transfer of CD44 from endothelial cells to target cells at distant sites including vessel wall cells and this could be a mechanism by which MVs may change the and thus contribute to the development and progression of atherosclerotic lesions.

Synaptic inputs to displaced intrinsically-photosensitive ganglion cells in macaque retina

Ganglion cells are the projection neurons of the retina. Intrinsically photosensitive retinal ganglion cells (ipRGCs) express the photopigment melanopsin and also receive input from rods and cones via bipolar cells and amacrine cells. In primates, multiple types of ipRGCs have been identified. The ipRGCs with somas in the ganglion cell layer have been studied extensively, but less is known about those with somas in the inner nuclear layer, the “displaced” cells. To investigate their synaptic inputs, three sets of horizontal, ultrathin sections through central macaque retina were collected using serial block-face scanning electron microscopy. One displaced ipRGC received nearly all of its excitatory inputs from ON bipolar cells and would therefore be expected to have ON responses to light. In each of the three volumes, there was also at least one cell that had a large soma in the inner nuclear layer, varicose axons and dendrites with a large diameter that formed large, extremely sparse arbor in the outermost stratum of the inner plexiform layer. They were identified as the displaced M1 type of ipRGCs based on this morphology and on the high density of granules in their somas. They received extensive input from amacrine cells, including the dopaminergic type. The vast majority of their excitatory inputs were from OFF bipolar cells, including two subtypes with extensive input from the primary rod pathway. They would be expected to have OFF responses to light stimuli below the threshold for melanopsin or soon after the offset of a light stimulus.