Serial section electron tomography of a Leishmania haptomonad-like cell attached to plastic
Ontology highlight
ABSTRACT:
SUBMITTER: Jack Daniel Sunter
PROVIDER: EMPIAR-11468 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell level.
| S-EPMC3369938 | biostudies-literature
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| EMPIAR-11464 | biostudies-other
Project description:Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identify neurons, and developed a protocol that enhances the electron-density of the labeled cells while retaining the quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by two orders of magnitude, facilitating directed inquiry of circuit motifs.
| S-EPMC4959841 | biostudies-literature
Project description:Automated tape-collecting ultramicrotomy in conjunction with scanning electron microscopy (SEM) is a powerful approach for volume electron microscopy and three-dimensional neuronal circuit analysis. Current tapes are limited by section wrinkle formation, surface scratches and sample charging during imaging. Here we show that a plasma-hydrophilized carbon nanotube (CNT)-coated polyethylene terephthalate (PET) tape effectively resolves these issues and produces SEM images of comparable quality to those from transmission electron microscopy. CNT tape can withstand multiple rounds of imaging, offer low surface resistance across the entire tape length and generate no wrinkles during the collection of ultrathin sections. When combined with an enhanced en bloc staining protocol, CNT tape-processed brain sections reveal detailed synaptic ultrastructure. In addition, CNT tape is compatible with post-embedding immunostaining for light and electron microscopy. We conclude that CNT tape can enable high-resolution volume electron microscopy for brain ultrastructure analysis.
| S-EPMC5789869 | biostudies-other
Project description:Dendrites, axons, and synapses are dynamic during circuit development; however, changes in microcircuit connections as branches stabilize have not been directly demonstrated. By combining in vivo time-lapse imaging of Xenopus tectal neurons with electron microscope reconstructions of imaged neurons, we report the distribution and ultrastructure of synapses on individual vertebrate neurons and relate these synaptic properties to dynamics in dendritic and axonal arbor structure over hours or days of imaging. Dynamic dendrites have a high density of immature synapses, whereas stable dendrites have sparser, mature synapses. Axons initiate contacts from multisynapse boutons on stable branches. Connections are refined by decreasing convergence from multiple inputs to postsynaptic dendrites and by decreasing divergence from multisynapse boutons to postsynaptic sites. Visual deprivation or NMDAR antagonists decreased synapse maturation and elimination, suggesting that coactive input activity promotes microcircuit development by concurrently regulating synapse elimination and maturation of remaining contacts.
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Project description:In order to understand the degradation potential of plastics in the marine environment, microorganisms that preferentially colonize and interact with plastic surfaces, as opposed to generalists potentially colonising everything, need to be identified. Accordingly, it was hypothesized that i.) plastic "specific" microorganisms are closely attached to the polymeric surface and ii.) that specificity of plastics biofilms are rather related to members of the rare biosphere. To answer these hypotheses, a three phased experiment to stepwise uncover closely attached microbes was conducted. In Phase 1, nine chemically distinct plastic films and glass were incubated in situ for 21 months in a seawater flow through system. In Phase 2, a high-pressure water jet treatment technique was used to remove the upper biofilm layers to further, in Phase 3, enrich a plastic "specific" community. To proof whether microbes colonizing different plastics are distinct from each other and from other inert hard substrates, the bacterial communities of these different substrates were analysed using 16S rRNA gene tag sequencing. Our findings indicate that tightly attached microorganisms account to the rare biosphere and suggest the presence of plastic "specific" microorganisms/assemblages which could benefit from the given plastic properties or at least grow under limited carbon resources.
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Project description: Not available
| EMPIAR-10353 | biostudies-other
Project description:The analysis of microcircuitry (the connectivity at the level of individual neuronal processes and synapses), which is indispensable for our understanding of brain function, is based on serial transmission electron microscopy (TEM) or one of its modern variants. Due to technical limitations, most previous studies that used serial TEM recorded relatively small stacks of individual neurons. As a result, our knowledge of microcircuitry in any nervous system is very limited. We applied the software package TrakEM2 to reconstruct neuronal microcircuitry from TEM sections of a small brain, the early larval brain of Drosophila melanogaster. TrakEM2 enables us to embed the analysis of the TEM image volumes at the microcircuit level into a light microscopically derived neuro-anatomical framework, by registering confocal stacks containing sparsely labeled neural structures with the TEM image volume. We imaged two sets of serial TEM sections of the Drosophila first instar larval brain neuropile and one ventral nerve cord segment, and here report our first results pertaining to Drosophila brain microcircuitry. Terminal neurites fall into a small number of generic classes termed globular, varicose, axiform, and dendritiform. Globular and varicose neurites have large diameter segments that carry almost exclusively presynaptic sites. Dendritiform neurites are thin, highly branched processes that are almost exclusively postsynaptic. Due to the high branching density of dendritiform fibers and the fact that synapses are polyadic, neurites are highly interconnected even within small neuropile volumes. We describe the network motifs most frequently encountered in the Drosophila neuropile. Our study introduces an approach towards a comprehensive anatomical reconstruction of neuronal microcircuitry and delivers microcircuitry comparisons between vertebrate and insect neuropile.
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Project description:We describe an approach for automation of the process of reconstruction of neural tissue from serial section transmission electron micrographs. Such reconstructions require 3D segmentation of individual neuronal processes (axons and dendrites) performed in densely packed neuropil. We first detect neuronal cell profiles in each image in a stack of serial micrographs with multi-scale ridge detector. Short breaks in detected boundaries are interpolated using anisotropic contour completion formulated in fuzzy-logic framework. Detected profiles from adjacent sections are linked together based on cues such as shape similarity and image texture. Thus obtained 3D segmentation is validated by human operators in computer-guided proofreading process. Our approach makes possible reconstructions of neural tissue at final rate of about 5 microm3/manh, as determined primarily by the speed of proofreading. To date we have applied this approach to reconstruct few blocks of neural tissue from different regions of rat brain totaling over 1000microm3, and used these to evaluate reconstruction speed, quality, error rates, and presence of ambiguous locations in neuropil ssTEM imaging data.
| S-EPMC2948845 | biostudies-literature