562 research outputs found
A convolutional autoencoder approach for mining features in cellular electron cryo-tomograms and weakly supervised coarse segmentation
Cellular electron cryo-tomography enables the 3D visualization of cellular
organization in the near-native state and at submolecular resolution. However,
the contents of cellular tomograms are often complex, making it difficult to
automatically isolate different in situ cellular components. In this paper, we
propose a convolutional autoencoder-based unsupervised approach to provide a
coarse grouping of 3D small subvolumes extracted from tomograms. We demonstrate
that the autoencoder can be used for efficient and coarse characterization of
features of macromolecular complexes and surfaces, such as membranes. In
addition, the autoencoder can be used to detect non-cellular features related
to sample preparation and data collection, such as carbon edges from the grid
and tomogram boundaries. The autoencoder is also able to detect patterns that
may indicate spatial interactions between cellular components. Furthermore, we
demonstrate that our autoencoder can be used for weakly supervised semantic
segmentation of cellular components, requiring a very small amount of manual
annotation.Comment: Accepted by Journal of Structural Biolog
3D ultrastructural organization of whole Chlamydomonas reinhardtii cells studied by nanoscale soft x-ray tomography
The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology
The promise and the challenges of cryo-electron tomography
Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-electron tomography (Cryo-ET) combines the power of 3D molecular-level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here, we review state-of-the-art Cryo-ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo-ET
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3D Ultrastructure of the Cochlear Outer Hair Cell Lateral Wall Revealed By Electron Tomography.
Outer Hair Cells (OHCs) in the mammalian cochlea display a unique type of voltage-induced mechanical movement termed electromotility, which amplifies auditory signals and contributes to the sensitivity and frequency selectivity of mammalian hearing. Electromotility occurs in the OHC lateral wall, but it is not fully understood how the supramolecular architecture of the lateral wall enables this unique form of cellular motility. Employing electron tomography of high-pressure frozen and freeze-substituted OHCs, we visualized the 3D structure and organization of the membrane and cytoskeletal components of the OHC lateral wall. The subsurface cisterna (SSC) is a highly prominent feature, and we report that the SSC membranes and lumen possess hexagonally ordered arrays of particles. We also find the SSC is tightly connected to adjacent actin filaments by short filamentous protein connections. Pillar proteins that join the plasma membrane to the cytoskeleton appear as variable structures considerably thinner than actin filaments and significantly more flexible than actin-SSC links. The structurally rich organization and rigidity of the SSC coupled with apparently weaker mechanical connections between the plasma membrane (PM) and cytoskeleton reveal that the membrane-cytoskeletal architecture of the OHC lateral wall is more complex than previously appreciated. These observations are important for our understanding of OHC mechanics and need to be considered in computational models of OHC electromotility that incorporate subcellular features
3D-surface reconstruction of cellular cryo-soft X-ray microscopy tomograms using semi-supervised deep learning
Cryo-soft X-ray tomography (cryo-SXT) is a powerful method to investigate the ultrastructure of cells, offering resolution in the tens of nm range and strong contrast for membranous structures without requirement for labeling or chemical fixation. The short acquisition time and the relatively large volumes acquired allow for fast acquisition of large amounts of tomographic image data. Segmentation of these data into accessible features is a necessary step in gaining biologically relevant information from cryo-soft X-ray tomograms. However, manual image segmentation still requires several orders of magnitude more time than data acquisition. To address this challenge, we have here developed an end-to-end automated 3D-segmentation pipeline based on semi-supervised deep learning. Our approach is suitable for high-throughput analysis of large amounts of tomographic data, while being robust when faced with limited manual annotations and variations in the tomographic conditions. We validate our approach by extracting three-dimensional information on cellular ultrastructure and by quantifying nanoscopic morphological parameters of filopodia in mammalian cells
Compressed sensing electron tomography of needle-shaped biological specimens--Potential for improved reconstruction fidelity with reduced dose.
Electron tomography is an invaluable method for 3D cellular imaging. The technique is, however, limited by the specimen geometry, with a loss of resolution due to a restricted tilt range, an increase in specimen thickness with tilt, and a resultant need for subjective and time-consuming manual segmentation. Here we show that 3D reconstructions of needle-shaped biological samples exhibit isotropic resolution, facilitating improved automated segmentation and feature detection. By using scanning transmission electron tomography, with small probe convergence angles, high spatial resolution is maintained over large depths of field and across the tilt range. Moreover, the application of compressed sensing methods to the needle data demonstrates how high fidelity reconstructions may be achieved with far fewer images (and thus greatly reduced dose) than needed by conventional methods. These findings open the door to high fidelity electron tomography over critically relevant length-scales, filling an important gap between existing 3D cellular imaging techniques.The research leading to these results has received funding from the European Union Seventh Framework Programme under Grant Agreement 312483 - ESTEEM2 (Integrated Infrastructure Initiative–I3), as well as from the European Research Council under the European Union’s Seventh Framework Programme (FP/2007-2013)/ERC grant agreement 291522 - 3DIMAGE. B.W. and E.S. acknowledge financial support from the Deutsche Forschungsgemeinschaft (DFG) within the framework of the SPP 1570 as well as through the Cluster of Excellence “Engineering of Advanced Materials” at the Friedrich-Alexander-Universität ErlangenNürnberg. G.D. and C.D. acknowledge funding from the ERC under grant number 259619 PHOTO EM. B.W. acknowledges the Research Training Group “Disperse Systems for Electronic Applications” (DFG GEPRIS GRK 1161). R.L. acknowledges a Junior Research Fellowship from Clare College.This is the final version of the article. It first appeared from Elsevier via http://dx.doi.org/10.1016/j.ultramic.2015.10.02
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