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FAST-EM array tomography data of rat pancreas prepared with rOTO protocol and stained with neodymium acetate

ABSTRACT:

SUBMITTER: Arent Kievits

PROVIDER: EMPIAR-12193 | biostudies-other |

REPOSITORIES: biostudies-other

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FAST-EM array tomography: a workflow for multibeam volume electron microscopy.

Kievits Arent J AJ Duinkerken B H Peter BHP Lane Ryan R de Heus Cecilia C van Beijeren Bergen En Henegouwen Daan D Höppener Tibbe T Wolters Anouk H G AHG Liv Nalan N Giepmans Ben N G BNG Hoogenboom Jacob P JP

Methods in microscopy 20240401 1

Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel wit ...[more]

PMID: 39119255

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FAST-EM array tomography data of rat pancreas prepared with rOTO protocol and stained with neodymium acetate

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FAST-EM array tomography: a workflow for multibeam volume electron microscopy.

Project description:Elucidating the 3D nanoscale structure of tissues and cells is essential for understanding the complexity of biological processes. Electron microscopy (EM) offers the resolution needed for reliable interpretation, but the limited throughput of electron microscopes has hindered its ability to effectively image large volumes. We report a workflow for volume EM with FAST-EM, a novel multibeam scanning transmission electron microscope that speeds up acquisition by scanning the sample in parallel with 64 electron beams. FAST-EM makes use of optical detection to separate the signals of the individual beams. The acquisition and 3D reconstruction of ultrastructural data from multiple biological samples is demonstrated. The results show that the workflow is capable of producing large reconstructed volumes with high resolution and contrast to address biological research questions within feasible acquisition time frames.

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Fast acquisition protocol for X-ray scattering tensor tomography.

Project description:Microstructural information over an entire sample is important to understand the macroscopic behaviour of materials. X-ray scattering tensor tomography facilitates the investigation of the microstructural organisation in statistically large sample volumes. However, established acquisition protocols based on scanning small-angle X-ray scattering and X-ray grating interferometry inherently require long scan times even with highly brilliant X-ray sources. Recent developments in X-ray diffractive optics towards circular pattern arrays enable fast single-shot acquisition of the sample scattering properties with 2D omnidirectional sensitivity. X-ray scattering tensor tomography with the use of this circular grating array has been demonstrated. We propose here simple yet inherently rapid acquisition protocols for X-ray scattering tensor tomography leveraging on these new optical elements. Results from both simulation and experimental data, supported by a null space analysis, suggest that the proposed acquisition protocols are not only rapid but also corroborate that sufficient information for the accurate volumetric reconstruction of the scattering properties is provided. The proposed acquisition protocols will build the basis for rapid inspection and/or time-resolved tensor tomography of the microstructural organisation over an extended field of view.

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Electron tomography of negatively stained complex viruses: application in their diagnosis.

Project description:BackgroundElectron tomographic analysis can be combined with the simple and rapid negative staining technique used in electron microscopy based virus diagnosis.MethodsStandard negative staining of representative examples of parapoxviruses and paramyxoviruses was combined with electron tomographic analysis.ResultsDigital sectioning of reconstructions of these viruses at a selected height demonstrated the viral ultrastructure in detail, including the characteristic diagnostic features like the surface threads on C-particles of a parapoxvirus and individual glycoproteins and the internal nucleoprotein strand of Newcastle disease virus. For both viruses, deformation and flattening were observed.ConclusionThe combination of negative staining of complex viruses with electron tomographic analysis, allows visualizing and measuring artifacts typical for negative staining. This approach allows sharp visualisation of structures in a subnanometer-thick plane, avoiding blurring due to superposition which is inherent to TEM. In selected examples, such analyses can improve diagnosis of viral agents.

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Porosome in the Exocrine Pancreas: A Detailed EM Study suppressor.

Project description:A major question in cell biology that accumulation of partially empty vesicles in cells following secretion is seen, while it is believed that secretion occurs via the complete merger of secretory vesicles with the cell plasma membrane. This important question was solved nearly two decades ago, with the discovery of the Porosome. Porosomes are cup-shaped lipoprotein structures found at the plasma membrane of all cells. Secretory vesicles dock and transiently fuse at the porosome base to form a continuous channel or fusion pore to release the pressurized vesicle contents to the outside. In a decade-long study by our group, we carefully examined using electron microscopy, the detailed structure of the porosome complex in acinar cells of the exocrine pancreas. Besides conformation of earlier findings, our study provides in much greater detail, the in situ morphology of the porosome complex in the exocrine pancreas. The discovery of the detailed morphology of the exocrine pancreas porosome complex in my laboratory is one of the major highlights of my academic career spanning nearly 50 years. Results from our EM studies, reveal for the first time the presence of tethers or cables, which are likely t-SNAREs, present at the porosome base. These EM studies further demonstrate for the first time the docking of a single secretory vesicle or zymogen granule at the base of more than one porosome complex. Detailed spoke-like elements lining the porosome cup were also observed for the first time in these studies, greatly advancing our understanding of the molecular architecture and physiology of the porosome in the exocrine pancreas.

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Project description:Pancreas divisum (PD) is the most common developmental anatomic variant of pancreatic duct. Endoscopic ultrasound (EUS) is often performed to evaluate idiopathic pancreatitis and has been shown to have high accuracy in diagnosis of PD. The different techniques to identify PD by linear EUS have been described differently by different authors. If EUS is done with a proper technique it can be a valuable tool in the diagnosis of PD. The anatomical and technical background of different signs has not been described so far. This article summarizes the different techniques of imaging of pancreatic duct in a suspected case of PD and gives a technical explanation of various signs. The common signs seen during evaluation of pancreatic duct in PD are stack sign of linear EUS, crossed duct sign on linear EUS, the dominant duct and ventral dorsal duct (VD) transition. Few other signs are described which include duct above duct, short ventral duct /absent ventral duct, separate opening of ducts with no communication, separate opening of ducts with filamentous communication, stacking of duct of Santorini and indirect signs like santorinecele. The principles of the sign have been explained on an anatomical basis and the techniques and the principles described in the review will be helpful in technical evaluation of PD during EUS.

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Identification of leishmania species using PCR assay on giemsa-stained slides prepared from cutaneous leishmaniasis patients.

Project description:BackgroundLeishmaniasis is a group of diseases that are created by intracellular parasites of Leishmania. Cutaneous leishmaniasis is considered as one of the health problems in some provinces of Iran.MethodsIn this study, a total of 178 Giemsa-stained slides from confirmed cases of cutaneous leishmaniasis were examined. The slides were prepared from the patients with cutaneous leishmaniasis that referred to health centers and infected during the epidemic of cutaneous leishmaniasis in Poldokhtar city, Lorestan Province, Iran in 2006.Genomic DNA from each slide was extracted. After DNA extraction, ITS-PCR was used.ResultsOut of 178 slides, 129 (72.47%) samples had a band in the range of 485 bp and 49 (27.53%) samples 626 bp that matched L. tropica and L. major standard samples, respectively.ConclusionThis study showed that Leishmania DNA could be efficiently extracted and amplified even from old Giemsa-stained microscopic slides that were stored more than 6 yr. In this study was shown that both L. tropica and L. major species exist in Lorestan Province.

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Fast segmentation of stained nuclei in terabyte-scale, time resolved 3D microscopy image stacks.

Project description:Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu's method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm's superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results.

| S-EPMC3937404 | biostudies-literature

Fast viral dynamics revealed by microsecond time-resolved cryo-EM.

Project description:Observing proteins as they perform their tasks has largely remained elusive, which has left our understanding of protein function fundamentally incomplete. To enable such observations, we have recently proposed a technique that improves the time resolution of cryo-electron microscopy (cryo-EM) to microseconds. Here, we demonstrate that microsecond time-resolved cryo-EM enables observations of fast protein dynamics. We use our approach to elucidate the mechanics of the capsid of cowpea chlorotic mottle virus (CCMV), whose large-amplitude motions play a crucial role in the viral life cycle. We observe that a pH jump causes the extended configuration of the capsid to contract on the microsecond timescale. While this is a concerted process, the motions of the capsid proteins involve different timescales, leading to a curved reaction path. It is difficult to conceive how such a detailed picture of the dynamics could have been obtained with any other method, which highlights the potential of our technique. Crucially, our experiments pave the way for microsecond time-resolved cryo-EM to be applied to a broad range of protein dynamics that previously could not have been observed. This promises to fundamentally advance our understanding of protein function.

| S-EPMC10499870 | biostudies-literature

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