Subtomogram analysis using the Volta phase plate.

Quantifying resolution limiting factors in subtomogram averaged cryo-electron tomography using simulations.

Single particle tomography in EMAN2.

Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR—a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent (“white box”) approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available.

47] Preparation of a nuclear matrix-pore complex-lamina fraction from embryos of Drosophila melanogaster

Workplace Monitoring of Refractory Ceramic Fiber in the United States

11 Drug-induced and toxic granulomatous hepatitis

Microscopy has always been an important tool in the investigation of the structure and growth of bacteria that possess a developmental life cycle. Prokaryotic predators are no exception. Phase contrast light microscopy and transmission electron microscopy were utilized in the 1960s to describe Bdellovibrio bacteriovorus, the first Bdellovibrio and like organism (BALO). New prokaryotic predators continue to be isolated to the present day. At the same time, new techniques in microscopy have been developed that allow researchers to ask ever more fundamental questions about the periplasmic and epibiotic life cycles of BALOs. This review outlines current methodologies available in light, electron and fluorescence microscopy and provides examples of their use in the study of BALOs. It is our fitting tribute to R.G.E. Murray.

In this work, non-Lorentz, Lorentz transmission electron microscopy (LTEM) and differential phase contrast (DPC) methods were used to observe the phase and magnetic structure of the alnico magnet. All observations were performed on alnico-5 in transverse orientation. The results showed the magnetic contrast of the domain walls in the conventional TEM images despite the effects of the lens magnetic field, which was verified and explained using DPC. Experimental methodology and sample results are summarized with advice for further research.

Morphologic, cytometric and functional characterisation of abalone ( Haliotis tuberculata) haemocytes

On the life history and functional morphology of Halipegus occidualis (Trematoda: Hemiuridae), with emphasis on the cystophorous cercaria stage

There is increasing evidence that fibre size and shape play an important part in the production of health effects related to asbestos. The dimensions of airborne fibres collected at various stages of fibre processing in three mines and six mills producing three types of asbestos were measured using phase contrast light microscopy and transmission electron microscopy. Airborne fibres of different asbestos types had appreciably different size and shape distributions. For a given asbestos type, fibres collected at different stages of processing differed in their size distributions but the differences were considerably less than between fibre types. Most of the airborne fibres to which miners and millers were exposed were short and thin and thus respirable. The physical properties which best differentiated crocidolite fibres from other types of asbestos and which had higher values determined for crocidolite fibres than those obtained for the other types, were median aspect ratio and the proportion of long thin fibres--that is, fibres less than or equal to 0.2 micron in diameter and greater than 5 micron in length as the percentage of total fibres. The median true diameter of amosite fibres was about four and three times higher than the median true diameters of chrysotile and crocidolite fibers respectively. The median true length of amosite fibres was more than four and two times higher than the median true lengths of chrysotile and crocidolite fibres respectively. The observed differences in size and shape of airborne fibres have important implications for the setting of work environmental standards and in explaining differences in health risks associated with different types of fibre.

Cryo-electron tomography (cryo-ET) has emerged as a powerful technique for elucidating the structures of cellular organelles and large protein complexes in their native environments. Unlike conventional room-temperature electron microscopy, cryo-ET minimizes radiation-induced and preparation-induced damage to internal structures. Moreover, in contrast to single-particle analysis (SPA), cryo-ET reduces the need for repetitive purification, thereby preserving the most authentic structural information of the samples. To achieve high-resolution imaging, cryo-ET also imposes stringent requirements on the imaging conditions of the TEM. Despite the continuous improvement in cryo-ET data acquisition efficiency and the emergence of numerous software platforms, most solutions are proprietary, vendor-specific, and tightly coupled to certain electron microscope models, leading to high implementation costs. In this study, we installed the open-source software SerialEM on a 200 kV electron microscope (Glacios 2 cryo-TEM) with a dual condenser lens system and performed comprehensive calibrations to enable high-quality tomographic data acquisition. Using apoferritin as a model specimen, we describe the entire workflow-from cryo-sample preparation to automated cryo-ET data collection, followed by tomogram reconstruction and sub-tomogram analysis. This method offers a cost-effective and accessible solution for cryo-ET data collection, particularly for laboratories using Glacios microscopes without access to proprietary software.

Human Immunodeficiency virus type 1 (HIV-1) is a lentivirus that infects non-dividing cells of the immune system. In non-dividing cells, nuclear import of the viral genome occurs through the nuclear pore complex (NPC), a large macromolecular assembly that forms a channel of ~40 nm across the nuclear envelope. This process requires interactions between NPC components known as nucleoporins and CA (capsid) proteins of HIV-1 post-fusion complexes. In mature HIV-1 virions, multiple copies of CA assemble into a lattice of hexamers and pentamers, forming a cone-shaped capsid core of ~60 nm in width and ~110 nm in length encasing the viral genome. Because of its large size, it was generally believed that the capsid core entirely or partially disassembled prior to its translocation through the NPC. However, nuclear entry and uncoating are rare events and challenging to characterize and therefore subject to a long-standing debate. Here, cryo-electron tomography (cryo-ET) on cryo-focused ion beam (FIB) milled T-cells was combined with subtomogram averaging (SA) to study the ultrastructure of HIV-1 capsids in the process of nuclear entry. Using a HIV-1 variant arrested at the nuclear pore due to a defect in binding of the CA lattice to the host cell protein CPSF6, snapshots of HIV-1 nuclear entry at multiple stages were captured. Reverse transcription-competent HIV-1 complexes were identified in the cytosol, docking to and within the NPC, and in the nucleoplasm of T cells. Surprisingly, in the cytosol and at the NPC, the viral complexes retained cone-shaped capsids highly resembling in size and geometry the mature capsid cores within intact virions. The density at their interior suggested that they were associated with the viral genome. The cone-shape capsids deeply enter into the NPC central channel with their narrow ends. The hexameric lattices of these capsids were intact or nearly intact. These findings argued against the current models of uncoating in the cytosol or at the NPC and rather supported translocation of intact HIV-1 capsids through the NPC. Instead, HIV-1 capsids uncoat in the nucleus of T cells. Inside the nucleus, tube-shape fragments still containing few lattice elements were observed. The lack of density at their inside suggested that the viral genome was released from these complexes. Uncoating may thus occur through the partial opening and remodeling of the hexameric lattices of HIV-1 capsids, rather than a step-wise disassembly. To address how an intact HIV-1 capsid can enter the central channel of NPC, the human NPC from T cells was structurally analyzed in cellulo by SA. The NPC overall structure was not altered upon infection but rather dilated in comparison to the structure previously obtained from purified envelopes. The diameter of the central channel of NPC in T cells corresponded to ~64 nm, suggesting that the translocation of intact HIV-1 capsid through the NPC is geometrically possible. The dilated conformation of NPCs in T cells is independent of HIV-1 infection, but represents a physiological condition of actively transporting NPCs.

Positioning of CCA-arms of the A- and the P-tRNAs towards the 28S rRNA in the human ribosome

Electron tomography is a method for three-dimensional reconstruction of a specimen, based on its electron microscopical projections which are produced by tilting the specimen in the microscope about a fixed axis. After determining a common coordinate frame, a tomogram is computed by weighted backprojection. Higher resolution can be obtained by applying a contrast transfer function (CTF) correction to the projections. The tomographic data is further analyzed by extracting structural motifs from the raw tomograms, by selective alignment, multivariate statistical analysis, classification, and class-averaging, resulting in an increased signal-to-noise ratio and substantial data reduction. The volumetric data analysis facilitates the separation of a heterogeneous population of motifs into its constituents, and the visualization of the motifs in the raw tomograms by reassembly, replacing low signal-to-noise instances with higher signal-to-noise representations