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Wild-type, full-length (40- and 42-residue) amyloid β-peptide (Aβ) fibrils have been shown by a variety of magnetic resonance techniques to contain cross-β structures in which the β-sheets have an in-register parallel supramolecular organization. In contrast, recent studies of fibrils formed in vitro by the Asp23-to-Asn mutant of 40-residue Aβ (D23N-Aβ1–40), which is associated with early onset neurodegeneration, indicate that D23N-Aβ1–40 fibrils can contain either parallel or antiparallel β-sheets. We report a protocol for producing structurally pure antiparallel D23N-Aβ1–40 fibril samples and a series of solid state nuclear magnetic resonance and electron microscopy measurements that lead to a specific model for the antiparallel D23N-Aβ1–40 fibril structure. This model reveals how both parallel and antiparallel cross-β structures can be constructed from similar peptide monomer conformations and stabilized by similar sets of interactions, primarily hydrophobic in nature. We find that antiparallel D23N-Aβ1–40 fibrils are thermodynamically metastable with respect to conversion to parallel structures, propagate less efficiently than parallel fibrils in seeded fibril growth, and therefore must nucleate more efficiently than parallel fibrils in order to be observable. Experiments in neuronal cell cultures indicate that both antiparallel and parallel D23N-Aβ1–40 fibrils are cytotoxic. Thus, our antiparallel D23N-Aβ1–40 fibril model represents a specific \"toxic intermediate\" in the aggregation process of a disease-associated Aβ mutant.

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins.

Cryoelectron tomography provides unprecedented insights into the macromolecular and supramolecular organization of cells in a close-to-living state. However because of the limited thickness range (< 0.5–1 μm) that is accessible with today’s intermediate voltage electron microscopes only small prokaryotic cells or peripheral regions of eukaryotic cells can be examined directly. Key to overcoming this limitation is the ability to prepare sufficiently thin samples. Cryosectioning can be used to prepare thin enough sections but suffers from severe artefacts, such as substantial compression. Here we describe a procedure, based upon focused ion beam (FIB) milling for the preparation of thin (200–500 nm) lamellae from vitrified cells grown on electron microscopy (EM) grids. The self-supporting lamellae are apparently free of distortions or other artefacts and open up large windows into the cell’s interior allowing tomographic studies to be performed on any chosen part of the cell. We illustrate the quality of sample preservation with a structure of the nuclear pore complex obtained from a single tomogram.

Modeling the 3D structures of cells and tissues is crucial in biology. Sequential cross-sectional images from electron microscopy provide high-resolution intracellular structure information. The segmentation of complex cell structures remains a laborious manual task for experts, demanding time and effort. This bottleneck in analyzing biological images requires efficient and automated solutions. In this study, the deep learning-based automated segmentation of biological images was explored to enable accurate reconstruction of the 3D structures of cells and organelles. An analysis system for the cell images of Cyanidioschyzon merolae , a primitive unicellular red algae, was constructed. This system utilizes sequential cross-sectional images captured by a focused ion beam scanning electron microscope (FIB-SEM). A U-Net was adopted and training was performed to identify and segment cell organelles from single-cell images. In addition, the segment anything model (SAM) and 3D watershed algorithm were employed to extract individual 3D images of each cell from large-scale microscope images containing numerous cells. Finally, the trained U-Net was applied to segment each structure within these 3D images. Through this procedure, the creation of 3D cell models could be fully automated. The adoption of other deep learning techniques and combinations of image processing methods will also be explored to enhance the segmentation accuracy further.

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results. Preparing biological material for electron microscopy (EM) involves harsh processing steps that can poorly preserve cellular ultrastructure. Here the authors apply a single layer of graphene onto wet cells to enable direct EM using low voltage, and correlate actin filaments and mitochondria using super-resolution microscopy.

The fluorescent proteins mEos4a and mEos4b maintain their fluorescence and photoconversion after fixation with osmium. This property enables applications such as correlative super-resolution and electron microscopy. Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they lose their fluorescence after heavy fixation, hindering applications such as correlative light and electron microscopy (CLEM). Here we report engineered variants of the photoconvertible Eos fluorescent protein that fluoresce and photoconvert normally in heavily fixed (0.5–1% OsO 4 ), plastic resin–embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy.

Mitochondrial functions are intrinsically linked to their morphology and membrane ultrastructure. Characterizing abnormal mitochondrial structural features may thus provide insight into the underlying pathogenesis of inherited and acquired mitochondrial diseases. Following a systematic literature review on ultrastructural defects in mitochondrial myopathy, we investigated skeletal muscle biopsies from seven subjects with genetically defined mtDNA mutations. Mitochondrial ultrastructure and morphology were characterized using two complimentary approaches: transmission electron microscopy (TEM) and serial block face scanning EM (SBF-SEM) with 3D reconstruction. Six ultrastructural abnormalities were identified including i) paracrystalline inclusions, ii) linearization of cristae and abnormal angular features, iii) concentric layering of cristae membranes, iv) matrix compartmentalization, v) nanotunelling and vi) donut-shaped mitochondria. In light of recent molecular advances in mitochondrial biology, these findings reveal novel aspects of mitochondrial ultrastructure and morphology in human tissues with implications for understanding the mechanisms linking mitochondrial dysfunction to disease.

The pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has impacted public health and the world economy and fueled a worldwide race to approve therapeutic and prophylactic agents, but so far there are no specific antiviral drugs. Understanding the biology of the virus is the first step in structuring strategies to combat it, and in this context several studies have been conducted with the aim of understanding the replication mechanism of SARS-CoV-2 in vitro systems. In this work, studies using transmission and scanning electron microscopy and 3D electron microscopy modeling were performed with the goal of characterizing the morphogenesis of SARS-CoV-2 in Vero-E6 cells. Several ultrastructural changes were observed—such as syncytia formation, cytoplasmic membrane projections, lipid droplets accumulation, proliferation of double-membrane vesicles derived from the rough endoplasmic reticulum, and alteration of mitochondria. The entry of the virus into cells occurred through endocytosis. Viral particles were observed attached to the cell membrane and in various cellular compartments, and extrusion of viral progeny took place by exocytosis. These findings allow us to infer that Vero-E6 cells are highly susceptible to SARS-CoV-2 infection as described in the literature and their replication cycle is similar to that described with SARS-CoV and MERS-CoV in vitro models.

Currently, lead (Pb) has become a severe environmental pollutant and fungi hold a promising potential for the remediation of Pb-containing wastewater. The present study showed that Penicillium polonicum was able to tolerate 4 mmol/L Pb(II), and remove 90.3% of them in 12 days through three mechanisms: extracellular immobilization, cell wall adsorption, and intracellular bioaccumulation. In this paper. the three mechanisms were studied by Raman, X-ray diffraction analysis (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM). The results indicated that Pb(II) was immobilized as lead oxalate outside the fungal cell, bound with phosphate, nitro, halide, hydroxyl, amino, and carboxyl groups on the cell wall, precipitated as pyromorphite [Pb 5 (PO 4 ) 3 Cl] on the cell wall, and reduced to Pb(0) inside the cell. These combined results provide a basis for additionally understanding the mechanisms of Pb(II) removal by P. polonicum and developing remediation strategies using this fungus for lead-polluted water.

The extraordinary properties of graphene and carbon nanotubes motivate the development of methods for their use in producing continuous, strong, tough fibres. Previous work has shown that the toughness of the carbon nanotube-reinforced polymer fibres exceeds that of previously known materials. Here we show that further increased toughness results from combining carbon nanotubes and reduced graphene oxide flakes in solution-spun polymer fibres. The gravimetric toughness approaches 1,000 J g −1 , far exceeding spider dragline silk (165 J g −1 ) and Kevlar (78 J g −1 ). This toughness enhancement is consistent with the observed formation of an interconnected network of partially aligned reduced graphene oxide flakes and carbon nanotubes during solution spinning, which act to deflect cracks and allow energy-consuming polymer deformation. Toughness is sensitive to the volume ratio of the reduced graphene oxide flakes to the carbon nanotubes in the spinning solution and the degree of graphene oxidation. The hybrid fibres were sewable and weavable, and could be shaped into high-modulus helical springs. Composite fibres made of polymers reinforced by carbon nanotubes are known for their exceptional toughness. Shin et al . make these composites even tougher, by self-aligning carbon nanotubes and reduced graphene oxide flakes within the polymer matrix.