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ELife Oct 2022Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks...
Electron microscopy of biological tissue has recently seen an unprecedented increase in imaging throughput moving the ultrastructural analysis of large tissue blocks such as whole brains into the realm of the feasible. However, homogeneous, high-quality electron microscopy staining of large biological samples is still a major challenge. To date, assessing the staining quality in electron microscopy requires running a sample through the entire staining protocol end-to-end, which can take weeks or even months for large samples, rendering protocol optimization for such samples to be inefficient. Here, we present an in situ time-lapsed X-ray-assisted staining procedure that opens the 'black box' of electron microscopy staining and allows observation of individual staining steps in real time. Using this novel method, we measured the accumulation of heavy metals in large tissue samples immersed in different staining solutions. We show that the measured accumulation of osmium in fixed tissue obeys empirically a quadratic dependence between the incubation time and sample size. We found that potassium ferrocyanide, a classic reducing agent for osmium tetroxide, clears the tissue after osmium staining and that the tissue expands in osmium tetroxide solution, but shrinks in potassium ferrocyanide reduced osmium solution. X-ray-assisted staining gave access to the in situ staining kinetics and allowed us to develop a diffusion-reaction-advection model that accurately simulates the measured accumulation of osmium in tissue. These are first steps towards staining experiments and simulation-guided optimization of staining protocols for large samples. Hence, X-ray-assisted staining will be a useful tool for the development of reliable staining procedures for large samples such as entire brains of mice, monkeys, or humans.
Topics: Humans; Mice; Animals; Osmium Tetroxide; Osmium; X-Rays; Staining and Labeling; Microscopy, Electron
PubMed: 36263931
DOI: 10.7554/eLife.72147 -
Membranes Oct 2022Liquid membranes based on nanoparticles follow a continuous development, both from obtaining methods and characterization of techniques points of view. Lately, osmium...
Liquid membranes based on nanoparticles follow a continuous development, both from obtaining methods and characterization of techniques points of view. Lately, osmium nanoparticles have been deposited either on flat membranes, with the aim of initiating some reaction processes, or on hollow fiber membranes, with the aim of increasing the contact surface with the phases of the membrane system. This paper presents the obtainment and characterization of a liquid membrane based on osmium nanoparticles (Os-NP) dispersed in decanol (Dol) for the realization of a membrane system with a large contact surface between the phases, but without using a liquid membrane support. The dispersion of osmium nanoparticles in -decanol is carried out by the method of reducing osmium tetroxide with 1-undecenoic acid (UDA). The resulting membrane was characterized by transmission electron microscopy (TEM), scanning electron microscopy (SEM), energy-dispersive spectroscopy analysis (EDAX), thermoanalysis (TG, DSC), Fourier transform infra-red (FTIR) spectroscopy and dynamic light scattering (DLS). In order to increase the mass transfer surface, a design for the membrane system was realized with the dispersion of the membrane through the receiving phase and the dispersion of the source phase through the membrane (DBLM-dispersion bulk liquid membrane). The process performance was tested for the reduction of -nitrophenol (pNP) from the source phase, using sodium tetra-borohydride (NaBH), to -aminophenol (pAP), which was transported and collected in the receiving phase. The obtained results show that membranes based on the dispersion of osmium nanoparticles in -decanol can be used with an efficiency of over 90% for the reduction of -nitrophenol and the separation of -aminophenol.
PubMed: 36295782
DOI: 10.3390/membranes12101024 -
Frontiers in Cell and Developmental... 2022Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must... (Review)
Review
Sample preparation is the novel bottleneck for high throughput correlative light and electron microscopy (CLEM). Protocols suitable for both imaging methods must therefore balance the requirements of each technique. For fluorescence light microscopy, a structure of interest can be targeted using: 1) staining, which is often structure or tissue specific rather than protein specific, 2) dye-coupled proteins or antibodies, or 3) genetically encoded fluorescent proteins. Each of these three methods has its own advantages. For ultrastructural investigation by electron microscopy (EM) resin embedding remains a significant sample preparation approach, as it stabilizes the sample such that it withstands the vacuum conditions of the EM, and enables long-term storage. Traditionally, samples are treated with heavy metal salts prior to resin embedding, in order to increase imaging contrast for EM. This is particularly important for volume EM (vEM) techniques. Yet, commonly used contrasting agents (e.g., osmium tetroxide, uranyl acetate) tend to impair fluorescence. The discovery that fluorescence can be preserved in resin-embedded specimens after mild heavy metal staining was a game changer for CLEM. These so-called in-resin fluorescence protocols present a significant leap forward for CLEM approaches towards high precision localization of a fluorescent signal in (volume) EM data. Integrated microscopy approaches, combining LM and EM detection into a single instrument certainly require such an "all in one" sample preparation. Preserving, or adding, dedicated fluorescence prior to resin embedding requires a compromise, which often comes at the expense of EM imaging contrast and membrane visibility. Especially vEM can be strongly hampered by a lack of heavy metal contrasting. This review critically reflects upon the fundamental aspects of resin embedding with regard to 1) specimen fixation and the physics and chemistry underlying the preservation of protein structure with respect to fluorescence and antigenicity, 2) optimization of EM contrast for transmission or scanning EM, and 3) the choice of embedding resin. On this basis, various existing workflows employing in-resin fluorescence are described, highlighting their common features, discussing advantages and disadvantages of the respective approach, and finally concluding with promising future developments for in-resin CLEM.
PubMed: 35846358
DOI: 10.3389/fcell.2022.866472 -
Applied Microscopy Jun 2020Scanning electron microscopy (SEM) plays a central role in analyzing structures by imaging a large area of brain tissue at nanometer scales. A vast amount of data in the...
Scanning electron microscopy (SEM) plays a central role in analyzing structures by imaging a large area of brain tissue at nanometer scales. A vast amount of data in the large area are required to study structural changes of cellular organelles in a specific cell, such as neurons, astrocytes, oligodendrocytes, and microglia among brain tissue, at sufficient resolution. Array tomography is a useful method for large-area imaging, and the osmium-thiocarbohydrazide-osmium (OTO) and ferrocyanide-reduced osmium methods are commonly used to enhance membrane contrast.Because many samples prepared using the conventional technique without en bloc staining are considered inadequate for array tomography, we suggested an alternative technique using post-staining conventional samples and compared the advantages.
PubMed: 33580409
DOI: 10.1186/s42649-020-00033-8 -
Royal Society Open Science Nov 2017We report an evaluation of the cytotoxicity of a series of electron-deficient (16-electron) half-sandwich precious metal complexes of ruthenium, osmium and iridium...
We report an evaluation of the cytotoxicity of a series of electron-deficient (16-electron) half-sandwich precious metal complexes of ruthenium, osmium and iridium ([Os/Ru(--cymene)(1,2-dicarba--dodecarborane-1,2-dithiolato)] (), [Ir(-pentamethylcyclopentadiene)(1,2-dicarba--dodecarborane-1,2-dithiolato)] (), [Os/Ru(--cymene)(benzene-1,2-dithiolato)] () and [Ir(-pentamethylcyclopentadiene)(benzene-1,2-dithiolato)] ()) towards RAW 264.7 murine macrophages and MRC-5 fibroblast cells. Complexes and were found to be non-cytotoxic. The anti-inflammatory activity of was evaluated in both cell lines after nitric oxide (NO) production and inflammation response induced by bacterial endotoxin lipopolysaccharide (LPS) as the stimulus. All metal complexes were shown to exhibit dose-dependent inhibitory effects on LPS-induced NO production on both cell lines. Remarkably, the two iridium complexes and trigger a full anti-inflammatory response against LPS-induced NO production, which opens up new avenues for the development of non-cytotoxic anti-inflammatory drug candidates with distinct structures and solution chemistry from that of organic drugs, and as such with potential novel mechanisms of action.
PubMed: 29291071
DOI: 10.1098/rsos.170786 -
Bioinorganic Chemistry and Applications 2024X-ray crystallography, spectroscopy, computational methods, molecular docking studies, and DNA-binding studies have been useful in the investigations of intermolecular...
Molecular Structure, Spectroscopic, Frontier Molecular Orbital Analysis, Molecular Docking Studies, and DNA-Binding Studies of Osmium(II)-Cymene Complexes with Aryl Phosphine and Aryl Phosphonium Assemblies.
X-ray crystallography, spectroscopy, computational methods, molecular docking studies, and DNA-binding studies have been useful in the investigations of intermolecular and intramolecular interactions of osmium-cymene oxalato complexes with aryl phosphine and aryl phosphonium groups in both primary and secondary coordination spheres, respectively. Molecular structures of the novel complexes PPh[Os(--cymene)Br(--CO)] () and [Os(--cymene) (--CO)PPh] () were resolved by single-crystal X-ray diffraction (XRD). Primary and secondary coordination sphere contacts were investigated using Hirshfeld surface analysis which was supported by molecular docking (MD) studies. The MD data obtained predicted significant differences in binding energy across three receptors for the two osmium complexes. An DNA-binding study was accomplished using UV-Vis spectroscopy which showed that both and bond with DNA through an intercalation approach. The optimized molecular geometry, frontier molecular orbital (E and E) energies, global electrophilicity index (), chemical hardness (), chemical potential (), and the energy band gap (E-E) were calculated utilizing density functional theory (DFT) methods. Computed structural parameters (bond lengths and angles) support the experimental single-crystal XRD data.
PubMed: 38840845
DOI: 10.1155/2024/6697523 -
Biology May 2021The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates...
The glycocalyx (GCX), a pericellular carbohydrate rich hydrogel, forms a selective barrier that shields the cellular membrane, provides mechanical support, and regulates the transport and diffusion of molecules. The GCX is a fragile structure, making it difficult to study by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM). Sample preparation by conventional chemical fixation destroys the GCX, giving a false impression of its organization. An additional challenge is to process the GCX in a way that preserves its morphology and enhanced antigenicity to study its cell-specific composition. The aim of this study was to provide a protocol to preserve both antigen accessibility and the unique morphology of the GCX. We established a combined high pressure freezing (HPF), osmium-free freeze substitution (FS), rehydration, and pre-embedding immunogold labeling method for TEM. Our results showed specific immunogold labeling of GCX components expressed in human monocytic THP-1 cells, hyaluronic acid receptor (CD44) and chondroitin sulfate (CS), and maintained a well-preserved GCX morphology. We adapted the protocol for antigen localization by CLSM and confirmed the specific distribution pattern of GCX components. The presented combination of HPF, FS, rehydration, and immunolabeling for both TEM and CLSM offers the possibility for analyzing the morphology and composition of the unique GCX structure.
PubMed: 34064459
DOI: 10.3390/biology10050402 -
Materials (Basel, Switzerland) Jun 2022Lead sludge from copper production is a source of rare metals, such as rhenium and osmium, whose content reaches 0.06-0.08% and 0.0025-0.0050%, respectively. The base of...
Lead sludge from copper production is a source of rare metals, such as rhenium and osmium, whose content reaches 0.06-0.08% and 0.0025-0.0050%, respectively. The base of the sludge consists of lead sulfate. A method of reductive smelting of lead sludge from copper smelting production at 1000-1100 °C has been developed. Coke was used as a reducing agent and sodium sulfate as a slag-forming material. Optimal conditions for selective extraction of rare metals in smelting products were found: osmium in the form of metallic form into raw lead and rhenium in the form of perrhenate compound NaReO into sodium-sulfate slag. The developed technology makes it possible to extract rhenium with a high degree of extraction in the form of water-soluble compounds for the subsequent production of commercial salts of rhenium by the known hydrometallurgical methods. The content of rhenium in the slag phase is 0.18-0.25%, with its initial content in the slime of 0.06-0.08%. The degree of rhenium concentration at the first stage of processing is 3-3.2 times in the form of water-soluble perrhenate. Osmium and lead do not form solid solutions; osmium in crude lead is mainly concentrated in the lower zones of lead. A method of obtaining a concentrate containing 53-67% osmium from raw lead with an initial content of 0.0025-0.0050% in the slurry and a concentration number of 13,000-21,000 times has been proposed.
PubMed: 35744129
DOI: 10.3390/ma15124071 -
Orthopaedic Surgery Apr 2020The morphological characteristics of tendons have been thoroughly evaluated via microscopy. Optical microscopy and electron microscopy are the most commonly used... (Review)
Review
The morphological characteristics of tendons have been thoroughly evaluated via microscopy. Optical microscopy and electron microscopy are the most commonly used techniques for tendon tissue observation. According to the principles of both microscopy types, preparation and evaluation methods vary. Simple optical microscopy is commonly used in the observation of cells and extracellular matrix, and many stains, including hematoxylin-eosin, Van Gieson, Prussian blue, Alcian blue, and toluidine blue, are used for evaluating cells, collagen fiber arrangement, and noncollagenous proteins. Histological scoring systems have been used in many studies for semi-quantification. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most commonly used electron microscopy types, and special consideration is needed for the fixation and embedding protocols. Glutaraldehyde followed by osmium is most commonly used in the chemical fixation of tendon tissue, followed by epoxy resin embedment. Longitudinal sections captured in SEM images show the arrangement of collagen fibrils and the cells and lipid drops among them, while cross sections captured in TEM images show the diameter and distribution of collagen fibrils. SEM and TEM are used together for comprehensive evaluations. This mini review is focused on the preparation methodology and related evaluation indexes for the morphological evaluation of tendons.
Topics: Humans; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Staining and Labeling; Tendons; Tissue Fixation
PubMed: 32096911
DOI: 10.1111/os.12637 -
Current Eye Research Aug 2018To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology...
PURPOSE
To examine the ultrastructure of lipofuscin bodies and melanosomes in retinal epithelium of elderly rhesus monkeys and determines changes in their number and morphology as a function of retinal eccentricity.
METHODS
Electron microscopy was used to describe and quantify two major organelles in elderly monkey retinal epithelium, lipofuscin bodies and melanosomes, at different retinal loci extending from the macula to the peri-macula, equator, periphery and ora serrata. Osmium tetroxide was used to distinguish lipofuscin bodies from melanosomes.
RESULTS
Lipofuscin bodies and melanosomes diminished in number with advanced age but there was an inverse relationship between these two organelles. Lipofuscin bodies were more numerous in the macula and melanosomes more numerous in the peripheral retina. Three types of lipofuscin bodies were identified: 1) smaller and tending to locate in the middle third of the epithelial cell, 2) larger, less common, and located more basally, and 3) extremely rare, melano-lipofuscin, containing a melanosome. When osmicated, all lipofuscin bodies contained electron dense materials. When osmium tetroxide was not used for fixation, the first two types of lipofuscin bodies lost their electron densities while the third type retained its electron density due to the melanosome it contained.
CONCLUSION
As previously reported for human retina, lipofuscin is most abundant in the macular and peri-macular epithelium and least abundant in the periphery, whereas melanosomes show the opposite relationship. This distribution pattern could contribute to the macula's greater vulnerability to photo-toxicity. Three types of lipofuscin bodies are found in aging monkey retinal epithelium. All types contain electron dense material, but the most prominent two types lose their densities in the absence of osmium tetroxide during fixation. Most of the electron densities in lipofuscin bodies must contain a material that binds strongly to osmium tetroxide such as polyunsaturated fatty acids.
Topics: Aging; Animals; Electron Probe Microanalysis; Female; Lipofuscin; Macaca mulatta; Male; Melanosomes; Microscopy, Electron; Models, Animal; Osmium Tetroxide; Retinal Pigment Epithelium
PubMed: 29641909
DOI: 10.1080/02713683.2018.1464194