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Scientific Reports May 2020Fat embolism is the mechanical blockage of blood vessels by circulating fat particles. It is frequently related to traumas involving soft tissues and fat-containing... (Comparative Study)
Comparative Study
Fat embolism is the mechanical blockage of blood vessels by circulating fat particles. It is frequently related to traumas involving soft tissues and fat-containing bones. Different techniques have been used for decades to demonstrate histologically fat emboli, being the extremely toxic post-fixation with osmium tetroxide one of the most used techniques in the last decades. In the present study, the osmium tetroxide technique was compared qualitatively and quantitatively, for the first time, with chromic acid and Oil Red O frozen techniques for histological fat emboli detection in the lungs of eight sperm whales that died due to ship strikes. This was also the first time that chromic acid technique was tested in cetaceans. Results showed that the three techniques were valuable for the histological detection of fat embolism in cetaceans, even when tissues presented advanced autolysis and had been stored in formaldehyde for years. Although quantitative differences could not be established, the Oil Red O frozen technique showed the lowest quality for fat emboli staining. On the contrary, the chromic acid technique was proven to be a good alternative to osmium tetroxide due to its slightly lower toxicity, its equivalent or even superior capacity of fat emboli detection, and its significantly lower economic cost.
Topics: Animals; Cetacea; Embolism, Fat; Histological Techniques; Lung; Pulmonary Embolism; Staining and Labeling
PubMed: 32427895
DOI: 10.1038/s41598-020-64821-8 -
European Journal of Endocrinology Feb 2016Visceral obesity is independently related to numerous cardiometabolic alterations, with adipose tissue dysfunction as a central feature.
BACKGROUND
Visceral obesity is independently related to numerous cardiometabolic alterations, with adipose tissue dysfunction as a central feature.
OBJECTIVE
To examine whether omental (OM) and subcutaneous (SC) adipocyte size populations in women relate to visceral obesity, cardiometabolic risk factors and adipocyte lipolysis independent of total adiposity.
DESIGN AND METHODS
OM and SC fat samples were obtained during gynecological surgery in 60 women (mean age, 46.1±5.9 years; mean BMI, 27.1±4.5 kg/m² (range, 20.3-41. kg/m²)). Fresh samples were treated with osmium tetroxide and were analyzed with a Multisizer Coulter. Cell size distributions were computed for each sample with exponential and Gaussian function fits.
RESULTS
Computed tomography-measured visceral fat accumulation was the best predictor of larger cell populations as well as the percentage of small cells in both OM and SC fat (P<0.0001 for all). Accordingly, women with visceral obesity had larger cells in the main population and higher proportion of small adipocytes independent of total adiposity (P≤0.05). Using linear regression analysis, we found that women characterized by larger-than-predicted adipocytes in either OM or SC adipose tissue presented higher visceral adipose tissue area, increased percentage of small cells and homeostasis model assessment insulin resistance index as well as higher OM adipocyte isoproterenol-, forskolin- and dbcAMP-stimulated lipolysis compared to women with smaller-than-predicted adipocytes, independent of total adiposity (P≤0.05).
CONCLUSION
Excess visceral adipose tissue accumulation is a strong marker of both adipocyte hypertrophy and increased number of small cells in either fat compartment, which relates to higher insulin resistance index and lipolytic response, independent of total adiposity.
Topics: Adipocytes; Adult; Female; Humans; Intra-Abdominal Fat; Middle Aged; Obesity, Abdominal; Radiography; Subcutaneous Fat, Abdominal
PubMed: 26578637
DOI: 10.1530/EJE-15-0822 -
The Journal of Organic Chemistry Nov 2022Reactions that result in the oxy-functionalization of sp C-H bonds to give phenols are relatively rare. Here we report experiments and density functional theory (DFT)...
Experimental Demonstration and Density Functional Theory Mechanistic Analysis of Arene C-H Bond Oxidation and Product Protection by Osmium Tetroxide in a Strongly Basic/Nucleophilic Solvent.
Reactions that result in the oxy-functionalization of sp C-H bonds to give phenols are relatively rare. Here we report experiments and density functional theory (DFT) calculations that demonstrate selective C-H bond hydroxylation of nitroarenes to their corresponding mono-phenoxide as the exclusive product using OsO in a highly basic solvent mixture of water, hydroxide, and pyridine. DFT calculations using a mixed explicit/continuum solvent approach indicate that there is likely a mixture of OsO-hydroxide/pyridine ground-state structures that have competitive reactivity and that the mechanism involves the nucleophilic addition of an anionic metal-oxo species to the arene followed by a hydride transfer process that is different from the standard [3 + 2] mechanism often invoked for the OsO oxidation of σ and π bonds. This work demonstrates the utility of using a strongly basic solvent for C-H bond oxidation reactions as this effectively converts any reactive phenolic product into the corresponding phenoxide, which is protected and essentially inert to further oxidation by the nucleophilic metal-oxo species.
PubMed: 36191170
DOI: 10.1021/acs.joc.2c01159 -
Journal of Microscopy Jun 2018Non-invasive imaging techniques like X-ray computed tomography have become very popular in zoology, as they allow for simultaneous imaging of the internal and external... (Comparative Study)
Comparative Study
Non-invasive imaging techniques like X-ray computed tomography have become very popular in zoology, as they allow for simultaneous imaging of the internal and external morphology of organisms. Nevertheless, the effect of different staining approaches required for this method on samples lacking mineralized tissues, such as soft-bodied invertebrates, remains understudied. Herein, we used synchrotron radiation-based X-ray micro-computed tomography to compare the effects of commonly used contrasting approaches on onychophorans - soft-bodied invertebrates important for studying animal evolution. Representatives of Euperipatoides rowelli were stained with osmium tetroxide (vapour or solution), ruthenium red, phosphotungstic acid, or iodine. Unstained specimens were imaged using both standard attenuation-based and differential phase-contrast setups to simulate analyses with museum material. Our comparative qualitative analyses of several tissue types demonstrate that osmium tetroxide provides the best overall tissue contrast in onychophorans, whereas the remaining staining agents rather favour the visualisation of specific tissues and/or structures. Quantitative analyses using signal-to-noise ratio measurements show that the level of image noise may vary according to the staining agent and scanning medium selected. Furthermore, box-and-whisker plots revealed substantial overlap in grey values among structures in all datasets, suggesting that a combination of semiautomatic and manual segmentation of structures is required for comprehensive 3D reconstructions of Onychophora, irrespective of the approach selected. Our results show that X-ray micro-computed tomography is a promising technique for studying onychophorans and, despite the benefits and disadvantages of different staining agents for specific tissues/structures, this method retrieves informative data that may eventually help address evolutionary questions long associated with Onychophora.
Topics: Animals; Helminths; Image Processing, Computer-Assisted; Iodine; Osmium Tetroxide; Phosphotungstic Acid; Ruthenium Red; Staining and Labeling; X-Ray Microtomography
PubMed: 29469207
DOI: 10.1111/jmi.12688 -
Journal of Microscopy Jul 2021Imaging the visual systems of bumblebees and other pollinating insects may increase understanding of their dependence on specific habitats and how they will be affected...
Imaging the visual systems of bumblebees and other pollinating insects may increase understanding of their dependence on specific habitats and how they will be affected by climate change. Current high-resolution imaging methods are either limited to two dimensions (light- and electron microscopy) or have limited access (synchrotron radiation x-ray tomography). For x-ray imaging, heavy metal stains are often used to increase contrast. Here, we present micron-resolution imaging of compound eyes of buff-tailed bumblebees (Bombus terrestris) using a table-top x-ray nanotomography (nano-CT) system. By propagation-based phase-contrast imaging, the use of stains was avoided and the microanatomy could more accurately be reconstructed than in samples stained with phosphotungstic acid or osmium tetroxide. The findings in the nano-CT images of the compound eye were confirmed by comparisons with light- and transmission electron microscopy of the same sample and finally, comparisons to synchrotron radiation tomography as well as to a commercial micro-CT system were done.
Topics: Animals; Bees; Laboratories; Microscopy, Phase-Contrast; Osmium Tetroxide; Synchrotrons; Tomography, X-Ray Computed; X-Ray Microtomography
PubMed: 33822371
DOI: 10.1111/jmi.13005 -
Chembiochem : a European Journal of... Mar 2019Staining compounds containing heavy elements (electron dyes) can facilitate the visualization of DNA and related biomolecules by using TEM. However, research into the...
Staining compounds containing heavy elements (electron dyes) can facilitate the visualization of DNA and related biomolecules by using TEM. However, research into the synthesis and utilization of alternative electron dyes has been limited. Here, we report the synthesis of a novel DNA intercalator molecule, bis-acridine uranyl (BAU). NMR spectroscopy and MS confirmed the validity of the synthetic strategy and gel electrophoresis verified the binding of BAU to DNA. For TEM imaging of DNA, two-dimensional DNA origami nanostructures were used as a robust microscopy test object. By using scanning transmission electron microscopy (STEM) imaging, which is favored over conventional wide-field TEM for improved contrast, and therefore, quantitative image analysis, it is found that the synthesized BAU intercalator can render DNA visible, even at the single-molecule scale. For comparison, other staining compounds with a purported affinity towards DNA, such as dichloroplatinum, cisplatin, osmium tetroxide, and uranyl acetate, have been evaluated. The STEM contrast is discussed in terms of the DNA-dye association constants, number of dye molecules bound per base pair, and the electron-scattering capacity of the metal-containing ligands. These findings pave the way for the future development of electron dyes with specific DNA-binding motifs for high-resolution TEM imaging.
Topics: Acridines; Coordination Complexes; DNA; Intercalating Agents; Microscopy, Electron, Scanning Transmission; Nucleic Acid Conformation; Single Molecule Imaging; Uranium
PubMed: 30501011
DOI: 10.1002/cbic.201800638 -
Protoplasma Nov 2020Bird feather lipids are usually attributed to the oily secretion product of the uropygial (preen) gland. We have observed, however, that feathers exhibit a strong...
Bird feather lipids are usually attributed to the oily secretion product of the uropygial (preen) gland. We have observed, however, that feathers exhibit a strong reaction with osmium tetroxide (OsO), even after treatment with detergents. This leads us to postulate the existence of endogenous feather lipids distinct from preen gland lipids. In order to substantiate our hypothesis, we investigated down feathers from a 1-day-old chicken as their uropgygial gland is not functionally active. The results confirmed the osmiophilic reaction, which was concentrated in the center of barbs and strongly reduced after lipid extraction. In these lipid extracts, we identified using thin layer chromatography, cholesterol, various ceramides, glycolipids, phospholipids, and fatty acids, which closely resembled the lipid composition of the water barrier in the chicken-cornified epidermal envelope. This composition is clearly distinct from chicken uropygeal gland secretion (UGS) known to consist of fatty alcohols as part of aliphatic monoester waxes and of free, predominantly saturated, fatty acids. A filter assay showed a strong reactivity between OsO and the fatty acids C18:1 and C18:2 and with feather lipid extracts, but not with UGS. These observations were confirmed by gas chromatography detecting unsaturated fatty acids including C18:1 and C18:2 as well as cholesterol exclusively in chicken feathers. Our results indicate that (1) endogenous lipids are detectable in chicken feathers and distinct from UGS and (2) in analogy to the morphogenesis of the cornified envelope of chicken feather lipids that may have derived from cellular feather-precursors, apparently enduring the specific cell death during developmental feather cornification.
Topics: Animals; Chickens; Feathers; Lipids; Sebaceous Glands
PubMed: 32851422
DOI: 10.1007/s00709-020-01544-7 -
Frontiers in Cardiovascular Medicine 2021Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining...
Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope.
PubMed: 34760942
DOI: 10.3389/fcvm.2021.739549 -
Journal of the Association For Research... Oct 2022The sensory end-organs responsible for hearing and balance in the mammalian inner ear are connected via a small membranous duct known as the ductus reuniens (also known...
The sensory end-organs responsible for hearing and balance in the mammalian inner ear are connected via a small membranous duct known as the ductus reuniens (also known as the reuniting duct (DR)). The DR serves as a vital nexus linking the hearing and balance systems by providing the only endolymphatic connection between the cochlea and vestibular labyrinth. Recent studies have hypothesized new roles of the DR in inner ear function and disease, but a lack of knowledge regarding its 3D morphology and spatial configuration precludes testing of such hypotheses. We reconstructed the 3D morphology of the DR and surrounding anatomy using osmium tetroxide micro-computed tomography and digital visualizations of three human inner ear specimens. This provides a detailed, quantitative description of the DR's morphology, spatial relationships to surrounding structures, and an estimation of its orientation relative to head position. Univariate measurements of the DR, inner ear, and cranial planes were taken using the software packages 3D Slicer and Zbrush. The DR forms a narrow, curved, flattened tube varying in lumen size, shape, and wall thickness, with its middle third being the narrowest. The DR runs in a shallow bony sulcus superior to the osseus spiral lamina and adjacent to a ridge of bone that we term the "crista reuniens" oriented posteromedially within the cranium. The DR's morphology and structural configuration relative to surrounding anatomy has important implications for understanding aspects of inner ear function and disease, particularly after surgical alteration of the labyrinth and potential causative factors for Ménière's disease.
Topics: Humans; Hearing; Meniere Disease; Vestibule, Labyrinth; X-Ray Microtomography
PubMed: 35804276
DOI: 10.1007/s10162-022-00858-y -
Rapid preparation of adherent mammalian cells for basic scanning electron microscopy (SEM) analysis.Analytical Biochemistry Oct 2017Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention...
Sample preparation for scanning electron microscope analysis involves reagents and equipment that are expensive and often hazardous. Here we demonstrate a circumvention of Osmium tetroxide and critical point drying, greatly reducing the duration, complexity and cost of the process. We captured early stage interactions of invasive-bacteria and HeLa cells during the process of bacteria-mediated gene delivery and illustrate sufficient clarity can be obtained using this procedure to preserve and clearly visualize relevant cellular structures. This protocol is significantly cheaper and easier to adapt compared to conventional methods, and will allow routine preparation/viewing of eukaryotic or bacterial samples for basic morphological studies.
Topics: Escherichia coli; Gene Transfer Techniques; Genetic Vectors; HeLa Cells; Humans; Microscopy, Electron, Scanning; Osmium Tetroxide
PubMed: 28693990
DOI: 10.1016/j.ab.2017.07.008