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Skin Pharmacology and Physiology 2018In the mid-1950s and 1960s, transmission electron microscopes became widely available, leading to many studies of the ultrastructure of various tissues including the... (Review)
Review
In the mid-1950s and 1960s, transmission electron microscopes became widely available, leading to many studies of the ultrastructure of various tissues including the epidermis. Most of these studies involved tissue fixation with formaldehyde and postfixation with osmium tetroxide. A few studies employed freeze-fracture electron microscopy. One set of these studies identified a small organelle variously called lamellar granules (LGs), lamellar bodies, membrane-coating granules, cementsomes, and Odland bodies. LGs are round to ovoid in shape, with a diameter of about 200 nm. They have a bounding membrane surrounding a stack of internal lipid lamellae. These small organelles are first seen in the spinous layer and accumulate with differentiation in the granular layer. In the uppermost granular cells, the bounding membrane of the LG fuses into the cell plasma membrane, and the internal contents are extruded into the intercellular space. The initially extruded contents of the LG then rearrange to form the intercellular lamellae of the stratum corneum. In this context, LGs serve as the precursor to the permeability barrier of the skin. Various studies have provided evidence that they are derived from the Golgi apparatus, specifically the trans-Golgi. Isolated LGs contain phosphoglycerides, sphingomyelin, and glucosylceramides. The most unusual lipid component is a linoleate-containing glucosylceramide comprising 30- to 34-carbon ω-hydroxy-acids. Isolated granules also contain acid hydrolases including glucocerebrosidase, sphingomyelinase, and phospholipase A. They also contain proteases and antimicrobial peptides. Defective LGs have been associated with a number of skin diseases including ichthyotic conditions and defective barrier function. Recently, studies employing cryo-transmission electron microscopy have called into question the validity of observations on LGs with more conventional electron microscopic techniques. These studies suggest a continuity of the membrane structure from the Golgi through the intercellular lamellae of the stratum corneum.
Topics: Animals; Epidermis; Glucosylceramides; Golgi Apparatus; Humans; Microscopy, Electron, Transmission; Skin Diseases
PubMed: 30110701
DOI: 10.1159/000491757 -
Internal Medicine (Tokyo, Japan) 2023A 32-year-old man was admitted for the evaluation of proteinuria (5.69 g/day). A light microscopic examination showed markedly dilated glomerular capillary loops with...
A 32-year-old man was admitted for the evaluation of proteinuria (5.69 g/day). A light microscopic examination showed markedly dilated glomerular capillary loops with vacuolated areas in many glomeruli, and vacuolated areas were seen on peritubular capillaries in the tubulointerstitium. When electron microscopy specimens prepared by pre-fixation with glutaraldehyde and post-fixation with osmium tetroxide were used for oil red staining, the deposition was confirmed on the affected areas. A genetic analysis of apoE showed that the lipoprotein glomerulopathy was due to apoE-Sendai (Arg145Pro, p.R163P) heterozygosity, which was found in not only the patient but also his mother and twin brother.
Topics: Male; Humans; Adult; Apolipoproteins E; Kidney Diseases; Kidney Glomerulus; Proteinuria; Heterozygote
PubMed: 37532513
DOI: 10.2169/internalmedicine.0834-22 -
Nature Methods Dec 2022We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that...
We report the rational engineering of a remarkably stable yellow fluorescent protein (YFP), 'hyperfolder YFP' (hfYFP), that withstands chaotropic conditions that denature most biological structures within seconds, including superfolder green fluorescent protein (GFP). hfYFP contains no cysteines, is chloride insensitive and tolerates aldehyde and osmium tetroxide fixation better than common fluorescent proteins, enabling its use in expansion and electron microscopies. We solved crystal structures of hfYFP (to 1.7-Å resolution), a monomeric variant, monomeric hyperfolder YFP (1.6 Å) and an mGreenLantern mutant (1.2 Å), and then rationally engineered highly stable 405-nm-excitable GFPs, large Stokes shift (LSS) monomeric GFP (LSSmGFP) and LSSA12 from these structures. Lastly, we directly exploited the chemical stability of hfYFP and LSSmGFP by devising a fluorescence-assisted protein purification strategy enabling all steps of denaturing affinity chromatography to be visualized using ultraviolet or blue light. hfYFP and LSSmGFP represent a new generation of robustly stable fluorescent proteins developed for advanced biotechnological applications.
Topics: Luminescent Proteins; Microscopy; Green Fluorescent Proteins; Fluorescence Resonance Energy Transfer; Light
PubMed: 36344833
DOI: 10.1038/s41592-022-01660-7 -
Applied Microscopy May 2020Plant specimens for scanning electron microscopy (SEM) are commonly treated using standard protocols. Conventional fixatives consist of toxic chemicals such as... (Review)
Review
Plant specimens for scanning electron microscopy (SEM) are commonly treated using standard protocols. Conventional fixatives consist of toxic chemicals such as glutaraldehyde, paraformaldehyde, and osmium tetroxide. In 1996, methanol fixation was reported as a rapid alternative to the standard protocols. If specimens are immersed in methanol for 30 s or longer and critical-point dried, they appear to be comparable in preservation quality to those treated with the chemical fixatives. A modified version that consists of methanol fixation and ethanol dehydration was effective at preserving the tissue morphology and dimensions. These solvent-based fixation and dehydration protocols are regarded as rapid and simple alternatives to standard protocols for SEM of plants.
PubMed: 33580311
DOI: 10.1186/s42649-020-00028-5 -
Frontiers in Cell and Developmental... 2022Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a...
Volume electron microscopy, a powerful approach to generate large three-dimensional cell and tissue volumes at electron microscopy resolutions, is rapidly becoming a routine tool for understanding fundamental and applied biological questions. One of the enabling factors for its adoption has been the development of conventional fixation protocols with improved heavy metal staining. However, freeze-substitution with organic solvent-based fixation and staining has not realized the same level of benefit. Here, we report a straightforward approach including osmium tetroxide, acetone and up to 3% water substitution fluid (compatible with traditional or fast freeze-substitution protocols), warm-up and transition from organic solvent to aqueous 2% osmium tetroxide. Once fully hydrated, samples were processed in aqueous based potassium ferrocyanide, thiocarbohydrazide, osmium tetroxide, uranyl acetate and lead acetate before resin infiltration and polymerization. We observed a consistent and substantial increase in heavy metal staining across diverse and difficult-to-fix test organisms and tissue types, including plant tissues (), nematode () and yeast (). Our approach opens new possibilities to combine the benefits of cryo-preservation with enhanced contrast for volume electron microscopy in diverse organisms.
PubMed: 36003147
DOI: 10.3389/fcell.2022.933376 -
Basic & Clinical Pharmacology &... Nov 2020Osmium tetroxide is a strong oxidizing agent used in electron microscopy. Eye exposure may cause severe burns, and after inhalation or ingestion damage to the...
Osmium tetroxide is a strong oxidizing agent used in electron microscopy. Eye exposure may cause severe burns, and after inhalation or ingestion damage to the respiratory or gastrointestinal tract occurs. Exposure to osmium and its compounds is extremely rare. We present a case of a 32-year-old female stained by 9 mL of 2% osmium tetroxide in acetone during an accident in the laboratory, with rare dermal and ocular findings. Due to lack of data in toxicological databases and the absence of antidote, the therapy was symptomatic. Osmium was detected in serum 19 hours later (0.22 μg/L) and in urine during the 15-hour collection (three samples-7.05, 1.65 and 8.45 μg/L). In blood serum on admission, after 1 and 2 days after exposure, the levels of iron (28.2, 39.8 and 50.5 μmol/L; reference range 5.8-34.5 μmol/L) and transferrin receptor/ferritine were elevated. To our knowledge, this is the first paper documenting a significant absorption from the skin and potentially from the eye conjunctiva, based on serum and urine analysis. The relationship between increased iron in blood and exposure has not been described yet, and the mechanism remains unknown. The patient is being followed up for the unknown long-term effects.
Topics: Adult; Eye; Female; Humans; Osmium Tetroxide; Skin
PubMed: 32524772
DOI: 10.1111/bcpt.13450 -
Membranes Dec 2021The recovery of osmium from residual osmium tetroxide (OsO) is a necessity imposed by its high toxicity, but also by the technical-economic value of metallic osmium. An...
The recovery of osmium from residual osmium tetroxide (OsO) is a necessity imposed by its high toxicity, but also by the technical-economic value of metallic osmium. An elegant and extremely useful method is the recovery of osmium as a membrane catalytic material, in the form of nanoparticles obtained on a polymeric support. The subject of the present study is the realization of a composite membrane in which the polymeric matrix is the polypropylene hollow fiber, and the active component consists of the osmium nanoparticles obtained by reducing an alcoholic solution of osmium tetroxides directly on the polymeric support. The method of reducing osmium tetroxide on the polymeric support is based on the use of 10-undecenoic acid (10-undecylenic acid) (UDA) as a reducing agent. The osmium tetroxide was solubilized in -butanol and the reducing agent, 10-undecenoic acid (UDA), in -propanol, -butanol or -decanol solution. The membranes containing osmium nanoparticles (Os-NP) were characterized morphologically by the following: scanning electron microscopy (SEM), high-resolution SEM (HR-SEM), structurally: energy-dispersive spectroscopy analysis (EDAX), Fourier transform infrared (FTIR) spectroscopy. In terms of process performance, thermal gravimetric analysis was performed by differential scanning calorimetry (TGA, DSC) and in a redox reaction of an organic marker, -nitrophenol (PNP) to -aminophenol (PAP). The catalytic reduction reaction with sodium tetraborate solution of PNP to PAP yielded a constant catalytic rate between 2.04 × 10 mmol s and 8.05 × 10 mmol s.
PubMed: 35054577
DOI: 10.3390/membranes12010051 -
Microscopy (Oxford, England) Aug 2022Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone...
Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone pigments in Boraginaceae. The new fixation method combined glutaraldehyde containing malachite green, imidazole-osmium and p-phenylenediamine treatments, and cells were then observed with a transmission electron microscope. The method prevented the extraction of lipids, including shikonin derivatives, and improved the visualization of subcellular structures, especially the membrane system, when compared with that of conventional fixation. The improved quality of the transmission electron micrographs is because malachite green ionically binds to the plasma membrane, organelles and lipids and acts as a mordant for electron staining with osmium tetroxide. Imidazole promotes the reaction of osmium tetroxide, leading to enhanced electron staining. p-Phenylenediamine reduces osmium tetroxide bound to cellular materials and increases the electron density. This protocol requires only three additional reagents over conventional chemical fixation using glutaraldehyde and osmium tetroxide.
Topics: Glutaral; Imidazoles; Lipids; Microscopy, Electron; Microscopy, Electron, Transmission; Osmium Tetroxide; Plant Cells
PubMed: 35388424
DOI: 10.1093/jmicro/dfac018 -
Academic Forensic Pathology Sep 2019Fat embolism is common following trauma and is a common autopsy finding in these cases. It may also be seen in non-traumatic cases and is seen in children as well as... (Review)
Review
Fat embolism is common following trauma and is a common autopsy finding in these cases. It may also be seen in non-traumatic cases and is seen in children as well as adults. In comparison fat embolism syndrome (FES) only occurs in a small number of trauma and non-trauma cases. Clinical diagnosis is based on characteristic clinical and laboratory findings. Fat embolism exerts its effect by mechanical blockage of vessels and/or by biochemical means including breakdown of fat to free fatty acids causing an inflammatory response. Fat embolism can be identified at autopsy on microscopy of the lungs using fat stains conducted on frozen tissue, including on formalin fixed but not processed tissue. With FES fat emboli can be seen in other organs including the brain, kidney and myocardium. Fat can also be identified with post-fixation staining, typically with osmium tetroxide. Scoring systems have been developed to try and determine the severity of fat embolism in lung tissue. Fat embolism is also common following resuscitation. When no resuscitation has taken place, the presence of fat on lung histology has been used as proof of vitality. Diagnosis of fat embolism syndrome at autopsy requires analysis of the history, clinical and laboratory findings along with autopsy investigations to determine its relevance, but is an important diagnosis to make which is not always identified clinically. This paper reviews the history, clinical and laboratory findings and diagnosis of fat embolism and fat embolism syndrome at autopsy.
PubMed: 32110249
DOI: 10.1177/1925362119896351 -
Journal of Neural Engineering Nov 2022Vagus nerve stimulation (VNS) is Food and Drug Administration-approved for epilepsy, depression, and obesity, and stroke rehabilitation; however, the morphological...
Vagus nerve stimulation (VNS) is Food and Drug Administration-approved for epilepsy, depression, and obesity, and stroke rehabilitation; however, the morphological anatomy of the vagus nerve targeted by stimulatation is poorly understood. Here, we used microCT to quantify the fascicular structure and neuroanatomy of human cervical vagus nerves (cVNs).We collected eight mid-cVN specimens from five fixed cadavers (three left nerves, five right nerves). Analysis focused on the 'surgical window': 5 cm of length, centered around the VNS implant location. Tissue was stained with osmium tetroxide, embedded in paraffin, and imaged on a microCT scanner. We visualized and quantified the merging and splitting of fascicles, and report a morphometric analysis of fascicles: count, diameter, and area.In our sample of human cVNs, a fascicle split or merge event was observed every ∼560m (17.8 ± 6.1 events cm). Mean morphological outcomes included: fascicle count (6.6 ± 2.8 fascicles; range 1-15), fascicle diameter (514 ± 142m; range 147-1360m), and total cross-sectional fascicular area (1.32 ± 0.41 mm; range 0.58-2.27 mm).The high degree of fascicular splitting and merging, along with wide range in key fascicular morphological parameters across humans may help to explain the clinical heterogeneity in patient responses to VNS. These data will enable modeling and experimental efforts to determine the clinical effect size of such variation. These data will also enable efforts to design improved VNS electrodes.
Topics: Humans; Cross-Sectional Studies; Vagus Nerve; Vagus Nerve Stimulation; Epilepsy; Cadaver
PubMed: 36174538
DOI: 10.1088/1741-2552/ac9643