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Scientific Reports Jul 2022Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity...
Biotin ligases have been developed as proximity biotinylation enzymes for analyses of the interactome. However, there has been no report on the application of proximity labeling for in-resin correlative light-electron microscopy of Epon-embedded cells. In this study, we established a proximity-labeled in-resin CLEM of Epon-embedded cells using miniTurbo, a biotin ligase. Biotinylation by miniTurbo was observed in cells within 10 min following the addition of biotin to the medium. Using fluorophore-conjugated streptavidin, intracellular biotinylated proteins were labeled after fixation of cells with a mixture of paraformaldehyde and glutaraldehyde. Fluorescence of these proteins was resistant to osmium tetroxide staining and was detected in 100-nm ultrathin sections of Epon-embedded cells. Ultrastructures of organelles were preserved well in the same sections. Fluorescence in sections was about 14-fold brighter than that in the sections of Epon-embedded cells expressing mCherry2 and was detectable for 14 days. When mitochondria-localized miniTurbo was expressed in the cells, mitochondria-like fluorescent signals were detected in the sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. Proximity labeling using miniTurbo led to more stable and brighter fluorescent signals in the ultrathin sections of Epon-embedded cells, resulting in better performance of in-resin CLEM.
Topics: Biotin; Microscopy, Electron, Scanning; Organelles; Osmium Tetroxide; Resins, Plant; Staining and Labeling
PubMed: 35778550
DOI: 10.1038/s41598-022-15438-6 -
Experimental Eye Research Aug 2022The distal outflow pathway of the human eye consists of the outer wall of Schlemm's canal, collector channels, and the deep-scleral, mid-scleral and episcleral vessels.... (Review)
Review
The distal outflow pathway of the human eye consists of the outer wall of Schlemm's canal, collector channels, and the deep-scleral, mid-scleral and episcleral vessels. It is the last region of transit for aqueous humor before returning to the venous system. While the trabecular meshwork, scleral spur, and inner wall of Schlemm's canal have been extensively analyzed to define their contributions to aqueous outflow, the role of the distal outflow pathway is not completely understood. Collector channels, emanating from Schlemm's canal were previously thought to be passive conduits for aqueous humor. However, recent studies have shown many collector channels contain flap-like appendages which move with changes in pressure. These findings, along with studies demonstrating innervation of episcleral vessels, have led to questions regarding whether other structures in the distal outflow pathway are under neural regulation and how this may influence aqueous humor outflow. This study evaluates the innervation of the outer wall of Schlemm's canal and collector channels, along with the deep-scleral, mid-scleral and episcleral vasculature with microcomputed tomography and 3-dimensional reconstruction, correlative light microscopy, immunohistochemistry, and transmission electron microscopy. Peripheral, autonomic, and sensory nerve fibers were found to be present adjacent to Schlemm's canal outer wall endothelium, collector channel endothelium, and in the different regions of the distal outflow vasculature. Nerves were more commonly identified in regions that contained collector channels when compared to regions without collector channels. These findings regarding the neural anatomy suggest an active neural regulation of aqueous humor outflow throughout the proximal and distal regions of the conventional outflow pathway.
Topics: Aqueous Humor; Humans; Intraocular Pressure; Microscopy, Electron, Transmission; Sclera; Trabecular Meshwork; X-Ray Microtomography
PubMed: 35636488
DOI: 10.1016/j.exer.2022.109132 -
The Journal of Histochemistry and... Jun 2022Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM)...
Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM) staining at a higher magnification, simply by removing the coverslip. However, it is not suitable for observation of immunohistochemistry (IHC) using 3,3'-diaminobenzidine (DAB) due to insufficient backscattered electron image. Traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium tetroxide and iron, (2) cobalt, (3) methenamine silver (Ag), (4) gold chloride (Gold), and (5) both Ag and Gold (Ag + Gold), were examined by LV-SEM. Tissue specimens from Thy1.1 glomerulonephritis rat kidney stained with α-smooth muscle actin and visualized with DAB were enhanced by each of these enhancement methods. We found, in light microscopic and LV-SEM, that the enhancement with Ag, Gold, or Ag + Gold had better intensity and contrast than others. At a higher magnification, Ag + Gold enhancement showed high intensity and low background, although only Ag or Gold enhancement had nonspecific background. Even after observation by LV-SEM, the quality of specimens was maintained after remounting the coverslip. It was also confirmed that Ag + Gold enhancement could be useful for IHC using clinical human renal biopsy. These findings indicate that Ag + Gold provided an adequate enhancement in IHC for both LM and LV SEM observation.
Topics: Animals; Gold; Immunohistochemistry; Microscopy, Electron, Scanning; Osmium Tetroxide; Rats; Vacuum
PubMed: 35611640
DOI: 10.1369/00221554221102996 -
Scientific Reports Apr 2022We aimed to develop and validate a deep learning model for automated segmentation and histomorphometry of myelinated peripheral nerve fibers from light microscopic...
We aimed to develop and validate a deep learning model for automated segmentation and histomorphometry of myelinated peripheral nerve fibers from light microscopic images. A convolutional neural network integrated in the AxonDeepSeg framework was trained for automated axon/myelin segmentation using a dataset of light-microscopic cross-sectional images of osmium tetroxide-stained rat nerves including various axonal regeneration stages. In a second dataset, accuracy of automated segmentation was determined against manual axon/myelin labels. Automated morphometry results, including axon diameter, myelin sheath thickness and g-ratio were compared against manual straight-line measurements and morphometrics extracted from manual labels with AxonDeepSeg as a reference standard. The neural network achieved high pixel-wise accuracy for nerve fiber segmentations with a mean (± standard deviation) ground truth overlap of 0.93 (± 0.03) for axons and 0.99 (± 0.01) for myelin sheaths, respectively. Nerve fibers were identified with a sensitivity of 0.99 and a precision of 0.97. For each nerve fiber, the myelin thickness, axon diameter, g-ratio, solidity, eccentricity, orientation, and individual x -and y-coordinates were determined automatically. Compared to manual morphometry, automated histomorphometry showed superior agreement with the reference standard while reducing the analysis time to below 2.5% of the time needed for manual morphometry. This open-source convolutional neural network provides rapid and accurate morphometry of entire peripheral nerve cross-sections. Given its easy applicability, it could contribute to significant time savings in biomedical research while extracting unprecedented amounts of objective morphologic information from large image datasets.
Topics: Animals; Artificial Intelligence; Axons; Microscopy; Myelin Sheath; Nerve Fibers, Myelinated; Rats
PubMed: 35396530
DOI: 10.1038/s41598-022-10066-6 -
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 -
Microscopy (Oxford, England) Apr 2022Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be...
Serial ultrathin sections to identify ultrastructural localization of GLUT1 molecules in vesicles in brain endothelial cells-correlative light and electron microscopy in depth.
Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be included in tiny ultrathin sections randomly selected for electron microscopy (EM). Glucose transporter 1 (GLUT1) is in charge of transporting glucose across brain capillary endothelial cells (BCECs). Paraformaldehyde-fixed floating sections (50 μm thick) of mouse brain were immunolabeled with anti-GLUT1 antibody and visualized with fluoronanogold. Fluorescent images encompassing the entire hemisphere were tiled to enable selection of GLUT1-positive BCECs suitable for subsequent EM and landmark placement with laser microdissection to guide trimming. Sections were then fixed with glutaraldehyde, gold enhanced to intensify the labeling and fixed with osmium tetroxide to facilitate ultrastructural recognition. Even though a region that contained target BCECs was successfully trimmed in the resin block, it was only after observation of serial ultrathin sections that GLUT1 signals in coated vesicles on the same cross section corresponding to the cross section preidentified by confocal laser microscope. This is the first ultrastructural demonstration of GLUT1 molecules in coated vesicles, which may well explain its functional relevance to transport glucose across BCECs. Successful ultrastructural localization of molecules in relation to well-preserved target structure in native tissue samples, as achieved in this study, will pave the way to understand the functional relevance of molecules and their relation to ultrastructural details.
Topics: Animals; Brain; Endothelial Cells; Glucose Transporter Type 1; Mice; Microscopy, Electron; Osmium Tetroxide
PubMed: 35157050
DOI: 10.1093/jmicro/dfac005 -
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 -
Histochemistry and Cell Biology Apr 2022High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid...
High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.
Topics: Freeze Substitution; Freezing; Lipids; Osmium Tetroxide; Potassium Permanganate
PubMed: 34984524
DOI: 10.1007/s00418-021-02070-0 -
Chemical & Pharmaceutical Bulletin 2022Osmium is defined in the international council for harmonization (ICH-Q3D) guidelines as an element whose concentration can be determined by validated methods including...
Osmium is defined in the international council for harmonization (ICH-Q3D) guidelines as an element whose concentration can be determined by validated methods including microwave-assisted nitric acid digestion and inductively coupled plasma mass spectrometry. However, microwave digestion using nitric acid is known to result in osmium recoveries higher than the theoretical values in spiked tests because of the formation of highly volatile osmium tetroxide in an oxidation reaction. To stabilize osmium, the addition of thiourea as a complexing agent has been tested and proved its utility. It remains unclear whether other compounds can prevent the over-recovery of osmium. In this study, we investigated four compounds, thiourea, ascorbic acid, sodium sulfite, and potassium metabisulfite, that could reduce the overestimation of osmium isotopes. The minimum amounts of thiourea, ascorbic acid, sodium sulfite, and potassium metabisulfite required to stabilize 10 ng/mL osmium in blank matrix were 1.0, 1.0, 2.5, and 2.5 g/L, respectively. The relative standard deviations obtained from 12 analyses for each stabilization solution were less than 3.3% in thiourea, 12.7% in ascorbic acid, 9.0% in sodium sulfite, and 10.6% in potassium metabisulfite. The stabilization solutions were investigated in a digested tablet matrix and were found to be effective. The impact of adding stabilization solutions on the determination of all ICH-Q3D element concentrations was also evaluated. As stabilization solutions had a small or significant impact on the determination of some elements, it was concluded that osmium determination should be conducted independently.
Topics: Hydrogen-Ion Concentration; Isotopes; Mass Spectrometry; Microwaves; Osmium
PubMed: 34980735
DOI: 10.1248/cpb.c21-00739 -
Sovremennye Tekhnologii V Meditsine 2021was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal...
UNLABELLED
was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal implants or their prototypes for their subsequent examination using scanning electron microscopy in the backscattered electron mode.
MATERIALS AND METHODS
After fixation in 10% formalin (24 h), the biomaterial (a titanium nickelide plate with the surrounding tissues after subcutaneous implantation, patented titanium alloy plates with the surrounding tissues after cranioplasty, primary and secondary calcified atherosclerotic plaques) were fixed with 1% osmium tetroxide (12 h) and then stained with 2% aqueous solution of osmium tetroxide (48 h). The samples were further stained with 2% alcoholic uranyl acetate (5 h), dehydrated with isopropanol (5 h) and acetone (1 h), impregnated with a mixture of acetone and epoxy resin Epon (1:1, 6 h) and then embedded into a fresh portion of epoxy resin (24 h), which was followed by polymerization at 60°C. After grinding and polishing, epoxy blocks were counterstained with lead citrate (7 min) and sputter-coated with carbon, then the samples were visualized by scanning electron microscopy in the backscattered electron mode. The elemental composition was studied using X-ray microanalysis.
RESULTS
The developed technique allows obtaining high-quality images at five thousand-fold magnifications, provides the possibility to identify the shape and structure of intact metal and mineral inclusions, and to type the surrounding cells, distinguishing mesenchymal and immunocompetent cells by shape and cytoplasmic content. Apart from connective tissue capsule thickness and leukocyte infiltration, this technique makes it possible to estimate the number and area of newly formed small-caliber vessels representing a surrogate marker of inflammation.
CONCLUSION
The proposed technique provides the possibility to investigate adequately the structure of samples when their sectioning is impossible or significantly complicated, with image quality remarkably higher than that obtained by light microscopy.
Topics: Alloys; Metals; Microscopy, Electron, Scanning; Osmium Tetroxide; Staining and Labeling
PubMed: 34795988
DOI: 10.17691/stm2020.12.4.02