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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 -
Neuropathology and Applied Neurobiology Aug 2021The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease.
AIMS
The objective of this study was to elucidate the early white matter changes in CADASIL small vessel disease.
METHODS
We used high-pressure freezing and freeze substitution (HPF/FS) in combination with high-resolution electron microscopy (EM), immunohistochemistry and confocal microscopy of brain specimens from control and CADASIL (TgNotch3 ) mice aged 4-15 months to study white matter lesions in the corpus callosum.
RESULTS
We first optimised the HPF/FS protocol in which samples were chemically prefixed, frozen in a sample carrier filled with 20% polyvinylpyrrolidone and freeze-substituted in a cocktail of tannic acid, osmium tetroxide and uranyl acetate dissolved in acetone. EM analysis showed that CADASIL mice exhibit significant splitting of myelin layers and enlargement of the inner tongue of small calibre axons from the age of 6 months, then vesiculation of the inner tongue and myelin sheath thinning at 15 months of age. Immunohistochemistry revealed an increased number of oligodendrocyte precursor cells, although only in older mice, but no reduction in the number of mature oligodendrocytes at any age. The number of Iba1 positive microglial cells was increased in older but not in younger CADASIL mice, but the number of activated microglial cells (Iba1 and CD68 positive) was unchanged at any age.
CONCLUSION
We conclude that early WM lesions in CADASIL affect first and foremost the myelin sheath and the inner tongue, suggestive of a primary myelin injury. We propose that those defects are consistent with a hypoxic/ischaemic mechanism.
Topics: Animals; CADASIL; Corpus Callosum; Freeze Substitution; Mice; Myelin Sheath; White Matter
PubMed: 33483954
DOI: 10.1111/nan.12697 -
Basic & Clinical Pharmacology &... Apr 2021
Topics: Osmium; Osmium Tetroxide; Skin
PubMed: 33459507
DOI: 10.1111/bcpt.13562 -
Biology Jan 2021Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used... (Review)
Review
Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.
PubMed: 33445707
DOI: 10.3390/biology10010051 -
Scientific Reports Nov 2020Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies, and repurposed it to detect any...
Nanopores can serve as single molecule sensors. We exploited the MinION, a portable nanopore device from Oxford Nanopore Technologies, and repurposed it to detect any DNA/RNA oligo (target) in a complex mixture by conducting voltage-driven ion-channel measurements. The detection and quantitation of the target is enabled by the use of a unique complementary probe. Using a validated labeling technology, probes are tagged with a bulky Osmium tag (Osmium tetroxide 2,2'-bipyridine), in a way that preserves strong hybridization between probe and target. Intact oligos traverse the MinION's nanopore relatively quickly compared to the device's acquisition rate, and exhibit count of events comparable to the baseline. Counts are reported by a publicly available software, OsBp_detect. Due to the presence of the bulky Osmium tag, probes traverse more slowly, produce multiple counts over the baseline, and are even detected at single digit attomole (amole) range. In the presence of the target the probe is "silenced". Silencing is attributed to a 1:1 double stranded (ds) complex that does not fit and cannot traverse this nanopore. This ready-to-use platform can be tailored as a diagnostic test to meet the requirements for point-of-care cell-free tumor DNA (ctDNA) and microRNA (miRNA) detection and quantitation in body fluids.
Topics: DNA; Diagnostic Tests, Routine; MicroRNAs; Nanopores; Nanotechnology; Osmium; Sequence Analysis, DNA
PubMed: 33188229
DOI: 10.1038/s41598-020-76667-1 -
Frontiers in Endocrinology 2020The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse...
The bone marrow (BM) exists heterogeneously as hematopoietic/red or adipocytic/yellow marrow depending on skeletal location, age, and physiological condition. Mouse models and patients undergoing radio/chemotherapy or suffering acute BM failure endure rapid adipocytic conversion of the marrow microenvironment, the so-called "red-to-yellow" transition. Following hematopoietic recovery, such as upon BM transplantation, a "yellow-to-red" transition occurs and functional hematopoiesis is restored. Gold Standards to estimate BM cellular composition are pathologists' assessment of hematopoietic cellularity in hematoxylin and eosin (H&E) stained histological sections as well as volumetric measurements of marrow adiposity with contrast-enhanced micro-computerized tomography (CE-μCT) upon osmium-tetroxide lipid staining. Due to user-dependent variables, reproducibility in longitudinal studies is a challenge for both methods. Here we report the development of a semi-automated image analysis plug-in, , which employs the open-source software QuPath, to systematically quantify multiple bone components in H&E sections in an unbiased manner. discerns and quantifies the areas occupied by bone, adipocyte ghosts, hematopoietic cells, and the interstitial/microvascular compartment. A separate feature, , fragments adipocyte ghosts in H&E-stained sections of extramedullary adipose tissue to render adipocyte area and size distribution. Quantification of BM hematopoietic cellularity with lies within the range of scoring by four independent pathologists, while quantification of the total adipocyte area in whole bone sections compares with volumetric measurements. Employing our tool, we were able to develop a standardized map of BM hematopoietic cellularity and adiposity in mid-sections of murine C57BL/6 bones in homeostatic conditions, including quantification of the highly predictable red-to-yellow transitions in the proximal section of the caudal tail and in the proximal-to-distal tibia. Additionally, we present a comparative skeletal map induced by lethal irradiation, with longitudinal quantification of the "red-to-yellow-to-red" transition over 2 months in C57BL/6 femurs and tibiae. We find that, following BM transplantation, BM adiposity inversely correlates with kinetics of hematopoietic recovery and that a proximal to distal gradient is conserved. Analysis of recovery through magnetic resonance imaging (MRI) reveals comparable kinetics. On human trephine biopsies successfully recognizes the BM compartments, opening avenues for its application in experimental, or clinical contexts that require standardized human BM evaluation.
Topics: Adipocytes; Aging; Animals; Bone Marrow Cells; Bone Marrow Diseases; Bone and Bones; Female; Mice; Mice, Inbred C57BL; Staining and Labeling; Workflow
PubMed: 33071956
DOI: 10.3389/fendo.2020.00480 -
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 -
Biomedical Research (Tokyo, Japan) 2020The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the...
The osmium maceration method is a powerful technique for observing the three-dimensional ultrastructure of cellular organelles by scanning electron microscopy. In the conventional osmium maceration method, tissues are immersed in a diluted osmium tetroxide solution for several days at 20°C to remove soluble cytosolic proteins from the freeze-cracked surface of cells, and the optimal duration of this process is dependent on the cell type. To improve the efficiency of the osmium maceration procedure, we have examined systematically the relationship between the reaction temperature and time of the osmium maceration procedure. Treatment at temperatures higher than 20°C drastically shortened the time required to remove cytosolic proteins from the freeze-cracked surface of specimens with optimal durations for the osmium maceration of hepatocytes at 30, 40, 50 and 60°C being 30, 15, 5 and 1 h, respectively. Considering the stability and reproducibility of the macerated specimens, we concluded that the most appropriate temperature was 30 to 40°C. This rapid osmium maceration procedure was used successfully to observe the 3D ultrastructure of Purkinje cells in the cerebellum and proximal convoluted tubule cells in the kidney. This simple and reproducible rapid osmium maceration protocol should find wide appeal for the 3D analysis of cellular organelles in various cell types.
Topics: Animals; Cryopreservation; Cryoprotective Agents; Dimethyl Sulfoxide; Formaldehyde; Glutaral; Hepatocytes; Liver; Male; Microscopy, Electron, Scanning; Osmium Tetroxide; Polymers; Rats; Rats, Wistar; Temperature; Time Factors; Tissue Fixation
PubMed: 32801265
DOI: 10.2220/biomedres.41.161 -
Visualization of cytoplasmic organelles via in-resin CLEM using an osmium-resistant far-red protein.Scientific Reports Jul 2020Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular...
Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular membranous structures during electron microscopic analyses. These treatments, however, can significantly diminish the fluorescent intensity of most fluorescent proteins in cells, which creates an obstacle for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded cells. In this study, we used a far-red fluorescent protein that retains fluorescence after osmium staining and Epon embedding to perform an in-resin CLEM of Epon-embedded samples. The fluorescence of this protein was detected in 100 nm thin sections of the cells in Epon-embedded samples after fixation with 2.5% glutaraldehyde and post-fixation with 1% osmium tetroxide. We performed in-resin CLEM of the mitochondria in Epon-embedded cells using a mitochondria-localized fluorescent protein. Using this protein, we achieved in-resin CLEM of the Golgi apparatus and the endoplasmic reticulum in thin sections of the cells in Epon-embedded samples. To our knowledge, this is the first reported use of a far-red fluorescent protein retains its fluorescence after osmium staining and Epon-embedding, and it represents the first achievement of in-resin CLEM of both the Golgi apparatus and the endoplasmic reticulum in Epon-embedded samples.
Topics: Animals; COS Cells; Chlorocebus aethiops; Endoplasmic Reticulum; Fluorescence; Fluorescent Dyes; Golgi Apparatus; HEK293 Cells; HeLa Cells; Humans; Luminescent Proteins; Mitochondria; Osmium Tetroxide; Staining and Labeling; Red Fluorescent Protein
PubMed: 32647231
DOI: 10.1038/s41598-020-68191-z -
Molecules (Basel, Switzerland) Jun 2020Lignans are bioactive compounds that are especially abundant in the Norway spruce ( L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and...
Lignans are bioactive compounds that are especially abundant in the Norway spruce ( L. Karst.) knotwood. By combining a variety of chromatographic, spectroscopic and imaging techniques, we were able to quantify, qualify and localise the easily extractable lignans in the xylem tissue. The knotwood samples contained 15 different lignans according to the gas chromatography-mass spectrometry analysis. They comprised 16% of the knotwood dry weight and 82% of the acetone extract. The main lignans were found to be hydroxymatairesinols HMR1 and HMR2. Cryosectioned and resin-embedded ultrathin sections of the knotwood were analysed with scanning transmission X-ray microscopy (STXM). Cryosectioning was found to retain only lignan residues inside the cell lumina. In the resin-embedded samples, lignan was interpreted to be unevenly distributed inside the cell lumina, and partially confined in deposits which were either readily present in the lumina or formed when OsO used in staining reacted with the lignans. Furthermore, the multi-technique characterisation enabled us to obtain information on the chemical composition of the structural components of knotwood. A simple spectral analysis of the STXM data gave consistent results with the gas chromatographic methods about the relative amounts of cell wall components (lignin and polysaccharides). The STXM analysis also indicated that a torus of a bordered pit contained aromatic compounds, possibly lignin.
Topics: Lignans; Microscopy, Electron, Scanning Transmission; Picea; Spectrometry, X-Ray Emission; X-Ray Microtomography
PubMed: 32630014
DOI: 10.3390/molecules25132997