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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 -
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 -
Biosensors & Bioelectronics May 2021Wearable devices that generate power using sweat have garnered much attention in the field of skin electronics. These devices require high performance with a small...
Wearable devices that generate power using sweat have garnered much attention in the field of skin electronics. These devices require high performance with a small volume and low production rate of sweat by living organisms. Here we demonstrate a high-power biofuel cell bracelet based on the lactate in human sweat. The biofuel cell was developed by using a lactate oxidase/osmium-based mediator/carbon nanotube fiber for lactate oxidation and a bilirubin oxidase/carbon nanotube fiber for oxygen reduction; the fibers were woven into a hydrophilic supportive textile for sweat storage. The storage textile was sandwiched between a hydrophobic textile for sweat absorption from the skin and a hydrophilic textile for water evaporation to improve sweat collection. The performance of the layered cell was 74 μW at 0.39 V in 20 mM artificial sweat lactate, and its performance was maintained at over 80% for 12 h. Furthermore, we demonstrated a series-connection between anode/cathode fibers by tying them up to wrap the bracelet-type biofuel cell on the wrist. The booster six-cell bracelet generated power at 2.0 V that is sufficient for operating digital wrist watches.
Topics: Bioelectric Energy Sources; Biofuels; Biosensing Techniques; Electronics; Humans; Wearable Electronic Devices
PubMed: 33640657
DOI: 10.1016/j.bios.2021.113107 -
Archives Italiennes de Biologie Dec 2022Spinal cord injury (SCI) causes various neurological consequences that disrupt the structure of axons. The C/EBP Homologous Protein (CHOP) acts in neuronal death by...
PURPOSE
Spinal cord injury (SCI) causes various neurological consequences that disrupt the structure of axons. The C/EBP Homologous Protein (CHOP) acts in neuronal death by apoptosis has been demonstrated in experimental models. Rosmarinic acid (RA) is a phenolic compound used for therapeutic purposes in many diseases. In this study, we investigated the therapeutic effect of Rosmarinic acid application on inflammation and apoptotic development after spinal cord injury.
METHODS
Male Wistar albino rats (n: 24) were assigned to three group: control, SCI and SCI+ RA. All rats were fixed on the operating table after anesthesia, the skin of the thoracic region was opened with a midline incision and the paravertebral muscles were dissected and T10-T11 laminas were exposed. A cylindrical tube of 10 cm length was fixed to the area to be laminectomy. A metal weight of 15 grams was left down the tube. Spinal damage was created, skin incisions were sutured. 50 mg/kg rosmarinic acid was given orally for 7 days after the spinal injury. Spinal tissues were fixed in formaldehyde solution and processed for paraffin wax tissue protocol and 4-5 μm sections were taken with microtome for further immunohistochemical examination. Caspase-12 and CHOP antibodies were applied to sections. Remaining tissues were carried out in glutaraldehyde for the first fixation then in osmium tetroxide for the second. Tissues were kept in pure araldite and thin sections were taken for transmission electron microscope.
RESULTS
Values of malondialdehyde (MDA), myeloperoxidase (MPO), glutathione peroxidase (GSH), neuronal degeneration, vascular dilation, inflammation, CHOP and Caspase-12 expression were increased in SCI group compared to control group. Only glutathione peroxidase content was decreased in SCI group. In SCI group, disruption of basement membrane structure in canalis ependymalis, degeneration in structures of unipolar bipolar and multipolar neurons, and apoptotic changes were seen with increased inflammation in the piamater region and positive CHOP expression in vascular endothelial cells. In SCI+RA group, reorganization of basement membrane pill in canalis ependymalis were observed with mild Caspase-12 activity in some canalis ependymal and glial cells. Also, moderate CHOP expression in multipolar and bipolar neurons and glia cells were observed.
CONCLUSIONS
The application of RA has a significant effect on preventing damage in SCI. It was thought that CHOP and Caspase-12 mediated oxidative stress could be a guide in showing the potential and therapeutic target to stop the apoptotic course after SCI injury.
Topics: Male; Rats; Animals; Rats, Wistar; Caspase 12; Endothelial Cells; Spinal Cord Injuries; Rosmarinic Acid
PubMed: 36881913
DOI: 10.12871/000398292022341 -
Frontiers in Endocrinology 2020The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis,... (Review)
Review
Reporting Guidelines, Review of Methodological Standards, and Challenges Toward Harmonization in Bone Marrow Adiposity Research. Report of the Methodologies Working Group of the International Bone Marrow Adiposity Society.
The interest in bone marrow adiposity (BMA) has increased over the last decade due to its association with, and potential role, in a range of diseases (osteoporosis, diabetes, anorexia, cancer) as well as treatments (corticosteroid, radiation, chemotherapy, thiazolidinediones). However, to advance the field of BMA research, standardization of methods is desirable to increase comparability of study outcomes and foster collaboration. Therefore, at the 2017 annual BMA meeting, the International Bone Marrow Adiposity Society (BMAS) founded a working group to evaluate methodologies in BMA research. All BMAS members could volunteer to participate. The working group members, who are all active preclinical or clinical BMA researchers, searched the literature for articles investigating BMA and discussed the results during personal and telephone conferences. According to the consensus opinion, both based on the review of the literature and on expert opinion, we describe existing methodologies and discuss the challenges and future directions for (1) histomorphometry of bone marrow adipocytes, (2) BMA imaging, (3) BMA imaging, (4) cell isolation, culture, differentiation and modulation of primary bone marrow adipocytes and bone marrow stromal cell precursors, (5) lineage tracing and BMA modulation, and (6) BMA biobanking. We identify as accepted standards in BMA research: manual histomorphometry and osmium tetroxide 3D contrast-enhanced μCT for quantification, specific MRI sequences (WFI and H-MRS) for studies, and RT-qPCR with a minimal four gene panel or lipid-based assays for quantification of bone marrow adipogenesis. Emerging techniques are described which may soon come to complement or substitute these gold standards. Known confounding factors and minimal reporting standards are presented, and their use is encouraged to facilitate comparison across studies. In conclusion, specific BMA methodologies have been developed. However, important challenges remain. In particular, we advocate for the harmonization of methodologies, the precise reporting of known confounding factors, and the identification of methods to modulate BMA independently from other tissues. Wider use of existing animal models with impaired BMA production (e.g., , Kit) and development of specific BMA deletion models would be highly desirable for this purpose.
Topics: Adipogenesis; Adiposity; Animals; Bone Marrow; Guidelines as Topic; Humans; International Agencies; Obesity; Research Design; Research Report; Societies, Scientific
PubMed: 32180758
DOI: 10.3389/fendo.2020.00065 -
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 -
Anatomical Record (Hoboken, N.J. : 2007) Aug 2023The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve...
The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve dissection. Little data have been reported on the normality of these nerves. The vestibular nerve is composed of the superior vestibular, inferior vestibular, and branch nerves. This study aimed to study the microscopic anatomy of the superior vestibular nerve (SVN) of guinea pigs using light microscopy and to search for normality patterns for use in experimental models in basic otologic research. We used eight male albino guinea pigs (Cavia porcellus, English strain), weighing between 400 and 500 g. After anesthetizing, the animals were perfused with a fixative solution of 2.5% glutaraldehyde. Dissection was performed by the access method to the temporal bone, coming to the rock and exposing the cochlea and vestibular nerve. The NVS fragments were removed, postfixed in osmium tetroxide, and embedded in the epoxy plastic resin Poly/Bed 812® (Polysciences Inc., Warrington, PA). Semi-thin transverse serial sections (0.5 μm) were made using a microtome MT6000-XL, RMC, Inc. and stained with toluidine blue. Morphology and morphometry were described and evaluated using the KS 400 application (Kontron 2.0, EchingBei, Munich, Germany) by macro, a computer program specially designed and developed for the study of the VIII nerve. The SVN was found to be devoid of epineurium, with only a thin conjunctive tissue layer. The myelin sheath of guinea pigs is relatively thin compared to the sensory and motor nerves found in mammals. The average fascicular area SVN was 0.19 ± 0.05 mm , with the largest area found to be 0.24 mm and the lowest was 0.12 mm . The average number of fibers was 5,753.00 ± 538 fibers. The density of myelinated fibers reached 32,316.08 ± 11,375.29 fibers/mm . Its diameter ranged from 1.0 to 9 μm and its peak was 3 μm. The measured results confirm the results of another study, indicating that the methodology is appropriate and reproducible. These findings are important for the evaluation of injured nerves in experimental models of peripheral neuropathy and basic ear disease.
Topics: Animals; Guinea Pigs; Vestibular Nerve; Male; Myelin Sheath; Cochlea
PubMed: 37461264
DOI: 10.1002/ar.25053 -
European Journal of Histochemistry : EJH Feb 2022The spread technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and...
The spread technique proposed by Miller and Beatty in 1969 allowed for the first time the visualization at transmission electron microscopy of nucleic acids and chromatin in an isolated and distended conformation. The final step of staining the spread chromatin is of critical importance because it can strongly influence the interpretation of the results. We evaluated different staining techniques and the most part of them provided a good result. Specifically, well contrasted micrographs were obtained when staining with H3PW12O40 (PTA), as originally proposed by Miller and Beatty, and with two alternatives proposed here: uranyl acetate or terbium citrate staining. Quite good contrast of the spread DNA could be achieved also by using Osmium Ammine; while no or few contrast of nucleic acids was observed by staining with KMnO₄ and H3PMo12O40 (PMA) respectively.
Topics: Chromatin; Coloring Agents; Microscopy, Electron; Nucleic Acids; Staining and Labeling
PubMed: 35212500
DOI: 10.4081/ejh.2022.3364