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Developmental Dynamics : An Official... Nov 2017Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may...
BACKGROUND
Spinal motor nerves are essential for relaying information between the central and peripheral nervous systems. Perturbations to cell types that comprise these nerves may impair rapid and efficient transmission of action potentials and alter nerve function. Identifying ultrastructural changes resulting from defects to these cellular components via transmission electron microscopy (TEM) can provide valuable insight into nerve function and disease. However, efficiently locating spinal motor nerves in adult zebrafish for TEM is challenging and time-consuming. Because of this, we developed a protocol that allows us to quickly and precisely locate spinal motor nerve roots in adult zebrafish for TEM processing.
RESULTS
Following fixation, a transverse slab of adult zebrafish dissected from the trunk region was mounted in embedding media, sectioned, and secondary fixation with osmium tetroxide performed. Transverse sections containing motor nerves were selected for TEM ultrathin sectioning and imaging.
CONCLUSIONS
We developed an efficient protocol for locating spinal motor nerves in adult zebrafish to allow for ultrastructural characterization. Although our work focuses on spinal motor nerves, this protocol may be useful for efficiently identifying other discrete, repeated structures within the developing and mature nervous system that are difficult to find via traditional, whole organism TEM processing. Developmental Dynamics 246:956-962, 2017. © 2017 Wiley Periodicals, Inc.
Topics: Animals; Histological Techniques; Microscopy, Electron, Transmission; Spinal Nerve Roots; Zebrafish
PubMed: 28598521
DOI: 10.1002/dvdy.24529 -
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 -
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 -
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 -
Beilstein Journal of Nanotechnology 2016Osmium tetroxide 2,2'-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product,...
Osmium tetroxide 2,2'-bipyridine (OsBp) is known to react with pyrimidines in ssDNA and preferentially label deoxythymine (T) over deoxycytosine (C). The product, osmylated DNA, was proposed as a surrogate for nanopore-based DNA sequencing due to OsBp's "perfect" label attributes. Osmylated deoxyoligos translocate unassisted and measurably slow via sub-2 nm SiN solid-state nanopores, as well as via the alpha-hemolysin (α-HL) pore. Both nanopores discriminate clearly between osmylated and intact nucleobase; α-HL was also shown to discriminate between osmylated T and osmylated C. Experiments presented here confirm that the kinetics of osmylation are comparable for short oligos and long ssDNA and show that pyrimidine osmylation is practically complete in two hours at room temperature with less than 15 mM OsBp. Under the proposed labeling conditions: deoxyoligo backbone degradation measures less than 1/1,000,000; false positives such as osmylated deoxyadenine (A) and osmylated deoxyguanine (G) measure less than 1/100,000; false negatives, i.e., unosmylated C measure less than 1/10,000; and unosmylated T must measure substantially lower than 1/10,000 due to the 27-fold higher reactivity of T compared to C. However, osmylated C undergoes degradation that amounts to about 1-2% for the duration of the labeling protocol. This degradation may be further characterized, possibly suppressed, and the properties of the degradation products via nanopore translocation can be evaluated to assure base calling quality in a DNA sequencing effort.
PubMed: 27826518
DOI: 10.3762/bjnano.7.135 -
Annals of Medicine and Surgery (2012) Oct 2021Electron microscopy is a powerful tool to study biological samples at higher magnification. The higher magnifications achieved by the electron microscopes are helpful to...
Electron microscopy is a powerful tool to study biological samples at higher magnification. The higher magnifications achieved by the electron microscopes are helpful to the researchers to study surface morphology as well as cellular morphology of the samples. The blood sample surface morphology can be visualized at higher magnification by scanning electron microscope (SEM). For the examination of the blood cells at the cellular level, transmission electron microscopes (TEM) are used. In this article, we have described the step-by-step standard protocol for the preparation of blood samples for electron microscopy. The prepared blood samples can be visualized under SEM and TEM. The obtained electron micrographs of blood cells can be used for differential diagnosis of various diseases at the cellular level.
PubMed: 34691432
DOI: 10.1016/j.amsu.2021.102895 -
Basic & Clinical Pharmacology &... Apr 2021
Topics: Osmium; Osmium Tetroxide; Skin
PubMed: 33459507
DOI: 10.1111/bcpt.13562 -
Ecology and Evolution Apr 2024Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly...
Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly mapping the microscale anatomies of related species poses a major impediment to using comparative anatomy approaches for identifying evolutionary adaptations. We describe a method using synchrotron source micro-x-ray computed tomography (syn-μXCT) combined with machine learning algorithms for high-throughput imaging of Lepidoptera (i.e., butterfly and moth) eyes. Our pipeline allows for imaging at rates of ~15 min/mm at 600 nm resolution. Image contrast is generated using standard electron microscopy labeling approaches (e.g., osmium tetroxide) that unbiasedly labels all cellular membranes in a species-independent manner thus removing any barrier to imaging any species of interest. To demonstrate the power of the method, we analyzed the 3D morphologies of butterfly crystalline cones, a part of the visual system associated with acuity and sensitivity and found significant variation within six butterfly individuals. Despite this variation, a classic measure of optimization, the ratio of interommatidial angle to resolving power of ommatidia, largely agrees with early work on eye geometry across species. We show that this method can successfully be used to determine compound eye organization and crystalline cone morphology. Our novel pipeline provides for fast, scalable visualization and analysis of eye anatomies that can be applied to any arthropod species, enabling new questions about evolutionary adaptations of compound eyes and beyond.
PubMed: 38571794
DOI: 10.1002/ece3.11137 -
Molecules (Basel, Switzerland) May 2024-Tetrahexylporphyrin was converted to its corresponding 7,8-dihydroxychlorin using an osmium tetroxide-mediated dihydroxylation strategy. Its diol moiety was shown to be...
-Tetrahexylporphyrin was converted to its corresponding 7,8-dihydroxychlorin using an osmium tetroxide-mediated dihydroxylation strategy. Its diol moiety was shown to be able to undergo a number of subsequent oxidation reactions to form a chlorin dione and porpholactone, the first -alkylporphyrin-based porphyrinoid containing a non-pyrrolic building block. Further, the diol chlorin was shown to be susceptible to dehydration, forming the porphyrin enol that is in equilibrium with its keto-chlorin form. The -hexylchlorin dione could be reduced and it underwent mono- and bis-methylation reactions using methyl-Grignard reagents, and trifluoromethylation using the Ruppert-Prakash reagent. The optical and spectroscopic properties of the products are discussed and contrasted to their corresponding -aryl derivatives (where known). This contribution establishes -tetrahexyl-7,8-dihydroxychlorins as a new and versatile class of chlorins that is susceptible to a broad range of conversions to generate functionalized chlorins and a pyrrole-modified chlorin analogue.
PubMed: 38731635
DOI: 10.3390/molecules29092144