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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 -
Scientific Reports Oct 2022Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of...
Characterization of brain infarct lesions in rodent models of stroke is crucial to assess stroke pathophysiology and therapy outcome. Until recently, the analysis of brain lesions was performed using two techniques: (1) histological methods, such as TTC (Triphenyltetrazolium chloride), a time-consuming and inaccurate process; or (2) MRI imaging, a faster, 3D imaging method, that comes at a high cost. In the last decade, high-resolution micro-CT for 3D sample analysis turned into a simple, fast, and cheaper solution. Here, we successfully describe the application of brain contrasting agents (Osmium tetroxide and inorganic iodine) for high-resolution micro-CT imaging for fine location and quantification of ischemic lesion and edema in mouse preclinical stroke models. We used the intraluminal transient MCAO (Middle Cerebral Artery Occlusion) mouse stroke model to identify and quantify ischemic lesion and edema, and segment core and penumbra regions at different time points after ischemia, by manual and automatic methods. In the transient-ischemic-attack (TIA) mouse model, we can quantify striatal myelinated fibers degeneration. Of note, whole brain 3D reconstructions allow brain atlas co-registration, to identify the affected brain areas, and correlate them with functional impairment. This methodology proves to be a breakthrough in the field, by providing a precise and detailed assessment of stroke outcomes in preclinical animal studies.
Topics: Animals; Mice; Osmium Tetroxide; X-Ray Microtomography; Stroke; Infarction, Middle Cerebral Artery; Disease Models, Animal; Iodine
PubMed: 36261475
DOI: 10.1038/s41598-022-21494-9 -
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 -
Angewandte Chemie (International Ed. in... Nov 2022Recent interest in skeletal editing necessitates the continued development of reagent classes with the ability to transfer single atoms. Terminal transition metal...
Recent interest in skeletal editing necessitates the continued development of reagent classes with the ability to transfer single atoms. Terminal transition metal nitrides hold immense promise for single-atom transfer, though their use in organic synthesis has so far been limited. Here we demonstrate a synthetic cycle with associated detailed mechanistic studies that primes the development of terminal transition metal nitrides as valuable single-atom transfer reagents. Specifically, we show [cis-terpyOsNCl ]PF inserts nitrogen into indenes to afford isoquinolines. Mechanistic studies for each step (insertion, aromatization, product release, and nitride regeneration) are reported, including crystallographic characterization of diverted intermediates, kinetics, and computational studies. The mechanistic foundation set by this synthetic cycle opens the door to the further development of nitrogen insertion heteroarene syntheses promoted by late transition metal nitrides.
PubMed: 36148482
DOI: 10.1002/anie.202213041 -
Bioelectrochemistry (Amsterdam,... Dec 2022A novel membraneless β-glucan/O enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose...
A novel membraneless β-glucan/O enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose dehydrogenase (AmPDH) and Rhodothermus marinus β-glucosidase (RmBgl3B) (RmBgl3B-AmPDH/buckypaper) with a biocathode based on solid graphite modified with Myrothecium verrucaria bilirubin oxidase (MvBOx/graphite). AmPDH was connected electrochemically with the buckypaper using an osmium redox polymer in a mediated reaction, whereas MvBOx was connected with graphite in a direct electron transfer reaction. The fuel for the bioanode was produced by enzymatic hydrolysis of β-glucan by the exoglucanase RmBgl3B into d-glucose, which in turn was enzymatically oxidised by AmPDH to generate a current response. This design allows to obtain an efficient enzymatic fuel cell, where the chemical energy converted into electrical energy is higher than the chemical energy stored in complex carbohydrate based fuel. The maximum power density of the assembled β-glucan/O biofuel cell reached 26.3 ± 4.6 μWcm at 0.36 V in phosphate buffer containing 0.5 % (w/v) β-glucan at 40 °C with excellent stability retaining 68.6 % of its initial performance after 5 days. The result confirms that β-glucan can be employed as fuel in an enzymatic biofuel cell.
Topics: Agaricales; Bioelectric Energy Sources; Electrodes; Enzymes, Immobilized; Glucose; Graphite; Osmium; Phosphates; Polymers; Rhodothermus; beta-Glucans; beta-Glucosidase
PubMed: 36122427
DOI: 10.1016/j.bioelechem.2022.108254 -
Inorganic Chemistry Sep 2022The synthesis and photophysical characterization of two osmium(II) polypyridyl complexes, [Os(TAP)dppz] () and [Os(TAP)dppp2] () containing dppz...
The synthesis and photophysical characterization of two osmium(II) polypyridyl complexes, [Os(TAP)dppz] () and [Os(TAP)dppp2] () containing dppz (dipyrido[3,2-:2',3'-]phenazine) and dppp2 (pyrido[2',3':5,6]pyrazino[2,3-][1,10]phenanthroline) intercalating ligands and TAP (1,4,5,8-tetraazaphenanthrene) ancillary ligands, are reported. The complexes exhibit complex electrochemistry with five distinct reductive redox couples, the first of which is assigned to a TAP-based process. The complexes emit in the near-IR ( at 761 nm and at 740 nm) with lifetimes of >35 ns with a low quantum yield of luminescence in aqueous solution (∼0.25%). The Δ and Λ enantiomers of and are found to bind to natural DNA and with AT and GC oligodeoxynucleotides with high affinities. In the presence of natural DNA, the visible absorption spectra are found to display significant hypochromic shifts, which is strongly evident for the ligand-centered π-π* dppp2 transition at 355 nm, which undergoes 46% hypochromism. The emission of both complexes increases upon DNA binding, which is observed to be sensitive to the Δ or Λ enantiomer and the DNA composition. A striking result is the sensitivity of Λ- to the presence of AT DNA, where a 6-fold enhancement of luminescence is observed and reflects the nature of the binding for the enantiomer and the protection from solution. Thermal denaturation studies show that both complexes are found to stabilize natural DNA. Finally, cellular studies show that the complexes are internalized by cultured mammalian cells and localize in the nucleus.
Topics: Animals; DNA; Intercalating Agents; Ligands; Mammals; Oligodeoxyribonucleotides; Osmium; Phenanthrolines; Phenazines; Ruthenium
PubMed: 36094851
DOI: 10.1021/acs.inorgchem.2c01231 -
Turkiye Parazitolojii Dergisi Sep 2022This study aimed to identify the larval form of sp. isolated from the visceral organs of (Bloch, 1793) using morphological and molecular methods.
OBJECTIVE
This study aimed to identify the larval form of sp. isolated from the visceral organs of (Bloch, 1793) using morphological and molecular methods.
METHODS
Fishes were collected from fish farms in Nadia and North 24 Paraganas for the collection of nematodes. The visceral organs were dissected and kept in 0.67% normal saline. Nematodes collected from the abdominal regions and visceral organs for light microscopy study were fixed in 70% ethanol. Morphological features were studied by placing the nematodes in lactophenol. The specimens were later preserved in 70% ethanol containing 5% glycerine. Specimens processed for scanning electron microscopy were fixed in 2.5% glutaraldehyde and postfixed in 1% osmium tetraoxide. Proper identification was done by using standard methology. Molecular studies were performed for the and gene fragments using polymerase chain reaction amplification, sequencing and phylogenetic analysis.
RESULTS
The morphological characteristics of nematodes were described with the help of light and scanning electron microscopy. Additional features not described earlier like dimensions and shape of the cephalic papillae, absence of somatic papillae, presence of caudal papillae, were identified for the first time. Moreover, molecular studies with ITS regions further confirmed the identification of the nematode.
CONCLUSION
Thus the use of morphotaxonomy along with molecular techniques would help in proper identification of sp infecting edible fish. Studies on the nematode would help toexplore the intermediate as well as paratenic hosts of the parasite. Data in this regard would contribute significantly to the fish database in regard to parasites infesting edible fishes.
Topics: Animals; Dioctophymatoidea; Fishes; Larva; Nematoda; Phylogeny
PubMed: 36094123
DOI: 10.4274/tpd.galenos.2022.41736 -
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 -
Ultramicroscopy Nov 2022Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde...
Muscle samples are commonly chemically fixed or frozen immediately upon collection for biochemical and morphological analysis. Certain fixatives such as glutaraldehyde and osmium tetroxide are widely used for transmission electron microscopy (TEM) and lead to adequate preservation of muscle ultrastructure, but do not preserve the molecular features of samples. Methacarn is suggested to be a preferable chemical fixative for light microscopy because it maintains immunohistological features of samples. However, the efficacy of methacarn to preserve ultrastructural features as a primary chemical fixative for TEM is currently unclear. Additionally, cryo-preservation of samples for TEM analysis involves freezing processes such as plunge freezing, slam freezing, or high pressure freezing. High pressure freezing is the considered the gold standard but requires costly equipment and may not be a viable option for many labs collecting tissue samples from remote locations. Dimethyl sulfoxide (DMSO) is a commonly used cryoprotectant that may allow for better structural preservation of samples by impairing ice damage that occurs during plunge/snap freezing. We aimed to assess the effectiveness of methacarn as a primary chemical fixative and determine the effect of pre-coating samples with DMSO before plunge/snap freezing tissues to be prepared for TEM. The micrographs of the methcarn-fixed samples indicate a loss of Z-disk integrity, intermyofibrillar space, mitochondria structure, and lipids. Ultimately, methacarn is not a viable primary fixative for tissue sample preparation for TEM. Similarly, liquid nitrogen freezing of samples wrapped in aluminum foil produced non-uniform Z-disk alignments that appeared smeared with swollen mitochondria. DMSO coating before freezing appears to lessen the alterations to contractile and mitochondrial morphological structures. DMSO appears to be useful for preserving the ultrastructure of sarcomeres if samples are covered before freezing.
Topics: Acetic Acid; Aluminum; Chloroform; Cryopreservation; Dimethyl Sulfoxide; Fixatives; Glutaral; Ice; Methanol; Microscopy, Electron, Transmission; Muscles; Osmium Tetroxide
PubMed: 35988477
DOI: 10.1016/j.ultramic.2022.113600 -
RSC Advances Jun 2022Studies of multiple bonding between transition metal complexes offer fundamental insight into the nature of bonding between metal ions and facilitate predictions of the...
Studies of multiple bonding between transition metal complexes offer fundamental insight into the nature of bonding between metal ions and facilitate predictions of the physical properties and the reactivities of metal complexes containing metal-metal multiple bonds. Here we report a computational interrogation on the nature of the metal-metal bonding for neutral, oxidized, and reduced forms of dinuclear rhenium and osmium corrole complexes, [{Re[TpXPC]}] and [{Os[TpXPC]}], using a complete active space self-consistent (CASSCF) methodology and density functional theory (DFT) calculations. For [{Re[TpXPC]}], [{Ru[TpXPC]}], and [{Os[TpXPC]}], CASSCF calculations shows that the effective bond order is 3.29, 2.63, and 2.73, respectively. On their oxidized forms, [{Re[TpXPC]}], [{Ru[TpXPC]}], and [{Os[TpXPC]}] molecules, the results indicate an electron removal from a ligand-based orbital, where [{Re[TpXPC]}] gives slightly different geometry from its neutral form due to populating the δ* orbital. In this regard, the CASSCF calculations give an effective bond order of 3.25 which is slightly lower than in the [{Re[TpXPC]}]. On their reduced forms, the electron addition appears to be in the metal-based orbital for [{Re[TpXPC]}] and [{Ru[TpXPC]}] whereas in the ligand-based orbital for the Os-analogue which has no effect on the Os-Os bonding, an effective bond order of 3.18 and 2.17 is presented for the [{Re[TpXPC]}] and [{Ru[TpXPC]}], respectively, within the CASSCF simulations. These results will further encourage theoreticians and experimentalists to design metalloporphyrin dimers with distinct metal-metal bonding.
PubMed: 35873315
DOI: 10.1039/d2ra03004g