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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 -
MethodsX 2022Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the...
Toxicity evaluations involve the analysis of multiple biomarkers. In this study, the liver, target organ analyzed by treatments with iron concentrations, indicated the accumulation of lipids as a response. Considering that the distribution of lipids in an organ is directly related to the induction of inflammatory processes by aquatic contaminants, this study proposes to carry out an integrative investigation of the behavior and the distribution of lipids in the liver tissue. Techniques of light and electron microscopy were performed in order to propose a new way of assessing and quantifying the distribution of lipid droplets, also presenting methodological alternatives that can be chosen by the reader according to the interests and resources available. Thus, it is assumed that the method begins with the fixation of the liver with Glutaraldehyde 2,5% in PBS 0,1 M and continues with post fixation with osmium tretoxide 1%, which marks lipids. For this proposition, two inclusion methodologies were performed to histological analyses in Historesin and ultrastructural analyses in EMBeed 812. For light microscopy (LM) analyses, cuts were obtained with 2,5 micrometers thickness, which were stained with (1) Mayers hematoxylin and (2) toluidine blue. The images obtained were processed in software Image J Fiji to evidence the lipid distribution in liver.•Cytological reactions with osmium tetroxide constitute low complexity methods that allow the optimization of the localization, identification and quantification of lipid droplets in the liver tissue when analyzed under the conventional light microscope.•Samples included in EMBeed 812 resin commonly used in Transmission Electron Microscopy can be analyzed by SEM-BEC, as complementary analyses for the detection of lipids.•Using SEM-BEC and conventional light microscopy, it is possible to quantify the area occupied by lipid droplets using Image J Fiji software, as these are contrasted due to the reaction with osmium tetroxide.
PubMed: 35818446
DOI: 10.1016/j.mex.2022.101769 -
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 -
Microscopy (Oxford, England) Dec 2022X-ray microscopes adopting computed tomography enable nondestructive 3D visualization of biological specimens at micron-level resolution without conventional 2D serial...
X-ray microscopes adopting computed tomography enable nondestructive 3D visualization of biological specimens at micron-level resolution without conventional 2D serial sectioning that is a destructive/laborious method and is routinely used for analyzing renal biopsy in clinical diagnosis of kidney diseases. Here we applied a compact commercial system of laboratory-based X-ray microscope to observe a resin-embedded osmium-stained 1-mm strip of a mouse kidney piece as a model of renal biopsy, toward a more efficient diagnosis of kidney diseases. A reconstructed computed tomography image from several hours of data collection using CCD detector allowed us to unambiguously segment a single nephron connected to a renal corpuscle, which was consistent with previous reports using serial sectioning. Histogram analysis on the segmented nephron confirmed that the proximal and distal tubules were distinguishable on the basis of their X-ray opacities. A 3D rendering model of the segmented nephron visualized a convoluted structure of renal tubules neighboring the renal corpuscle and a branched structure of efferent arterioles. Furthermore, another data collection using scientific complementary metal-oxide semiconductor detector with a much shorter data acquisition time of 15 min provided similar results from the same samples. These results suggest a potential application of the compact laboratory-based X-ray microscope to analyze mouse renal biopsy.
Topics: Mice; Animals; X-Rays; Microscopy; Kidney Diseases
PubMed: 35778966
DOI: 10.1093/jmicro/dfac033 -
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 -
RSC Advances Jun 2022This study reports the stereoselective total syntheses of the antipodes of the unique 3/10 bicyclic skeletal sesquiterpenoids, namely, hypocoprin A and hypocoprin B. The...
This study reports the stereoselective total syntheses of the antipodes of the unique 3/10 bicyclic skeletal sesquiterpenoids, namely, hypocoprin A and hypocoprin B. The synthesis involved conjugate addition accelerated by trimethylsilyl chloride, construction of the ten-membered ring the intramolecular S2 reaction promoted by 1,8-diazabicyclo[5.4.0]undec-7-ene, and osmium-mediated π-facial selective dihydroxylation to functionalize the 1,1-disubstituted alkene.
PubMed: 35754888
DOI: 10.1039/d2ra02891c -
Materials (Basel, Switzerland) Jun 2022Lead sludge from copper production is a source of rare metals, such as rhenium and osmium, whose content reaches 0.06-0.08% and 0.0025-0.0050%, respectively. The base of...
Lead sludge from copper production is a source of rare metals, such as rhenium and osmium, whose content reaches 0.06-0.08% and 0.0025-0.0050%, respectively. The base of the sludge consists of lead sulfate. A method of reductive smelting of lead sludge from copper smelting production at 1000-1100 °C has been developed. Coke was used as a reducing agent and sodium sulfate as a slag-forming material. Optimal conditions for selective extraction of rare metals in smelting products were found: osmium in the form of metallic form into raw lead and rhenium in the form of perrhenate compound NaReO into sodium-sulfate slag. The developed technology makes it possible to extract rhenium with a high degree of extraction in the form of water-soluble compounds for the subsequent production of commercial salts of rhenium by the known hydrometallurgical methods. The content of rhenium in the slag phase is 0.18-0.25%, with its initial content in the slime of 0.06-0.08%. The degree of rhenium concentration at the first stage of processing is 3-3.2 times in the form of water-soluble perrhenate. Osmium and lead do not form solid solutions; osmium in crude lead is mainly concentrated in the lower zones of lead. A method of obtaining a concentrate containing 53-67% osmium from raw lead with an initial content of 0.0025-0.0050% in the slurry and a concentration number of 13,000-21,000 times has been proposed.
PubMed: 35744129
DOI: 10.3390/ma15124071 -
International Journal of Molecular... Jun 2022(1) Background: Since the discovery of cisplatin’s cytotoxic properties, platinum(II) compounds have attracted much interest in the field of anticancer drug...
Novel Nickel(II), Palladium(II), and Platinum(II) Complexes with , Bidendate Cinnamic Acid Ester Derivatives: An In Vitro Cytotoxic Comparison to Ruthenium(II) and Osmium(II) Analogues.
(1) Background: Since the discovery of cisplatin’s cytotoxic properties, platinum(II) compounds have attracted much interest in the field of anticancer drug development. Over the last few years, classical structure−activity relationships (SAR) have been broken by some promising new compounds based on platinum or other metals. We focus on the synthesis and characterization of 17 different complexes with β-hydroxydithiocinnamic acid esters as O,S bidendate ligands for nickel(II), palladium(II), and platinum(II) complexes. (2) Methods: The bidendate compounds were synthesized and characterized using classical methods including NMR spectroscopy, MS spectrometry, elemental analysis, and X-ray crystallography, and their cytotoxic potential was assessed using in vitro cell culture assays. Data were compared with other recently reported platinum(II), ruthenium(II), and osmium(II) complexes based on the same main ligand system. (3) Results: SAR analyses regarding the metal ion (M), and the alkyl-chain position (P) and length (L), revealed the following order of the effect strength for in vitro activity: M > P > L. The highest activities have Pd complexes and ortho-substituted compounds. Specific palladium(II) complexes show lower IC50 values compared to cisplatin, are able to elude cisplatin resistance mechanisms, and show a higher cancer cell specificity. (4) Conclusion: A promising new palladium(II) candidate (Pd3) should be evaluated in further studies using in vivo model systems, and the identified SARs may help to target platinum-resistant tumors.
Topics: Antineoplastic Agents; Cell Line, Tumor; Cinnamates; Cisplatin; Coordination Complexes; Esters; Ligands; Nickel; Osmium; Palladium; Platinum; Ruthenium
PubMed: 35743112
DOI: 10.3390/ijms23126669 -
Organometallics May 2021An unusual 1,3-hydrogen shift from the metal center to the C atom of the C-chain of the allenylidene ligand in a hydride-osmium(II)-allenylidene complex is the beginning...
An unusual 1,3-hydrogen shift from the metal center to the C atom of the C-chain of the allenylidene ligand in a hydride-osmium(II)-allenylidene complex is the beginning of several interesting transformations in the cumulene. The hydride-osmium(II)-allenylidene complex was prepared in two steps, starting from the tetrahydride dimer [(Os(H···H){κ-,,-[xant(P Pr)]})(μ-Cl)][BF] (). Complex reacts with 1,1-diphenyl-2-propyn-1-ol to give the hydride-osmium(II)-alkenylcarbyne [OsHCl(≡CCH=CPh){κ-,,-[xant(P Pr)]}]BF (), which yields OsHCl(=C=C=CPh){κ-,,-[xant(P Pr)]} () by selective abstraction of the C-H hydrogen atom of the alkenylcarbyne ligand with K BuO. Complex is metastable. According to results of DFT calculations, the migration of the hydride ligand to the C atom of the cumulene has an activation energy too high to occur in a concerted manner. However, the migration can be catalyzed by water, alcohols, and aldehydes. The resulting alkenylcarbyne-osmium(0) intermediate is unstable and evolves into a 7:3 mixture of the hydride-osmium(II)-indenylidene OsHCl(=C){κ-,,-[xant(P Pr)]} () and the osmanaphthalene OsCl(CHPh){κ-,,-[xant(P Pr)]} (). Protonation of with HBF leads to the elongated dihydrogen complex [OsCl(η-H)(=C){κ-,,-[xant(P Pr)]}]BF (), while the protonation of regenerates . In contrast to , complex evolves to a half-sandwich indenyl derivative, [Os(η-IndPh)H{κ-,,-[xant(P Pr)]}][BF]Cl (). Phenylacetylene also provokes the 1,3-hydrogen shift in . However, it does not participate in the migration. In contrast to water, alcohols, and aldehydes, it stabilizes the resulting alkenylcarbyne to afford [Os(≡CCH=CPh)(η-HC≡CPh){κ-,,-[xant(P Pr)]}]Cl ().
PubMed: 35693112
DOI: 10.1021/acs.organomet.1c00176 -
Journal of the Electrochemical Society Jan 2022Electrochemical sensors that utilize enzymes are a sensitive, inexpensive means of detecting biologically relevant analytes. These sensors are categorized based on their...
Electrochemical sensors that utilize enzymes are a sensitive, inexpensive means of detecting biologically relevant analytes. These sensors are categorized based on their construction and method of signal transport. Type I sensors consist of a crosslinked enzyme on an electrode surface and are potentially subject to interference from byproducts and other biological analytes. However, type II sensors help alleviate this problem with the addition of a redox polymer layer that assists in signal transduction, thus minimizing interferences. An osmium-loaded poly(vinylimidazole) polymer (Os-PVI) is commonly used with successful results, and when combined with an enzyme yields a type II sensor. Our initial attempts at the synthesis of this polymer resulted in an unexpected osmium precursor, which had fluorescent and redox properties that did not match with the desired Os-PVI polymer. Careful exclusion of oxygen during the Os complex precursor synthesis was necessary to avoid this unexpected oxygen containing Os-precursor, which had been seen previously in mass spectrometry studies. All precursors and osmium polymers were characterized with H NMR, fluorescence, mass spectrometry, and cyclic voltammetry to provide a better understanding of these compounds and assist in the building of new sensors.
PubMed: 35692370
DOI: 10.1149/1945-7111/ac42a0