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Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM) staining at a higher magnification, simply by removing the coverslip. However, it is not suitable for observation of immunohistochemistry (IHC) using 3,3′-diaminobenzidine (DAB) due to insufficient backscattered electron image. Traditional heavy metal enhancement techniques for DAB in IHC, (1) osmium tetroxide and iron, (2) cobalt, (3) methenamine silver (Ag), (4) gold chloride (Gold), and (5) both Ag and Gold (Ag + Gold), were examined by LV-SEM. Tissue specimens from Thy1.1 glomerulonephritis rat kidney stained with α-smooth muscle actin and visualized with DAB were enhanced by each of these enhancement methods. We found, in light microscopic and LV-SEM, that the enhancement with Ag, Gold, or Ag + Gold had better intensity and contrast than others. At a higher magnification, Ag + Gold enhancement showed high intensity and low background, although only Ag or Gold enhancement had nonspecific background. Even after observation by LV-SEM, the quality of specimens was maintained after remounting the coverslip. It was also confirmed that Ag + Gold enhancement could be useful for IHC using clinical human renal biopsy. These findings indicate that Ag + Gold provided an adequate enhancement in IHC for both LM and LV SEM observation.

Cultured Lithospermum erythrorhizon cells were fixed with a new fixation method to visualize the metabolism of shikonin derivatives, the lipophilic naphthoquinone pigments in Boraginaceae. The new fixation method combined glutaraldehyde containing malachite green, imidazole–osmium and p-phenylenediamine treatments, and cells were then observed with a transmission electron microscope. The method prevented the extraction of lipids, including shikonin derivatives, and improved the visualization of subcellular structures, especially the membrane system, when compared with that of conventional fixation. The improved quality of the transmission electron micrographs is because malachite green ionically binds to the plasma membrane, organelles and lipids and acts as a mordant for electron staining with osmium tetroxide. Imidazole promotes the reaction of osmium tetroxide, leading to enhanced electron staining. p-Phenylenediamine reduces osmium tetroxide bound to cellular materials and increases the electron density. This protocol requires only three additional reagents over conventional chemical fixation using glutaraldehyde and osmium tetroxide.

We demonstrate a facile method to fabricate a recyclable cell-alignment scaffold using nanogrooves based on sublimable liquid crystal (LC) material. Randomly and uniaxially arranged smectic LC structures are obtained, followed by sublimation and recondensation processes, which directly produce periodic nanogrooves with dimensions of a couple of hundreds of nanometers. After treatment with osmium tetroxide (OsO

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.

This communication reports experimental and theoretical evidences of σ‐hole interactions in adducts between nitrogen or oxygen nucleophiles and tetroxides of osmium or other group 8 elements. Cocrystals between pyridine or pyridine N‐oxide derivatives and osmium tetroxide are characterized through various techniques and rationalized as σ‐hole interactions using DFT calculations and several other computational tools. We propose the term “osme bond” (OmB, Om=Fe, Ru, Os, (Hs)) for naming the noncovalent interactions wherein group 8 elements have the role of the electrophile. The word osme is the transcription of ὀσμή, the ancient Greek word for smell that was used to name the heaviest group 8 element in relation to the smoky odor of its tetroxide., σ‐Hole interactions in adducts between nitrogen or oxygen nucleophiles and tetroxides of osmium or other group 8 elements are reported. Cocrystals between pyridine or pyridine N‐oxide derivatives and osmium tetroxide are characterized through various techniques including computational tools. We propose the term “osme bond” (OmB, Om=Fe, Ru, Os, (Hs)) for naming the noncovalent interactions wherein group 8 elements have the role of the electrophile.

Histochemical and fluorescence-based techniques enable the specific identification of myelin by bright-field or fluorescence microscopy. In this chapter, we describe four histological methods for the evaluation of myelin on peripheral nerve tissue sections. The first method combines the Luxol fast blue (LFB) technique with a modified Picrosirius staining contrasted with Harris hematoxylin, called MCOLL. This method simultaneously stains myelin, collagen fibers, and cell nuclei, thus giving an integrated overview of the histology, collagen network, and myelin content of the tissue in paraffin-embedded or cryosectioned samples. Secondly, we describe the osmium tetroxide method, which provides a permanent positive reaction for myelin as well as other lipids present in the tissue. The third method is the immunofluorescence-based detection of myelin proteins that allows to combine information about their expression status with other proteins of interest. Finally, the FluoroMyelin™ stains enable a fast detection of the myelin content that can be easily implemented in immunofluorescence staining panels for cryosectioned tissues. Together, this chapter provides a variety of methods to accurately identify myelin in different experimental approaches.

Postfixation with osmium tetroxide and Epon embedding are essential for the preservation and visualization of subcellular ultrastructures via electron microscopy. These chemical treatments diminish the fluorescent intensity of most fluorescent proteins in cells, creating a problem for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded mammalian cultured cells. We found that two green and two far-red fluorescent proteins retain their fluorescence after chemical fixation with glutaraldehyde, osmium tetroxide-staining, dehydration, and polymerization of Epon resins. Consequently, we could observe the fluorescence of fluorescent proteins in ultrathin sections of Epon-embedded cells via fluorescence microscopy, investigate ultrastructures of the cells in the same sections via electron microscopy, and correlate the fluorescent image with the electron microscopic image without chemical or physical distortion of the cells. In other words, referred as "in-resin CLEM" of Epon-embedded samples. This technique also improves the Z-axis resolution of fluorescent images. In this chapter, we introduce the detailed protocol for in-resin CLEM of Epon-embedded mammalian cultured cells using these fluorescent proteins.

BACKGROUND: Dicrocoeliasis is caused by trematode Dicrocoelium lanceatum from the family Dicrocoeliidae, a parasite in the bile ducts of the liver of domestic and wild animals. Dicrocoeliasis mainly affects sheep, cattle, camels, zebu, deer, fallow deer, argali, less often – horses, donkeys, dogs, rabbits, hares and bears, as well as humans. Dicrocoeliasis of ruminants is widespread across the whole Kazakhstan. Invasive diseases represent a significant obstacle in the development of domestic maral breeding, among which trematodoses, and particularly dicrocoeliasis of maral play a major role. AIM: The aim of the research was to study the influence of dicrocoelia on the ultrastructural organization of the liver of maral. MATERIALS AND METHODS: For examination under electron microscope, biopsy pieces of liver tissue of maral were fixed in 2.5% solution of glutaraldehyde with post-fixation in 1% solution of osmium tetroxide, conducted according to a conventional method, and enclosed in epon. Semi-thin and ultra-thin sections were prepared on the ultra-microtome Leica. Semi-thin sections were stained with methylene blue, azure 2, and studied at a high-resolution light optical level. The ultra-thin sections were contrasted with uranyl acetate and lead citrate according Reynolds method and examined under electron microscope Libra 120 (C. Zeiss). RESULTS: The light optical examination of half-thin sections revealed that the morphological pattern of pathological changes in liver tissue was polymorphic, even within a single hepatic lobe. CONCLUSIONS: In the liver of maral infected with chronic dicrocoeliasis, dystrophic and destructive pathological changes developed in all the cellular structures of the hepatic lobules: In the form of plethora and vast enlargement of sinusoids, vacuolar and lipodegeneration of hepatocytes, destruction of the hepatic tissue with edema, hemorrhages, in the appearance of cells associated with inflammation, and the deposition of hematin crystals.

Correlative light and electron microscopy (CLEM) is an excellent approach for examining the cellular localization of biomolecules. Here, we developed a simple method for CLEM by combining pre-embedding immunohistochemistry with a novel fluorescent probe, namely Fluolid NS Orange, and an embedding resin called ʻDurcupan™ʼ. Specimens were embedded in Durcupan™ or LR White after immunolabeling and post-fixation using glutaraldehyde and osmium tetroxide. Next, ultrathin sections were prepared on a finder grid with navigation markers. The section of the specimen embedded in Durcupan™ was found to be more stable against electron beam irradiation than specimens embedded in LR White. A fluorescence light microscopy image and a transmission electron microscopy (TEM) image, at wide-field, and low magnification, were independently obtained with the same ultrathin section. Using the three corners between finder grid bars as landmarks, fluorescence light microscopy images were superimposed with wide-field, low-magnification TEM images to identify the region of interest, which was subsequently enlarged to ascertain cellular structures localized beneath fluorescent signals. However, the enlarged TEM images appeared blurred, and fluorescence signals had a hazy appearance. To resolve this, the enlarged TEM images were replaced by high-resolution TEM images focused directly on the region of interest, thereby facilitating the collection of high-resolution CLEM images. The simple sample processing method for CLEM using osmium-resistant Fluolid NS Orange and electron beam damage-resistant Durcupan™ allowed the determination of the precise localization of fluorescence signals at subcellular levels.

The aim of the present study was to describe the morphology of the eggs of Culex (Culex) saltanensis Dyar that occurs in the Neotropical region. Eggs of the Cx. (Cux.) saltanensis were collected at the Mata Atlântica FIOCRUZ campus, fixed in 1% osmium tetroxide, prepared for mounting on metal supports, observed under a scanning electron microscope, and described morphologically. The eggs had a coniform shape with a length of approximately 0.5 mm (505–510 µm) and a width in the median portion of 117 µm (113–123 µm). Upper portion is lined with tubers of irregular shape and varying sizes (0.64–1.31 µm), located on a cross-linked matrix forming bands observed under optical microscopy. The micropyle is encased in a necklace of approximately 6.6-µm plates arranged in a flower-like shape. Comparing Cx. (Cux.) saltanensis eggs with several species of different genera, important divergent characteristics can be observed. However, this study points to the need for new descriptions of eggs of species belonging to the same subgenus in order to analyze if there will be differences between them. Culex (Cux.) saltanensis eggs have particular characteristics not observed in eggs of other Culicidae genera.

Lobomycosis is a skin infection produced by the fungus Lacazia loboi, which mainly affects some indigenous and afro-descendant populations in Tropical America. We previously reported the comparative effect of osmium tetroxide (OsO4 ) and ruthenium tetroxide (RuO4 ) in the electron microscopy (EM) of other related microorganisms. The objective of this study is to compare the effect of postfixation with OsO4 and RuO4 in the ultrastructure of L. loboi yeasts. Skin biopsies on patients diagnosed with lobomycosis were fixed in glutaraldehyde at 3% and postfixed in the following solutions: (a) 1% OsO4 , (b) 0.2% RuO4 , and (c) OsO4 at 1% followed by RuO4 at 0.2%. They were then processed using the conventional method for EM. Unlike OsO4, the treatment with RuO4 revealed different shades of gray and electron dense bands in the cell wall and other cell components of L. loboi. The most notable finding was the presence of radial filamentous structures around the yeast, which made the image look like the sun. Postfixation with RuO4 revealed ultrastructural details that had not been previously reported for L loboi. The combined use of OsO4 and RuO4 in EM of microorganisms with cell walls can be useful to evaluate the effect of microbicide substances.

Epoxidation of olefinic heterocyclic amines and subsequent acid-catalyzed hydrolysis or alternatively the direct dihydroxylation with the help of osmium tetroxide led to diols, which underwent ring cleavage in the presence of lead tetraacetate to give 3-isopropyl-2,2,4,4-tetramethyl-3-azahexanedial and 3-tert-butyl-2,2,4,4-tetramethyl-3-azapentanedial. Whereas the former dialdehyde is a highly unstable model compound because of a rapid intramolecular aldol reaction, the latter product proves to be isolable at room temperature. Furthermore, this compound is the first open-chain tri-tert-alkylamine establishing in a new record of steric crowding in tertiary amines. Strong tendencies to a Hofmann-like elimination reaction or to ring-closing reactions were observed when the aldehyde units of 3-tert-butyl-2,2,4,4-tetramethyl-3-azapentanedial were transformed into other functionalities, since both types of reactions led to a significantly decrease of the steric stress.

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 using biotin ligase 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 mins 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 including mitochondria and endoplasmic reticulum 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 ultrathin sections, and ultrastructures of mitochondria were observed as fluorescence-positive structures in the same sections by scanning electron microscopy. These results indicated that in-resin correlative light-electron microscopy of Epon-embedded cells using proximity labeling was achieved.

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.

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.

Introduction: The use of electron microscopy in the study of the inner ear has allowed us to observe minute details of the hair cells, especially in ototoxicity studies; however, the preparation of this material is a difficult and delicate task. In an attempt to simplify the handling of these materials, two agents, toluidine blue and ethylenediamine tetra-acetic acid were tested, in addition to the elimination of osmium tetroxide during the preparation of albino guinea pig cochleae. We also tested the applicability of these methodologies in an ototoxicity protocol. Objective: To verify the quality of the images obtained with and without the use of ethylenediamine tetra-acetic acid, toluidine blue and osmium tetroxide in the preparation of cochleae of albino guinea pigs for the scanning electron microscopy. Methods: Three groups of cochleae were used. In Group 1, 10 cochleae were prepared with the usual methodology, dissecting the optical capsule without decalcification and using osmium tetroxide as a post-fixative agent. In Group 2, we prepared 10 cochleae decalcified with ethylenediamine tetra-acetic acid, injecting toluidine blue in the endolymphatic space to facilitate the identification of the organ of Corti. In Group 3, we used 4 cochleae of guinea pigs that received 3 doses of cisplatin (7.5 mg/kg, D1–D5–D6), two prepared according to the methodology used in Group 1 and two with that used in Group 2. Scanning electron microscopy images were obtained from the organ of Corti region of the basal turn of each cochlea. Results: The organ of Corti was more easily identified with the use of toluidine blue. The dissection of the cochlea was more accurate in the decalcified cochleae. The quality of the images and the preservation of the organ of Corti obtained with the two methodologies were similar. Conclusion: The proposed modifications resulted in images of similar quality as those observed using the traditional methodology. Resumo: Introdução: O emprego da microscopia eletrônica no estudo da orelha interna permitiu observar detalhes minuciosos das células ciliadas especialmente em estudos de ototoxicidade. Entretanto, o preparo desse material é trabalhoso e delicado. Para simplificar a manipulação desses materiais, testou-se o uso de dois agentes, azul de toluidina e ácido etilenodiamino tetra-acético, além da retirada do tetróxido de ósmio na preparação de cócleas de cobaias albinas. Testamos também a aplicabilidade dessas metodologias em um protocolo de ototoxicidade. Objetivo: Verificar a qualidade das imagens obtidas com e sem o uso de ácido etilenodiamino tetra-acético, azul de toluidina e tetróxido de ósmio na preparação de cócleas de cobaias albinas para a microscopia eletrônica de varredura. Método: Foram utilizados três grupos de cócleas. No Grupo 1 preparou-se 10 cócleas com a metodologia usual, dissecando a cápsula ótica sem descalcificação e utilizando tetróxido de ósmio como pós-fixador. No Grupo 2 preparamos 10 cócleas descalcificadas com ácido etilenodiamino tetra-acético, injetando azul de toluidina no espaço endolinfático para facilitar a identificação do órgão de Corti. No Grupo 3 utilizamos 4 cócleas de cobaias que receberam 3 doses de cisplatina (7,5 mg/kg, D1–D5–D6), duas preparadas com a metodologia do Grupo 1 e duas com a do Grupo 2. Foram obtidas imagens da microscopia eletrônica de varredura da região do órgão de Corti do giro basal de cada cóclea. Resultados: O órgão de Corti foi mais facilmente identificado com o azul de touidina. A dissecção da cóclea foi mais precisa nas cócleas descalcificadas A qualidade das imagens e a preservação do órgão de Corti obtidas com as duas metodologias foi similar. Conclusão: As modificações propostas resultaram em imagens de qualidade similar as observadas com o uso da metodologia tradicional. Keywords: Electron microscopy, Inner ear, Cisplatin, EDTA, Palavras-chave: Microscopia eletrônica, Ouvido interno, Cisplatina, EDTA

The views of A.B. Zezin and V.A. Kabanov on the structure of polyelectrolyte complexes of linear polymers are considered and confirmed by studying chitosan hydrogels with lambda-carrageenan formed by regulated gelation. The complexes are visualized by transmission electron microscopy using osmium tetroxide for staining.

Epoxy-embedded semithin sections are useful for the analysis of cell and tissue organization, as well as for the processing of samples for transmission electron microscopy. Because only a very limited number of staining protocols have been developed for epoxy-embedded sections; semithin sections are used infrequently compared to conventional paraffin sections. Here, we describe a simple and reproducible polychromatic protocol for the routine staining of epoxy-embedded semithin sections by adapting Twort's staining method (mixture of neutral red and fast green FCF). The method can be used for the visualization of cellular organization as well as for the detection of elastic and collagen fibers. The proposed protocol demonstrated the best results for samples fixed for transmission electron microscopy, which suggests, as we demonstrated here, that this staining protocol can also be used for correlative light and electron microscopy.

Osmium tetroxide and TMEDA form stable crystalline adducts with alkenes. The structure of liquid alkenes can be determined through X-ray analysis of these derivatives. Osmium, a heavy atom, facilitates the crystallographic analysis and the determination of the absolute configuration using common Mo X-ray sources. The utility of this method for assigning structures and absolute configurations was demonstrated on a number of unsaturated substrates that include simple alkenes, enones, enol ethers, and silyl enol ethers.

We examined the functional morphology of loach Misgurnus anguillicaudatus skin by using synchrotron X-ray micro-computed tomography (SR-μCT) and high-contrast staining using osmium tetroxide or phosphotungstic acid (PTA), which enhances the image contrast of soft tissues. The captured high-spatial resolution images revealed that the surface ornamentations were stuck in the basement membrane of the loach scales. The ornamentations consisting of grooves (radii) and ridges (circuli) that can move freely and bend flexibly. The cross-sectional lateral microstructures of flat, concave and convex loach skins were observed from a live image of loach skin obtained through dark-field optical coherence tomography (OCT) imaging. The thickness of loach skin was changed with varying empty space between the mucous-cell layer and the scales by bending motion of loach. In addition, through direct measurement of drag reduction of loach skin, the mucous layer was found to have a strong influence on the reduction of skin friction. The present results enhance the understanding of the functional morphologies of mucous layer of loach to secrete mucus for skin friction reduction.

Super-resolution correlative light and electron microscopy (SR-CLEM) is a powerful approach for imaging specific molecules at the nanoscale in the context of the cellular ultrastructure. Epon epoxy resin embedding offers advantages for SR-CLEM, including ultrastructural preservation and high quality sectioning. However, Epon embedding eliminates fluorescence from most fluorescent proteins. We describe a photocontrollable fluorescent protein, mEosEM, that can survive Epon embedding after osmium tetroxide (OsO4) treatment for improved SR-CLEM.

Background: The ability of women to reproduce is well known to decline with age. Advanced maternal age is related to reduce fertility and adverse pregnancy outcomes. There is evident decline in human fertility with age due to several histological changes occur with progressive age. Objective: The research was intended to know that some cellular modifications happen during the reproductive and post-menopause phases in human endometrial cells. Materials and methods: 30 womb samples each from reproductive and postmenopausal women were collected from unembalmed human cadavers. Before being taken to electron microscopy, samples were resolved in glutaraldehyde solution and osmium tetroxide as fixative. Small sizes of endometrial specimens were drawn into a unique fixative (i.e., 5% glutaraldehyde in a buffer of 0.1μphosphate (PH 7.4), then washed three times in a buffer solution. The moved to prepare thin sections for electron microscopy. Samples were integrated at 60co for two days in Arledit –Rezen capsule. Thin parts for E.M. were then ready study Ultra-structural cell of endometrium Results: Many differences were clear and evident when comparison was made between reproductive and postmenopause stages of endometrial cell ultrastructurally. The structural changes involved shape, density, cell organelles aggregation in general, and were assigned to age, as well as degeneration, deposition, and irregularity. Conclusion: Present work results indicated that, increase the amount of cell organelles in uterine cells of young women, while the old ages characterize by less number of organelles and degenerated.

High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation.

Precise immunolocalization of molecules in relation to ultrastructural features is challenging, especially when the target is small and not frequent enough to be included in tiny ultrathin sections randomly selected for electron microscopy (EM). Glucose transporter 1 (GLUT1) is in charge of transporting glucose across brain capillary endothelial cells (BCECs). Paraformaldehyde-fixed floating sections (50 μm thick) of mouse brain were immunolabeled with anti-GLUT1 antibody and visualized with fluoronanogold. Fluorescent images encompassing the entire hemisphere were tiled to enable selection of GLUT1-positive BCECs suitable for subsequent EM and landmark placement with laser microdissection to guide trimming. Sections were then fixed with glutaraldehyde, gold enhanced to intensify the labeling and fixed with osmium tetroxide to facilitate ultrastructural recognition. Even though a region that contained target BCECs was successfully trimmed in the resin block, it was only after observation of serial ultrathin sections that GLUT1 signals in coated vesicles on the same cross section corresponding to the cross section preidentified by confocal laser microscope. This is the first ultrastructural demonstration of GLUT1 molecules in coated vesicles, which may well explain its functional relevance to transport glucose across BCECs. Successful ultrastructural localization of molecules in relation to well-preserved target structure in native tissue samples, as achieved in this study, will pave the way to understand the functional relevance of molecules and their relation to ultrastructural details.

In the current research, catalytic effectiveness of synchrotron and synchrocyclotron radiations on Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) nano capsules delivery in DNA/RNA of cancer cells is investigated. The calculation of thickness and optical constants of Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) catalytic effectiveness of synchrotron and synchrocyclotron radiations on Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) nano capsules delivery in DNA/RNA of cancer cells produced using sol-gel method over glassy medium through a single reflection spectrum is presented. To obtain an appropriate fit for reflection spectrum, the classic Drudge-Lorentz model for parametric di–electric function is used. The best fitting parameters are determined to simulate the reflection spectrum using Levenberg-Marquardt optimization method. The simulated reflectivity from the derived optical constants and thickness are in good agreement with experimental results. Keywords:Catalytic Effectiveness; Synchrotron and Synchrocyclotron Radiations; Osmium Dioxide (OsO2) and Osmium Tetroxide (OsO4) Nano Capsules; Delivery; DNA/RNA; Cancer Cells https://www.raftpubs.com/ijho-hematology-and-oncology/articles/ijho_raft1014.php

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.

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 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 in silico 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.

SUMMARY Leaves of Lolium multiflorum Lam. and Zea mays L. of different ages were prepared for TEM by a number of cytochemical methods. After post-fixation in potassium ferricyanide with osmium tetroxide the peripheral reticulum in both mesophyll and bundle sheath chloroplasts of maize stain very clearly. Post-fixation with uranyl acetate, copper and lead salts also stains the chloroplast envelope rather than the internal thylakoid system. Differences between the mesophyll and bundle sheath chloroplasts of maize and between those of ryegrass are revealed after post-fixation in zinc iodide with osmium tetroxide. After post-fixation in potassium iodide with osmium tetroxide the loculi stain less intensely than in zinc iodide. Malachite green added to glutaraldehyde at pH 7 0 enhances membrane staining in general and reveals large lipid drops in the stroma. At pH below 6 0 only the 'A' space is stained. The 'A' space and the loculus are stained when ethidium bromide is added to glutaraldehyde. The different reactions of the chloroplast membranes probably reflect differences in chemical composition.

Leafy liverwort is one of the most abundant and diverse plants in Indonesia. Their high variation and beneficial secondary metabolites contained in the oil bodies have attracted researchers' attention. The ultrastructural analysis of leafy liverworts is important as a means of species identification and also for further exploration of their oil bodies. However, the optimization of the preparation steps for observing leafy liverworts by SEM is necessary to avoid sample destruction. Fixation and drying play important roles in maintaining a sample's structure as close to its natural state as possible. Thus, in this study, we evaluated the effect of 4% Osmium tetroxide (OsO4 ) and drying on leafy liverworts ultrastructure. Microlejeunea, Acrolejeunea, and Frullania were fixed with 2.5% glutaraldehyde. Some samples were then post-fixed with 4% OsO4 , while the rest were directly dehydrated with an ethanol series and then subjected to different drying methods, i.e. air drying, freeze drying, and drying with hexamethyldisilazane (HMDS). According to the data obtained, post-fixation with 4% OsO4 could better maintain the integrity of the samples and enhance the contrast of leafy liverwort SEM images. In addition, samples dried with HMDS showed more detailed structures compared to those that were air dried. Different ultrastructure were found among the different leafy liverworts observed by SEM. Our data suggested the advantages of SEM in providing ultrastructure information on leafy liverworts as well as the optimum conditions to observe them with less deformation. OsO4 post-fixation could enhance the contrast of leafy liverwort SEM images and maintain the structure of the samples. Drying with HMDS provided a convenient way for rapid SEM preparation with less structural distortion.

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.

Receptor occupancy, the ratio between amount of drug bound and amount of total receptor on single cells, is a biomarker for treatment response to therapeutic monoclonal antibodies. Receptor occupancy is traditionally measured by flow cytometry. However, spectral overlap in flow cytometry limits the number of markers that can be measured simultaneously. This restricts receptor occupancy assays to the analysis of major cell types, although rare cell populations are of potential therapeutic relevance. We therefore developed a receptor occupancy assay suitable for mass cytometry. Measuring more markers than currently available in flow cytometry allows simultaneous receptor occupancy assessment and high‐parameter immune phenotyping in whole blood, which should yield new insights into disease activity and therapeutic effects. However, varying sensitivity across the mass cytometer detection range may lead to misinterpretation of the receptor occupancy when drug and receptor are detected in different channels. In this report, we describe a method for optimization of mass cytometry receptor occupancy measurements by using antibody‐binding quantum simply cellular (QSC) beads for standardization across channels with different sensitivities. We evaluated the method in a mass cytometry‐based receptor occupancy assay for natalizumab, a therapeutic antibody used in multiple sclerosis treatment that binds to α4‐integrin, which is expressed on leukocyte cell surfaces. Peripheral blood leukocytes from a treated patient were stained with a panel containing metal‐conjugated antibodies for detection of natalizumab and α4‐integrin. QSC beads with known antibody binding capacity were stained with the same metal‐conjugated antibodies and were used to standardize the signal intensity in the leukocyte sample before calculating receptor occupancy. We found that QSC bead standardization across channels corrected for sensitivity differences for detection of drug and receptor and generated more accurate results than observed without standardization. © 2019 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

O emprego do microscópio eletrônico de transmissão (MET) é bastante difundido no estudo de materiais biológicos, pois ele permite definição de imagens intracelulares, permitindo estudos de morfologia celular e de aspectos gerais das organelas. As imagens são produzidas por meio da interação inicial de um feixe de elétrons com a amostra, que é mantida sob condições de alto vácuo. Tanto o ambiente de vácuo como a própria irradiação dos elétrons durante a observação, representam condições adversar para a amostra, especialmente no caso de materiais orgânicos ou biológicos, que são por natureza hidratados, frágeis e elétricamente isolantes. Considerando que existem várias técnicas de preparo para MET, mas pouca descrição detalhada destas técnicas na literatura, principalmente para o preparo de raízes de plântulas, assim, esse trabalho teve como objetivo aplicar e adaptar essas técnicas para o estudo de boa qualidade de raízes de plântulas. As amostras primeiramente foram lavadas em tampão fosfato salina, passando pela fixação primária com glutaraldeído, depois pós-fixação com tetróxido de Ósmio, onde posteriormente foram desidratadas com acetona e infiltradas com resina Epon, finalizando com a polimerização, ultramicrotomia e contrastação da amostra. Contudo, essa descrição detalhada da metodologia aplicada para raiz de plântulas, pode ser utilizada com segurança e eficácia por outras pessoas, pois quase não existem descrições adaptadas de metodologia de MET na literatura, principalmente para raiz de plântulas.

Background: Glioblastoma (GB) is the most commonly diagnosed brain tumor. Its management involves adjuvant therapies, such as radiation. The cause of high probability of GB local relapse is its radioresistance related to hypoxia arising from abnormal blood-brain barrier permeability in GB vessels and in the peritumoral zone (PZ). Aim: To study pathophysiology of hypoxia in the residual GB based on the abnormalities of the morphological elements of the capillary walls building up the blood-brain barrier in GB and PZ capillaries. Materials and methods: Samples for morphological evaluation were taken during surgery for GB in 5 patients. The samples were prepared for transmission electron microscopy according to the standard technique with fixation in 2% glutaraldehyde in phosphate buffer, post-fixation with osmium tetroxide, embedding in the epon-araldite mixture, and contrast staining of ultrathin sections with uranylacetate and lead citrate. Abnormalities of the capillary cells (mitochondrial vacuolization and vacuolization of endoplasmic reticulum in endothelial cells, pericytes and astrocytes), as well as of the acellular element of the capillary wall, i.e. basement membrane, were assessed in two groups of capillaries – those of GB (n = 38) and those of PZ (n = 32). Results: Abnormalities characteristic for apoptosis and oncosis were found in the cells of the GB and PZ capillaries of the blood-brain barrier, such as endothelial cells and pericytes. However, in the GB capillaries these abnormalities were signifcantly more frequent (р < 0.001). Only half (52.6%) of the GB capillaries had an edematous pericapillary astrocyte layer. In all other capillaries, astrocyte sprouts either were visualized as separate morphological elements (13.2%) or were not visualized at all (34.2%). All PZ capillaries had the astrocyte layer, being edematous in 68.8% of the capillaries and totally edematous only in 25%. Thickened basement membrane was found in the vast majority (89.5%) of the GB capillaries and only in 25% of the PZ capillaries (р < 0.001). Conclusion: Findings of abnormal cell elements in the GB capillaries leading to peritumoral edema and consequent hypoxia are highly likely to be the cause of the remnant GB radioresistance.

The novel aspect of this study is to contextualize the co-localization of biomolecular expression in widened and narrowed periodontal ligament (PDL) space within a mechanically activated periodontal complex. The PDL is unique as it is the only ligament with both innervation and vascularization. Maxillary molars in 6-week old male C57BL/6 mice (N=5) were experimentally translated for two weeks using an elastic spacer. Contralateral teeth were used as controls. Mechanical testing of the periodontal complex of a mouse in situ and imaging using an X-ray micro-computed tomography (micro-XCT) illustrated deformations within blood vessels (BV) of the PDL under load. PDL-bone and PDL-cementum entheses at the widened and narrowed PDL-spaces following experimental tooth movement (ETM) illustrated osterix (OSX), bone sialoprotein (BSP), cluster of differentiation 146 (CD146), and protein gene product 9.5 (PGP9.5), indicating active remodeling at these sites. PGP9.5 positive nerve bundles (NBs) were co-localized with multinucleated cells (MCs), Howship’s resorption lacunae, and CD146 positive BVs. Association between nerves and MC was complemented by visualizing the proximity of osmium tetroxide stained NBs with the ultrastructure of MCs by performing scanning transmission electron microscopy. Spatial association of NB with BV, and NB with MC, provided insights into the plausible co-activation of NBs to initiate osteoclastic activity. Resorption of mineral occurs as an attempt to restore PDL-space of the load bearing complex, specifically at the PDL-entheses. Mapping of anatomy-specific structural elements and their association with regenerative molecules by correlating light and electron micrographs can provide insights into the use of these extracellular matrix molecules as plausible targets for pharmacological interventions related to tooth movement. Within the realm of tissue regeneration, modulation of load can reverse naturally occurring mineral formation to experimentally-induced resorption, and naturally occurring mineral resorption to experimentally-induced formation at the enthesial sites in order to permit tooth translation.

This study presents an X-ray computed nanotomography (nano-CT) based, high-resolution imaging technique. Thanks to a voxel resolution of 540 nm, this novel technique is suitable for observing the 3D morphology of soft biopolymeric scaffolds seeded with stem cells. A sample of highly porous collagen scaffold seeded with contrasted mesenchymal stem cells (MSC) was investigated by using lab-based nano-CT. The whole volume of the sample was analysed without its destruction. To evaluate the potential of nano-CT, a comparison measurement was done using a standard microscopy technique. Scanning electron microscopy (SEM) combined with energy dispersive X-ray analysis (EDX) established an extension and local accumulation of the contrasting agent - heavy metallic osmium tetroxide. The presented imaging technique is novel as it will help to understand better the behaviour of cells while interacting with three-dimensional biomaterials. This is crucial for both experimental and clinical tissue engineering applications in order to limit the risk of uncontrolled cell growth, and potentially tumour formation. LAY DESCRIPTION: Biomaterials play a crucial role in tissue engineering by serving as 3D scaffolds for cellular attachment, proliferation, and in growth ultimately leading to new tissue formation. Cell morphology and proliferation inside the 3D scaffold are necessary to know for assessing cell viability. However, these studies are usually negatively affected by the limitations of imaging techniques. We demonstrate that X-ray computed nanotomography (nano-CT), based on high-resolution imaging technique providing voxel resolution of 540 nm, is a suitable method for observing the 3D morphology of soft biopolymeric scaffolds seeded with stem cells. A sample of highly porous collagen scaffold seeded with contrasted mesenchymal stem cells (MSC) was investigated by using a lab-based nano-CT. The whole volume of the sample was analysed without its destruction. To evaluate the potential of nano-CT, a comparison measurement was done using a standard microscopy technique. Scanning electron microscopy in a combination with energy dispersive X-ray analysis established an extension and local accumulation of the contrasting agent - heavy metallic osmium tetroxide. The presented imaging technique is novel as it will help to understand better the behaviour of cells while interacting with three-dimensional biomaterials. This is crucial for both experimental and clinical tissue engineering applications in order to limit the risk of uncontrolled cell growth, and potentially tumour formation.

X-ray micro-CT is one of the most useful techniques to examine 3D cellular architecture inside dry seeds. However, the examination of imbibed seeds is difficult because immersion in water causes a decline in the image quality. Here, we examined the use of ionic liquids for specimen preparation of chemically fixed imbibed seeds of Arabidopsis. We found that treatment with high concentrations of ionic liquids after osmium tetroxide fixation helped not only to prevent the structural damage caused by seed shrinkage, but also to preserve the image quality. Under these conditions, the cellular architecture of seeds was also well maintained.

Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00799-2.

Parasitic infections can be challenging to study, because light and electron microscopy are often limited in visualising complex and inaccessible attachment sites. Exemplifying this, Trichuris spp. inhabits a tunnel of epithelial cells within the host caecum and colon. A significant global burden of this infection persists partly because available anthelminthics lack efficacy, although the mechanisms underlying this remain unknown. Consequently, there is a need to pioneer new approaches to better characterize the parasite niche within the host and investigate how variation in its morphology and integrity may contribute to resistance to therapeutic intervention. To address these aims, we exploited 3D X-ray micro-computed tomography (microCT) to image the mouse whipworm T. muris in caeca of wild-type C57BL/6 and SCID mice ex vivo. Using osmium tetroxide staining to effectively enhance contrast of worms, we found that a subset exhibited preferential positioning towards the bases of the intestinal crypts. Moreover, in one rare event, we demonstrate whipworm traversal of the lamina propria. This morphological variability contradicts widely accepted conclusions from conventional microscopy of the parasite niche, showing Trichuris in close contact with the host proliferative and immune compartments that may facilitate immunomodulation. Furthermore, by using a skeletonization-based approach we demonstrate considerable variation in tunnel length and integrity which may represent an indicator of tunnel “health”. The qualitative and quantitative observations provide a new morphological point of reference for future in vitro study of Trichuris-host interactions and highlight the potential of microCT to more accurately characterise enigmatic host-parasite interactions.Author SummaryParasites are often difficult to observe once established within host tissues, presenting a barrier to biological understanding and therapeutic innovation. Whipworms (Trichuris spp.) affect 500 million people worldwide, causing significant disability, and appear partially resistant to widely used “deworming” drugs. However, the inaccessibility of worms within the cells of the host intestine makes them highly challenging to image and study. By investigating Trichuris attachment sites in 3D, using X-ray micro-computed tomography, we found that the niche is highly variable in size and, contrary to reports in all previous studies, can also penetrate different layers of intestinal tissue. By showing that worms are positioned much closer to host immune cells that previously appreciated, we provide a morphological reference point for future studies on how Trichuris effectively avoids clearance by the host. The non-invasive imaging approach used represents an excellent opportunity to clarify the lifecycles of other difficult-to-study parasites.

Transmission electron microscopy of central nervous system white matter has provided unparalleled access to the ultrastructural features of axons, their myelin sheaths, and the major cells of white matter; namely, oligodendrocytes, oligodendrocyte precursors, astrocytes, and microglia. In particular, it has been invaluable in elucidating pathological changes in axons and myelin following experimentally induced injury or genetic alteration, in animal models. While also of value in the examination of human white matter, the tissue is rarely fixed adequately for the types of detailed analyses that can be performed on well-preserved samples from animal models, perfusion fixed at the time of death. In this chapter we describe methods for obtaining, processing, and visualizing white matter samples using transmission electron microscopy of perfusion fixed tissue and for unbiased morphometry of white matter, with particular emphasis on axon and myelin pathology. Several advanced electron microscopy techniques are now available, but this method remains the most expedient and accessible for routine ultrastructural examination and morphometry.

Lignans are bioactive compounds that are especially abundant in the Norway spruce (Picea abies 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 OsO4 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.

Osmium tetroxide is a strong oxidizing agent used in electron microscopy. Eye exposure may cause severe burns, and after inhalation or ingestion damage to the respiratory or gastrointestinal tract occurs. Exposure to osmium and its compounds is extremely rare. We present a case of a 32-year-old female stained by 9 mL of 2% osmium tetroxide in acetone during an accident in the laboratory, with rare dermal and ocular findings. Due to lack of data in toxicological databases and the absence of antidote, the therapy was symptomatic. Osmium was detected in serum 19 hours later (0.22 μg/L) and in urine during the 15-hour collection (three samples-7.05, 1.65 and 8.45 μg/L). In blood serum on admission, after 1 and 2 days after exposure, the levels of iron (28.2, 39.8 and 50.5 μmol/L; reference range 5.8-34.5 μmol/L) and transferrin receptor/ferritine were elevated. To our knowledge, this is the first paper documenting a significant absorption from the skin and potentially from the eye conjunctiva, based on serum and urine analysis. The relationship between increased iron in blood and exposure has not been described yet, and the mechanism remains unknown. The patient is being followed up for the unknown long-term effects.

The use of high resolution, three-dimensional visualization has been receiving growing interest within life sciences, with non-invasive imaging tools becoming more readily accessible. Although initially useful for visualizing mineralized tissues, recent developments are promising for studying soft tissues as well. Especially for micro-CT scanning, several X-ray contrast enhancers are performant in sufficiently contrasting soft tissue organ systems by a different attenuation strength of X-rays. Overall visualization of soft tissue organs has proven to be possible, although the tissue-specific capacities of these enhancers remain unclear. In this study, we tested several contrast agents for their usefulness to discriminate between tissue types and organs, using three model organisms (mouse, zebrafish and Xenopus). Specimens were stained with osmium tetroxide (OsO4), phosphomolybdic acid (PMA) and phosphotungstic acid (PTA), and were scanned using high resolution microtomography. The contrasting potentials between tissue types and organs are described based on volume renderings and virtual sections. In general, PTA and PMA appeared to allow better discrimination. Especially epithelial structures, cell-dense brain regions, liver, lung and blood could be easily distinguished. The PMA yielded the best results, allowing discrimination even at the level of cell layers. Our results show that those staining techniques combined with micro-CT imaging have good potential for use in future research in life sciences.

The high-resolution technique transmission electron microscopy (TEM), with OsO4 as the traditional fixative, is an essential tool for cell biology and medicine. Although OsO4 has been extensively used, it is far from perfect because of its high volatility and toxicity. Os(II) polypyridyl complexes like [Os(phen)2(dppz)]2+ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) are not only the well-known molecular DNA “light-switches” but also the potential ideal candidates for TEM studies. Here, we report that the cell-impermeable cationic [Os(phen)2(dppz)]2+ can be preferentially delivered into the live-cell nucleus through ion-pairing with chlorophenolate counter-anions, where it functions as an unparalleled enantioselective nuclear DNA imaging reagent especially suitable for correlative light and electron microscopy (CLEM) studies in both living and fixed cells, which can clearly visualize chromosome aggregation and decondensation during mitosis simultaneously. We propose that the chiral Os(II) polypyr...

Aim of the study was to investigate the atherosclerotic calcification employing our original technique of tissue staining, embedding, and backscattered scanning electron microscopy. Materials and methods . Atherosclerotic plaques excised during the carotid endarterectomy have been fixed in 10 % neutral phosphate buffered saline formalin for 24 hours, stained in 2 % osmium tetroxide for 60 hours and in alcoholic uranyl acetate for 5 hours with the subsequent epoxy resin embedding, grinding, polishing, lead citrate counterstaining for 7 minutes, sputter coating with carbon and backscattered scanning electron microscopy. We then analysed localisation, structure, and microenvironment of the calcium deposits. Results . Within the atherosclerotic plaques, we identified 3 distinct calcification morphologies: compact homogenous macrocalcifications, sheet-like heterogeneous macrocalcifications along the destructed collagen and elastin fibers, and microcalcifications around large calcium deposits with the sharp margins. However, even relatively homogenous compact macrocalcifications had uneven distribution of electron density. In conjunction with groups of mineral deposits of distinct calcium phosphate phases including nascent calcified loci, this testified to ongoing calcium phosphate maturation while multiple microcalcifications merging into the single macrocalcification indicated ossification. Compact ossifying macrocalcifications with smooth margins frequently had a dense connective tissue capsule. The microenvironment of calcium deposits was often characterised by leaky neovessels. Conclusion . Diversity of calcification morphologies and ossification in atherosclerotic plaques is similar to those observed in calcific aortic valve disease and bioprosthetic heart valve failure. Combined with atherosclerotic plaque disaggregation followed by flow cytometry and singlecell RNA sequencing, our technique may improve our understanding of atherosclerotic calcification and lead to the identification of appropriate therapeutic targets to prevent or retard this process.

OBJECTIVE A preliminary study to determine collagen fibril diameter (CF-ED) distribution on medial and lateral sides of cleft lip (CL). MATERIAL AND METHODS Tissue samples from medial and lateral sides of CL were fixed in 2.5% glutaraldehyde and 1% osmium tetroxide and embedded in Araldite CY212 resin for transmission electron microscopy. The analysis of CF-ED was performed using the ImageJ program. To characterize the packaging of collagen fibrils (CFs) in the two tissues, we estimated the collagen number density (CF-ND) and fibril-area-fraction (FAF). Differences in measurements across the two sides were calculated using Wilcoxon signed-rank test. RESULTS The CF-ED was statistically significantly (p

Detection of single-base mismatches with respect to a probe strand has been a predominant pursuit in electrochemical biosensor efforts, due to links found between single nucleotide polymorphisms (SNPs) and the predisposition to various diseases. We report an osmium tetroxide bipyridine-based, thermally-controlled, competitive-strand electrochemical assay to allow amplified detection of single-base mismatches. Optimally designed competitive-strand displacement and hybridization temperature allows us to distinguish the single-mismatched-target from the fully complementary sequence with unambiguous, highly reproducible, robust signal differences of over 90%. Furthermore, we find a complex interplay between the position of the redox label, variations in strand displacement kinetics due to mismatches incorporated into the competitive strand, and alterations in the melting temperature of DNA duplexes tethered on the gold surface, when probed by square-wave voltammetry. These insights will apply to any surface-tethered DNA-based electrochemical biosensor, and can help with understanding complex phenomena involved in these types of assays.

For nearly 50 years immunogold labeling on ultrathin sections has been successfully used for protein localization in laboratories worldwide. In theory and in practice, this method has undergone continual improvement over time. In this study, we carefully analyzed circulating protocols for postembedding labeling to find out if they are still valid under modern laboratory conditions, and in addition, we tested unconventional protocols. For this, we investigated immunolabeling of Epon-embedded cells, immunolabeling of cells treated with osmium, and the binding behavior of differently sized gold particles. Here we show that (in contrast to widespread belief) immunolabeling of Epon-embedded cells and of cells treated with osmium tetroxide is actually working. Furthermore, we established a "speed protocol" for immunolabeling by reducing antibody incubation times. Finally, we present our results on three-dimensional immunogold labeling.

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.

The development of histological techniques for the past five centuries was propelled by the invention of the microscope and the improvement of its magnification and resolution. The greatest improvement of methods, allowing observation of plant and animal tissues, dates from the 18th and 19th centuries; whereas in the 20th century mainly the development of electron microscopical and other methods such as fluorescence and freezing techniques, as well as histochemistry and immunohistochemistry proceeded. Independent of the microscope type used for the observation each sample requires specific histoprocessing techniques including fixation (chemical or physical), dehydration (e.g. in series of alcohols), embedding (e.g. in paraffin wax or plastic media) and staining (e.g. hematoxylin and eosin, trichrome methods, toluidine, and methylene blue etc.). High resolution and magnification achieved in transmission electron microscopy requires specific methods of fixation (e.g. double fixation with glutaraldehyde and osmium tetroxide), infiltration and embedding in polymerizing plastics like epoxy or acrylic resins, cutting for ultrathin sections with diamond knifes and contrasting with uranyl acetate and lead citrate, whereas samples for scanning electron microscopy require drying after dehydration and vacuum sputtering with carbon or gold before observation.