-
Food Chemistry Jul 2019The current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of...
The current methods used to routinely assess freshness in the fishing industry reflect more a state of spoilage than a state of freshness. Mitochondria, the seat of cellular respiration, undergo profound changes in post mortem tissues. The objective of this study was to demonstrate that mitochondrial activity constitutes a putative early fish freshness marker. The structure of gilthead sea bream (Sparus aurata) muscle tissue was evaluated over time by transmission electron microscopy. Respiration was assessed in mitochondria isolated from sea bream fillets using oxygraphy. Membrane potential (ΔΨ) was determined by fluorescence (Rhodamine 123). Mitochondrial activity of fillets stored at +4 °C was studied for 6 days. Changes in mitochondrial cristae structure appeared from Day 3 highlighting the presence of dense granules. ΔΨ and mitochondrial activity were significantly disrupted in sea bream fillets after 96 h of storage at +4 °C. Mitochondrial activity constituted a reliable and early indicator of fish freshness.
Topics: Animals; Mitochondria; Sea Bream; Seafood
PubMed: 30857714
DOI: 10.1016/j.foodchem.2019.02.076 -
Ecology and Evolution Apr 2024Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly...
Comparative anatomy is an important tool for investigating evolutionary relationships among species, but the lack of scalable imaging tools and stains for rapidly mapping the microscale anatomies of related species poses a major impediment to using comparative anatomy approaches for identifying evolutionary adaptations. We describe a method using synchrotron source micro-x-ray computed tomography (syn-μXCT) combined with machine learning algorithms for high-throughput imaging of Lepidoptera (i.e., butterfly and moth) eyes. Our pipeline allows for imaging at rates of ~15 min/mm at 600 nm resolution. Image contrast is generated using standard electron microscopy labeling approaches (e.g., osmium tetroxide) that unbiasedly labels all cellular membranes in a species-independent manner thus removing any barrier to imaging any species of interest. To demonstrate the power of the method, we analyzed the 3D morphologies of butterfly crystalline cones, a part of the visual system associated with acuity and sensitivity and found significant variation within six butterfly individuals. Despite this variation, a classic measure of optimization, the ratio of interommatidial angle to resolving power of ommatidia, largely agrees with early work on eye geometry across species. We show that this method can successfully be used to determine compound eye organization and crystalline cone morphology. Our novel pipeline provides for fast, scalable visualization and analysis of eye anatomies that can be applied to any arthropod species, enabling new questions about evolutionary adaptations of compound eyes and beyond.
PubMed: 38571794
DOI: 10.1002/ece3.11137 -
Arkhiv Patologii 2022To compare the neointima structure in conduits for coronary bypass grafting, bioprosthetic heart valves, tissue-engineered vascular grafts, and metal stents.
OBJECTIVE
To compare the neointima structure in conduits for coronary bypass grafting, bioprosthetic heart valves, tissue-engineered vascular grafts, and metal stents.
MATERIAL AND METHODS
The objects of the study were the fragments of the human internal thoracic artery, experimental biodegradable vascular prostheses, leaflets of xenopericardial bioprostheses of heart valves, and fragments of stented vessels. Tissue samples were fixed in formalin and post-fixed in osmium tetroxide. After dehydration and epoxy resin embedding, the samples were ground and polished. Samples were counterstained with uranyl acetate and lead citrate and visualized by means of backscattered scanning electron microscopy.
RESULTS
Neointimal pattern in all samples was similar. Neointima was comprised of endothelial cells, smooth muscle cells, fibroblasts, and the extracellular matrix. Endothelial cells showed significant diversity both between different elements of the circulatory system and within the same tissue, having either elongated or polygonal shape. Adhesion of leukocytes testified to the endothelial cell activation. In the absence of inflammation in the superficial layer of the neointima, the arrangement of smooth muscle cells and extracellular matrix fibers was parallel to the endothelium. Clusters of foam cells were frequently detected around the neointimal layers with solid inclusions (metal stents or calcium deposits). Thickening of the neointima was accompanied by the presence of capillaries and capillary-like structures.
CONCLUSION
Neointima formation is a typical response to the damage inflicted to the elements of the circulatory system. Neointima underwent a constant remodeling characterized by an altered cellular composition, macrophage invasion, neovascularization, and calcification.
Topics: Bioprosthesis; Endothelial Cells; Heart Valves; Humans; Myocytes, Smooth Muscle; Neointima
PubMed: 35639839
DOI: 10.17116/patol20228403114 -
Angewandte Chemie (International Ed. in... Sep 2021This communication reports experimental and theoretical evidences of σ-hole interactions in adducts between nitrogen or oxygen nucleophiles and tetroxides of osmium or...
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.
PubMed: 34260810
DOI: 10.1002/anie.202107978 -
Methods in Molecular Biology (Clifton,... 2020Transmission electron microscopy of central nervous system white matter has provided unparalleled access to the ultrastructural features of axons, their myelin sheaths,...
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.
Topics: Animals; Axons; Cacodylic Acid; Dissection; Epoxy Resins; Formaldehyde; Glutaral; Humans; Microscopy, Electron, Transmission; Microtomy; Myelin Sheath; Neuroglia; Osmium Tetroxide; Phthalic Anhydrides; Polymers; Staining and Labeling; Tissue Embedding; Tissue Fixation; Wallerian Degeneration; White Matter
PubMed: 32524485
DOI: 10.1007/978-1-0716-0585-1_18 -
Sovremennye Tekhnologii V Meditsine 2021was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal...
UNLABELLED
was to evaluate the efficacy of a novel technique for preparation, staining, and visualization of tissues containing extra-skeletal mineralization areas, all-metal implants or their prototypes for their subsequent examination using scanning electron microscopy in the backscattered electron mode.
MATERIALS AND METHODS
After fixation in 10% formalin (24 h), the biomaterial (a titanium nickelide plate with the surrounding tissues after subcutaneous implantation, patented titanium alloy plates with the surrounding tissues after cranioplasty, primary and secondary calcified atherosclerotic plaques) were fixed with 1% osmium tetroxide (12 h) and then stained with 2% aqueous solution of osmium tetroxide (48 h). The samples were further stained with 2% alcoholic uranyl acetate (5 h), dehydrated with isopropanol (5 h) and acetone (1 h), impregnated with a mixture of acetone and epoxy resin Epon (1:1, 6 h) and then embedded into a fresh portion of epoxy resin (24 h), which was followed by polymerization at 60°C. After grinding and polishing, epoxy blocks were counterstained with lead citrate (7 min) and sputter-coated with carbon, then the samples were visualized by scanning electron microscopy in the backscattered electron mode. The elemental composition was studied using X-ray microanalysis.
RESULTS
The developed technique allows obtaining high-quality images at five thousand-fold magnifications, provides the possibility to identify the shape and structure of intact metal and mineral inclusions, and to type the surrounding cells, distinguishing mesenchymal and immunocompetent cells by shape and cytoplasmic content. Apart from connective tissue capsule thickness and leukocyte infiltration, this technique makes it possible to estimate the number and area of newly formed small-caliber vessels representing a surrogate marker of inflammation.
CONCLUSION
The proposed technique provides the possibility to investigate adequately the structure of samples when their sectioning is impossible or significantly complicated, with image quality remarkably higher than that obtained by light microscopy.
Topics: Alloys; Metals; Microscopy, Electron, Scanning; Osmium Tetroxide; Staining and Labeling
PubMed: 34795988
DOI: 10.17691/stm2020.12.4.02 -
Plant Methods 2020Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the...
BACKGROUND
Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented.
RESULTS
Different algae (, ) and higher plant tissues ( sp., , ) were successfully frozen and prepared for HPF at freezing temperatures (- 2 °C, - 5 °C, - 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature.
CONCLUSIONS
The method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques.
PubMed: 32280364
DOI: 10.1186/s13007-020-00586-5 -
Journal of Chemical Neuroanatomy Apr 2021Acrylamide is a fundamental cause of accidental toxicity in humans. This study aimed to investigate the neuroprotective effect of vitamin E (Vit. E), 5-amino salicylic...
Acrylamide is a fundamental cause of accidental toxicity in humans. This study aimed to investigate the neuroprotective effect of vitamin E (Vit. E), 5-amino salicylic acid (5-ASA), and their combination against acrylamide-induced sciatic nerve toxicity. For this purpose, 25 male Wister rats were divided into 5 groups: control, acrylamide, acrylamide + Vit. E, acrylamide + 5-ASA, and acrylamide + Vit. E + 5-ASA. Food intake and body weight were assessed after 7 days. Furthermore, the gait score was also evaluated for each rat. The sciatic nerve was dissected, fixed, and processed for routine light and electron microscopic examination. Haematoxylin and eosin, osmium tetroxide for myelin sheath, and toluidine blue for semithin section were used. In addition, immunohistochemistry for caspase-3 and inducible nitric oxide synthase (iNOS) were performed. The results showed reduced food intake and body weight in acrylamide rats. Abnormal gait score was also recorded in acrylamide rats with significant improvement in Vit. E, and Vit. E + 5-ASA groups. Histologically, Vit. E and 5-ASA provided potential protection against decreased sciatic nerve axon density, disrupted myelination, and the alteration in the immunohistochemistry induced by acrylamide. Vit. E and its combination with 5-ASA provided more evident protection compared to 5-ASA alone. 5-ASA significantly decreased apoptotic cell death (caspase-3 immunoexpression) while Vit. E failed. Both Vit. E and 5-ASA significantly decreased iNOS immunoexpression in the sciatic nerve, where 5-ASA was superior to Vit. E. These findings concluded that both Vit. E and 5-ASA protect against acrylamide-induced peripheral neuropathy through downregulation of both caspase-3 and iNOS immunoexpression.
Topics: Acrylamide; Animals; Caspase 3; Immunohistochemistry; Male; Mesalamine; Neuroprotective Agents; Nitric Oxide Synthase Type II; Peripheral Nervous System Diseases; Rats; Rats, Wistar; Sciatic Nerve; Vitamin E
PubMed: 33588031
DOI: 10.1016/j.jchemneu.2021.101935 -
The Journal of Histochemistry and... Jun 2022Low-vacuum scanning electron microscopy (LV-SEM) is a powerful tool that allows to observe light microscopic specimens with periodic acid-silver methenamine (PAM)...
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.
Topics: Animals; Gold; Immunohistochemistry; Microscopy, Electron, Scanning; Osmium Tetroxide; Rats; Vacuum
PubMed: 35611640
DOI: 10.1369/00221554221102996 -
Photochemistry and Photobiology Sep 2021Peripheral injuries constitute a substantial clinical problem with unsatisfactory treatment. The study's objective was to analyze the effects of photobiomodulation...
Peripheral injuries constitute a substantial clinical problem with unsatisfactory treatment. The study's objective was to analyze the effects of photobiomodulation therapy (PBMT) on median nerve regeneration and muscle recovery after axonotmesis. Twenty-four rats were randomized into three groups: control (CG), injury (IG), and LED therapy (LEDG). A 630 ± 20 nm (300-mW) LED was placed in contact with the skin. One point over the injury site was irradiated for 30 s, delivering 9 J (9 J cm ). PBMT irradiation was performed once daily for 5 days followed by two-day interval and then more five consecutive days of treatment. Proximal and distal segments of the nerve and flexors muscles were removed for histomorphometric analysis using H&E staining for muscles and osmium tetroxide for nerves. The myelinated fiber and axon diameter and the myelin sheath thickness were greater in the proximal and distal nerve segments in the LEDG compared to the IG (P ≤ 0.05). The number of myelinated fibers was greater in the distal segment of the LEDG (P ≤ 0.05). The area, circumference, and diameter of the muscle fibers were larger in the LEDG than in the IG (P ≤ 0.05). The PBMT protocol used favored axonal regeneration and muscle recovery.
Topics: Animals; Low-Level Light Therapy; Muscle, Skeletal; Nerve Regeneration; Rats; Trauma, Nervous System
PubMed: 33714216
DOI: 10.1111/php.13415