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Heliyon Jun 2023In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same...
In-resin CLEM (Correlative Light and Electron Microscopy) of Epon-embedded cells involves correlating fluorescence microscopy with electron microscopy in the same Epon-embedded ultrathin section. This method offers the advantage of high positional accuracy compared to standard CLEM. However, it requires the expression of recombinant proteins. In order to detect the localization of endogenous target(s) and their localized ultrastructures of Epon-embedded samples using in-resin CLEM, we investigated whether immunological and affinity-labeling using fluorescent dyes applied to in-resin CLEM of Epon-embedded cells. The orange fluorescent (λ ∼550 nm) and far-red (λ ∼650 nm) fluorescent dyes examined maintained a sufficient level of fluorescent intensity after staining with osmium tetroxide and subsequent dehydration treatment with ethanol. Immunological in-resin CLEM of mitochondria and the Golgi apparatus was achieved using anti-TOM20, anti-GM130 antibodies, and fluorescent dyes. Two-color in-resin CLEM revealed that wheat germ agglutinin-puncta showed the ultrastructures of multivesicular body-like structures. Finally, taking the advantage of high positional accuracy, volume in-resin CLEM of mitochondria in the semi-thin section (2 μm thick) of Epon-embedded cells was performed by focused ion beam scanning electron microscopy. These results suggested that the application of immunological reaction and affinity-labeling with fluorescent dyes to in-resin CLEM of Epon-embedded cells is suitable for analyzing the localization of endogenous targets and their ultrastructures by scanning and transmission electron microscopy.
PubMed: 37389060
DOI: 10.1016/j.heliyon.2023.e17394 -
Journal of Visualized Experiments : JoVE Jan 2024Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence microscopy (FM) and...
Correlative light and electron microscopy (CLEM) is a comprehensive microscopy that combines the localization information provided by fluorescence microscopy (FM) and the context of cellular ultrastructure acquired by electron microscopy (EM). CLEM is a trade-off between fluorescence and ultrastructure, and usually, ultrastructure compromises fluorescence. Compared with other hydrophilic embedding resins, such as glycidyl methacrylate, HM20, or K4M, Epon is superior in ultrastructure preservation and sectioning properties. Previously, we had demonstrated that mEosEM can survive osmium tetroxide fixation and Epon embedding. Using mEosEM, we achieved, for the first time, Epon post embedding CLEM, which maintains the fluorescence and the ultrastructure simultaneously. Here, we provide step-by-step details about the EM sample preparation, the FM imaging, the EM imaging, and the image alignment. We also improve the procedures for identifying the same cell imaged by FM imaging during the EM imaging and detail the registration between the FM and EM images. We believe one can easily achieve Epon post embedding correlative light and electron microscopy following this new protocol in traditional EM facilities.
Topics: Microscopy, Electron; Microscopy, Fluorescence
PubMed: 38284521
DOI: 10.3791/66141 -
Brazilian Journal of Otorhinolaryngology 2020The 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...
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 (7.5mg/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.
Topics: Animals; Cisplatin; Cochlea; Edetic Acid; Female; Guinea Pigs; Hair Cells, Auditory; Microscopy, Electron, Scanning; Organ of Corti; Osmium Tetroxide; Tolonium Chloride
PubMed: 30797727
DOI: 10.1016/j.bjorl.2018.11.008 -
Generation and characterization of two strains of transgene mice expressing chimeric MiniSOG-MusPrP.Journal of Neuroscience Methods Jul 2020Although the presences of scrapie associated fibril in the brain tissues is a ultrastructural hallmark for prion diseases, the exact morphological structure of prion...
BACKGROUND
Although the presences of scrapie associated fibril in the brain tissues is a ultrastructural hallmark for prion diseases, the exact morphological structure of prion during the progression of the disease is still unclear. The host prion protein (PrP) is encoded by PrP gene (PRNP) locating on the chromosome 20 in human and the chromosome 2 in mouse. Recently, a novel correlative light and electron microscopy with Mini Singlet Oxygen Generator (miniSOG) was generated. MiniSOG, a small protein of 106 amino acids, can absorb blue light and emit green fluorescence that is detectable under the fluorescence microscope. MiniSOG can also partially catalyze the polymerization of DAB to form black stained structures in the presence of osmium tetroxide, which is able to be observed under transmission electron microscope.
NEW METHODS
Two kinds of miniSOG-PrP expressing recombinant plasmids were generated. Correlative photooxidation and transmission electron microscope were used to detect these plasmids. The plasmids were microinjected into fertilized FVB/NJ eggs and Tg mice expressing miniSOG-PrP fusion proteins were selected after successive bred withPRNP KO Tg mice.
RESULTS
Those two strains of Tg mice, Tg and Tg, developed normally and maintained healthy without detectable abnormality after one-year observation. Western blots and immunohistochemical assays with PrP- and miniSOG-specific antibodies confirmed that the chimeric miniSOG-PrP proteins were expressed in the brain tissues of Tg mice. Digital PCR assays proposed that the copy numbers of the inserted external gene in Tg and Tg were 2 and 12, respectively.
COMPARISON WITH EXISTING METHOD(S)
Compared with GFP tag miniSOG is significantly smaller, which makes it easy be operated experimentally and possibly has less influence on the biological function of the labeled protein. Additionally, GFP tag is an ideal marker for immunofluorescent assays, but may not be suitable for ultrastructural assays for prion morphology.
CONCLUSION
Those Tg mice may supply novel and useful experimental animals for further study on the potential morphological structure formation and deposits of prion in the brain tissues during prion infection.
Topics: Animals; Blotting, Western; Mice; Mice, Transgenic; Prions; Recombinant Proteins; Transgenes
PubMed: 32416277
DOI: 10.1016/j.jneumeth.2020.108764 -
Biology Jan 2021Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used... (Review)
Review
Several imaging methodologies have been used in biofilm studies, contributing to deepening the knowledge on their structure. This review illustrates the most widely used microscopy techniques in biofilm investigations, focusing on traditional and innovative scanning electron microscopy techniques such as scanning electron microscopy (SEM), variable pressure SEM (VP-SEM), environmental SEM (ESEM), and the more recent ambiental SEM (ASEM), ending with the cutting edge Cryo-SEM and focused ion beam SEM (FIB SEM), highlighting the pros and cons of several methods with particular emphasis on conventional SEM and VP-SEM. As each technique has its own advantages and disadvantages, the choice of the most appropriate method must be done carefully, based on the specific aim of the study. The evaluation of the drug effects on biofilm requires imaging methods that show the most detailed ultrastructural features of the biofilm. In this kind of research, the use of scanning electron microscopy with customized protocols such as osmium tetroxide (OsO), ruthenium red (RR), tannic acid (TA) staining, and ionic liquid (IL) treatment is unrivalled for its image quality, magnification, resolution, minimal sample loss, and actual sample structure preservation. The combined use of innovative SEM protocols and 3-D image analysis software will allow for quantitative data from SEM images to be extracted; in this way, data from images of samples that have undergone different antibiofilm treatments can be compared.
PubMed: 33445707
DOI: 10.3390/biology10010051 -
Visualization of cytoplasmic organelles via in-resin CLEM using an osmium-resistant far-red protein.Scientific Reports Jul 2020Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular...
Post-fixation with osmium tetroxide staining and the embedding of Epon are robust and essential treatments that are used to preserve and visualize intracellular membranous structures during electron microscopic analyses. These treatments, however, can significantly diminish the fluorescent intensity of most fluorescent proteins in cells, which creates an obstacle for the in-resin correlative light-electron microscopy (CLEM) of Epon-embedded cells. In this study, we used a far-red fluorescent protein that retains fluorescence after osmium staining and Epon embedding to perform an in-resin CLEM of Epon-embedded samples. The fluorescence of this protein was detected in 100 nm thin sections of the cells in Epon-embedded samples after fixation with 2.5% glutaraldehyde and post-fixation with 1% osmium tetroxide. We performed in-resin CLEM of the mitochondria in Epon-embedded cells using a mitochondria-localized fluorescent protein. Using this protein, we achieved in-resin CLEM of the Golgi apparatus and the endoplasmic reticulum in thin sections of the cells in Epon-embedded samples. To our knowledge, this is the first reported use of a far-red fluorescent protein retains its fluorescence after osmium staining and Epon-embedding, and it represents the first achievement of in-resin CLEM of both the Golgi apparatus and the endoplasmic reticulum in Epon-embedded samples.
Topics: Animals; COS Cells; Chlorocebus aethiops; Endoplasmic Reticulum; Fluorescence; Fluorescent Dyes; Golgi Apparatus; HEK293 Cells; HeLa Cells; Humans; Luminescent Proteins; Mitochondria; Osmium Tetroxide; Staining and Labeling; Red Fluorescent Protein
PubMed: 32647231
DOI: 10.1038/s41598-020-68191-z -
Anatomical Record (Hoboken, N.J. : 2007) Aug 2023The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve...
The guinea pig has been chosen as a research model for otologic or neuropathic studies due to the relative ease of the cochlea, cochlear nerve, and vestibular nerve dissection. Little data have been reported on the normality of these nerves. The vestibular nerve is composed of the superior vestibular, inferior vestibular, and branch nerves. This study aimed to study the microscopic anatomy of the superior vestibular nerve (SVN) of guinea pigs using light microscopy and to search for normality patterns for use in experimental models in basic otologic research. We used eight male albino guinea pigs (Cavia porcellus, English strain), weighing between 400 and 500 g. After anesthetizing, the animals were perfused with a fixative solution of 2.5% glutaraldehyde. Dissection was performed by the access method to the temporal bone, coming to the rock and exposing the cochlea and vestibular nerve. The NVS fragments were removed, postfixed in osmium tetroxide, and embedded in the epoxy plastic resin Poly/Bed 812® (Polysciences Inc., Warrington, PA). Semi-thin transverse serial sections (0.5 μm) were made using a microtome MT6000-XL, RMC, Inc. and stained with toluidine blue. Morphology and morphometry were described and evaluated using the KS 400 application (Kontron 2.0, EchingBei, Munich, Germany) by macro, a computer program specially designed and developed for the study of the VIII nerve. The SVN was found to be devoid of epineurium, with only a thin conjunctive tissue layer. The myelin sheath of guinea pigs is relatively thin compared to the sensory and motor nerves found in mammals. The average fascicular area SVN was 0.19 ± 0.05 mm , with the largest area found to be 0.24 mm and the lowest was 0.12 mm . The average number of fibers was 5,753.00 ± 538 fibers. The density of myelinated fibers reached 32,316.08 ± 11,375.29 fibers/mm . Its diameter ranged from 1.0 to 9 μm and its peak was 3 μm. The measured results confirm the results of another study, indicating that the methodology is appropriate and reproducible. These findings are important for the evaluation of injured nerves in experimental models of peripheral neuropathy and basic ear disease.
Topics: Animals; Guinea Pigs; Vestibular Nerve; Male; Myelin Sheath; Cochlea
PubMed: 37461264
DOI: 10.1002/ar.25053 -
Journal of Biomolecular Structure &... Feb 2023The concentrations of specific macromolecular species can be quantified using diagnostic tools that rely on molecular recognition by nucleic acid aptamers. One such...
The concentrations of specific macromolecular species can be quantified using diagnostic tools that rely on molecular recognition by nucleic acid aptamers. One such approach involves the formation of osmium tetroxide 2,2'-bipyridine protein adducts, followed by electrochemical detection of analytes that bind specifically to electrode-tethered aptamers. In conjunction with a 27-mer DNA aptamer that binds specifically to exosite II on human alpha thrombin, this technique permits, in theory, a highly sensitive diagnostic tool for the quantification of serum thrombin levels. However, thrombin's aptamer binding site is lined by two tryptophan residues and the conjugation of bulky osmium groups to these residues weakens aptamer binding by an estimated 4 to 12 kcal/mol, undermining detection sensitivity. Therefore, we have rationally modified this DNA aptamer to strengthen its thrombin binding in the presence of conjugated osmium. Specifically, aptamers carrying long hydrophobic thymine derivatives in place of guanine 21 have binding affinities for osmium-conjugated thrombin that are enhanced by 10 to 15 kcal/mol, suggesting that these modified aptamers may be effective in a highly sensitive electrochemical sensor for the quantification of low concentrations of thrombin. Our approach of using molecular simulation to subtly re-engineer a DNA aptamer may be generally applicable for the optimization of other macromolecular binding interfaces.Communicated by Ramaswamy H. Sarma.
Topics: Humans; Aptamers, Nucleotide; Thrombin; Osmium; Computer Simulation; Binding Sites
PubMed: 34895068
DOI: 10.1080/07391102.2021.2011414 -
Wiadomosci Lekarskie (Warsaw, Poland :... 2022The aim: The objective of our study was to evaluate the features of ultramorphometric characteristics of exocrine parenchyma and microvasculature of the pancreas in the...
OBJECTIVE
The aim: The objective of our study was to evaluate the features of ultramorphometric characteristics of exocrine parenchyma and microvasculature of the pancreas in the presence of moderate dehydration by means of an experiment in laboratory rats.
PATIENTS AND METHODS
Materials and methods: The experiment involved 20 mature white male rats divided into 2 groups: control and experimental (10 rats each). In the experimental group, moderate dehydration was simulated, i.e. the animals were deprived of water for 7 days, while the control rats were provided with a normal water supply during the study. Pancreatic parenchyma samples were fixed in phosphate-buffered glutaraldehyde solution and post-fixed in osmium tetroxide solution, dehydrated and embedded in a mixture of epoxy resins. Ultrastructural analysis was performed using JEOL JEM-1230 transmission electron microscope (Japan).
RESULTS
Results: Pancreatic electron microscopy in the presence of moderate dehydration demonstrated statistically significant changes in exocrinocytes area and exocrinocyte nucleus area which increased by 8.02% (p = 0.028) and 40.28% (p < 0.001), respectively. Among the vessels of microcirculation, the largest changes occurred in the capillaries: their lumen narrowed by 22.34% (p = 0.002) as compared with the control group. The cytoplasm of endothelial cells contained a large number of vacuoles and micropinocytotic vesicles.
CONCLUSION
Conclusions: Among the organelles of exocrinocytes, mitochondria appeared the most vulnerable to the effects of dehydration. They demonstrated polymorphic changes: a part of the mitochondria was hypotrophic and had partially reduced cristae, and another part was hypertrophic.
Topics: Animals; Dehydration; Endothelial Cells; Humans; Male; Microscopy, Electron; Microvessels; Pancreas; Rats
PubMed: 35633332
DOI: 10.36740/WLek202204214 -
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