scholarly journals Just Seeing Is Not Enough for Believing: Immunolabelling as Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue

Viruses ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1816
Author(s):  
Andreja Erman ◽  
Karmen Wechtersbach ◽  
Daniel Velkavrh ◽  
Jerica Pleško ◽  
Maja Frelih ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Protein Identification ◽  
Light And Electron Microscopy ◽  
Immunogold Labelling ◽  
Correlative Microscopy ◽  
Nucleocapsid Proteins ◽  
Similar Morphology ◽  
Cell Structures ◽  
Transmission Electron ◽  
Nasopharyngeal Tissue

Background: There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. Methods: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. Results: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. Conclusions: Immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

scholarly journals Three-dimensional nanostructure of an intact microglia cell

2018 ◽  
Author(s):  
Giulia Bolasco ◽  
Laetitia Weinhard ◽  
Tom Boissonnet ◽  
Ralph Neujahr ◽  
Cornelius T. Gross
Keyword(s):  
Electron Microscopy ◽  
Cell Biology ◽  
Light And Electron Microscopy ◽  
Three Dimensional ◽  
Microglia Cell ◽  
Cell Structures ◽  
Transmission Electron ◽  
Morphological Criteria ◽  
Membrane Protrusions ◽  
Microscopy Techniques

Microglia are non-neuronal cells of the myeloid lineage that invade and take up long-term residence in the brain during development (Ginhoux et al. 2010) and are increasingly implicated in neuronal maturation, homeostasis, and pathology (Bessis et al. 2007; Paolicelli et al. 2011; Li et al. 2012; Aguzzi et al. 2013, Cunningham 2013, Cunningham et al. 2013). Since the early twentieth century several methods for staining and visualizing microglia have been developed. Scientists in Ramón y Cajal’s group (Achúcarro 1913, Río-Hortega 1919) pioneered these methods and their work led to the christening of microglia as the third element of the nervous system, distinct from astrocytes and neurons. More recently, a combination of imaging, genetic, and immunological tools has been used to visualize microglia in living brain (Davalos et al. 2005; Nimmerjahn et al. 2005). It was found that microglia are highly motile under resting conditions and rapidly respond to injuries (Kettenmann et al. 2011) suggesting a role for microglia in both brain homeostasis and pathology. Transmission Electron microscopy (TEM) has provided crucial complementary information on microglia morphology and physiology but until recently EM analyses have been limited to single or limited serial section studies (Tremblay et al. 2010; Paolicelli et al. 2011; Schafer et al. 2012; Tremblay et al. 2012; Sipe et al. 2016). TEM studies were successful in defining a set of morphological criteria for microglia: a polygonal nucleus with peripheral condensed chromatin, a relatively small cytoplasm with abundant presence of rough endoplasmic reticulum (RER), and a large volume of lysosomes and inclusions in the perikaryon. Recent advances in volumetric electron microscopy techniques allow for 3D reconstruction of large samples at nanometer-resolution, thus opening up new avenues for the understanding of cell biology and architecture in intact tissues. At the same time, correlative light and electron microscopy (CLEM) techniques have been extended to 3D brain samples to help navigate and identify critical molecular landmarks within large EM volumes (Briggman and Denk 2006; Maco et al. 2013; Blazquez-Llorca et al. 2015, Bosch et al. 2015). Here we present the first volumetric ultrastructural reconstruction of an entire mouse hippocampal microglia using serial block face scanning electron microscopy (SBEM). Using CLEM we have ensured the inclusion of both large, small, and filopodial microglia processes. Segmentation of the dataset allowed us to carry out a comprehensive inventory of microglia cell structures, including vesicles, organelles, membrane protrusions, and processes. This study provides a reference that can serve as a data mining resource for investigating microglia cell biology.

Investigation of Tickborne Pathogens within Naturally Infected Brown Dog Tick (Ixodidae: Rhipicephalus Sanguineus) in Egypt by Light and Electron Microscopy

2020 ◽  
Keyword(s):  
Electron Microscopy ◽  
Salivary Gland ◽  
Light And Electron Microscopy ◽  
Rhipicephalus Sanguineus ◽  
Mammalian Host ◽  
Babesia Canis ◽  
Brown Dog Tick ◽  
Transmission Electron ◽  
Dog Tick ◽  
Theileria Spp

Tick borne pathogens present a significant health challenge to animals and human because a single tick may transmit multiple pathogens to a mammalian host during feeding. The present study detected tick-borne pathogens from pet dogs. A total of 666 ticks were collected from 144 pet and sheltered dogs in Egypt from April to September 2018. For hemolymph, midgut and salivary gland smears 546 ticks were used as well as 360 egg smears from 120 female tick were examined by light microscope. The infected ticks were prepared for transmission electron microscopy (TEM). Ticks were identified; Rhipicephalus sanguineus. Light microscopy showed infection rates of 44.69%, 68.50% & 15.75%, in hemolymph, midgut and salivary gland, respectively. H. canis recorded the highest rates in hemolymph and midgut (35.89% & 49.82%, respectively), but Theileria spp. was the lowest (0.73% & 2.93%, respectively). In salivary gland smears, Babesia canis. was detected in 13.55% and Theileria spp. in 1.83%. Mixed infection in same tick was recorded in 4.76% &0.37% in midgut and salivary gland smears, respectively. Babesia canis stages were recovered from 15% of egg smears. R. sanguineus was natural infected by Babesia, Theileria, Hepatozoon and Anaplasma phagocytophilum as well as mixed infections of protozoa accompanied by a complicated sign of diseases and failure in accurate diagnosis.

scholarly journals Localization of ‘Candidatus Liberibacter solanacearum’ and Evidence for Surface Appendages in the Potato Psyllid Vector

2016 ◽  
Vol 106 (2) ◽  
pp. 142-154 ◽  
Author(s):  
J. M. Cicero ◽  
T. W. Fisher ◽  
J. K. Brown
Keyword(s):  
Electron Microscopy ◽  
Light And Electron Microscopy ◽  
Fluorescence Labeling ◽  
Potato Psyllid ◽  
Bactericera Cockerelli ◽  
Visual Identification ◽  
Transmission Electron ◽  
Candidatus Liberibacter ◽  
Identification Of Bacteria

The potato psyllid Bactericera cockerelli is implicated as the vector of the causal agent of zebra chip of potato and vein-greening of tomato diseases. Until now, visual identification of bacteria in the genus ‘Candidatus Liberibacter’ has relied on direct imaging by light and electron microscopy without labeling, or with whole-organ fluorescence labeling only. In this study, aldehyde fixative followed by a coagulant fixative, was used to process adult psyllids for transmission electron microscopy (TEM) colloidal gold in situ hybridization experiments. Results indicated that ‘Ca. Liberibacter solanacearum’ (CLso)-specific DNA probes annealed to a bacterium that formed extensive, monocultural biofilms on gut, salivary gland, and oral region tissues, confirming that it is one morphotype of potentially others, that is rod-shaped, approximately 2.5 µm in diameter and of variable length, and has a rough, granular cytosol. In addition, CLso, prepared from shredded midguts, and negatively stained for TEM, possessed pili- and flagella-like surface appendages. Genes implicating coding capacity for both types of surface structures are encoded in the CLso genome sequence. Neither type was seen for CLso associated with biofilms within or on digestive organs, suggesting that their production is stimulated only in certain environments, putatively, in the gut during adhesion leading to multiplication, and in hemolymph to afford systemic invasion.

scholarly journals Correlative Microscopy of Bone in Implant Osteointegration Studies

2010 ◽  
Vol 10 ◽  
pp. 2238-2247 ◽  
Author(s):  
Alessandra Triré ◽  
Désirée Martini ◽  
Ester Orsini ◽  
Marco Franchi ◽  
Viviana De Pasquale ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Specific Information ◽  
Experimental Sample ◽  
Correlative Microscopy ◽  
Bone Morphology ◽  
Bone Implant ◽  
Technical Procedure ◽  
Transmission Electron ◽  
Bone Section ◽  
Scanning Electron

Routine morphological analyses usually include investigations by light microscopy (LM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Each of these techniques provides specific information on tissue morphology and all the obtained results are then combined to give an in-depth morphological overview of the examined sample. The limitations of this traditional comparative microscopy lie in the fact that each technique requires a different experimental sample, so that many specimens are necessary and the combined results come from different samples. The present study describes a technical procedure of correlative microscopy, which allows us to examine the same bone section first by LM and then, after appropriate processing, by SEM or TEM. Thanks to the possibility of analyzing the same undecalcified bone sections both by LM and SEM, the approach described in the present study allows us to make very accurate evaluations of old/new bone morphology at the bone-implant interface.

scholarly journals Ultrastructural analysis of the spermatogenesis in the guinea pig (Cavia porcellus)

2016 ◽  
Vol 36 (suppl 1) ◽  
pp. 89-94 ◽  
Author(s):  
Luciana S. Simões ◽  
Rose E.G. Rici ◽  
Phelipe O. Favaron ◽  
Taís Harumi de Castro Sasahara ◽  
Rodrigo S.N. Barreto ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Guinea Pig ◽  
Sexual Development ◽  
Morphological Changes ◽  
Light And Electron Microscopy ◽  
Management Systems ◽  
Cavia Porcellus ◽  
Reproductive Parameters ◽  
Development Stages ◽  
Transmission Electron

Abstract: al for both, the establishment of appropriate management systems, and for the use of new species as animal models. In this study, we used light and electron microscopy to characterize the sexual development stages of the guinea pig (Cavia porcellus) in specimens of 30, 45 and 90 days of age. We observed the differentiation of spermatocytes only through transmission electron microscopy in the leptotene, zygotene and pachytene phases of meiosis, in 30-day-old animals. During puberty, there was differentiation of the germinative epithelium and formation of the acrosome. Spermatozoa, however, were not detected. Thus, we could infer that puberty happens after 45 days of age. Sexual maturity was evident in 90-day-old specimens. Our results showed that changes in the testicular germinative epithelium during the postnatal sexual development in guinea pig led to morphological changes, including the ones related to the development of Leydig and Sertoli cells, which are directly related to puberty. In this work, we provide new morphological subsidies for a better understanding of reproductive parameters of this species, enabling its use as an animal model in the field of the reproductive biology.

scholarly journals The osmium tetroxide-p-phenylenediamine procedure reveals the chromatid cores and kinetochores of meiotic chromosomes by light and electron microscopy.

1996 ◽  
Vol 44 (11) ◽  
pp. 1279-1288 ◽  
Author(s):  
C Antonio ◽  
J M González-García ◽  
J Page ◽  
J A Suja ◽  
J C Stockert ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Reaction Mechanism ◽  
Osmium Tetroxide ◽  
Light And Electron Microscopy ◽  
X Ray ◽  
Meiotic Chromosomes ◽  
Transmission Electron ◽  
Ethanol Acetic Acid ◽  
Electron Imaging ◽  
Z Contrast

We analyzed first-metaphase meiotic chromosomes of the grasshopper Chorthippus jucundus by two different methods, i.e., a silver impregnation technique and the osmium tetroxide-p-phenylenediamine (Os-PPD) procedure. The former was applied on squashed testes previously fixed in ethanol-acetic acid, whereas for Os-PPD the material was not subjected to any previous extraction treatment but was fixed in OsO4, treated with PPD, and embedded in Epon 812. Both techniques revealed chromatid cores and kinetochores regardless of the processing of the material (squashed or sectioned). Unstained Os-PPD sections were analyzed by light microscopy and transmission electron microscopy (TEM). The Os-PPD technique provided a high contrast of chromatid cores and kinetochores in relation to the chromatin, which revealed a low electron density. To determine the Os-PPD reaction mechanism, the PAS procedure, as well as scanning electron microscopy (SEM) backscattering and SEM X-ray microanalysis, was performed on sections. By use of the Os-PPD-PAS procedure, glycol groups formed by oxidation of osmium bound to aromatic substrates were detected in chromatid cores and kinetochores by brightfield and fluorescence microscopy. A high Z contrast was detected in these structures by backscattered electron imaging. SEM X-ray microanalysis showed osmium and phosphorus to be the main elements present on the chromatid cores. Taking into account the known reactivity of OsO4 and the present results, the possible participation of nucleic acids as well as proteins in the Os-PPD reaction mechanism and in the composition of chromatid cores and kinetochores is discussed.

Fabrication and Characterization of Nanostructured Surface Layer of 38CrSi Steel

2007 ◽  
Author(s):  
Shi-Ning Ma ◽  
De-Ma Ba ◽  
Chang-Qing Li ◽  
Fan-Jun Meng
Keyword(s):  
Electron Microscopy ◽  
Grain Size ◽  
Surface Layer ◽  
Grain Refinement ◽  
Fine Particles ◽  
Nanostructured Surface ◽  
Cell Structures ◽  
Strain And Strain Rate ◽  
Transmission Electron ◽  
Average Grain Size

A nanocrystalline surface layer was fabricated on a 38CrSi Steel with tempered sorbite structure by using Supersonic Fine Particles Bombarding (SFPB). The microstructural evolution of SFPB-treated specimens under different processing conditions was characterized by using X-ray diffraction, scanning electron microscopy and transmission electron microscopy. Experimental evidence showed severe plastic deformation and obvious grains refinement were observed and a nanocrystalline surface layer (grain size < 100nm) was found after SFPB treatment. The thickness of nanostructured surface layer varies from a few to about 25μm as treated time increasing from 80s to 240s, but the grain size varies slightly. For the sample treated for 240s, the average grain size of equiaxed nanocrystallites with random crystallographic orientations on the top surface layer is about 16nm. The indexing of diffraction rings indicates nanostructured surface layer consists of ferrite and cementite phases without any evidence of a new phase. The structure size increases gradually from nano-scale to original-scale with an increase of the distance from the top surface layer. In the region about 20–30μm deep from the top surface, the microstructures are mainly composed of 60–100nm roughly equiaxed grains and subgrains. Some subbounsaries are composed of dense dislocation walls (DDWs). In this regime some cell structures are also seen, which are separated by dislocation lines (DTs) and some DDWs. Experimental analysis indicate coarse-grains are gradually refined into nano-sized grains by dislocations activity with gradual increase of strain and strain rate from matrix to treated surface. Both ferrite and cementite phases occur grain refinement. Grain refinement of 38CrSi sample is mainly attributed to the movement of dislocation.

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