scholarly journals Systematic Transmission Electron Microscopy-Based Identification of Cellular Degradation Machinery

2021 ◽  
Author(s):  
Kit Neikirk ◽  
Zer Vue ◽  
Prasanna Katti ◽  
Jianqiang Shao ◽  
Trace A. Christensen ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Inclusion Bodies ◽  
Metabolic Diseases ◽  
Imaging Techniques ◽  
Intracellular Degradation ◽  
Fixed Sample ◽  
Transmission Electron ◽  
Cellular Organelles

Autophagosomes and lysosomes work in tandem to conduct autophagy, an intracellular degradation system which is crucial for cellular homeostasis. Altered autophagy contributes to the pathophysiology of various diseases, including cancers and metabolic diseases. Although many studies have investigated autophagy to elucidate disease pathogenesis, specific identification of the various components of the cellular degradation machinery remains difficult. The goal of this paper is to describe an approach to reproducibly identify and distinguish subcellular structures involved in autophagy. We provide methods that avoid common pitfalls, including a detailed explanation for how to distinguish lysosomes and lipid droplets and discuss the differences between autophagosomes and inclusion bodies. These methods are based on using transmission electron microscopy (TEM), capable of generating nanometer-scale micrographs of cellular degradation components in a fixed sample. In addition to TEM, we discuss other imaging techniques, such as immunofluorescence and immunogold labeling, which can be utilized for the reliable and accurate classification of cellular organelles. Our results show how these methods may be employed to accurately quantify the cellular degradation machinery under various conditions, such as treatment with the endoplasmic reticulum stressor thapsigargin or the ablation of dynamin-related protein 1.

scholarly journals Unraveling the complexity of amyloid polymorphism using gold nanoparticles and cryo-EM

2020 ◽  
Vol 117 (12) ◽  
pp. 6866-6874 ◽  
Author(s):  
Urszula Cendrowska ◽  
Paulo Jacob Silva ◽  
Nadine Ait-Bouziad ◽  
Marie Müller ◽  
Zekiye Pelin Guven ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Gold Nanoparticles ◽  
Amyloid Fibrils ◽  
Imaging Techniques ◽  
Postmortem Brain ◽  
Structural Basis ◽  
Transmission Electron

Increasing evidence suggests that amyloid polymorphism gives rise to different strains of amyloids with distinct toxicities and pathology-spreading properties. Validating this hypothesis is challenging due to a lack of tools and methods that allow for the direct characterization of amyloid polymorphism in hydrated and complex biological samples. Here, we report on the development of 11-mercapto-1-undecanesulfonate-coated gold nanoparticles (NPs) that efficiently label the edges of synthetic, recombinant, and native amyloid fibrils derived from different amyloidogenic proteins. We demonstrate that these NPs represent powerful tools for assessing amyloid morphological polymorphism, using cryogenic transmission electron microscopy (cryo-EM). The NPs allowed for the visualization of morphological features that are not directly observed using standard imaging techniques, including transmission electron microscopy with use of the negative stain or cryo-EM imaging. The use of these NPs to label native paired helical filaments (PHFs) from the postmortem brain of a patient with Alzheimer’s disease, as well as amyloid fibrils extracted from the heart tissue of a patient suffering from systemic amyloid light-chain amyloidosis, revealed a high degree of homogeneity across the fibrils derived from human tissue in comparison with fibrils aggregated in vitro. These findings are consistent with, and strongly support, the emerging view that the physiologic milieu is a key determinant of amyloid fibril strains. Together, these advances should not only facilitate the profiling and characterization of amyloids for structural studies by cryo-EM, but also pave the way to elucidate the structural basis of amyloid strains and toxicity, and possibly the correlation between the pathological and clinical heterogeneity of amyloid diseases.

Review and Detail Classification of Stacking Faults in 4H-SiC Epitaxial Layer by Mirror Projection Electron Microscopy

2020 ◽  
Vol 1004 ◽  
pp. 314-320
Author(s):  
Kentaro Ohira ◽  
Toshiyuki Isshiki ◽  
Hideki Sako ◽  
Masaki Hasegawa ◽  
Kenji Kobayashi ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Cross Section ◽  
Epitaxial Layer ◽  
Stacking Faults ◽  
Crystal Defects ◽  
Prismatic Plane ◽  
Classification Result ◽  
Transmission Electron

Stacking faults (SFs) in 4H-SiC epitaxial wafers were inspected by using a mirror projection electron microscope (MPJ) [1, 2]. Dark and bright line contrasts of SFs in MPJ images represent surface morphology and crystal defects located in the epitaxial layer. Inspected SFs were classified into three types of SFs on the basis of the MPJ images. After classification, a cross section of each type of SF was observed by transmission electron microscopy (TEM) to verify the classification result. Complex SFs classified by MPJ images consisted of prismatic plane and basal plane SFs.

scholarly journals Association between Caveolin-1 expression and pathophysiological progression of femoral nerves in diabetic foot amputation patients

2017 ◽  
Vol 12 (1) ◽  
pp. 308-314
Author(s):  
Min Ding ◽  
Xianjun Zhu ◽  
Lei Ye ◽  
Meijun Wang ◽  
Yuantao Cui ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Diabetic Foot ◽  
Metabolic Diseases ◽  
Nerve Fibers ◽  
Key Factors ◽  
Fiber Density ◽  
Caveolin 1 ◽  
Transmission Electron ◽  
Clinical Scoring

AbstractObjectiveTo evaluate the pathological changes of femoral nerves and the levels of caveolin-1 in diabetic foot amputation patients with neuropathy, and evaluate the association between caveolin-1 and neuropathy development.MethodThirty seven diabetic foot amputation patients were consecutively recruited from inpatients of Tianjin Metabolic Diseases Hospital between Jan 2003 and Nov 2005. Symptoms and signs of neuropathy, and scores of Toronto Clinical Scoring System (TCSS) were recorded. The nerve conduction velocity and HbA1c were measured. Femoral nerves were obtained 2-3 minutes after amputation. HE, Masson staining and transmission electron microscopy were used for pathological observation. Immunohistochemistry was used to observe changes of axons and count of nerve fiber density (NFD) and detect the levels of caveolin-1.ResultsHE, Masson and transmission electron microscopy showed nerve fibers were asymmetrical, the degenerated axons part had stronger staining and typical demyelinating changes. Stepwise regression models showed that HbA1c and NFD were the independent factors of caveolin-1 (F=45.090, p<0.001, R2=0.790) expression, and Caveolin-1, diabetes duration were independent factors of NFD (F=27.911, p<0.001, R2=0.691).ConclusionCaveolin-1 may be one of the key factors related to pathophysiological progression of femoral nerves in diabetic foot amputation patients.

The ultrastructure of the tegument of adult Schistosoma haematobium

Parasitology ◽  
1984 ◽  
Vol 89 (1) ◽  
pp. 71-78 ◽  
Author(s):  
B. Leitch ◽  
A. J. Probert ◽  
N. W. Runham
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Inclusion Bodies ◽  
Schistosoma Haematobium ◽  
Uranyl Acetate ◽  
En Bloc ◽  
Male Worm ◽  
Elliptical Inclusion ◽  
Transmission Electron ◽  
First Time

SummaryThe ultrastructure of the tegument of Schistosoma haematobium was examined using scanning and transmission electron microscopy. The surface of the male worm is characterized by numerous raised tubercles bearing apically directed spines. The female in contrast to the male is cylindrical and relatively smooth. Details of oral and ventral suckers are given. The use of uranyl acetate as a tertiary fixative and en bloc stain has revealed the heptalaminate nature of the outer membrane. Tegumental mitochondria are shown to be morphologically more complex than those of S. mansoni. Spherical and elliptical inclusion bodies are also described. The ultrastructure of the oesophageal tegument of S. haematobium is described for the first time and corresponds with earlier observations of S. mansoni.

Transmission Electron Microscopy Characterization of FinFET – Understanding the 3D Structure by 2D Imaging Technique

2011 ◽  
Author(s):  
Yu Zhu ◽  
Jemima Gonsalves
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Process Development ◽  
Imaging Techniques ◽  
3D Structure ◽  
Fully Depleted ◽  
Critical Dimensions ◽  
Transmission Electron ◽  
Projection Effect ◽  
Microscopy Characterization

Abstract For 22nm technology node and beyond, fully depleted devices such as FinFET and ETSOI are leading candidates. Certain critical dimensions of such devices are well below 10nm, and only transmission electron microscopy (TEM) has the resolution to provide measurement with sub-nanometer accuracy. Due to the projection effect of TEM technique, comprehensive understanding of the 3D structure from 2D images is needed for process development of FinFET. This paper will address sample preparations and TEM imaging techniques for FinFET device at sub-100nm pitch.

scholarly journals Rabies-Like Inclusions in Dogs

1989 ◽  
Vol 1 (4) ◽  
pp. 333-338 ◽  
Author(s):  
Jerome C. Nietfeld ◽  
Pauline M. Rakich ◽  
David E. Tyler ◽  
Rudy W. Bauer
Keyword(s):  
Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Rabies Virus ◽  
Inclusion Bodies ◽  
Fluorescent Antibody ◽  
Immunoperoxidase Staining ◽  
Immunoperoxidase Method ◽  
Transmission Electron ◽  
The Brain ◽  
Brain Tissues

Inclusion bodies, indistinguishable from rabies inclusion bodies (Negri bodies), were found in the brains of 8 nonrabid dogs. The inclusions were compared to Negri bodies present in neurons of rabies-positive animals and examined for the presence of rabies virus by a combination of immunoperoxidase staining (7 cases), fluorescent antibody (FA) staining (1 case), and transmission electron microscopy (4 cases). Positive immunoperoxidase staining for rabies was obtained in brain tissues from FA rabies-positive animals. All brain tissues from the 7 dogs stained by the immunoperoxidase method and the brain from the 1 dog stained by the FA method were negative for rabies. Rabies virus was not found in inclusion-containing neurons in the cases examined by transmission electron microscopy. These results emphasize the importance of FA testing and mouse inoculation for the diagnosis of rabies.

scholarly journals Sensitivity of Polyvoltine Thai Strains of Bombyx mori to a BmNPV Isolate From Mahasarakham

2020 ◽  
Vol 20 (2) ◽  
Author(s):  
Siripuk Suraporn ◽  
Olle Terenius
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Transmission Electron Microscopy ◽  
Bombyx Mori ◽  
Inclusion Bodies ◽  
Severe Disease ◽  
Bombyx Mori Nucleopolyhedrovirus ◽  
Time To Death ◽  
Transmission Electron ◽  
Scanning Electron

Abstract Virus infection by the Bombyx mori nucleopolyhedrovirus (BmNPV) is the most severe disease in Thai sericultural practice of polyvoltine silkworms. Here, we characterized a newly isolated BmNPV isolated from the Mahasarakham province in Thailand (BmNPV-MSU). The purity and morphology of BmNPV-MSU were examined using light microscopy and scanning electron microscopy. The polyhedral inclusion bodies (PIBs) of BmNPV-MSU appeared in tetragonal, hexagonal, octagonal, and globular forms. The virions were both single and multiple embedded as observed by transmission electron microscopy. We also determined the virulence of BmNPV-MSU for six different Thai polyvoltine strains by LC50 and time to death after infection. The LC50 values of Nang Lai, NK04, and Sam Rong strains were 5.05–1.52 × 107 PIBs per ml and mortality peaked 7- to 8-d after inoculation. For Nang Noi, SP2, and RE05 strains the LC50 values were 7.91–1.82 × 106 PIBs/ml and mortality peaked 4–5 d after inoculation, thus having lower chance of survival to infection by BmNPV-MSU.

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