Correlative fractography: combining scanning electron microscopy and light microscopes for qualitative and quantitative analysis of fracture surfaces.

PubMed

Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

2013-04-01

Correlative fractography is a new expression proposed here to describe a new method for the association between scanning electron microscopy (SEM) and light microscopy (LM) for the qualitative and quantitative analysis of fracture surfaces. This article presents a new method involving the fusion of one elevation map obtained by extended depth from focus reconstruction from LM with exactly the same area by SEM and associated techniques, as X-ray mapping. The true topographic information is perfectly associated to local fracture mechanisms with this new technique, presented here as an alternative to stereo-pair reconstruction for the investigation of fractured components. The great advantage of this technique resides in the possibility of combining any imaging methods associated with LM and SEM for the same observed field from fracture surface.

Emodin affects biofilm formation and expression of virulence factors in Streptococcus suis ATCC700794.

PubMed

Yang, Yan-Bei; Wang, Shuai; Wang, Chang; Huang, Quan-Yong; Bai, Jing-Wen; Chen, Jian-Qing; Chen, Xue-Ying; Li, Yan-Hua

2015-12-01

Streptococcus suis (S. suis) is a swine pathogen and also a zoonotic agent. In this study, the effects of subinhibitory concentrations (sub-MICs) of emodin on biofilm formation by S. suis ATCC700794 were evaluated. As quantified by crystal violet staining, biofilm formation by S. suis ATCC700794 was dose-dependently decreased after growth with 1/2 MIC, 1/4 MIC, or 1/8 MIC of emodin. By scanning electron microscopy, the structural architecture of the S. suis ATCC700794 biofilms was examined following growth in culture medium supplemented with 1/2 MIC, 1/4 MIC, 1/8 MIC, or 1/16 MIC of emodin. Scanning electron microscopy analysis revealed the potential effect of emodin on biofilm formation by S. suis ATCC700794. The expression of luxS gene and virulence genes in S. suis ATCC700794 was investigated by quantitative RT-PCR. It was found that sub-MICs of emodin significantly decreased the expression of gapdh, sly, fbps, ef, and luxS. However, it was found that sub-MICs of emodin significantly increased the expression of cps2J, mrp, and gdh. These findings showed that sub-MICs of emodin could cause the difference in the expression level of the virulence genes.

Autophagy in anti-apoptotic effect of augmenter of liver regeneration in HepG2 cells.

PubMed

Shi, Hong-Bo; Sun, Hai-Qing; Shi, Hong-Lin; Ren, Feng; Chen, Yu; Chen, De-Xi; Lou, Jin-Li; Duan, Zhong-Ping

2015-05-07

To investigate the role of autophagy in the anti-apoptotic effect of augmenter of liver regeneration (ALR). Autophagy was induced through serum deprivation. An ALR-expressing plasmid was transfected into HepG2 cells, and autophagic flux was determined using fluorescence microscopy, electron microscopy, Western blot and quantitative polymerase chain reaction (qPCR) assays. After ALR-expressing plasmid transfection, an autophagy inhibitor [3-methyladenine (3-MA)] was added to HepG2 cells, and apoptosis was observed using fluorescence microscopy and flow cytometry. Autophagy was activated in HepG2 cells, peaking at 24 h after serum deprivation. Microtubule-associated protein light chain three-II levels were higher in HepG2 cells treated with ALR than in control cells, fluorescence microscopy, electron microscopy and qPCR studies showed the similar trend, and p62 levels showed the opposite trend, which indicated that ALR may play an important role in increasing autophagy flux. The numbers of apoptotic cells were substantially higher in HepG2 cells treated with both ALR and 3-MA than in cells treated with ALR alone. Therefore, the protective effect of ALR was significantly attenuated or abolished when autophagy was inhibited, indicating that the anti-apoptotic effect of ALR may be related to autophagy. ALR protects cells from apoptosis partly through increased autophagy in HepG2 cells and may be valuable as a new therapeutic treatment for liver disease.

Rapid fibroblast activation in mammalian cells induced by silicon nanowire arrays.

PubMed

Ha, Qing; Yang, Gao; Ao, Zhuo; Han, Dong; Niu, Fenglan; Wang, Shutao

2014-07-21

Activated tumor-associated fibroblasts (TAFs) with abundant fibroblast activation protein (FAP) expression attract tremendous attention in tumor progression studies. In this work, we report a rapid 24 h FAP activation method for fibroblasts using silicon nanowires (SiNWs) as culture substrates instead of growth factors or chemokines. In contrast with cells cultured on flat silicon which rarely express FAP, SiNW cultivated cells exhibit FAP levels similar to those found in cancerous tissue. We demonstrated that activated cells grown on SiNWs maintain their viability and proliferation in a time-dependent manner. Moreover, environmental scanning electron microscopy (ESEM) and focused ion beam and scanning electron microscopy (FIB-SEM) analysis clearly revealed that activated cells on SiNWs adapt to the structure of their substrates by filling inter-wire cavities via filopodia in contrast to cells cultured on flat silicon which spread freely. We further illustrated that the expression of FAP was rarely detected in activated cells after being re-cultured in Petri dishes, suggesting that the unique structure of SiNWs may have a certain influence on FAP activation.

Phytosynthesis of silver nanoparticles using Artemisia marschalliana Sprengel aerial part extract and assessment of their antioxidant, anticancer, and antibacterial properties

PubMed Central

Salehi, Soheil; Shandiz, Seyed Ataollah Sadat; Ghanbar, Farinaz; Darvish, Mohammad Raouf; Ardestani, Mehdi Shafiee; Mirzaie, Amir; Jafari, Mohsen

2016-01-01

A rapid phytosynthesis of silver nanoparticles (AgNPs) using an extract from the aerial parts of Artemisia marschalliana Sprengel was investigated in this study. The synthesized AgNPs using A. marschalliana extract was analyzed by UV–visible spectroscopy, X-ray diffraction, and Fourier transform infrared spectroscopy and further characterized by transmission electron microscopy, scanning electron microscopy, zeta potential, and energy-dispersive spectroscopy. Characteristic absorption bands of AgNPs were found near 430 nm in the UV–vis spectrum. Energy-dispersive spectroscopy analysis of AgNPs in the energy range 2–4 keV confirmed the silver signal due to surface plasmon resonance. Scanning electron microscopy and transmission electron microscopy results revealed that the AgNPs were mostly spherical with an average size ranging from 5 nm to 50 nm. The zeta potential value of −31 mV confirmed the stability of the AgNPs. AgNPs produced using the aqueous A. marschalliana extract might serve as a potent in vitro antioxidant, as revealed by 2,2-diphenyl-1-picryl hydrazyl assay. The present study demonstrates the anticancer properties of phytosynthesized AgNPs against human gastric carcinoma AGS cells. AgNPs exerted a dose-dependent inhibitory effect on the viability of cells. Real-time polymerase chain reaction was used for the investigation of Bax and Bcl-2 gene expression in cancer and normal cell lines. Our findings show that the mRNA levels of pro-apoptotic Bax gene expression were significantly upregulated, while the expression of anti-apoptotic Bcl-2 was declined in cells treated with AgNPs compared to normal cells. In addition, flow cytometric analysis showed that the number of early and late apoptotic AGS cells was significantly enhanced following treatment with AgNPs as compared to untreated cells. In addition, the AgNPs showed strong antibacterial properties against tested pathogenic bacteria such as Staphylococcus aureus, Bacillus cereus, Acinetobacter baumannii, and Pseudomonas aeruginosa. Based on the obtained data, we suggest that phytosynthesized AgNPs are good alternatives in the treatment of diseases because of the presence of bioactive agents. PMID:27199558

Integrated light and scanning electron microscopy of GFP-expressing cells.

PubMed

Peddie, Christopher J; Liv, Nalan; Hoogenboom, Jacob P; Collinson, Lucy M

2014-01-01

Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes. © 2014 Elsevier Inc. All rights reserved.

Examination of enterotoxigenic Escherichia coli H10407 (colonization factor antigen I+) by scanning electron microscopy with conductive staining.

PubMed Central

Sherburne, R; Armstrong, G D

1989-01-01

We have used the scanning electron microscope to examine enterotoxigenic Escherichia coli H10407, which expresses colonization factor antigen I pili. The use of low accelerating voltages and conductive staining procedures allowed us to obtain images of colonization factor antigen I pili and other structural details which were obscured by conventional gold-coating techniques. Images PMID:2570062

SRRF: Universal live-cell super-resolution microscopy.

PubMed

Culley, Siân; Tosheva, Kalina L; Matos Pereira, Pedro; Henriques, Ricardo

2018-08-01

Super-resolution microscopy techniques break the diffraction limit of conventional optical microscopy to achieve resolutions approaching tens of nanometres. The major advantage of such techniques is that they provide resolutions close to those obtainable with electron microscopy while maintaining the benefits of light microscopy such as a wide palette of high specificity molecular labels, straightforward sample preparation and live-cell compatibility. Despite this, the application of super-resolution microscopy to dynamic, living samples has thus far been limited and often requires specialised, complex hardware. Here we demonstrate how a novel analytical approach, Super-Resolution Radial Fluctuations (SRRF), is able to make live-cell super-resolution microscopy accessible to a wider range of researchers. We show its applicability to live samples expressing GFP using commercial confocal as well as laser- and LED-based widefield microscopes, with the latter achieving long-term timelapse imaging with minimal photobleaching. Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.

Phenotypically heterogeneous deletion of the ABH antigen from the transformed bladder urothelium. A scanning electron microscope study.

PubMed

De Harven, E; He, S; Hanna, W; Bootsma, G; Connolly, J G

1987-10-01

The deletion of ABH blood group antigens from the luminal surface of the bladder mucosa in cases of well differentiated transitional cell carcinomata, and the formation of pleomorphic microvilli have both been associated with aggressive biological behaviour and invasiveness of the tumors. We have studied cold cup biopsies from 8 normal mucosae and 17 papillary transitional cell carcinomata of the urinary bladder. The aim of our study was to correlate the formation of uniform or pleomorphic microvilli with the extent of deletion of the ABH blood group antigens on the surface of normal and transformed bladder urothelium. Immunogold scanning electron microscopy (SEM) in the backscattered electron (BE) imaging mode was used for this purpose. In the normal urothelium, uniform labeling of the luminal cells was demonstrated. In well differentiated tumors, the superficial cells exhibited uniform microvilli and a heterogeneous expression of the ABH antigens, giving characteristic 'mosaic' patterns of the antigenic labeling across the mucosal surface. These patterns were sharply delimitated at cell junctions when viewed by SEM; these observations were confirmed by transmission electron microscopy. In higher grade tumors, decreased ABH antigen expression, pleomorphic microvilli and/or featureless luminal cells were observed. In the transformed urothelium, the formation of uniform microvilli appeared to precede the loss of ABH antigen in most cases.

Dual-labeling method for electron microscopy to characterize synaptic connectivity using genetically encoded fluorescent reporters in Drosophila

PubMed Central

Tanaka, Nobuaki K.; Dye, Louis; Stopfer, Mark

2010-01-01

Light and electron microscopy (LM and EM) both offer important advantages for characterizing neuronal circuitry in intact brains: LM can reveal the general patterns neurons trace between brain areas, and EM can confirm synaptic connections between identified neurons within a small area. In a few species, genetic labeling with fluorescent proteins has been used with LM to visualize many kinds of neurons and to analyze their morphologies and projection patterns. However, combining these large-scale patterns with the fine detail available in EM analysis has been a technical challenge. To analyze the synaptic connectivity of neurons expressing fluorescent markers with EM, we developed a dual-labeling method for use with pre-embedded brains. In Drosophila expressing genetic labels and also injected with markers we visualized synaptic connections among two populations of neurons in the AL, one of which has been shown to mediate a specific function, odor evoked neural oscillation. PMID:21074556

Use of electron microscopy to classify canine perivascular wall tumors.

PubMed

Palmieri, C; Avallone, G; Cimini, M; Roccabianca, P; Stefanello, D; Della Salda, L

2013-03-01

The histologic classification of canine perivascular wall tumors (PWTs) is controversial. Many PWTs are still classified as hemangiopericytomas (HEPs), and the distinction from peripheral nerve sheath tumors (PNSTs) is still under debate. A recent histologic classification of canine soft tissue sarcomas included most histologic types of PWT but omitted those that were termed undifferentiated. Twelve cases of undifferentiated canine PWTs were evaluated by transmission electron microscopy. The ultrastructural findings supported a perivascular wall origin for all cases with 4 categories of differentiation: myopericytic (n = 4), myofibroblastic (n = 1), fibroblastic (n = 2), and undifferentiated (n = 5). A PNST was considered unlikely in each case based on immunohistochemical expression of desmin and/or the lack of typical ultrastructural features, such as basal lamina. Electron microscopy was pivotal for the subclassification of canine PWTs, and the results support the hypothesis that canine PWTs represent a continuum paralleling the phenotypic plasticity of vascular mural cells. The hypothesis that a subgroup of PWTs could arise from a pluripotent mesenchymal perivascular wall cell was also considered and may explain the diverse differentiation of canine PWTs.

Distinct Particle Morphologies Revealed through Comparative Parallel Analyses of Retrovirus-Like Particles.

PubMed

Martin, Jessica L; Cao, Sheng; Maldonado, Jose O; Zhang, Wei; Mansky, Louis M

2016-09-15

The Gag protein is the main retroviral structural protein, and its expression alone is usually sufficient for production of virus-like particles (VLPs). In this study, we sought to investigate-in parallel comparative analyses-Gag cellular distribution, VLP size, and basic morphological features using Gag expression constructs (Gag or Gag-YFP, where YFP is yellow fluorescent protein) created from all representative retroviral genera: Alpharetrovirus, Betaretrovirus, Deltaretrovirus, Epsilonretrovirus, Gammaretrovirus, Lentivirus, and Spumavirus. We analyzed Gag cellular distribution by confocal microscopy, VLP budding by thin-section transmission electron microscopy (TEM), and general morphological features of the VLPs by cryogenic transmission electron microscopy (cryo-TEM). Punctate Gag was observed near the plasma membrane for all Gag constructs tested except for the representative Beta- and Epsilonretrovirus Gag proteins. This is the first report of Epsilonretrovirus Gag localizing to the nucleus of HeLa cells. While VLPs were not produced by the representative Beta- and Epsilonretrovirus Gag proteins, the other Gag proteins produced VLPs as confirmed by TEM, and morphological differences were observed by cryo-TEM. In particular, we observed Deltaretrovirus-like particles with flat regions of electron density that did not follow viral membrane curvature, Lentivirus-like particles with a narrow range and consistent electron density, suggesting a tightly packed Gag lattice, and Spumavirus-like particles with large envelope protein spikes and no visible electron density associated with a Gag lattice. Taken together, these parallel comparative analyses demonstrate for the first time the distinct morphological features that exist among retrovirus-like particles. Investigation of these differences will provide greater insights into the retroviral assembly pathway. Comparative analysis among retroviruses has been critically important in enhancing our understanding of retroviral replication and pathogenesis, including that of important human pathogens such as human T-cell leukemia virus type 1 (HTLV-1) and HIV-1. In this study, parallel comparative analyses have been used to study Gag expression and virus-like particle morphology among representative retroviruses in the known retroviral genera. Distinct differences were observed, which enhances current knowledge of the retroviral assembly pathway. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Expression and characterization of HPV-16 L1 capsid protein in Pichia pastoris

PubMed Central

Bazan, Silvia Boschi; de Alencar Muniz Chaves, Agtha; Aires, Karina Araújo; Cianciarullo, Aurora Marques; Garcea, Robert L.; Ho, Paulo Lee

2013-01-01

Human papillomaviruses (HPVs) are responsible for the most common human sexually transmitted viral infections. Infection with high-risk HPVs, particularly HPV16, is associated with the development of cervical cancer. The papillomavirus L1 major capsid protein, the basis of the currently marketed vaccines, self-assembles into virus-like particles (VLPs). Here, we describe the expression, purification and characterization of recombinant HPV16 L1 produced by a methylotrophic yeast. A codon-optimized HPV16 L1 gene was cloned into a non-integrative expression vector under the regulation of a methanol-inducible promoter and used to transform competent Pichia pastoris cells. Purification of L1 protein from yeast extracts was performed using heparin–sepharose chromatography, followed by a disassembly/reassembly step. VLPs could be assembled from the purified L1 protein, as demonstrated by electron microscopy. The display of conformational epitopes on the VLPs surface was confirmed by hemagglutination and hemagglutination inhibition assays and by immuno-electron microscopy. This study has implications for the development of an alternative platform for the production of a papillomavirus vaccine that could be provided by public health programs, especially in resource-poor areas, where there is a great demand for low-cost vaccines. PMID:19756360

Targeted Cancer Therapy: Correlative Light-Electron Microscopy Shows RGD-Targeted ZnO Nanoparticles Dissolve in the Intracellular Environment of Triple Negative Breast Cancer Cells and Cause Apoptosis with Intratumor Heterogeneity (Adv. Healthcare Mater. 11/2016).

PubMed

Othman, Basmah A; Greenwood, Christina; Abuelela, Ayman F; Bharath, Anil A; Chen, Shu; Theodorou, Ioannis; Douglas, Trevor; Uchida, Maskai; Ryan, Mary; Merzaban, Jasmeen S; Porter, Alexandra E

2016-06-01

On page 1310 J. S. Merzaban, A. E. Porter, and co-workers present fluorescently labeled RGD-targeted ZnO nanoparticles (NPs; green) for the targeted delivery of cytotoxic ZnO to integrin αvβ3 receptors expressed on triple negative breast cancer cells. Correlative light-electron microscopy shows that NPs dissolve into ionic Zn(2+) (blue) upon uptake and cause apoptosis (red) with intra-tumor heterogeneity, thereby providing a possible strategy for targeted breast cancer therapy. Cover design by Ivan Gromicho. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deep-sea spherules from Pacific clay - Mass distribution and influx rate. [extraterrestrial origins from optical and electron microscopy

NASA Technical Reports Server (NTRS)

Murrell, M. T.; Davis, P. A., Jr.; Nishiizumi, K.; Millard, H. T., Jr.

1980-01-01

From 411 kg of Pacific clay, 22 mg of stony spherules and 50 mg of iron spherules larger than 150 microns were concentrated. The extraterrestrial origin of these particles was evaluated with the aid of optical and electron microscopy and atomic absorption elemental analysis. An expression for the integral number of stony particles from this sediment in the mass range 20-300 micrograms was derived. The world-wide influx rate of stony particles in the mass range which survive atmospheric heating and ocean sediment storage is calculated to be 90 tons/yr. The relative contributions of ablation debris vs fused interplanetary dust to the influx of stony spherules is discussed, but no conclusions could be made.

Morphological observation of the stria vascularis in midkine and pleiotrophin knockout mice.

PubMed

Sone, Michihiko; Muramatsu, Hisako; Muramatsu, Takashi; Nakashima, Tsutomu

2011-02-01

Midkine and Pleiotrophin are low molecular weight basic proteins with closely related structures and serve as growth/differentiation factors. They have been reported to be expressed in the cochlea during the embryonic and perinatal periods. In the present study, we focused on the roles of midkine and pleiotrophin in the stria vascularis and investigated morphological changes using mice deficient in these genes. Midkine knockout, pleiotrophin knockout, and double knockout mice were used and compared to wild-type mice. Auditory brain stem responses (ABRs) and cochlear blood flows were measured in each type of mice. Pathological changes in the stria vascularis were examined by light microscopy, including immunohistochemical staining with anti-Kir4.1 antibody, and electron microscopy. Hearing thresholds examined by ABRs were significantly higher in midkine knockout and pleiotrophin knockout mice than in wild-type mice. Double knockout mice showed higher thresholds compared to midkine knockout and pleiotrophin knockout mice. Blood flow in the lateral walls did not significantly differ and light microscopy examination showed an almost normal appearance of the stria vascularis in these knockout mice. However, the expression of Kir4.1 was weak in the knockout mice and severe vacuolar degeneration was observed by electron microscopy in the intermediate cells of the double knockout mice. The present study demonstrates that midkine and pleiotrophin play some roles for the morphological maintenance of intermediate cell in the stria vascularis. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

PubMed

Loussert Fonta, Celine; Leis, Andrew; Mathisen, Cliff; Bouvier, David S; Blanchard, Willy; Volterra, Andrea; Lich, Ben; Humbel, Bruno M

2015-01-01

Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of antigenicity. Finally, an important feature of this approach is that the fluorescence of the GFP signal is preserved throughout the entire preparation process until the last step before electron microscopy. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

A versatile nanobody-based toolkit to analyze retrograde transport from the cell surface.

PubMed

Buser, Dominik P; Schleicher, Kai D; Prescianotto-Baschong, Cristina; Spiess, Martin

2018-06-18

Retrograde transport of membranes and proteins from the cell surface to the Golgi and beyond is essential to maintain homeostasis, compartment identity, and physiological functions. To study retrograde traffic biochemically, by live-cell imaging or by electron microscopy, we engineered functionalized anti-GFP nanobodies (camelid VHH antibody domains) to be bacterially expressed and purified. Tyrosine sulfation consensus sequences were fused to the nanobody for biochemical detection of trans -Golgi arrival, fluorophores for fluorescence microscopy and live imaging, and APEX2 (ascorbate peroxidase 2) for electron microscopy and compartment ablation. These functionalized nanobodies are specifically captured by GFP-modified reporter proteins at the cell surface and transported piggyback to the reporters' homing compartments. As an application of this tool, we have used it to determine the contribution of adaptor protein-1/clathrin in retrograde transport kinetics of the mannose-6-phosphate receptors from endosomes back to the trans -Golgi network. Our experiments establish functionalized nanobodies as a powerful tool to demonstrate and quantify retrograde transport pathways.

The role of immunohistochemistry, electron microscopy, and ultrastructural cytochemistry in the diagnosis of mixed carcinoma-neuroendocrine neoplasms.

PubMed

Graham, A R; Payne, C M; Nagle, R B; Angel, E

1987-02-01

We studied four mixed carcinoma-neuroendocrine neoplasms from gastrointestinal tract and pancreas by routine light microscopy (LM), immunohistochemistry (IH), electron microscopy (EM), and ultrastructural cytochemistry (UC). By LM, the individual tumors showed fairly pure neuroendocrine (carcinoid) or epithelial (papillary) patterns, mixed neuroendocrine-carcinoma features and poorly-differentiated tumor in sheets and nests which did not lend itself to morphologic characterization. IH demonstrated mixed expression, within different areas of the same neoplasm, of epithelial antigens (keratins and carcinoembryonic antigen [CEA]) and neuroendocrine markers (neuron-specific enolase [NSE], bombesin and neurohormonal peptides). By EM, each tumor showed ultrastructural features of epithelial and neuroendocrine differentiation which varied substantially in terms of number of cells involved and their distribution; two of the neoplasms showed biphasic differentiation within single cells. The nature of the neurosecretory granules was verified with the uranaffin reaction (UR). This study illustrates the value of combining LM, IH, EM and UC for the identification of mixed carcinoma-neuroendocrine lesions.

Attachment, proliferation and collagen type I mRNA expression of human gingival fibroblasts on different biodegradable membranes.

PubMed

Hakki, Sema S; Korkusuz, Petek; Purali, Nuhan; Bozkurt, Buket; Kus, Mahmut; Duran, Ismet

2013-01-01

The purpose of this study was to investigate adhesion, proliferation and type I collagen (COL I) mRNA expression of gingival fibroblasts on different membranes used in periodontal applications. Collagen (C), acellular dermal matrix (ADM) and polylactic acid; polyglycolic acid; lactide/glycolide copolymer (PLGA) biodegradable membranes were combined with gingival fibroblasts in culture and incubated for 48 h. Cell adhesion was examined with scanning electron and confocal microscopy. MTT assay was used to measure proliferation. COL I mRNA expression was assessed using quantitative-polymerase chain reaction (QPCR). The PLGA group exhibited the lowest cell survival on day 5 and 10, and lowest cell proliferation on days 5, 10 and 14. While cell proliferation was similar in C and ADM groups, the C membrane showed a slightly greater increase in viable cells to day 10. Confocal and scanning electron microscopy confirmed the results of proliferation and MTT assays. The highest COL I mRNA expression was noted in the PLGA membrane group when compared to the C (p < 0.01) and ADM (p < 0.05) membrane groups. These data revealed that adherence and proliferation of primary gingival fibroblasts on collagen-based C and ADM membranes is better than that seen with PLGA membranes, and thus may be preferable in the treatment of gingival recession defects.

Subcellular localization of leptin and leptin receptor in breast cancer detected in an electron microscopic study.

PubMed

Al-Shibli, Saad M; Amjad, Nasser M; Al-Kubaisi, Muna K; Mizan, Shaikh

2017-01-22

Leptin (LEP) and leptin receptor (LEPR) have long been found associated with breast cancer. So far no high-resolution method such as electron microscopy has been used to investigate the subcellular localization of leptin and leptin receptor in breast cancer. We collected cancer and non-cancer breast tissues from 51 women with invasive ductal breast cancer. Leptin and leptin receptor in the tissues were estimated using immunohistochemistry (IHC). LEP and LEPR were localized at subcellular level by immunocytochemistry (ICC) using ultra-fine gold particle conjugated antibody, and visualized with transmission electron microscopy (TEM). IHC showed high presence of LEP and LEPR in 65% and 67% respectively of the breast cancer samples, 100% and 0% respectively of the adipose tissue samples, and no high presence in the non-cancer breast tissue samples. On TEM views both LEP and LEPR were found highly concentrated within the nucleus of the cancer cells, indicating that nucleus is the principal seat of action. However, presence of high concentration of LEP does not necessarily prove its over-expression, as often concluded, because LEP could be internalized from outside by LEPR in the cells. In contrast, LEPR is definitely over-expressed in the ductal breast cancer cells. Therefore, we hypothesize that over-expression of LEPR, rather than that of LEP has a fundamental role in breast carcinogenesis in particular, and probably for LEP-LEPR associated tumors in general. Copyright © 2016 Elsevier Inc. All rights reserved.

Focus on membrane differentiation and membrane domains in the prokaryotic cell.

PubMed

Boekema, Egbert J; Scheffers, Dirk-Jan; van Bezouwen, Laura S; Bolhuis, Henk; Folea, I Mihaela

2013-01-01

A summary is presented of membrane differentiation in the prokaryotic cell, with an emphasis on the organization of proteins in the plasma/cell membrane. Many species belonging to the Eubacteria and Archaea have special membrane domains and/or membrane proliferation, which are vital for different cellular processes. Typical membrane domains are found in bacteria where a specific membrane protein is abundantly expressed. Lipid rafts form another example. Despite the rareness of conventional organelles as found in eukaryotes, some bacteria are known to have an intricate internal cell membrane organization. Membrane proliferation can be divided into curvature and invaginations which can lead to internal compartmentalization. This study discusses some of the clearest examples of bacteria with such domains and internal membranes. The need for membrane specialization is highest among the heterogeneous group of bacteria which harvest light energy, such as photosynthetic bacteria and halophilic archaea. Most of the highly specialized membranes and domains, such as the purple membrane, chromatophore and chlorosome, are found in these autotrophic organisms. Otherwise the need for membrane differentiation is lower and variable, except for those structures involved in cell division. Microscopy techniques have given essential insight into bacterial membrane morphology. As microscopy will further contribute to the unraveling of membrane organization in the years to come, past and present technology in electron microscopy and light microscopy is discussed. Electron microscopy was the first to unravel bacterial morphology because it can directly visualize membranes with inserted proteins, which no other technique can do. Electron microscopy techniques developed in the 1950s and perfected in the following decades involve the thin sectioning and freeze fractioning of cells. Several studies from the golden age of these techniques show amazing examples of cell membrane morphology. More recently, light microscopy in combination with the use of fluorescent dyes has become an attractive technique for protein localization with the natural membrane. However, the resolution problem in light microscopy remains and overinterpretation of observed phenomena is a pitfall. Thus, light microscopy as a stand-alone technique is not sufficient to prove, for instance, the long-range helical distribution of proteins in membrane such as MinD spirals in Bacillus subtilis. Electron tomography is an emerging electron microscopy technique that can provide three-dimensional reconstructions of small, nonchemically fixed bacteria. It will become a useful tool for studying prokaryotic membranes in more detail and is expected to collect information complementary to those of advanced light microscopy. Together, microscopy techniques can meet the challenge of the coming years: to specify membrane structures in more detail and to bring them to the level of specific protein-protein interactions. Copyright © 2013 S. Karger AG, Basel.

Elevated hydrostatic pressure triggers release of OPA1 and cytochrome C, and induces apoptotic cell death in differentiated RGC-5 cells

PubMed Central

Kim, Keun-Young; Lindsey, James D.; Angert, Mila; Patel, Ankur; Scott, Ray T.; Liu, Quan; Crowston, Jonathan G.; Ellisman, Mark H.; Perkins, Guy A.; Weinreb, Robert N.

2009-01-01

Purpose This study was conducted to determine whether elevated hydrostatic pressure alters mitochondrial structure, triggers release of the dynamin-related guanosine triphosphatase (GTPase) optic atrophy type 1 (OPA1) or cytochrome C from mitochondria, alters OPA1 gene expression, and can directly induce apoptotic cell death in cultured retinal ganglion cell (RGC)-5 cells. Methods Differentiated RGC-5 cells were exposed to 30 mmHg for three days in a pressurized incubator. As a control, differentiated RGC-5 cell cultures were incubated simultaneously in a conventional incubator. Live RGC-5 cells were then labeled with MitoTracker Red and mitochondrial morphology was assessed by fluorescence microscopy. Mitochondrial structural changes were also assessed by electron microscopy and three-dimenstional (3D) electron microscope tomography. OPA1 mRNA was measured by Taqman quantitative PCR. The cellular distribution of OPA1 protein and cytochrome C was assessed by immunocytochemistry and western blot. Caspase-3 activation was examined by western blot. Apoptotic cell death was evaluated by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Results Mitochondrial fission, characterized by the conversion of tubular fused mitochondria into isolated small organelles, was triggered after three days exposure to elevated hydrostatic pressure. Electron microscopy confirmed the fission and noted no changes to mitochondrial architecture, nor outer membrane rupture. Electron microscope tomography showed that elevated pressure depleted mitochondrial cristae content by fourfold. Elevated hydrostatic pressure increased OPA1 gene expression by 35±14% on day 2, but reduced expression by 36±4% on day 3. Total OPA1 protein content was not changed on day 2 or 3. However, pressure treatment induced release of OPA1 and cytochrome C from mitochondria to the cytoplasm. Elevated pressure also activated caspase-3 and induced apoptotic cell death. Conclusions Elevated hydrostatic pressure triggered mitochondrial changes including mitochondrial fission and abnormal cristae depletion, alteration of OPA1 gene expression, and release of OPA1 and cytochrome C into the cytoplasm before the onset of apoptotic cell death in differentiated RGC-5 cells. These results suggest that sustained moderate pressure elevation may directly damage RGC integrity by injuring mitochondria. PMID:19169378

Liver cell-derived microparticles activate hedgehog signaling and alter gene expression in hepatic endothelial cells.

PubMed

Witek, Rafal P; Yang, Liu; Liu, Renshui; Jung, Youngmi; Omenetti, Alessia; Syn, Wing-Kin; Choi, Steve S; Cheong, Yeiwon; Fearing, Caitlin M; Agboola, Kolade M; Chen, Wei; Diehl, Anna Mae

2009-01-01

Angiogenesis contributes to vascular remodeling during cirrhosis. In cirrhotic livers, cholangiocytes, and myofibroblastic hepatic stellate cells (MF-HSC) produce Hedgehog (Hh) ligands. During embryogenesis Hh ligands are released from ligand-producing cells in microparticles and activate Hh signaling in endothelial cells. We studied whether adult liver cell-derived microparticles contain Hh ligands that alter hepatic sinusoidal endothelial cells (SEC). MF-HSC and cholangiocytes were exposed to platelet-derived growth factor to induce Hh ligands; microparticles were isolated from medium, analyzed by transmission electron microscopy and immunoblots, and applied to Hh-reporter-containing cells. Microparticles were obtained from serum and bile of rats after bile duct ligation (BDL) or sham surgery and applied to normal primary liver SEC with or without cyclopamine, an Hh signaling inhibitor. Effects on SEC gene expression were evaluated by quantitative reverse-transcription polymerase chain reaction and immunoblotting. Hh target gene expression and SEC activation markers were compared in primary SEC and in liver sections from healthy and BDL rats. Platelet-derived growth factor-treated MF-HSC and cholangiocytes released exosome-enriched microparticles containing biologically-active Hh ligands. BDL increased release of Hh-containing exosome-enriched microparticles into plasma and bile. Transmission electron microscopy and immunoblots revealed similarities among microparticles from all sources; all microparticles induced similar Hh-dependent changes in SEC gene expression. SEC from healthy livers did not express Hh target genes or activation markers, but both were up-regulated in SEC after BDL. Hh-containing exosome-enriched microparticles released from liver cells alter hepatic SEC gene expression, suggesting a novel mechanism for cirrhotic vasculopathy.

New developments in electron microscopy for serial image acquisition of neuronal profiles.

PubMed

Kubota, Yoshiyuki

2015-02-01

Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Evaluation of Magnetic Nanoparticle-Labeled Chondrocytes Cultivated on a Type II Collagen–Chitosan/Poly(Lactic-co-Glycolic) Acid Biphasic Scaffold

PubMed Central

Su, Juin-Yih; Chen, Shi-Hui; Chen, Yu-Pin; Chen, Wei-Chuan

2017-01-01

Chondral or osteochondral defects are still controversial problems in orthopedics. Here, chondrocytes labeled with magnetic nanoparticles were cultivated on a biphasic, type II collagen–chitosan/poly(lactic-co-glycolic acid) scaffold in an attempt to develop cultures with trackable cells exhibiting growth, differentiation, and regeneration. Rabbit chondrocytes were labeled with magnetic nanoparticles and characterized by scanning electron microscopy (SEM), transmission electron (TEM) microscopy, and gene and protein expression analyses. The experimental results showed that the magnetic nanoparticles did not affect the phenotype of chondrocytes after cell labeling, nor were protein and gene expression affected. The biphasic type II collagen–chitosan/poly(lactic-co-glycolic) acid scaffold was characterized by SEM, and labeled chondrocytes showed a homogeneous distribution throughout the scaffold after cultivation onto the polymer. Cellular phenotype remained unaltered but with increased gene expression of type II collagen and aggrecan, as indicated by cell staining, indicating chondrogenesis. Decreased SRY-related high mobility group-box gene (Sox-9) levels of cultured chondrocytes indicated that differentiation was associated with osteogenesis. These results are encouraging for the development of techniques for trackable cartilage regeneration and osteochondral defect repair which may be applied in vivo and, eventually, in clinical trials. PMID:28054960

The Human “Cochlear Battery” – Claudin-11 Barrier and Ion Transport Proteins in the Lateral Wall of the Cochlea

PubMed Central

Liu, Wei; Schrott-Fischer, Annelies; Glueckert, Rudolf; Benav, Heval; Rask-Andersen, Helge

2017-01-01

Background: The cochlea produces an electric field potential essential for hair cell transduction and hearing. This biological “battery” is situated in the lateral wall of the cochlea and contains molecular machinery that secretes and recycles K+ ions. Its functioning depends on junctional proteins that restrict the para-cellular escape of ions. The tight junction protein Claudin-11 has been found to be one of the major constituents of this barrier that maintains ion gradients (Gow et al., 2004; Kitajiri et al., 2004a). We are the first to elucidate the human Claudin-11 framework and the associated ion transport machinery using super-resolution fluorescence illumination microscopy (SR-SIM). Methods: Archival cochleae obtained during meningioma surgery were used for SR-SIM together with transmission electron microscopy after ethical consent. Results: Claudin-11-expressing cells formed parallel tight junction lamellae that insulated the epithelial syncytium of the stria vascularis and extended to the suprastrial region. Intercellular gap junctions were found between the barrier cells and fibrocytes. Conclusion: Transmission electron microscopy, confocal microscopy and SR-SIM revealed exclusive cell specialization in the various subdomains of the lateral wall of the human cochlea. The Claudin-11-expressing cells exhibited both conductor and isolator characteristics, and these micro-porous separators may selectively mediate the movement of charged units to the intrastrial space in a manner that is analogous to a conventional electrochemical “battery.” The function and relevance of this battery for the development of inner ear disease are discussed. PMID:28848383

Electron microscopic, genetic and protein expression analyses of Helicobacter acinonychis strains from a Bengal tiger.

PubMed

Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L; Fox, James G; Berg, Douglas E; Backert, Steffen

2013-01-01

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5-6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections.

Electron Microscopic, Genetic and Protein Expression Analyses of Helicobacter acinonychis Strains from a Bengal Tiger

PubMed Central

Tegtmeyer, Nicole; Rivas Traverso, Francisco; Rohde, Manfred; Oyarzabal, Omar A.; Lehn, Norbert; Schneider-Brachert, Wulf; Ferrero, Richard L.; Fox, James G.; Berg, Douglas E.; Backert, Steffen

2013-01-01

Colonization by Helicobacter species is commonly noted in many mammals. These infections often remain unrecognized, but can cause severe health complications or more subtle host immune perturbations. The aim of this study was to isolate and characterize putative novel Helicobacter spp. from Bengal tigers in Thailand. Morphological investigation (Gram-staining and electron microscopy) and genetic studies (16SrRNA, 23SrRNA, flagellin, urease and prophage gene analyses, RAPD DNA fingerprinting and restriction fragment polymorphisms) as well as Western blotting were used to characterize the isolated Helicobacters. Electron microscopy revealed spiral-shaped bacteria, which varied in length (2.5–6 µm) and contained up to four monopolar sheathed flagella. The 16SrRNA, 23SrRNA, sequencing and protein expression analyses identified novel H. acinonychis isolates closely related to H. pylori. These Asian isolates are genetically very similar to H. acinonychis strains of other big cats (cheetahs, lions, lion-tiger hybrid and other tigers) from North America and Europe, which is remarkable in the context of the great genetic diversity among worldwide H. pylori strains. We also found by immunoblotting that the Bengal tiger isolates express UreaseA/B, flagellin, BabA adhesin, neutrophil-activating protein NapA, HtrA protease, γ-glutamyl-transpeptidase GGT, Slt lytic transglycosylase and two DNA transfer relaxase orthologs that were known from H. pylori, but not the cag pathogenicity island, nor CagA, VacA, SabA, DupA or OipA proteins. These results give fresh insights into H. acinonychis genetics and the expression of potential pathogenicity-associated factors and their possible pathophysiological relevance in related gastric infections. PMID:23940723

Annexin-V/quantum dot probes for multimodal apoptosis monitoring in living cells: improving bioanalysis using electrochemistry

NASA Astrophysics Data System (ADS)

Montón, Helena; Parolo, Claudio; Aranda-Ramos, Antonio; Merkoçi, Arben; Nogués, Carme

2015-02-01

There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry.There is a great demand to develop novel techniques that allow useful and complete monitoring of apoptosis, which is a key factor of several diseases and a target for drug development. Here, we present the use of a novel dual electrochemical/optical label for the detection and study of apoptosis. We combined the specificity of Annexin-V for phosphatidylserine, a phospholipid expressed in the outer membrane of apoptotic cells, with the optical and electrochemical properties of quantum dots to create a more efficient label. Using this conjugate we addressed three important issues: (i) we made the labeling of apoptotic cells faster (30 min) and easier; (ii) we fully characterized the samples by common cell biological techniques (confocal laser scanning microscopy, scanning electron microscopy and flow cytometry); and (iii) we developed a fast, cheap and quantitative electrochemical detection method for apoptotic cells with results in full agreement with those obtained by flow cytometry. Electronic supplementary information (ESI) available: Optical microscopy images of apoptotic induced cell cultures at different times and negative control of flow cytometry. See DOI: 10.1039/c4nr07191c

Assessment and characterization of biofilm formation among human isolates of Streptococcus dysgalactiae subsp. equisimilis.

PubMed

Genteluci, Gabrielle Limeira; Silva, Ligia Guedes; Souza, Maria Clara; Glatthardt, Thaís; de Mattos, Marcos Corrêa; Ejzemberg, Regina; Alviano, Celuta Sales; Figueiredo, Agnes Marie Sá; Ferreira-Carvalho, Bernadete Teixeira

2015-12-01

The capacity to form biofilm is considered a protective mechanism that allows the bacteria to survive and proliferate in hostile environments, facilitating the maintenance of the infectious process. Recently, biofilm has become a topic of interest in the study of the human pathogen group A Streptococcus (GAS). Although GAS has not been associated with infection on medical implants, the presence of microcolonies embedded in an extracellular matrix on infected tissues has been reported. Despite the similarity between GAS and Streptococcus dysgalactiae subspecies equisimilis (SDSE), there are no studies in the literature describing the production of biofilm by SDSE. In this work, we assessed and characterized biofilm development among SDSE human isolates of group C. The in vitro data showed that 59.3% of the 118 isolates tested were able to form acid-induced biofilm on glass, and 28% formed it on polystyrene surfaces. More importantly, biofilm was also formed in a foreign body model in mice. The biofilm structure was analyzed by confocal laser scanning microscopy, transmission electron microscopy, and scanning electron microscopy. Long fibrillar-like structures were observed by scanning electron microscopy. Additionally, the expression of a pilus associated gene of SDSE was increased for in vitro sessile cells compared with planktonics, and when sessile cells were collected from biofilms formed in the animal model compared with that of in vitro model. Results obtained from the immunofluorescence microscopy indicated the biofilm was immunogenic. Our data also suggested a role for proteins, exopolysaccharide and extracellular DNA in the formation and accumulation of biofilm by SDSE. Copyright © 2015 Elsevier GmbH. All rights reserved.

Colonization of epidermal tissue by Staphylococcus aureus produces localized hypoxia and stimulates secretion of antioxidant and caspase-14 proteins

USDA-ARS?s Scientific Manuscript database

A partial-thickness epidermal explant model was colonized with GFP-expressing S. aureus and the pattern of S. aureus biofilm growth was characterized using electron and confocal laser scanning microscopy. Oxygen concentration in explants and H2O2 in media was quantified using microelectrodes. The re...

Structural analysis of the PSD-95 cluster by electron tomography and CEMOVIS: a proposal for the application of the genetically encoded metallothionein tag.

PubMed

Hirabayashi, Ai; Fukunaga, Yuko; Miyazawa, Atsuo

2014-06-01

Postsynaptic density-95 (PSD-95) accumulates at excitatory postsynapses and plays important roles in the clustering and anchoring of numerous proteins at the PSD. However, a detailed ultrastructural analysis of clusters exclusively consisting of PSD-95 has never been performed. Here, we employed a genetically encoded tag, three tandem repeats of metallothionein (3MT), to study the structure of PSD-95 clusters in cells by electron tomography and cryo-electron microscopy of vitreous sections. We also performed conventional transmission electron microscopy (TEM). Cultured hippocampal neurons expressing a fusion protein of PSD-95 coupled to 3MT (PDS-95-3MT) were incubated with CdCl2 to result in the formation of Cd-bound PSD-95-3MT. Two types of electron-dense deposits composed of Cd-bound PSD-95-3MT were observed in these cells by TEM, as reported previously. Electron tomography revealed the presence of membrane-shaped structures representing PSD-95 clusters at the PSD and an ellipsoidal structure located in the non-synaptic cytoplasm. By TEM, the PSD-95 clusters appeared to be composed of a number of dense cores. In frozen hydrated sections, these dense cores were also found beneath the postsynaptic membrane. Taken together, our findings suggest that dense cores of PSD-95 aggregate to form the larger clusters present in the PSD and the non-synaptic cytoplasm. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Ghost cells in pilomatrixoma, craniopharyngioma, and calcifying cystic odontogenic tumor: histological, immunohistochemical, and ultrastructural study.

PubMed

Rumayor, Alicia; Carlos, Román; Kirsch, Hernán Molina; de Andrade, Bruno A Benevenuto; Romañach, Mario J; de Almeida, Oslei Paes

2015-04-01

Pilomatrixoma, craniopharyngioma, and calcifying cystic odontogenic tumor are the main entities presenting ghost cells as an important histological feature, in spite their quite different clinical presentation; it seems that they share a common pathway in the formation of these cells. The aim of this study is to examine and compare the characteristics of ghost and other cells that form these lesions. Forty-three cases including 21 pilomatrixomas, 14 craniopharyngiomas, and eight calcifying cystic odontogenic tumors were evaluated by immunohistochemistry for cytokeratins, CD138, β-catenin, D2-40, Glut-1, FAS, CD10 and also by scanning electron microscopy. The CKs, CD138, β-catenin, Glut-1, FAS, and CD10 were more often expressed by transitional cells of craniopharyngioma and calcifying cystic odontogenic tumor, compared with pilomatrixoma. Basaloid cells of pilomatrixoma showed strong positivity for CD138 and CD10. Differences on expression pattern were identified in transitional and basal cells, as ghost cells were negative for most antibodies used, except by low expression for cytokeratins. By scanning electron microscopy, the morphology of ghost cells were similar in their fibrillar cytoplasm, but their pattern varied from sheets in pilomatrixoma to small clusters in craniopharyngioma and calcifying cystic odontogenic tumor. Mechanisms involved in formation of ghost cells are unknown, but probably they follow different pathways as protein expression in the basal/transitional cells was not uniform in the three tumors studied. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2.

PubMed

Xin, Jia-Xuan; Yue, Zhen; Zhang, Shuai; Jiang, Zhong-Hua; Wang, Ping-Yu; Li, You-Jie; Pang, Min; Xie, Shu-Yang

2013-10-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3'-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics.

Impact of traffic-related air pollution on the expression of Platanus orientalis pollen allergens.

PubMed

Sedghy, Farnaz; Sankian, Mojtaba; Moghadam, Maliheh; Ghasemi, Ziba; Mahmoudi, Mahmoud; Varasteh, Abdol-Reza

2017-01-01

Air pollutants and their interaction with environmental allergens have been considered as an important reason for the recent increase in the prevalence of allergic diseases. The aim of this study was to investigate the traffic pollution effect, as a stressor, on Platanus orientalis pollen allergens messenger RNA (mRNA) and protein expression. P. orientalis pollen grains were collected along main streets of heavy traffic and from unpolluted sites in Mashhad city, in northeast Iran. The pollen samples were examined by scanning electron microscopy. To assess the abundance of pollen allergens (Pla or 1, Pla or 2, and Pla or 3) from polluted and unpolluted sites, immunoblotting was performed. Moreover, the sequences encoding P. orientalis allergens were amplified using real-time PCR. Scanning electron microscopy showed a number of particles of 150-550 nm on the surface of pollen from polluted sites. Also, protein and gene expression levels of Pla or 1 and Pla or 3 were considerably greater in pollen samples from highly polluted areas than in pollen from unpolluted areas (p < 0.05). In contrast, no statically significant difference in Pla or 2 protein and mRNA expression level was found between samples from the two areas. We found greater expression of allergens involved in plant defense mechanisms (Pla or 1 and Pla or 3) in polluted sites than in unpolluted ones. The high expression of these proteins can lead to an increase in the prevalence of allergic diseases. These findings suggest the necessity of supporting public policies aimed at controlling traffic pollution to improve air quality and prevent the subsequent clinical outcomes and new cases of asthma.

Mitochondria-targeted antioxidants protect against amyloid-beta toxicity in Alzheimer's disease neurons.

PubMed

Manczak, Maria; Mao, Peizhong; Calkins, Marcus J; Cornea, Anda; Reddy, Arubala P; Murphy, Michael P; Szeto, Hazel H; Park, Byung; Reddy, P Hemachandra

2010-01-01

The purpose of our study was to investigate the effects of the mitochondria-targeted antioxidants, MitoQ and SS31, and the anti-aging agent resveratrol on neurons from a mouse model (Tg2576 line) of Alzheimer's disease (AD) and on mouse neuroblastoma (N2a) cells incubated with the amyloid-beta (Abeta) peptide. Using electron and confocal microscopy, gene expression analysis, and biochemical methods, we studied mitochondrial structure and function and neurite outgrowth in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta. In N2a cells only incubated with the Abeta, we found increased expressions of mitochondrial fission genes and decreased expression of fusion genes and also decreased expression of peroxiredoxins. Electron microscopy of the N2a cells incubated with Abeta revealed a significantly increased number of mitochondria, indicating that Abeta fragments mitochondria. Biochemical analysis revealed that function is defective in mitochondria. Neurite outgrowth was significantly decreased in Abeta-incubated N2a cells, indicating that Abeta affects neurite outgrowth. However, in N2a cells treated with MitoQ, SS31, and resveratrol, and then incubated with Abeta, abnormal expression of peroxiredoxins and mitochondrial structural genes were prevented and mitochondrial function was normal; intact mitochondria were present and neurite outgrowth was significantly increased. In primary neurons from amyloid-beta precursor protein transgenic mice that were treated with MitoQ and SS31, neurite outgrowth was significantly increased and cyclophilin D expression was significantly decreased. These findings suggest that MitoQ and SS31 prevent Abeta toxicity, which would warrant the study of MitoQ and SS31 as potential drugs to treat patients with AD.

Enhanced differentiation of human embryonic stem cells into cardiomyocytes by combining hanging drop culture and 5-azacytidine treatment.

PubMed

Yoon, Byung Sun; Yoo, Seung Jun; Lee, Jeoung Eun; You, Seungkwon; Lee, Hoon Taek; Yoon, Hyun Soo

2006-04-01

Cell replacement therapy is a promising approach for the treatment of cardiac diseases. It is, however, challenged by a limited supply of appropriate cells. Therefore, we have investigated whether functional cardiomyocytes can be efficiently generated from human embryonic stem cells (hESCs). In this study, we developed an efficient protocol for the generation of functional cardiomyocytes from hESCs by combining hanging drop culture and 5-azacytidine, a well-known demethylating agent, and then evaluated the expression of cardiac-specific markers. hESCs were cultured both in the medium without or with 0.1, 1, or 10 microM of 5-azacytidine under a hanging drop culture. The expression of several cardiac-specific markers was determined by real-time PCR, RT-PCR, immunofluorescence, and confocal microscopy. To verify the structural and functional properties of hESC-derived cardiomyocytes, we performed electron microscopy and electrophysiological recording. The efficiency of beating cell generation was significantly improved in the hanging drop culture compared with that in suspension culture. Treatment of hESCs with 0.1 microM of 5-azacytidine for 1-3 days significantly increased the number of beating cells and simultaneously enhanced the expression of cardiac-specific markers. Transmission electron microscopy and electrophysiological recording showed that hESC-derived cardiomyocytes acquired structural and functional properties of cardiomyocytes. In conclusion, these results suggest that differentiation of hESCs into cardiomyocytes can be enhanced by the combination of hanging drop culture and 5-azacytidine treatment. Also the methylation status of genes related to cardiomyocyte development may play an important role in the differentiation of hESCs into cardiomyocytes.

Up-regulation of mRNA ventricular PRNP prion protein gene expression in air pollution highly exposed young urbanites: endoplasmic reticulum stress, glucose regulated protein 78, and nanosized particles.

PubMed

Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

2013-11-28

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role.

Up-Regulation of mRNA Ventricular PRNP Prion Protein Gene Expression in Air Pollution Highly Exposed Young Urbanites: Endoplasmic Reticulum Stress, Glucose Regulated Protein 78, and Nanosized Particles

PubMed Central

Villarreal-Calderon, Rodolfo; Franco-Lira, Maricela; González-Maciel, Angélica; Reynoso-Robles, Rafael; Harritt, Lou; Pérez-Guillé, Beatriz; Ferreira-Azevedo, Lara; Drecktrah, Dan; Zhu, Hongtu; Sun, Qiang; Torres-Jardón, Ricardo; Aragón-Flores, Mariana; Calderón-Garcidueñas, Ana; Diaz, Philippe; Calderón-Garcidueñas, Lilian

2013-01-01

Mexico City Metropolitan Area children and young adults exposed to high concentrations of air pollutants including fine and ultrafine particulate matter (PM) vs. clean air controls, exhibit myocardial inflammation and inflammasome activation with a differential right and left ventricular expression of key inflammatory genes and inflammasomes. We investigated the mRNA expression levels of the prion protein gene PRNP, which plays an important role in the protection against oxidative stress and metal toxicity, and the glucose regulated protein 78, a key protein in endoplasmic reticulum (ER) stress signaling, in ventricular autopsy samples from 30 children and young adults age 19.97 ± 6.8 years with a lifetime of low (n:4) vs. high (n:26) air pollution exposures. Light microscopy and transmission electron microscopy studies were carried out in human ventricles, and electron microscopy studies were also done in 5 young, highly exposed Mexico City dogs. There was significant left ventricular PRNP and bi-ventricular GRP78 mRNA up-regulation in Mexico City young urbanites vs. controls. PRNP up-regulation in the left ventricle was significantly different from the right, p < 0.0001, and there was a strong left ventricular PRNP and GRP78 correlation (p = 0.0005). Marked abnormalities in capillary endothelial cells, numerous nanosized particles in myocardial ER and in abnormal mitochondria characterized the highly exposed ventricles. Early and sustained cardiac ER stress could result in detrimental irreversible consequences in urban children, and while highly complex systems maintain myocardial homeostasis, failure to compensate for chronic myocardial inflammation, oxidative and ER stress, and particles damaging myocardial organelles may prime the development of pathophysiological cardiovascular states in young urbanites. Nanosized PM could play a key cardiac myocyte toxicity role. PMID:24287918

In vitro Paracoccidioides brasiliensis biofilm and gene expression of adhesins and hydrolytic enzymes.

PubMed

Sardi, Janaina de Cássia Orlandi; Pitangui, Nayla de Souza; Voltan, Aline Raquel; Braz, Jaqueline Derissi; Machado, Marcelo Pelajo; Fusco Almeida, Ana Marisa; Mendes Giannini, Maria Jose Soares

2015-01-01

Paracoccidioides species are dimorphic fungi that initially infect the lungs but can also spread throughout the body. The spreading infection is most likely due to the formation of a biofilm that makes it difficult for the host to eliminate the infection. Biofilm formation is crucial for the development of infections and confines the pathogen to an extracellular matrix. Its presence is associated with antimicrobial resistance and avoidance of host defenses. This current study provides the first description of biofilm formation by Paracoccidioides brasiliensis (Pb18) and an analysis of gene expression, using real-time PCR, associated with 3 adhesins and 2 hydrolytic enzymes that could be associated with the virulence profile. Biofilm formation was analyzed using fluorescence microscopy, scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Metabolic activity was determined using the XTT reduction assay. P. brasiliensis was able to form mature biofilm in 144 h with a thickness of 100 μm. The presence of a biofilm was found to be associated with an increase in the expression of adhesins and enzymes. GP43, enolase, GAPDH and aspartyl proteinase genes were over-expressed, whereas phospholipase was down-regulated in biofilm. The characterization of biofilm formed by P. brasiliensis may contribute to a better understanding of the pathogenesis of paracoccidioidomycosis as well as the search for new therapeutic alternatives; while improving the effectiveness of treatment.

Cell adhesion on nanotextured slippery superhydrophobic substrates.

PubMed

Di Mundo, Rosa; Nardulli, Marina; Milella, Antonella; Favia, Pietro; d'Agostino, Riccardo; Gristina, Roberto

2011-04-19

In this work, the response of Saos2 cells to polymeric surfaces with different roughness/density of nanometric dots produced by a tailored plasma-etching process has been studied. Topographical features have been evaluated by atomic force microscopy, while wetting behavior, in terms of water-surface adhesion energy, has been evaluated by measurements of drop sliding angle. Saos2 cytocompatibility has been investigated by scanning electron microscopy, fluorescent microscopy, and optical microscopy. The similarity in outer chemical composition has allowed isolation of the impact of the topographical features on cellular behavior. The results indicate that Saos2 cells respond differently to surfaces with different nanoscale topographical features, clearly showing a certain inhibition in cell adhesion when the nanoscale is particularly small. This effect appears to be attenuated in surfaces with relatively bigger nanofeatures, though these express a more pronounced slippery/dry wetting character. © 2011 American Chemical Society

Silver stain for electron microscopy

NASA Technical Reports Server (NTRS)

Corbett, R. L.

1972-01-01

Ammoniacal silver stain used for light microscopy was adapted advantageously for use with very thin biological sections required for electron microscopy. Silver stain can be performed in short time, has more contrast, and is especially useful for low power electron microscopy.

Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

PubMed Central

Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

2009-01-01

Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881

Neutrophils Express Distinct RNA Receptors in a Non-canonical Way*

PubMed Central

Berger, Michael; Hsieh, Chin-Yuan; Bakele, Martina; Marcos, Veronica; Rieber, Nikolaus; Kormann, Michael; Mays, Lauren; Hofer, Laura; Neth, Olaf; Vitkov, Ljubomir; Krautgartner, Wolf Dietrich; von Schweinitz, Dietrich; Kappler, Roland; Hector, Andreas; Weber, Alexander; Hartl, Dominik

2012-01-01

RNAs are capable of modulating immune responses by binding to specific receptors. Neutrophils represent the major fraction of circulating immune cells, but receptors and mechanisms by which neutrophils sense RNA are poorly defined. Here, we analyzed the mRNA and protein expression patterns and the subcellular localization of the RNA receptors RIG-I, MDA-5, TLR3, TLR7, and TLR8 in primary neutrophils and immortalized neutrophil-like differentiated HL-60 cells. Our results demonstrate that both neutrophils and differentiated HL-60 cells express RIG-I, MDA-5, and TLR8 at the mRNA and protein levels, whereas TLR3 and TLR7 are not expressed at the protein level. Subcellular fractionation, flow cytometry, confocal laser scanning microscopy, and immuno-transmission electron microscopy provided evidence that, besides the cytoplasm, RIG-I and MDA-5 are stored in secretory vesicles of neutrophils and showed that RIG-I and its ligand, 3p-RNA, co-localize at the cell surface without triggering neutrophil activation. In summary, this study demonstrates that neutrophils express a distinct pattern of RNA recognition receptors in a non-canonical way, which could have essential implications for future RNA-based therapeutics. PMID:22532562

Graphene-enabled electron microscopy and correlated super-resolution microscopy of wet cells.

PubMed

Wojcik, Michal; Hauser, Margaret; Li, Wan; Moon, Seonah; Xu, Ke

2015-06-11

The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results.

Hyaluronic Acid Surface Modified Liposomes Prepared via Orthogonal Aminoxy Coupling: Synthesis of Nontoxic Aminoxylipids Based on Symmetrically α-Branched Fatty Acids, Preparation of Liposomes by Microfluidic Mixing, and Targeting to Cancer Cells Expressing CD44.

PubMed

Bartheldyová, Eliška; Effenberg, Roman; Mašek, Josef; Procházka, Lubomír; Knötigová, Pavlína Turánek; Kulich, Pavel; Hubatka, František; Velínská, Kamila; Zelníčková, Jaroslava; Zouharová, Darina; Fojtíková, Martina; Hrebík, Dominik; Plevka, Pavel; Mikulík, Robert; Miller, Andrew D; Macaulay, Stuart; Zyka, Daniel; Drož, Ladislav; Raška, Milan; Ledvina, Miroslav; Turánek, Jaroslav

2018-06-25

New synthetic aminoxy lipids are designed and synthesized as building blocks for the formulation of functionalized nanoliposomes by microfluidization using a NanoAssemblr. Orthogonal binding of hyaluronic acid onto the outer surface of functionalized nanoliposomes via aminoxy coupling ( N-oxy ligation) is achieved at hemiacetal function of hyaluronic acid and the structure of hyaluronic acid-liposomes is visualized by transmission electron microscopy and cryotransmission electron microscopy. Observed structures are in a good correlation with data obtained by dynamic light scattering (size and ζ-potential). In vitro experiments on cell lines expressing CD44 receptors demonstrate selective internalization of fluorochrome-labeled hyaluronic acid-liposomes, while cells with down regulated CD44 receptor levels exhibit very low internalization of hyaluronic acid-liposomes. A method based on microfluidization mixing was developed for preparation of monodispersive unilamellar liposomes containing aminoxy lipids and orthogonal binding of hyaluronic acid onto the liposomal surface was demonstrated. These hyaluronic acid-liposomes represent a potentially new drug delivery platform for CD44-targeted anticancer drugs as well as for immunotherapeutics and vaccines.

Biosynthesis of silver nanoparticles using ethanolic petals extract of Rosa indica and characterization of its antibacterial, anticancer and anti-inflammatory activities

NASA Astrophysics Data System (ADS)

Manikandan, Ramar; Manikandan, Beulaja; Raman, Thiagarajan; Arunagirinathan, Koodalingam; Prabhu, Narayanan Marimuthu; Jothi Basu, Muthuramalingam; Perumal, Muthulakshmi; Palanisamy, Subramanian; Munusamy, Arumugam

2015-03-01

The present study was aimed at biosynthesis of silver nanoparticles (AgNPs) using ethanolic extract of rose (Rosa indica) petals and testing their potential antibacterial activity using selective human pathogenic microbes, anticancer activity using human colon adenocarcinoma cancer cell line HCT 15 as well as anti-inflammatory activity using rat peritoneal macrophages in vitro. The biologically synthesized AgNPs were also characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The characterized AgNPs showed an effective antibacterial activity against Gram negative (Escherichia coli, Klebsiella pneumoniae) than Gram positive (Streptococcus mutans, Enterococcus faecalis) bacteria. MTT assay, analysis of nuclear morphology, mRNA expression of Bcl-2, Bax and protein expression of caspase 3 as well as 9, indicated potential anticancer activity. In addition, green synthesized AgNPs also attenuated cytotoxicity, nuclear morphology and free radical generation (O2- and NO) by rat peritoneal macrophages in vitro. The results of our study show the potential green synthesis of silver nanoparticles in mitigating their toxicity while retaining their antibacterial activities.

Primitive Sca-1 Positive Bone Marrow HSC in Mouse Model of Aplastic Anemia: A Comparative Study through Flowcytometric Analysis and Scanning Electron Microscopy

PubMed Central

Chatterjee, Sumanta; Basak, Pratima; Das, Prosun; Das, Madhurima; Pereira, Jacintha Archana; Dutta, Ranjan Kumar; Chaklader, Malay; Chaudhuri, Samaresh; Law, Sujata

2010-01-01

Self-renewing Hematopoietic Stem Cells (HSCs) are responsible for reconstitution of all blood cell lineages. Sca-1 is the “stem cell antigen” marker used to identify the primitive murine HSC population, the expression of which decreases upon differentiation to other mature cell types. Sca-1+ HSCs maintain the bone marrow stem cell pool throughout the life. Aplastic anemia is a disease considered to involve primary stem cell deficiency and is characterized by severe pancytopenia and a decline in healthy blood cell generation system. Studies conducted in our laboratory revealed that the primitive Sca-1+ BM-HSCs (bone marrow hematopoietic stem cell) are significantly affected in experimental Aplastic animals pretreated with chemotherapeutic drugs (Busulfan and Cyclophosphamide) and there is increased Caspase-3 activity with consecutive high Annexin-V positivity leading to premature apoptosis in the bone marrow hematopoietic stem cell population in Aplastic condition. The Sca-1bright, that is, “more primitive” BM-HSC population was more affected than the “less primitive” BM-HSC Sca-1dim  population. The decreased cell population and the receptor expression were directly associated with an empty and deranged marrow microenvironment, which is evident from scanning electron microscopy (SEM). The above experimental evidences hint toward the manipulation of receptor expression for the benefit of cytotherapy by primitive stem cell population in Aplastic anemia cases. PMID:21048851

Scanning ultrafast electron microscopy.

PubMed

Yang, Ding-Shyue; Mohammed, Omar F; Zewail, Ahmed H

2010-08-24

Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability.

Expression, Biochemistry, and Stabilization with Camel Antibodies of Membrane Proteins: Case Study of the Mouse 5-HT3 Receptor.

PubMed

Hassaïne, Ghérici; Deluz, Cédric; Grasso, Luigino; Wyss, Romain; Hovius, Ruud; Stahlberg, Henning; Tomizaki, Takashi; Desmyter, Aline; Moreau, Christophe; Peclinovska, Lucie; Minniberger, Sonja; Mebarki, Lamia; Li, Xiao-Dan; Vogel, Horst; Nury, Hugues

2017-01-01

There is growing interest in the use of mammalian protein expression systems, and in the use of antibody-derived chaperones, for structural studies. Here, we describe protocols ranging from the production of recombinant membrane proteins in stable inducible cell lines to biophysical characterization of purified membrane proteins in complex with llama antibody domains. These protocols were used to solve the structure of the mouse 5-HT3 serotonin receptor but are of broad applicability for crystallization or cryo-electron microscopy projects.

Gene expression and morphogenesis during the deposition of Drosophila wing cuticle

PubMed Central

Adler, Paul N.

2017-01-01

ABSTRACT The exoskeleton of insects and other arthropods is a very versatile material that is characterized by a complex multilayer structure. In Sobala and Adler (2016) we analyzed the process of wing cuticle deposition by RNAseq and electron microscopy. In this extra view we discuss the unique aspects of the envelope the first and most outermost layer and the gene expression program seen at the end of cuticle deposition. We discussed the role of undulae in the deposition of cuticle and how the hydrophobicity of wing cuticle arises. PMID:28631994

Gene expression and morphogenesis during the deposition of Drosophila wing cuticle.

PubMed

Adler, Paul N

2017-07-03

The exoskeleton of insects and other arthropods is a very versatile material that is characterized by a complex multilayer structure. In Sobala and Adler (2016) we analyzed the process of wing cuticle deposition by RNAseq and electron microscopy. In this extra view we discuss the unique aspects of the envelope the first and most outermost layer and the gene expression program seen at the end of cuticle deposition. We discussed the role of undulae in the deposition of cuticle and how the hydrophobicity of wing cuticle arises.

Osteograft, plastic material for regenerative medicine

NASA Astrophysics Data System (ADS)

Zaidman, A. M.; Korel, A. V.; Shevchenko, A. I.; Shchelkunova, E. I.; Sherman, K. M.; Predein, Yu. A.; Kosareva, O. S.

2016-08-01

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14-30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissue specificity of the developed osteograft.

Fourier-space TEM reconstructions with symmetry adapted functions for all rotational point groups.

PubMed

Trapani, Stefano; Navaza, Jorge

2013-05-01

A general-purpose and simple expression for the coefficients of symmetry adapted functions referred to conveniently oriented symmetry axes is given for all rotational point groups. The expression involves the computation of reduced Wigner-matrix elements corresponding to an angle specific to each group and has the computational advantage of leading to Fourier-space TEM (transmission electron microscopy) reconstruction procedures involving only real valued unknowns. Using this expression, a protocol for ab initio view and center assignment and reconstruction so far used for icosahedral particles has been tested with experimental data in other point groups. Copyright © 2013 Elsevier Inc. All rights reserved.

High-Resolution of Electron Microscopy of Montmorillonite and Montmorillonite/Epoxy Nanocomposites

DTIC Science & Technology

2005-01-01

AFRL-ML-WP-TP-2006-464 HIGH-RESOLUTION OF ELECTRON MICROSCOPY OF MONTMORILLONITE AND MONTMORILLONITE /EPOXY NANOCOMPOSITES Lawrence F...HIGH-RESOLUTION OF ELECTRON MICROSCOPY OF MONTMORILLONITE AND MONTMORILLONITE /EPOXY NANOCOMPOSITES 5c. PROGRAM ELEMENT NUMBER 62102F 5d...transmission electron microscopy the structure and morphology of montmorillonite (MMT), a material of current interest for use in polymer nanocomposites, was

The detailed analysis of the changes of murine dendritic cells (DCs) induced by thymic peptide

PubMed Central

Hu, Xiaofang; Zheng, Wei; Wang, Lu; Wan, Nan; Wang, Bing; Li, Weiwei; Hua, Hui; Hu, Xu; Shan, Fengping

2012-01-01

The aim of present research is to analyze the detailed changes of dendritic cells (DCs) induced by pidotimod(PTD). These impacts on DCs of both bone marrow derived DCs and established DC2.4 cell line were assessed with use of conventional scanning electron microscopy (SEM), flow cytometry (FCM), transmission electron microscopy (TEM), cytochemistry assay FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We demonstrated the ability of PTD to induce DC phynotypic and functional maturation as evidenced by higher expression of key surface molecules such as MHC II, CD80 and CD86. The functional tests proved the downregulation of ACP inside the DCs, occurred when phagocytosis of DCs decreased, with simultaneously antigen presentation increased toward maturation. Finally, PTD also stimulated production of more cytokine IL-12 and less TNF-α. Therefore it is concluded that PTD can markedly exert positive induction to murine DCs. PMID:22863756

Proteomic profile of Mycobacterium tuberculosis after eupomatenoid-5 induction reveals potential drug targets.

PubMed

Ghiraldi-Lopes, Luciana D; Campanerut-Sá, Paula Az; Meneguello, Jean E; Seixas, Flávio Av; Lopes-Ortiz, Mariana A; Scodro, Regiane Bl; Pires, Claudia Ta; da Silva, Rosi Z; Siqueira, Vera Ld; Nakamura, Celso V; Cardoso, Rosilene F

2017-08-01

We investigated a proteome profile, protein-protein interaction and morphological changes of Mycobacterium tuberculosis after different times of eupomatenoid-5 (EUP-5) induction to evaluate the cellular response to the drug-induced damages. The bacillus was induced to sub-minimal inhibitory concentration of EUP-5 at 12 h, 24 h and 48 h. The proteins were separated by 2D gel electrophoresis, identified by LC/MS-MS. Scanning electron microscopy and Search Tool for the Retrieval of Interacting Genes/Proteins analyses were performed. EUP-5 impacts mainly in M. tuberculosis proteins of intermediary metabolism and interactome suggests a multisite disturbance that contributes to bacilli death. Scanning electron microscopy revealed the loss of bacillary form. Some of the differentially expressed proteins have the potential to be drug targets such as citrate synthase (Rv0896), phosphoglycerate kinase (Rv1437), ketol-acid reductoisomerase (Rv3001c) and ATP synthase alpha chain (Rv1308).

Vesicular Location and Transport of S100A8 and S100A9 Proteins in Monocytoid Cells

PubMed Central

Chakraborty, Paramita; Bjork, Per; Källberg, Eva; Olsson, Anders; Riva, Matteo; Mörgelin, Matthias; Liberg, David; Ivars, Fredrik; Leanderson, Tomas

2015-01-01

We show here, by using surface biotinylation, followed by Western blotting or surface plasmon resonance analysis, that very low levels of S100A8 and/or S100A9 can be detected on the surface of THP-1 cells or freshly isolated human monocytes. This was supported by immune-electron microscopy where we observed membrane-associated expression of the proteins restricted to small patches. By using confocal microscopy we could determine that S100A8 and S100A9 protein in THP-1 cells or freshly isolated human monocytes was mostly present in vesicular structures. This finding was confirmed using immune-electron microscopy. Subcellular fractionation and confocal microscopy showed that these vesicular structures are mainly early endosomes and endolysosomes. Our subsequent studies showed that accumulation of S100A8 and S100A9 in the endolysosomal compartment is associated with induction of their release from the cells. Furthermore, an inhibitor of lysosomal activity could modulate the release of S100A8 and S100A9 in the extracellular milieu. Our current results suggest that the S100A8 and S100A9 proteins are primarily associated with certain kinds of cytosolic vesicles and may be secreted via an endolysosomal pathway. PMID:26661255

Localization of latency-associated nuclear antigen (LANA) on mitotic chromosomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rahayu, Retno; Ohsaki, Eriko; Omori, Hiroko

In latent infection of Kaposi's sarcoma-associated herpesvirus (KSHV), viral gene expression is extremely limited and copy numbers of viral genomes remain constant. Latency-associated nuclear antigen (LANA) is known to have a role in maintaining viral genome copy numbers in growing cells. Several studies have shown that LANA is localized in particular regions on mitotic chromosomes, such as centromeres/pericentromeres. We independently examined the distinct localization of LANA on mitotic chromosomes during mitosis, using super-resolution laser confocal microscopy and correlative fluorescence microscopy–electron microscopy (FM-EM) analyses. We found that the majority of LANA were not localized at particular regions such as telomeres/peritelomeres, centromeres/pericentromeres,more » and cohesion sites, but at the bodies of condensed chromosomes. Thus, LANA may undergo various interactions with the host factors on the condensed chromosomes in order to tether the viral genome to mitotic chromosomes and realize faithful viral genome segregation during cell division. - Highlights: • This is the first report showing LANA dots on mitotic chromosomes by fluorescent microscopy followed by electron microscopy. • LANA dots localized randomly on condensed chromosomes other than centromere/pericentromere and telomere/peritelomre. • Cellular mitotic checkpoint should not be always involved in the segregation of KSHV genomes in the latency.« less

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

Electron Microscopy.

ERIC Educational Resources Information Center

Beer, Michael

1980-01-01

Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

Meckelin 3 Is Necessary for Photoreceptor Outer Segment Development in Rat Meckel Syndrome

PubMed Central

Tiwari, Sarika; Hudson, Scott; Gattone, Vincent H.; Miller, Caroline; Chernoff, Ellen A. G.; Belecky-Adams, Teri L.

2013-01-01

Ciliopathies lead to multiorgan pathologies that include renal cysts, deafness, obesity and retinal degeneration. Retinal photoreceptors have connecting cilia joining the inner and outer segment that are responsible for transport of molecules to develop and maintain the outer segment process. The present study evaluated meckelin (MKS3) expression during outer segment genesis and determined the consequences of mutant meckelin on photoreceptor development and survival in Wistar polycystic kidney disease Wpk/Wpk rat using immunohistochemistry, analysis of cell death and electron microscopy. MKS3 was ubiquitously expressed throughout the retina at postnatal day 10 (P10) and P21. However, in the mature retina, MKS3 expression was restricted to photoreceptors and the retinal ganglion cell layer. At P10, both the wild type and homozygous Wpk mutant retina had all retinal cell types. In contrast, by P21, cells expressing rod- and cone-specific markers were fewer in number and expression of opsins appeared to be abnormally localized to the cell body. Cell death analyses were consistent with the disappearance of photoreceptor-specific markers and showed that the cells were undergoing caspase-dependent cell death. By electron microscopy, P10 photoreceptors showed rudimentary outer segments with an axoneme, but did not develop outer segment discs that were clearly present in the wild type counterpart. At p21 the mutant outer segments appeared much the same as the P10 mutant outer segments with only a short axoneme, while the wild-type controls had developed outer segments with many well-organized discs. We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments, but is critical for the maturation of outer segment processes. PMID:23516626

Imaging of endodontic biofilms by combined microscopy (FISH/cLSM - SEM).

PubMed

Schaudinn, C; Carr, G; Gorur, A; Jaramillo, D; Costerton, J W; Webster, P

2009-08-01

Scanning electron microscopy is a useful imaging approach for the visualization of bacterial biofilms in their natural environments including their medical and dental habitats, because it allows for the exploration of large surfaces with excellent resolution of topographic features. Most biofilms in nature, however, are embedded in a thick layer of extracellular matrix that prevents a clear identification of individual bacteria by scanning electron microscopy. The use of confocal laser scanning microscopy on the other hand in combination with fluorescence in situ hybridization enables the visualization of matrix embedded bacteria in multi-layered biofilms. In our study, fluorescence in situ hybridization/confocal laser scanning microscopy and scanning electron microscopy were applied to visualize bacterial biofilm in endodontic root canals. The resulting fluorescence in situ hybridization /confocal laser scanning microscopy and scanning electron microscopy and pictures were subsequently combined into one single image to provide high-resolution information on the location of hidden bacteria. The combined use of scanning electron microscopy and fluorescence in situ hybridization / confocal laser scanning microscopy has the potential to overcome the limits of each single technique.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

PubMed

Schorb, Martin; Gaechter, Leander; Avinoam, Ori; Sieckmann, Frank; Clarke, Mairi; Bebeacua, Cecilia; Bykov, Yury S; Sonnen, Andreas F-P; Lihl, Reinhard; Briggs, John A G

2017-02-01

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Scanning ultrafast electron microscopy

PubMed Central

Yang, Ding-Shyue; Mohammed, Omar F.; Zewail, Ahmed H.

2010-01-01

Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability. PMID:20696933

miR-99 inhibits cervical carcinoma cell proliferation by targeting TRIB2

PubMed Central

XIN, JIA-XUAN; YUE, ZHEN; ZHANG, SHUAI; JIANG, ZHONG-HUA; WANG, PING-YU; LI, YOU-JIE; PANG, MIN; XIE, SHU-YANG

2013-01-01

MicroRNAs (miRNAs) have significant roles in cell processes, including proliferation, apoptosis and stress responses. To investigate the involvement of miR-99 in the inhibition of HeLa cell proliferation, an miR-99 gene expression vector (pU6.1/miR-99), which overexpressed miR-99 in HeLa cells after transient transfection, was constructed. The expression of miR-99 was detected by qPCR. Cell proliferation and apoptosis were analyzed by cell viability, proliferation and apoptosis assays, as well as by electron microscopy. The results showed that overexpression of miR-99 in HeLa cells increased the HeLa cell mortality rate. Moreover, miR-99 overexpression was able to markedly inhibit HeLa cell proliferation according to the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The cell apoptosis rate was significantly higher in pU6.1/miR-99-treated cells compared with that in the control cultures. Increases in intracellular electron density, as well as the proportion of nuclear plasma, blebbing phenomena and apoptotic bodies were observed in pU6.1/miR-99-treated cells compared with control cultures according to electron microscopy analysis. The Tribbles 2 (TRIB2) 3′-untranslated region was also observed to be targeted by miR-99 and the results further demonstrated that miR-99 was able to negatively regulate TRIB2 expression in HeLa cells The results indicate that miR-99 acts as a tumor suppressor gene in HeLa cells, establishing a theoretical basis for its application in cancer therapeutics. PMID:24137458

Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

PubMed

Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

2014-03-01

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

Applications of microscopy to genetic therapy of cystic fibrosis and other human diseases.

PubMed

Moninger, Thomas O; Nessler, Randy A; Moore, Kenneth C

2006-01-01

Gene therapy has become an extremely important and active field of biomedical research. Microscopy is an integral component of this effort. This chapter presents an overview of imaging techniques used in our facility in support of cystic fibrosis gene therapy research. Instrumentation used in these studies includes light and confocal microscopy, transmission electron microscopy, and scanning electron microscopy. Techniques outlined include negative staining, cryo-electron microscopy, three-dimentional reconstruction, enzyme cytochemistry, immunocytochemistry, and fluorescence imaging.

Application of environmental scanning electron microscopy to determine biological surface structure.

PubMed

Kirk, S E; Skepper, J N; Donald, A M

2009-02-01

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Electron Microscopist | Center for Cancer Research

Cancer.gov

PROGRAM DESCRIPTION The Cancer Research Technology Program (CRTP) develops and implements emerging technology, cancer biology expertise and research capabilities to accomplish NCI research objectives. The CRTP is an outward-facing, multi-disciplinary hub purposed to enable the external cancer research community and provides dedicated support to NCI’s intramural Center for Cancer Research (CCR). The dedicated units provide electron microscopy, protein characterization, protein expression, optical microscopy and genetics. These research efforts are an integral part of CCR at the Frederick National Laboratory for Cancer Research (FNLCR). CRTP scientists also work collaboratively with intramural NCI investigators to provide research technologies and expertise. KEY ROLES/RESPONSIBILITIES - THIS POSITION IS CONTINGENT UPON FUNDING APPROVAL The Electron Microscopist will: Operate ultramicrotomes (Leica) and other instrumentation related to the preparation of embedded samples for EM (TEM and SEM) Operate TEM microscopes, (specifically Hitachi, FEI T20 and FEI T12) as well as SEM microscopes (Hitachi); task will include loading samples, screening, and performing data collection for a variety of samples: from cells to proteins Manage maintenance for the TEM and SEM microscopes Provide technical advice to investigators on sample preparation and data collection

Mass Determination of Rous Sarcoma Virus Virions by Scanning Transmission Electron Microscopy

PubMed Central

Vogt, Volker M.; Simon, Martha N.

1999-01-01

The internal structural protein of retroviruses, Gag, comprises most of the mass of the virion, and Gag itself can give rise to virus-like particles when expressed in appropriate cells. Previously the stoichiometry of Gag in virions was inferred from indirect measurements carried out 2 decades ago. We now have directly determined the masses of individual particles of the prototypic avian retrovirus, Rous sarcoma virus (RSV), by using scanning transmission electron microscopy. In this technique, the number of scattered electrons in the dark-field image integrated over an individual freeze-dried virus particle on a grid is directly proportional to its mass. The RSV virions had a mean mass of 2.5 × 108 Da, corresponding to about 1,500 Gag molecules per virion. The population of virions was not homogeneous, with about one-third to two-thirds of the virions deviating from the mean by more than 10% of the mass in two respective preparations. The mean masses for virions carrying genomes of 7.4 or 9.3 kb were indistinguishable, suggesting that mass variability is not due to differences in RNA incorporation. PMID:10400808

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Morphological changes of cerebral vessels and expression patterns of MMP-2 and MMP-9 on cerebrovascular wall of alcoholic rats.

PubMed

Qi, Qian; Liu, Xia; Zhang, Guozhong; He, Wenjing; Ma, Rufei; Cong, Bin; Li, Yingmin

2014-01-01

Alcohol abuse increases the incidence of cerebral accidents, which correlates with cerebrovascular structural changes. The present study was designed to observe the cerebrovascular remodeling of drinking rats with light microscopy and transmission electron microscopy (TEM). Short-term alcohol administration induced apparent amplification of perivascular spaces around small vessels in brain tissue, while long-term administration caused pathological changes of basilar arteries (BAs), including endothelial exfoliation, inner elastic lamina (IEL) fragmentation and thickening of tunica media and adventitia. In addition, the relationship between cerebrovascular remodeling and MMP-2 and MMP-9 synthesized by endothelial cells and vascular smooth muscle cells was explored by immunohistochemistry. The two protein expression in cerebral vessels changed dynamically, peaking at 1-2 weeks after treatment, and decreasing as treatment continued. These results suggest that MMP-2 and MMP-9 may play a significant role in blood-brain barrier disruption after alcohol abuse. But the chronic changes of cerebral arteries resulted from drinking are not coincident with time course of MMP-2 and MMP-9 expression in situ.

Transcriptome and Degradome Sequencing Reveals Dormancy Mechanisms of Cunninghamia lanceolata Seeds.

PubMed

Cao, Dechang; Xu, Huimin; Zhao, Yuanyuan; Deng, Xin; Liu, Yongxiu; Soppe, Wim J J; Lin, Jinxing

2016-12-01

Seeds with physiological dormancy usually experience primary and secondary dormancy in the nature; however, little is known about the differential regulation of primary and secondary dormancy. We combined multiple approaches to investigate cytological changes, hormonal levels, and gene expression dynamics in Cunninghamia lanceolata seeds during primary dormancy release and secondary dormancy induction. Light microscopy and transmission electron microscopy revealed that protein bodies in the embryo cells coalesced during primary dormancy release and then separated during secondary dormancy induction. Transcriptomic profiling demonstrated that expression of genes negatively regulating gibberellic acid (GA) sensitivity reduced specifically during primary dormancy release, whereas the expression of genes positively regulating abscisic acid (ABA) biosynthesis increased during secondary dormancy induction. Parallel analysis of RNA ends revealed uncapped transcripts for ∼55% of all unigenes. A negative correlation between fold changes in expression levels of uncapped versus capped mRNAs was observed during primary dormancy release. However, this correlation was loose during secondary dormancy induction. Our analyses suggest that the reversible changes in cytology and gene expression during dormancy release and induction are related to ABA/GA balance. Moreover, mRNA degradation functions as a critical posttranscriptional regulator during primary dormancy release. These findings provide a mechanistic framework for understanding physiological dormancy in seeds. © 2016 American Society of Plant Biologists. All Rights Reserved.

Transcriptome and Degradome Sequencing Reveals Dormancy Mechanisms of Cunninghamia lanceolata Seeds1

PubMed Central

Xu, Huimin; Liu, Yongxiu; Soppe, Wim J.J.; Lin, Jinxing

2016-01-01

Seeds with physiological dormancy usually experience primary and secondary dormancy in the nature; however, little is known about the differential regulation of primary and secondary dormancy. We combined multiple approaches to investigate cytological changes, hormonal levels, and gene expression dynamics in Cunninghamia lanceolata seeds during primary dormancy release and secondary dormancy induction. Light microscopy and transmission electron microscopy revealed that protein bodies in the embryo cells coalesced during primary dormancy release and then separated during secondary dormancy induction. Transcriptomic profiling demonstrated that expression of genes negatively regulating gibberellic acid (GA) sensitivity reduced specifically during primary dormancy release, whereas the expression of genes positively regulating abscisic acid (ABA) biosynthesis increased during secondary dormancy induction. Parallel analysis of RNA ends revealed uncapped transcripts for ∼55% of all unigenes. A negative correlation between fold changes in expression levels of uncapped versus capped mRNAs was observed during primary dormancy release. However, this correlation was loose during secondary dormancy induction. Our analyses suggest that the reversible changes in cytology and gene expression during dormancy release and induction are related to ABA/GA balance. Moreover, mRNA degradation functions as a critical posttranscriptional regulator during primary dormancy release. These findings provide a mechanistic framework for understanding physiological dormancy in seeds. PMID:27760880

Osteograft, plastic material for regenerative medicine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zaidman, A. M., E-mail: AZaydman@niito.ru; Korel, A. V., E-mail: AKorel@niito.ru; Shchelkunova, E. I., E-mail: EShelkunova@niito.ru

Creating tissue-engineering constructs based on the mechanism of cartilage-bone evolution is promising for traumatology and orthopedics. Such a graft was obtained from a chondrograft by transdifferentiation. The hondrograft placed in osteogenic medium is undergoing osteogenic differentiation for 14–30 days. Tissue specificity of the osteograft was studied by morphology, immunohistochemistry, electron microscopy, and the expression of the corresponding genes was estimated. The expression of osteonectin, fibronectin, collagen of type I, izolektin and CD 44 is determined. Alkaline phosphatase and matrix vesicles are determined in osteoblasts. Calcificates are observed in the matrix. Chondrogenic proteins expression is absent. These findings evidence the tissuemore » specificity of the developed osteograft.« less

Demonstration of transmission high energy electron microscopy

DOE PAGES

Merrill, F. E.; Goett, J.; Gibbs, J. W.; ...

2018-04-06

High energy electrons have been used to investigate an extension of transmission electron microscopy. This technique, transmission high energy electron microscopy (THEEM), provides two additional capabilities to electron microscopy. First, high energy electrons are more penetrating than low energy electrons, and thus, they are able to image through thicker samples. Second, the accelerating mode of a radio-frequency linear accelerator provides fast exposures, down to 1 ps, which are ideal for flash radiography, making THEEM well suited to study the evolution of fast material processes under dynamic conditions. Lastly, initial investigations with static objects and during material processing have been performedmore » to investigate the capabilities of this technique.« less

Introduction: A Symposium in Honor of Professor Sir John Meurig Thomas

NASA Astrophysics Data System (ADS)

Gai, P. L.; Saka, H.; Tomokiyo, Y.; Boyes, E. D.

2002-02-01

This issue is dedicated to Professor Sir John Meurig Thomas for his renowned contributions to electron microscopy in the chemical sciences. It is a collection of peer-reviewed leading articles in electron microscopy, based on the presentations at the Microscopy and Microanalysis (M&M) 2000 symposium, which was held to honor Professor Thomas's exceptional scientific leadership and wide-ranging fundamental contributions in the chemical applications of electron microscopy.The issue contains key papers by leading international researchers on the recent developments and applications of electron microscopy in the solid state and liquid state sciences. They include synthesis and characterization of silicon nitride nanorods, nanostructures of amorphous silica, electron microscopy studies of nanoscale structure and chemistry of Pt-Ru electrocatalysts of interest in direct methanol fuel cells, development of in situ wet-environmental transmission electron microscopy for the first nanoscale studies of dynamic liquid-catalyst reactions, strain analysis of silicon by finite element method and energy filtering convergent beam electron diffraction, applications of chemistry with electron microscopy, bismuth nanowires for applications in nanoelectronics technology, synthesis and characterization of quantum dots for superlattices and in situ electron microscopy at very high temperatures to study the motion of W5Si3 on [alpha][beta]-SiN3 substrates.We thank all the participants, including the invited speakers, contributors, and session chairs, who made the symposium successful. We also thank the authors and reviewers of the papers who worked assiduously towards the publication of this issue.We are very grateful to the Microscopy Society of America (MSA) for providing the opportunity to honor Professor Sir John Meurig Thomas. Organizational support from the MSA is also gratefully acknowledged.We thank Charles E. Lyman, editor in chief of Microscopy and Microanalysis for coordinating the publication of this issue and the entire journal staff for their efforts.

[Construction of Lactobacillus rhamnosus GG particles surface display system].

PubMed

Su, Runyu; Nie, Boyao; Yuan, Shengling; Tao, Haoxia; Liu, Chunjie; Yang, Bailiang; Wang, Yanchun

2017-01-25

To describe a novel particles surface display system which is consisted of gram-positive enhancer matrix (GEM) particles and anchor proteins for bacteria-like particles vaccines, we treated Lactobacillus rhamnosus GG bacteria with 10% heated-TCA for preparing GEM particles, and then identified the harvested GEM particles by electron microscopy, RT-PCR and SDS-PAGE. Meanwhile, Escherichia coli was induced to express hybrid proteins PA3-EGFP and P60-EGFP, and GEM particles were incubated with them. Then binding of anchor proteins were determined by Western blotting, transmission electron microscopy, fluorescence microscopy and spectrofluorometry. GEM particles preserved original size and shape, and proteins and DNA contents of GEM particles were released substantially. The two anchor proteins both had efficiently immobilized on the surface of GEM. GEM particles that were bounded by anchor proteins were brushy. The fluorescence of GEM particles anchoring PA3 was slightly brighter than P60, but the difference was not significant (P>0.05). GEM particles prepared from L. rhamnosus GG have a good binding efficiency with anchor proteins PA3-EGFP and P60-EGFP. Therefore, this novel foreign protein surface display system could be used for bacteria-like particle vaccines.

Preparation and evaluation of MS2 bacteriophage-like particles packaging hepatitis E virus RNA.

PubMed

Wang, Shen; Liu, Ying; Li, Dandan; Zhou, Tiezhong; Gao, Shenyang; Zha, Enhui; Yue, Xiqing

2016-10-01

Hepatitis E virus (HEV) is the pathogen causing hepatitis E (HE). It arouses global public health concern since it is a zoonotic disease. The objective of this letter is to report a cost-effective internal control prepared for monitoring procedures of HEV reverse transcriptase (RT)-PCR detection. A selected conserved HEV RNA fragment was integrated into the downstream of the truncated MS2 bacteriophage genome based on Armored RNA technology. The resulting MS2-HEV gene harbored by the pET-28b-MS2-HEV plasmid was transformed into E. coli BL21(DE3) for expression analysis by SDS-PAGE. The expression products were purified and concentrated by ultrasonication and ultrafiltration separation. The morphology and stability properties of the virus-like particles (VLPs) were evaluated by electron microscopy scanning and nuclease challenges, respectively. SDS-PAGE results showed that the constructed MS2-HEV gene expressed efficiently and the purity of the VLPs was highly consistent with the result in electron microscopy. Stability evaluation results demonstrated that the prepared VLPs exhibited strong resistance to DNase I and RNase A attacks and also performed long-lasting protection of coated HEV RNA for at least 4 months at -20°C. These data revealed that the prepared VLPs meet the basic requirements of use as internal control material in the HEV RNA amplification assay. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Role of heat shock transcription factor 1(HSF1)-upregulated macrophage in ameliorating pressure overload-induced heart failure in mice.

PubMed

Du, Peizhao; Chang, Yaowei; Dai, Fangjie; Wei, Chunyan; Zhang, Qi; Li, Jiming

2018-08-15

In order to explore the role of macrophages in HSF1-mediated alleviation of heart failure, mice model of pressure overload-induced heart failure was established using transverse aortic constriction (TAC). Changes in cardiac function and morphology were studied in TAC and SHAM groups using ultrasonic device, tissue staining, electron microscopy, real-time quantitative polymerase chain reaction (RT-QPCR), and Western blotting. We found that mice in the TAC group showed evidence of impaired cardiac function and aggravation of fibrosis on ultrasonic and histopathological examination when compared to those in the SHAM group. The expressions of HSF1, LC3II/LC3I, Becline-1 and HIF-1, as well as autophagosome formation in TAC group were greater than that in SHAM group. On sub-group analyses in the TAC group, improved cardiac function and alleviation of fibrosis was observed in the HSF1 TG subgroup as compared to that in the wild type subgroup. Expressions of LC3II/LC3I, Becline-1 and HIF-1, too showed an obvious increase; and increased autophagosome formation was observed on electron microscopy. Opposite results were observed in the HSF1 KO subgroup. These results collectively suggest that in the pressure overload heart failure model, HSF1 promoted formation of macrophages by inducing upregulation of HIF-1 expression, through which heart failure was ameliorated. Copyright © 2018 Elsevier B.V. All rights reserved.

Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate.

PubMed

Karuppasamy, Manikandan; Karimi Nejadasl, Fatemeh; Vulovic, Milos; Koster, Abraham J; Ravelli, Raimond B G

2011-05-01

Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from parameters including incident flux, beam energy, beam size, sample composition and sample size. In here, the use of dose is reintroduced for electron microscopy, accounting for the electron energy, incident flux and measured sample thickness and composition. Knowledge of the amount of energy deposited allowed us to compare doses with experimental limits in macromolecular X-ray crystallography, to obtain an upper estimate of radical concentrations that build up in the vitreous sample, and to translate heat-transfer simulations carried out for macromolecular X-ray crystallography to cryo-electron microscopy. Stroboscopic exposure series of 50-250 images were collected for different incident flux densities and integration times from Lumbricus terrestris extracellular hemoglobin. The images within each series were computationally aligned and analyzed with similarity metrics such as Fourier ring correlation, Fourier ring phase residual and figure of merit. Prior to gas bubble formation, the images become linearly brighter with dose, at a rate of approximately 0.1% per 10 MGy. The gradual decomposition of a vitrified hemoglobin sample could be visualized at a series of doses up to 5500 MGy, by which dose the sample was sublimed. Comparison of equal-dose series collected with different incident flux densities showed a dose-rate effect favoring lower flux densities. Heat simulations predict that sample heating will only become an issue for very large dose rates (50 e(-)Å(-2) s(-1) or higher) combined with poor thermal contact between the grid and cryo-holder. Secondary radiolytic effects are likely to play a role in dose-rate effects. Stroboscopic data collection combined with an improved understanding of the effects of dose and dose rate will aid single-particle cryo-electron microscopists to have better control of the outcome of their experiments.

Radiation damage in single-particle cryo-electron microscopy: effects of dose and dose rate

PubMed Central

Karuppasamy, Manikandan; Karimi Nejadasl, Fatemeh; Vulovic, Milos; Koster, Abraham J.; Ravelli, Raimond B. G.

2011-01-01

Radiation damage is an important resolution limiting factor both in macromolecular X-ray crystallography and cryo-electron microscopy. Systematic studies in macromolecular X-ray crystallography greatly benefited from the use of dose, expressed as energy deposited per mass unit, which is derived from parameters including incident flux, beam energy, beam size, sample composition and sample size. In here, the use of dose is reintroduced for electron microscopy, accounting for the electron energy, incident flux and measured sample thickness and composition. Knowledge of the amount of energy deposited allowed us to compare doses with experimental limits in macromolecular X-ray crystallography, to obtain an upper estimate of radical concentrations that build up in the vitreous sample, and to translate heat-transfer simulations carried out for macromolecular X-ray crystallography to cryo-electron microscopy. Stroboscopic exposure series of 50–250 images were collected for different incident flux densities and integration times from Lumbricus terrestris extracellular hemoglobin. The images within each series were computationally aligned and analyzed with similarity metrics such as Fourier ring correlation, Fourier ring phase residual and figure of merit. Prior to gas bubble formation, the images become linearly brighter with dose, at a rate of approximately 0.1% per 10 MGy. The gradual decomposition of a vitrified hemoglobin sample could be visualized at a series of doses up to 5500 MGy, by which dose the sample was sublimed. Comparison of equal-dose series collected with different incident flux densities showed a dose-rate effect favoring lower flux densities. Heat simulations predict that sample heating will only become an issue for very large dose rates (50 e−Å−2 s−1 or higher) combined with poor thermal contact between the grid and cryo-holder. Secondary radiolytic effects are likely to play a role in dose-rate effects. Stroboscopic data collection combined with an improved understanding of the effects of dose and dose rate will aid single-particle cryo-electron microscopists to have better control of the outcome of their experiments. PMID:21525648

Light and electron microscopic analyses of Vasa expression in adult germ cells of the fish medaka.

PubMed

Yuan, Yongming; Li, Mingyou; Hong, Yunhan

2014-07-15

Germ cells of diverse animal species have a unique membrane-less organelle called germ plasm (GP). GP is usually associated with mitochondria and contains RNA binding proteins and mRNAs of germ genes such as vasa. GP has been described as the mitochondrial cloud (MC), intermitochondrial cement (IC) and chromatoid body (CB). The mechanism underlying varying GP structures has remained incompletely understood. Here we report the analysis of GP through light and electron microscopy by using Vasa as a marker in adult male germ cells of the fish medaka (Oryzias latipes). Immunofluorescence light microscopy revealed germ cell-specific Vasa expression. Vasa is the most abundant in mitotic germ cells (oogonia and spermatogonia) and reduced in meiotic germ cells. Vasa in round spermatids exist as a spherical structure reminiscent of CB. Nanogold immunoelectron microscopy revealed subcellular Vasa redistribution in male germ cells. Vasa in spermatogonia concentrates in small areas of the cytoplasm and is surrounded by mitochondria, which is reminiscent of MC. Vasa is intermixed with mitochondria to form IC in primary spermatocytes, appears as the free cement (FC) via separation from mitochondria in secondary spermatocyte and becomes condensed in CB at the caudal pole of round spermatids. During spermatid morphogenesis, Vasa redistributes and forms a second CB that is a ring-like structure surrounding the dense fiber of the flagellum in the midpiece. These structures resemble those described for GP in various species. Thus, Vasa identifies GP and adopts varying structures via dynamic reorganization at different stages of germ cell development. Copyright © 2014 Elsevier B.V. All rights reserved.

[Morphological features of the myometrium in connective tissue dysplasia in women with uterine inertia].

PubMed

Konovalov, P V; Mitrofanova, L B; Gorshkov, A N; Ovsyannikov, F A

2015-01-01

to reveal the morphological features of the lower uterine segment myometrium in connective tissue dysplasia (CTD) in women with uterine inertia. Histological, immunohistochemical (with antibodies against collagen types I and III, matrix metalloproteinases 1 and 9 (MMR-1, MMP-9), tissue inhibitor of metalloproteinase 1 (TIMP-1), fibronectin; fibulin-5, connexin-43), electron microscopic, and electron immunocytochemical studies with morphometry of myometrial fragments from 15 parturient women with CTD and uterine inertia (a study group) and those from 10 women without CTD (a control group). The myometrium in CTD exhibited the decreased expression of connextin-43, fibulin-5, TIMP-1, collagens types I and III with collagen type III predominance and the unchanged levels of fibronectin and MMP-1 and MMP-9. Electron microscopy and immunocytochemistry showed fewer intercellular contacts and the dramatically lower expression of connexin-43 than in the control. A set of found myometrial changes in women with uterine inertia is a manifestation of CTD.

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

PubMed

Kempen, Paul J; Kircher, Moritz F; de la Zerda, Adam; Zavaleta, Cristina L; Jokerst, Jesse V; Mellinghoff, Ingo K; Gambhir, Sanjiv S; Sinclair, Robert

2015-01-01

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buck, E.C.; Dietz, N.L.; Bates, J.K.

Uranium contaminated soils from the Fernald Operation Site, Ohio, have been examined by a combination of optical microscopy, scanning electron microscopy with backscattered electron detection (SEM/BSE), and analytical electron microscopy (AEM). A method is described for preparing of transmission electron microscopy (TEM) thin sections by ultramicrotomy. By using these thin sections, SEM and TEM images can be compared directly. Uranium was found in iron oxides, silicates (soddyite), phosphates (autunites), and fluorite. Little uranium was associated with clays. The distribution of uranium phases was found to be inhomogeneous at the microscopic level.

Electron Microscopy of Ebola Virus-Infected Cells.

PubMed

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

The functional interrelationship between gap junctions and fenestrae in endothelial cells of the liver organoid.

PubMed

Saito, Masaya; Matsuura, Tomokazu; Nagatsuma, Keisuke; Tanaka, Ken; Maehashi, Haruka; Shimizu, Keiko; Hataba, Yoshiaki; Kato, Fumitaka; Kashimori, Isao; Tajiri, Hisao; Braet, Filip

2007-06-01

Functional intact liver organoid can be reconstructed in a radial-flow bioreactor when human hepatocellular carcinoma (FLC-5), mouse immortalized sinusoidal endothelial M1 (SEC) and A7 (HSC) hepatic stellate cell lines are cocultured. The structural and functional characteristics of the reconstructed organoid closely resemble the in vivo liver situation. Previous liver organoid studies indicated that cell-to-cell communications might be an important factor for the functional and structural integrity of the reconstructed organoid, including the expression of fenestrae. Therefore, we examined the possible relationship between functional intact gap junctional intercellular communication (GJIC) and fenestrae dynamics in M1-SEC cells. The fine morphology of liver organoid was studied in the presence of (1) irsogladine maleate (IM), (2) oleamide and (3) oleamide followed by IM treatment. Fine ultrastructural changes were studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and compared with control liver organoid data. TEM revealed that oleamide affected the integrity of cell-to-cell contacts predominantly in FLC-5 hepatocytes. SEM observation showed the presence of fenestrae on M1-SEC cells; however, oleamide inhibited fenestrae expression on the surface of endothelial cells. Interestingly, fenestrae reappeared when IM was added after initial oleamide exposure. GJIC mediates the number of fenestrae in endothelial cells of the liver organoid.

Biosynthesis of silver nanoparticles using ethanolic petals extract of Rosa indica and characterization of its antibacterial, anticancer and anti-inflammatory activities.

PubMed

Manikandan, Ramar; Manikandan, Beulaja; Raman, Thiagarajan; Arunagirinathan, Koodalingam; Prabhu, Narayanan Marimuthu; Jothi Basu, Muthuramalingam; Perumal, Muthulakshmi; Palanisamy, Subramanian; Munusamy, Arumugam

2015-03-05

The present study was aimed at biosynthesis of silver nanoparticles (AgNPs) using ethanolic extract of rose (Rosa indica) petals and testing their potential antibacterial activity using selective human pathogenic microbes, anticancer activity using human colon adenocarcinoma cancer cell line HCT 15 as well as anti-inflammatory activity using rat peritoneal macrophages in vitro. The biologically synthesized AgNPs were also characterized by UV-visible spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), energy-dispersive X-ray spectroscopy (EDX) and X-ray diffraction (XRD). The characterized AgNPs showed an effective antibacterial activity against Gram negative (Escherichia coli, Klebsiella pneumoniae) than Gram positive (Streptococcus mutans, Enterococcus faecalis) bacteria. MTT assay, analysis of nuclear morphology, mRNA expression of Bcl-2, Bax and protein expression of caspase 3 as well as 9, indicated potential anticancer activity. In addition, green synthesized AgNPs also attenuated cytotoxicity, nuclear morphology and free radical generation (O2(-) and NO) by rat peritoneal macrophages in vitro. The results of our study show the potential green synthesis of silver nanoparticles in mitigating their toxicity while retaining their antibacterial activities. Copyright © 2014 Elsevier B.V. All rights reserved.

Formation and differentiation of three-dimensional rat marrow stromal cell culture on microcarriers in a rotating-wall vessel

NASA Technical Reports Server (NTRS)

Qiu, Q.; Ducheyne, P.; Gao, H.; Ayyaswamy, P.

1998-01-01

Using a high aspect ratio vessel (HARV), this study investigated the formation of 3-D rat marrow stromal cell culture on microcarriers and the expression of bone-related biochemical markers under conditions of simulated microgravity. In addition, it calculated the shear stresses imparted on the surface of microcarriers of different densities by the medium fluid in an HARV. Secondary rat marrow stromal cells were cultured on two types of microcarriers, Cytodex-3 beads and modified bioactive glass particles. Examination of cellular morphology by scanning electron microscopy revealed the presence of three-dimensional multicellular aggregates consisting of multiple cell-covered Cytodex-3 microcarriers bridged together. Mineralization was observed in the aggregates. Spherical cell-bead aggregates were observed in an HARV, while cell-bead assemblies were mostly loosely packed in a chain-like or branched structure in a cell bag. The expressions of alkaline phosphatase activity, collagen type I, and osteopontin were shown via the use of histochemical staining, immunolabeling, and confocal scanning electron microscopy. Using a numerical approach, it was found that at a given rotational speed and for a given culture medium, a larger density difference between the microcarrier and the culture medium (e.g., a modified bioactive glass particle) imparted a higher maximum shear stress on the microcarrier.

Venlafaxine inhibits apoptosis of hippocampal neurons by up-regulating brain-derived neurotrophic factor in a rat depression model

PubMed Central

Huang, Xiao; Mao, Yue-Shi; Li, Chao; Wang, Hao; Ji, Jian-Lin

2014-01-01

Objective: To study the effect of venlafaxine on the expression of brain-derived neurotrophic factor (BDNF) in rat hippocampal neurons, as well as its inhibitory effect on apoptosis of hippocampal neurons. Methods: Differences in behavioral ability between the depression model group and the Venlafaxine treatment group were observed using behavioral, sucrose-water and open field tests. The rat hippocampal tissue was sliced, stained and observed for BDNF distribution by immunohistochemistry. Apoptosis of hippocampal neurons was detected by TUNEL. BDNF expression in the hippocampal tissue was detected by Western blot. Injury and apoptosis of the hippocampal tissue were observed by electron microscopy. Results: Behavioral test showed that venlafaxine effectively improved the behavioral abilities of depressed rats. Immunohistochemistry showed that venlafaxine markedly increased the BDNF expression in the rat hippocampus. TUNEL showed that venlafaxine markedly inhibited apoptosis of hippocampal neurons, which was also confirmed by electron microscopic observation of the pathologic sections. Conclusion: Venlafaxine improved the expression of BDNF through working on PI3k/PKB/eNOS pathway and repressed the apoptosis of hippocampal neurons. PMID:25197330

TRPV2 expression in rat oral mucosa.

PubMed

Shimohira, Daiji; Kido, Mizuho A; Danjo, Atsushi; Takao, Tomoka; Wang, Bing; Zhang, Jing-Qi; Yamaza, Takayoshi; Masuko, Sadahiko; Goto, Masaaki; Tanaka, Teruo

2009-10-01

The oral mucosa is a highly specialised, stratified epithelium that confers protection from infection and physical, chemical and thermal stimuli. The non-keratinised junctional epithelium surrounds each tooth like a collar and is easily attacked by foreign substances from the oral sulcus. We found that TRPV2, a temperature-gated channel, is highly expressed in junctional epithelial cells, but not in oral sulcular epithelial cells or oral epithelial cells. Dual or triple immunolabelling with immunocompetent cell markers also revealed TRPV2 expression in Langerhans cells and in dendritic cells and macrophages. Electron microscopy disclosed TRPV2 immunoreactivity in the unmyelinated and thinly myelinated axons within the connective tissue underlying the epithelium. TRPV2 labelling was also observed in venule endothelial cells. The electron-dense immunoreaction in junctional epithelial cells, macrophages and neural axons occurred on the plasma membrane, on invaginations of the plasma membrane and in vesicular structures. Because TRPV2 has been shown to respond to temperature, hypotonicity and mechanical stimuli, gingival cells expressing TRPV2 may act as sensor cells, detecting changes in the physical and chemical environment, and may play a role in subsequent defence mechanisms.

Histophilus somni biofilm formation in cardiopulmonary tissue of the bovine host following respiratory challenge.

PubMed

Sandal, Indra; Shao, Jian Q; Annadata, Satish; Apicella, Michael A; Boye, Mette; Jensen, Tim K; Saunders, Geoffrey K; Inzana, Thomas J

2009-02-01

Biofilms form in a variety of host sites following infection with many bacterial species. However, the study of biofilms in a host is hindered due to the lack of protocols for the proper experimental investigation of biofilms in vivo. Histophilus somni is an agent of respiratory and systemic diseases in bovines, and readily forms biofilms in vitro. In the present study the capability of H. somni to form biofilms in cardiopulmonary tissue following experimental respiratory infection in the bovine host was examined by light microscopy, transmission electron microscopy, immunoelectron microscopy of ultrathin cryosections, scanning electron microscopy of freeze-fractured samples, and fluorescent in situ hybridization. Biofilms were evident and most prominent in the myocardium, and were associated with a large amount of amorphous extracellular material. Furthermore, Pasteurella multocida was often cultured with H. somni from heart and lung samples. Transposon mutagenesis of H. somni strain 2336 resulted in the generation of mutants that expressed more or less biofilm than the parent strain. Six mutants deficient in biofilm formation had an insertion in the gene encoding for a homolog of filamentous haemagglutinin (FHA), predicted to be involved in attachment. Thus, this investigation demonstrated that H. somni is capable of forming a biofilm in its natural host, that such a biofilm may be capable of harboring other bovine respiratory disease pathogens, and that the genes responsible for biofilm formation can be identified by transposon mutagenesis.

A direct electron detector for time-resolved MeV electron microscopy

DOE PAGES

Vecchione, T.; Denes, P.; Jobe, R. K.; ...

2017-03-15

The introduction of direct electron detectors enabled the structural biology revolution of cryogenic electron microscopy. Direct electron detectors are now expected to have a similarly dramatic impact on time-resolved MeV electron microscopy, particularly by enabling both spatial and temporal jitter correction. Here in this paper, we report on the commissioning of a direct electron detector for time-resolved MeV electron microscopy. The direct electron detector demonstrated MeV single electron sensitivity and is capable of recording megapixel images at 180 Hz. The detector has a 15-bit dynamic range, better than 30-μm spatial resolution and less than 20 analogue-to-digital converter count RMS pixelmore » noise. The unique capabilities of the direct electron detector and the data analysis required to take advantage of these capabilities are presented. The technical challenges associated with generating and processing large amounts of data are also discussed.« less

A direct electron detector for time-resolved MeV electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vecchione, T.; Denes, P.; Jobe, R. K.

The introduction of direct electron detectors enabled the structural biology revolution of cryogenic electron microscopy. Direct electron detectors are now expected to have a similarly dramatic impact on time-resolved MeV electron microscopy, particularly by enabling both spatial and temporal jitter correction. Here we report on the commissioning of a direct electron detector for time-resolved MeV electron microscopy. The direct electron detector demonstrated MeV single electron sensitivity and is capable of recording megapixel images at 180 Hz. The detector has a 15-bit dynamic range, better than 30-μmμm spatial resolution and less than 20 analogue-to-digital converter count RMS pixel noise. The uniquemore » capabilities of the direct electron detector and the data analysis required to take advantage of these capabilities are presented. The technical challenges associated with generating and processing large amounts of data are also discussed.« less

A direct electron detector for time-resolved MeV electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vecchione, T.; Denes, P.; Jobe, R. K.

The introduction of direct electron detectors enabled the structural biology revolution of cryogenic electron microscopy. Direct electron detectors are now expected to have a similarly dramatic impact on time-resolved MeV electron microscopy, particularly by enabling both spatial and temporal jitter correction. Here in this paper, we report on the commissioning of a direct electron detector for time-resolved MeV electron microscopy. The direct electron detector demonstrated MeV single electron sensitivity and is capable of recording megapixel images at 180 Hz. The detector has a 15-bit dynamic range, better than 30-μm spatial resolution and less than 20 analogue-to-digital converter count RMS pixelmore » noise. The unique capabilities of the direct electron detector and the data analysis required to take advantage of these capabilities are presented. The technical challenges associated with generating and processing large amounts of data are also discussed.« less

Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

PubMed Central

Kim, Doory; Deerinck, Thomas J.; Sigal, Yaron M.; Babcock, Hazen P.; Ellisman, Mark H.; Zhuang, Xiaowei

2015-01-01

Correlative fluorescence light microscopy and electron microscopy allows the imaging of spatial distributions of specific biomolecules in the context of cellular ultrastructure. Recent development of super-resolution fluorescence microscopy allows the location of molecules to be determined with nanometer-scale spatial resolution. However, correlative super-resolution fluorescence microscopy and electron microscopy (EM) still remains challenging because the optimal specimen preparation and imaging conditions for super-resolution fluorescence microscopy and EM are often not compatible. Here, we have developed several experiment protocols for correlative stochastic optical reconstruction microscopy (STORM) and EM methods, both for un-embedded samples by applying EM-specific sample preparations after STORM imaging and for embedded and sectioned samples by optimizing the fluorescence under EM fixation, staining and embedding conditions. We demonstrated these methods using a variety of cellular targets. PMID:25874453

High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.

PubMed

Koga, Daisuke; Kusumi, Satoshi; Shodo, Ryusuke; Dan, Yukari; Ushiki, Tatsuo

2015-12-01

In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

On the Progress of Scanning Transmission Electron Microscopy (STEM) Imaging in a Scanning Electron Microscope.

PubMed

Sun, Cheng; Müller, Erich; Meffert, Matthias; Gerthsen, Dagmar

2018-04-01

Transmission electron microscopy (TEM) with low-energy electrons has been recognized as an important addition to the family of electron microscopies as it may avoid knock-on damage and increase the contrast of weakly scattering objects. Scanning electron microscopes (SEMs) are well suited for low-energy electron microscopy with maximum electron energies of 30 keV, but they are mainly used for topography imaging of bulk samples. Implementation of a scanning transmission electron microscopy (STEM) detector and a charge-coupled-device camera for the acquisition of on-axis transmission electron diffraction (TED) patterns, in combination with recent resolution improvements, make SEMs highly interesting for structure analysis of some electron-transparent specimens which are traditionally investigated by TEM. A new aspect is correlative SEM, STEM, and TED imaging from the same specimen region in a SEM which leads to a wealth of information. Simultaneous image acquisition gives information on surface topography, inner structure including crystal defects and qualitative material contrast. Lattice-fringe resolution is obtained in bright-field STEM imaging. The benefits of correlative SEM/STEM/TED imaging in a SEM are exemplified by structure analyses from representative sample classes such as nanoparticulates and bulk materials.

Role of gap junctions in the contractile response to agonists in the mesenteric artery of spontaneously hypertensive rats.

PubMed

Ma, Ke-Tao; Li, Xin-Zhi; Li, Li; Jiang, Xue-Wei; Chen, Xin-Yan; Liu, Wei-Dong; Zhao, Lei; Zhang, Zhong-Shuang; Si, Jun-Qiang

2014-02-01

To investigate the effects of hypertension on the changes in gap junctions between vascular smooth muscle cells (VSMCs) in the mesenteric artery (MA) of spontaneously hypertensive rats (SHRs). Whole-cell patch clamp, pressure myography, real-time quantitative reverse transcription PCR (qRT-PCR), western blot analysis and transmission electron microscopy were used to examine the differences in expression and function of the gap junction between MA VSMCs of SHR and control normotensive Wistar-Kyoto (WKY) rats. (1) Whole-cell patch clamp measurements showed that the membrane capacitance and conductance of in-situ MA VSMCs of SHR were significantly greater than those of WKY rats (P<0.05), suggesting enhanced gap junction coupling between MA VSMCs of SHR. (2) The administration of phenylephrine (PE) and KCl (an endothelium-independent vasoconstrictor) initiated more pronounced vasoconstriction in SHR versus WKY rats (P<0.05). Furthermore, 2-APB (a gap junction inhibitor) attenuated PE- and KCl-induced vasoconstriction, and the inhibitory effects of 2-APB were significantly greater in SHR (P<0.05). (3) The expression of connexin 45 (Cx45) mRNA and protein in the MA was greater in SHR versus WKY rats (P<0.05). The level of phosphorylated Cx43 was significantly higher in SHR versus WKY rats (P<0.05), although the expression of total Cx43 mRNA and protein in the MA was equivalent between SHR and WKY rats. Electron microscopy revealed that the gap junctions were significantly larger in SHR versus WKY rats. Increases in the expression of Cx45 and phosphorylation of Cx43 may contribute to the enhancement of communication across gap junctions between MA VSMCs of SHR, which may increase the contractile response to agonists.

Nature and mechanisms of hepatocyte apoptosis induced by D-galactosamine/lipopolysaccharide challenge in mice.

PubMed

Wu, Yi-Hang; Hu, Shao-Qing; Liu, Jun; Cao, Hong-Cui; Xu, Wei; Li, Yong-Jun; Li, Lan-Juan

2014-06-01

Apoptosis plays a role in the normal development of liver. However, overactivation thereof may lead to hepatocellular damage. The aim of this study was to assess D-galactosamine (D-GalN)/lipopolysaccharide (LPS)-induced hepatocyte apoptotic changes in mice and clarify the mechanisms involved in this process. DNA ladder detection was employed to determine the induction condition of hepatic apoptosis. An initial test indicated that typical hepatocyte apoptosis was observed at 6-10 h after the intraperitoneal injection of D-GalN (700 mg/kg) and LPS (10 µg/kg). Subsequently, we evaluated hepatocyte apoptosis at 8 h after administering D-GalN/LPS by histopathological analysis, terminal deoxynucleotidyl transferase-mediated dUTP nick end‑labeling (TUNEL) detection, flow cytometry and electron microscopy analysis. To clarify the apoptosis-related gene expression, the expression levels of tumor necrosis factor-α (TNF-α), transforming growth factor-β1 (TGF-β1), caspase-3, and Fas/Fas ligand (FasL) were determined by serum enzyme immunoassay, immunohistochemistry and western blot analysis. Strong apoptotic positive signals following D-GalN/LPS injection were observed from the results of the serum analysis, histopathological and immunohistochemical analyses, DNA ladder detection, TUNEL detection, flow cytometry and electron microscopy analysis. Additionally, apoptotic hepatocytes were mainly at the late stage of cell apoptosis. The expression of TNF-α, TGF-β1, caspase-3 and Fas/FasL was significantly increased. In conclusion, this study evaluated the D-GalN/LPS-induced hepatocyte apoptotic changes and clarified the apoptosis-related gene expression in mice. The hepatocyte apoptosis induced by D-GalN/LPS may be mainly regulated by the death receptor pathway. TGF-β signaling pathway may also play a vital role in this process of hepatocyte apoptosis.

PM2.5 exposure-induced autophagy is mediated by lncRNA loc146880 which also promotes the migration and invasion of lung cancer cells.

PubMed

Deng, Xiaobei; Feng, Nannan; Zheng, Min; Ye, Xiaofei; Lin, Hongyan; Yu, Xiao; Gan, Zhihua; Fang, Zheng; Zhang, Huan; Gao, Ming; Zheng, Zhi-Jie; Yu, Herbert; Ding, Wenjun; Qian, Biyun

2017-02-01

Evidence shows that individuals who are under long-term exposure to environmental PM 2.5 are at increased risk of lung cancer. Various laboratory experiments also suggest several mechanistic links between PM 2.5 exposure and lung carcinogenesis. However, a long non-coding RNA (lncRNA) mediated pathogenic change after PM 2.5 exposure and its potential roles in tumorigenesis and disease progression have not been reported. Cytotoxicity induced by PM 2.5 was assessed by using scanning electron microscopy and transmission electron microscopy. ROS generation, autophagy, and metastasis induced by PM 2.5 were detected by using comprehensive approaches. Expression of lncRNA-loc146880 and lc3b (autophagy marker) in A549 cells, lung tissue and serum were determined by RT-PCR and Western blotting. PM 2.5 could be internalized into lung cancer cells, resulting in marked increases in ROS levels and autophagy. ROS may be responsible for increased expression of loc146880 which further up-regulates autophagy. Both loc146880 and autophagy could promote lung tumor cell migration, invasion and EMT. In addition, a positive correlation was observed between loc146880 expression and lc3b levels in tumor tissues and serum of lung cancer patients. Taken together, our data suggest that PM 2.5 exposure induces ROS, which activates loc146880 expression. The lncRNA, in turn, up-regulates autophagy and promotes the malignant behaviors of lung cancer cells. The results show the toxicological effects of PM 2.5 in lung tumor progression and metastasis. Copyright © 2016 Elsevier B.V. All rights reserved.

Synthesis of silver nanoparticles using Solanum trilobatum fruits extract and its antibacterial, cytotoxic activity against human breast cancer cell line MCF 7

NASA Astrophysics Data System (ADS)

Ramar, Manikandan; Manikandan, Beulaja; Marimuthu, Prabhu Narayanan; Raman, Thiagarajan; Mahalingam, Anjugam; Subramanian, Palanisamy; Karthick, Saravanan; Munusamy, Arumugam

2015-04-01

In the present study, we have synthesized silver nanoparticles by a simple and eco-friendly method using unripe fruits of Solanum trilobatum. The aqueous silver ions when exposed to unripe fruits extract were reduced and stabilized over long time resulting in biosynthesis of surface functionalized silver nanoparticles. The bio-reduced silver nanoparticles were characterized by UV-visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), energy-dispersive spectroscopy (EDX) and X-ray diffraction (XRD). These biologically synthesized silver nanoparticles were tested for its antibacterial activity against few human pathogenic bacteria including Gram-positive (Streptococcus mutans, Enterococcus faecalis) and Gram-negative (Escherichia coli, Klebsiella pneumoniae) bacteria. In addition, we also demonstrated anticancer activity of these nanoparticles in vitro against human breast cancer cell line (MCF 7) using MTT, nuclear morphology assay, Western blot and RT-PCR expression. These results taken together show the potential applications of biosynthesized silver nanoparticles using S. trilobatum fruits.

A novel gelatin hydrogel carrier sheet for corneal endothelial transplantation.

PubMed

Watanabe, Ryou; Hayashi, Ryuhei; Kimura, Yu; Tanaka, Yuji; Kageyama, Tomofumi; Hara, Susumu; Tabata, Yasuhiko; Nishida, Kohji

2011-09-01

We examined the feasibility of using gelatin hydrogels as carrier sheets for the transplantation of cultivated corneal endothelial cells. The mechanical properties, transparency, and permeability of gelatin hydrogel sheets were compared with those of atelocollagen sheets. Immunohistochemistry (ZO-1, Na(+)/K(+)-ATPase, and N-cadherin), hematoxylin and eosin staining, and scanning electron microscopy were performed to assess the integrity of corneal endothelial cells that were cultured on gelatin hydrogel sheets. The gelatin hydrogel sheets displayed greater transparency, elastic modulus, and albumin permeability compared to those of atelocollagen sheets. The corneal endothelial cells on gelatin hydrogel sheets showed normal expression levels of ZO-1, Na(+)/K(+)-ATPase, and N-cadherin. Hematoxylin and eosin staining revealed the formation of a continuous monolayer of cells attached to the gelatin hydrogel sheet. Scanning electron microscopy observations showed that the corneal endothelial cells were arranged in a regular, mosaic, and polygonal pattern with normal cilia. These results indicate that the gelatin hydrogel sheet is a promising material to transport corneal endothelial cells during transplantation.

Effects of microgravity on muscle and cerebral cortex: a suggested interaction

NASA Astrophysics Data System (ADS)

D'Amelio, F.; Fox, R. A.; Wu, L. C.; Daunton, N. G.; Corcoran, M. L.

The ``slow'' antigravity muscle adductor longus was studied in rats after 14 days of spaceflight (SF). The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light and electron microscopy revealed myofiber atrophy, segmental necrosis and regenerative myofibers. Regenerative myofibers were N-CAM immunoreactive (N-CAM-IR). The neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles, degenerative changes, vacant axonal spaces and changes suggestive of axonal sprouting. No alterations of muscle spindles was seen either by light or electron microscopy. These observations suggest that muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight. In a separate study, GABA immunoreactivity (GABA-IR) was evaluated at the level of the hindlimb representation of the rat somatosensory cortex after 14 days of hindlimb unloading by tail suspension (``simulated'' microgravity). A reduction in number of GABA-immunoreactive cells with respect to the control animals was observed in layer Va and Vb. GABA-IR terminals were also reduced in the same layers, particularly those terminals surrounding the soma and apical dendrites of pyramidal cells in layer Vb. On the basis of previous morphological and behavioral studies of the neuromuscular system after spaceflight and hindlimb suspension it is suggested that after limb unloading there are alterations of afferent signaling and feedback information from intramuscular receptors to the cerebral cortex due to modifications in the reflex organization of hindlimb muscle groups. We propose that the changes observed in GABA immunoreactivity of cells and terminals is an expression of changes in their modulatory activity to compensate for the alterations in the afferent information.

Molecular and ultrastructural analysis of forisome subunits reveals the principles of forisome assembly

PubMed Central

Müller, Boje; Groscurth, Sira; Menzel, Matthias; Rüping, Boris A.; Twyman, Richard M.; Prüfer, Dirk; Noll, Gundula A.

2014-01-01

Background and Aims Forisomes are specialized structural phloem proteins that mediate sieve element occlusion after wounding exclusively in papilionoid legumes, but most studies of forisome structure and function have focused on the Old World clade rather than the early lineages. A comprehensive phylogenetic, molecular, structural and functional analysis of forisomes from species covering a broad spectrum of the papilionoid legumes was therefore carried out, including the first analysis of Dipteryx panamensis forisomes, representing the earliest branch of the Papilionoideae lineage. The aim was to study the molecular, structural and functional conservation among forisomes from different tribes and to establish the roles of individual forisome subunits. Methods Sequence analysis and bioinformatics were combined with structural and functional analysis of native forisomes and artificial forisome-like protein bodies, the latter produced by expressing forisome genes from different legumes in a heterologous background. The structure of these bodies was analysed using a combination of confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and the function of individual subunits was examined by combinatorial expression, micromanipulation and light microscopy. Key Results Dipteryx panamensis native forisomes and homomeric protein bodies assembled from the single sieve element occlusion by forisome (SEO-F) subunit identified in this species were structurally and functionally similar to forisomes from the Old World clade. In contrast, homomeric protein bodies assembled from individual SEO-F subunits from Old World species yielded artificial forisomes differing in proportion to their native counterparts, suggesting that multiple SEO-F proteins are required for forisome assembly in these plants. Structural differences between Medicago truncatula native forisomes, homomeric protein bodies and heteromeric bodies containing all possible subunit combinations suggested that combinations of SEO-F proteins may fine-tune the geometric proportions and reactivity of forisomes. Conclusions It is concluded that forisome structure and function have been strongly conserved during evolution and that species-dependent subsets of SEO-F proteins may have evolved to fine-tune the structure of native forisomes. PMID:24694827

Electron transparent graphene windows for environmental scanning electron microscopy in liquids and dense gases.

PubMed

Stoll, Joshua D; Kolmakov, Andrei

2012-12-21

Due to its ultrahigh electron transmissivity in a wide electron energy range, molecular impermeability, high electrical conductivity and excellent mechanical stiffness, suspended graphene membranes appear to be a nearly ideal window material for in situ (in vivo) environmental electron microscopy of nano- and mesoscopic objects (including bio-medical samples) immersed in liquids and/or in dense gaseous media. In this paper, taking advantage of a small modification of the graphene transfer protocol onto metallic and SiN supporting orifices, reusable environmental cells with exchangeable graphene windows have been designed. Using colloidal gold nanoparticles (50 nm) dispersed in water as model objects for scanning electron microscopy in liquids as proof of concept, different conditions for imaging through the graphene membrane were tested. Limiting factors for electron microscopy in liquids, such as electron beam induced water radiolysis and damage of the graphene membrane at high electron doses, are discussed.

Ultrafast Science Opportunities with Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Durr, Hermann

X-rays and electrons are two of the most fundamental probes of matter. When the Linac Coherent Light Source (LCLS), the world’s first x-ray free electron laser, began operation in 2009, it transformed ultrafast science with the ability to generate laser-like x-ray pulses from the manipulation of relativistic electron beams. This document describes a similar future transformation. In Transmission Electron Microscopy, ultrafast relativistic (MeV energy) electron pulses can achieve unsurpassed spatial and temporal resolution. Ultrafast temporal resolution will be the next frontier in electron microscopy and can ideally complement ultrafast x-ray science done with free electron lasers. This document describes themore » Grand Challenge science opportunities in chemistry, material science, physics and biology that arise from an MeV ultrafast electron diffraction & microscopy facility, especially when coupled with linac-based intense THz and X-ray pump capabilities.« less

Microstructure of milk

USDA-ARS?s Scientific Manuscript database

The fat and protein in milk may be examined by scanning electron microscopy, transmission electron microscopy, and confocal laser scanning microscopy, and any bacteria present may be viewed by light microscopy. The fat exists as globules, the bulk of the protein is in the form of casein micelles, a...

Fabrication of [001]-oriented tungsten tips for high resolution scanning tunneling microscopy

PubMed Central

Chaika, A. N.; Orlova, N. N.; Semenov, V. N.; Postnova, E. Yu.; Krasnikov, S. A.; Lazarev, M. G.; Chekmazov, S. V.; Aristov, V. Yu.; Glebovsky, V. G.; Bozhko, S. I.; Shvets, I. V.

2014-01-01

The structure of the [001]-oriented single crystalline tungsten probes sharpened in ultra-high vacuum using electron beam heating and ion sputtering has been studied using scanning and transmission electron microscopy. The electron microscopy data prove reproducible fabrication of the single-apex tips with nanoscale pyramids grained by the {011} planes at the apexes. These sharp, [001]-oriented tungsten tips have been successfully utilized in high resolution scanning tunneling microscopy imaging of HOPG(0001), SiC(001) and graphene/SiC(001) surfaces. The electron microscopy characterization performed before and after the high resolution STM experiments provides direct correlation between the tip structure and picoscale spatial resolution achieved in the experiments. PMID:24434734

Towards native-state imaging in biological context in the electron microscope

PubMed Central

Weston, Anne E.; Armer, Hannah E. J.

2009-01-01

Modern cell biology is reliant on light and fluorescence microscopy for analysis of cells, tissues and protein localisation. However, these powerful techniques are ultimately limited in resolution by the wavelength of light. Electron microscopes offer much greater resolution due to the shorter effective wavelength of electrons, allowing direct imaging of sub-cellular architecture. The harsh environment of the electron microscope chamber and the properties of the electron beam have led to complex chemical and mechanical preparation techniques, which distance biological samples from their native state and complicate data interpretation. Here we describe recent advances in sample preparation and instrumentation, which push the boundaries of high-resolution imaging. Cryopreparation, cryoelectron microscopy and environmental scanning electron microscopy strive to image samples in near native state. Advances in correlative microscopy and markers enable high-resolution localisation of proteins. Innovation in microscope design has pushed the boundaries of resolution to atomic scale, whilst automatic acquisition of high-resolution electron microscopy data through large volumes is finally able to place ultrastructure in biological context. PMID:19916039

An overview of state-of-the-art image restoration in electron microscopy.

PubMed

Roels, J; Aelterman, J; Luong, H Q; Lippens, S; Pižurica, A; Saeys, Y; Philips, W

2018-06-08

In Life Science research, electron microscopy (EM) is an essential tool for morphological analysis at the subcellular level as it allows for visualization at nanometer resolution. However, electron micrographs contain image degradations such as noise and blur caused by electromagnetic interference, electron counting errors, magnetic lens imperfections, electron diffraction, etc. These imperfections in raw image quality are inevitable and hamper subsequent image analysis and visualization. In an effort to mitigate these artefacts, many electron microscopy image restoration algorithms have been proposed in the last years. Most of these methods rely on generic assumptions on the image or degradations and are therefore outperformed by advanced methods that are based on more accurate models. Ideally, a method will accurately model the specific degradations that fit the physical acquisition settings. In this overview paper, we discuss different electron microscopy image degradation solutions and demonstrate that dedicated artefact regularisation results in higher quality restoration and is applicable through recently developed probabilistic methods. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

X-ray photoelectron spectroscopic analysis of rice kernels and flours: Measurement of surface chemical composition.

PubMed

Nawaz, Malik A; Gaiani, Claire; Fukai, Shu; Bhandari, Bhesh

2016-12-01

The objectives of this study were to evaluate the ability of X-ray photoelectron spectroscopy (XPS) to differentiate rice macromolecules and to calculate the surface composition of rice kernels and flours. The uncooked kernels and flours surface composition of the two selected rice varieties, Thadokkham-11 (TDK11) and Doongara (DG) demonstrated an over-expression of lipids and proteins and an under-expression of starch compared to the bulk composition. The results of the study showed that XPS was able to differentiate rice polysaccharides (mainly starch), proteins and lipids in uncooked rice kernels and flours. Nevertheless, it was unable to distinguish components in cooked rice samples possibly due to complex interactions between gelatinized starch, denatured proteins and lipids. High resolution imaging methods (Scanning Electron Microscopy and Confocal Laser Scanning Microscopy) were employed to obtain complementary information about the properties and location of starch, proteins and lipids in rice kernels and flours. Copyright © 2016. Published by Elsevier Ltd.

In vitro activity of chlorogenic acid against Aspergillus fumigatus biofilm and gliotoxin production.

PubMed

Kong, Jin-Liang; Luo, Jing; Li, Bing; Dong, Bi-Ying; Huang, Hong; Wang, Ke; Wu, Li-Hong; Chen, Yi-Qiang

2017-06-01

Aspergillus ( A .) fumigatus , one of the most common causes of life-threatening fungal infections in immunocompromised patients, shows resistance to antifungal agents as has a high propensity to forming a biofilm. The present study aimed to investigate the effects of chlorogenic acid (CRA) on A. fumigatus biofilm formation and integrity. Confocal laser scanning microscopy was performed to determine the inhibitory effects of CRA against A. fumigatus biofilm formation. Transmission electron microscopy was performed to investigate the ultrastructural changes of A. fumigatus biofilm after CRA exposure. High-performance liquid chromatography and reverse-transcription quantitative PCR were performed to determine the expression of gliotoxin production in biofilm culture. The results showed that CRA at sub-minimum inhibitory concentrations inhibited A. fumigatus biofilm formation. In addition, CRA could decreased the gliotoxin production in the biofilm culture supernatant through inhibiting the expression of master genes involved in gliotoxin biosynthesis. The present study provided useful information for the development of novel strategies to reduce the incidence of A. fumigatus biofilm-associated diseases.

PREFERENTIAL SECRETION OF INDUCIBLE HSP70 BY VITILIGO MELANOCYTES UNDER STRESS

PubMed Central

Mosenson, Jeffrey A.; Flood, Kelsey; Klarquist, Jared; Eby, Jonathan M.; Koshoffer, Amy; Boissy, Raymond E.; Overbeck, Andreas; C.Tung, Rebecca; Poole, I. Caroline Le

2014-01-01

SUMMARY Inducible HSP70 (HSP70i) chaperones peptides from stressed cells, protecting them from apoptosis. Upon extracellular release, HSP70i serves an adjuvant function, enhancing immune responses to bound peptides. We questioned whether HSP70i differentially protects control and vitiligo melanocytes from stress and subsequent immune responses. We compared expression of HSP70i in skin samples, evaluated the viability of primary vitiligo and control melanocytes exposed to bleaching phenols, and measured secreted HSP70i. We determined whether HSP70i traffics to melanosomes to contact immunogenic proteins by cell fractionation, western blotting, electron microscopy and confocal microscopy. Viability of vitiligo and control melanocytes was equally affected under stress. However, vitiligo melanocytes secreted increased amounts of HSP70i in response to MBEH, corroborating with aberrant HSP70i expression in patient skin. Intracellular HSP70i colocalized with melanosomes, and more so in response to MBEH in vitiligo melanocytes. Thus whereas either agent is cytotoxic to melanocytes, MBEH preferentially induces immune responses to melanocytes. PMID:24354861

Multi-modal Registration for Correlative Microscopy using Image Analogies

PubMed Central

Cao, Tian; Zach, Christopher; Modla, Shannon; Powell, Debbie; Czymmek, Kirk; Niethammer, Marc

2014-01-01

Correlative microscopy is a methodology combining the functionality of light microscopy with the high resolution of electron microscopy and other microscopy technologies for the same biological specimen. In this paper, we propose an image registration method for correlative microscopy, which is challenging due to the distinct appearance of biological structures when imaged with different modalities. Our method is based on image analogies and allows to transform images of a given modality into the appearance-space of another modality. Hence, the registration between two different types of microscopy images can be transformed to a mono-modality image registration. We use a sparse representation model to obtain image analogies. The method makes use of corresponding image training patches of two different imaging modalities to learn a dictionary capturing appearance relations. We test our approach on backscattered electron (BSE) scanning electron microscopy (SEM)/confocal and transmission electron microscopy (TEM)/confocal images. We perform rigid, affine, and deformable registration via B-splines and show improvements over direct registration using both mutual information and sum of squared differences similarity measures to account for differences in image appearance. PMID:24387943

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens.

PubMed

Phelps, Jamie P; Dao, Philip; Jin, Hongfan; Rasochova, Lada

2007-02-01

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.

[application of the analytical transmission electron microscopy techniques for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in mammalian cells].

PubMed

Shebanova, A S; Bogdanov, A G; Ismagulova, T T; Feofanov, A V; Semenyuk, P I; Muronets, V I; Erokhina, M V; Onishchenko, G E; Kirpichnikov, M P; Shaitan, K V

2014-01-01

This work represents the results of the study on applicability of the modern methods of analytical transmission electron microscopy for detection, identification and visualization of localization of nanoparticles of titanium and cerium oxides in A549 cell, human lung adenocarcinoma cell line. A comparative analysis of images of the nanoparticles in the cells obtained in the bright field mode of transmission electron microscopy, under dark-field scanning transmission electron microscopy and high-angle annular dark field scanning transmission electron was performed. For identification of nanoparticles in the cells the analytical techniques, energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy, were compared when used in the mode of obtaining energy spectrum from different particles and element mapping. It was shown that the method for electron tomography is applicable to confirm that nanoparticles are localized in the sample but not coated by contamination. The possibilities and fields of utilizing different techniques for analytical transmission electron microscopy for detection, visualization and identification of nanoparticles in the biological samples are discussed.

Targeted therapy of glioblastoma stem-like cells and tumor non-stem cells using cetuximab-conjugated iron-oxide nanoparticles

PubMed Central

Kaluzova, Milota; Bouras, Alexandros; Machaidze, Revaz; Hadjipanayis, Costas G.

2015-01-01

Malignant gliomas remain aggressive and lethal primary brain tumors in adults. The epidermal growth factor receptor (EGFR) is frequently overexpressed in the most common malignant glioma, glioblastoma (GBM), and represents an important therapeutic target. GBM stem-like cells (GSCs) present in tumors are felt to be highly tumorigenic and responsible for tumor recurrence. Multifunctional magnetic iron-oxide nanoparticles (IONPs) can be directly imaged by magnetic resonance imaging (MRI) and designed to therapeutically target cancer cells. The targeting effects of IONPs conjugated to the EGFR inhibitor, cetuximab (cetuximab-IONPs), were determined with EGFR- and EGFRvIII-expressing human GBM neurospheres and GSCs. Transmission electron microscopy revealed cetuximab-IONP GBM cell binding and internalization. Fluorescence microscopy and Prussian blue staining showed increased uptake of cetuximab-IONPs by EGFR- as well as EGFRvIII-expressing GSCs and neurospheres in comparison to cetuximab or free IONPs. Treatment with cetuximab-IONPs resulted in a significant antitumor effect that was greater than with cetuximab alone due to more efficient, CD133-independent cellular targeting and uptake, EGFR signaling alterations, EGFR internalization, and apoptosis induction in EGFR-expressing GSCs and neurospheres. A significant increase in survival was found after cetuximab-IONP convection-enhanced delivery treatment of 3 intracranial rodent GBM models employing human EGFR-expressing GBM xenografts. PMID:25871395

Chemistry Viewed through the Eyes of High-Resolution Microscopy.

ERIC Educational Resources Information Center

Beer, Michael; And Others

1981-01-01

This special report, prepared by several chemists working in the field of electron microscopy, provides information regarding the most recent developments in transmission and scanning electron microscopy that have chemical significance. (CS)

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

PubMed

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Correlative Scanning-Transmission Electron Microscopy Reveals that a Chimeric Flavivirus Is Released as Individual Particles in Secretory Vesicles

PubMed Central

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations. PMID:24681578

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

Development and characterization of a eukaryotic expression system for human type II procollagen.

PubMed

Wieczorek, Andrew; Rezaei, Naghmeh; Chan, Clara K; Xu, Chuan; Panwar, Preety; Brömme, Dieter; Merschrod S, Erika F; Forde, Nancy R

2015-12-15

Triple helical collagens are the most abundant structural protein in vertebrates and are widely used as biomaterials for a variety of applications including drug delivery and cellular and tissue engineering. In these applications, the mechanics of this hierarchically structured protein play a key role, as does its chemical composition. To facilitate investigation into how gene mutations of collagen lead to disease as well as the rational development of tunable mechanical and chemical properties of this full-length protein, production of recombinant expressed protein is required. Here, we present a human type II procollagen expression system that produces full-length procollagen utilizing a previously characterized human fibrosarcoma cell line for production. The system exploits a non-covalently linked fluorescence readout for gene expression to facilitate screening of cell lines. Biochemical and biophysical characterization of the secreted, purified protein are used to demonstrate the proper formation and function of the protein. Assays to demonstrate fidelity include proteolytic digestion, mass spectrometric sequence and posttranslational composition analysis, circular dichroism spectroscopy, single-molecule stretching with optical tweezers, atomic-force microscopy imaging of fibril assembly, and transmission electron microscopy imaging of self-assembled fibrils. Using a mammalian expression system, we produced full-length recombinant human type II procollagen. The integrity of the collagen preparation was verified by various structural and degradation assays. This system provides a platform from which to explore new directions in collagen manipulation.

Writing silica structures in liquid with scanning transmission electron microscopy.

PubMed

van de Put, Marcel W P; Carcouët, Camille C M C; Bomans, Paul H H; Friedrich, Heiner; de Jonge, Niels; Sommerdijk, Nico A J M

2015-02-04

Silica nanoparticles are imaged in solution with scanning transmission electron microscopy (STEM) using a liquid cell with silicon nitride (SiN) membrane windows. The STEM images reveal that silica structures are deposited in well-defined patches on the upper SiN membranes upon electron beam irradiation. The thickness of the deposits is linear with the applied electron dose. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate that the deposited patches are a result of the merging of the original 20 nm-diameter nanoparticles, and that the related surface roughness depends on the electron dose rate used. Using this approach, sub-micrometer scale structures are written on the SiN in liquid by controlling the electron exposure as function of the lateral position. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Canine Clitoral Carcinoma: A Clinical, Cytologic, Histopathologic, Immunohistochemical, and Ultrastructural Study.

PubMed

Verin, Ranieri; Cian, Francesco; Stewart, Jennifer; Binanti, Diana; MacNeill, Amy L; Piviani, Martina; Monti, Paola; Baroni, Gianna; Le Calvez, Sophie; Scase, Timothy J; Finotello, Riccardo

2018-01-01

Vaginal and vulvar tumors are uncommon in dogs. Knowledge of canine primary clitoral neoplasia is restricted to a few case reports, and only carcinomas have been reported. Cytologic and histologic features reported in the literature seem to overlap with those of canine apocrine gland anal sac adenocarcinoma (AGASA). Clinical features also recall those of canine AGASA, such as locoregional metastases and hypercalcemia of malignancy (HM). In this study, 6 cases of primary canine clitoral carcinomas (CCCs), with and without HM, were investigated by means of cytology, histopathology, electron microscopy, and immunohistochemistry for neuroendocrine markers including chromogranin A (CGA), synaptophysin (SYN), neuron-specific enolase (NSE), and S-100. In all 6 tumors, cytologic findings were consistent with malignant epithelial neoplasia of apocrine gland origin. The tumors examined were classified into 3 different histological patterns representing different degrees of differentiation: tubular, solid, and rosette type. Both CGA and SYN were mildly expressed in 2 of 6 tumors, while NSE was consistently expressed in all 6 cases. None of the tumors were S-100 positive. Transmission electron microscopy revealed electron-dense cytoplasmic granules compatible with neuroendocrine granules in all 6 cases. CCCs presented clinicopathologic features resembling AGASAs with neuroendocrine characteristics, and 2 of 6 neoplasms were considered as carcinomas with neuroendocrine differentiation and were positive for 3 neuroendocrine markers. CCCs can often present with HM, and long-term outcome is likely poor. Our study concludes that CCC seems to be a rare tumor, but it might be underestimated because of the overlapping features with AGASA. Further studies should aim to define the true incidence of this disease.

X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.

PubMed

Bushong, Eric A; Johnson, Donald D; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H

2015-02-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

PubMed Central

Bushong, Eric A.; Johnson, Donald D.; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T.; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H.

2015-01-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging. PMID:25392009

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

Increasing RpoS expression causes cell death in Borrelia burgdorferi.

PubMed

Chen, Linxu; Xu, Qilong; Tu, Jiagang; Ge, Yihe; Liu, Jun; Liang, Fang Ting

2013-01-01

RpoS, one of the two alternative σ factors in Borrelia burgdorferi, is tightly controlled by multiple regulators and, in turn, determines expression of many critical virulence factors. Here we show that increasing RpoS expression causes cell death. The immediate effect of increasing RpoS expression was to promote bacterial division and as a consequence result in a rapid increase in cell number before causing bacterial death. No DNA fragmentation or degradation was observed during this induced cell death. Cryo-electron microscopy showed induced cells first formed blebs, which were eventually released from dying cells. Apparently blebbing initiated cell disintegration leading to cell death. These findings led us to hypothesize that increasing RpoS expression triggers intracellular programs and/or pathways that cause spirochete death. The potential biological significance of induced cell death may help B. burgdorferi regulate its population to maintain its life cycle in nature.

The Corneal Epithelial Barrier and Its Developmental Role in Isolating Corneal Epithelial and Conjunctival Cells From One Another

PubMed Central

Kubilus, James K.; Zapater i Morales, Carolina; Linsenmayer, Thomas F.

2017-01-01

Purpose During development, the corneal epithelium (CE) and the conjunctiva are derived from the surface ectoderm. Here we have examined how, during development, the cells of these two issues become isolated from each other. Methods Epithelia from the anterior eyes of chicken embryos were labeled with the fluorescent, lipophilic dye, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). DiI was placed on the epithelial surface of the developing anterior eye and its diffusion was monitored by fluorescence microscopy. Concomitant morphologic changes in the surface cells of these epithelial were examined by scanning electron microscopy. Immunofluorescence was used to analyze the expression of cytokeratin K3, ZO-1, N-cadherin and Connexin-43 and the function of gap junctions was analyzed using a cut-loading with the fluorescent dye rhodamine-dextran. Results Prior to embryonic day 8 (E8), DiI placed on the surface of the CE spreads throughout all the epithelial cells of the anterior eye. When older eyes were similarly labeled, dye diffusion was restricted to the CE. Similarly, diffusion of DiI placed on the conjunctival surface after E8 was restricted to the conjunctiva. Scanning electron microscopy showed that developmentally (1) physical separations progressively form between the cells of the CE and those of the conjunctiva, and (2) by E8 these separations form a ring that completely encompasses the cornea. The functional restriction of gap junctions between these tissues did not occur until E14. Conclusions During ocular development, a barrier to the diffusion of DiI forms between the contiguous CE and conjunctiva prior to the differential expression of gap junctions within these tissues. PMID:28319640

A Versatile High-Vacuum Cryo-transfer System for Cryo-microscopy and Analytics

PubMed Central

Tacke, Sebastian; Krzyzanek, Vladislav; Nüsse, Harald; Wepf, Roger Albert; Klingauf, Jürgen; Reichelt, Rudolf

2016-01-01

Cryogenic microscopy methods have gained increasing popularity, as they offer an unaltered view on the architecture of biological specimens. As a prerequisite, samples must be handled under cryogenic conditions below their recrystallization temperature, and contamination during sample transfer and handling must be prevented. We present a high-vacuum cryo-transfer system that streamlines the entire handling of frozen-hydrated samples from the vitrification process to low temperature imaging for scanning transmission electron microscopy and transmission electron microscopy. A template for cryo-electron microscopy and multimodal cryo-imaging approaches with numerous sample transfer steps is presented. PMID:26910419

Graphene Oxide–Silver Nanoparticles Nanocomposite Stimulates Differentiation in Human Neuroblastoma Cancer Cells (SH-SY5Y)

PubMed Central

Gurunathan, Sangiliyandi

2017-01-01

Recently, graphene and graphene related nanocomposite receive much attention due to high surface-to-volume ratio, and unique physiochemical and biological properties. The combination of metallic nanoparticles with graphene-based materials offers a promising method to fabricate novel graphene–silver hybrid nanomaterials with unique functions in biomedical nanotechnology, and nanomedicine. Therefore, this study was designed to prepare graphene oxide (GO) silver nanoparticles (AgNPs) nanocomposite (GO-AgNPs) containing two different nanomaterials in single platform with distinctive properties using luciferin as reducing agents. In addition, we investigated the effect of GO-AgNPs on differentiation in SH-SY5Y cells. The synthesized GO-AgNPs were characterized by ultraviolet-visible absorption spectroscopy (UV-vis), X-ray diffraction (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and Raman spectroscopy. The differentiation was confirmed by series of cellular and biochemical assays. The AgNPs were distributed uniformly on the surface of graphene oxide with an average size of 25 nm. As prepared GO-AgNPOs induces differentiation by increasing the expression of neuronal differentiation markers and decreasing the expression of stem cell markers. The results indicated that the redox biology involved the expression of various signaling molecules, which play an important role in differentiation. This study suggests that GO-AgNP nanocomposite could stimulate differentiation of SH-SY5Y cells. Furthermore, understanding the mechanisms of differentiation of neuroblastoma cells could provide new strategies for cancer and stem cell therapies. Therefore, these studies suggest that GO-AgNPs could target specific chemotherapy-resistant cells within a tumor. PMID:29182571

Modeling pulmonary fibrosis by abnormal expression of telomerase/apoptosis/collagen V in experimental usual interstitial pneumonia

PubMed Central

Parra, E.R.; Pincelli, M.S.; Teodoro, W.R.; Velosa, A.P.P.; Martins, V.; Rangel, M.P.; Barbas-Filho, J.V.; Capelozzi, V.L.

2014-01-01

Limitations on tissue proliferation capacity determined by telomerase/apoptosis balance have been implicated in pathogenesis of idiopathic pulmonary fibrosis. In addition, collagen V shows promise as an inductor of apoptosis. We evaluated the quantitative relationship between the telomerase/apoptosis index, collagen V synthesis, and epithelial/fibroblast replication in mice exposed to butylated hydroxytoluene (BHT) at high oxygen concentration. Two groups of mice were analyzed: 20 mice received BHT, and 10 control mice received corn oil. Telomerase expression, apoptosis, collagen I, III, and V fibers, and hydroxyproline were evaluated by immunohistochemistry, in situ detection of apoptosis, electron microscopy, immunofluorescence, and histomorphometry. Electron microscopy confirmed the presence of increased alveolar epithelial cells type 1 (AEC1) in apoptosis. Immunostaining showed increased nuclear expression of telomerase in AEC type 2 (AEC2) between normal and chronic scarring areas of usual interstitial pneumonia (UIP). Control lungs and normal areas from UIP lungs showed weak green birefringence of type I and III collagens in the alveolar wall and type V collagen in the basement membrane of alveolar capillaries. The increase in collagen V was greater than collagens I and III in scarring areas of UIP. A significant direct association was found between collagen V and AEC2 apoptosis. We concluded that telomerase, collagen V fiber density, and apoptosis evaluation in experimental UIP offers the potential to control reepithelization of alveolar septa and fibroblast proliferation. Strategies aimed at preventing high rates of collagen V synthesis, or local responses to high rates of cell apoptosis, may have a significant impact in pulmonary fibrosis. PMID:24919172

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro

PubMed Central

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M.; Kumar, Manish; Nixon, B. Tracy; Bulone, Vincent; Zimmer, Jochen

2016-01-01

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme’s N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils. PMID:27647898

A single heterologously expressed plant cellulose synthase isoform is sufficient for cellulose microfibril formation in vitro.

PubMed

Purushotham, Pallinti; Cho, Sung Hyun; Díaz-Moreno, Sara M; Kumar, Manish; Nixon, B Tracy; Bulone, Vincent; Zimmer, Jochen

2016-10-04

Plant cell walls are a composite material of polysaccharides, proteins, and other noncarbohydrate polymers. In the majority of plant tissues, the most abundant polysaccharide is cellulose, a linear polymer of glucose molecules. As the load-bearing component of the cell wall, individual cellulose chains are frequently bundled into micro and macrofibrils and are wrapped around the cell. Cellulose is synthesized by membrane-integrated and processive glycosyltransferases that polymerize UDP-activated glucose and secrete the nascent polymer through a channel formed by their own transmembrane regions. Plants express several different cellulose synthase isoforms during primary and secondary cell wall formation; however, so far, none has been functionally reconstituted in vitro for detailed biochemical analyses. Here we report the heterologous expression, purification, and functional reconstitution of Populus tremula x tremuloides CesA8 (PttCesA8), implicated in secondary cell wall formation. The recombinant enzyme polymerizes UDP-activated glucose to cellulose, as determined by enzyme degradation, permethylation glycosyl linkage analysis, electron microscopy, and mutagenesis studies. Catalytic activity is dependent on the presence of a lipid bilayer environment and divalent manganese cations. Further, electron microscopy analyses reveal that PttCesA8 produces cellulose fibers several micrometers long that occasionally are capped by globular particles, likely representing PttCesA8 complexes. Deletion of the enzyme's N-terminal RING-finger domain almost completely abolishes fiber formation but not cellulose biosynthetic activity. Our results demonstrate that reconstituted PttCesA8 is not only sufficient for cellulose biosynthesis in vitro but also suffices to bundle individual glucan chains into cellulose microfibrils.

The New Electron Microscopy: Cells and Molecules in Three Dimensions | Poster

Cancer.gov

NCI recently announced the launch of the new National Cryo-Electron Microscopy Facility (NCEF) at the Frederick National Laboratory for Cancer Research (FNLCR). The launch comes while cryo-electron microscopy (cryo-EM) is enjoying the spotlight as a newly emerging, rapidly evolving technology with the potential to revolutionize the field of structural biology. Read more...

Crystal structure of stacking faults in InGaAs/InAlAs/InAs heterostructures

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trunkin, I. N.; Presniakov, M. Yu.; Vasiliev, A. L., E-mail: a.vasiliev56@gmail.com

Stacking faults and dislocations in InGaAs/InAlAs/InAs heterostructures have been studied by electron microscopy. The use of different techniques of transmission electron microscopy (primarily, highresolution dark-field scanning transmission electron microscopy) has made it possible to determine the defect structure at the atomic level.

The Expression of Dectin-1, Irak1 and Rip2 During the Host Response to Aspergillus fumigatus.

PubMed

Liu, Jinguo; Yu, Lin; Chen, Cuicui; Zhou, Jian; Gong, Xin; Li, Dandan; Hou, Dongni; Song, Yuanlin; Shao, Changzhou

2018-04-01

C-type lectin receptors (CLRs), Toll-like receptors (TLRs), and Nod-like receptors (NLRs) have the ability to recognize Aspergillus fumigatus (A. fumigates) and induce innate immune response. Dectin-1 is a well-described CLR, while interleukin-1 receptor-associated kinase 1 (Irak1) and receptor-interacting protein 2 (Rip2) are pivotal adaptor proteins of TLRs and NLRs signaling pathways, respectively. Our primary aim is to elucidate whether Dectin-1 regulates the expression of Irak1 and Rip2, and confirm that CLRs, TLRs, and NLRs pathways act synergistically in response to A. fumigatus infection. Pulmonary infection mouse models were established. Myeloid cells were differentiated in cell culture and examined by inverted microscopy, flow cytometry, and scanning electron microscopy. The relative mRNA levels were determined by qRT-PCR. The protein expression levels were determined by immunohistochemistry and Western blot. The expression of Dectin-1, Irak1, Rip2, and phosphorylation level of nuclear factor (NF)-κB p65 were induced by conidia in immunocompetent mice, while their expression and phosphorylation level were inhibited in immunocompromised mice after the administration of conidia. Conidia increased the expression of Dectin-1, Irak1, and Rip2 in myeloid cells, while Dectin-1 silencing significantly reduced their expression. Our findings demonstrate that Dectin-1, Irak1, and Rip2 are involved in response to A. fumigatus infection. Dectin-1 modulates the expression of Irak1 and Rip2. Additionally, these three signaling pathways are interconnected, and CLRs pathway plays a dominant role against A. fumigatus invasion.

Near-infrared branding efficiently correlates light and electron microscopy.

PubMed

Bishop, Derron; Nikić, Ivana; Brinkoetter, Mary; Knecht, Sharmon; Potz, Stephanie; Kerschensteiner, Martin; Misgeld, Thomas

2011-06-05

The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

Pharmacological blocking of the osteoclastic biocorrosion of surgical stainless steel in vitro.

PubMed

Lionetto, S; Little, A; Moriceau, G; Heymann, D; Decurtins, M; Plecko, M; Filgueira, L; Cadosch, D

2013-04-01

In vitro studies suggest that human osteoclasts (OC) are able to corrode surgical stainless steel 316L (SS). The aim of this study was to investigate whether osteoclastic biocorrosion can be blocked pharmacologically. Human OCs were generated in vitro from peripheral blood monocytic cells (PBMCs) in the presence of OC differentiation cytokines. The osteoclastic viability, differentiation, and resorptive function (on both bone and SS) were assessed using standard colorimetric cell viability assay 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenil)-2H-tetrazolium, inner salt (MTS), fluorescence microscopy, tartrate-resistant acid phosphatase expression (flow cytometry), and scanning electron microscopy. OCs cultured on SS were exposed to nontoxic concentrations of bafilomycin A1, amiloride hydrochloride, or zoledronic acid. The extent of biocorrosion was quantified using atomic emission spectrometry (to measure the concentration of metal ions released into the supernatant) and scanning electron microscopy. PBMCs differentiated into mature and functional OC in the presence of all the drugs used. Osteoclastic resorption of SS was noted with differences in the resorption pattern for all drug treatments. Under the drug treatments, single areas of osteoclastic resorption were larger in size but less abundant when compared with positive controls. None of the drugs used were able to inhibit osteoclastic biocorrosion of SS. Copyright © 2012 Wiley Periodicals, Inc.

Synergistic effect of methionine encephalin (MENK) combined with pidotimod(PTD) on the maturation of murine dendritic cells (DCs)

PubMed Central

Meng, Yiming; Wang, Qiushi; Zhang, Zhenjie; Wang, Enhua; Plotnikoff, Nicollas P.; Shan, Fengping

2013-01-01

To gain new insight into the functional interaction between dendritic cells and methionine encephalin (MENK) combined with pidotimod (PTD), we have analyzed the effect of MENK plus PTD on the morphology, phenotype and functions of murine bone-marrow derived dendritic cells (BMDCs) in vitro. The maturation of BMDCs cultured in the presence of either MENK or PTD alone, or MENK in combination with PTD, was detected. The cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt/phenazinemethosulphate (MTS/PMS). The changes of BMDCs morphology were confirmed with light microscopy, transmission electron microscopy (TEM) and scanning electron microscopy (SEM). The BMDCs treated with MENK combined with PTD displayed a higher expression of typical maturation markers of CD40, CD80, CD83, CD86 and MHC-IIidentified by fluorescence activated cell sorting (FACS), and stronger ability to drive T cells. The decrease of the endocytic ability was assayed by DAB kit, FITC-dextran and cellular immunohistochemistry. Finally upregulation of cytokines production of IL-12 and TNF-α was determined by ELISA. These data indicate that MENK combined with PTD could exert synergistic action on BMDC maturation. PMID:23470544

Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors

PubMed Central

de Souza, Devandir Antonio; Borges, Antonio Carlos; Santana, Ana Carolina; Oliver, Constance; Jamur, Maria Célia

2015-01-01

Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7) in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization. PMID:26633538

Hepatitis C Virus Induces the Mitochondrial Translocation of Parkin and Subsequent Mitophagy

PubMed Central

Kim, Seong-Jun; Syed, Gulam H.; Siddiqui, Aleem

2013-01-01

Hepatitis C Virus (HCV) induces intracellular events that trigger mitochondrial dysfunction and promote host metabolic alterations. Here, we investigated selective autophagic degradation of mitochondria (mitophagy) in HCV-infected cells. HCV infection stimulated Parkin and PINK1 gene expression, induced perinuclear clustering of mitochondria, and promoted mitochondrial translocation of Parkin, an initial event in mitophagy. Liver tissues from chronic HCV patients also exhibited notable levels of Parkin induction. Using multiple strategies involving confocal and electron microscopy, we demonstrated that HCV-infected cells display greater number of mitophagosomes and mitophagolysosomes compared to uninfected cells. HCV-induced mitophagy was evidenced by the colocalization of LC3 puncta with Parkin-associated mitochondria and lysosomes. Ultrastructural analysis by electron microscopy and immunoelectron microscopy also displayed engulfment of damaged mitochondria in double membrane vesicles in HCV-infected cells. The HCV-induced mitophagy occurred irrespective of genotypic differences. Silencing Parkin and PINK1 hindered HCV replication suggesting the functional relevance of mitophagy in HCV propagation. HCV-mediated decline of mitochondrial complex I enzyme activity was rescued by chemical inhibition of mitophagy or by Parkin silencing. Overall our results suggest that HCV induces Parkin-dependent mitophagy, which may have significant contribution in mitochondrial liver injury associated with chronic hepatitis C. PMID:23555273

New Tools for Comparing Microscopy Images: Quantitative Analysis of Cell Types in Bacillus subtilis

PubMed Central

van Gestel, Jordi; Vlamakis, Hera

2014-01-01

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed. PMID:25448819

New tools for comparing microscopy images: quantitative analysis of cell types in Bacillus subtilis.

PubMed

van Gestel, Jordi; Vlamakis, Hera; Kolter, Roberto

2015-02-15

Fluorescence microscopy is a method commonly used to examine individual differences between bacterial cells, yet many studies still lack a quantitative analysis of fluorescence microscopy data. Here we introduce some simple tools that microbiologists can use to analyze and compare their microscopy images. We show how image data can be converted to distribution data. These data can be subjected to a cluster analysis that makes it possible to objectively compare microscopy images. The distribution data can further be analyzed using distribution fitting. We illustrate our methods by scrutinizing two independently acquired data sets, each containing microscopy images of a doubly labeled Bacillus subtilis strain. For the first data set, we examined the expression of srfA and tapA, two genes which are expressed in surfactin-producing and matrix-producing cells, respectively. For the second data set, we examined the expression of eps and tapA; these genes are expressed in matrix-producing cells. We show that srfA is expressed by all cells in the population, a finding which contrasts with a previously reported bimodal distribution of srfA expression. In addition, we show that eps and tapA do not always have the same expression profiles, despite being expressed in the same cell type: both operons are expressed in cell chains, while single cells mainly express eps. These findings exemplify that the quantification and comparison of microscopy data can yield insights that otherwise would go unnoticed. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy

PubMed Central

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM). PMID:29568263

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy.

PubMed

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

PubMed

Pluk, H; Stokes, D J; Lich, B; Wieringa, B; Fransen, J

2009-03-01

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Ectopic Expression of BnaC.CP20.1 Results in Premature Tapetal Programmed Cell Death in Arabidopsis.

PubMed

Song, Liping; Zhou, Zhengfu; Tang, Shan; Zhang, Zhiqiang; Xia, Shengqian; Qin, Maomao; Li, Bao; Wen, Jing; Yi, Bin; Shen, Jinxiong; Ma, Chaozhi; Fu, Tingdong; Tu, Jinxing

2016-09-01

Tapetal programmed cell death (PCD) is essential in pollen grain development, and cysteine proteases are ubiquitous enzymes participating in plant PCD. Although the major papain-like cysteine proteases (PLCPs) have been investigated, the exact functions of many PLCPs are still poorly understood in PCD. Here, we identified a PLCP gene, BnaC.CP20.1, which was closely related to XP_013596648.1 from Brassica oleracea. Quantitative real-time PCR analysis revealed that BnaC.CP20.1 expression was down-regulated in male-sterile lines in oilseed rape, suggesting a connection between this gene and male sterility. BnaC.CP20.1 is especially active in the tapetum and microspores in Brassica napus from the uninucleate stage until formation of mature pollen grains during anther development. On expression of BnaC.CP20.1 prior to the tetrad stage, BnA9::BnaC.CP20.1 transgenic lines in Arabidopsis thaliana showed a male-sterile phenotype with shortened siliques containing fewer or no seeds by self-crossing. Scanning electron microscopy indicated that the reticulate exine was defective in aborted microspores. Callose degradation was delayed and microspores were not released from the tetrad in a timely fashion. Additionally, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay indicated that BnaC.CP20.1 ectopic expression led to premature tapetal PCD. Transmission electron microscopy analyses further demonstrated that the pollen abortion was due to the absence of tectum connections to the bacula in the transgenic anthers. These findings suggest that timely expression of BnaC.CP20.1 is necessary for tapetal degeneration and pollen wall formation. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Two clonal cell lines of immortalized human corneal endothelial cells show either differentiated or precursor cell characteristics.

PubMed

Valtink, Monika; Gruschwitz, Rita; Funk, Richard H W; Engelmann, Katrin

2008-01-01

Access to primary human corneal endothelial cells (HCEC) is limited and donor-derived differences between cultures exacerbate the issue of data reproducibility, whereas cell lines can provide sufficient numbers of homogenous cells for multiple experiments. An immortalized HCEC population was adapted to serum-free culture medium and repeated cloning was performed. Clonally grown cells were propagated under serum-free conditions and growth curves were recorded. Cells were characterized immunocytochemically for junctional proteins, collagens, Na,K-ATPase and HCEC-specific 9.3.E-antigen. Ultrastructure was monitored by scanning and transmission electron microscopy. Two clonal cell lines, HCEC-B4G12 and HCEC-H9C1, could be isolated and expanded, which differed morphologically: B4G12 cells were polygonal, strongly adherent and formed a strict monolayer, H9C1 cells were less adherent and formed floating spheres. The generation time of B4G12 cells was 62.26 +/- 14.5 h and that of H9C1 cells 44.05 +/- 5.05 h. Scanning electron microscopy revealed that B4G12 cells had a smooth cell surface, while H9C1 cells had numerous thin filopodia. Both cell lines expressed ZO-1 and occludin adequately, and little but well detectable amounts of connexin-43. Expression of HCEC-specific 9.3.E-antigen was found commensurately in both cell lines, while expression of Na,K-ATPase alpha1 was higher in H9C1 cells than in B4G12 cells. B4G12 cells expressed collagen IV abundantly and almost no collagen III, while H9C1 cells expressed both collagens at reasonable amounts. It is concluded that the clonal cell line B4G12 represents an ideal model of differentiated HCEC, while H9C1 may reflect features of developing or transitional HCEC. Copyright 2008 S. Karger AG, Basel.

l-Homocarnosine attenuates inflammation in cerebral ischemia-reperfusion injury through inhibition of nod-like receptor protein 3 inflammasome.

PubMed

Huang, Jing; Wang, Tao; Yu, Daorui; Fang, Xingyue; Fan, Haofei; Liu, Qiang; Yi, Guohui; Yi, Xinan; Liu, Qibin

2018-06-08

We investigated the therapeutic effects of l-homocarnosine against inflammation in a rat model of cerebral ischemia-reperfusion injury. Rats were grouped into control, middle cerebral artery occlusion (MCAO), 0.5 mM l-homocarnosine + MCAO, and 1 mM l-homocarnosine + MCAO treatment groups. Superoxide dismutase (SOD), glutathione peroxidase (Gpx), catalase, lipid peroxidation, and reduced glutathione (GSH) levels were measured. Neurological scores were assessed, and histopathology, scanning electron microscopy (SEM), and fluorescence microscopy analyses were conducted. The mRNA expression levels of nod-like receptor protein 3 (NLRP3), tumor necrosis factor alpha (TNF-α), and interleukin-6 (IL-6) and protein expression levels of NLRP3 were assessed. l-Homocarnosine supplementation substantially increased SOD, catalase, Gpx, and GSH levels, whereas it reduced the levels of lipid peroxidation relative to MCAO rats. l-Homocarnosine significantly reduced the infarct area and neurological deficit score, as well as histopathological alteration, apoptosis, and necrosis in brain tissue. The mRNA expression levels of NLRP3, TNF-α, and IL-6 were increased in MCAO rats, whereas l-homocarnosine supplementation reduced mRNA expression by >40%, and NLRP3 protein expression was reduced by >30% in 1 mM l-homocarnosine-treated MCAO rats. We propose that l-homocarnosine exerts a protective effect in cerebral ischemia-reperfusion injury-induced rats by downregulating NLRP3 expression. Copyright © 2017. Published by Elsevier B.V.

Retraction: Using the Medipix3 detector for direct electron imaging in the range 60 keV to 200 keV in electron microscopy Retraction: Using the Medipix3 detector for direct electron imaging in the range 60 keV to 200 keV in electron microscopy

NASA Astrophysics Data System (ADS)

Mir, J. A.; Plackett, R.; Shipsey, I.; dos Santos, J. M. F.

2018-01-01

The paper "Using the Medipix3 detector for direct electron imaging in the range 60keV to 200keV in electron microscopy" by J.A. Mir, R. Plackett, I. Shipsey and J.M.F. dos Santos has been retracted following the authors' request on the basis of the existence of a disagreement about the ownership of the data, to prevent conflict between collaborators.

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber.

PubMed

Nguyen, Kayla X; Holtz, Megan E; Richmond-Decker, Justin; Muller, David A

2016-08-01

A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope's objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Monte Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400 μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens and in situ chemical and electrochemical processes.

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Kayla X.; Holtz, Megan E.; Richmond-Decker, Justin

2016-07-25

Abstract A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope’s objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Montemore » Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens andin situchemical and electrochemical processes.« less

The Effect of Electron Beam Irradiation in Environmental Scanning Transmission Electron Microscopy of Whole Cells in Liquid.

PubMed

Hermannsdörfer, Justus; Tinnemann, Verena; Peckys, Diana B; de Jonge, Niels

2016-06-01

Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels.

Chapter 14: Electron Microscopy on Thin Films for Solar Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Romero, Manuel; Abou-Ras, Daniel; Nichterwitz, Melanie

2016-07-22

This chapter overviews the various techniques applied in scanning electron microscopy (SEM) and transmission electron microscopy (TEM), and highlights their possibilities and also limitations. It gives the various imaging and analysis techniques applied on a scanning electron microscope. The chapter shows that imaging is divided into that making use of secondary electrons (SEs) and of backscattered electrons (BSEs), resulting in different contrasts in the images and thus providing information on compositions, microstructures, and surface potentials. Whenever aiming for imaging and analyses at scales of down to the angstroms range, TEM and its related techniques are appropriate tools. In many cases,more » also SEM techniques provide the access to various material properties of the individual layers, not requiring specimen preparation as time consuming as TEM techniques. Finally, the chapter dedicates to cross-sectional specimen preparation for electron microscopy. The preparation decides indeed on the quality of imaging and analyses.« less

Analysis of the neuronal marker protein gene product 9.5 in internal limiting membranes after indocyanine-green assisted peeling.

PubMed

Peters, Swaantje; Tatar, Olcay; Spitzer, Martin S; Szurman, Peter; Aisenbrey, Sabine; Lüke, Matthias; Adam, Annemarie; Yoeruek, Efdal; Grisanti, Salvatore

2009-02-01

Indocyanine green-assisted internal limiting membrane (ILM) peeling was suspected to disrupt the innermost layer of the neural retina. We examined whether surgically excised specimens contain remnants of neuronal tissue. Ten patients with macular hole underwent pars plana vitrectomy and indocyanine green-assisted ILM peeling. A total of 0.1 mL of a 0.5% indocyanine green solution was applied for 15 seconds. The ILM specimens were prepared for immunohistochemistry, using a polyclonal antibody against protein gene product 9.5. Protein gene product 9.5 is a pan-neuronal marker labeling human neuronal cells. Appropriate controls to show selectivity of the antibody were performed on neuronal tissue of donor eyes. One ILM was prepared for electron microscopy. A selective expression of protein gene product 9.5 was found in neuronal fibers of the retina and optic nerve of donor eyes. Only 1 of the 10 surgical ILM specimens showed a minimal focal positivity for protein gene product 9.5. No neuronal tissue was detected on the ILM by electron microscopy. Focal expression of protein gene product 9.5 in only 1 of 10 surgical ILM specimens argues against a general indocyanine green-related disruption of the innermost retinal layers. However, higher concentrations of the dye, longer incubation times or different solvents than used in this study may lead to different results.

A Long Terminal Repeat-Containing Retrotransposon of Schizosaccharomyces pombe Expresses a Gag-Like Protein That Assembles into Virus-Like Particles Which Mediate Reverse Transcription

PubMed Central

Teysset, Laure; Dang, Van-Dinh; Kim, Min Kyung; Levin, Henry L.

2003-01-01

The Tf1 element of Schizosaccharomyces pombe is a long terminal repeat-containing retrotransposon that encodes functional protease, reverse transcriptase, and integrase proteins. Although these proteins are known to be necessary for protein processing, reverse transcription, and integration, respectively, the function of the protein thought to be Gag has not been determined. We present here the first electron microscopy of Tf1 particles. We tested whether the putative Gag of Tf1 was required for particle formation, packaging of RNA, and reverse transcription. We generated deletions of 10 amino acids in each of the four hydrophilic domains of the protein and found that all four mutations reduced transposition activity. The N-terminal deletion removed a nuclear localization signal and inhibited nuclear import of the transposon. The two mutations in the center of Gag destabilized the protein and resulted in no virus-like particles. The C-terminal deletion caused a defect in RNA packaging and, as a result, low levels of cDNA. The electron microscopy of cells expressing a truncated Tf1 showed that Gag alone was sufficient for the formation of virus-like particles. Taken together, these results indicate that Tf1 encodes a Gag protein that is a functional equivalent of the Gag proteins of retroviruses. PMID:12692246

Chlorhexidine-induced apoptosis or necrosis in L929 fibroblasts: A role for endoplasmic reticulum stress

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faria, Gisele; Cardoso, Cristina R.B.; Department of Biological Sciences, Federal University of Triangulo Mineiro, Uberaba, Minas Gerais

Chlorhexidine (CHX), widely used as antiseptic and therapeutic agent in medicine and dentistry, has a toxic effect both in vivo and in vitro. The intrinsic mechanism underlying CHX-induced cytotoxicity in eukaryotic cells is, however, still unknown. A recent study from our laboratory has suggested that CHX may induce death in cultured L929 fibroblasts via endoplasmic reticulum (ER) stress. This hypothesis was further tested by means of light and electron microscopy, quantification of apoptosis and necrosis by flow cytometry, fluorescence visualization of the cytoskeleton and endoplasmic reticulum, and evaluation of the expression of 78-kDa glucose-regulated protein 78 (Grp78), a marker ofmore » activation of the unfolded protein response (UPR) in cultured L929 fibroblasts. Our finding showing increased Grp 78 expression in CHX-treated cells and the results of flow cytometry, cytoskeleton and endoplasmic reticulum fluorescence visualization, and scanning and transmission electron microscopy allowed us to suggest that CHX elicits accumulation of proteins in the endoplasmic reticulum, which causes ER overload, resulting in ER stress and cell death either by necrosis or apoptosis. It must be pointed out, however, that this does not necessarily mean that ER stress is the only way that CHX kills L929 fibroblasts, but rather that ER stress is an important target or indicator of cell death induced by this drug.« less

Electron microscopy methods in studies of cultural heritage sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasiliev, A. L., E-mail: a.vasiliev56@gmail.com; Kovalchuk, M. V.; Yatsishina, E. B.

The history of the development and application of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray microanalysis (EDXMA) in studies of cultural heritage sites is considered. In fact, investigations based on these methods began when electron microscopes became a commercial product. Currently, these methods, being developed and improved, help solve many historical enigmas. To date, electron microscopy combined with microanalysis makes it possible to investigate any object, from parchment and wooden articles to pigments, tools, and objects of art. Studies by these methods have revealed that some articles were made by ancient masters using ancient “nanotechnologies”; hence,more » their comprehensive analysis calls for the latest achievements in the corresponding instrumental methods and sample preparation techniques.« less

Electron microscopy methods in studies of cultural heritage sites

NASA Astrophysics Data System (ADS)

Vasiliev, A. L.; Kovalchuk, M. V.; Yatsishina, E. B.

2016-11-01

The history of the development and application of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray microanalysis (EDXMA) in studies of cultural heritage sites is considered. In fact, investigations based on these methods began when electron microscopes became a commercial product. Currently, these methods, being developed and improved, help solve many historical enigmas. To date, electron microscopy combined with microanalysis makes it possible to investigate any object, from parchment and wooden articles to pigments, tools, and objects of art. Studies by these methods have revealed that some articles were made by ancient masters using ancient "nanotechnologies"; hence, their comprehensive analysis calls for the latest achievements in the corresponding instrumental methods and sample preparation techniques.

A scanning electron microscopy study of the macro-crystalline structure of 2-(2,4-dinitrobenzyl) pyridine

NASA Technical Reports Server (NTRS)

Ware, Jacqueline; Hammond, Ernest C., Jr.

1989-01-01

The compound, 2-(2,4-dinitrobenzyl) pyridine, was synthesized in the laboratory; an introductory level electron microscopy study of the macro-crystalline structure was conducted using the scanning electron microscope (SEM). The structure of these crystals was compared with the macrostructure of the crystal of 2-(2,4-dinitrobenzyl) pyridinium bromide, the hydrobromic salt of the compound which was also synthesized in the laboratory. A scanning electron microscopy crystal study was combined with a study of the principle of the electron microscope.

Stromal fibroblasts are associated with collagen IV in scar tissues of alkali-burned and lacerated corneas.

PubMed

Ishizaki, M; Shimoda, M; Wakamatsu, K; Ogro, T; Yamanaka, N; Kao, C W; Kao, W W

1997-04-01

Corneal wound healing frequently leads to the formation of opaque scar tissue. We examined whether stromal fibroblastic cells of injured corneas express collagen IV and contributes to the formation of a basal lamina-like structure. Rabbits were anesthetized, and central corneal alkali burn (8 mm in diameter; 1 M NaOH, 1 min) or laceration (8 mm long) were produced. The injured corneas, which had healed for 1, 7, 21 and 45 days, were subjected to histological and immunohistochemical studies with goat anti-collagen IV antibodies, using light and electron microscopy, and in situ hybridization with an antisense digoxigenin-labeled riboprobe of collagen alpha 1(IV) mRNA. For comparison, twenty-day-old fetal corneas were subjected to immunohistochemical study and transmission electron microscopy (TEM). TEM examinations revealed that the stromal collagenous matrix was organized in orthogonal lamellae during corneal development, whereas that of alkali-burned cornea, which had healed for 3 weeks, was disorganized. The stroma of twenty-day-old fetal cornea was not labeled by the anti-collagen IV antibodies. In contrast, one week after injury, specific collagen IV immunostaining was detected in the injured stroma. As the healing proceeded (21-45 days), the antibodies reacted with fibroblastic cells and the extracellular matrix of scar tissues located in the anterior portion of alkali-burned corneas, as well as the posterior portion of lacerated corneas. The middle portion of the stromal tissues was weakly labeled by the anti-collagen IV antibodies with the exception of the blood vessel wall. Immuno-electron microscopic study showed that collagen IV and fibronectin were closely associated with the fibroblastic cells. In situ hybridization demonstrated that epithelial and endothelial cells and fibroblastic cells in the wounded corneal stroma and retro-corneal membrane expressed alpha 1(IV) mRNA, whereas in normal corneas the expression of alpha 1(IV) mRNA was limited to epithelial and endothelial cells. The enhanced expression of collagen IV by the fibroblastic cells in the stroma of injured corneas is consistent with the notion that they may contribute to the formation of basal lamina-like structures in injured corneas.

Tissue and cellular localization of tannins in Tunisian dates (Phoenix dactylifera L.) by light and transmission electron microscopy.

PubMed

Hammouda, Hédi; Alvarado, Camille; Bouchet, Brigitte; Kalthoum-Chérif, Jamila; Trabelsi-Ayadi, Malika; Guyot, Sylvain

2014-07-16

A histological approach including light microscopy and transmission electron microscopy (TEM) was used to provide accurate information on the localization of condensed tannins in the edible tissues and in the stone of date fruits (Phoenix dactylifera L.). Light microscopy was carried out on fresh tissues after staining by 4-dimethylaminocinnamaldehyde (DMACA) for a specific detection of condensed tannins. Thus, whether under light microscopy or transmission electron microscopy (TEM), results showed that tannins are not located in the epidermis but more deeply in the mesocarp in the vacuole of very large cells. Regarding the stones, tannins are found in a specific cell layer located at 50 μm from the sclereid cells of the testa.

Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-04-01

Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

Atomic force microscopy on plasma membranes from Xenopus laevis oocytes containing human aquaporin 4.

PubMed

Orsini, Francesco; Santacroce, Massimo; Cremona, Andrea; Gosvami, Nitya N; Lascialfari, Alessandro; Hoogenboom, Bart W

2014-11-01

Atomic force microscopy (AFM) is a unique tool for imaging membrane proteins in near-native environment (embedded in a membrane and in buffer solution) at ~1 nm spatial resolution. It has been most successful on membrane proteins reconstituted in 2D crystals and on some specialized and densely packed native membranes. Here, we report on AFM imaging of purified plasma membranes from Xenopus laevis oocytes, a commonly used system for the heterologous expression of membrane proteins. Isoform M23 of human aquaporin 4 (AQP4-M23) was expressed in the X. laevis oocytes following their injection with AQP4-M23 cRNA. AQP4-M23 expression and incorporation in the plasma membrane were confirmed by the changes in oocyte volume in response to applied osmotic gradients. Oocyte plasma membranes were then purified by ultracentrifugation on a discontinuous sucrose gradient, and the presence of AQP4-M23 proteins in the purified membranes was established by Western blotting analysis. Compared with membranes without over-expressed AQP4-M23, the membranes from AQP4-M23 cRNA injected oocytes showed clusters of structures with lateral size of about 10 nm in the AFM topography images, with a tendency to a fourfold symmetry as may be expected for higher-order arrays of AQP4-M23. In addition, but only infrequently, AQP4-M23 tetramers could be resolved in 2D arrays on top of the plasma membrane, in good quantitative agreement with transmission electron microscopy analysis and the current model of AQP4. Our results show the potential and the difficulties of AFM studies on cloned membrane proteins in native eukaryotic membranes. Copyright © 2014 John Wiley & Sons, Ltd.

In Vitro Evaluation of the Biological Responses of Canine Macrophages Challenged with PLGA Nanoparticles Containing Monophosphoryl Lipid A.

PubMed

Guldner, Delphine; Hwang, Julianne K; Cardieri, Maria Clara D; Eren, Meaghan; Ziaei, Parissa; Norton, M Grant; Souza, Cleverson D

2016-01-01

Poly (lactic-co-glycolic acid) nanoparticles (PLGA NPs) have been considerably studied as a promising biodegradable delivery system to induce effective immune responses and to improve stability, safety, and cost effectiveness of vaccines. The study aimed at evaluating early inflammatory effects and cellular safety of PLGA NPs, co-encapsulating ovalbumin (PLGA/OVA NPs), as a model antigen and the adjuvant monophosphoryl lipid A (PLGA/MPLA NPs) as an adjuvant, on primary canine macrophages. The PLGA NPs constructs were prepared following the emulsion-solvent evaporation technique and further physic-chemically characterized. Peripheral blood mononuclear cells were isolated from canine whole blood by magnetic sorting and further cultured to generate macrophages. The uptake of PLGA NP constructs by macrophages was demonstrated by flow cytometry, transmission electron microscopy and confocal microscopy. Macrophage viability and morphology were evaluated by trypan blue exclusion and light microscopy. Macrophages were immunophenotyped for the expression of MHC-I and MHC-II and gene expression of Interleukin-10 (IL-10), Interleukin-12 (IL-12p40), and tumor necrosis factor alpha (TNF-α) were measured. The results showed that incubation of PLGA NP constructs with macrophages revealed effective early uptake of the PLGA NPs without altering the viability of macrophages. PLGA/OVA/MPLA NPs strongly induced TNF-α and IL-12p40 expression by macrophages as well as increase relative expression of MHC-I but not MHC-II molecules. Taken together, these results indicated that PLGA NPs with addition of MPLA represent a good model, when used as antigen carrier, for further, in vivo, work aiming to evaluate their potential to induce strong, specific, immune responses in dogs.

Advanced Nanoscale Thin Film & Bulk Materials Towards Thermoelectric Power Conversion Efficiencies of 30%

DTIC Science & Technology

2014-02-27

Electron Microscopy. Detailed Kronig -Penny (K-P)) modeling of electron transport through these superlattices suggests an estimated e-h transition energy...superalttices was confirmed by Transmission Electron Microscopy. Detailed Kronig -Penny (K-P)) modeling of electron transport through these superlattices

New modes of electron microscopy for materials science enabled by fast direct electron detectors

NASA Astrophysics Data System (ADS)

Minor, Andrew

There is an ongoing revolution in the development of electron detector technology that has enabled modes of electron microscopy imaging that had only before been theorized. The age of electron microscopy as a tool for imaging is quickly giving way to a new frontier of multidimensional datasets to be mined. These improvements in electron detection have enabled cryo-electron microscopy to resolve the three-dimensional structures of non-crystalized proteins, revolutionizing structural biology. In the physical sciences direct electron detectors has enabled four-dimensional reciprocal space maps of materials at atomic resolution, providing all the structural information about nanoscale materials in one experiment. This talk will highlight the impact of direct electron detectors for materials science, including a new method of scanning nanobeam diffraction. With faster detectors we can take a series of 2D diffraction patterns at each position in a 2D STEM raster scan resulting in a four-dimensional data set. For thin film analysis, direct electron detectors hold the potential to enable strain, polarization, composition and electrical field mapping over relatively large fields of view, all from a single experiment.

Increased AAA-TOB3 correlates with lymph node metastasis and advanced stage of lung adenocarcinoma.

PubMed

Liu, Yanfeng; Bu, Lina; Li, Wei; Wu, Wei; Wang, Shengyu; Diao, Xin; Zhou, Jing; Chen, Guoan; Yang, Shuanying

2017-07-24

This study was to investigate the differential mitochondrial protein expressions in human lung adenocarcinoma and provide preliminary data for further exploration of the carcinogenic mechanism. Total proteins of A549 and 16HBE mitochondria were extracted through 2D polyacrylamide gel electrophoresis (2-DE). The differential mitochondria proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and were further confirmed by Western blot, immunoelectron microscopy and immunohistochemistry (IHC) in A549 cells as well as lung adenocarcinoma tissues. A total of 41 differentially expressed protein spots were found in A549 mitochondria. Of them, 15 proteins were highly expressed and 26 proteins were lowly expressed in the mitochondria of A549 (by more than 1.5 times). Among the 15 more highly expressed proteins, AAA-TOB3 (by more than 3 times) was highly expressed in the mitochondria of A549 compared with the 16HBE, by LC-MS/MS identification. High electron density and clear circular colloidal gold-marked AAA-TOB3 particles were observed in the A549 cells via immunoelectron microscopy. Besides, AAA-TOB3 was confirmed to be elevated in lung adenocarcinoma by Western blot and IHC. Moreover, increased AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma (p<0.05). AAA-TOB3 was highly expressed in lung adenocarcinoma, and the up-regulation of AAA-TOB3 correlated with lymph node metastasis and advanced stage of lung adenocarcinoma, which suggested that it could serve as a potential molecular marker for lung adenocarcinoma.

Three-Dimensional Intercalated Porous Graphene on Si(111)

NASA Astrophysics Data System (ADS)

Pham, Trung T.; Sporken, Robert

2018-02-01

Three-dimensional intercalated porous graphene has been formed on Si(111) by electron beam evaporation under appropriate conditions and its structural and electronic properties investigated in detail by reflection high-energy electron diffraction, x-ray photoemission spectroscopy, Raman spectroscopy, high-resolution scanning electron microscopy, atomic force microscopy, and scanning tunneling microscopy. The results show that the crystalline quality of the porous graphene depended not only on the substrate temperature but also on the SiC layer thickness during carbon atom deposition.

Scanning Transmission Electron Microscopy | Materials Science | NREL

Science.gov Websites

mode by collecting the EDS and EELS signals point-by-point as one scans the electron probe across the . Examples of Scanning Transmission Electron Microscopy Capabilities Z-contrast image microphoto taken by

Diagnostic electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dickersin, G.R.

1988-01-01

In this book the author presents a comprehensive reference text on diagnostic electron microscopy. Throughout the book he illustrates how ultrastructural identification can be helpful for the recognition of cell type and the identification of mechanisms of pathogenesis in various diseases. In addition to electron microscopy photographs, there are also numerous light microscopy photographs for comparison. This text presents the classification of neoplasms in the order and arrangement most familiar to the pathologist. Contents: Introduction; Diagram of a Normal Cell; Normal Cell Function; Embryology; Neoplasms; Infectious Agents; Metabolic Diseases; Renal Diseases; Skeletal Muscle and Peripheral Nerve Diseases; Index.

HALE STAIN FOR SIALIC ACID-CONTAINING MUCINS. ADAPTATION TO ELECTRON MICROSCOPY.

PubMed

GASIC, G; BERWICK, L

1963-10-01

The feasibility of using the Hale stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Three kinds of mouse ascites tumor cells were fixed in neutral buffered formalin, exposed to fresh colloidal ferric oxide, treated with potassium ferrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acetate and potassium permanganate was done after sectioning in order to increase contrast. Those cells known to be coated with sialomucin showed deposits of electron-opaque ferric ferrocyanide crystals in the areas where sialomucin concentrations were expected. When these cells were treated with neuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with electron microscopy to identify sialomucin.

Perspectives on in situ electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Haimei; Zhu, Yimei

In situ transmission electron microscopy (TEM) with the ability to reveal materials dynamic processes with high spatial and temporal resolution has attracted significant interest. The recent advances in in situ methods, including liquid and gas sample environment, pump-probe ultrafast microscopy, nanomechanics and ferroelectric domain switching the aberration corrected electron optics as well as fast electron detector has opened new opportunities to extend the impact of in situ TEM in broad areas of research ranging from materials science to chemistry, physics and biology. Here in this paper, we highlight the development of liquid environment electron microscopy and its applications in themore » study of colloidal nanoparticle growth, electrochemical processes and others; in situ study of topological vortices in ferroelectric and ferromagnetic materials. At the end, perspectives of future in situ TEM are provided.« less

Perspectives on in situ electron microscopy

DOE PAGES

Zheng, Haimei; Zhu, Yimei

2017-03-29

In situ transmission electron microscopy (TEM) with the ability to reveal materials dynamic processes with high spatial and temporal resolution has attracted significant interest. The recent advances in in situ methods, including liquid and gas sample environment, pump-probe ultrafast microscopy, nanomechanics and ferroelectric domain switching the aberration corrected electron optics as well as fast electron detector has opened new opportunities to extend the impact of in situ TEM in broad areas of research ranging from materials science to chemistry, physics and biology. Here in this paper, we highlight the development of liquid environment electron microscopy and its applications in themore » study of colloidal nanoparticle growth, electrochemical processes and others; in situ study of topological vortices in ferroelectric and ferromagnetic materials. At the end, perspectives of future in situ TEM are provided.« less

Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

PubMed

Wille, Guillaume; Hellal, Jennifer; Ollivier, Patrick; Richard, Annie; Burel, Agnes; Jolly, Louis; Crampon, Marc; Michel, Caroline

2017-12-01

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

Expression of the core antigen gene of hepatitis B virus (HBV) in Acetobacter methanolicus using broad-host-range vectors.

PubMed

Schröder, R; Maassen, A; Lippoldt, A; Börner, T; von Baehr, R; Dobrowolski, P

1991-08-01

Using the broad-host-range promoter probe vector pRS201 for cloning of phage Acm1 promoters, we established a convenient vector system for expression of heterologous genes in different Gram-negative bacteria. The usefulness of this system was demonstrated by expression of the HBV core gene in Acetobacter methanolicus. Plasmids carrying the HBV core gene downstream of different Acm1-phage promoters were transferred to A. methanolicus, a new potential host for recombinant DNA expression. Using enzyme immunoassay and immunoblot techniques, the amount and composition of core antigen produced in A. methanolicus were compared with that derived from Escherichia coli. The expression of immunoreactive core antigen in A. methanolicus exceeds by sevenfold that in E. coli using an expression system with tandemly arranged promoters. Morphological observations by electron microscopy show that the HBV core gene products isolated from both hosts are assembled into regular spherical particles with a diameter of about 28 nm that are comparable to original viral nucleocapsids.

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells.

PubMed

Koh, Ai Leen; Shachaf, Catherine M; Elchuri, Sailaja; Nolan, Garry P; Sinclair, Robert

2008-12-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

Three-dimensional electron microscopy simulation with the CASINO Monte Carlo software.

PubMed

Demers, Hendrix; Poirier-Demers, Nicolas; Couture, Alexandre Réal; Joly, Dany; Guilmain, Marc; de Jonge, Niels; Drouin, Dominique

2011-01-01

Monte Carlo softwares are widely used to understand the capabilities of electron microscopes. To study more realistic applications with complex samples, 3D Monte Carlo softwares are needed. In this article, the development of the 3D version of CASINO is presented. The software feature a graphical user interface, an efficient (in relation to simulation time and memory use) 3D simulation model, accurate physic models for electron microscopy applications, and it is available freely to the scientific community at this website: www.gel.usherbrooke.ca/casino/index.html. It can be used to model backscattered, secondary, and transmitted electron signals as well as absorbed energy. The software features like scan points and shot noise allow the simulation and study of realistic experimental conditions. This software has an improved energy range for scanning electron microscopy and scanning transmission electron microscopy applications. Copyright © 2011 Wiley Periodicals, Inc.

Three-Dimensional Electron Microscopy Simulation with the CASINO Monte Carlo Software

PubMed Central

Demers, Hendrix; Poirier-Demers, Nicolas; Couture, Alexandre Réal; Joly, Dany; Guilmain, Marc; de Jonge, Niels; Drouin, Dominique

2011-01-01

Monte Carlo softwares are widely used to understand the capabilities of electron microscopes. To study more realistic applications with complex samples, 3D Monte Carlo softwares are needed. In this paper, the development of the 3D version of CASINO is presented. The software feature a graphical user interface, an efficient (in relation to simulation time and memory use) 3D simulation model, accurate physic models for electron microscopy applications, and it is available freely to the scientific community at this website: www.gel.usherbrooke.ca/casino/index.html. It can be used to model backscattered, secondary, and transmitted electron signals as well as absorbed energy. The software features like scan points and shot noise allow the simulation and study of realistic experimental conditions. This software has an improved energy range for scanning electron microscopy and scanning transmission electron microscopy applications. PMID:21769885

Chemical mapping and quantification at the atomic scale by scanning transmission electron microscopy.

PubMed

Chu, Ming-Wen; Chen, Cheng Hsuan

2013-06-25

With innovative modern material-growth methods, a broad spectrum of fascinating materials with reduced dimensions-ranging from single-atom catalysts, nanoplasmonic and nanophotonic materials to two-dimensional heterostructural interfaces-is continually emerging and extending the new frontiers of materials research. A persistent central challenge in this grand scientific context has been the detailed characterization of the individual objects in these materials with the highest spatial resolution, a problem prompting the need for experimental techniques that integrate both microscopic and spectroscopic capabilities. To date, several representative microscopy-spectroscopy combinations have become available, such as scanning tunneling microscopy, tip-enhanced scanning optical microscopy, atom probe tomography, scanning transmission X-ray microscopy, and scanning transmission electron microscopy (STEM). Among these tools, STEM boasts unique chemical and electronic sensitivity at unparalleled resolution. In this Perspective, we elucidate the advances in STEM and chemical mapping applications at the atomic scale by energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy with a focus on the ultimate challenge of chemical quantification with atomic accuracy.

Neurite density from magnetic resonance diffusion measurements at ultrahigh field: Comparison with light microscopy and electron microscopy

PubMed Central

Jespersen, Sune N.; Bjarkam, Carsten R.; Nyengaard, Jens R.; Chakravarty, M. Mallar; Hansen, Brian; Vosegaard, Thomas; Østergaard, Leif; Yablonskiy, Dmitriy; Nielsen, Niels Chr.; Vestergaard-Poulsen, Peter

2010-01-01

Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. PMID:19732836

Application of high-angle annular dark field scanning transmission electron microscopy, scanning transmission electron microscopy-energy dispersive X-ray spectrometry, and energy-filtered transmission electron microscopy to the characterization of nanoparticles in the environment.

PubMed

Utsunomiya, Satoshi; Ewing, Rodney C

2003-02-15

A major challenge to the development of a fundamental understanding of transport and retardation mechanisms of trace metal contaminants (<10 ppm) is their identification and characterization at the nanoscale. Atomic-scale techniques, such as conventional transmission electron microscopy, although powerful, are limited by the extremely small amounts of material that are examined. However, recent advances in electron microscopy provide a number of new analytical techniques that expand its application in environmental studies, particularly those concerning heavy metals on airborne particulates or water-borne colloids. High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), STEM-energy-dispersive X-ray spectrometry (EDX), and energy-filtered TEM (EFTEM) can be effectively used to identify and characterize nanoparticles. The image contrast in HAADF-STEM is strongly correlated to the atomic mass: heavier elements contribute to brighter contrast. Gold nanocrystals in pyrite and uranium nanocrystals in atmospheric aerosols have been identified by HAADF-STEM and STEM-EDX mapping and subsequently characterized by high-resolution TEM (HRTEM). EFTEM was used to identify U and Fe nanocrystals embedded in an aluminosilicate. A rare, As-bearing nanophase, westerveldite (FeAs), was identified by STEM-EDX and HRTEM. The combined use of these techniques greatly expands the effective application of electron microscopy in environmental studies, especially when applied to metals of very low concentrations. This paper describes examples of how these electron microbeam techniques can be used in combination to characterize a low concentration of heavy metals (a few ppm) on nanoscale particles.

Consecutive light microscopy, scanning-transmission electron microscopy and transmission electron microscopy of traumatic human brain oedema and ischaemic brain damage.

PubMed

Castejon, O J; Castejon, H V; Diaz, M; Castellano, A

2001-10-01

Cortical biopsies of 11 patients with traumatic brain oedema were consecutively studied by light microscopy (LM) using thick plastic sections, scanning-transmission electron microscopy ((S)TEM) using semithin plastic sections and transmission electron microscopy (TEM) using ultrathin sections. Samples were glutaraldehyde-osmium fixed and embedded in Araldite or Epon. Thick sections were stained with toluidine-blue for light microscopy. Semithin sections were examined unstained and uncoated for (S)TEM. Ultrathin sections were stained with uranyl and lead. Perivascular haemorrhages and perivascular extravasation of proteinaceous oedema fluid were observed in both moderate and severe oedema. Ischaemic pyramidal and non-pyramidal nerve cells appeared shrunken, electron dense and with enlargement of intracytoplasmic membrane compartment. Notably swollen astrocytes were observed in all samples examined. Glycogen-rich and glycogen-depleted astrocytes were identified in anoxic-ischaemic regions. Dark and hydropic satellite, interfascicular and perivascular oligodendrocytes were also found. The status spongiosus of severely oedematous brain parenchyma observed by LM and (S)TEM was correlated with the enlarged extracellular space and disrupted neuropil observed by TEM. The (S)TEM is recommended as a suitable technique for studying pathological processes in the central nervous system and as an informative adjunct to LM and TEM.

Contributed review: Review of integrated correlative light and electron microscopy.

PubMed

Timmermans, F J; Otto, C

2015-01-01

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

Protective effect of L-arginine against necrosis and apoptosis induced by experimental ischemic and reperfusion in rat liver.

PubMed

Chattopadhyay, Pronobesh; Shukla, Gunjan; Wahi, Arun Kumar

2009-01-01

To study the effect of L-arginine on apoptosis and necrosis induced by 1-h ischemia followed by 3-h reperfusion. Adult Wistar rats underwent 60 min of partial liver ischemia followed by 3-h reperfusion. Eighteen Wistar rats were divided into sham-operated control group (I) (n = 6), ischemia and reperfusion (I/R) group (0.9 % saline (5 mL/kg, orally) for 7 days) (II) (n = 6), and L-arginine-treated group (10 mg/kg body weight daily orally for 7 days before inducing ischemia-reperfusion maneuver) (III) (n = 6). Apoptotic and necrotic hepatocytes, nitric oxide levels in hepatocytes, Bcl-2 mRNA, and Bcl-2 protein were measured. Liver injury was assessed by plasma alanine transaminases (ALT), aspartate transaminases (AST), liver histopathology, and electron microscopy. An ischemic and reperfusion hepatocellular injury occurred as was indicated by increased serum ALT, AST, histopathology, and electron microscopy. Apoptosis and necrosis associated marker gene Bcl-2 mRNA and protein expression were decreased in I/R group. Pretreatment with L-arginine significantly decreased serum ALT and AST level and apoptotic and necrotic cells after 1 h ischemia followed by 3 h of reperfusion. Nitric oxide production in hepatocytes was increased twofold by L-arginine treatment when compared with I/R group. Histopathology and transmission electron microscopy (TEM) studies showed markedly diminished hepatocellular injury in L-arginine-pretreated rats during the hepatic I/R. Thus, it may be concluded that L-arginine afforded significant protection from necrosis and apoptosis in I/R injury by upregulated Bcl-2 gene and nitric oxide production.

A standardized extract of Butea monosperma (Lam.) flowers suppresses the IL-1β-induced expression of IL-6 and matrix-metalloproteases by activating autophagy in human osteoarthritis chondrocytes.

PubMed

Ansari, Mohammad Y; Khan, Nazir M; Haqqi, Tariq M

2017-12-01

Osteoarthritis (OA) is a leading cause of joint dysfunction, disability and poor quality of life in the affected population. The underlying mechanism of joint dysfunction involves increased oxidative stress, inflammation, high levels of cartilage extracellular matrix degrading proteases and decline in autophagy-a mechanism of cellular defense. There is no disease modifying therapies currently available for OA. Different parts of the Butea monosperma (Lam.) plant have widely been used in the traditional Indian Ayurvedic medicine system for the treatment of various human diseases including inflammatory conditions. Here we studied the chondroprotective effect of hydromethanolic extract of Butea monosperma (Lam.) flowers (BME) standardized to the concentration of Butein on human OA chondrocytes stimulated with IL-1β. The hydromethanolic extract of Butea monosperma (Lam.) (BME) was prepared with 70% methanol-water mixer using Soxhlet. Chondrocytes viability after BME treatment was measured by MTT assay. Gene expression levels were determined by quantitative polymerase chain reaction (qPCR) using TaqMan assays and immunoblotting with specific antibodies. Autophagy activation was determined by measuring the levels of microtubule associated protein 1 light chain 3-II (LC3-II) by immunoblotting and visualization of autophagosomes by transmission electron and confocal microscopy. BME was non-toxic to the OA chondrocytes at the doses employed and suppressed the IL-1β induced expression of inerleukin-6 (IL-6) and matrix metalloprotease-3 (MMP-3), MMP-9 and MMP-13. BME enhanced autophagy in chondrocytes as determined by measuring the levels of LC3-II by immunoblotting and increased number of autophagosomes in BME treated chondrocytes by transmission electron microscopy and confocal microscopy. BME upregulated the expression of several autophagy related genes and increased the autophagy flux in human OA chondrocytes under pathological conditions. Further analysis revealed that BME activated autophagy in chondrocytes via inhibition of mammalian target of rapamycin (mTOR) pathway. Of importance is our finding that BME-mediated suppression of IL-1β induced expression of IL-6, MMP-3, -9, and -13 was autophagy dependent and was abrogated by inhibition of autophagy. The above results show that the Butea monosperma (Lam.) extract has strong potential to activate autophagy and suppress IL-1β induced expression of IL-6 and MMP-3, -9 and -13 in human OA chondrocytes. This study shows that BME or compounds derived from BME can be developed as safe and effective chondroprotective agent(s) that function by activating autophagy to suppress the expression of inflammatory and catabolic factors associated with OA pathogenesis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified aromatic-turmerone (AR).

PubMed

Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-10-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone (ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs.

Maturation and upregulation of functions of murine dendritic cells (DCs) under the influence of purified Aromatic-Turmerone (AR)

PubMed Central

Yonggang, Tan; Yiming, Meng; Heying, Zhang; Cheng, Sun; Qiushi, Wang; Xianghong, Yang; Wei, Zheng; Huawei, Zhou; Shan, Fengping

2012-01-01

The aim of this work is to evaluate the effects of purified aromatic-turmerone(ar-turmerione, AR) on murine dendritic cells (DCs). These impacts of AR on DCs from bone marrow derived DCs(BMDCs) were assessed with use of conventional scanning electron microscopy (SEM), fluorescence activated cell sorting (FACS), transmission electron microscopy (TEM), cytochemistry assay, FITC-dextran, bio-assay and enzyme linked immunosorbent assay (ELISA). We found that AR induced phenotypic maturation as evidenced by increased expression of CD86, CD40, CD83, CD80 and major histocompatibility complex II (MHC II). The functional tests showed the activity of acidic phosphatase (ACP) inside the DCs were downregulated after treatment with AR (which occurs when phagocytosis of DCs were decreased). Finally, we proved that AR increased the production of IL-12 and tumor necrosis factor α (TNF-α). These data suggested that AR could promote phenotypic and functional maturation of DCs and this adjuvant-like activity may have potential therapeutic value. It is therefore concluded that AR could exert positive modulation on murine DCs. PMID:23095866

Internalization of silver nanoparticles into mouse spermatozoa results in poor fertilization and compromised embryo development

PubMed Central

Yoisungnern, Ton; Choi, Yun-Jung; Woong Han, Jae; Kang, Min-Hee; Das, Joydeep; Gurunathan, Sangiliyandi; Kwon, Deug-Nam; Cho, Ssang-Goo; Park, Chankyu; Kyung Chang, Won; Chang, Byung-Soo; Parnpai, Rangsun; Kim, Jin-Hoi

2015-01-01

Silver nanoparticles (AgNPs) have many features that make them attractive as medical devices, especially in therapeutic agents and drug delivery systems. Here we have introduced AgNPs into mouse spermatozoa and then determined the cytotoxic effects of AgNPs on sperm function and subsequent embryo development. Scanning electron microscopy and transmission electron microscopy analyses showed that AgNPs could be internalized into sperm cells. Furthermore, exposure to AgNPs inhibited sperm viability and the acrosome reaction in a dose-dependent manner, whereas sperm mitochondrial copy numbers, morphological abnormalities, and mortality due to reactive oxygen species were significantly increased. Likewise, sperm abnormalities due to AgNPs internalization significantly decreased the rate of oocyte fertilization and blastocyst formation. Blastocysts obtained from AgNPs-treated spermatozoa showed lower expression of trophectoderm-associated and pluripotent marker genes. Overall, we propose that AgNPs internalization into spermatozoa may alter sperm physiology, leading to poor fertilization and embryonic development. Such AgNPs-induced reprotoxicity may be a valuable tool as models for testing the safety and applicability of medical devices using AgNPs. PMID:26054035

Modular Detection of GFP-Labeled Proteins for Rapid Screening by Electron Microscopy in Cells and Organisms.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Rae, James; Ferguson, Charles; McMahon, Kerrie-Ann; Martel, Nick; Webb, Robyn E; Webb, Richard I; Teasdale, Rohan D; Parton, Robert G

2015-11-23

Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines. Copyright © 2015 Elsevier Inc. All rights reserved.

Compartmentalization of membrane trafficking, glucose transport, glycolysis, actin, tubulin and the proteasome in the cytoplasmic droplet/Hermes body of epididymal sperm.

PubMed

Au, Catherine E; Hermo, Louis; Byrne, Elliot; Smirle, Jeffrey; Fazel, Ali; Kearney, Robert E; Smith, Charles E; Vali, Hojatollah; Fernandez-Rodriguez, Julia; Simon, Paul H G; Mandato, Craig; Nilsson, Tommy; Bergeron, John J M

2015-08-01

Discovered in 1909 by Retzius and described mainly by morphology, the cytoplasmic droplet of sperm (renamed here the Hermes body) is conserved among all mammalian species but largely undefined at the molecular level. Tandem mass spectrometry of the isolated Hermes body from rat epididymal sperm characterized 1511 proteins, 43 of which were localized to the structure in situ by light microscopy and two by quantitative electron microscopy localization. Glucose transporter 3 (GLUT-3) glycolytic enzymes, selected membrane traffic and cytoskeletal proteins were highly abundant and concentrated in the Hermes body. By electron microscope gold antibody labelling, the Golgi trafficking protein TMED7/p27 localized to unstacked flattened cisternae of the Hermes body, as did GLUT-3, the most abundant protein. Its biogenesis was deduced through the mapping of protein expression for all 43 proteins during male germ cell differentiation in the testis. It is at the terminal step 19 of spermiogenesis that the 43 characteristic proteins accumulated in the nascent Hermes body. © 2015 The Authors.

An evaluation of chondrocyte morphology and gene expression on superhydrophilic vertically-aligned multi-walled carbon nanotube films.

PubMed

Antonioli, Eliane; Lobo, Anderson O; Ferretti, Mario; Cohen, Moisés; Marciano, Fernanda R; Corat, Evaldo J; Trava-Airoldi, Vladimir J

2013-03-01

Cartilage serves as a low-friction and wear-resistant articulating surface in diarthrodial joints and is also important during early stages of bone remodeling. Recently, regenerative cartilage research has focused on combinations of cells paired with scaffolds. Superhydrophilic vertically aligned carbon nanotubes (VACNTs) are of particular interest in regenerative medicine. The aim of this study is to evaluate cell expansion of human articular chondrocytes on superhydrophilic VACNTs, as well as their morphology and gene expression. VACNT films were produced using a microwave plasma chamber on Ti substrates and submitted to an O2 plasma treatment to make them superhydrophilic. Human chondrocytes were cultivated on superhydrophilic VACNTs up to five days. Quantitative RT-PCR was performed to measure type I and type II Collagen, Sox9, and Aggrecan mRNA expression levels. The morphology was analyzed by scanning electron microscopy (SEM) and confocal microscopy. SEM images demonstrated that superhydrophilic VACNTs permit cell growth and adhesion of human chondrocytes. The chondrocytes had an elongated morphology with some prolongations. Chondrocytes cultivated on superhydrophilic VACNTs maintain the level expression of Aggrecan, Sox9, and Collagen II determined by qPCR. This study was the first to indicate that superhydrophilic VACNTs may be used as an efficient scaffold for cartilage or bone repair. Copyright © 2012 Elsevier B.V. All rights reserved.

Artemisia princeps Inhibits Biofilm Formation and Virulence-Factor Expression of Antibiotic-Resistant Bacteria

PubMed Central

Kang, Sun-Young; Kim, Kang-Ju

2015-01-01

In this study, we used ethanol extract of A. princeps and investigated its antibacterial effects against MRSA. Ethanol extract of A. princeps significantly inhibited MRSA growth and organic acid production during glucose metabolism at concentrations greater than 1 mg/mL (P < 0.05). MRSA biofilm formation was observed using scanning electron microscopy (SEM) and safranin staining. A. princeps extract was found to inhibit MRSA biofilm formation at concentrations higher than 2 mg/mL significantly (P < 0.05). Bactericidal effects of the A. princeps were observed using confocal laser microscopy, which showed that A. princeps was bactericidal in a dose-dependent manner. Using real-time PCR, expression of mecA, an antibiotic-resistance gene of MRSA, was observed, along with that of sea, agrA, and sarA. A. princeps significantly inhibited mecA, sea, agrA, and sarA, mRNA expression at the concentrations greater than 1 mg/mL (P < 0.05). The phytochemical analysis of A. princeps showed a relatively high content of organic acids and glycosides. The results of this study suggest that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract. PMID:26247012

Artemisia princeps Inhibits Biofilm Formation and Virulence-Factor Expression of Antibiotic-Resistant Bacteria.

PubMed

Choi, Na-Young; Kang, Sun-Young; Kim, Kang-Ju

2015-01-01

In this study, we used ethanol extract of A. princeps and investigated its antibacterial effects against MRSA. Ethanol extract of A. princeps significantly inhibited MRSA growth and organic acid production during glucose metabolism at concentrations greater than 1 mg/mL (P < 0.05). MRSA biofilm formation was observed using scanning electron microscopy (SEM) and safranin staining. A. princeps extract was found to inhibit MRSA biofilm formation at concentrations higher than 2 mg/mL significantly (P < 0.05). Bactericidal effects of the A. princeps were observed using confocal laser microscopy, which showed that A. princeps was bactericidal in a dose-dependent manner. Using real-time PCR, expression of mecA, an antibiotic-resistance gene of MRSA, was observed, along with that of sea, agrA, and sarA. A. princeps significantly inhibited mecA, sea, agrA, and sarA, mRNA expression at the concentrations greater than 1 mg/mL (P < 0.05). The phytochemical analysis of A. princeps showed a relatively high content of organic acids and glycosides. The results of this study suggest that the ethanol extract of A. princeps may inhibit proliferation, acid production, biofilm formation, and virulence gene expressions of MRSA, which may be related to organic acids and glycosides, the major components in the extract.

Carbon contamination in scanning transmission electron microscopy and its impact on phase-plate applications.

PubMed

Hettler, Simon; Dries, Manuel; Hermann, Peter; Obermair, Martin; Gerthsen, Dagmar; Malac, Marek

2017-05-01

We analyze electron-beam induced carbon contamination in a transmission electron microscope. The study is performed on thin films potentially suitable as phase plates for phase-contrast transmission electron microscopy. Electron energy-loss spectroscopy and phase-plate imaging is utilized to analyze the contamination. The deposited contamination layer is identified as a graphitic carbon layer which is not prone to electrostatic charging whereas a non-conductive underlying substrate charges. Several methods that inhibit contamination are evaluated and the impact of carbon contamination on phase-plate imaging is discussed. The findings are in general interesting for scanning transmission electron microscopy applications. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Effect of the sample annealing temperature and sample crystallographic orientation on the charge kinetics of MgO single crystals subjected to keV electron irradiation.

PubMed

Boughariou, A; Damamme, G; Kallel, A

2015-04-01

This paper focuses on the effect of sample annealing temperature and crystallographic orientation on the secondary electron yield of MgO during charging by a defocused electron beam irradiation. The experimental results show that there are two regimes during the charging process that are better identified by plotting the logarithm of the secondary electron emission yield, lnσ, as function of the total trapped charge in the material QT. The impact of the annealing temperature and crystallographic orientation on the evolution of lnσ is presented here. The slope of the asymptotic regime of the curve lnσ as function of QT, expressed in cm(2) per trapped charge, is probably linked to the elementary cross section of electron-hole recombination, σhole, which controls the trapping evolution in the reach of the stationary flow regime. © 2014 The Authors Journal of Microscopy © 2014 Royal Microscopical Society.

Evaluation of anterior lenticonus in alport syndrome using tracey wavefront aberrometry and transmission electron microscopy.

PubMed

Kim, Kwan Soo; Kim, Mo Sae; Kim, Joon Mo; Choi, Chul Young

2010-01-01

To evaluate the efficacy of Tracey wavefront aberrometry (Tracey Technologies, Houston, TX) and transmission electron microscopy for the detection of anterior lenticonus in Alport syndrome. Tracey wavefront aberrometry was used to treat a patient with bilateral anterior lenticonus who had a history of Alport syndrome. For transmission electron microscopic examination, anterior lens capsules were obtained during clear lens phacoemulsification and intraocular lens implantation. Spherical aberrations were the predominant higher-order aberrations in the internal optics of both eyes. The Tracey wavefront aberrometer showed that most of the irregular astigmatism originated from the lenticular portion. Transmission electron microscopy of the specimens showed anterior lens capsules with decreased thickness and multiple dehiscences. Tracey wavefront aberrometry and transmission electron microscopy are effective tools for evaluation of anterior lenticonus in Alport syndrome. Copyright 2010, SLACK Incorporated.

On the state of crystallography at the dawn of the electron microscopy revolution.

PubMed

Higgins, Matthew K; Lea, Susan M

2017-10-01

While protein crystallography has, for many years, been the most used method for structural analysis of macromolecular complexes, remarkable recent advances in high-resolution electron cryo-microscopy led to suggestions that 'the revolution will not be crystallised'. Here we highlight the current success rate, speed and ease of modern crystallographic structure determination and some recent triumphs of both 'classical' crystallography and the use of X-ray free electron lasers. We also outline fundamental differences between structure determination using X-ray crystallography and electron microscopy. We suggest that crystallography will continue to co-exist with electron microscopy as part of an integrated array of methods, allowing structural biologists to focus on fundamental biological questions rather than being constrained by the methods available. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

A Simple Transmission Electron Microscopy Method for Fast Thickness Characterization of Suspended Graphene and Graphite Flakes.

PubMed

Rubino, Stefano; Akhtar, Sultan; Leifer, Klaus

2016-02-01

We present a simple, fast method for thickness characterization of suspended graphene/graphite flakes that is based on transmission electron microscopy (TEM). We derive an analytical expression for the intensity of the transmitted electron beam I 0(t), as a function of the specimen thickness t (t<λ; where λ is the absorption constant for graphite). We show that in thin graphite crystals the transmitted intensity is a linear function of t. Furthermore, high-resolution (HR) TEM simulations are performed to obtain λ for a 001 zone axis orientation, in a two-beam case and in a low symmetry orientation. Subsequently, HR (used to determine t) and bright-field (to measure I 0(0) and I 0(t)) images were acquired to experimentally determine λ. The experimental value measured in low symmetry orientation matches the calculated value (i.e., λ=225±9 nm). The simulations also show that the linear approximation is valid up to a sample thickness of 3-4 nm regardless of the orientation and up to several ten nanometers for a low symmetry orientation. When compared with standard techniques for thickness determination of graphene/graphite, the method we propose has the advantage of being simple and fast, requiring only the acquisition of bright-field images.

Fabrication of ZnS nanoparticle chains on a protein template

PubMed Central

Hulleman, J.; Kim, S. M.; Tumkur, T.; Rochet, J.-C.; Stach, E.; Stanciu, L.

2011-01-01

In the present study, we have exploited the properties of a fibrillar protein for the template synthesis of zinc sulfide (ZnS) nanoparticle chains. The diameter of the ZnS nanoparticle chains was tuned in range of ~30 to ~165 nm by varying the process variables. The nanoparticle chains were characterized by field emission scanning electron microscopy, UV–Visible spectroscopy, transmission electron microscopy, electron energy loss spectroscopy, and high-resolution transmission electron microscopy. The effect of incubation temperature on the morphology of the nanoparticle chains was also studied. PMID:21804765

Correlation of live-cell imaging with volume scanning electron microscopy.

PubMed

Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

2017-01-01

Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic research.

PubMed

Moretti, Elena; Sutera, Gaetano; Collodel, Giulia

2016-06-01

This review is aimed at discussing the role of ultrastructural studies on human spermatozoa and evaluating transmission electron microscopy as a diagnostic tool that can complete andrology protocols. It is clear that morphological sperm defects may explain decreased fertilizing potential and acquire particular value in the field of male infertility. Electron microscopy is the best method to identify systematic or monomorphic and non-systematic or polymorphic sperm defects. The systematic defects are characterized by a particular anomaly that affects the vast majority of spermatozoa in a semen sample, whereas a heterogeneous combination of head and tail defects found in variable percentages are typically non-systematic or polymorphic sperm defects. A correct diagnosis of these specific sperm alterations is important for choosing the male infertility's therapy and for deciding to turn to assisted reproduction techniques. Transmission electron microscopy (TEM) also represents a valuable method to explore the in vitro effects of different compounds (for example drugs with potential spermicidal activity) on the morphology of human spermatozoa. Finally, TEM used in combination with immunohistochemical techniques, integrates structural and functional aspects that provide a wide horizon in the understanding of sperm physiology and pathology. transmission electron microscopy: TEM; World Health Organization: WHO; light microscopy: LM; motile sperm organelle morphology examination: MSOME; intracytoplasmic morphologically selected sperm injection: IMSI; intracytoplasmic sperm injection: ICSI; dysplasia of fibrous sheath: DFS; primary ciliary dyskinesia: PCD; outer dense fibers: ODF; assisted reproduction technologies: ART; scanning electron microscopy: SEM; polyvinylpirrolidone: PVP; tert-butylhydroperoxide: TBHP.

Primary skeletal muscle cells cultured on gelatin bead microcarriers develop structural and biochemical features characteristic of adult skeletal muscle.

PubMed

Kubis, Hans-Peter; Scheibe, Renate J; Decker, Brigitte; Hufendiek, Karsten; Hanke, Nina; Gros, Gerolf; Meissner, Joachim D

2016-04-01

A primary skeletal muscle cell culture, in which myoblasts derived from newborn rabbit hindlimb muscles grow on gelatin bead microcarriers in suspension and differentiate into myotubes, has been established previously. In the course of differentiation and beginning spontaneous contractions, these multinucleated myotubes do not detach from their support. Here, we describe the development of the primary myotubes with respect to their ultrastructural differentiation. Scanning electron microscopy reveals that myotubes not only grow around the surface of one carrier bead but also attach themselves to neighboring carriers, forming bridges between carriers. Transmission electron microscopy demonstrates highly ordered myofibrils, T-tubules, and sarcoplasmic reticulum. The functionality of the contractile apparatus is evidenced by contractile activity that occurs spontaneously or can be elicited by electrostimulation. Creatine kinase activity increases steadily until day 20 of culture. Regarding the expression of isoforms of myosin heavy chains (MHC), we could demonstrate that from day 16 on, no non-adult MHC isoform mRNAs are present. Instead, on day 28 the myotubes express predominantly adult fast MHCIId/x mRNA and protein. This MHC pattern resembles that of fast muscles of adult rabbits. In contrast, primary myotubes grown on matrigel-covered culture dishes express substantial amounts of non-adult MHC protein even on day 21. To conclude, primary myotubes grown on microcarriers in their later stages exhibit many features of adult skeletal muscle and characteristics of fast type II fibers. Thus, the culture represents an excellent model of adult fast skeletal muscle, for example, when investigating molecular mechanisms of fast-to-slow fiber-type transformation. © 2015 International Federation for Cell Biology.

A Novel Antifibrotic Mechanism of Nintedanib and Pirfenidone. Inhibition of Collagen Fibril Assembly.

PubMed

Knüppel, Larissa; Ishikawa, Yoshihiro; Aichler, Michaela; Heinzelmann, Katharina; Hatz, Rudolf; Behr, Jürgen; Walch, Axel; Bächinger, Hans Peter; Eickelberg, Oliver; Staab-Weijnitz, Claudia A

2017-07-01

Idiopathic pulmonary fibrosis (IPF) is characterized by excessive deposition of extracellular matrix, in particular, collagens. Two IPF therapeutics, nintedanib and pirfenidone, decelerate lung function decline, but their underlying mechanisms of action are poorly understood. In this study, we sought to analyze their effects on collagen synthesis and maturation at important regulatory levels. Primary human fibroblasts from patients with IPF and healthy donors were treated with nintedanib (0.01-1.0 μM) or pirfenidone (100-1,000 μM) in the absence or presence of transforming growth factor-β1. Effects on collagen, fibronectin, FKBP10, and HSP47 expression, and collagen I and III secretion, were analyzed by quantitative polymerase chain reaction and Western blot. The appearance of collagen fibrils was monitored by scanning electron microscopy, and the kinetics of collagen fibril assembly was assessed using a light-scattering approach. In IPF fibroblasts, nintedanib reduced the expression of collagen I and V, fibronectin, and FKBP10 and attenuated the secretion of collagen I and III. Pirfenidone also down-regulated collagen V but otherwise showed fewer and less pronounced effects. By and large, the effects were similar in donor fibroblasts. For both drugs, electron microscopy of IPF fibroblast cultures revealed fewer and thinner collagen fibrils compared with untreated controls. Finally, both drugs dose-dependently delayed fibril formation of purified collagen I. In summary, both drugs act on important regulatory levels in collagen synthesis and processing. Nintedanib was more effective in down-regulating profibrotic gene expression and collagen secretion. Importantly, both drugs inhibited collagen I fibril formation and caused a reduction in and an altered appearance of collagen fibril bundles, representing a completely novel mechanism of action for both drugs.

Study of damage to red blood cells exposed to different doses of γ-ray irradiation.

PubMed

Xu, Deyi; Peng, Mingxi; Zhang, Zhe; Dong, Guofei; Zhang, Yiqin; Yu, Hongwei

2012-07-01

The aims of this research were to study alterations in the ultrastructure of red blood cells, the changes in concentrations of plasma electrolytes and the killing effect of lymphocytes in samples of blood exposed to different doses of γ-ray irradiation. Blood samples were treated with different doses of γ-ray irradiation and then preserved for different periods. Specimens were prepared for standard electron microscopy and transmission electron microscopy. At the same time, changes in the concentrations of Na(+), K(+) and Cl(-) and pH values in the plasma as well as Fas and FasL expression of lymphocytes before and after irradiation were determined. The proportions of reversibly and irreversibly transformed cells, for example, echinocytes, sphero-echinocytes, and degenerated forms, increased with increasing doses of irradiation and storage period, while the number of discocyte shaped red blood cells decreased. The change in K(+) concentration was greater than that of Na+ or Cl(-) after irradiation and was dosage-dependent. Plasma pH was influenced by different doses of radiation and storage time. After exposure to (137)Cs γ-irradiation, the expression of both Fas and FasL in lymphocytes differed significantly from that in the control group: the expression was positively correlated with irradiation dose (r=0.95, 0.96), but no significant difference in the Fas/FasL ratio was observed (P>0.05). We conclude that the ultrastructure of red blood cells is not changed obviously by irradiation with some doses of γ-rays and various periods of storage. However, irradiation does have some dose-dependent and time-dependent adverse effects on the erythrocytes.

Albumin Overload and PINK1/Parkin Signaling-Related Mitophagy in Renal Tubular Epithelial Cells.

PubMed

Tan, Jin; Xie, Qi; Song, Shuling; Miao, Yuyang; Zhang, Qiang

2018-03-01

BACKGROUND Albumin, as a major urinary protein component, is a risk factor for chronic kidney disease progression. Mitochondrial dysfunction is one of the main causes of albumin-induced proximal tubule cells injury. Mitophagy is considered as a pivotal protective mechanism for the elimination of dysfunctional mitochondria. The objective of this research was to determine whether albumin overload-induced mitochondrial dysfunction can activate PINK1/Parkin-mediated mitophagy in renal tubular epithelial cells (TECs). MATERIAL AND METHODS Immunofluorescence assay and Western blot assay were used to detect the effects of albumin overload on autophagy marker protein LC3. Transmission electron microscopy and Western blot assay were used to investigate the role of albumin in mitochondrial injury. Western blot assay and co-localization of acidic lysosomes and mitochondria assay were employed to detect the activation of mitophagy induced by albumin. Finally, we explored the role of PINK1/Parkin signaling in albumin-induced mitophagy by inhibiting mitophagy by knockdown of PARK2 (Parkin) level. RESULTS Immunofluorescence and Western blot results showed that the expression level of LC3-II increased, and the maximum increase point was observed after 8 h of albumin treatment. Transmission electron microscopy results demonstrated that albumin overload-induced mitochondrial injury and quantity of autophagosomes increased. Additionally, expression of PINK1 and cytosolic cytochrome C increased and mitochondria cytochrome C decreased in the albumin group. The co-localization of acidic lysosomes and mitochondria demonstrated that the number of albumin overload-induced mitophagy-positive dots increased. The transient transfection of PARK2 siRNA result showed knockdown of the expression level of PARK2 can inhibit mitophagy induced by albumin. CONCLUSIONS In conclusion, our study suggests that mitochondrial dysfunction activates the PINK1/Parkin signaling and mitophagy in renal tubular epithelial cells under albumin overload condition.

Somato-Dendritic Localization and Signaling by Leptin Receptors in Hypothalamic POMC and AgRP Neurons

PubMed Central

Ha, Sangdeuk; Baver, Scott; Huo, Lihong; Gata, Adriana; Hairston, Joyce; Huntoon, Nicholas; Li, Wenjing; Zhang, Thompson; Benecchi, Elizabeth J.; Ericsson, Maria; Hentges, Shane T.; Bjørbæk, Christian

2013-01-01

Leptin acts via neuronal leptin receptors to control energy balance. Hypothalamic pro-opiomelanocortin (POMC) and agouti-related peptide (AgRP)/Neuropeptide Y (NPY)/GABA neurons produce anorexigenic and orexigenic neuropeptides and neurotransmitters, and express the long signaling form of the leptin receptor (LepRb). Despite progress in the understanding of LepRb signaling and function, the sub-cellular localization of LepRb in target neurons has not been determined, primarily due to lack of sensitive anti-LepRb antibodies. Here we applied light microscopy (LM), confocal-laser scanning microscopy (CLSM), and electron microscopy (EM) to investigate LepRb localization and signaling in mice expressing a HA-tagged LepRb selectively in POMC or AgRP/NPY/GABA neurons. We report that LepRb receptors exhibit a somato-dendritic expression pattern. We further show that LepRb activates STAT3 phosphorylation in neuronal fibers within several hypothalamic and hindbrain nuclei of wild-type mice and rats, and specifically in dendrites of arcuate POMC and AgRP/NPY/GABA neurons of Leprb +/+ mice and in Leprb db/db mice expressing HA-LepRb in a neuron specific manner. We did not find evidence of LepRb localization or STAT3-signaling in axon-fibers or nerve-terminals of POMC and AgRP/NPY/GABA neurons. Three-dimensional serial EM-reconstruction of dendritic segments from POMC and AgRP/NPY/GABA neurons indicates a high density of shaft synapses. In addition, we found that the leptin activates STAT3 signaling in proximity to synapses on POMC and AgRP/NPY/GABA dendritic shafts. Taken together, these data suggest that the signaling-form of the leptin receptor exhibits a somato-dendritic expression pattern in POMC and AgRP/NPY/GABA neurons. Dendritic LepRb signaling may therefore play an important role in leptin’s central effects on energy balance, possibly through modulation of synaptic activity via post-synaptic mechanisms. PMID:24204898

Diffraction and microscopy with attosecond electron pulse trains

NASA Astrophysics Data System (ADS)

Morimoto, Yuya; Baum, Peter

2018-03-01

Attosecond spectroscopy1-7 can resolve electronic processes directly in time, but a movie-like space-time recording is impeded by the too long wavelength ( 100 times larger than atomic distances) or the source-sample entanglement in re-collision techniques8-11. Here we advance attosecond metrology to picometre wavelength and sub-atomic resolution by using free-space electrons instead of higher-harmonic photons1-7 or re-colliding wavepackets8-11. A beam of 70-keV electrons at 4.5-pm de Broglie wavelength is modulated by the electric field of laser cycles into a sequence of electron pulses with sub-optical-cycle duration. Time-resolved diffraction from crystalline silicon reveals a < 10-as delay of Bragg emission and demonstrates the possibility of analytic attosecond-ångström diffraction. Real-space electron microscopy visualizes with sub-light-cycle resolution how an optical wave propagates in space and time. This unification of attosecond science with electron microscopy and diffraction enables space-time imaging of light-driven processes in the entire range of sample morphologies that electron microscopy can access.

HANFORD WASTE MINERALOGY REFERENCE REPORT

DOE Office of Scientific and Technical Information (OSTI.GOV)

DISSELKAMP RS

2010-06-29

This report lists the observed mineral phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports that used experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases observed in Hanford waste.

HANFORD WASTE MINEROLOGY REFERENCE REPORT

DOE Office of Scientific and Technical Information (OSTI.GOV)

DISSELKAMP RS

2010-06-18

This report lists the observed mineral phase phases present in the Hanford tanks. This task was accomplished by performing a review of numerous reports using experimental techniques including, but not limited to: x-ray diffraction, polarized light microscopy, scanning electron microscopy, transmission electron microscopy, energy dispersive spectroscopy, electron energy loss spectroscopy, and particle size distribution analyses. This report contains tables that can be used as a quick reference to identify the crystal phases present observed in Hanford waste.

Three dimensional electron microscopy and in silico tools for macromolecular structure determination

PubMed Central

Borkotoky, Subhomoi; Meena, Chetan Kumar; Khan, Mohammad Wahab; Murali, Ayaluru

2013-01-01

Recently, structural biology witnessed a major tool - electron microscopy - in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it. PMID:27092033

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are imaged in these same sections using a scanning electron microscope. Fifth, the fluorescence and electron micrographs are aligned using gold particles as fiducial markers. In summary, the subcellular localization of fluorescently tagged proteins can be determined at nanometer resolution in approximately one week.

Small molecule PZL318: forming fluorescent nanoparticles capable of tracing their interactions with cancer cells and activated platelets, slowing tumor growth and inhibiting thrombosis

PubMed Central

Li, Shan; Wang, Yuji; Wang, Feng; Wang, Yaonan; Zhang, Xiaoyi; Zhao, Ming; Feng, Qiqi; Wu, Jianhui; Zhao, Shurui; Wu, Wei; Peng, Shiqi

2015-01-01

Low selectivity of chemotherapy correlates with poor outcomes of cancer patients. To improve this issue, a novel agent, N-(1-[3-methoxycarbonyl-4-hydroxyphenyl]-β-carboline-3-carbonyl)-Trp-Lys-OBzl (PZL318), was reported here. The transmission electron microscopy, scanning electron microscopy, and atomic force microscopy images demonstrated that PZL318 can form nanoparticles. Fluorescent and confocal images visualized that PZL318 formed fluorescent nanoparticles capable of targeting cancer cells and tracing their interactions with cancer cells. In vitro, 40 μM of PZL318 inhibited the proliferation of tumorigenic cells, but not nontumorigenic cells. In vivo, 10 nmol/kg of PZL318 slowed the tumor growth of S180 mice and alleviated the thrombosis of ferric chloride-treated ICR mice, while 100 μmol/kg of PZL318 did not injure healthy mice and they exhibited no liver toxicity. By analyzing Fourier transform–mass spectrometry and rotating-frame Overhauser spectroscopy (ROESY) two-dimensional nuclear magnetic resonance spectra, the chemical mechanism of PZL318-forming trimers and nanoparticles was explored. By using mesoscale simulation, a nanoparticle of 3.01 nm in diameter was predicted containing 13 trimers. Scavenging free radicals, downregulating sP-selectin expression and intercalating toward DNA were correlated with the antitumor mechanism of PZL318. PMID:26345234

Reconstitution of Homomeric GluA2flop Receptors in Supported Lipid Membranes

PubMed Central

Baranovic, Jelena; Ramanujan, Chandra S.; Kasai, Nahoko; Midgett, Charles R.; Madden, Dean R.; Torimitsu, Keiichi; Ryan, John F.

2013-01-01

AMPA receptors (AMPARs) are glutamate-gated ion channels ubiquitous in the vertebrate central nervous system, where they mediate fast excitatory neurotransmission and act as molecular determinants of memory formation and learning. Together with detailed analyses of individual AMPAR domains, structural studies of full-length AMPARs by electron microscopy and x-ray crystallography have provided important insights into channel assembly and function. However, the correlation between the structure and functional states of the channel remains ambiguous particularly because these functional states can be assessed only with the receptor bound within an intact lipid bilayer. To provide a basis for investigating AMPAR structure in a membrane environment, we developed an optimized reconstitution protocol using a receptor whose structure has previously been characterized by electron microscopy. Single-channel recordings of reconstituted homomeric GluA2flop receptors recapitulate key electrophysiological parameters of the channels expressed in native cellular membranes. Atomic force microscopy studies of the reconstituted samples provide high-resolution images of membrane-embedded full-length AMPARs at densities comparable to those in postsynaptic membranes. The data demonstrate the effect of protein density on conformational flexibility and dimensions of the receptors and provide the first structural characterization of functional membrane-embedded AMPARs, thus laying the foundation for correlated structure-function analyses of the predominant mediators of excitatory synaptic signals in the brain. PMID:23382380

Non-Cytotoxic Quantum Dot–Chitosan Nanogel Biosensing Probe for Potential Cancer Targeting Agent

PubMed Central

Maxwell, Tyler; Banu, Tahmina; Price, Edward; Tharkur, Jeremy; Campos, Maria Gabriela Nogueira; Gesquiere, Andre; Santra, Swadeshmukul

2015-01-01

Quantum dot (Qdot) biosensors have consistently provided valuable information to researchers about cellular activity due to their unique fluorescent properties. Many of the most popularly used Qdots contain cadmium, posing the risk of toxicity that could negate their attractive optical properties. The design of a non-cytotoxic probe usually involves multiple components and a complex synthesis process. In this paper, the design and synthesis of a non-cytotoxic Qdot-chitosan nanogel composite using straight-forward cyanogen bromide (CNBr) coupling is reported. The probe was characterized by spectroscopy (UV-Vis, fluorescence), microscopy (Fluorescence, Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering. This activatable (“OFF”/“ON”) probe contains a core–shell Qdot (CdS:Mn/ZnS) capped with dopamine, which acts as a fluorescence quencher and a model drug. Dopamine capped “OFF” Qdots can undergo ligand exchange with intercellular glutathione, which turns the Qdots “ON” to restore fluorescence. These Qdots were then coated with chitosan (natural biocompatible polymer) functionalized with folic acid (targeting motif) and Fluorescein Isothiocyanate (FITC; fluorescent dye). To demonstrate cancer cell targetability, the interaction of the probe with cells that express different folate receptor levels was analyzed, and the cytotoxicity of the probe was evaluated on these cells and was shown to be nontoxic even at concentrations as high as 100 mg/L. PMID:28347126

Backscattered electron SEM imaging of resin sections from plant specimens: observation of histological to subcellular structure and CLEM.

PubMed

Rizzo, N W; Duncan, K E; Bourett, T M; Howard, R J

2016-08-01

We have refined methods for biological specimen preparation and low-voltage backscattered electron imaging in the scanning electron microscope that allow for observation at continuous magnifications of ca. 130-70 000 X, and documentation of tissue and subcellular ultrastructure detail. The technique, based upon early work by Ogura & Hasegawa (1980), affords use of significantly larger sections from fixed and resin-embedded specimens than is possible with transmission electron microscopy while providing similar data. After microtomy, the sections, typically ca. 750 nm thick, were dried onto the surface of glass or silicon wafer and stained with heavy metals-the use of grids avoided. The glass/wafer support was then mounted onto standard scanning electron microscopy sample stubs, carbon-coated and imaged directly at an accelerating voltage of 5 kV, using either a yttrium aluminum garnet or ExB backscattered electron detector. Alternatively, the sections could be viewed first by light microscopy, for example to document signal from a fluorescent protein, and then by scanning electron microscopy to provide correlative light/electron microscope (CLEM) data. These methods provide unobstructed access to ultrastructure in the spatial context of a section ca. 7 × 10 mm in size, significantly larger than the typical 0.2 × 0.3 mm section used for conventional transmission electron microscopy imaging. Application of this approach was especially useful when the biology of interest was rare or difficult to find, e.g. a particular cell type, developmental stage, large organ, the interface between cells of interacting organisms, when contextual information within a large tissue was obligatory, or combinations of these factors. In addition, the methods were easily adapted for immunolocalizations. © 2015 The Author. Journal of Microscopy published by John Wiley & Sons, Ltd on behalf of the Royal Microscopical Society.

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila melanogaster.

PubMed

Cantera, Rafael; Lüer, Karin; Rusten, Tor Erik; Barrio, Rosa; Kafatos, Fotis C; Technau, Gerhard M

2002-12-01

The gene spalt is expressed in the embryonic central nervous system of Drosophila melanogaster but its function in this tissue is still unknown. To investigate this question, we used a combination of techniques to analyse spalt mutant embryos. Electron microscopy showed that in the absence of spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway which influences the expression of the beta-catenin Armadillo, its ligand N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organisation of the central nervous system.

Expression and arrangement of extracellular matrix proteins in the lungs of mice infected with Paracoccidioides brasiliensis conidia

PubMed Central

González, Angel; Lenzi, Henrique Leonel; Motta, Ester Maria; Caputo, Luzia; Restrepo, Angela; Cano, Luz Elena

2008-01-01

Extracellular matrix (ECM) proteins are important modulators of migration, differentiation and proliferation for the various cell types present in the lungs; they influence the immune response as well as participate in the adherence of several fungi including Paracoccidioides brasiliensis. The expression, deposition and arrangement of ECM proteins such as laminin, fibronectin, fibrinogen, collagen and proteoglycans in the lungs of mice infected with P. brasiliensis conidia has been evaluated in this study, together with the elastic fibre system. Lungs of BALB/c mice infected with P. brasiliensis conidia were analysed for the different ECM proteins by histological and immunohistochemical procedures at different times of infection. In addition, laser scanning confocal microscopy and scanning electron microscopy were used. During the early periods, the lungs of infected animals showed an inflammatory infiltrate composed mainly of polymorphonuclear neutrophils (PMNs) and macrophages, while during the later periods, mice presented a chronic inflammatory response with granuloma formation. Re-arrangement and increased expression of all ECM proteins tested were observed throughout all studied periods, especially during the occurrence of inflammatory infiltration and formation of the granuloma. The elastic fibre system showed an elastolysis process in all experiments. In conclusion, this study provides new details of pulmonary ECM distribution during the course of paracoccidioidomycosis. PMID:18336528

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

PubMed

Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

2015-08-01

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Integration of a high-NA light microscope in a scanning electron microscope.

PubMed

Zonnevylle, A C; Van Tol, R F C; Liv, N; Narvaez, A C; Effting, A P J; Kruit, P; Hoogenboom, J P

2013-10-01

We present an integrated light-electron microscope in which an inverted high-NA objective lens is positioned inside a scanning electron microscope (SEM). The SEM objective lens and the light objective lens have a common axis and focal plane, allowing high-resolution optical microscopy and scanning electron microscopy on the same area of a sample simultaneously. Components for light illumination and detection can be mounted outside the vacuum, enabling flexibility in the construction of the light microscope. The light objective lens can be positioned underneath the SEM objective lens during operation for sub-10 μm alignment of the fields of view of the light and electron microscopes. We demonstrate in situ epifluorescence microscopy in the SEM with a numerical aperture of 1.4 using vacuum-compatible immersion oil. For a 40-nm-diameter fluorescent polymer nanoparticle, an intensity profile with a FWHM of 380 nm is measured whereas the SEM performance is uncompromised. The integrated instrument may offer new possibilities for correlative light and electron microscopy in the life sciences as well as in physics and chemistry. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

BcMF8, a putative arabinogalactan protein-encoding gene, contributes to pollen wall development, aperture formation and pollen tube growth in Brassica campestris

PubMed Central

Lin, Sue; Dong, Heng; Zhang, Fang; Qiu, Lin; Wang, Fangzhan; Cao, Jiashu; Huang, Li

2014-01-01

Background and Aims The arabinogalactan protein (AGP) gene family is involved in plant reproduction. However, little is known about the function of individual AGP genes in pollen development and pollen tube growth. In this study, Brassica campestris male fertility 8 (BcMF8), a putative AGP-encoding gene previously found to be pollen specific in Chinese cabbage (B. campestris ssp. chinensis), was investigated. Methods Real-time reverse transcription–PCR and in situ hybridization were used to analyse the expression pattern of BcMF8 in pistils. Prokaryotic expression and western blots were used to ensure that BcMF8 could encode a protein. Antisense RNA technology was applied to silence gene expression, and morphological and cytological approaches (e.g. scanning electron microscopy and transmission electron microscopy) were used to reveal abnormal phenotypes caused by gene silencing. Key Results The BcMF8 gene encoded a putative AGP protein that was located in the cell wall, and was expressed in pollen grains and pollen tubes. The functional interruption of BcMF8 by antisense RNA technology resulted in slipper-shaped and bilaterally sunken pollen with abnormal intine development and aperture formation. The inhibition of BcMF8 led to a decrease in the percentage of in vitro pollen germination. In pollen that did germinate, the pollen tubes were unstable, abnormally shaped and burst more frequently relative to controls, which corresponded to an in vivo arrest of pollen germination at the stigma surface and retarded pollen tube growth in the stylar transmitting tissues. Conclusions The phenotypic defects of antisense BcMF8 RNA lines (bcmf8) suggest a crucial function of BcMF8 in modulating the physical nature of the pollen wall and in helping in maintaining the integrity of the pollen tube wall matrix. PMID:24489019

Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.

Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251

Electron microscopy approach for the visualization of the epithelial and endothelial glycocalyx.

PubMed

Chevalier, L; Selim, J; Genty, D; Baste, J M; Piton, N; Boukhalfa, I; Hamzaoui, M; Pareige, P; Richard, V

2017-06-01

This study presents a methodological approach for the visualization of the glycocalyx by electron microscopy. The glycocalyx is a three dimensional network mainly composed of glycolipids, glycoproteins and proteoglycans associated with the plasma membrane. Since less than a decade, the epithelial and endothelial glycocalyx proved to play an important role in physiology and pathology, increasing its research interest especially in vascular functions. Therefore, visualization of the glycocalyx requires reliable techniques and its preservation remains challenging due to its fragile and dynamic organization, which is highly sensitive to the different process steps for electron microscopy sampling. In this study, chemical fixation was performed by perfusion as a good alternative to conventional fixation. Additional lanthanum nitrate in the fixative enhances staining of the glycocalyx in transmission electron microscopy bright field and improves its visualization by detecting the elastic scattered electrons, thus providing a chemical contrast. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Scanning electron microscopy of cells and tissues under fully hydrated conditions

PubMed Central

Thiberge, Stephan; Nechushtan, Amotz; Sprinzak, David; Gileadi, Opher; Behar, Vered; Zik, Ory; Chowers, Yehuda; Michaeli, Shulamit; Schlessinger, Joseph; Moses, Elisha

2004-01-01

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is ≈100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers. PMID:14988502

Sirolimus damages podocytes in rats with protein overload nephropathy.

PubMed

Cai, Yong; Chen, Yan; Zheng, Shaoling; Chen, Bicheng; Yang, Yirong; Xia, Peng

2011-01-01

Conversion from calcineurin inhibitors (CNIs) to sirolimus could significantly improve long-term graft survival after kidney transplantation. Proteinuria was found in some recipients after the switch, which could be alleviated by an angiotensin II receptor blocker (ARB). But the mechanisms for this have remained unclear. In this study, we utilized a rat model with protein overload nephropathy to explore the mechanisms of sirolimus-related proteinuria. A rat model with protein overload nephropathy was induced by repeated injections of bovine serum albumin. Model rats also received sirolimus (rapamycin) treatment or ARB agent (losartan) pretreatment. Urinary protein excretion from 24-hour urine specimens was calculated, and the morphological changes of renal tissues were analyzed by hematoxylin and eosin staining and electron microscopy. The expression of desmin, a sensitive marker of podocyte injury, was detected by immunohistochemical staining. Rapamycin increased urinary protein excretion and intratubular protein cast formation in rats with protein overload nephropathy. The foot process effacement of podocytes was found by electron microscopy after rapamycin treatment. The expression of desmin was up-regulated after rapamycin treatment. However, losartan pretreatment could attenuate proteinuria in spite of rapamycin treatment. Sirolimus aggravates proteinuria in rats with protein overload nephropathy by damaging podocytes, a barrier of glomerular filtration. Furthermore, angiotensin II receptor blocker can counteract the effect of sirolimus, not only through hemodynamic changes but also partly by repairing the injury of podocytes. This study might be useful for understanding the mechanism of sirolimus-related proteinuria and guiding clinical treatments.

Interleukin-13 conjugated quantum dots for identification of glioma initiating cells and their extracellular vesicles.

PubMed

Madhankumar, A B; Mrowczynski, Oliver D; Patel, Suhag R; Weston, Cody L; Zacharia, Brad E; Glantz, Michael J; Siedlecki, Christopher A; Xu, Li-Chong; Connor, James R

2017-08-01

Cadmium selenide (CdSe) based quantum dots modified with polyethylene glycol and chemically linked to interleukin-13 (IL13) were prepared with the aim of identifying the high affinity receptor (IL13Rα2) which is expressed in glioma stem cells and exosomes secreted by these cancer stem cells. IL13 conjugated quantum dots (IL13QD) were thoroughly characterized for their physicochemical properties including particle size and surface morphology. Furthermore, the specific binding of the IL13QD to glioma cells and to glioma stem cells (GSC) was verified using a competitive binding study. The exosomes were isolated from the GSC conditioned medium and the expression of IL13Rα2 in the GSC and exosomes was verified. The binding property of IL13QD to the tumor associated exosomes was initially confirmed by transmission electron microscopy. The force of attraction between the quantum dots and U251 glioma cells and the exosomes was investigated by atomic force microscopy, which indicated a higher force of binding interaction between the IL13QD and IL13Rα2 expressing glioma cells and exosomes secreted by glioma stem cells. Flow cytometry of the IL13QD and exosomes from the culture media and cerebrospinal fluid (CSF) of patients with glioma tumors indicated a distinctly populated complex pattern different from that of non-targeted quantum dots and bovine serum albumin (BSA) conjugated quantum dots confirming specific binding potential of the IL13QD to the tumor associated exosomes. The results of this study demonstrate that IL13QD can serve as an ex vivo marker for glioma stem cells and exosomes that can inform diagnosis and prognosis of patients harboring malignant disease. Functionalized quantum dots are flexible semiconductor nanomaterials which have an immense application in biomedical research. In particular, when they are functionalized with biomolecules like proteins or antibodies, they have the specialized ability to detect the expression of receptors and antigens in cells and tissues. In this study we designed a cytokine (interleukin-13) functionalized quantum dot to detect a cancer associated receptor expressed in cancer stem cells and the extracellular vesicles (exosomes) secreted by the cancer cells themselves. The binding pattern of these cytokine modified quantum dots to the cancer stem cells and exosomes alters the physical properties of the complex in the fixed and suspended form. This altered binding pattern can be monitored by a variety of techniques, including transmission electron microscopy, atomic force microscopy and flow cytometry, and subsequent characterization of this quantum dot binding profile provides useful data that can be utilized as a fingerprint to detect cancer disease progression. This type of functionalized quantum dot fingerprint is especially useful for invasive cancers including brain and other metastatic cancers and may allow for earlier detection of disease progression or recurrence, thus saving the lives of patients suffering from this devastating disease. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Optical properties of the magnetic monopole field applied to electron microscopy and spectroscopy

NASA Astrophysics Data System (ADS)

Kruit, P.; Lenc, M.

1992-11-01

An analytical treatment of the electron's motion in a magnetic monopole field results in useful expressions for both the lens action and the mirror action of the field. Using an appropriate definition of the magnetic moment of the electron, it is shown that there is an exact conservation of this parameter in the monopole field, implying that the motion is perfectly adiabatic. This property is important when the field is used for directing Auger electrons from a target to a detector; that is, when it is used as a parallelizer in a through-the-lens detection scheme. Regarding the monopole field as an electron lens, the image position and magnification are derived for an arbitrary object position. Expressions for both the axial aberrations (chromatic and spherical) and the image aberrations (coma, field curvature, astigmatism, distortion, and transverse chromatic) are derived for an arbitrary number of intermediate images between object and final image. The chromatic aberration turns out to be independent of the number of intermediate images and the spherical aberration decreases slightly with this number. This property is important when an electron beam must be focused to a small probe in a strong magnetic field. It is shown that if a certain combination of deflectors is used in conjunction with the monopole field, an ideal swinging objective lens is obtained: All image aberrations except field curvature disappear. Designs are presented in which the monopole field is used in the objective lenses of a transmission electron microscope and a scanning electron microscope.

Attosecond electron pulse trains and quantum state reconstruction in ultrafast transmission electron microscopy

NASA Astrophysics Data System (ADS)

Priebe, Katharina E.; Rathje, Christopher; Yalunin, Sergey V.; Hohage, Thorsten; Feist, Armin; Schäfer, Sascha; Ropers, Claus

2017-12-01

Ultrafast electron and X-ray imaging and spectroscopy are the basis for an ongoing revolution in the understanding of dynamical atomic-scale processes in matter. The underlying technology relies heavily on laser science for the generation and characterization of ever shorter pulses. Recent findings suggest that ultrafast electron microscopy with attosecond-structured wavefunctions may be feasible. However, such future technologies call for means to both prepare and fully analyse the corresponding free-electron quantum states. Here, we introduce a framework for the preparation, coherent manipulation and characterization of free-electron quantum states, experimentally demonstrating attosecond electron pulse trains. Phase-locked optical fields coherently control the electron wavefunction along the beam direction. We establish a new variant of quantum state tomography—`SQUIRRELS'—for free-electron ensembles. The ability to tailor and quantitatively map electron quantum states will promote the nanoscale study of electron-matter entanglement and new forms of ultrafast electron microscopy down to the attosecond regime.

Nanowire growth kinetics in aberration corrected environmental transmission electron microscopy

DOE PAGES

Chou, Yi -Chia; Panciera, Federico; Reuter, Mark C.; ...

2016-03-15

Here, we visualize atomic level dynamics during Si nanowire growth using aberration corrected environmental transmission electron microscopy, and compare with lower pressure results from ultra-high vacuum microscopy. We discuss the importance of higher pressure observations for understanding growth mechanisms and describe protocols to minimize effects of the higher pressure background gas.

Electronic Blending in Virtual Microscopy

ERIC Educational Resources Information Center

Maybury, Terrence S.; Farah, Camile S.

2010-01-01

Virtual microscopy (VM) is a relatively new technology that transforms the computer into a microscope. In essence, VM allows for the scanning and transfer of glass slides from light microscopy technology to the digital environment of the computer. This transition is also a function of the change from print knowledge to electronic knowledge, or as…

Structural and Ultrastructural Characteristics of Bone-Tendon Junction of the Calcaneal Tendon of Adult and Elderly Wistar Rats

PubMed Central

Cury, Diego Pulzatto; Dias, Fernando José; Miglino, Maria Angélica; Watanabe, Ii-sei

2016-01-01

Tendons are transition tissues that transfer the contractile forces generated by the muscles to the bones, allowing movement. The region where the tendon attaches to the bone is called bone-tendon junction or enthesis and may be classified as fibrous or fibrocartilaginous. This study aims to analyze the collagen fibers and the cells present in the bone-tendon junction using light microscopy and ultrastructural techniques as scanning electron microscopy and transmission electron microscopy. Forty male Wistar rats were used in the experiment, being 20 adult rats at 4 months-old and 20 elderly rats at 20 months-old. The hind limbs of the rats were removed, dissected and prepared to light microscopy, transmission electron microscopy and scanning electron microscopy. The aging process showed changes in the collagen fibrils, with a predominance of type III fibers in the elderly group, in addition to a decrease in the amount of the fibrocartilage cells, fewer and shorter cytoplasmic processes and a decreased synthetic capacity due to degradation of the organelles involved in synthesis. PMID:27078690

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

PubMed Central

HÖHN, K.; FUCHS, J.; FRÖBER, A.; KIRMSE, R.; GLASS, B.; ANDERS‐ÖSSWEIN, M.; WALTHER, P.; KRÄUSSLICH, H.‐G.

2015-01-01

Summary In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells. PMID:25786567

Structural and Ultrastructural Characteristics of Bone-Tendon Junction of the Calcaneal Tendon of Adult and Elderly Wistar Rats.

PubMed

Cury, Diego Pulzatto; Dias, Fernando José; Miglino, Maria Angélica; Watanabe, Ii-sei

2016-01-01

Tendons are transition tissues that transfer the contractile forces generated by the muscles to the bones, allowing movement. The region where the tendon attaches to the bone is called bone-tendon junction or enthesis and may be classified as fibrous or fibrocartilaginous. This study aims to analyze the collagen fibers and the cells present in the bone-tendon junction using light microscopy and ultrastructural techniques as scanning electron microscopy and transmission electron microscopy. Forty male Wistar rats were used in the experiment, being 20 adult rats at 4 months-old and 20 elderly rats at 20 months-old. The hind limbs of the rats were removed, dissected and prepared to light microscopy, transmission electron microscopy and scanning electron microscopy. The aging process showed changes in the collagen fibrils, with a predominance of type III fibers in the elderly group, in addition to a decrease in the amount of the fibrocartilage cells, fewer and shorter cytoplasmic processes and a decreased synthetic capacity due to degradation of the organelles involved in synthesis.

Colony-Forming Progenitor Cells in the Postnatal Mouse Liver and Pancreas Give Rise to Morphologically Distinct Insulin-Expressing Colonies in 3D Cultures

PubMed Central

Jin, Liang; Feng, Tao; Chai, Jing; Ghazalli, Nadiah; Gao, Dan; Zerda, Ricardo; Li, Zhuo; Hsu, Jasper; Mahdavi, Alborz; Tirrell, David A.; Riggs, Arthur D.; Ku, Hsun Teresa

2014-01-01

In our previous studies, colony-forming progenitor cells isolated from murine embryonic stem cell-derived cultures were differentiated into morphologically distinct insulin-expressing colonies. These colonies were small and not light-reflective when observed by phase-contrast microscopy (therefore termed “Dark” colonies). A single progenitor cell capable of giving rise to a Dark colony was termed a Dark colony-forming unit (CFU-Dark). The goal of the current study was to test whether endogenous pancreas, and its developmentally related liver, harbored CFU-Dark. Here we show that dissociated single cells from liver and pancreas of one-week-old mice give rise to Dark colonies in methylcellulose-based semisolid culture media containing either Matrigel or laminin hydrogel (an artificial extracellular matrix protein). CFU-Dark comprise approximately 0.1% and 0.03% of the postnatal hepatic and pancreatic cells, respectively. Adult liver also contains CFU-Dark, but at a much lower frequency (~0.003%). Microfluidic qRT-PCR, immunostaining, and electron microscopy analyses of individually handpicked colonies reveal the expression of insulin in many, but not all, Dark colonies. Most pancreatic insulin-positive Dark colonies also express glucagon, whereas liver colonies do not. Liver CFU-Dark require Matrigel, but not laminin hydrogel, to become insulin-positive. In contrast, laminin hydrogel is sufficient to support the development of pancreatic Dark colonies that express insulin. Postnatal liver CFU-Dark display a cell surface marker CD133+CD49flowCD107blow phenotype, while pancreatic CFU-Dark are CD133-. Together, these results demonstrate that specific progenitor cells in the postnatal liver and pancreas are capable of developing into insulin-expressing colonies, but they differ in frequency, marker expression, and matrix protein requirements for growth. PMID:25148366

Biological applications of phase-contrast electron microscopy.

PubMed

Nagayama, Kuniaki

2014-01-01

Here, I review the principles and applications of phase-contrast electron microscopy using phase plates. First, I develop the principle of phase contrast based on a minimal model of microscopy, introducing a double Fourier-transform process to mathematically formulate the image formation. Next, I explain four phase-contrast (PC) schemes, defocus PC, Zernike PC, Hilbert differential contrast, and schlieren optics, as image-filtering processes in the context of the minimal model, with particular emphases on the Zernike PC and corresponding Zernike phase plates. Finally, I review applications of Zernike PC cryo-electron microscopy to biological systems such as protein molecules, virus particles, and cells, including single-particle analysis to delineate three-dimensional (3D) structures of protein and virus particles and cryo-electron tomography to reconstruct 3D images of complex protein systems and cells.

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

PubMed

Ansardi, D C; Porter, D C; Morrow, C D

1991-04-01

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.

Fast electron microscopy via compressive sensing

DOEpatents

Larson, Kurt W; Anderson, Hyrum S; Wheeler, Jason W

2014-12-09

Various technologies described herein pertain to compressive sensing electron microscopy. A compressive sensing electron microscope includes a multi-beam generator and a detector. The multi-beam generator emits a sequence of electron patterns over time. Each of the electron patterns can include a plurality of electron beams, where the plurality of electron beams is configured to impart a spatially varying electron density on a sample. Further, the spatially varying electron density varies between each of the electron patterns in the sequence. Moreover, the detector collects signals respectively corresponding to interactions between the sample and each of the electron patterns in the sequence.

Modelling relativistic effects in momentum-resolved electron energy loss spectroscopy of graphene

NASA Astrophysics Data System (ADS)

Lyon, K.; Mowbray, D. J.; Miskovic, Z. L.

2018-02-01

We present an analytical model for the electron energy loss through a two-dimensional (2D) layer of graphene, fully taking into account relativistic effects. Using two different models for graphene's 2D conductivity, one a two-fluid hydrodynamic model with an added correction to account for the inter-band electron transitions near the Dirac point in undoped graphene, the other derived from ab initio plane-wave time-dependent density functional theory in the frequency domain (PW-TDDFT-ω) calculations applied on a graphene superlattice, we derive various different expressions for the probability density of energy and momentum transfer from the incident electron to graphene. To further compare with electron energy loss spectroscopy (EELS) experiments that use setups like scanning Transmission Electron Microscopy, we integrated our energy loss functions over a range of wavenumbers, and compared how the choice of range directly affects the shape, position, and relative heights of graphene's π → π* and σ → σ* transition peaks. Comparisons were made with experimental EELS data under different model inputs, revealing again the strong effect that the choice of wavenumber range has on the energy loss.

Photon gating in four-dimensional ultrafast electron microscopy.

PubMed

Hassan, Mohammed T; Liu, Haihua; Baskin, John Spencer; Zewail, Ahmed H

2015-10-20

Ultrafast electron microscopy (UEM) is a pivotal tool for imaging of nanoscale structural dynamics with subparticle resolution on the time scale of atomic motion. Photon-induced near-field electron microscopy (PINEM), a key UEM technique, involves the detection of electrons that have gained energy from a femtosecond optical pulse via photon-electron coupling on nanostructures. PINEM has been applied in various fields of study, from materials science to biological imaging, exploiting the unique spatial, energy, and temporal characteristics of the PINEM electrons gained by interaction with a "single" light pulse. The further potential of photon-gated PINEM electrons in probing ultrafast dynamics of matter and the optical gating of electrons by invoking a "second" optical pulse has previously been proposed and examined theoretically in our group. Here, we experimentally demonstrate this photon-gating technique, and, through diffraction, visualize the phase transition dynamics in vanadium dioxide nanoparticles. With optical gating of PINEM electrons, imaging temporal resolution was improved by a factor of 3 or better, being limited only by the optical pulse widths. This work enables the combination of the high spatial resolution of electron microscopy and the ultrafast temporal response of the optical pulses, which provides a promising approach to attain the resolution of few femtoseconds and attoseconds in UEM.

Photon gating in four-dimensional ultrafast electron microscopy

PubMed Central

Hassan, Mohammed T.; Liu, Haihua; Baskin, John Spencer; Zewail, Ahmed H.

2015-01-01

Ultrafast electron microscopy (UEM) is a pivotal tool for imaging of nanoscale structural dynamics with subparticle resolution on the time scale of atomic motion. Photon-induced near-field electron microscopy (PINEM), a key UEM technique, involves the detection of electrons that have gained energy from a femtosecond optical pulse via photon–electron coupling on nanostructures. PINEM has been applied in various fields of study, from materials science to biological imaging, exploiting the unique spatial, energy, and temporal characteristics of the PINEM electrons gained by interaction with a “single” light pulse. The further potential of photon-gated PINEM electrons in probing ultrafast dynamics of matter and the optical gating of electrons by invoking a “second” optical pulse has previously been proposed and examined theoretically in our group. Here, we experimentally demonstrate this photon-gating technique, and, through diffraction, visualize the phase transition dynamics in vanadium dioxide nanoparticles. With optical gating of PINEM electrons, imaging temporal resolution was improved by a factor of 3 or better, being limited only by the optical pulse widths. This work enables the combination of the high spatial resolution of electron microscopy and the ultrafast temporal response of the optical pulses, which provides a promising approach to attain the resolution of few femtoseconds and attoseconds in UEM. PMID:26438835

High yield production of long branched Au nanoparticles characterized by atomic resolution transmission electron microscopy

PubMed Central

Mayoral, Alvaro; Magen, Cesar; Jose-Yacaman, Miguel

2011-01-01

Long multi-branched gold nanoparticles have been synthesized in a very high yield through a facile synthesis combining two different capping agents. The stability of these materials with the time has been tested and their characterization have been performed by diverse advanced electron microscopy techniques, paying special attention to aberration corrected transmission electron microscopy in order to unambiguously analyze the surface structure of the branches and provide insights for the formation of stellated gold nanoparticles. PMID:22125420

Use of scanning electron microscopy and microanalysis to determine chloride content of concrete and raw materials.

DOT National Transportation Integrated Search

2013-02-01

Standard sample sets of cement and mortar formulations with known levels of Cl as well as concrete samples subject to Cl diffusion were all prepared for and analyzed with scanning electron microscopy (SEM) and electron microprobe (EPMA). Using x-ray ...

Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: Light and electron microscopy

USGS Publications Warehouse

Walker, G.K.; Black, M.G.; Edwards, C.A.

1996-01-01

Adult zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondrial "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria.

Advances in high-resolution imaging--techniques for three-dimensional imaging of cellular structures.

PubMed

Lidke, Diane S; Lidke, Keith A

2012-06-01

A fundamental goal in biology is to determine how cellular organization is coupled to function. To achieve this goal, a better understanding of organelle composition and structure is needed. Although visualization of cellular organelles using fluorescence or electron microscopy (EM) has become a common tool for the cell biologist, recent advances are providing a clearer picture of the cell than ever before. In particular, advanced light-microscopy techniques are achieving resolutions below the diffraction limit and EM tomography provides high-resolution three-dimensional (3D) images of cellular structures. The ability to perform both fluorescence and electron microscopy on the same sample (correlative light and electron microscopy, CLEM) makes it possible to identify where a fluorescently labeled protein is located with respect to organelle structures visualized by EM. Here, we review the current state of the art in 3D biological imaging techniques with a focus on recent advances in electron microscopy and fluorescence super-resolution techniques.

Microchemical Analysis Of Space Operation Debris

NASA Technical Reports Server (NTRS)

Cummings, Virginia J.; Kim, Hae Soo

1995-01-01

Report discusses techniques used in analyzing debris relative to space shuttle operations. Debris collected from space shuttle, expendable launch vehicles, payloads carried by space shuttle, and payloads carried by expendable launch vehicles. Optical microscopy, scanning electron microscopy with energy-dispersive spectrometry, analytical electron microscopy with wavelength-dispersive spectrometry, and X-ray diffraction chosen as techniques used in examining samples of debris.

[Thrombopenia and radial aplasia: 2 cases with platelet function and ultrastructural studies of megakaryocytes and platelets (author's transl)].

PubMed

Juhan, I; Bayle, J; Mattei, J F; Thevenieau, D; Perrimond, H; Muratore, R

1979-10-01

The authors report on two cases of congenital thrombopenia with radial aplasia. Both children display several formative abnormalities and a mild thrombopenia; hemorragic manifestations occurred in the first case only. Megacryoblastic to platelets series, as studied with electronic microscopy, show small-sized, "microcytic" and hypogranular megacaryocytes, displaying a maturative disorder (dysmegacaryocytopoiesis). In functional studies, platelets of the first patient show an imperfect nucleotidic release and do not agregate normally with ristocetin. The second case exhibits mostly a PF3 reduction. The variety of expression of the megacaryocytic-platelets disorders appears likewise in the squelettal and visceral malformations. The whole disorder could be ascribed to a pleiotropic abnormal gene with a variable expressivity.

Publications - GMC 357 | Alaska Division of Geological & Geophysical

Science.gov Websites

DGGS GMC 357 Publication Details Title: Thin Section and Scanning Electron Microscopy summary Laboratories, Inc., 2008, Thin Section and Scanning Electron Microscopy summary photographs from plugs taken

Structure of the cell envelope of corynebacteria: importance of the non-covalently bound lipids in the formation of the cell wall permeability barrier and fracture plane.

PubMed

Puech, V; Chami, M; Lemassu, A; Lanéelle, M A; Schiffler, B; Gounon, P; Bayan, N; Benz, R; Daffé, M

2001-05-01

With the recent success of the heterologous expression of mycobacterial antigens in corynebacteria, in addition to the importance of these bacteria in biotechnology and medicine, a better understanding of the structure of their cell envelopes was needed. A combination of molecular compositional analysis, ultrastructural appearance and freeze-etch electron microscopy study was used to arrive at a chemical model, unique to corynebacteria but consistent with their phylogenetic relatedness to mycobacteria and other members of the distinctive suprageneric actinomycete taxon. Transmission electron microscopy and chemical analyses showed that the cell envelopes of the representative strains of corynebacteria examined consisted of (i) an outer layer composed of polysaccharides (primarily a high-molecular-mass glucan and arabinomannans), proteins, which include the mycoloyltransferase PS1, and lipids; (ii) a cell wall glycan core of peptidoglycan-arabinogalactan which may contain other sugar residues and was usually esterified by corynomycolic acids; and (iii) a typical plasma membrane bilayer. Freeze-etch electron microscopy showed that most corynomycolate-containing strains exhibited a main fracture plane in their cell wall and contained low-molecular-mass porins, while the fracture occurred within the plasma membrane of strains devoid of both corynomycolate and pore-forming proteins. Importantly, in most strains, the amount of cell wall-linked corynomycolates was not sufficient to cover the bacterial surface; interestingly, the occurrence of a cell wall fracture plane correlated with the amount of non-covalently bound lipids of the strains. Furthermore, these lipids were shown to spontaneously form liposomes, indicating that they may participate in a bilayer structure. Altogether, the data suggested that the cell wall permeability barrier in corynebacteria involved both covalently linked corynomycolates and non-covalently bound lipids of their cell envelopes.

Electron microscopy of electromagnetic waveforms.

PubMed

Ryabov, A; Baum, P

2016-07-22

Rapidly changing electromagnetic fields are the basis of almost any photonic or electronic device operation. We report how electron microscopy can measure collective carrier motion and fields with subcycle and subwavelength resolution. A collimated beam of femtosecond electron pulses passes through a metamaterial resonator that is previously excited with a single-cycle electromagnetic pulse. If the probing electrons are shorter in duration than half a field cycle, then time-frozen Lorentz forces distort the images quasi-classically and with subcycle time resolution. A pump-probe sequence reveals in a movie the sample's oscillating electromagnetic field vectors with time, phase, amplitude, and polarization information. This waveform electron microscopy can be used to visualize electrodynamic phenomena in devices as small and fast as available. Copyright © 2016, American Association for the Advancement of Science.

Murphy's law-if anything can go wrong, it will: Problems in phage electron microscopy.

PubMed

Ackermann, Hans-W; Tiekotter, Kenneth L

2012-04-01

The quality of bacteriophage electron microscopy appears to be on a downward course since the 1980s. This coincides with the introduction of digital electron microscopes and a general lowering of standards, possibly due to the disappearance of several world-class electron microscopists The most important problem seems to be poor contrast. Positive staining is frequently not recognized as an undesirable artifact. Phage parts, bacterial debris, and aberrant or damaged phage particles may be misdiagnosed as bacterial viruses. Digital electron microscopes often seem to be operated without magnification control because this is difficult and inconvenient. In summary, most phage electron microscopy problems may be attributed to human failure. Journals are a last-ditch defense and have a heavy responsibility in selecting competent reviewers and rejecting, or not, unsatisfactory articles.

[Blood cerebrospinal fluid barrier damage of rats induced by lead acetate or nano-lead exposure].

PubMed

Feng, P P; Zhai, F J; Jiang, S F; Wu, J Z; Xue, L; Zheng, M M; Zhou, L L; Meng, C Y; Cao, M Y; Zhang, Y S

2016-05-20

To investigate the damage of blood-cerebrospinal fluid barrier (BCB) of rats induced by lead and nano-lead exposure in order to provide the basis for mechanism study of lead neurotoxicity. 39 male rats were randomly divided into control group, lead acetate exposed group and nano-lead exposed group. Rats in lead acetate exposed group and nano-lead exposed group were given 20 mg/kg lead acetate or nano-lead by oral gavage and rats in control groups were given the same amount saline for 9 weeks.Morris maze was used to test the learning function, serum albumin and CSF albumin were determined by ELISA. Confocal laser scanning microscope was applied to detect ZO-1 and Occludin protein expression in choroid plexus, real time-PCR was used to test the expression of ZO-1 and Occludin mRNA expression. Pathological changes of choroid plexus cells were observed by the electron microscopy. Compared with the control group, the escape latency of rats in lead acetate or nano-lead exposure group were longer and times of across platform were less. The levels of CSF albumin and the CSF albumin index in lead acetate or nano-lead exposed rats were obviously higher, and the fluorescence intensity of ZO-1, Occludin as well as mRNA expressions were lower than those in control group(P<0.05). Compared with lead acetate exposed group, the levels of CSF albumin and the CSF albumin index in nano-lead exposure group were higher. The fluorescence intensity and mRNA expressions of ZO-1, Occludin in nano-lead exposure group were than those in lead acetate group(P<0.05). Electron microscopy revealed that lead acetate or nano-lead exposure could induce shorter microvillus of choroid plexus epithelial cells, mitochondrion destruction and partial disconnection in intracellular junctions between two adjacent epithelial cells. Lead acetate and nano-lead exposed can result in the blood-cerebrospinal fluid barrier damage, which may involve in the process of lead induced neurotoxicity. Meanwhile, nano-lead exposure can induced in more worse damage in terms of blood-results in blood-cerebrospinal fluid barrier function.

75 FR 34096 - Application(s) for Duty-Free Entry of Scientific Instruments

Federal Register 2010, 2011, 2012, 2013, 2014

2010-06-16

... dynamin, using negative stain nad cryo-electron microscopy methods. Justification for Duty-Free Entry..., using negative stain nad cryo-electron microscopy methods. Justification for Duty-Free Entry: There are...

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

NASA Astrophysics Data System (ADS)

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-08-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium-oxygen, lithium-sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

PubMed Central

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-01-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium–oxygen, lithium–sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

The Electron Microscopy Outreach Program: A Web-based resource for research and education.

PubMed

Sosinsky, G E; Baker, T S; Hand, G; Ellisman, M H

1999-01-01

We have developed a centralized World Wide Web (WWW)-based environment that serves as a resource of software tools and expertise for biological electron microscopy. A major focus is molecular electron microscopy, but the site also includes information and links on structural biology at all levels of resolution. This site serves to help integrate or link structural biology techniques in accordance with user needs. The WWW site, called the Electron Microscopy (EM) Outreach Program (URL: http://emoutreach.sdsc.edu), provides scientists with computational and educational tools for their research and edification. In particular, we have set up a centralized resource containing course notes, references, and links to image analysis and three-dimensional reconstruction software for investigators wanting to learn about EM techniques either within or outside of their fields of expertise. Copyright 1999 Academic Press.

A compilation of cold cases using scanning electron microscopy at the University of Rhode Island

NASA Astrophysics Data System (ADS)

Platek, Michael J.; Gregory, Otto J.

2015-10-01

Scanning electron microscopy combined with microchemical analysis has evolved into one of the most widely used instruments in forensic science today. In particular, the environmental scanning electron microscope (SEM) in conjunction with energy dispersive spectroscopy (EDS), has created unique opportunities in forensic science in regard to the examination of trace evidence; i.e. the examination of evidence without altering the evidence with conductive coatings, thereby enabling criminalists to solve cases that were previously considered unsolvable. Two cold cases were solved at URI using a JEOL 5900 LV SEM in conjunction with EDS. A cold case murder and a cold missing person case will be presented from the viewpoint of the microscopist and will include sample preparation, as well as image and chemical analysis of the trace evidence using electron microscopy and optical microscopy.

The EIGER detector for low-energy electron microscopy and photoemission electron microscopy.

PubMed

Tinti, G; Marchetto, H; Vaz, C A F; Kleibert, A; Andrä, M; Barten, R; Bergamaschi, A; Brückner, M; Cartier, S; Dinapoli, R; Franz, T; Fröjdh, E; Greiffenberg, D; Lopez-Cuenca, C; Mezza, D; Mozzanica, A; Nolting, F; Ramilli, M; Redford, S; Ruat, M; Ruder, Ch; Schädler, L; Schmidt, Th; Schmitt, B; Schütz, F; Shi, X; Thattil, D; Vetter, S; Zhang, J

2017-09-01

EIGER is a single-photon-counting hybrid pixel detector developed at the Paul Scherrer Institut, Switzerland. It is designed for applications at synchrotron light sources with photon energies above 5 keV. Features of EIGER include a small pixel size (75 µm × 75 µm), a high frame rate (up to 23 kHz), a small dead-time between frames (down to 3 µs) and a dynamic range up to 32-bit. In this article, the use of EIGER as a detector for electrons in low-energy electron microscopy (LEEM) and photoemission electron microscopy (PEEM) is reported. It is demonstrated that, with only a minimal modification to the sensitive part of the detector, EIGER is able to detect electrons emitted or reflected by the sample and accelerated to 8-20 keV. The imaging capabilities are shown to be superior to the standard microchannel plate detector for these types of applications. This is due to the much higher signal-to-noise ratio, better homogeneity and improved dynamic range. In addition, the operation of the EIGER detector is not affected by radiation damage from electrons in the present energy range and guarantees more stable performance over time. To benchmark the detector capabilities, LEEM experiments are performed on selected surfaces and the magnetic and electronic properties of individual iron nanoparticles with sizes ranging from 8 to 22 nm are detected using the PEEM endstation at the Surface/Interface Microscopy (SIM) beamline of the Swiss Light Source.

Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-03-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. (c) 2010 Elsevier Inc. All rights reserved.

Study of thermal-field emission properties and investigation of temperature dependent noise in the field emission current from vertical carbon nanotube emitters

NASA Astrophysics Data System (ADS)

Kolekar, Sadhu; Patole, S. P.; Patil, Sumati; Yoo, J. B.; Dharmadhikari, C. V.

2017-10-01

We have investigated temperature dependent field electron emission characteristics of vertical carbon nanotubes (CNTs). The generalized expression for electron emission from well-defined cathode surface is given by Millikan and Lauritsen [1] for the combination of temperature and electric field effect. The same expression has been used to explain the electron emission characteristics from vertical CNT emitters. Furthermore, this has been applied to explain the electron emission for different temperatures ranging from room temperature to 1500 K. The real-time field electron emission images at room temperature and 1500 K are recorded by using Charge Coupled Device (CCD) in order to understand the effect of temperature on distribution of electron emission spots and ring like structures in Field Emission Microscope (FEM) image. The FEM images could be used to calculate the total number of emitters per cm2 for electron emission. The calculated number of emitters per cm2 from FEM image is typically, 4.5 × 107 and the actual number emitters per cm2 present as per Atomic Force Microscopy (AFM) data is 1.2 × 1012. The measured Current-Voltage (I-V) characteristics exhibit non linear Folwer-Nordheim (F-N) type behavior. The fluctuations in the emission current were recorded at different temperatures and Fast Fourier transformed into temperature dependent power spectral density. The latter was found to obey power law relation S(f) = A(Iδ/fξ), where δ and ξ are temperature dependent current and frequency exponents respectively.

Room temperature chemical synthesis of lead selenide thin films with preferred orientation

NASA Astrophysics Data System (ADS)

Kale, R. B.; Sartale, S. D.; Ganesan, V.; Lokhande, C. D.; Lin, Yi-Feng; Lu, Shih-Yuan

2006-11-01

Room temperature chemical synthesis of PbSe thin films was carried out from aqueous ammoniacal solution using Pb(CH3COO)2 as Pb2+ and Na2SeSO3 as Se2- ion sources. The films were characterized by a various techniques including, X-ray diffraction (XRD), energy dispersive X-ray analysis (EDAX), scanning electron microscopy (SEM), transmission electron microscopy (TEM), high resolution transmission electron microscopy (HR-TEM), selected area electron diffraction (SAED), Fast Fourier transform (FFT) and UV-vis-NIR techniques. The study revealed that the PbSe thin film consists of preferentially oriented nanocubes with energy band gap of 0.5 eV.

Imaging of high-angle annular dark-field scanning transmission electron microscopy and observations of GaN-based violet laser diodes.

PubMed

Shiojiri, M; Saijo, H

2006-09-01

The first part of this paper is devoted to physics, to explain high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) imaging and to interpret why HAADF-STEM imaging is incoherent, instructing a strict definition of interference and coherence of electron waves. Next, we present our recent investigations of InGaN/GaN multiple quantum wells and AlGaN/GaN strained-layer superlattice claddings in GaN-based violet laser diodes, which have been performed by HAADF-STEM and high-resolution field-emission gun scanning electron microscopy.

Application of He ion microscopy for material analysis

NASA Astrophysics Data System (ADS)

Altmann, F.; Simon, M.; Klengel, R.

2009-05-01

Helium ion beam microscopy (HIM) is a new high resolution imaging technique. The use of Helium ions instead of electrons enables none destructive imaging combined with contrasts quite similar to that from Gallium ion beam imaging. The use of very low probe currents and the comfortable charge compensation using low energy electrons offer imaging of none conductive samples without conductive coating. An ongoing microelectronic sample with Gold/Aluminum interconnects and polymer electronic devices were chosen to evaluate HIM in comparison to scanning electron microscopy (SEM). The aim was to look for key applications of HIM in material analysis. Main focus was on complementary contrast mechanisms and imaging of none conductive samples.

Catalytic chemical vapor deposition synthesis and electron microscopy observation of coiled carbon nanotubes

NASA Astrophysics Data System (ADS)

Xie, Jining; Mukhopadyay, K.; Yadev, J.; Varadan, V. K.

2003-10-01

Coiled carbon nanotubes exhibit excellent mechanical and electrical properties because of the combination of coil morphology and properties of nanotubes. They could have potential novel applications in nanocomposites and nano-electronic devices as well as nano-electromechanical systems. In this work, synthesis of regularly coiled carbon nanotubes is presented. It involves pyrolysis of hydrocarbon gas over metal/support catalyst by both thermal filament and microwave catalytic chemical vapor deposition methods. Scanning electron microscopy and transmission electron microscopy were performed to observe the coil morphology and nanostructure of coiled nanotubes. The growth mechanism and structural and electrical properties of coiled carbon nanotubes are also discussed.

Use of light, scanning electron microscopy and bioassays to evaluate parasitism by entomopathogenic fungi of the red scale insect of palms (Phoenicococcus marlatti Ckll., 1899).

PubMed

Asensio, L; Lopez-Llorca, L V; López-Jiménez, J A

2005-01-01

We have evaluated the parasitism of the red scale insect of the date palm (Phoenicococcus marlatti) by entomopathogenic fungi, using light microscopy (LM), scanning electron microscopy (SEM) and low temperature scanning electron microscopy (LTSEM). Beauveria bassiana, Lecanicillium dimorphum and Lecanicillium cf. psalliotae, were inoculated directly on the scale insects or on insect infested plant material. We found that L. dimorphum and L. cf. psalliotae developed on plant material and on scale insects, making infection structures. B. bassiana was a bad colonizer of date palm leaves (Phoenix dactylifera L.) and did not parasite the scale insects.

Airborne asbestos in Colorado public schools.

PubMed

Chadwick, D A; Buchan, R M; Beaulieu, H J

1985-02-01

Levels of airborne asbestos for six Colorado public school facilities with sprayed-on asbestos materials were documented using three analytical techniques. Phase contrast microscopy showed levels up to the thousandths of a fiber per cubic centimeter (f/cc), scanning electron microscopy (SEM) up to the hundredths of a f/cc, and transmission electron microscopy coupled to selected area electron diffraction and energy dispersive X-ray analysis (TEM-SAED-EDXA) up to the tenths of an asbestos f/cc. Phase contrast microscopy was found to be an inadequate analytical technique for documenting the levels of airborne asbestos fibers in the schools: only large fibers which were not embedded in the filter were counted, and asbestos fibers were not distinguished from nonasbestos.

Further description of Cruzia tentaculata (Rudolphi, 1819) Travassos, 1917 (Nematoda: Cruzidae) by light and scanning electron microscopy.

PubMed

Adnet, F A O; Anjos, D H S; Menezes-Oliveira, A; Lanfredi, R M

2009-04-01

Species of Cruzia are parasites of the large intestine of marsupials, reptiles, amphibians, and mammalians. Cruzia tentaculata specimens were collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Colombia (new geographical record) and from Brazil and analyzed by light and scanning electron microscopy. The morphology of males and females by light microscopy corroborated most of the previous description and the ultrastructure by scanning electron microscopy evidence: the topography of the cuticle, deirids, amphids, phasmids in both sexes, a pair of papillae near the vulva opening, and the number and location of male caudal papillae, adding new features for species identification only observed by this technique.

Requirements for the identification of dense-core granules.

PubMed

Meldolesi, Jacopo; Chieregatti, Evelina; Luisa Malosio, Maria

2004-01-01

Dense-core granules (DCGs), cytoplasmic organelles competent for regulated exocytosis, show considerable heterogeneity depending upon the specificity of their expressing cells--primarily neurons and neurosecretory cells. DCGs have been mainly identified by detecting their cargo molecules, often members of the granin family, and using conventional electron microscopy and immunocytochemistry. However, by a critical analysis of the various stages of DCG "life" within neurosecretory cells, we have highlighted several specific molecular and functional properties that are common to all these organelles. We propose that these properties be considered as strict requirements for the identification of DCGs.

Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

PubMed

Lyrio, Eloah C D; Campos-Souza, Ivy C; Corrêa, Luiz C D; Lechuga, Guilherme C; Verícimo, Maurício; Castro, Helena C; Bourguignon, Saulo C; Côrte-Real, Suzana; Ratcliffe, Norman; Declercq, Wim; Santos, Dilvani O

2015-07-01

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1β production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Magnetism of epitaxial Tb films on W(110) studied by spin-polarized low-energy electron microscopy

NASA Astrophysics Data System (ADS)

Prieto, J. E.; Chen, Gong; Schmid, A. K.; de la Figuera, J.

2016-11-01

Thin epitaxial films of Tb metal were grown on a clean W(110) substrate in ultrahigh vacuum and studied in situ by low-energy electron microscopy. Annealed films present magnetic contrast in spin-polarized low-energy electron microscopy. The energy dependence of the electron reflectivity was determined and a maximum value of its spin asymmetry of about 1% was measured. The magnetization direction of the Tb films is in-plane. Upon raising the temperature, no change in the domain distribution is observed, while the asymmetry in the electron reflectivity decreases when approaching the critical temperature, following a power law ˜(1-T /TC) β with a critical exponent β of 0.39.

Effects of Renal Ischemic Postconditioning on Myocardial Ultrastructural Organization and Myocardial Expression of Bcl-2/Bax in Rabbits

PubMed Central

Zhang, Wen-zhong; Li, Rong; Liu, Song; Ning, Xian-feng; Cai, Shang-lang

2016-01-01

We investigated the cardioprotective effect of renal ischemic postconditioning (RI-PostC) and its mechanisms in a rabbit model. Rabbits underwent 60 min of left anterior descending coronary artery occlusion (LADO) and 6 h of reperfusion. The ischemia-reperfusion (IR) group underwent LADO and reperfusion only. In the RI-PostC group, the left renal artery underwent 3 cycles of occlusion for 30 seconds and release for 30 seconds, before the coronary artery was reperfused. In the RI-PostC + GF109203X group, the rabbits received 0.05 mg/kg GF109203X (protein kinase C inhibitor) intravenously for 10 min followed by RI-PostC. Light microscopy and electron microscopy demonstrated that the RI-PostC group showed less pronounced changes, a smaller infarct region, and less apoptosis than the other two groups. Bcl-2 and Bax protein expression did not differ between the IR and RI-PostC + GF109203X groups. However, in the RI-PostC group, Bcl-2 protein expression was significantly higher and Bax protein expression was significantly lower than in the other two groups (P < 0.05). Changes in heart rate and mean arterial pressure were also smaller in the RI-PostC group than in the other two groups. These results indicate that RI-PostC can ameliorate myocardial ischemia-reperfusion injury and increase the Bcl-2/Bax ratio through a mechanism involving protein kinase C. PMID:28097153

Development of Thin Films as Potential Structural Cathodes to Enable Multifunctional Energy-Storage Structural Composite Batteries for the U.S. Army’s Future Force

DTIC Science & Technology

2011-09-01

glancing angle X - ray diffraction (GAXRD), atomic force microscopy (AFM), scanning electron microscopy (SEM), and electrochemical...Emission SEM FWHM full width at half maximum GAXRD glancing angle X - ray diffraction H3COCH2CH2OH 2-methoxyethanol LiMn2O4 lithium manganese oxide...were characterized by scanning electron microscopy (SEM), X - ray diffraction (XRD), and atomic force microscopy (AFM). In addition,

Realistic representation of Bacillus subtilis biofilms architecture using combined microscopy (CLSM, ESEM and FESEM).

PubMed

Bridier, A; Meylheuc, T; Briandet, R

2013-05-01

In this contribution, we used a set of microscopic techniques including confocal laser scanning microscopy (CLSM), environmental scanning electron microscopy (ESEM) and field emission scanning electron microscopy (FESEM) to analyze the three-dimensional spatial arrangement of cells and their surrounding matrix in Bacillus subtilis biofilm. The combination of the different techniques enabled a deeper and realistic deciphering of biofilm architecture by providing the opportunity to overcome the limits of each single technique. Copyright © 2013 Elsevier Ltd. All rights reserved.

Fingerprint-Based Structure Retrieval Using Electron Density

PubMed Central

Yin, Shuangye; Dokholyan, Nikolay V.

2010-01-01

We present a computational approach that can quickly search a large protein structural database to identify structures that fit a given electron density, such as determined by cryo-electron microscopy. We use geometric invariants (fingerprints) constructed using 3D Zernike moments to describe the electron density, and reduce the problem of fitting of the structure to the electron density to simple fingerprint comparison. Using this approach, we are able to screen the entire Protein Data Bank and identify structures that fit two experimental electron densities determined by cryo-electron microscopy. PMID:21287628

Fingerprint-based structure retrieval using electron density.

PubMed

Yin, Shuangye; Dokholyan, Nikolay V

2011-03-01

We present a computational approach that can quickly search a large protein structural database to identify structures that fit a given electron density, such as determined by cryo-electron microscopy. We use geometric invariants (fingerprints) constructed using 3D Zernike moments to describe the electron density, and reduce the problem of fitting of the structure to the electron density to simple fingerprint comparison. Using this approach, we are able to screen the entire Protein Data Bank and identify structures that fit two experimental electron densities determined by cryo-electron microscopy. Copyright © 2010 Wiley-Liss, Inc.

Ultra-thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures.

PubMed

Belu, A; Schnitker, J; Bertazzo, S; Neumann, E; Mayer, D; Offenhäusser, A; Santoro, F

2016-07-01

The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin-infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge-like morphology of nondistinguishable intracellular compartments. Resin-infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell-cell and cell-surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra-thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three-dimensional features by scanning electron microscopy. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

A national facility for biological cryo-electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saibil, Helen R., E-mail: h.saibil@mail.cryst.bbk.ac.uk; Grünewald, Kay; Stuart, David I.

2015-01-01

This review provides a brief update on the use of cryo-electron microscopy for integrated structural biology, along with an overview of the plans for the UK national facility for electron microscopy being built at the Diamond synchrotron. Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided ofmore » the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.« less

Electron Energy Loss Spectral Imaging of TiC Formed by Supernovae: A Scanning Transmission Electron Microscopy Study of Grain Formation and Alteration Mechanisms

NASA Astrophysics Data System (ADS)

Daulton, T. L.; Bernatowicz, T. J.; Croat, T. K.

2012-03-01

Micrometer-sized spherules of graphite formed by supernovae contain numerous TiC and Fe-Ni subgrains. These subgrains often have disordered surface rims. The mechanism(s) of rim formation on these subgrains is studied by transmission electron microscopy.

Characterization of Discontinuous Coarsening Reaction Products in INCONEL® Alloy 740H® Fusion Welds

NASA Astrophysics Data System (ADS)

Bechetti, Daniel H.; Dupont, John N.; Watanabe, Masashi; de Barbadillo, John J.

2017-04-01

Characterization of γ' coarsened zones (CZs) in alloy 740H fusion welds via a variety of electron microscopy techniques was conducted. The effects of solute partitioning during nonequilibrium solidification on the amount of strengthening precipitates along the grain boundaries were evaluated via electron-probe microanalysis and scanning electron microscopy. Electron backscatter diffraction was used to present evidence for the preferential growth of CZs toward regions of lower γ' content, even if growth in that direction increases grain boundary area. Scanning electron microscopy and image analysis were used to quantify the propensity for CZs to develop along certain segments of the grain boundaries, as governed by the local variations in γ' content. Scanning transmission electron microscopy with X-ray energy-dispersive spectrometry (XEDS) was used to assess the compositions of the matrix and precipitate phases within the CZs and to quantify the segregation of alloying components to the reaction front. Thermodynamic and kinetic modeling were used to compare calculated and experimental compositions. The work presented here provides new insight into the progression of the discontinuous coarsening (DC) reaction in a complex engineering alloy.

Characterization of Sulfur and Nanostructured Sulfur Battery Cathodes in Electron Microscopy Without Sublimation Artifacts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levin, Barnaby D. A.; Zachman, Michael J.; Werner, Jörg G.

Abstract Lithium sulfur (Li–S) batteries have the potential to provide higher energy storage density at lower cost than conventional lithium ion batteries. A key challenge for Li–S batteries is the loss of sulfur to the electrolyte during cycling. This loss can be mitigated by sequestering the sulfur in nanostructured carbon–sulfur composites. The nanoscale characterization of the sulfur distribution within these complex nanostructured electrodes is normally performed by electron microscopy, but sulfur sublimates and redistributes in the high-vacuum conditions of conventional electron microscopes. The resulting sublimation artifacts render characterization of sulfur in conventional electron microscopes problematic and unreliable. Here, we demonstratemore » two techniques, cryogenic transmission electron microscopy (cryo-TEM) and scanning electron microscopy in air (airSEM), that enable the reliable characterization of sulfur across multiple length scales by suppressing sulfur sublimation. We use cryo-TEM and airSEM to examine carbon–sulfur composites synthesized for use as Li–S battery cathodes, noting several cases where the commonly employed sulfur melt infusion method is highly inefficient at infiltrating sulfur into porous carbon hosts.« less

Insights into radiation damage from atomic resolution scanning transmission electron microscopy imaging of mono-layer CuPcCl16 films on graphene.

PubMed

Mittelberger, Andreas; Kramberger, Christian; Meyer, Jannik C

2018-03-19

Atomically resolved images of monolayer organic crystals have only been obtained with scanning probe methods so far. On the one hand, they are usually prepared on surfaces of bulk materials, which are not accessible by (scanning) transmission electron microscopy. On the other hand, the critical electron dose of a monolayer organic crystal is orders of magnitudes lower than the one for bulk crystals, making (scanning) transmission electron microscopy characterization very challenging. In this work we present an atomically resolved study on the dynamics of a monolayer CuPcCl 16 crystal under the electron beam as well as an image of the undamaged molecules obtained by low-dose electron microscopy. The results show the dynamics and the radiation damage mechanisms in the 2D layer of this material, complementing what has been found for bulk crystals in earlier studies. Furthermore, being able to image the undamaged molecular crystal allows the characterization of new composites consisting of 2D materials and organic molecules.

Environmental scanning electron microscopy in cell biology.

PubMed

McGregor, J E; Staniewicz, L T L; Guthrie Neé Kirk, S E; Donald, A M

2013-01-01

Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

Optimisations and Challenges Involved in the Creation of Various Bioluminescent and Fluorescent Influenza A Virus Strains for In Vitro and In Vivo Applications

PubMed Central

Herfst, Sander; Bestebroer, Theo M.; Vaes, Vincent P.; van der Hoeven, Barbara; Koster, Abraham J.; Kremers, Gert-Jan; Scott, Dana P.; Gultyaev, Alexander P.; Sorell, Erin M.; de Graaf, Miranda; Bárcena, Montserrat; Rimmelzwaan, Guus F.; Fouchier, Ron A.

2015-01-01

Bioluminescent and fluorescent influenza A viruses offer new opportunities to study influenza virus replication, tropism and pathogenesis. To date, several influenza A reporter viruses have been described. These strategies typically focused on a single reporter gene (either bioluminescent or fluorescent) in a single virus backbone. However, whilst bioluminescence is suited to in vivo imaging, fluorescent viruses are more appropriate for microscopy. Therefore, the idea l reporter virus varies depending on the experiment in question, and it is important that any reporter virus strategy can be adapted accordingly. Herein, a strategy was developed to create five different reporter viruses in a single virus backbone. Specifically, enhanced green fluorescent protein (eGFP), far-red fluorescent protein (fRFP), near-infrared fluorescent protein (iRFP), Gaussia luciferase (gLUC) and firefly luciferase (fLUC) were inserted into the PA gene segment of A/PR/8/34 (H1N1). This study provides a comprehensive characterisation of the effects of different reporter genes on influenza virus replication and reporter activity. In vivo reporter gene expression, in lung tissues, was only detected for eGFP, fRFP and gLUC expressing viruses. In vitro, the eGFP-expressing virus displayed the best reporter stability and could be used for correlative light electron microscopy (CLEM). This strategy was then used to create eGFP-expressing viruses consisting entirely of pandemic H1N1, highly pathogenic avian influenza (HPAI) H5N1 and H7N9. The HPAI H5N1 eGFP-expressing virus infected mice and reporter gene expression was detected, in lung tissues, in vivo. Thus, this study provides new tools and insights for the creation of bioluminescent and fluorescent influenza A reporter viruses. PMID:26241861

Sensitivity of Aspergillus nidulans to the Cellulose Synthase Inhibitor Dichlobenil: Insights from Wall-Related Genes’ Expression and Ultrastructural Hyphal Morphologies

PubMed Central

Obersriebnig, Michael; Salerno, Marco; Pum, Dietmar; Strauss, Joseph

2013-01-01

The fungal cell wall constitutes an important target for the development of antifungal drugs, because of its central role in morphogenesis, development and determination of fungal-specific molecular features. Fungal walls are characterized by a network of interconnected glycoproteins and polysaccharides, namely α-, β-glucans and chitin. Cell walls promptly and dynamically respond to environmental stimuli by a signaling mechanism, which triggers, among other responses, modulations in wall biosynthetic genes’ expression. Despite the absence of cellulose in the wall of the model filamentous fungus Aspergillus nidulans, we found in this study that fungal growth, spore germination and morphology are affected by the addition of the cellulose synthase inhibitor dichlobenil. Expression analysis of selected genes putatively involved in cell wall biosynthesis, carried out at different time points of drug exposure (i.e. 0, 1, 3, 6 and 24 h), revealed increased expression for the putative mixed linkage β-1,3;1,4 glucan synthase celA together with the β-1,3-glucan synthase fksA and the Rho-related GTPase rhoA. We also compared these data with the response to Congo Red, a known plant/fungal drug affecting both chitin and cellulose biosynthesis. The two drugs exerted different effects at the cell wall level, as shown by gene expression analysis and the ultrastructural features observed through atomic force microscopy and scanning electron microscopy. Although the concentration of dichlobenil required to affect growth of A. nidulans is approximately 10-fold higher than that required to inhibit plant cellulose biosynthesis, our work for the first time demonstrates that a cellulose biosynthesis inhibitor affects fungal growth, changes fungal morphology and expression of genes connected to fungal cell wall biosynthesis. PMID:24312197

Angiogenesis and phenotypic alteration of alveolar capillary endothelium in areas of neoplastic cell spread in primary lung adenocarcinoma.

PubMed

Jin, E; Ghazizadeh, M; Fujiwara, M; Nagashima, M; Shimizu, H; Ohaki, Y; Arai, S; Gomibuchi, M; Takemura, T; Kawanami, O

2001-09-01

Normal alveolar capillary endothelium is quiescent in nature and displays anticoagulant thrombomodulin (TM) on its surface. The cytoplasms of these endothelial cells are ultrastructurally non-fenestrated type, and they barely express von Willebrand factor (vWf). Alveolar fibrosis is accompanied by a capillary endothelium reactive for vWf, and a loss of TM expression. In primary lung adenocarcinoma, neovascularization occurs in association with alveolar fibrosis. In order to study basic factors related to angiogenesis and phenotypic changes of the capillaries located in tumor-bearing alveolar walls, we examined 37 primary lung adenocarcinomas with electron microscopy and confocal laser scanning microscopy with antibodies for TM, vWf, vascular endothelial growth factor (VEGF), and its receptors (KDR and Flt-1), and proliferating markers (Ki-67/proliferating cell nuclear antigen). Tissues microdissected specifically from alveolar walls were used for reverse transcription-polymerase chain reaction (RT-PCR) to assess expressions of mRNA isoforms of VEGF and its receptors. New capillary branching was found by ultrastructural study in the alveolar walls in 12% of the patients. Nuclei of the capillary endothelial cells were reactive for proliferating cell markers. Endothelial fenestrae were developed in 65% of the patients, TM reactivity was lost in the alveolar capillaries, and their cell cytoplasms obtained a reactivity for vWf through a transitional mosaic-like distribution pattern of both antigens. Besides cytoplasmic VEGF expression in neoplastic cells, tumor-bearing alveolar walls showed significant expression of mRNA of VEGF165 and KDR. These findings imply that angiogenesis and phenotypic changes of the alveolar capillaries are closely related to a higher expression of tumor-associated VEGF165 and of KDR in the alveolar walls in primary lung adenocarcinoma.

Software electron counting for low-dose scanning transmission electron microscopy.

PubMed

Mittelberger, Andreas; Kramberger, Christian; Meyer, Jannik C

2018-05-01

The performance of the detector is of key importance for low-dose imaging in transmission electron microscopy, and counting every single electron can be considered as the ultimate goal. In scanning transmission electron microscopy, low-dose imaging can be realized by very fast scanning, however, this also introduces artifacts and a loss of resolution in the scan direction. We have developed a software approach to correct for artifacts introduced by fast scans, making use of a scintillator and photomultiplier response that extends over several pixels. The parameters for this correction can be directly extracted from the raw image. Finally, the images can be converted into electron counts. This approach enables low-dose imaging in the scanning transmission electron microscope via high scan speeds while retaining the image quality of artifact-free slower scans. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

[Structure and function of suburothelial myofibroblasts in the human urinary bladder under normal and pathological conditions].

PubMed

Neuhaus, J; Heinrich, M; Schlichting, N; Oberbach, A; Fitzl, G; Schwalenberg, T; Horn, L-C; Stolzenburg, J-U

2007-09-01

Myofibroblasts play a pivotal role in numerous pathological alterations. Clarification of the structure and function and of the cellular plasticity of this cell type in the bladder may lead to new insights into the pathogenesis of lower urinary tract disorders. Bladder biopsies from patients with bladder carcinoma and interstitial cystitis were used to analyse the morphology and receptor expression using confocal immunofluorescence and electron microscopy. Cytokine effects and coupling behavior were tested in cultured myofibroblasts and detrusor smooth muscle cells. Myofibroblasts are in close contact with the suburothelial capillary network. They express Cx43 and form functional syncytia. The expression of muscarinic and purinergic receptors is highly variable. Dye coupling experiments showed differences to detrusor myocytes. Upregulation of smooth muscle cell alpha-actin and/or transdifferentiation into smooth muscle cells may contribute to the etiology of urge incontinence. A multi-step model is presented as a working hypothesis.

High ordered biomineralization induced by carbon nanoparticles in the sea urchin Paracentrotus lividus.

PubMed

Manno, Daniela; Carata, Elisabetta; Tenuzzo, Bernadetta A; Panzarini, Elisa; Buccolieri, Alessandro; Filippo, Emanuela; Rossi, Marco; Serra, Antonio; Dini, Luciana

2012-12-14

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

High ordered biomineralization induced by carbon nanoparticles in the sea urchin Paracentrotus lividus

NASA Astrophysics Data System (ADS)

Manno, Daniela; Carata, Elisabetta; Tenuzzo, Bernadetta A.; Panzarini, Elisa; Buccolieri, Alessandro; Filippo, Emanuela; Rossi, Marco; Serra, Antonio; Dini, Luciana

2012-12-01

A surprising and unexpected biomineralization process was observed during toxicological assessment of carbon nanoparticles on Paracentrotus lividus (sea urchin) pluteus larvae. The larvae activate a process of defense against external material, by incorporating the nanoparticles into microstructures of aragonite similarly to pearl oysters. Aiming at a better understanding of this phenomenon, the larvae were exposed to increasing concentrations of carbon nanoparticles and the biomineralization products were analyzed by electron microscopy, x-ray diffraction and Raman spectroscopy. In order to evaluate the possible influence of Sp-CyP-1 expression on this biomineralization process by larvae, analyses of gene expression (Sp-CyP-1) and calcein labeling were performed. Overall, we report experimental evidence about the capability of carbon nanoparticles to induce an increment of Sp-CyP-1 expression with the consequent activation of a biomineralization process leading to the production of a new pearl-like biomaterial never previously observed in sea urchins.

Mycoparasitism studies of Trichoderma harzianum against Sclerotinia sclerotiorum: evaluation of antagonism and expression of cell wall-degrading enzymes genes.

PubMed

Troian, Rogério Fraga; Steindorff, Andrei Stecca; Ramada, Marcelo Henrique Soller; Arruda, Walquiria; Ulhoa, Cirano José

2014-10-01

Trichoderma spp. are known for their biocontrol activity against several plant pathogens. A specific isolate of Trichoderma harzianum, 303/02, has the potential to inhibit the growth of Sclerotinia sclerotiorum, an important agent involved in several crop diseases. In this study, the interaction between T. harzianum 303/02 and mycelia, sclerotia and apothecia of S. sclerotiorum was studied by scanning electron microscopy. RT-qPCR was used to examine the expression of 11 genes potentially involved in biocontrol. T. harzianum 303/02 parasitizes S. sclerotiorum by forming branches that coil around the hyphae. The fungus multiplied abundantly at the sclerotia and apothecia surface, forming a dense mycelium that penetrated the inner surface of these structures. The levels of gene expression varied according to the type of structure with which T. harzianum was interacting. The data also showed the presence of synergistic action between the cell-wall degrading enzymes.

Correlative light-electron fractography for fatigue striations characterization in metallic alloys.

PubMed

Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

2013-09-01

The correlative light-electron fractography technique combines correlative microscopy concepts to the extended depth-from-focus reconstruction method, associating the reliable topographic information of 3-D maps from light microscopy ordered Z-stacks to the finest lateral resolution and large focus depth from scanning electron microscopy. Fatigue striations spacing analysis can be precisely measured, by correcting the mean surface tilting with the knowledge of local elevation data from elevation maps. This new technique aims to improve the accuracy of quantitative fractography in fatigue fracture investigations. Copyright © 2013 Wiley Periodicals, Inc.

Transmission Electron Microscopy Analysis of Skin Lesions from Sporotrichosis Epidemic in Rio de Janeiro, Brazil

PubMed Central

Porto Ferreira, Cassio; Oliveira de Almeida, Ana Cristina; Corte-Real, Suzana

2015-01-01

Transmission electron microscopy can yield useful information in a range of scientific fields; it is capable of imaging at a significantly higher resolution than light microscopes and has been a very useful tool in the identification of morphological changes of the dermis as well as assessment of changes in the extracellular matrix. Our aim is to characterize by electron microscopy the cellular profile of lesions caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form that occurs in Rio de Janeiro, Brazil. PMID:25653392

Relative merits and limiting factors for x-ray and electron microscopy of thick, hydrated organic materials

DOE PAGES

Du, Ming; Jacobsen, Chris

2017-10-07

Electron and x-ray microscopes allow one to image the entire, unlabeled structure of hydrated materials at a resolution well beyond what visible light microscopes can achieve. However, both approaches involve ionizing radiation, so that radiation damage must be considered as one of the limits to imaging. Drawing upon earlier work, we describe here a unified approach to estimating the image contrast (and thus the required exposure and corresponding radiation dose) in both x-ray and electron microscopy. This approach accounts for factors such as plural and inelastic scattering, and (in electron microscopy) the use of energy filters to obtain so-called "zeromore » loss" images. As expected, it shows that electron microscopy offers lower dose for specimens thinner than about 1 mu m (such as for studies of macromolecules, viruses, bacteria and archaebacteria, and thin sectioned material), while x-ray microscopy offers superior characteristics for imaging thicker specimen such as whole eukaryotic cells, thick-sectioned tissues, and organs. The required radiation dose scales strongly as a function of the desired spatial resolution, allowing one to understand the limits of live and frozen hydrated specimen imaging. Lastly, we consider the factors limiting x-ray microscopy of thicker materials, suggesting that specimens as thick as a whole mouse brain can be imaged with x-ray microscopes without significant image degradation should appropriate image reconstruction methods be identified.« less

Relative merits and limiting factors for x-ray and electron microscopy of thick, hydrated organic materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Ming; Jacobsen, Chris

Electron and x-ray microscopes allow one to image the entire, unlabeled structure of hydrated materials at a resolution well beyond what visible light microscopes can achieve. However, both approaches involve ionizing radiation, so that radiation damage must be considered as one of the limits to imaging. Drawing upon earlier work, we describe here a unified approach to estimating the image contrast (and thus the required exposure and corresponding radiation dose) in both x-ray and electron microscopy. This approach accounts for factors such as plural and inelastic scattering, and (in electron microscopy) the use of energy filters to obtain so-called "zeromore » loss" images. As expected, it shows that electron microscopy offers lower dose for specimens thinner than about 1 mu m (such as for studies of macromolecules, viruses, bacteria and archaebacteria, and thin sectioned material), while x-ray microscopy offers superior characteristics for imaging thicker specimen such as whole eukaryotic cells, thick-sectioned tissues, and organs. The required radiation dose scales strongly as a function of the desired spatial resolution, allowing one to understand the limits of live and frozen hydrated specimen imaging. Lastly, we consider the factors limiting x-ray microscopy of thicker materials, suggesting that specimens as thick as a whole mouse brain can be imaged with x-ray microscopes without significant image degradation should appropriate image reconstruction methods be identified.« less

Microscopy with slow electrons: from LEEM to XPEEM

ScienceCinema

Bauer, Ernst [Arizona State University, Phoenix, Arizona, United States

2017-12-09

The short penetration and escape depth of electrons with energies below 1 keV make them ideally suited for the study of surfaces and ultrathin films. The combination of the low energy electrons and the high lateral resolution of a microscope produces a powerful method for the characterization of nanostructures on bulk samples, in particular if the microscope is equipped with an imaging energy filter and connected to a synchrotron radiation source. Comprehensive characterization by imaging, diffraction, and spectroscope of the structural, chemical, and magnetic properties is then possible. The Talk will describe the various imaging techniques in using reflected and emitted electrons in low-energy electron microscopy (LEEM) and x-ray photoemission electron microscopy (XPEEM), with an emphasis on magnetic materials with spin-polarized LEEM and x-ray magnetic circular dichroism PEEM. The talk with end with an outlook on future possibilities.

Structure refinement of the δ1p phase in the Fe-Zn system by single-crystal X-ray diffraction combined with scanning transmission electron microscopy.

PubMed

Okamoto, Norihiko L; Tanaka, Katsushi; Yasuhara, Akira; Inui, Haruyuki

2014-04-01

The structure of the δ1p phase in the iron-zinc system has been refined by single-crystal synchrotron X-ray diffraction combined with scanning transmission electron microscopy. The large hexagonal unit cell of the δ1p phase with the space group of P63/mmc comprises more or less regular (normal) Zn12 icosahedra, disordered Zn12 icosahedra, Zn16 icosioctahedra and dangling Zn atoms that do not constitute any polyhedra. The unit cell contains 52 Fe and 504 Zn atoms so that the compound is expressed with the chemical formula of Fe13Zn126. All Fe atoms exclusively occupy the centre of normal and disordered icosahedra. Iron-centred normal icosahedra are linked to one another by face- and vertex-sharing forming two types of basal slabs, which are bridged with each other by face-sharing with icosioctahedra, whereas disordered icosahedra with positional disorder at their vertex sites are isolated from other polyhedra. The bonding features in the δ1p phase are discussed in comparison with those in the Γ and ζ phases in the iron-zinc system.

Electron microscopy of lamin and the nuclear lamina in Caenorhabditis elegans.

PubMed

Cohen, Merav; Santarella, Rachel; Wiesel, Naama; Mattaj, Iain; Gruenbaum, Yosef

2008-01-01

The nuclear lamina is found between the inner nuclear membrane and the peripheral chromatin. Lamins are the main components of the nuclear lamina, where they form protein complexes with integral proteins of the inner nuclear membrane, transcriptional regulators, histones and chromatin modifiers. Lamins are required for mechanical stability, chromatin organization, Pol II transcription, DNA replication, nuclear assembly, and nuclear positioning. Mutations in human lamins cause at least 13 distinct human diseases, collectively termed laminopathies, affecting muscle, adipose, bone, nerve and skin cells, and range from muscular dystrophies to accelerated aging. Caenorhabditis elegans has unique advantages in studying lamins and nuclear lamina genes including low complexity of lamina genes and the unique ability of bacterially expressed C. elegans lamin protein to form stable 10 nm fibers. In addition, transgenic techniques, simple application of RNA interference, sophisticated genetic analyses, and the production of a large collection of mutant lines, all make C. elegans especially attractive for studying the functions of its nuclear lamina genes. In this chapter we will include a short review of our current knowledge of nuclear lamina in C. elegans and will describe electron microscopy techniques used for their analyses.

Direct extraction of lead (II) from untreated human blood serum using restricted access carbon nanotubes and its determination by atomic absorption spectrometry.

PubMed

Barbosa, Valéria Maria Pereira; Barbosa, Adriano Francisco; Bettini, Jefferson; Luccas, Pedro Orival; Figueiredo, Eduardo Costa

2016-01-15

Oxidized carbon nanotubes were covered with layers of bovine serum albumin to result in so-called restricted-access carbon nanotubes (RACNTs). This material can extract Pb(2+) ions directly from untreated human blood serum while excluding all the serum proteins. The RACNTs have a protein exclusion capacity of almost 100% and a maximum Pb(2+) adsorption capacity of 34.5mg g(-1). High resolution transmission electron microscopy, scanning transmission electron microscopy and energy dispersive spectroscopy were used to confirm the BSA layer and Pb(2+) adsorption sites. A mini-column filled with RACNTs was used in an on-line solid phase extraction system coupled to a thermospray flame furnace atomic absorption spectrometry. At optimized experimental conditions, the method has a detection limit as low as 2.1µg L(-1), an enrichment factor of 5.5, and inter- and intra-day precisions (expressed as relative standard deviation) of <8.1%. Recoveries of the Pb(2+) spiked samples ranged from 89.4% to 107.3% for the extraction from untreated human blood serum. Copyright © 2015 Elsevier B.V. All rights reserved.

Petrological features of selected components of the Cergowa sandstones (Outer Carpathians) recorded by scanning electron microscopy - preliminary study

NASA Astrophysics Data System (ADS)

Pszonka, Joanna

2017-11-01

The scanning electron microscope analysis of the Cergowa sandstones brings new data on their petrological features and chemical composition. Previous work in standard petrographic examination, e.g. polarising (PL) or cathodoluminescence (CL) microscopy, displayed limited information on grain surface topography and only assumptions to their geochemistry. Both identification and characterisation of minerals are fundamental in the progress of mining and minerals processing systems. Detrital grains of the Cergowa sandstones are bound by calcite and dolomitic cement and commonly corroded by diagenetic fluids, however, in varying degrees, which is illustrated here by feldspar, quartz and dolomite minerals. Dissolution processes of marginal parts of these mineral grains resulted in corrosion, which increased the contact surface between the grains and the cement. The difference in resistance to these processes was observed not only among distinct groups of minerals, but also within the group of feldspars: between K-feldspars and minerals of plagioclase. That combination resulted in exceptionally strong cementation of the Cergowa sandstones, which is expressed by their high hardness and resistance to abrasion, freezing, and thawing. Inherent parameters of sandstones are characterised by their petrographical properties.

Microscopy image segmentation tool: Robust image data analysis

NASA Astrophysics Data System (ADS)

Valmianski, Ilya; Monton, Carlos; Schuller, Ivan K.

2014-03-01

We present a software package called Microscopy Image Segmentation Tool (MIST). MIST is designed for analysis of microscopy images which contain large collections of small regions of interest (ROIs). Originally developed for analysis of porous anodic alumina scanning electron images, MIST capabilities have been expanded to allow use in a large variety of problems including analysis of biological tissue, inorganic and organic film grain structure, as well as nano- and meso-scopic structures. MIST provides a robust segmentation algorithm for the ROIs, includes many useful analysis capabilities, and is highly flexible allowing incorporation of specialized user developed analysis. We describe the unique advantages MIST has over existing analysis software. In addition, we present a number of diverse applications to scanning electron microscopy, atomic force microscopy, magnetic force microscopy, scanning tunneling microscopy, and fluorescent confocal laser scanning microscopy.

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

PubMed Central

Karreman, Matthia A.; Mercier, Luc; Schieber, Nicole L.; Shibue, Tsukasa; Schwab, Yannick; Goetz, Jacky G.

2014-01-01

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. PMID:25479106

Host Cell Responses to Persistent Mycoplasmas - Different Stages in Infection of HeLa Cells with Mycoplasma hominis

PubMed Central

Hopfe, Miriam; Deenen, René; Degrandi, Daniel; Köhrer, Karl; Henrich, Birgit

2013-01-01

Mycoplasma hominis is a facultative human pathogen primarily associated with bacterial vaginosis and pelvic inflammatory disease, but it is also able to spread to other sites, leading to arthritis or, in neonates, meningitis. With a minimal set of 537 annotated genes, M. hominis is the second smallest self-replicating mycoplasma and thus an ideal model organism for studying the effects of an infectious agent on its host more closely. M. hominis adherence, colonisation and invasion of HeLa cells were characterised in a time-course study using scanning electron microscopy, confocal microscopy and microarray-based analysis of the HeLa cell transcriptome. At 4 h post infection, cytoadherence of M. hominis to the HeLa cell surface was accompanied by differential regulation of 723 host genes (>2 fold change in expression). Genes associated with immune responses and signal transduction pathways were mainly affected and components involved in cell-cycle regulation, growth and death were highly upregulated. At 48 h post infection, when mycoplasma invasion started, 1588 host genes were differentially expressed and expression of genes for lysosome-specific proteins associated with bacterial lysis was detected. In a chronically infected HeLa cell line (2 weeks), the proportion of intracellular mycoplasmas reached a maximum of 10% and M. hominis-filled protrusions of the host cell membrane were seen by confocal microscopy, suggesting exocytotic dissemination. Of the 1972 regulated host genes, components of the ECM-receptor interaction pathway and phagosome-related integrins were markedly increased. The immune response was quite different to that at the beginning of infection, with a prominent induction of IL1B gene expression, affecting pathways of MAPK signalling, and genes connected with cytokine-cytokine interactions and apoptosis. These data show for the first time the complex, time-dependent reaction of the host directed at mycoplasmal clearance and the counter measures of this pestering pathogen. PMID:23326599

Publications - GMC 58 | Alaska Division of Geological & Geophysical Surveys

Science.gov Websites

DGGS GMC 58 Publication Details Title: X-ray diffraction and scanning electron microscopy mineral , Michael, and Core Laboratories, 1985, X-ray diffraction and scanning electron microscopy mineral analyses

Nanodiamond Landmarks for Subcellular Multimodal Optical and Electron Imaging

PubMed Central

Zurbuchen, Mark A.; Lake, Michael P.; Kohan, Sirus A.; Leung, Belinda; Bouchard, Louis-S.

2013-01-01

There is a growing need for biolabels that can be used in both optical and electron microscopies, are non-cytotoxic, and do not photobleach. Such biolabels could enable targeted nanoscale imaging of sub-cellular structures, and help to establish correlations between conjugation-delivered biomolecules and function. Here we demonstrate a sub-cellular multi-modal imaging methodology that enables localization of inert particulate probes, consisting of nanodiamonds having fluorescent nitrogen-vacancy centers. These are functionalized to target specific structures, and are observable by both optical and electron microscopies. Nanodiamonds targeted to the nuclear pore complex are rapidly localized in electron-microscopy diffraction mode to enable “zooming-in” to regions of interest for detailed structural investigations. Optical microscopies reveal nanodiamonds for in-vitro tracking or uptake-confirmation. The approach is general, works down to the single nanodiamond level, and can leverage the unique capabilities of nanodiamonds, such as biocompatibility, sensitive magnetometry, and gene and drug delivery. PMID:24036840

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

PubMed Central

Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong; Dillard, Rebecca S; Hammonds, Jason E; Alonas, Eric; Desai, Tanay M; Marin, Mariana; Storms, Rachel E; Leon, Fredrick; Melikyan, Gregory B; Santangelo, Philip J; Spearman, Paul W; Wright, Elizabeth R

2016-01-01

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM. PMID:27977021

In situ transmission electron microscopy of transistor operation and failure.

PubMed

Wang, Baoming; Islam, Zahabul; Haque, Aman; Chabak, Kelson; Snure, Michael; Heller, Eric; Glavin, Nicholas

2018-08-03

Microscopy is typically used as a post-mortem analytical tool in performance and reliability studies on nanoscale materials and devices. In this study, we demonstrate real time microscopy of the operation and failure of AlGaN/GaN high electron mobility transistors inside the transmission electron microscope. Loading until failure was performed on the electron transparent transistors to visualize the failure mechanisms caused by self-heating. At lower drain voltages, thermo-mechanical stresses induce irreversible microstructural deformation, mostly along the AlGaN/GaN interface, to initiate the damage process. At higher biasing, the self-heating deteriorates the gate and catastrophic failure takes place through metal/semiconductor inter-diffusion and/or buffer layer breakdown. This study indicates that the current trend of recreating the events, from damage nucleation to catastrophic failure, can be replaced by in situ microscopy for a quick and accurate account of the failure mechanisms.

The formation of the light-sensing compartment of cone photoreceptors coincides with a transcriptional switch

PubMed Central

Daum, Janine M; Keles, Özkan; Holwerda, Sjoerd JB; Kohler, Hubertus; Rijli, Filippo M

2017-01-01

High-resolution daylight vision is mediated by cone photoreceptors. The molecular program responsible for the formation of their light sensor, the outer segment, is not well understood. We correlated daily changes in ultrastructure and gene expression in postmitotic mouse cones, between birth and eye opening, using serial block-face electron microscopy (EM) and RNA sequencing. Outer segments appeared rapidly at postnatal day six and their appearance coincided with a switch in gene expression. The switch affected over 14% of all expressed genes. Genes that switched off were rich in transcription factors and neurogenic genes. Those that switched on contained genes relevant for cone function. Chromatin rearrangements in enhancer regions occurred before the switch was completed, but not after. We provide a resource comprised of correlated EM, RNAseq, and ATACseq data, showing that the growth of a key compartment of a postmitotic cell involves an extensive switch in gene expression and chromatin accessibility. PMID:29106373

Low-voltage electron microscopy of polymer and organic molecular thin films.

PubMed

Drummy, Lawrence F; Yang, Junyan; Martin, David C

2004-06-01

We have demonstrated the capabilities of a novel low-voltage electron microscope (LVEM) for imaging polymer and organic molecular thin films. The LVEM can operate in transmission electron microscopy, scanning transmission electron microscopy, scanning electron microscopy, and electron diffraction modes. The microscope operates at a nominal accelerating voltage of 5 kV and fits on a tabletop. A detailed discussion of the electron-sample interaction processes is presented, and the mean free path for total electron scattering was calculated to be 15 nm for organic samples at 5 kV. The total end point dose for the destruction of crystallinity at 5 kV was estimated at 5 x 10(-4) and 3.5 x 10(-2) C/cm2 for polyethylene and pentacene, respectively. These values are significantly lower than those measured at voltages greater than 100 kV. A defocus series of colloidal gold particles allowed us to estimate the experimental contrast transfer function of the microscope. Images taken of several organic materials have shown high contrast for low atomic number elements and a resolution of 2.5 nm. The materials studied here include thin films of the organic semiconductor pentacene, triblock copolymer films, single-molecule dendrimers, electrospun polymer fibers and gold nanoparticles. Copyright 2004 Elsevier B.V.

[THE CHARACTERISTICS OF MORPHOLOGY OF BIOFILM OF PERIODONTIUM UNDER INFLAMMATORY DISEASES OF GUMS (CHRONIC CATARRHAL GINGIVITIS, CHRONIC PERIODONTITIS, CANDIDA-ASSOCIATED PERIODONTITIS) ACCORDING RESULTS OF ELECTRONIC MICROSCOPY].

PubMed

Ippolitov, E V; Didenko, L V; Tzarev, V N

2015-12-01

The study was carried out to analyze morphology of biofilm of periodontium and to develop electronic microscopic criteria of differentiated diagnostic of inflammatory diseases of gums. The scanning electronic microscopy was applied to analyze samples of bioflm of periodont from 70 patients. Including ten patients with every nosologic form of groups with chronic catarrhal periodontitis. of light, mean and severe degree, chronic catarrhal gingivitis, Candida-associated paroperiodontitis and 20 healthy persons with intact periodontium. The analysis was implemented using dual-beam scanning electronic microscope Quanta 200 3D (FEI company, USA) and walk-through electronic micJEM 100B (JEOL, Japan). To detect marker DNA of periodont pathogenic bacteria in analyzed samples the kit of reagentsfor polymerase chain reaction "MultiDent-5" ("GenLab", Russia). The scanning electronic microscopy in combination with transmission electronic microscopy and polymerase chain reaction permits analyzing structure, composition and degree of development of biofilm of periodontium and to apply differentiated diagnostic of different nosologic forms of inflammatory diseases of periodontium, including light form of chronic periodontitis and gingivitis. The electronic microscopical indications of diseases ofperiodontium of inflammatory character are established: catarrhal gingivitis, (coccal morphological alternate), chronic periodontitis (bacillary morphological alternate), Candida-associated periodontitis (Candida morphological alternate of biofilm ofperiodontium).

Molecular expressions: exploring the world of optics and microscopy. http://microscopy.fsu.edu.

PubMed

Eliceiri, Kevin W

2004-08-01

Our knowledge of the structure, dynamics and physiology of a cell has increased significantly in the last ten years through the emergence of new optical imaging modalities such as optical sectioning microscopy, computer- enhanced video microscopy and laser-scanning microscopy. These techniques together with the use of genetically engineered fluorophores have helped scientists visualize the 3-dimensional dynamic processes of living cells. However as powerful as these imaging tools are, they can often be difficult to understand and fully utilize. Below I will discuss my favorite website: The Molecular Expressions Web Site that endeavors to present the power of microscopy to its visitors. The Molecular Expressions group does a remarkable job of not only clearly presenting the principles behind these techniques in a manner approachable by lay and scientific audiences alike but also provides representative data from each as well.

Ultrastructural instability of paired helical filaments from corticobasal degeneration as examined by scanning transmission electron microscopy.

PubMed Central

Ksiezak-Reding, H.; Tracz, E.; Yang, L. S.; Dickson, D. W.; Simon, M.; Wall, J. S.

1996-01-01

Paired helical filaments (PHFs) accumulate in the brains of subjects affected with Alzheimer's disease (AD) and certain other neurodegenerative disorders, including corticobasal degeneration (CBD). Electron microscope studies have shown that PHFs from CBD differ from those of AD by being wider and having a longer periodicity of the helical twist. Moreover, PHFs from CBD have been shown to be primarily composed of two rather than three highly phosphorylated polypeptides of tau (PHF-tau), with these polypeptides expressing no exons 3 and 10. To further explore the relationship between the heterogeneity of PHF-tau and the appearance of abnormal filaments, the ultrastructure and physical parameters such as mass per unit length and dimensions were compared in filaments from CBD and AD using high resolution scanning transmission electron microscopy (STEM). Filament-enriched fractions were isolated as Sarcosyl-insoluble pellets and for STEM studies, samples were freeze-dried without prior fixation or staining. Ultrastructurally, PHFs from CBD were shown to be a heterogeneous population as double- and single-stranded filaments could be identified based on their width and physical mass per unit length expressed in kilodaltons (kd) per nanometer (nm). Less abundant, double-stranded filaments had a maximal width of 29 nm and a mass per unit length of 133 kd/nm, whereas three times more abundant single-stranded filaments were 15 nm wide and bad a mass per unit length of 62 kd/nm. Double-stranded filaments also displayed a distinct axial region of less dense mass, which appeared to divide the PHFs into two protofilament-like strands. Furthermore, these filaments were frequently observed to physically separate along the long axis into two single strands or to break longitudinally. In contrast, PHFs from AD were ultrastructurally stable and uniform both in their width (22 nm) and physical mass per unit length (104 kd/nm). The ultrastructural features indicate that filaments of CBD and AD differ both in stability and packing of tau and that CBD filaments, composed of two distinct protofilaments, are more labile under STEM conditions. As fixed and stained filaments from CBD have been shown to be stable and uniform in size by conventional transmission electron microscopy, STEM studies may be particularly suitable for detecting instability of unstained and unfixed filaments. The results also suggest that molecular heterogeneity and/or post-translational modifications of tau may strongly influence the morphology and stability of abnormal filaments. Images Figure 1 Figure 2 Figure 3 PMID:8702002

Analysis of Long Bone and Vertebral Failure Patterns.

DTIC Science & Technology

1982-09-30

processes further supported the findings of • :the scanning electron microscopy studies . In the impacted animals, the cartilage surface was eroded... cartilage matrix. In the six years post-impaction group, the articular cartilage had converted to fibrocartilage instead of normal hyaline cartilage . The...columns of four rhesus monkeys have been collected and are being processed for study with light microscopy and scanning electron microscopy. The baboon

A simple method for maintaining relative positions of separate tissue elements during processing for electron microscopy.

PubMed

Stirling, C A

1978-09-01

Molten (328 K) 20% gelatin is used as a 'glue' to hold together separate tissue elements or tissue elements that may be separated when cutting small blocks of tissue for plastic embedding. Standard aldehyde and osmium fixation, dehydration and epoxy embedding are compatible with this as is semi-thin sectioning for light microscopy or thin sectioning for electron microscopy.

Symposium LL: Nanowires--Synthesis Properties Assembly and Application

DTIC Science & Technology

2010-09-10

dedicated hard x - ray microscopy beamline is operated in partnership with the Advanced Photon Source to provide fluorescence, diffraction, and...characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and X - ray diffraction (XRD) measurements, proving it to be...Investigation of Preferred Growth Direction of GaN Nanorods by Synchrotron X - ray Reciprocal Space Mapping. Yuri Sohn1, Sanghwa Lee1, Chinkyo Kim1 and Dong

STEM VQ Method, Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification

DTIC Science & Technology

2017-02-02

Corresponding Author Abstract Accurate virus quantification is sought, but a perfect method still eludes the scientific community. Electron...unlimited. UNCLASSIFIED 2 provides morphology data and counts all viral particles, including partial or noninfectious particles; however, EM methods ...consistent, reproducible virus quantification method called Scanning Transmission Electron Microscopy – Virus Quantification (STEM-VQ) which simplifies

Evaluation of a hybrid pixel detector for electron microscopy.

PubMed

Faruqi, A R; Cattermole, D M; Henderson, R; Mikulec, B; Raeburn, C

2003-04-01

We describe the application of a silicon hybrid pixel detector, containing 64 by 64 pixels, each 170 microm(2), in electron microscopy. The device offers improved resolution compared to CCDs along with faster and noiseless readout. Evaluation of the detector, carried out on a 120 kV electron microscope, demonstrates the potential of the device.

Mineralogy and Microstructures of Shock-Induced Melt Veins in Chondrites

NASA Technical Reports Server (NTRS)

Sharp, Thomas G.

2000-01-01

The applicability of phase equilibrium data to the interpretation of shock-induced melt veins can only be tested by a detailed study of melt- vein mineralogy to see how high-pressure assemblages vary as a function of shock conditions inferred from other indicators. We have used transmission electron microscopy (TEM), analytical electron microscopy (AEM), scanning electron microscopy (SEM), electron microprobe analysis (EMA) and optical petrography to characterize the mineralogy, microstructures, and compositions of melt veins and associated high-pressure minerals in shocked chondrites and SNC meteorites. In the processes, we have gained a better understanding of what melt veining can tell us about shock conditions and we have discovered new mineral phases in chondritic and SNC meteorites.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Schottky barrier height measurements of Cu/Si(001), Ag/Si(001), and Au/Si(001) interfaces utilizing ballistic electron emission microscopy and ballistic hole emission microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balsano, Robert; Matsubayashi, Akitomo; LaBella, Vincent P., E-mail: vlabella@albany.edu

2013-11-15

The Schottky barrier heights of both n and p doped Cu/Si(001), Ag/Si(001), and Au/Si(001) diodes were measured using ballistic electron emission microscopy and ballistic hole emission microscopy (BHEM), respectively. Measurements using both forward and reverse ballistic electron emission microscopy (BEEM) and (BHEM) injection conditions were performed. The Schottky barrier heights were found by fitting to a linearization of the power law form of the Bell-Kaiser BEEM model. The sum of the n-type and p-type barrier heights are in good agreement with the band gap of silicon and independent of the metal utilized. The Schottky barrier heights are found to bemore » below the region of best fit for the power law form of the BK model, demonstrating its region of validity.« less

Grinding and polishing instead of sectioning for the tissue samples with a graft: Implications for light and electron microscopy.

PubMed

Mukhamadiyarov, Rinat A; Sevostyanova, Victoria V; Shishkova, Daria K; Nokhrin, Andrey V; Sidorova, Olga D; Kutikhin, Anton G

2016-06-01

A broad use of the graft replacement requires a detailed investigation of the host-graft interaction, including both histological examination and electron microscopy. A high quality sectioning of the host tissue with a graft seems to be complicated; in addition, it is difficult to examine the same tissue area by both of the mentioned microscopy techniques. To solve these problems, we developed a new technique of epoxy resin embedding with the further grinding, polishing, and staining. Graft-containing tissues prepared by grinding and polishing preserved their structure; however, sectioning frequently required the explantation of the graft and led to tissue disintegration. Moreover, stained samples prepared by grinding and polishing may then be assessed by both light microscopy and backscattered scanning electron microscopy. Therefore, grinding and polishing outperform sectioning when applied to the tissues with a graft. Copyright © 2016 Elsevier Ltd. All rights reserved.

Injectable nanosilica-chitosan microparticles for bone regeneration applications.

PubMed

Gaihre, Bipin; Lecka-Czernik, Beata; Jayasuriya, Ambalangodage C

2018-01-01

This study was aimed at assessing the effects of silica nanopowder incorporation into chitosan-tripolyphosphate microparticles with the ultimate goal of improving their osteogenic properties. The microparticles were prepared by simple coacervation technique and silica nanopowder was added at 0% (C), 2.5% (S1), 5% (S2) and 10% (S3) (w/w) to chitosan. We observed that this simple incorporation of silica nanopowder improved the growth and proliferation of osteoblasts along the surface of the microparticles. In addition, the composite microparticles also showed the increased expression of alkaline phosphatase and osteoblast specific genes. We observed a significant increase ( p < 0.05) in the expression of alkaline phosphatase by the cells growing on all sample groups compared to the control (C) groups at day 14. The morphological characterization of these microparticles through scanning electron microscopy showed that these microparticles were well suited to be used as the injectable scaffolds with perfectly spherical shape and size. The incorporation of silica nanopowder altered the nano-roughness of the microparticles as observed through atomic force microscopy scans with roughness values going down from C to S3. The results in this study, taken together, show the potential of chitosan-tripolyphosphate-silica nanopowder microparticles for improved bone regeneration applications.

Nicotine Enhances Interspecies Relationship between Streptococcus mutans and Candida albicans.

PubMed

Liu, Shiyu; Qiu, Wei; Zhang, Keke; Zhou, Xuedong; Ren, Biao; He, Jinzhi; Xu, Xin; Cheng, Lei; Li, Mingyun

2017-01-01

Streptococcus mutans and Candida albicans are common microorganisms in the human oral cavity. The synergistic relationship between these two species has been deeply explored in many studies. In the present study, the effect of alkaloid nicotine on the interspecies between S. mutans and C. albicans is explored. We developed a dual-species biofilm model and studied biofilm biomass, biofilm structure, synthesis of extracellular polysaccharides (EPS), and expression of glucosyltransferases (Gtfs). Biofilm formation and bacterial and fungal cell numbers in dual-species biofilms increased in the presence of nicotine. More C. albicans cells were present in the dual-species biofilms in the nicotine-treated groups as determined by scanning electron microscopy. The synthesis of EPS was increased by 1 mg/ml of nicotine as detected by confocal laser scanning microscopy. The result of qRT-PCR showed gtfs expression was upregulated when 1 mg/ml of nicotine was used. We speculate that nicotine promoted the growth of S. mutans , and more S. mutans cells attracted more C. albicans cells due to the interaction between two species. Since S. mutans and C. albicans are putative pathogens for dental caries, the enhancement of the synergistic relationship by nicotine may contribute to caries development in smokers.

Neuroprotective effect of curcumin-loaded lactoferrin nano particles against rotenone induced neurotoxicity.

PubMed

Bollimpelli, V Satish; Kumar, Prashant; Kumari, Sonali; Kondapi, Anand K

2016-05-01

Curcumin is known to have neuroprotective role and possess antioxidant, anti-inflammatory activities. Rotenone, a flavonoid induced neurotoxicity in dopaminergic cells is being widely studied in Parkinson's Disease (PD) research. In the present study, curcumin loaded lactoferrin nano particles prepared by sol-oil chemistry were used to protect dopaminergic cell line SK-N-SH against rotenone induced neurotoxicity. These curcumin loaded nano particles were of 43-60 nm diameter size and around 100 nm hydrodynamic size as assessed by transmission electron microscopy, atomic force microscopy and dynamic light scattering analysis respectively. The encapsulation efficiency was 61.3% ± 2.4%. Cellular uptake of curcumin through these nano particles was confirmed by confocal imaging and spectrofluorimetric analysis. The curcumin loaded lactoferrin nanoparticles showed greater intracellular drug uptake, sustained retention and greater neuroprotection than soluble counterpart. Neuroprotective activity was characterized through viability assays and by estimating ROS levels. Furthermore rotenone induced PD like features were characterized by decrease in tyrosine hydroxylase expression and increase in α-synuclein expression. Taken together curcumin loaded lactoferrin nanoparticles could be a promising drug delivery strategy against neurotoxicity in dopaminergic neurons. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ultrastructural characterization of the new NG97ht human-derived glioma cell line using two different electron microscopy technical procedures.

PubMed

Machado, Camila Maria Longo; Zorzeto, Tatiane Queiroz; Bianco, Juares E Romero; Rosa, Renata Giardini; Genari, Selma Candelaria; Joazeiro, Paulo Pinto; Verinaud, Liana

2009-04-01

On the basis of transmission electron microscopy observations in tumor cell lines, oncologists have made innumerous diagnostic and therapeutical progresses. Following this path, the UNICAMP immunopathologies laboratory established the NG97 cell line derived from a human astrocytoma grade III, which when injected to the athymic nude mouse flank developed a grade IV astrocytoma. In this study, we focused on ultrastructural characterization of the NG97 cells after being recovered from xenotransplant (NG97ht). These cells in culture were assayed by two different electron microscopy procedures to characterize ultrastructures related to grade IV astrocytomas and to observe their structures through cell subcultivation. Additionally, comparative morphological descriptions of different cell passages in these technical procedures could be a useful tool for improving electron microscopy cell lineage protocols. Results from many cell passage observations showed ultrastructural similarities, which suggest malignant and glioblastoma phenotypes. In the first procedure, NG97ht cells were harvested and then incorporated into agarose before subjecting them to electron microscopy protocols, whereas in the second one, monolayer cells grew first on cover slides. Comparison among protocols revealed that organelles, cytoplasmatic extensions, spatial conformation of filopodia, and cell attachment to substrate were more preserved in the second procedure. Furthermore, in this latter procedure, a unique ellipsoidal structure was observed, which was already described when dealing with gliosarcoma cell line elsewhere. Therefore, these analyses demonstrated a morphological characterization of a new NG97ht cell line using electron transmission microscopy. Moreover, it has been shown that the second procedure provides more detailed information compared with the first.

Aharonov-Bohm Effect in the Photodetachment Microscopy of Hydrogen Negative Ions in an Electric Field

NASA Astrophysics Data System (ADS)

Wang, Dehua

2014-09-01

The Aharonov-Bohm (AB) effect in the photodetachment microscopy of the H- ions in an electric field has been studied on the basis of the semiclassical theory. After the H- ion is irradiated by a laser light, they provide a coherent electron source. When the detached electron is accelerated by a uniform electric field, two trajectories of a detached electron which run from the source to the same point on the detector, will interfere with each other and lead to an interference pattern in the photodetachment microscopy. After the solenoid is electrified beside the H- ion, even though no Lorentz force acts on the electron outside the solenoid, the photodetachment microscopy interference pattern on the detector is changed with the variation in the magnetic flux enclosed by the solenoid. This is caused by the AB effect. Under certain conditions, the interference pattern reaches the macroscopic dimensions and could be observed in a direct AB effect experiment. Our study can provide some predictions for the future experimental study of the AB effect in the photodetachment microscopy of negative ions.

Analysis of liquid suspensions using scanning electron microscopy in transmission: estimation of the water film thickness using Monte-Carlo simulations.

PubMed

Xiao, J; Foray, G; Masenelli-Varlot, K

2018-02-01

Environmental scanning electron microscopy (ESEM) allows the observation of liquids under specific conditions of pressure and temperature. Moreover, when working in the transmission mode, that is in scanning transmission electron microscopy (STEM), nano-objects can be analysed inside a liquid. The contrast in the images is mass-thickness dependent as in STEM-in-TEM (transmission electron microscopy) using closed cells. However, in STEM-in-ESEM, as the liquid-vapour equilibrium is kept dynamically, the thickness of the water droplet remains unknown. In this paper, the contrasts measured in the experimental images are compared with calculations using Monte-Carlo simulations in order to estimate the thickness of water. Two examples are given. On gold nanoparticles, the thickness of a thick film can be estimated thanks to a contrast inversion. On core-shell latex particles, the grey level of the shell compared with those of the core and of the water film gives a relatively precise measurement of the water film thickness. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Simulated electron beam trajectories toward a field ion microscopy specimen

NASA Astrophysics Data System (ADS)

Larson, D. J.; Camus, P. P.; Kelly, T. F.

1993-04-01

This article explores the conditions under which a directed electron beam originating nearly normal to the specimen axis can be made to impact the near-apex region of a field ion microscopy specimen in a high electric field. Electron trajectories were calculated using a modified Runge-Kutta numerical method. The results indicate that an electron beam can be directed to a specimen under typical field ion microscopy conditions using two methods: by varying initial beam tilt (less than 60 mrad) or by translating the initial beam position relative to the specimen apex (less than 5 mm). The net focusing effect of the high electric field on the electron beam can be treated, to first order, as an astigmatism and may be correctable by a post-lens deflection system.

Direct observation of iron-induced conformational changes of mitochondrial DNA by high-resolution field-emission in-lens scanning electron microscopy.

PubMed Central

Yaffee, M; Walter, P; Richter, C; Müller, M

1996-01-01

When respiring rat liver mitochondria are incubated in the presence of Fe(III) gluconate, their DNA (mtDNA) relaxes from the supercoiled to the open circular form dependent on the iron dose. Anaerobiosis or antioxidants fail to completely inhibit the unwinding. High-resolution field-emission in-lens scanning electron microscopy imaging, in concert with backscattered electron detection, pinpoints nanometer-range iron colloids bound to mtDNA isolated from iron-exposed mitochondria. High-resolution field-emission in-lens scanning electron microscopy with backscattered electron detection imaging permits simultaneous detailed visual analysis of DNA topology, iron dose-dependent mtDNA unwinding, and assessment of iron colloid formation on mtDNA strands. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 PMID:8643576

Nanosystems based on siRNA silencing HuR expression counteract diabetic retinopathy in rat.

PubMed

Amadio, Marialaura; Pascale, Alessia; Cupri, Sarha; Pignatello, Rosario; Osera, Cecilia; D Agata, Velia; D Amico, Agata Grazia; Leggio, Gian Marco; Ruozi, Barbara; Govoni, Stefano; Drago, Filippo; Bucolo, Claudio

2016-09-01

We evaluated whether specifically and directly targeting human antigen R (HuR), a member of embryonic lethal abnormal vision (ELAV) proteins family, may represent a new potential therapeutic strategy to manage diabetic retinopathy. Nanosystems loaded with siRNA silencing HuR expression (lipoplexes), consisting of solid lipid nanoparticles (SLN) and liposomes (SUV) were prepared. Photon correlation spectroscopy analysis, Zeta potential measurement and atomic force microscopy (AFM) studies were carried out to characterize the complexation of siRNA with the lipid nanocarriers. Nanosystems were evaluated by using AFM and scanning electron microscopy. The lipoplexes were injected into the eye of streptozotocin (STZ)-induced diabetic rats. Retinal HuR and VEGF levels were detected by Western blot and ELISA, respectively. Retinal histology was also carried out. The results demonstrated that retinal HuR and VEGF are significantly increased in STZ-rats and are blunted by HuR siRNA treatment. Lipoplexes with a weak positive surface charge and with a 4:1 N/P (cationic lipid nitrogen to siRNA phosphate) ratio exert a better transfection efficiency, significantly dumping retinal HuR and VEGF levels. In conclusion, we demonstrated that siRNA can be efficiently delivered into the rat retina using lipid-based nanocarriers, and some of the lipoplexes loaded with siRNA silencing HuR expression are potential candidates to manage retinal diseases. Copyright © 2016 Elsevier Ltd. All rights reserved.

The effect of collagen coating on titanium with nanotopography on in vitro osteogenesis.

PubMed

Costa, Daniel G; Ferraz, Emanuela P; Abuna, Rodrigo P F; de Oliveira, Paulo T; Morra, Marco; Beloti, Marcio M; Rosa, Adalberto L

2017-10-01

Several studies have shown the positive effects of Ti either with nanotopography or coated with collagen on osteoblast differentiation. Thus, we hypothesized that the association of nanotopography with collagen may increase the in vitro osteogenesis on Ti surface. Ti discs with nanotopography with or without collagen coating were characterized by scanning electron microscopy and atomic force microscopy. Rat calvaria-derived osteoblastic cells were cultured on both Ti surfaces for up to 14 days and the following parameters were evaluated: cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, protein expression of bone sialoprotein (BSP) and osteopontin (OPN), and gene expression of collagen type 1a (Coll1a), runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OC), Ki67, Survivin, and Bcl2-associated X protein (BAX). Surface characterization evidenced that collagen coating did not alter the nanotopography. Collagen coating increased cell proliferation, ALP activity, extracellular matrix mineralization, and Coll1a, OSX, OC, and BAX gene expression. Also, OPN and BSP proteins were strongly detected in cultures grown on both Ti surfaces. In conclusion, our results showed that the combination of nanotopography with collagen coating stimulates the early, intermediate, and final events of the in vitro osteogenesis and may be considered a potential approach to promote osseointegration of Ti implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2783-2788, 2017. © 2017 Wiley Periodicals, Inc.

MATER protein expression and intracellular localization throughout folliculogenesis and preimplantation embryo development in the bovine

PubMed Central

Pennetier, Sophie; Perreau, Christine; Uzbekova, Svetlana; Thélie, Aurore; Delaleu, Bernadette; Mermillod, Pascal; Dalbiès-Tran, Rozenn

2006-01-01

Background Mater (Maternal Antigen that Embryos Require), also known as Nalp5 (NACHT, leucine rich repeat and PYD containing 5), is an oocyte-specific maternal effect gene required for early embryonic development beyond the two-cell stage in mouse. We previously characterized the bovine orthologue MATER as an oocyte marker gene in cattle, and this gene was recently assigned to a QTL region for reproductive traits. Results Here we have analyzed gene expression during folliculogenesis and preimplantation embryo development. In situ hybridization and immunohistochemistry on bovine ovarian section revealed that both the transcript and protein are restricted to the oocyte from primary follicles onwards, and accumulate in the oocyte cytoplasm during follicle growth. In immature oocytes, cytoplasmic, and more precisely cytosolic localization of MATER was confirmed by immunohistochemistry coupled with confocal microscopy and immunogold electron microscopy. By real-time PCR, MATER messenger RNA was observed to decrease strongly during maturation, and progressively during the embryo cleavage stages; it was hardly detected in morulae and blastocysts. The protein persisted after fertilization up until the blastocyst stage, and was mostly degraded after hatching. A similar predominantly cytoplasmic localization was observed in blastomeres from embryos up to 8-cells, with an apparent concentration near the nuclear membrane. Conclusion Altogether, these expression patterns are consistent with bovine MATER protein being an oocyte specific maternal effect factor as in mouse. PMID:16753072

Cardiomyogenic Differentiation in Cardiac Myxoma Expressing Lineage-Specific Transcription Factors

PubMed Central

Kodama, Hiroaki; Hirotani, Takashi; Suzuki, Yusuke; Ogawa, Satoshi; Yamazaki, Kazuto

2002-01-01

We investigated five cases of cardiac myxoma and one case of cardiac undifferentiated sarcoma by light and electron microscopy, in situ hybridization, immunohistochemical staining, and reverse transcriptase-polymerase chain reaction for cardiomyocyte-specific transcription factors, Nkx2.5/Csx, GATA-4, MEF2, and eHAND. Conventional light microscopy revealed that cardiac myxoma and sarcoma cells presented variable cellular arrangements and different histological characteristics. Ultrastructurally, some of the myxoma cells exhibited endothelium-like or immature mesenchymal cell differentiation. Immunohistochemistry for Nkx2.5/Csx, GATA-4, and eHAND was slightly to intensely positive in all myxoma cases. MEF2 immunoreactivity was observed in all cases including the case of sarcoma, thus suggesting myogenic differentiation of myxoma or sarcoma cells. In situ hybridization for Nkx2.5/Csx also revealed that all myxoma cells, but not sarcoma cells, expressed mRNA of the cardiac homeobox gene, Nkx2.5/Csx. Furthermore, nested reverse transcriptase-polymerase chain reaction from formalin-fixed, paraffin-embedded tissue was performed and demonstrated that the Nkx2.5/Csx and eHAND gene product to be detected in all cases, and in three of six cases, respectively. In conclusion, cardiac myxoma cells were found to express various amounts of cardiomyocyte-specific transcription factor gene products at the mRNA and protein levels, thus suggesting cardiomyogenic differentiation. These results support the concept that cardiac myxoma might arise from mesenchymal cardiomyocyte progenitor cells. PMID:12163362

Reprint of: Atmospheric scanning electron microscope observes cells and tissues in open medium through silicon nitride film.

PubMed

Nishiyama, Hidetoshi; Suga, Mitsuo; Ogura, Toshihiko; Maruyama, Yuusuke; Koizumi, Mitsuru; Mio, Kazuhiro; Kitamura, Shinichi; Sato, Chikara

2010-11-01

Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry. Copyright © 2010 Elsevier Inc. All rights reserved.

Crystal growth and molecular modeling studies of inhibition of struvite by phosphocitrate.

PubMed

Wierzbicki, A; Sallis, J D; Stevens, E D; Smith, M; Sikes, C S

1997-09-01

The inhibition by phosphocitrate of struvite crystal formation and growth has been examined in the present study. Crystal growth in a gel matrix was controlled by phosphocitrate in a dose-dependent manner. The effects of inhibition were followed using scanning electron microscopy, optical microscopy, and single crystal X-ray analysis. The presence of phosphocitrate induced very strong, crystal face specific inhibition of struvite, leading to total cessation of crystal growth when sufficient concentration of the inhibitor was made available. Crystal growth studies and results from molecular modeling indicated strong affinity of phosphocitrate to (101) faces of struvite. This in turn led to an alteration in the expression of these faces and the development of a characteristic arrowhead struvite morphology. Similar changes were not observed in the presence of identical concentrations of citrate, acetohydroxamic acid, and N-sulfo-2 amino tricarballylate (an analog of phosphocitrate), emphasizing the unique interaction of phosphocitrate with the struvite crystal lattice.

Diffusion length measurements in bulk and epitaxially grown 3-5 semiconductors using charge collection microscopy

NASA Technical Reports Server (NTRS)

Leon, R. P.

1987-01-01

Diffusion lengths and surface recombination velocities were measured in GaAs diodes and InP finished solar cells. The basic techniques used was charge collection microscopy also known as electron beam induced current (EBIC). The normalized currents and distances from the pn junction were read directly from the calibrated curves obtained while using the line scan mode in an SEM. These values were then equated to integral and infinite series expressions resulting from the solution of the diffusion equation with both extended generation and point generation functions. This expands previous work by examining both thin and thick samples. The surface recombination velocity was either treated as an unknown in a system of two equations, or measured directly using low e(-) beam accelerating voltages. These techniques give accurate results by accounting for the effects of surface recombination and the finite size of the generation volume.

[Syphilis. Current physiobiological data. I. The bacteriological problem].

PubMed

Collart, P; Poitevin, M

For lack of being able to grow Treponema pallidum, the only method which allows us to study the biology of this germ and the physiopathology of this infection lies in researches in experimental syphilis. After pointing out the different aspects of Treponema pallidum, either with light microscopy or electron microscopy, the authors review the different kinds of reproduction suggested by syphiligraphs, the recent trials to cultivate the treponema, and the processes of elimination. Then, they examine the biological properties and the antigenic structure of T.p. as it has been established by comparison with cultivable spirochetes. To end with, the authors show that both the TPI test and the FTA test are two very specific reactions; these tests mean nothing but the fact that the patient has been in contact with the antigens of Treponema pallidum and the quantitative tests cannot be considered as expressing the infectious potential capacity.

Unfolded protein response activation in cataracts.

PubMed

Torres-Bernal, Beatriz E; Torres-Bernal, Luis Fernando; Gutiérrez-Campos, Rafael R; Kershenobich Stalnikowitz, David D; Barba-Gallardo, Luis Fernando; Chayet, Arturo A; Ventura-Juárez, Javier

2014-10-01

To analyze the expression of 78 kDa glucose-regulated protein (GRP78) and activating transcription factor 6 (ATF6), 2 factors in the unfolded protein response (UPR), in age-related and diabetes-associated cataract. Universidad Autónoma de Aguascalientes, Aguascalientes, México. Experimental study. The qualitative and quantitative expression of GRP78 and ATF6 were measured in surgical samples from 11 senile cataracts, 9 diabetic-associated cataracts, and 3 normal lenses. Both proteins were detected by immunofluorescence and immunogold-conjugated antibodies. Quantitative morphometry was used to analyze the differences in GRP78 and ATF6 between samples. The Mann-Whitney test was used for statistical analysis. Scanning electron microscopy showed the characteristic organization of fibers in normal lenses with regular alignment and interdigitation between them. On the other hand, lenses from eyes with senile or diabetic cataract showed the same pattern of misalignment and disorganization of the fibers. Both proteins were detected through immunofluorescence in senile and diabetic cataracts, but not in normal lenses. Immunogold-conjugated antibodies and transmission electron microscopy showed that GRP78 and ATF6 grains were 30% higher and 35% higher, respectively, in diabetic cataracts than in senile cataracts (P<.05). These data show for the first time in humans that GRP78 and ATF6 are present in lens fibers of senile cataracts and diabetic cataracts, establishing that the UPR may be important in the process of cataractogenesis. No author has a financial or proprietary interest in any material or method mentioned. Copyright © 2014 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

Inhibition of Vaccinia virus entry by a broad spectrum antiviral peptide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Altmann, S.E.; Jones, J.C.; Schultz-Cherry, S.

2009-06-05

Concerns about the possible use of Variola virus, the causative agent of smallpox, as a weapon for bioterrorism have led to renewed efforts to identify new antivirals against orthopoxviruses. We identified a peptide, EB, which inhibited infection by Vaccinia virus with an EC{sub 50} of 15 muM. A control peptide, EBX, identical in composition to EB but differing in sequence, was inactive (EC{sub 50} > 200 muM), indicating sequence specificity. The inhibition was reversed upon removal of the peptide, and EB treatment had no effect on the physical integrity of virus particles as determined by electron microscopy. Viral adsorption wasmore » unaffected by the presence of EB, and the addition of EB post-entry had no effect on viral titers or on early gene expression. The addition of EB post-adsorption resulted in the inhibition of beta-galactosidase expression from an early viral promoter with an EC{sub 50} of 45 muM. A significant reduction in virus entry was detected in the presence of the peptide when the number of viral cores released into the cytoplasm was quantified. Electron microscopy indicated that 88% of the virions remained on the surface of cells in the presence of EB, compared to 37% in the control (p < 0.001). EB also blocked fusion-from-within, suggesting that virus infection is inhibited at the fusion step. Analysis of EB derivatives suggested that peptide length may be important for the activity of EB. The EB peptide is, to our knowledge, the first known small molecule inhibitor of Vaccinia virus entry.« less

TGF-β1 induces the formation of vascular-like structures in embryoid bodies derived from human embryonic stem cells.

PubMed

Wang, Yan; Qian, DE-Jian; Zhong, Wen-Yu; Lu, Jun-Hong; Guo, Xiang-Kai; Cao, Yi-Lin; Liu, Ju

2014-07-01

Human embryonic stem cells (ESCs) can differentiate into endothelial cells in response to stimuli from extracellular cytokines. Transforming growth factor (TGF)-β1 signaling is involved in stem cell renewal and vascular development. Previously, human ESCs were isolated from inner cell mass and a stable ESC line was developed. In the present study, the effects of extracellular TGF-β1 were investigated on human ESC-derived embryoid bodies (EB) in suspension. The structures of the EBs were analyzed with light and electron microscopy, while the cellular composition of the EBs was examined via the expression levels of specific markers. Vascular-like tubular structures and cardiomyocyte-like beating cells were observed in the EBs at day 3 and 8, respectively. The frequencies of vascular-like structures and beating cells in the TGF-β1 treated group were significantly higher compared with the control group (84.31 vs. 12.77%; P<0.001; 37.25 vs. 8.51%; P<0.001, respectively). Electron microscopy revealed the presence of lumens and gap junctions in the sections of the tubular structures. Semiquantitative polymerase chain reaction revealed elevated expression levels of CD31 and fetal liver kinase-1 in EBs cultured with TGF-β1. In addition, extensive staining of von Willebrand factor was observed in the vascular-like structures of TGF-β1-treated EBs. Therefore, the results of the present study may aid the understanding of the underlying mechanisms of human ESC differentiation and improve the methods of propagating specific cell types for the clinical therapy of cardiovascular diseases.

Strategies for the structural analysis of multi-protein complexes: lessons from the 3D-Repertoire project.

PubMed

Collinet, B; Friberg, A; Brooks, M A; van den Elzen, T; Henriot, V; Dziembowski, A; Graille, M; Durand, D; Leulliot, N; Saint André, C; Lazar, N; Sattler, M; Séraphin, B; van Tilbeurgh, H

2011-08-01

Structural studies of multi-protein complexes, whether by X-ray diffraction, scattering, NMR spectroscopy or electron microscopy, require stringent quality control of the component samples. The inability to produce 'keystone' subunits in a soluble and correctly folded form is a serious impediment to the reconstitution of the complexes. Co-expression of the components offers a valuable alternative to the expression of single proteins as a route to obtain sufficient amounts of the sample of interest. Even in cases where milligram-scale quantities of purified complex of interest become available, there is still no guarantee that good quality crystals can be obtained. At this step, protein engineering of one or more components of the complex is frequently required to improve solubility, yield or the ability to crystallize the sample. Subsequent characterization of these constructs may be performed by solution techniques such as Small Angle X-ray Scattering and Nuclear Magnetic Resonance to identify 'well behaved' complexes. Herein, we recount our experiences gained at protein production and complex assembly during the European 3D Repertoire project (3DR). The goal of this consortium was to obtain structural information on multi-protein complexes from yeast by combining crystallography, electron microscopy, NMR and in silico modeling methods. We present here representative set case studies of complexes that were produced and analyzed within the 3DR project. Our experience provides useful insight into strategies that are more generally applicable for structural analysis of protein complexes. Copyright © 2011 Elsevier Inc. All rights reserved.

Identification and Characterization of lpfABCC′DE, a Fimbrial Operon of Enterohemorrhagic Escherichia coli O157:H7

PubMed Central

Torres, Alfredo G.; Giron, Jorge A.; Perna, Nicole T.; Burland, Valerie; Blattner, Fred R.; Avelino-Flores, Fabiola; Kaper, James B.

2002-01-01

The mechanisms underlying the adherence of Escherichia coli O157:H7 and other enterohemorrhagic E. coli (EHEC) strains to intestinal epithelial cells are poorly understood. We have identified a chromosomal region (designated lpfABCC′DE) in EHEC O157:H7 containing six putative open reading frames that was found to be closely related to the long polar (LP) fimbria operon (lpf) of Salmonella enterica serovar Typhimurium, both in gene order and in conservation of the deduced amino acid sequences. We show that lpfABCC′DE is organized as an operon and that its expression is induced during the exponential growth phase. The lpf genes from EHEC strain EDL933 were introduced into a nonfimbriated (Fim−) E. coli K-12 strain, and the transformed strain produced fimbriae as visualized by electron microscopy and adhered to tissue culture cells. Anti-LpfA antiserum recognized a ca. 16-kDa LpfA protein when expressed under regulation of the T7 promoter system. The antiserum also cross-reacted with the LP fimbriae in immunogold electron microscopy and Western blot experiments. Isogenic E. coli O157:H7 lpf mutants derived from strains 86-24 and AGT300 showed slight reductions in adherence to tissue culture cells and formed fewer microcolonies compared with their wild-type parent strains. The adherence and microcolony formation phenotypes were restored when the lpf operon was introduced on a plasmid. We propose that LP fimbriae participate in the interaction of E. coli O157:H7 with eukaryotic cells by assisting in microcolony formation. PMID:12228266

Dynamical electron diffraction simulation for non-orthogonal crystal system by a revised real space method.

PubMed

Lv, C L; Liu, Q B; Cai, C Y; Huang, J; Zhou, G W; Wang, Y G

2015-01-01

In the transmission electron microscopy, a revised real space (RRS) method has been confirmed to be a more accurate dynamical electron diffraction simulation method for low-energy electron diffraction than the conventional multislice method (CMS). However, the RRS method can be only used to calculate the dynamical electron diffraction of orthogonal crystal system. In this work, the expression of the RRS method for non-orthogonal crystal system is derived. By taking Na2 Ti3 O7 and Si as examples, the correctness of the derived RRS formula for non-orthogonal crystal system is confirmed by testing the coincidence of numerical results of both sides of Schrödinger equation; moreover, the difference between the RRS method and the CMS for non-orthogonal crystal system is compared at the accelerating voltage range from 40 to 10 kV. Our results show that the CMS method is almost the same as the RRS method for the accelerating voltage above 40 kV. However, when the accelerating voltage is further lowered to 20 kV or below, the CMS method introduces significant errors, not only for the higher-order Laue zone diffractions, but also for zero-order Laue zone. These indicate that the RRS method for non-orthogonal crystal system is necessary to be used for more accurate dynamical simulation when the accelerating voltage is low. Furthermore, the reason for the increase of differences between those diffraction patterns calculated by the RRS method and the CMS method with the decrease of the accelerating voltage is discussed. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Transmission electron microscopy of amyloid fibrils.

PubMed

Gras, Sally L; Waddington, Lynne J; Goldie, Kenneth N

2011-01-01

Transmission Electron Microscopy of negatively stained and cryo-prepared specimens allows amyloid fibrils to be visualised at high resolution in a dried or a hydrated state, and is an essential method for characterising the morphology of fibrils and pre-fibrillar species. We outline the key steps involved in the preparation and observation of samples using negative staining and cryo-electron preservation. We also discuss methods to measure fibril characteristics, such as fibril width, from electron micrographs.

Accurate Virus Quantitation Using a Scanning Transmission Electron Microscopy (STEM) Detector in a Scanning Electron Microscope

DTIC Science & Technology

2017-06-29

Accurate Virus Quantitation Using a Scanning Transmission Electron Microscopy (STEM) Detector in a Scanning Electron Microscope Candace D Blancett1...L Norris2, Cynthia A Rossi4 , Pamela J Glass3, Mei G Sun1,* 1 Pathology Division, United States Army Medical Research Institute of Infectious...Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Maryland, 21702 2Biostatistics Division, United States Army Medical Research Institute of

Preface: phys. stat. sol. (a) 202/12

NASA Astrophysics Data System (ADS)

Neumann, Wolfgang; Stutzmann, Martin; Hildebrandt, Stefan

2005-09-01

The present special issue contains a collection of Original Papers dedicated to Professor Johannes Heydenreich on the occasion of his 75th birthday.Johannes Heydenreich, born on 20 June 1930 in Plauen/Vogtland near Dresden, studied physics at the Pädagogische Hochschule Potsdam, where he obtained his first academic degree Dipl. Phys. in 1958. He received his doctoral degree at the Martin Luther University in Halle in 1961 and the Habilitation degree in 1969. Already during his studies in Potsdam, he showed an interest in electron microscopy due to the influence of his teacher and supervisor Prof. Picht, one of the pioneers in electron optics. His interests were strengthened when Johannes Heydenreich did the experimental work for his Diploma degree at the Institute for Experimental Physics of the University of Halle, where he met Prof. Heinz Bethge for the first time. This was the beginning of a fruitful and longstanding collaboration. In 1962 Johannes Heydenreich joined the team of the later Institute for Solid State Physics and Electron Microscopy of the Academy of Sciences of the GDR, in Halle, for which the basis was laid by Prof. Bethge in 1960.Heydenreich has been working as Assistant Director for many years and played a decisive role in introducing and organising the various techniques of electron microscopy in the institute.The research activities of Prof. Heydenreich covered a broad spectrum over the years. At the beginning of his career he made significant contributions in the field of electron mirror microscopy. After that, his main interests were focused on transmission electron microscopy, ranging from diffraction contrast analysis of crystal defects to high-resolution electron microscopy and image processing. His favourite field was studies of defect-induced phenomena in advanced materials. The so-called Bethge-Heydenreich, the book Electron Microscopy in Solid State Physics, published at first in a German edition in 1982 and later in a revised English edition by Elsevier in 1987, provides an excellent overview both of Heydenreich's work and of the spectrum of the Institute of Solid State Physics and Electron Micros-copy in Halle.The international reputation of this institute was the basis for the transformation of it, after the re-unification of Germany, into the Max Planck Institute of Microstructure Physics, the first institute of the Max Planck Society in the Eastern part of Germany. From the beginning, Prof. Heydenreich was on the Board of Directors and served as the Executive Director from 1993 until his retirement in 1995.During this year, the International Centre of Advanced Materials and Electron Microscopy celebrates its 30th anniversary. The Centre was founded in Halle in 1975 as a Centre of Electron Microscopy for the Eastern countries.A remarkable and time-consuming part of Heydenreich's work was associated with this centre. Young scientists from Eastern Europe were trained in the theoretical and practical aspects of electron microscopy during the annual spring and autumn meetings. All in all, Johannes Heydenreich conducted 35 of these schools! From the beginning, he was a member of the Scientific Council of the Centre, and from 1985 until his retirement he was the Director of the Centre. Nowadays, the Centre acts as a real bridge between East and West largely due to the efforts of Johannes Heydenreich.Furthermore, Johannes Heydenreich had been active as a leading member of various scientific boards for many years. He was a co-editor of several journals covering electron microscopy, solid state physics and crystallography. As a member of the Executive Committee of the European Society for Elec-tron Microscopy (CESEM) for many years, and as a member of the Executive board of the German Soci-ety for Electron Microscopy, Johannes Heydenreich has invested a great deal of time and effort in the welfare of the scientific community.Over the course of nearly three decades, Johannes Heydenreich worked as a Professor at the Universities of Halle and Leipzig. Generations of students have admired his excellent lectures with clear repre-sentation and rigorous scholarship.The scientific work of Johannes Heydenreich was honoured by his election to the Academy of Natural Scientists Leopoldina where he worked as a Secretary of Natural Science for many years. The Technical University of Chemnitz conferred an honory doctors degree on Prof. Heydenreich in recognition of his scientific work. The German Society for Electron Microscopy awarded him Honorary Membership.Wolfgang Neumann especially acknowledges with gratitude the time of inspiring collaboration in Halle.All his friends, colleagues, and students wish him many further years of good health. The Editors of physica status solidi join these wishes and add their gratefulness for his long-standing and continuous engagement as a member of the Advisory and Editorial Board of physica status solidi (a).

Electron Microscopy Localization and Characterization of Functionalized Composite Organic-Inorganic SERS Nanoparticles on Leukemia Cells

PubMed Central

Koh, Ai Leen; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P.; Sinclair, Robert

2008-01-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet Scanning Electron Microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron detector (BSE) was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution Transmission Electron Microscope (TEM) images and Scanning Auger Electron Spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens. PMID:18995965

Evaluations of carbon nanotube field emitters for electron microscopy

NASA Astrophysics Data System (ADS)

Nakahara, Hitoshi; Kusano, Yoshikazu; Kono, Takumi; Saito, Yahachi

2009-11-01

Brightness of carbon nanotube (CNT) emitters was already reported elsewhere. However, brightness of electron emitter is affected by a virtual source size of the emitter, which strongly depends on electron optical configuration around the emitter. In this work, I- V characteristics and brightness of a CNT emitter are measured under a practical field emission electron gun (e-gun) configuration to investigate availability of CNT for electron microscopy. As a result, it is obtained that an emission area of MWNT is smaller than its tip surface area, and the emission area corresponds to a five-membered-ring with 2nd nearest six-membered-rings on the MWNT cap surface. Reduced brightness of MWNT is measured as at least 2.6×109 A/m 2 sr V. It is concluded that even a thick MWNT has enough brightness under a practical e-gun electrode configuration and suitable for electron microscopy.

The application of scanning electron microscopy to fractography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brooks, C.R.; McGill, B.L.

1994-10-01

Many failures involve fracture, and determination of the fracture process is a key factor in understanding the failure. This is frequently accomplished by characterizing the topography of the fracture surface. Scanning electron microscopy has a prominent role in fractography due to three features of the scanning electron microscope (SEM): high resolution, great depth of field, and the ability to obtain chemical information via analysis of the X-rays generated by the electrons. A qualitative treatment is presented of the interaction of electrons with a sample and the effect of the SEM operating parameters on image formation, quality, and X-ray analysis. Fractographsmore » are presented to illustrate these features of scanning electron microscopy and to illustrate the limitations and precautions in obtaining fractographs and x-ray analyses. The review is concluded with examples of fracture surface features of metallic, ceramic, and polymeric materials.« less

The spatial coherence function in scanning transmission electron microscopy and spectroscopy.

PubMed

Nguyen, D T; Findlay, S D; Etheridge, J

2014-11-01

We investigate the implications of the form of the spatial coherence function, also referred to as the effective source distribution, for quantitative analysis in scanning transmission electron microscopy, and in particular for interpreting the spatial origin of imaging and spectroscopy signals. These questions are explored using three different source distribution models applied to a GaAs crystal case study. The shape of the effective source distribution was found to have a strong influence not only on the scanning transmission electron microscopy (STEM) image contrast, but also on the distribution of the scattered electron wavefield and hence on the spatial origin of the detected electron intensities. The implications this has for measuring structure, composition and bonding at atomic resolution via annular dark field, X-ray and electron energy loss STEM imaging are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

PubMed

Tylko, G; Karasiński, J; Wróblewski, R; Roomans, G M; Kilarski, W M

2000-01-01

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

Temporal expression and immunogold localization of Plodia interpunctella granulosis virus structural proteins

NASA Technical Reports Server (NTRS)

Funk, C. J.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

Monospecific antisera were produced against four structural proteins (VP12, VP17, VP31, and granulin) of the Plodia interpunctella granulosis virus using polypeptides derived by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or acid extraction. The antisera were shown to be specific on immunoblots of SDS-PAGE separated granulosis virus and were further used to detect structural proteins in infected fat body lysates. Immunoblots of fat body lysates from early stages of infection indicated that VP12, VP17, VP31, and granulin were expressed by 2.5 days post-infection. Immunogold labeling of the virus using the monospecific antisera and electron microscopy confirmed earlier reports that granulin is located in the protein matrix, V17 is an envelope protein, and VP31 is a capsid protein.

Construction and characterization of a human T-cell lymphotropic virus type 3 infectious molecular clone.

PubMed

Chevalier, Sébastien Alain; Ko, Nga Ling; Calattini, Sara; Mallet, Adeline; Prévost, Marie-Christine; Kehn, Kylene; Brady, John N; Kashanchi, Fatah; Gessain, Antoine; Mahieux, Renaud

2008-07-01

We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24(gag) protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.

Rhabdom evolution in butterflies: insights from the uniquely tiered and heterogeneous ommatidia of the Glacial Apollo butterfly, Parnassius glacialis.

PubMed

Matsushita, Atsuko; Awata, Hiroko; Wakakuwa, Motohiro; Takemura, Shin-ya; Arikawa, Kentaro

2012-09-07

The eye of the Glacial Apollo butterfly, Parnassius glacialis, a 'living fossil' species of the family Papilionidae, contains three types of spectrally heterogeneous ommatidia. Electron microscopy reveals that the Apollo rhabdom is tiered. The distal tier is composed exclusively of photoreceptors expressing opsins of ultraviolet or blue-absorbing visual pigments, and the proximal tier consists of photoreceptors expressing opsins of green or red-absorbing visual pigments. This organization is unique because the distal tier of other known butterflies contains two green-sensitive photoreceptors, which probably function in improving spatial and/or motion vision. Interspecific comparison suggests that the Apollo rhabdom retains an ancestral tiered pattern with some modification to enhance its colour vision towards the long-wavelength region of the spectrum.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Buck, E.C.; Cunnane, J.C.; Brown, N.R.

A combination of optical microscopy, scanning electron microscopy with backscattered electron detection (SEM/BSE), and analytical electron microscopy (AEM) is being used to determine the nature of uranium in soils from the Fernald Environmental Management Project. The information gained from these studies is being used to develop and test remediation technologies. Investigations using SEM have shown that uranium is contained within particles that are typically 1 to 100 {mu}m in diameter. Further analysis with AEM has shown that these uranium-rich regions are made up of discrete uranium-bearing phases. The distribution of these uranium phases was found to be inhomogeneous at themore » microscopic level.« less

Transmission electron microscopy analysis of skin lesions from sporotrichosis epidemic in Rio de Janeiro, Brazil.

PubMed

Ferreira, Cassio Porto; Oliveira de Almeida, Ana Cristina; Corte-Real, Suzana

2015-02-01

Transmission electron microscopy can yield useful information in a range of scientific fields; it is capable of imaging at a significantly higher resolution than light microscopes and has been a very useful tool in the identification of morphological changes of the dermis as well as assessment of changes in the extracellular matrix. Our aim is to characterize by electron microscopy the cellular profile of lesions caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form that occurs in Rio de Janeiro, Brazil. © The American Society of Tropical Medicine and Hygiene.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Timmermans, F. J.; Otto, C.

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemicallymore » or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.« less

U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prabhakaran, Ramprashad; Joshi, Vineet V.; Rhodes, Mark A.

2016-10-01

The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prabhakaran, Ramprashad; Joshi, Vineet V.; Rhodes, Mark A.

2016-03-30

The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

Symposium N: Materials and Devices for Thermal-to-Electric Energy Conversion

DTIC Science & Technology

2010-08-24

X - ray diffraction, transmission electron microscopy, scanning electron microscopy, and dynamic light scattering. Thermal conductivity measurements...SEM), X - ray diffraction (XRD) measurements as well as Raman spectroscopy. The results from these techniques indicate a clear modification...was examined by using scanning electron microscope (SEM; HITACHI S-4500 model) attached with an energy dispersive x - ray spectroscopy. The electrical

Quantitative Cryo-Scanning Transmission Electron Microscopy of Biological Materials.

PubMed

Elbaum, Michael

2018-05-11

Electron tomography provides a detailed view into the 3D structure of biological cells and tissues. Physical fixation by vitrification of the aqueous medium provides the most faithful preservation of biological specimens in the native, fully hydrated state. Cryo-microscopy is challenging, however, because of the sensitivity to electron irradiation and due to the weak electron scattering of organic material. Tomography is even more challenging because of the dependence on multiple exposures of the same area. Tomographic imaging is typically performed in wide-field transmission electron microscopy (TEM) mode with phase contrast generated by defocus. Scanning transmission electron microscopy (STEM) is an alternative mode based on detection of scattering from a focused probe beam, without imaging optics following the specimen. While careful configuration of the illumination and detectors is required to generate useful contrast, STEM circumvents the major restrictions of phase contrast TEM to very thin specimens and provides a signal that is more simply interpreted in terms of local composition and density. STEM has gained popularity in recent years for materials science. The extension of STEM to cryomicroscopy and tomography of cells and macromolecules is summarized herein. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Environmental scanning electron microscopy gold immunolabeling in cell biology.

PubMed

Rosso, Francesco; Papale, Ferdinando; Barbarisi, Alfonso

2013-01-01

Immunogold labeling (IGL) technique has been utilized by many authors in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to obtain the identification/localization of receptors and antigens, both in cells and tissues. Environmental scanning electron microscopy (ESEM) represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artifacts and interfere with the IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labeling detection is based on the atomic number difference between nanogold spheres and the biological material. Using the gaseous secondary electron detector, compositional contrast is easily revealed by the backscattered electron component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimized to improve the intensity and the specificity of the labeling signal, in order to obtain a semiquantitative evaluation of the labeling signal.In particular, we used a combination of IGL and ESEM to detect the presence of a protein on the cell surface. To achieve this purpose, we chose as an experimental system 3T3 Swiss albino mouse fibroblasts and galectin-3.

Visualization of HIV T Cell Virological Synapses and Virus-Containing Compartments by Three-Dimensional Correlative Light and Electron Microscopy

PubMed Central

Wang, Lili; Eng, Edward T.; Law, Kenneth; Gordon, Ronald E.; Rice, William J.

2016-01-01

ABSTRACT Virological synapses (VS) are adhesive structures that form between infected and uninfected cells to enhance the spread of HIV-1. During T cell VS formation, viral proteins are actively recruited to the site of cell-cell contact where the viral material is efficiently translocated to target cells into heterogeneous, protease-resistant, antibody-inaccessible compartments. Using correlative light and electron microscopy (CLEM), we define the membrane topography of the virus-containing compartments (VCC) where HIV is found following VS-mediated transfer. Focused ion beam scanning electron microscopy (FIB-SEM) and serial sectioning transmission electron microscopy (SS-TEM) were used to better resolve the fluorescent Gag-containing structures within the VCC. We found that small punctate fluorescent signals correlated with single viral particles in enclosed vesicular compartments or surface-localized virus particles and that large fluorescent signals correlated with membranous Gag-containing structures with unknown pathological function. CLEM imaging revealed distinct pools of newly deposited viral proteins within endocytic and nonendocytic compartments in VS target T cells. IMPORTANCE This study directly correlates individual virus-associated objects observed in light microscopy with ultrastructural features seen by electron microscopy in the HIV-1 virological synapse. This approach elucidates which infection-associated ultrastructural features represent bona fide HIV protein complexes. We define the morphology of some HIV cell-to-cell transfer intermediates as true endocytic compartments and resolve unique synapse-associated viral structures created by transfer across virological synapses. PMID:27847357

Relative merits and limiting factors for x-ray and electron microscopy of thick, hydrated organic materials.

PubMed

Du, Ming; Jacobsen, Chris

2018-01-01

Electron and x-ray microscopes allow one to image the entire, unlabeled structure of hydrated materials at a resolution well beyond what visible light microscopes can achieve. However, both approaches involve ionizing radiation, so that radiation damage must be considered as one of the limits to imaging. Drawing upon earlier work, we describe here a unified approach to estimating the image contrast (and thus the required exposure and corresponding radiation dose) in both x-ray and electron microscopy. This approach accounts for factors such as plural and inelastic scattering, and (in electron microscopy) the use of energy filters to obtain so-called "zero loss" images. As expected, it shows that electron microscopy offers lower dose for specimens thinner than about 1 µm (such as for studies of macromolecules, viruses, bacteria and archaebacteria, and thin sectioned material), while x-ray microscopy offers superior characteristics for imaging thicker specimen such as whole eukaryotic cells, thick-sectioned tissues, and organs. The required radiation dose scales strongly as a function of the desired spatial resolution, allowing one to understand the limits of live and frozen hydrated specimen imaging. Finally, we consider the factors limiting x-ray microscopy of thicker materials, suggesting that specimens as thick as a whole mouse brain can be imaged with x-ray microscopes without significant image degradation should appropriate image reconstruction methods be identified. Copyright © 2017 Elsevier B.V. All rights reserved.

Comparison of six bone-graft substitutes regarding to cell seeding efficiency, metabolism and growth behaviour of human mesenchymal stem cells (MSC) in vitro.

PubMed

Seebach, Caroline; Schultheiss, Judith; Wilhelm, Kerstin; Frank, Johannes; Henrich, Dirk

2010-07-01

Various synthetic bone-graft substitutes are used commercially as osteoconductive scaffolds in the treatment of bone defects and fractures. The role of bone-graft substitutes is changing from osteoconductive conduits for growth to an delivery system for biologic fracture treatments. Achieving optimal bone regeneration requires biologics (e.g. MSC) and using the correct scaffold incorporated into a local environment for bone regeneration. The need for an unlimited supply with high quality bone-graft substitutes continue to find alternatives for bone replacement surgery. This in vitro study investigates cell seeding efficiency, metabolism, gene expression and growth behaviour of MSC sown on six commercially clinical available bone-graft substitutes in order to define their biological properties: synthetic silicate-substituted porous hydroxyapatite (Actifuse ABX), synthetic alpha-TCP (Biobase), synthetic beta-TCP (Vitoss), synthetic beta-TCP (Chronos), processed human cancellous allograft (Tutoplast) and processed bovines hydroxyapatite ceramic (Cerabone). 250,000 MSC derived from human bone marrow (n=4) were seeded onto the scaffolds, respectively. On days 2, 6 and 10 the adherence of MSC (fluorescence microscopy) and cellular activity (MTT assay) were analysed. Osteogenic gene expression (cbfa-1) was analysed by RT-PCR and scanning electron microscopy was performed. The highest number of adhering cells was found on Tutoplast (e.g. day 6: 110.0+/-24.0 cells/microscopic field; p<0.05) followed by Chronos (47.5+/-19.5, p<0.05), Actifuse ABX (19.1+/-4.4), Biobase (15.7+/-9.9), Vitoss (8.8+/-8.7) and Cerabone (8.1+/-2.2). MSC seeded onto Tutoplast showed highest metabolic activity and gene expression of cbfa-1. These data are confirmed by scanning electron microscopy. The cell shapes varied from round-shaped cells to wide spread cells and cell clusters, depending on the bone-graft substitutes. Processed human cancellous allograft is a well-structured and biocompatible scaffold for ingrowing MSC in vitro. Of all other synthetical scaffolds, beta-tricalcium phosphate (Chronos) have shown the best growth behaviour for MSC. Our results indicate that various bone-graft substitutes influence cell seeding efficiency, metabolic activity and growth behaviour of MSC in different manners. We detected a high variety of cellular integration of MSC in vitro, which may be important for bony integration in the clinical setting. 2010 Elsevier Ltd. All rights reserved.

Specimen preparation by ion beam slope cutting for characterization of ductile damage by scanning electron microscopy.

PubMed

Besserer, Hans-Bernward; Gerstein, Gregory; Maier, Hans Jürgen; Nürnberger, Florian

2016-04-01

To investigate ductile damage in parts made by cold sheet-bulk metal forming a suited specimen preparation is required to observe the microstructure and defects such as voids by electron microscopy. By means of ion beam slope cutting both a targeted material removal can be applied and mechanical or thermal influences during preparation avoided. In combination with scanning electron microscopy this method allows to examine voids in the submicron range and thus to analyze early stages of ductile damage. In addition, a relief structure is formed by the selectivity of the ion bombardment, which depends on grain orientation and microstructural defects. The formation of these relief structures is studied using scanning electron microscopy and electron backscatter diffraction and the use of this side effect to interpret the microstructural mechanisms of voids formation by plastic deformation is discussed. A comprehensive investigation of the suitability of ion beam milling to analyze ductile damage is given at the examples of a ferritic deep drawing steel and a dual phase steel. © 2016 Wiley Periodicals, Inc.

Sparse imaging for fast electron microscopy

NASA Astrophysics Data System (ADS)

Anderson, Hyrum S.; Ilic-Helms, Jovana; Rohrer, Brandon; Wheeler, Jason; Larson, Kurt

2013-02-01

Scanning electron microscopes (SEMs) are used in neuroscience and materials science to image centimeters of sample area at nanometer scales. Since imaging rates are in large part SNR-limited, large collections can lead to weeks of around-the-clock imaging time. To increase data collection speed, we propose and demonstrate on an operational SEM a fast method to sparsely sample and reconstruct smooth images. To accurately localize the electron probe position at fast scan rates, we model the dynamics of the scan coils, and use the model to rapidly and accurately visit a randomly selected subset of pixel locations. Images are reconstructed from the undersampled data by compressed sensing inversion using image smoothness as a prior. We report image fidelity as a function of acquisition speed by comparing traditional raster to sparse imaging modes. Our approach is equally applicable to other domains of nanometer microscopy in which the time to position a probe is a limiting factor (e.g., atomic force microscopy), or in which excessive electron doses might otherwise alter the sample being observed (e.g., scanning transmission electron microscopy).

Domain imaging in ferroelectric thin films via channeling-contrast backscattered electron microscopy

DOE PAGES

Ihlefeld, Jon F.; Michael, Joseph R.; McKenzie, Bonnie B.; ...

2016-09-16

We report that ferroelastic domain walls provide opportunities for deterministically controlling mechanical, optical, electrical, and thermal energy. Domain wall characterization in micro- and nanoscale systems, where their spacing may be of the order of 100 nm or less is presently limited to only a few techniques, such as piezoresponse force microscopy and transmission electron microscopy. These respective techniques cannot, however, independently characterize domain polarization orientation and domain wall motion in technologically relevant capacitor structures or in a non-destructive manner, thus presenting a limitation of their utility. In this work, we show how backscatter scanning electron microscopy utilizing channeling contrast yieldmore » can image the ferroelastic domain structure of ferroelectric films with domain wall spacing as narrow as 10 nm.« less

TEM characterization of nanodiamond thin films.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qin, L.-C.; Zhou, D.; Krauss, A. R.

The microstructure of thin films grown by microwave plasma-enhanced chemical vapor deposition (MPCVD) from fullerene C{sub 60} precursors has been characterized by scanning electron microscopy (SEM), selected-area electron diffraction (SAED), bright-field electron microscopy, high-resolution electron microscopy (HREM), and parallel electron energy loss spectroscopy (PEELS). The films are composed of nanosize crystallites of diamond, and no graphitic or amorphous phases were observed. The diamond crystallite size measured from lattice images shows that most grains range between 3-5 nm, reflecting a gamma distribution. SAED gave no evidence of either sp2-bonded glassy carbon or sp3-bonded diamondlike amorphous carbon. The sp2-bonded configuration found inmore » PEELS was attributed to grain boundary carbon atoms, which constitute 5-10% of the total. Occasionally observed larger diamond grains tend to be highly faulted.« less

Nanoparticle discrimination based on wavelength and lifetime-multiplexed cathodoluminescence microscopy.

PubMed

Garming, Mathijs W H; Weppelman, I Gerward C; de Boer, Pascal; Martínez, Felipe Perona; Schirhagl, Romana; Hoogenboom, Jacob P; Moerland, Robert J

2017-08-31

Nanomaterials can be identified in high-resolution electron microscopy images using spectrally-selective cathodoluminescence. Capabilities for multiplex detection can however be limited, e.g., due to spectral overlap or availability of filters. Also, the available photon flux may be limited due to degradation under electron irradiation. Here, we demonstrate single-pass cathodoluminescence-lifetime based discrimination of different nanoparticles, using a pulsed electron beam. We also show that cathodoluminescence lifetime is a robust parameter even when the nanoparticle cathodoluminescence intensity decays over an order of magnitude. We create lifetime maps, where the lifetime of the cathodoluminescence emission is correlated with the emission intensity and secondary-electron images. The consistency of lifetime-based discrimination is verified by also correlating the emission wavelength and the lifetime of nanoparticles. Our results show how cathodoluminescence lifetime provides an additional channel of information in electron microscopy.

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

EPA Science Inventory

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum "cast" intended for examination by transmission electron microscopy. Specimens are subjected to u...

WEATHERING DEGRADATION OF A POLYURETHANE COATING. (R828081E01)

EPA Science Inventory

The degradation of polyurethane topcoat over a chromate pigmented epoxy primer was examined by atomic force microscopy (AFM), scanning electronic microscopy (SEM), X-ray photo-electron spectroscopy (XPS) and Fourier transform infra-red spectroscopy (FTIR) after the coated pane...

EVALUATION OF COMPUTER-CONTROLLED SCANNING ELECTRON MICROSCOPY APPLIED TO AN AMBIENT URBAN AEROSOL SAMPLE

EPA Science Inventory

Concerns about the environmental and public health effects of particulate matter (PM) have stimulated interest in analytical techniques capable of measuring the size and chemical composition of individual aerosol particles. Computer-controlled scanning electron microscopy (CCSE...

Direct observation of anti-phase boundaries in heteroepitaxy of GaSb thin films grown on Si(001) by transmission electron microscopy

NASA Astrophysics Data System (ADS)

Woo, S. Y.; Hosseini Vajargah, S.; Ghanad-Tavakoli, S.; Kleiman, R. N.; Botton, G. A.

2012-10-01

Unambiguous identification of anti-phase boundaries (APBs) in heteroepitaxial films of GaSb grown on Si has been so far elusive. In this work, we present conventional transmission electron microscopy (TEM) diffraction contrast imaging using superlattice reflections, in conjunction with convergent beam electron diffraction analysis, to determine a change in polarity across APBs in order to confirm the presence of anti-phase disorder. In-depth analysis of anti-phase disorder is further supported with atomic resolution high-angle annular dark-field scanning transmission electron microscopy. The nature of APBs in GaSb is further elucidated by a comparison to previous results for GaAs epilayers grown on Si.

A Survey of the Use of Iterative Reconstruction Algorithms in Electron Microscopy

PubMed Central

Otón, J.; Vilas, J. L.; Kazemi, M.; Melero, R.; del Caño, L.; Cuenca, J.; Conesa, P.; Gómez-Blanco, J.; Marabini, R.; Carazo, J. M.

2017-01-01

One of the key steps in Electron Microscopy is the tomographic reconstruction of a three-dimensional (3D) map of the specimen being studied from a set of two-dimensional (2D) projections acquired at the microscope. This tomographic reconstruction may be performed with different reconstruction algorithms that can be grouped into several large families: direct Fourier inversion methods, back-projection methods, Radon methods, or iterative algorithms. In this review, we focus on the latter family of algorithms, explaining the mathematical rationale behind the different algorithms in this family as they have been introduced in the field of Electron Microscopy. We cover their use in Single Particle Analysis (SPA) as well as in Electron Tomography (ET). PMID:29312997

Three-dimensional imaging of adherent cells using FIB/SEM and STEM.

PubMed

Villinger, Clarissa; Schauflinger, Martin; Gregorius, Heiko; Kranz, Christine; Höhn, Katharina; Nafeey, Soufi; Walther, Paul

2014-01-01

In this chapter we describe three different approaches for three-dimensional imaging of electron microscopic samples: serial sectioning transmission electron microscopy (TEM), scanning transmission electron microscopy (STEM) tomography, and focused ion beam/scanning electron microscopy (FIB/SEM) tomography. With these methods, relatively large volumes of resin-embedded biological structures can be analyzed at resolutions of a few nm within a reasonable expenditure of time. The traditional method is serial sectioning and imaging the same area in all sections. Another method is TEM tomography that involves tilting a section in the electron beam and then reconstruction of the volume by back projection of the images. When the scanning transmission (STEM) mode is used, thicker sections (up to 1 μm) can be analyzed. The third approach presented here is focused ion beam/scanning electron microscopy (FIB/SEM) tomography, in which a sample is repeatedly milled with a focused ion beam (FIB) and each newly produced block face is imaged with the scanning electron microscope (SEM). This process can be repeated ad libitum in arbitrary small increments allowing 3D analysis of relatively large volumes such as eukaryotic cells. We show that resolution of this approach is considerably improved when the secondary electron signal is used. However, the most important prerequisite for three-dimensional imaging is good specimen preparation. For all three imaging methods, cryo-fixed (high-pressure frozen) and freeze-substituted samples have been used.

The pathogenic relevance of αM-integrin in Guillain-Barré syndrome.

PubMed

Dong, Chaoling; Palladino, Steven P; Helton, Eric Scott; Ubogu, Eroboghene E

2016-11-01

The molecular determinants and mechanisms involved in leukocyte trafficking across the blood-nerve barrier (BNB) in the acute inflammatory demyelinating polyradiculoneuropathy (AIDP) variant of Guillain-Barré syndrome are incompletely understood. Prior work using a flow-dependent in vitro human BNB model demonstrated a crucial role for α M -integrin (CD11b)-intercellular adhesion molecule-1 interactions in AIDP patient leukocyte trafficking. The aim of this study is to directly investigate the biological relevance of CD11b in AIDP pathogenesis. Immunohistochemistry was performed on three AIDP patient sural nerve biopsies to evaluate endoneurial leukocyte CD11b expression. A severe murine experimental autoimmune neuritis (sm-EAN) model was utilized to determine the functional role of CD11b in leukocyte trafficking in vivo and determine its effect on neurobehavioral measures of disease severity, electrophysiological assessments of axonal integrity and myelination and histopathological measures of peripheral nerve inflammatory demyelination. Time-lapse video microscopy and electron microscopy were employed to observe structural alterations at the BNB during AIDP patient leukocyte trafficking in vitro and in situ, respectively. Large clusters of endoneurial CD11b+ leukocytes associated with demyelinating axons were observed in AIDP patient sural nerves. Leukocyte CD11b expression was upregulated during sm-EAN. 5 mg/kg of a function-neutralizing monoclonal rat anti-mouse CD11b antibody administered after sm-EAN disease onset significantly ameliorated disease severity, as well as electrophysiological and histopathological parameters of inflammatory demyelination compared to vehicle- and isotype antibody-treated mice. Consistent with in vitro observations of leukocyte trafficking at the BNB, electron micrographs of AIDP patient sural nerves demonstrated intact electron-dense endoneurial microvascular intercellular junctions during paracellular mononuclear leukocyte transmigration. Our data support a crucial pathogenic role of CD11b in AIDP leukocyte trafficking, providing a potential therapeutic target for demyelinating variants of Guillain-Barré syndrome.

Comparative Proteomic and Morphological Change Analyses of Staphylococcus aureus During Resuscitation From Prolonged Freezing

PubMed Central

Suo, Biao; Yang, Hua; Wang, Yuexia; Lv, Haipeng; Li, Zhen; Xu, Chao; Ai, Zhilu

2018-01-01

When frozen, Staphylococcus aureus survives in a sublethally injured state. However, S. aureus can recover at a suitable temperature, which poses a threat to food safety. To elucidate the resuscitation mechanism of freezing survived S. aureus, we used cells stored at -18°C for 90 days as controls. After resuscitating the survived cells at 37°C, the viable cell numbers were determined on tryptic soy agar with 0.6% yeast extract (TSAYE), and the non-injured-cell numbers were determined on TSAYE supplemented with 10% NaCl. The results showed that the total viable cell number did not increase within the first 3 h of resuscitation, but the osmotic regulation ability of freezing survived cells gradually recovered to the level of healthy cells, which was evidenced by the lack of difference between the two samples seen by differential cell enumeration. Scanning electron microscopy (SEM) showed that, compared to late exponential stage cells, some frozen survived cells underwent splitting and cell lysis due to deep distortion and membrane rupture. Transmission electron microscopy (TEM) showed that, in most of the frozen survived cells, the nucleoids (low electronic density area) were loose, and the cytoplasmic matrices (high electronic density area) were sparse. Additionally, a gap was seen to form between the cytoplasmic membranes and the cell walls in the frozen survived cells. The morphological changes were restored when the survived cells were resuscitated at 37°C. We also analyzed the differential proteome after resuscitation using non-labeled high-performance liquid chromatography–mass spectrometry (HPLC-MS). The results showed that, compared with freezing survived S. aureus cells, the cells resuscitated for 1 h had 45 upregulated and 73 downregulated proteins. The differentially expressed proteins were functionally categorized by gene ontology enrichment, KEGG pathway, and STRING analyses. Cell membrane synthesis-related proteins, oxidative stress resistance-related proteins, metabolism-related proteins, and virulence factors exhibited distinct expression patterns during resuscitation. These findings have implications in the understanding of the resuscitation mechanism of freezing survived S. aureus, which may facilitate the development of novel technologies for improved detection and control of foodborne pathogens in frozen food. PMID:29774015

DESIGN NOTE: A modified Nanosurf scanning tunnelling microscope for ballistic electron emission microscopy and spectroscopy

NASA Astrophysics Data System (ADS)

Appelbaum, Ian; Thompson, Pete; van Schendel, P. J. A.

2006-04-01

We describe the design and implementation of modifications to an ambient STM with a slip stick approach mechanism to create a system capable of ballistic electron emission microscopy (BEEM) and spectroscopy (BEES). These modifications require building a custom sample holder which operates as a high gain transimpedance preamplifier. Results of microscopy and spectroscopy using a Au/n-GaAs Schottky device demonstrate the effectiveness of our design.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

PubMed

Aqeel, Yousuf; Siddiqui, Ruqaiyyah; Ateeq, Muhammad; Raza Shah, Muhammad; Kulsoom, Huma; Khan, Naveed Ahmed

2015-01-01

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens. © 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists.

Viral assembly of oriented quantum dot nanowires

NASA Astrophysics Data System (ADS)

Mao, Chuanbin; Flynn, Christine E.; Hayhurst, Andrew; Sweeney, Rozamond; Qi, Jifa; Georgiou, George; Iverson, Brent; Belcher, Angela M.

2003-06-01

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Viral assembly of oriented quantum dot nanowires.

PubMed

Mao, Chuanbin; Flynn, Christine E; Hayhurst, Andrew; Sweeney, Rozamond; Qi, Jifa; Georgiou, George; Iverson, Brent; Belcher, Angela M

2003-06-10

The highly organized structure of M13 bacteriophage was used as an evolved biological template for the nucleation and orientation of semiconductor nanowires. To create this organized template, peptides were selected by using a pIII phage display library for their ability to nucleate ZnS or CdS nanocrystals. The successful peptides were expressed as pVIII fusion proteins into the crystalline capsid of the virus. The engineered viruses were exposed to semiconductor precursor solutions, and the resultant nanocrystals that were templated along the viruses to form nanowires were extensively characterized by using high-resolution analytical electron microscopy and photoluminescence. ZnS nanocrystals were well crystallized on the viral capsid in a hexagonal wurtzite or a cubic zinc blende structure, depending on the peptide expressed on the viral capsid. Electron diffraction patterns showed single-crystal type behavior from a polynanocrystalline area of the nanowire formed, suggesting that the nanocrystals on the virus were preferentially oriented with their [001] perpendicular to the viral surface. Peptides that specifically directed CdS nanocrystal growth were also engineered into the viral capsid to create wurtzite CdS virus-based nanowires. Lastly, heterostructured nucleation was achieved with a dual-peptide virus engineered to express two distinct peptides within the same viral capsid. This work represents a genetically controlled biological synthesis route to a semiconductor nanoscale heterostructure.

Purification and biochemical characterization of NpABCG5/NpPDR5, a plant pleiotropic drug resistance transporter expressed in Nicotiana tabacum BY-2 suspension cells.

PubMed

Toussaint, Frédéric; Pierman, Baptiste; Bertin, Aurélie; Lévy, Daniel; Boutry, Marc

2017-05-04

Pleiotropic drug resistance (PDR) transporters belong to the ABCG subfamily of ATP-binding cassette (ABC) transporters and are involved in the transport of various molecules across plasma membranes. During evolution, PDR genes appeared independently in fungi and in plants from a duplication of a half-size ABC gene. The enzymatic properties of purified PDR transporters from yeast have been characterized. This is not the case for any plant PDR transporter, or, incidentally, for any purified plant ABC transporter. Yet, plant PDR transporters play important roles in plant physiology such as hormone signaling or resistance to pathogens or herbivores. Here, we describe the expression, purification, enzymatic characterization and 2D analysis by electron microscopy of NpABCG5/NpPDR5 from Nicotiana plumbaginifolia , which has been shown to be involved in the plant defense against herbivores. We constitutively expressed NpABCG5/NpPDR5, provided with a His-tag in a homologous system: suspension cells from Nicotiana tabacum (Bright Yellow 2 line). NpABCG5/NpPDR5 was targeted to the plasma membrane and was solubilized by dodecyl maltoside and purified by Ni-affinity chromatography. The ATP-hydrolyzing specific activity (27 nmol min -1  mg -1 ) was stimulated seven-fold in the presence of 0.1% asolectin. Electron microscopy analysis indicated that NpABCG5/NpPDR5 is monomeric and with dimensions shorter than those of known ABC transporters. Enzymatic data (optimal pH and sensitivity to inhibitors) confirmed that plant and fungal PDR transporters have different properties. These data also show that N. tabacum suspension cells are a convenient host for the purification and biochemical characterization of ABC transporters. © 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.

Autophagy and skeletal muscles in sepsis.

PubMed

Mofarrahi, Mahroo; Sigala, Ioanna; Guo, Yeting; Godin, Richard; Davis, Elaine C; Petrof, Basil; Sandri, Marco; Burelle, Yan; Hussain, Sabah N A

2012-01-01

Mitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles. Adult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca(++) retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor. We conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor.

Autophagy and Skeletal Muscles in Sepsis

PubMed Central

Mofarrahi, Mahroo; Sigala, Ioanna; Guo, Yeting; Godin, Richard; Davis, Elaine C.; Petrof, Basil; Sandri, Marco

2012-01-01

Background Mitochondrial injury develops in skeletal muscles during the course of severe sepsis. Autophagy is a protein and organelle recycling pathway which functions to degrade or recycle unnecessary, redundant, or inefficient cellular components. No information is available regarding the degree of sepsis-induced mitochondrial injury and autophagy in the ventilatory and locomotor muscles. This study tests the hypotheses that the locomotor muscles are more prone to sepsis-induced mitochondrial injury, depressed biogenesis and autophagy induction compared with the ventilatory muscles. Methodology/Principal Findings Adult male C57/Bl6 mice were injected with i.p. phosphate buffered saline (PBS) or E. coli lipopolysaccharide (LPS, 20 mg/kg) and sacrificed 24 h later. The tibialis anterior (TA), soleus (SOLD) and diaphragm (DIA) muscles were quickly excised and examined for mitochondrial morphological injury, Ca++ retention capacity and biogenesis. Autophagy was detected with electron microscopy, lipidation of Lc3b proteins and by measuring gene expression of several autophagy-related genes. Electron microscopy revealed ultrastructural injuries in the mitochondria of each muscle, however, injuries were more severe in the TA and SOL muscles than they were in the DIA. Gene expressions of nuclear and mitochondrial DNA transcription factors and co-activators (indicators of biogenesis) were significantly depressed in all treated muscles, although to a greater extent in the TA and SOL muscles. Significant autophagosome formation, Lc3b protein lipidation and upregulation of autophagy-related proteins were detected to a greater extent in the TA and SOL muscles and less so in the DIA. Lipidation of Lc3b and the degree of induction of autophagy-related proteins were significantly blunted in mice expressing a muscle-specific IκBα superrepresor. Conclusion/Significance We conclude that locomotor muscles are more prone to sepsis-induced mitochondrial injury, decreased biogenesis and increased autophagy compared with the ventilatory muscles and that autophagy in skeletal muscles during sepsis is regulated in part through the NFκB transcription factor. PMID:23056618

Electron Diffraction Using Transmission Electron Microscopy

PubMed Central

Bendersky, Leonid A.; Gayle, Frank W.

2001-01-01

Electron diffraction via the transmission electron microscope is a powerful method for characterizing the structure of materials, including perfect crystals and defect structures. The advantages of electron diffraction over other methods, e.g., x-ray or neutron, arise from the extremely short wavelength (≈2 pm), the strong atomic scattering, and the ability to examine tiny volumes of matter (≈10 nm3). The NIST Materials Science and Engineering Laboratory has a history of discovery and characterization of new structures through electron diffraction, alone or in combination with other diffraction methods. This paper provides a survey of some of this work enabled through electron microscopy. PMID:27500060

A Dose-Rate Effect in Single-Particle Electron Microscopy

PubMed Central

Chen, James Z.; Sachse, Carsten; Xu, Chen; Mielke, Thorsten; Spahn, Christian M. T.; Grigorieff, Nikolaus

2008-01-01

A low beam-intensity, low electron-dose imaging method has been developed for single-particle electron cryo-microscopy (cryo-EM). Experiments indicate that the new technique can reduce beam-induced specimen movement and secondary radiolytic effects, such as “bubbling”. The improvement in image quality, especially for multiple-exposure data collection, will help single-particle cryo-EM to reach higher resolution. PMID:17977018

Recombinant Reflectin-Based Optical Materials

DTIC Science & Technology

2012-01-01

sili- con substrates were placed in a sealed plastic box. The RH was controlled using a Dydra electronic cigar humidifier and monitored using a Fisher...diffraction gratings to generate diffraction patterns. Nano-spheres and la- mellar microstructures of refCBA samples were observed by scanning electron ...samples were observed by scanning electron microscopy and atomic force microscopy. Despite the reduced complexity of the refCBA protein compared to natural

Analysis of electromagnetic forces and causality in electron microscopy.

PubMed

Reyes-Coronado, Alejandro; Ortíz-Solano, Carlos Gael; Zabala, Nerea; Rivacoba, Alberto; Esquivel-Sirvent, Raúl

2018-09-01

The non-physical effects on the transverse momentum transfer from fast electrons to gold nanoparticles associated to the use of non-causal dielectric functions are studied. A direct test of the causality based on the surface Kramers-Kronig relations is presented. This test is applied to the different dielectric function used to describe gold nanostructures in electron microscopy. Copyright © 2018. Published by Elsevier B.V.

The microscopic world: A demonstration of electron microscopy for younger students

NASA Technical Reports Server (NTRS)

Horton, Linda L.

1992-01-01

The purpose is to excite students about the importance of scientific investigation and demonstrate why they should look at things in greater detail, extending beyond superficial examination. The topics covered include: microscopy, scanning electron microscopes, high magnification, and the scientific method.

Scanning electron microscopy study of adhesion in sea urchin blastulae. M.S. Thesis

NASA Technical Reports Server (NTRS)

Crowther, Susan D.

1988-01-01

The dissociation supernatant (DS) isolated by disaggregating Strongylocentrotus purpuratus blastulae in calcium- and magnesium-free seawater specifically promotes reaggregation of S. purpuratus blastula cells. The purpose of this study was to use scanning electron microscopy to examine the gross morphology of aggregates formed in the presence of DS to see if it resembles adhesion in partially dissociated blastulae. A new reaggregation procedure developed here, using large volumes of cell suspension and a large diameter of rotation, was utilized to obtain sufficient quantities of aggregates for scanning electron microscopy. The results indicate that aggregates formed in the presence of DS resemble partially dissociated intact embryos in terms of the direct cell-cell adhesion observed. DS did not cause aggregation to form as a result of the entrapment of cells in masses of extracellular material. These studies provide the groundwork for further studies using transmission electron microscopy to more precisely define the adhesive contacts made by cells in the presence of the putative adhesion molecules present in DS.

Comparison of macroscopic and microscopic (stereomicroscopy and scanning electron microscopy) features of bone lesions due to hatchet hacking trauma.

PubMed

Nogueira, Luísa; Quatrehomme, Gérald; Bertrand, Marie-France; Rallon, Christophe; Ceinos, Romain; du Jardin, Philippe; Adalian, Pascal; Alunni, Véronique

2017-03-01

This experimental study examined the lesions produced by a hatchet on human bones (tibiae). A total of 30 lesions were produced and examined macroscopically (naked eye) and by stereomicroscopy. 13 of them were also analyzed using scanning electron microscopy. The general shape of the lesion, both edges, both walls, the kerf floor and the extremities were described. The length and maximum width of the lesions were also recorded. The microscopic analysis of the lesions led to the description of a sharp-blunt mechanism. Specific criteria were identified (lateral pushing back, fragmentation of the upraising, fossa dug laterally to the edge and vertical striae) enabling the forensic expert to conclude that a hacking instrument was used. These criteria are easily identifiable using scanning electron microscopy, but can also be observed with stereomicroscopy. Overall, lateral pushing back and vertical striae visible using stereomicroscopy and scanning electron microscopy signal the use of a hacking tool.

Intracerebral Injections and Ultrastructural Analysis of High-Pressure Frozen Brain Tissue.

PubMed

Weil, Marie-Theres; Ruhwedel, Torben; Möbius, Wiebke; Simons, Mikael

2017-01-03

Intracerebral injections are an invasive method to bypass the blood brain barrier and are widely used to study molecular and cellular mechanisms of the central nervous system. The administered substances are injected directly at the site of interest, executing their effect locally. By combining injections in the rat brain with state-of-the-art electron microscopy, subtle changes in ultrastructure of the nervous tissue can be detected prior to overt damage or disease. The protocol presented here involves stereotactic injection into the corpus callosum of Lewis rats and the cryopreparation of freshly dissected tissue for electron microscopy. The localization of the injection site in tissue sections during the sample preparation for transmission electron microscopy is explained and possible artifacts of the method are indicated. With the help of this powerful combination of injections and electron microscopy, subtle effects of the applied substances on the biology of neural cells can be identified and monitored over time. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Crystallographic features related to a van der Waals coupling in the layered chalcogenide FePS{sub 3}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Murayama, Chisato; Okabe, Momoko; Fukuda, Koichiro

We investigated the crystallographic structure of FePS{sub 3} with a layered structure using transmission electron microscopy and powder X-ray diffraction. We found that FePS{sub 3} forms a rotational twin structure with the common axis along the c*-axis. The high-resolution transmission electron microscopy images revealed that the twin boundaries were positioned at the van der Waals gaps between the layers. The narrow bands of dark contrast were observed in the bright-field transmission electron microscopy images below the antiferromagnetic transition temperature, T{sub N} ≈ 120 K. Low-temperature X-ray diffraction showed a lattice distortion; the a- and b-axes shortened and lengthened, respectively, as the temperature decreasedmore » below T{sub N.} We propose that the narrow bands of dark contrast observed in the bright-field transmission electron microscopy images are caused by the directional lattice distortion with respect to each micro-twin variant in the antiferromagnetic phase.« less

High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

PubMed Central

Boassa, Daniela; Hu, Junru; Romoli, Benedetto; Phan, Sebastien; Dulcis, Davide

2018-01-01

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications. PMID:29749931

Role for Dynamin in Late Endosome Dynamics and Trafficking of the Cation-independent Mannose 6-Phosphate Receptor

PubMed Central

Nicoziani, Paolo; Vilhardt, Frederik; Llorente, Alicia; Hilout, Leila; Courtoy, Pierre J.; Sandvig, Kirsten; van Deurs, Bo

2000-01-01

It is well established that dynamin is involved in clathrin-dependent endocytosis, but relatively little is known about possible intracellular functions of this GTPase. Using confocal imaging, we found that endogenous dynamin was associated with the plasma membrane, the trans-Golgi network, and a perinuclear cluster of cation-independent mannose 6-phosphate receptor (CI-MPR)–containing structures. By electron microscopy (EM), it was shown that these structures were late endosomes and that the endogenous dynamin was preferentially localized to tubulo-vesicular appendices on these late endosomes. Upon induction of the dominant-negative dynK44A mutant, confocal microscopy demonstrated a redistribution of the CI-MPR in mutant-expressing cells. Quantitative EM analysis of the ratio of CI-MPR to lysosome-associated membrane protein-1 in endosome profiles revealed a higher colocalization of the two markers in dynK44A-expressing cells than in control cells. Western blot analysis showed that dynK44A-expressing cells had an increased cellular procathepsin D content. Finally, EM revealed that in dynK44A-expressing cells, endosomal tubules containing CI-MPR were formed. These results are in contrast to recent reports that dynamin-2 is exclusively associated with endocytic structures at the plasma membrane. They suggest instead that endogenous dynamin also plays an important role in the molecular machinery behind the recycling of the CI-MPR from endosomes to the trans-Golgi network, and we propose that dynamin is required for the final scission of vesicles budding from endosome tubules. PMID:10679008

Morphological and molecular features of oral fluid-derived exosomes: oral cancer patients versus healthy individuals.

PubMed

Zlotogorski-Hurvitz, Ayelet; Dayan, Dan; Chaushu, Gavriel; Salo, Tuula; Vered, Marilena

2016-01-01

Oral cancer (OC) patients are at high risk to develop recurrent disease or secondary primary cancers with no available biomarkers to detect these events until a visible lesion is readily present and diagnosed by biopsy. Exosomes secreted by cancer cells are involved in tumor growth, invasion and metastasis. We aimed to determine morphological and molecular differences between oral fluid (OF)-derived exosomes of OC patients and those isolated from healthy individuals (HI). OF from OC patients (n = 36) and HI (n = 25) was initially assessed by nanoparticle tracking analysis (NTA). Following ultracentrifugation, exosomal pellets of OC patients and HI were morphologically examined by transmission electron microscopy and atomic force microscopy (AFM). Enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) were used to analyze the expression of exosomal markers--CD9, CD81 and CD63. NTA showed that OC samples of OF had a significantly higher concentration of nanoparticles/ml (p = 0.01) and modal nanoparticle size (p = 0.002) compared to HI. The difference in size was structurally highlighted by AFM three-dimensional images applied on exosomal pellets. ELISA and WB showed differential expression of exosomal markers in OC exosomes compared to HI: lower expression of CD81 and CD9 in contrast to a higher expression of CD63 (~53 kDa). OF-derived exosomes from OC patients differ both morphologically and molecularly from exosomes present in HI. This study is a baseline that provides a starting point for finding exosomal biomarkers for early detection of malignant changes in high-risk patients without overt clinical signs/lesions.

Nanodiamonds as multi-purpose labels for microscopy.

PubMed

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Retracing in correlative light electron microscopy: where is my object of interest?

PubMed

Hodgson, Lorna; Nam, David; Mantell, Judith; Achim, Alin; Verkade, Paul

2014-01-01

Correlative light electron microscopy (CLEM) combines the strengths of light and electron microscopy in a single experiment. There are many ways to perform a CLEM experiment and a variety of microscopy modalities can be combined either on separate instruments or as completely integrated solutions. In general, however, a CLEM experiment can be divided into three parts: probes, processing, and analysis. Most of the existing technologies are focussed around the development and use of probes or describe processing methodologies that explain or circumvent some of the compromises that need to be made when performing both light and electron microscopy on the same sample. So far, relatively little attention has been paid to the analysis part of CLEM experiments. Although it is an essential part of each CLEM experiment, it is usually a cumbersome manual process. Here, we briefly discuss each of the three above-mentioned steps, with a focus on the analysis part. We will also introduce an automated registration algorithm that can be applied to the analysis stage to enable the accurate registration of LM and EM images. This facilitates tracing back the right cell/object seen in the light microscope in the EM. © 2014 Elsevier Inc. All rights reserved.

Growth and Electronic Structure of Heusler Compounds for Use in Electron Spin Based Devices

DTIC Science & Technology

2015-06-01

either Co– or MnSi– initiated films on c(4x4) GaAs. Studies using x - ray photoemission spectroscopy (XPS), STM/STS, and transmission electron microscopy...Co– or MnSi– initiated films on c(4x4) GaAs. Studies using x - ray photoemission spectroscopy (XPS), STM/STS, and transmission electron microscopy (TEM...diagram of the Palmstrøm lab in-situ growth and char- acterization setup, with 6 MBE growth chambers, 3 scanning probe microscopes, an x - ray

Disorganization of cell division of methicillin-resistant Staphylococcus aureus by methanolic extract from Phyllanthus columnaris stem bark

DOE Office of Scientific and Technical Information (OSTI.GOV)

Adnalizawati, A. Siti Noor; Nazlina, I.; Yaacob, W. A.

The in vitro activity of methanolic extract from Phyllanthus columnaris stem bark was studied against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and MRSA BM1 (clinical strain) using time-kill curves in conjunction with scanning and transmission electron microscopy. The extract showed more markedly bactericidal activity in MRSA BM1 clinical strain within less than 4 h by 6.25-12.5 mg/mL and within 6 h by 1.56 mg/mL. Scanning electron microscopy of MRSA BM1 revealed distortion of cell whilst transmission electron microscopy revealed disruption in cell wall division.

Analytical electron microscopy of Mg-SiO smokes - A comparison with infrared and XRD studies

NASA Technical Reports Server (NTRS)

Rietmeijer, F. J. M.; Nuth, J. A.; Mackinnon, I. D. R.

1986-01-01

Analytical electron microscopy conducted for Mg-SiO smokes (experimentally obtained from samples previously characterized by IR spectroscopy) indicates that the microcrystallinity content of unannealed smokes increases with increased annealing for up to 30 hr. The growth of forsterite microcrystallites in the initially nonstoichiometric smokes may give rise to the contemporaneous growth of the SiO polymorph tridymite and MgO; after 4 hr of annealing, these react to form enstatite. It is suggested that XRD analysis and IR spectroscopy should be conducted in conjunction with detailed analytical electron microscopy for the detection of emerging crystallinity in vapor-phase condensates.

Disorganization of cell division of methicillin-resistant Staphylococcus aureus by methanolic extract from Phyllanthus columnaris stem bark

NASA Astrophysics Data System (ADS)

Adnalizawati, A. Siti Noor; Nazlina, I.; Yaacob, W. A.

2013-11-01

The in vitro activity of methanolic extract from Phyllanthus columnaris stem bark was studied against Methicillin-resistant Staphylococcus aureus (MRSA) ATCC 43300 and MRSA BM1 (clinical strain) using time-kill curves in conjunction with scanning and transmission electron microscopy. The extract showed more markedly bactericidal activity in MRSA BM1 clinical strain within less than 4 h by 6.25-12.5 mg/mL and within 6 h by 1.56 mg/mL. Scanning electron microscopy of MRSA BM1 revealed distortion of cell whilst transmission electron microscopy revealed disruption in cell wall division.

Surface Diagnostics in Tribology Technology and Advanced Coatings Development

NASA Technical Reports Server (NTRS)

Miyoshi, Kazuhisa

1999-01-01

This paper discusses the methodologies used for surface property measurement of thin films and coatings, lubricants, and materials in the field of tribology. Surface diagnostic techniques include scanning electron microscopy, transmission electron microscopy, atomic force microscopy, stylus profilometry, x-ray diffraction, electron diffraction, Raman spectroscopy, Rutherford backscattering, elastic recoil spectroscopy, and tribology examination. Each diagnostic technique provides specific measurement results in its own unique way. In due course it should be possible to coordinate the different pieces of information provided by these diagnostic techniques into a coherent self-consistent description of the surface properties. Examples are given on the nature and character of thin diamond films.

Advanced electron microscopy methods for the analysis of MgB2 superconductor

NASA Astrophysics Data System (ADS)

Birajdar, B.; Peranio, N.; Eibl, O.

2008-02-01

Advanced electron microscopy methods used for the analysis of superconducting MgB2 wires and tapes are described. The wires and tapes were prepared by the powder in tube method using different processing technologies and thoroughly characterised for their superconducting properties within the HIPERMAG project. Microstructure analysis on μm to nm length scales is necessary to understand the superconducting properties of MgB2. For the MgB2 phase analysis on μm scale an analytical SEM, and for the analysis on nm scale a energy-filtered STEM is used. Both the microscopes were equipped with EDX detector and field emission gun. Electron microscopy and spectroscopy of MgB2 is challenging because of the boron analysis, carbon and oxygen contamination, and the presence of large number of secondary phases. Advanced electron microscopy involves, combined SEM, EPMA and TEM analysis with artefact free sample preparation, elemental mapping and chemical quantification of point spectra. Details of the acquisition conditions and achieved accuracy are presented. Ex-situ wires show oxygen-free MgB2 colonies (a colony is a dense arrangement of several MgB2 grains) embedded in a porous and oxygen-rich matrix, introducing structural granularity. In comparison, in-situ wires are generally more dense, but show inhibited MgB2 phase formation with significantly higher fraction of B-rich secondary phases. SiC additives in the in-situ wires forms Mg2Si secondary phases. The advanced electron microscopy has been used to extract the microstructure parameters like colony size, B-rich secondary phase fraction, O mole fraction and MgB2 grain size, and establish a microstructure-critical current density model [1]. In summary, conventional secondary electron imaging in SEM and diffraction contrast imaging in the TEM are by far not sufficient and advanced electron microscopy methods are essential for the analysis of superconducting MgB2 wires and tapes.

Asbestos Analysis: What School Officials Should Know.

ERIC Educational Resources Information Center

Harris, Bonnie Lee

1984-01-01

Transmission electron microscopy and scanning electron microscopy are used to detect asbestos by analyzing filters from air tests. The modes of operation and types of samples examined by each are explained. Circumstances that a school board should consider when deciding whether to use these methods are discussed. (MLF)

Electron Microscopy Characterization of Aerosols Collected at Mauna Loa Observatory During Asian Dust Storm Event

EPA Science Inventory

Atmospheric aerosol particles have a significant influence on global climate due to their ability to absorb and scatter incoming solar radiation. Size, composition, and morphology affect a particle’s radiative properties and these can be characterized by electron microscopy. Lo...

Keggin-type polyoxometalate nanosheets: synthesis and characterization via scanning transmission electron microscopy.

PubMed

Hiyoshi, Norihito

2018-05-17

Polyoxometalate nanosheets were synthesized at the gas/liquid interface of an aqueous solution of Keggin-type silicotungstic acid, cesium chloride, and n-octylamine. The structure of the nanosheets was elucidated via aberration-corrected scanning transmission electron microscopy at the atomic and molecular levels.

Scanning electron microscopy analysis of corrosion degradation on tinplate substrates.

PubMed

Zumelzu, E; Cabezas, C; Vera, A

2003-01-01

The degradation of electrolytic tinplate used in food containers was analysed and evaluated, using scanning electron microscopy and electrochemical measurements of microcorrosion and ion dissolution by atomic absorption to prevent food contamination caused by metal traces and to increase the durability of such tinplates.

EVALUATION OF COMPUTER-CONTROLLED SCANNING ELECTRON MICROSCOPY APPLIED TO AN AMBIENT URBAN AEROSOL SAMPLE

EPA Science Inventory

Recent interest in monitoring and speciation of particulate matter has led to increased application of scanning electron microscopy (SEM) coupled with energy-dispersive x-ray analysis (EDX) to individual particle analysis. SEM/EDX provides information on the size, shape, co...

In-situ Isotopic Analysis at Nanoscale using Parallel Ion Electron Spectrometry: A Powerful New Paradigm for Correlative Microscopy

NASA Astrophysics Data System (ADS)

Yedra, Lluís; Eswara, Santhana; Dowsett, David; Wirtz, Tom

2016-06-01

Isotopic analysis is of paramount importance across the entire gamut of scientific research. To advance the frontiers of knowledge, a technique for nanoscale isotopic analysis is indispensable. Secondary Ion Mass Spectrometry (SIMS) is a well-established technique for analyzing isotopes, but its spatial-resolution is fundamentally limited. Transmission Electron Microscopy (TEM) is a well-known method for high-resolution imaging down to the atomic scale. However, isotopic analysis in TEM is not possible. Here, we introduce a powerful new paradigm for in-situ correlative microscopy called the Parallel Ion Electron Spectrometry by synergizing SIMS with TEM. We demonstrate this technique by distinguishing lithium carbonate nanoparticles according to the isotopic label of lithium, viz. 6Li and 7Li and imaging them at high-resolution by TEM, adding a new dimension to correlative microscopy.

Dynamic imaging with electron microscopy

ScienceCinema

Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

2018-02-13

Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses.

PubMed

Horne, R W; Wildy, P

1979-09-01

A brief historical account of the development and applications of the negative staining techniques to the study of the structure of viruses and their components as observed in the electron microscope is presented. Although the basic method of surrounding or embedding specimens in opaque dyes was used in light microscopy dating from about 1884, the equivalent preparative techniques applied to electron microscopy were comparatively recent. The combination of experiments on a sophisticated bacterial virus and the installation of a high resolution electron microscope in the Cavendish Laboratory, Cambridge, during 1954, subsequently led to the analysis of several important morphological features of animal, plant and bacterial viruses. The implications of the results from these early experiments on viruses and recent developments in negative staining methods for high resolution image analysis of electron micrographs are also discussed.

Possibilities and limitations of advanced transmission electron microscopy for carbon-based nanomaterials

PubMed Central

Bittencourt, Carla; Van Tendeloo, Gustaaf

2015-01-01

Summary A major revolution for electron microscopy in the past decade is the introduction of aberration correction, which enables one to increase both the spatial resolution and the energy resolution to the optical limit. Aberration correction has contributed significantly to the imaging at low operating voltages. This is crucial for carbon-based nanomaterials which are sensitive to electron irradiation. The research of carbon nanomaterials and nanohybrids, in particular the fundamental understanding of defects and interfaces, can now be carried out in unprecedented detail by aberration-corrected transmission electron microscopy (AC-TEM). This review discusses new possibilities and limits of AC-TEM at low voltage, including the structural imaging at atomic resolution, in three dimensions and spectroscopic investigation of chemistry and bonding. In situ TEM of carbon-based nanomaterials is discussed and illustrated through recent reports with particular emphasis on the underlying physics of interactions between electrons and carbon atoms. PMID:26425406

Quantifying the cellular uptake of semiconductor quantum dot nanoparticles by analytical electron microscopy.

PubMed

Hondow, Nicole; Brown, M Rowan; Starborg, Tobias; Monteith, Alexander G; Brydson, Rik; Summers, Huw D; Rees, Paul; Brown, Andy

2016-02-01

Semiconductor quantum dot nanoparticles are in demand as optical biomarkers yet the cellular uptake process is not fully understood; quantification of numbers and the fate of internalized particles are still to be achieved. We have focussed on the characterization of cellular uptake of quantum dots using a combination of analytical electron microscopies because of the spatial resolution available to examine uptake at the nanoparticle level, using both imaging to locate particles and spectroscopy to confirm identity. In this study, commercially available quantum dots, CdSe/ZnS core/shell particles coated in peptides to target cellular uptake by endocytosis, have been investigated in terms of the agglomeration state in typical cell culture media, the traverse of particle agglomerates across U-2 OS cell membranes during endocytosis, the merging of endosomal vesicles during incubation of cells and in the correlation of imaging flow cytometry and transmission electron microscopy to measure the final nanoparticle dose internalized by the U-2 OS cells. We show that a combination of analytical transmission electron microscopy and serial block face scanning electron microscopy can provide a comprehensive description of the internalization of an initial exposure dose of nanoparticles by an endocytically active cell population and how the internalized, membrane bound nanoparticle load is processed by the cells. We present a stochastic model of an endosome merging process and show that this provides a data-driven modelling framework for the prediction of cellular uptake of engineered nanoparticles in general. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Functionalization of a nanostructured hydroxyapatite with Cu(II) compounds as a pesticide: in situ transmission electron microscopy and environmental scanning electron microscopy observations of treated Vitis vinifera L. leaves.

PubMed

Battiston, Enrico; Salvatici, Maria C; Lavacchi, Alessandro; Gatti, Antonietta; Di Marco, Stefano; Mugnai, Laura

2018-02-19

The present study evaluated a biocompatible material for plant protection with the aim of reducing the amount of active substance applied. We used a synthetic hydroxyapatite (HA) that has been studied extensively as a consequence of its bioactivity and biocompatibility. An aggregation between HA nanoparticles and four Cu(II) compounds applied to Vitis vinifera L. leaves as a pesticide was studied. Formulations were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS) and electron microscopy and applied in planta to verify particle aggregation and efficiency in controlling the pathogen Plasmopara viticola. The XRD patterns showed different crystalline phases dependig on the Cu(II) compound formulated with HA particles, DLS showed that nanostructured particles are stable as aggregates out of the nanometer range and, in all formulations, transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM) microscopy showed large aggregates which were partially nanostructured and were recognized as stable in their micrometric dimensions. Such particles did not show phytotoxic effects after their application in planta. A formulation based on HA and a soluble Cu(II) compound showed promising results in the control of the fungal pathogen, confirming the potential role of HA as an innovative delivery system of Cu(II) ions. The present work indicates the possibility of improving the biological activity of a bioactive substance by modifying its structure through an achievable formulation with a biocompatible material. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis.

PubMed

Bonnell, B S; Larabell, C; Chandler, D E

1993-06-01

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a three-dimensional network of interconnected fibers extending nearly 50 microns from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a three-dimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50-60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix.

New Insights into Mutable Collagenous Tissue: Correlations between the Microstructure and Mechanical State of a Sea-Urchin Ligament

PubMed Central

Ribeiro, Ana R.; Barbaglio, Alice; Benedetto, Cristiano D.; Ribeiro, Cristina C.; Wilkie, Iain C.; Carnevali, Maria D. C.; Barbosa, Mário A.

2011-01-01

The mutable collagenous tissue (MCT) of echinoderms has the ability to undergo rapid and reversible changes in passive mechanical properties that are initiated and modulated by the nervous system. Since the mechanism of MCT mutability is poorly understood, the aim of this work was to provide a detailed morphological analysis of a typical mutable collagenous structure in its different mechanical states. The model studied was the compass depressor ligament (CDL) of a sea urchin (Paracentrotus lividus), which was characterized in different functional states mimicking MCT mutability. Transmission electron microscopy, histochemistry, cryo-scanning electron microscopy, focused ion beam/scanning electron microscopy, and field emission gun-environmental scanning electron microscopy were used to visualize CDLs at the micro- and nano-scales. This investigation has revealed previously unreported differences in both extracellular and cellular constituents, expanding the current knowledge of the relationship between the organization of the CDL and its mechanical state. Scanning electron microscopies in particular provided a three-dimensional overview of CDL architecture at the micro- and nano-scales, and clarified the micro-organization of the ECM components that are involved in mutability. Further evidence that the juxtaligamental cells are the effectors of these changes in mechanical properties was provided by a correlation between their cytology and the tensile state of the CDLs. PMID:21935473

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Evidence for Alzheimer's disease-linked synapse loss and compensation in mouse and human hippocampal CA1 pyramidal neurons.

PubMed

Neuman, Krystina M; Molina-Campos, Elizabeth; Musial, Timothy F; Price, Andrea L; Oh, Kwang-Jin; Wolke, Malerie L; Buss, Eric W; Scheff, Stephen W; Mufson, Elliott J; Nicholson, Daniel A

2015-11-01

Alzheimer's disease (AD) is associated with alterations in the distribution, number, and size of inputs to hippocampal neurons. Some of these changes are thought to be neurodegenerative, whereas others are conceptualized as compensatory, plasticity-like responses, wherein the remaining inputs reactively innervate vulnerable dendritic regions. Here, we provide evidence that the axospinous synapses of human AD cases and mice harboring AD-linked genetic mutations (the 5XFAD line) exhibit both, in the form of synapse loss and compensatory changes in the synapses that remain. Using array tomography, quantitative conventional electron microscopy, immunogold electron microscopy for AMPARs, and whole-cell patch-clamp physiology, we find that hippocampal CA1 pyramidal neurons in transgenic mice are host to an age-related synapse loss in their distal dendrites, and that the remaining synapses express more AMPA-type glutamate receptors. Moreover, the number of axonal boutons that synapse with multiple spines is significantly reduced in the transgenic mice. Through serial section electron microscopic analyses of human hippocampal tissue, we further show that putative compensatory changes in synapse strength are also detectable in axospinous synapses of proximal and distal dendrites in human AD cases, and that their multiple synapse boutons may be more powerful than those in non-cognitively impaired human cases. Such findings are consistent with the notion that the pathophysiology of AD is a multivariate product of both neurodegenerative and neuroplastic processes, which may produce adaptive and/or maladaptive responses in hippocampal synaptic strength and plasticity.

Juxtaglomerular cell tumor of the kidney: report of two cases with a papillary pattern.

PubMed

Têtu, B; Vaillancourt, L; Camilleri, J P; Bruneval, P; Bernier, L; Tourigny, R

1993-11-01

We report the clinicopathologic, immunohistochemical, and electron microscopic study of two cases of juxtaglomerular cell tumor of the kidney with a hitherto unreported dominant papillary pattern. Both tumors were associated with high blood pressure that did not respond to medical therapy, but that returned to normal after removal of the kidney. They were well delineated, tan, and had no necrosis. The cores of the papillary structures consisted of polygonal cells found to express renin by immunohistochemistry and to contain renin protogranules by electron microscopy. The papillary fronds were covered by one layer of cuboidal epithelial cells that did not stain for renin and had ultrastructural features reminiscent of the collecting duct epithelium. These tumors must be differentiated from malignant papillary tumors of the kidney, such as papillary clear cell carcinoma, transitional cell carcinoma, and collecting duct carcinoma.

ultraLM and miniLM: Locator tools for smart tracking of fluorescent cells in correlative light and electron microscopy.

PubMed

Brama, Elisabeth; Peddie, Christopher J; Wilkes, Gary; Gu, Yan; Collinson, Lucy M; Jones, Martin L

2016-12-13

In-resin fluorescence (IRF) protocols preserve fluorescent proteins in resin-embedded cells and tissues for correlative light and electron microscopy, aiding interpretation of macromolecular function within the complex cellular landscape. Dual-contrast IRF samples can be imaged in separate fluorescence and electron microscopes, or in dual-modality integrated microscopes for high resolution correlation of fluorophore to organelle. IRF samples also offer a unique opportunity to automate correlative imaging workflows. Here we present two new locator tools for finding and following fluorescent cells in IRF blocks, enabling future automation of correlative imaging. The ultraLM is a fluorescence microscope that integrates with an ultramicrotome, which enables 'smart collection' of ultrathin sections containing fluorescent cells or tissues for subsequent transmission electron microscopy or array tomography. The miniLM is a fluorescence microscope that integrates with serial block face scanning electron microscopes, which enables 'smart tracking' of fluorescent structures during automated serial electron image acquisition from large cell and tissue volumes.

The significance of Bragg's law in electron diffraction and microscopy, and Bragg's second law.

PubMed

Humphreys, C J

2013-01-01

Bragg's second law, which deserves to be more widely known, is recounted. The significance of Bragg's law in electron diffraction and microscopy is then discussed, with particular emphasis on differences between X-ray and electron diffraction. As an example of such differences, the critical voltage effect in electron diffraction is described. It is then shown that the lattice imaging of crystals in high-resolution electron microscopy directly reveals the Bragg planes used for the imaging process, exactly as visualized by Bragg in his real-space law. Finally, it is shown how in 2012, for the first time, on the centennial anniversary of Bragg's law, single atoms have been identified in an electron microscope using X-rays emitted from the specimen. Hence atomic resolution X-ray maps of a crystal in real space can be formed which give the positions and identities of the different atoms in the crystal, or of a single impurity atom in the crystal.

An inexpensive approach for bright-field and dark-field imaging by scanning transmission electron microscopy in scanning electron microscopy.

PubMed

Patel, Binay; Watanabe, Masashi

2014-02-01

Scanning transmission electron microscopy in scanning electron microscopy (STEM-in-SEM) is a convenient technique for soft materials characterization. Various specimen-holder geometries and detector arrangements have been used for bright-field (BF) STEM-in-SEM imaging. In this study, to further the characterization potential of STEM-IN-SEM, a new specimen holder has been developed to facilitate direct detection of BF signals and indirect detection of dark-field (DF) signals without the need for substantial instrument modification. DF imaging is conducted with the use of a gold (Au)-coated copper (Cu) plate attached to the specimen holder which directs highly scattered transmitted electrons to an off-axis yttrium-aluminum-garnet (YAG) detector. A hole in the copper plate allows for BF imaging with a transmission electron (TE) detector. The inclusion of an Au-coated Cu plate enhanced DF signal intensity. Experiments validating the acquisition of true DF signals revealed that atomic number (Z) contrast may be achieved for materials with large lattice spacing. However, materials with small lattice spacing still exhibit diffraction contrast effects in this approach. The calculated theoretical fine probe size is 1.8 nm. At 30 kV, in this indirect approach, DF spatial resolution is limited to 3.2 nm as confirmed experimentally.

Insights into the prominent effect of mahanimbine on Acanthamoeba castellanii: Cell profiling analysis based on microscopy techniques

NASA Astrophysics Data System (ADS)

Hashim, Fatimah; Amin, Nakisah Mat

2017-02-01

Mahanimbine (MH), has been shown to have antiamoeba properties. Therefore, the aim of this study was to assess the growth inhibitory mechanisms of MH on Acanthamoeba castellanii, a causative agents for Acanthamoeba keratitis. The IC50 value obtained for MH against A. castellanii was 1.18 µg/ml. Light and scanning electron microscopy observation showed that most cells were in cystic appearance. While transmission electron microscopy observation revealed changes at the ultrastructural level and fluorescence microscopy observation indicated the induction of apoptosis and autophagic activity in the amoeba cytoplasms. In conclusion, MH has very potent anti-amoebic properties on A. castellanii as is shown by cytotoxicity analyses based on microscopy techniques.

Mass transfer in fuel cells. [electron microscopy of components, thermal decomposition of Teflon, water transport, and surface tension of KOH solutions

NASA Technical Reports Server (NTRS)

Walker, R. D., Jr.

1973-01-01

Results of experiments on electron microscopy of fuel cell components, thermal decomposition of Teflon by thermogravimetry, surface area and pore size distribution measurements, water transport in fuel cells, and surface tension of KOH solutions are described.

The complete genome sequence of a second distinct betabaculovirus from the true armyworm, Mythimna unipuncta

USDA-ARS?s Scientific Manuscript database

The betabaculovirus Pseudaletia (Mythimna) sp. granulovirus #8 (MyspGV#8) was examined by electron microscopy, host barcoding PCR, and determination of the nucleotide sequence of its genome. Scanning and transmission electron microscopy revealed that the occlusion bodies of MyspGV#8 possessed the c...

An enteroendocrine cell-enteric glia connection revealed by 3D electron microscopy.

PubMed

Bohórquez, Diego V; Samsa, Leigh A; Roholt, Andrew; Medicetty, Satish; Chandra, Rashmi; Liddle, Rodger A

2014-01-01

The enteroendocrine cell is the cornerstone of gastrointestinal chemosensation. In the intestine and colon, this cell is stimulated by nutrients, tastants that elicit the perception of flavor, and bacterial by-products; and in response, the cell secretes hormones like cholecystokinin and peptide YY--both potent regulators of appetite. The development of transgenic mice with enteroendocrine cells expressing green fluorescent protein has allowed for the elucidation of the apical nutrient sensing mechanisms of the cell. However, the basal secretory aspects of the enteroendocrine cell remain largely unexplored, particularly because a complete account of the enteroendocrine cell ultrastructure does not exist. Today, the fine ultrastructure of a specific cell can be revealed in the third dimension thanks to the invention of serial block face scanning electron microscopy (SBEM). Here, we bridged confocal microscopy with SBEM to identify the enteroendocrine cell of the mouse and study its ultrastructure in the third dimension. The results demonstrated that 73.5% of the peptide-secreting vesicles in the enteroendocrine cell are contained within an axon-like basal process. We called this process a neuropod. This neuropod contains neurofilaments, which are typical structural proteins of axons. Surprisingly, the SBEM data also demonstrated that the enteroendocrine cell neuropod is escorted by enteric glia--the cells that nurture enteric neurons. We extended these structural findings into an in vitro intestinal organoid system, in which the addition of glial derived neurotrophic factors enhanced the development of neuropods in enteroendocrine cells. These findings open a new avenue of exploration in gastrointestinal chemosensation by unveiling an unforeseen physical relationship between enteric glia and enteroendocrine cells.

FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

PubMed

Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

2015-04-01

Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

Sparse sampling and reconstruction for electron and scanning probe microscope imaging

DOEpatents

Anderson, Hyrum; Helms, Jovana; Wheeler, Jason W.; Larson, Kurt W.; Rohrer, Brandon R.

2015-07-28

Systems and methods for conducting electron or scanning probe microscopy are provided herein. In a general embodiment, the systems and methods for conducting electron or scanning probe microscopy with an undersampled data set include: driving an electron beam or probe to scan across a sample and visit a subset of pixel locations of the sample that are randomly or pseudo-randomly designated; determining actual pixel locations on the sample that are visited by the electron beam or probe; and processing data collected by detectors from the visits of the electron beam or probe at the actual pixel locations and recovering a reconstructed image of the sample.

A simple energy filter for low energy electron microscopy/photoelectron emission microscopy instruments.

PubMed

Tromp, R M; Fujikawa, Y; Hannon, J B; Ellis, A W; Berghaus, A; Schaff, O

2009-08-05

Addition of an electron energy filter to low energy electron microscopy (LEEM) and photoelectron emission microscopy (PEEM) instruments greatly improves their analytical capabilities. However, such filters tend to be quite complex, both electron optically and mechanically. Here we describe a simple energy filter for the existing IBM LEEM/PEEM instrument, which is realized by adding a single scanning aperture slit to the objective transfer optics, without any further modifications to the microscope. This energy filter displays a very high energy resolution ΔE/E = 2 × 10(-5), and a non-isochromaticity of ∼0.5 eV/10 µm. The setup is capable of recording selected area electron energy spectra and angular distributions at 0.15 eV energy resolution, as well as energy filtered images with a 1.5 eV energy pass band at an estimated spatial resolution of ∼10 nm. We demonstrate the use of this energy filter in imaging and spectroscopy of surfaces using a laboratory-based He I (21.2 eV) light source, as well as imaging of Ag nanowires on Si(001) using the 4 eV energy loss Ag plasmon.

Direct observation of dopant distribution in GaAs compound semiconductors using phase-shifting electron holography and Lorentz microscopy.

PubMed

Sasaki, Hirokazu; Otomo, Shinya; Minato, Ryuichiro; Yamamoto, Kazuo; Hirayama, Tsukasa

2014-06-01

Phase-shifting electron holography and Lorentz microscopy were used to map dopant distributions in GaAs compound semiconductors with step-like dopant concentration. Transmission electron microscope specimens were prepared using a triple beam focused ion beam (FIB) system, which combines a Ga ion beam, a scanning electron microscope, and an Ar ion beam to remove the FIB damaged layers. The p-n junctions were clearly observed in both under-focused and over-focused Lorentz microscopy images. A phase image was obtained by using a phase-shifting reconstruction method to simultaneously achieve high sensitivity and high spatial resolution. Differences in dopant concentrations between 1 × 10(19) cm(-3) and 1 × 10(18) cm(-3) regions were clearly observed by using phase-shifting electron holography. We also interpreted phase profiles quantitatively by considering inactive layers induced by ion implantation during the FIB process. The thickness of an inactive layer at different dopant concentration area can be measured from the phase image. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Attosecond electron pulses for 4D diffraction and microscopy

PubMed Central

Baum, Peter; Zewail, Ahmed H.

2007-01-01

In this contribution, we consider the advancement of ultrafast electron diffraction and microscopy to cover the attosecond time domain. The concept is centered on the compression of femtosecond electron packets to trains of 15-attosecond pulses by the use of the ponderomotive force in synthesized gratings of optical fields. Such attosecond electron pulses are significantly shorter than those achievable with extreme UV light sources near 25 nm (≈50 eV) and have the potential for applications in the visualization of ultrafast electron dynamics, especially of atomic structures, clusters of atoms, and some materials. PMID:18000040

Inhibition of melanosome transfer results in skin lightening.

PubMed

Seiberg, M; Paine, C; Sharlow, E; Andrade-Gordon, P; Costanzo, M; Eisinger, M; Shapiro, S S

2000-08-01

The chemical basis of melanogenesis is well documented, but the mechanism of melanosome transfer and the regulation of pigmentation by keratinocyte-melanocyte interactions are not well understood. Therefore we examined the effects of serine protease inhibitors on skin pigmentation and found that the protease-activated receptor 2, expressed on keratinocytes, may regulate pigmentation via keratinocyte-melanocyte interactions. Here we show that modulation of protease-activated receptor 2 activation affects melanosome transfer into keratinocytes, resulting in changes in pigment production and deposition. SLIGRL, the protease-activated receptor 2 activating peptide, enhanced melanosome ingestion by keratinocytes, thus increasing pigment deposition. RWJ-50353, a serine protease inhibitor, led to reduced pigment deposition in melanocytes and depigmentation. Electron microscopy studies illustrated an accumulation of immature melanosomes inside melanocytes and abnormal dendrite dynamics in RWJ-50353-treated epidermal equivalents. RWJ-50353 induced a visible and dose-dependent skin lightening effect in the dark-skinned Yucatan swine. Examinations by electron microscopy indicated that the in vivo transfer of melanosomes from melanocytes to keratinocytes was affected. Our data suggest that modulation of keratinocyte-melanocyte interactions via the protease-activated receptor 2 pathway affects melanosome transfer. The use of RWJ-50353 to modulate protease-activated receptor 2 activation could lead to a new class of depigmenting agents.

Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells

PubMed Central

Ding, Ding; Xie, Youtao; Li, Kai; Huang, Liping; Zheng, Xuebin

2018-01-01

Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs), a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were comprehensively studied by employing scanning electron microscopy (SEM), X-ray diffraction (XRD) as well as transmission electron microscopy (TEM). The effects of hierarchical structures as well as micro-porous structure of tantalum coating on the behavior for human bone marrow stem cells (hBMSCs) were evaluated and compared at both cellular and molecular levels in vitro. The experimental results show that a hierarchical micro/nano structure with Ta2O5 nanotubes spread onto a micro-scale tantalum coating has been fabricated successfully, which is confirmed to promote cell adhesion and spreading. Besides, the hierarchical micro/nano tantalum coating can provide 1.5~2.1 times improvement in gene expression, compared with the micro-porous tantalum coating. It demonstrates that it can effectively enhance the proliferation and differentiation of hBMSCs in vitro. PMID:29614022

Micro/Nano Structural Tantalum Coating for Enhanced Osteogenic Differentiation of Human Bone Marrow Stem Cells.

PubMed

Ding, Ding; Xie, Youtao; Li, Kai; Huang, Liping; Zheng, Xuebin

2018-04-03

Recently, tantalum has been attracting much attention for its anticorrosion resistance and biocompatibility, and it has been widely used in surface modification for implant applications. To improve its osteogenic differentiation of human bone marrow stem cells (hBMSCs), a micro/nano structure has been fabricated on the tantalum coating surface through the combination of anodic oxidation and plasma spraying method. The morphology, composition, and microstructure of the modified coating were comprehensively studied by employing scanning electron microscopy (SEM), X-ray diffraction (XRD) as well as transmission electron microscopy (TEM). The effects of hierarchical structures as well as micro-porous structure of tantalum coating on the behavior for human bone marrow stem cells (hBMSCs) were evaluated and compared at both cellular and molecular levels in vitro. The experimental results show that a hierarchical micro/nano structure with Ta₂O₅ nanotubes spread onto a micro-scale tantalum coating has been fabricated successfully, which is confirmed to promote cell adhesion and spreading. Besides, the hierarchical micro/nano tantalum coating can provide 1.5~2.1 times improvement in gene expression, compared with the micro-porous tantalum coating. It demonstrates that it can effectively enhance the proliferation and differentiation of hBMSCs in vitro.

Pericellular innervation of neurons expressing abnormally hyperphosphorylated tau in the hippocampal formation of Alzheimer's disease patients.

PubMed

Blazquez-Llorca, Lidia; Garcia-Marin, Virginia; Defelipe, Javier

2010-01-01

Neurofibrillary tangles (NFT) represent one of the main neuropathological features in the cerebral cortex associated with Alzheimer's disease (AD). This neurofibrillary lesion involves the accumulation of abnormally hyperphosphorylated or abnormally phosphorylated microtubule-associated protein tau into paired helical filaments (PHF-tau) within neurons. We have used immunocytochemical techniques and confocal microscopy reconstructions to examine the distribution of PHF-tau-immunoreactive (ir) cells, and their perisomatic GABAergic and glutamatergic innervations in the hippocampal formation and adjacent cortex of AD patients. Furthermore, correlative light and electron microscopy was employed to examine these neurons and the perisomatic synapses. We observed two patterns of staining in PHF-tau-ir neurons, pattern I (without NFT) and pattern II (with NFT), the distribution of which varies according to the cortical layer and area. Furthermore, the distribution of both GABAergic and glutamatergic terminals around the soma and proximal processes of PHF-tau-ir neurons does not seem to be altered as it is indistinguishable from both control cases and from adjacent neurons that did not contain PHF-tau. At the electron microscope level, a normal looking neuropil with typical symmetric and asymmetric synapses was observed around PHF-tau-ir neurons. These observations suggest that the synaptic connectivity around the perisomatic region of these PHF-tau-ir neurons was apparently unaltered.

PECTATE LYASE-LIKE 9 from Brassica campestris is associated with intine formation.

PubMed

Jiang, Jingjing; Yao, Lina; Yu, Youjian; Liang, Ying; Jiang, Jianxia; Ye, Nenghui; Miao, Ying; Cao, Jiashu

2014-12-01

Brassica campestris pectate lyase-like 9 (BcPLL9) was previously identified as a differentially expressed gene both in buds during late pollen developmental stage and in pistils during fertilization in Chinese cabbage. To characterize the gene's function, antisense-RNA lines of BcPLL9 (bcpll9) were constructed in Chinese cabbage. Self- and cross-fertilization experiments harvested half seed yields when bcpll9 lines were used as pollen donors. In vivo and in vitro pollen germination assays showed that nearly half of the pollen tubes in bcpll9 were irregular with shorter length and uneven surface. Aniline blue staining identified abnormal accumulation of a specific bright blue unknown material in the bcpll9 pollen portion. Scanning electron microscopy observation verified the abnormal outthrust material to be near the pollen germinal furrows. Transmission electron microscopy observation revealed the internal endintine layer was overdeveloped and predominantly occupied the intine. This abnormally formed intine likely induced the wavy structure and growth arrest of the pollen tube in half of the bcpll9 pollen grains, which resulted in less seed yields. Collectively, this study presented a novel PLL gene that has an important function in B. campestris intine formation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Malignant peripheral nerve sheath tumor of the uterine cervix expressing both S-100 protein and HMB-45.

PubMed

Kim, Na Rae; Chung, Dong-Hae; Park, Chan Yong; Ha, Seung Yeon

2009-12-01

A 50-year-old woman presented with a large cervical polypoid mass. Grossly, the mass occupied a substantial proportion of the cervical canal, measuring 6 cm. Histologically, the mass showed a spindle cell malignancy arranged in large fascicles that penetrated deeply into the fibromuscular wall of the cervix. The spindle cells were immunoreactive for both S-100 protein and HMB-45 antigen, but were negative for Melan-A. Electron microscopy showed that cytoplasmic processes of the spindle to oval tumor cells contained microtubules and were lined by basal lamina and abundant intercellular collagen spacing with no melanosomes in any stage. As far as we are aware, this is the ninth reported case of cervical malignant peripheral nerve sheath tumor (MPNST), and the second reported case of MPNST expressing HMB-45 antigen.

Heterochrony in mandible development of larval shrimp (Decapoda: Caridea)--a comparative morphological SEM study of two carideans.

PubMed

Batel, Annika; Melzer, Roland R; Anger, Klaus; Geiselbrecht, Hannes

2014-11-01

Mandible development in the larval stages I-V of two palaemonid shrimp species, Palaemon elegans and Macrobrachium amazonicum, was analyzed using scanning electron microscopy, light microscopy, and confocal laser scanning microscopy. In contrast to the zoea I of P. elegans, first-stage larvae of M. amazonicum are nonfeeding. At hatching, the morphology of the mandibles is fully expressed in P. elegans, while it appears underdeveloped in M. amazonicum, presenting only small precursors of typical caridean features. In successive zoeal stages, both species show similar developmental changes, but the mandibular characters of the larvae in M. amazonicum were delayed compared to the equivalent stages in P. elegans, especially in the development of submarginal setae and mandible size. In conclusion, our results indicate heterochrony (postdisplacement) of mandible development in M. amazonicum compared to that in P. elegans, which is related to initial lack of mandible functionality or planktivorous feeding at hatching, respectively. This conclusion is supported by comparison with other palaemonid zoeae exhibiting different feeding modes. Our data suggest that an evolutionary ground pattern of mandible morphology is present even in species with nonfeeding first-stage larvae. © 2014 Wiley Periodicals, Inc.

Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps.

PubMed

Lees, Robert M; Peddie, Christopher J; Collinson, Lucy M; Ashby, Michael C; Verkade, Paul

2017-01-01

Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM). This provides a snapshot of high resolution structural information pertaining to the shape, organization, and localization of multiple subcellular structures at the same time. The pairing of these two imaging modalities in the same specimen provides key information to relate cellular dynamics to the ultrastructural environment. Until recently, approaches to relocate a region of interest (ROI) in tissue from 2P microscopy for SBF-SEM have been inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by using the laser from a multiphoton microscope to create fiducial markers for accurate correlation of 2P and electron microscopy (EM) imaging volumes. The process is quick and can be user defined for each sample. Here, to increase the efficiency of ROI relocation, multiple NIRB marks are used in 3D to target ultramicrotomy. A workflow is described and discussed to obtain a data set for 3D correlated light and electron microscopy, using three different preparations of brain tissue as examples. Copyright © 2017 Elsevier Inc. All rights reserved.

Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy

PubMed Central

Liv, Nalan; van Oosten Slingeland, Daan S. B.; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W.; Hoogenboom, Jacob P.

2016-01-01

We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization. PMID:26580231

A549 lung epithelial cells grown as three-dimensional aggregates: alternative tissue culture model for Pseudomonas aeruginosa pathogenesis.

PubMed

Carterson, A J; Höner zu Bentrup, K; Ott, C M; Clarke, M S; Pierson, D L; Vanderburg, C R; Buchanan, K L; Nickerson, C A; Schurr, M J

2005-02-01

A three-dimensional (3-D) lung aggregate model was developed from A549 human lung epithelial cells by using a rotating-wall vessel bioreactor to study the interactions between Pseudomonas aeruginosa and lung epithelial cells. The suitability of the 3-D aggregates as an infection model was examined by immunohistochemistry, adherence and invasion assays, scanning electron microscopy, and cytokine and mucoglycoprotein production. Immunohistochemical characterization of the 3-D A549 aggregates showed increased expression of epithelial cell-specific markers and decreased expression of cancer-specific markers compared to their monolayer counterparts. Immunohistochemistry of junctional markers on A549 3-D cells revealed that these cells formed tight junctions and polarity, in contrast to the cells grown as monolayers. Additionally, the 3-D aggregates stained positively for the production of mucoglycoprotein while the monolayers showed no indication of staining. Moreover, mucin-specific antibodies to MUC1 and MUC5A bound with greater affinity to 3-D aggregates than to the monolayers. P. aeruginosa attached to and penetrated A549 monolayers significantly more than the same cells grown as 3-D aggregates. Scanning electron microscopy of A549 cells grown as monolayers and 3-D aggregates infected with P. aeruginosa showed that monolayers detached from the surface of the culture plate postinfection, in contrast to the 3-D aggregates, which remained attached to the microcarrier beads. In response to infection, proinflammatory cytokine levels were elevated for the 3-D A549 aggregates compared to monolayer controls. These findings suggest that A549 lung cells grown as 3-D aggregates may represent a more physiologically relevant model to examine the interactions between P. aeruginosa and the lung epithelium during infection.

Development of lacrimal gland spheroids for lacrimal gland tissue regeneration.

PubMed

Massie, Isobel; Spaniol, Kristina; Barbian, Andreas; Geerling, Gerd; Metzger, Marco; Schrader, Stefan

2018-04-01

Severe dry eye syndrome resulting from lacrimal gland (LG) dysfunction can cause blindness, yet treatments remain palliative. In vitro reconstruction of LG tissue could provide a curative treatment. We aimed to combine epithelial cells with endothelial cells and mesenchymal stem cells (MSCs) to form a 3D functional unit. Epithelial cells and MSCs were isolated from porcine LG; endothelial cells were isolated from human foreskin. MSCs were characterised (flow cytometry and differentiation potential assays). All 3 cell types were combined on Matrigel and spheroid formation observed. Spheroids were characterised [immunohistochemistry (IHC) and transmission electron microscopy] and function assessed (β-hexosaminidase assay). Spheroids were transferred to decellularised jejunum (SIS-Muc) in dynamic cultures for 1 week before further characterisation. MSCs did not express CD31 but expressed CD44 and CD105 and differentiated towards osteogenic and adipogenic lineages. Spheroids formed on Matrigel within 18 hr, contracting to ~10% of the well area (p < .005). IHC revealed presence of all 3 cells within spheroids. Transmission electron microscopy revealed cell-cell contacts and polarisation at the apical surface. In static cultures, function was increased in spheroids cf. monolayer controls (p < .05) but over 72 hr, spheroid function (p < .05), viability (p < .05), and proliferation decreased, whilst apoptosis increased. On SIS-Muc under dynamic culture, however, spheroids continued to proliferate to repopulate SIS-Muc. IHC revealed LG epithelial cells coexpressing pan-cytokeratin and lysozyme, as well as endothelial cells and MSCs and cells remained capable of responding to carbachol (p < .05). These spheroids could form the basis of a regenerative medicine treatment approach for dry eye syndrome. In vivo studies are required to evaluate this further. Copyright © 2017 John Wiley & Sons, Ltd.

A recombined fusion protein PTD-Grb2-SH2 inhibits the proliferation of breast cancer cells in vitro.

PubMed

Yin, Jikai; Cai, Zhongliang; Zhang, Li; Zhang, Jian; He, Xianli; Du, Xilin; Wang, Qing; Lu, Jianguo

2013-03-01

The growth factor receptor bound protein 2 (Grb2) is one of the affirmative targets for cancer therapy, especially for breast cancer. In this study, we hypothesized the Src-homology 2 (SH2) domain in Grb2 may serve as a competitive protein-binding agent to interfere with the proliferation of breast cancer cells in vitro. We designed, constructed, expressed and purified a novel fusion protein containing the protein transduction domain (PTD) and Grb2-SH2 domain (we named it after PTD-Grb2-SH2). An immunofluorescence assay was used to investigate the location of PTD-Grb2-SH2 in cells. MTT assay and EdU experiments were applied to detect the proliferation of breast cancer cells. The ultra-structure was observed using transmission electron microscopy. Flow cytometry was used to determine the cytotoxicity of PTD-Grb2-SH2 on cell proliferation. We successfully obtained the PTD-Grb2-SH2 fusion protein in soluble form using a prokaryotic expression system. The new fusion protein successfully passed through both the cellular and nuclear membranes of breast cancer cells. The MTT assay showed that PTD-Grb2-SH2 exhibited significant toxicity to breast cancer cells in a dose- and time-dependent manner in vitro. EdU identified the decreased proliferation rates in treated MDA-MB-231 and SK-BR-3 cells. Observation by transmission electron microscopy and flow cytometry further confirmed the cytotoxicity as apoptosis. Our results show that the HIV1-TAT domain is a useful tool for transporting a low molecular weight protein across the cell membrane in vitro. The PTD-Grb2-SH2 may be a novel agent for breast cancer therapy.

Physico-chemical properties and biological effects of diesel and biomass particles.

PubMed

Longhin, Eleonora; Gualtieri, Maurizio; Capasso, Laura; Bengalli, Rossella; Mollerup, Steen; Holme, Jørn A; Øvrevik, Johan; Casadei, Simone; Di Benedetto, Cristiano; Parenti, Paolo; Camatini, Marina

2016-08-01

Diesel combustion and solid biomass burning are the major sources of ultrafine particles (UFP) in urbanized areas. Cardiovascular and pulmonary diseases, including lung cancer, are possible outcomes of combustion particles exposure, but differences in particles properties seem to influence their biological effects. Here the physico-chemical properties and biological effects of diesel and biomass particles, produced under controlled laboratory conditions, have been characterized. Diesel UFP were sampled from a Euro 4 light duty vehicle without DPF fuelled by commercial diesel and run over a chassis dyno. Biomass UFP were collected from a modern automatic 25 kW boiler propelled by prime quality spruce pellet. Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images of both diesel and biomass samples showed aggregates of soot particles, but in biomass samples ash particles were also present. Chemical characterization showed that metals and PAHs total content was higher in diesel samples compared to biomass ones. Human bronchial epithelial (HBEC3) cells were exposed to particles for up to 2 weeks. Changes in the expression of genes involved in xenobiotic metabolism were observed after exposure to both UFP already after 24 h. However, only diesel particles modulated the expression of genes involved in inflammation, oxidative stress and epithelial-to-mesenchymal transition (EMT), increased the release of inflammatory mediators and caused phenotypical alterations, mostly after two weeks of exposure. These results show that diesel UFP affected cellular processes involved in lung and cardiovascular diseases and cancer. Biomass particles exerted low biological activity compared to diesel UFP. This evidence emphasizes that the study of different emission sources contribution to ambient PM toxicity may have a fundamental role in the development of more effective strategies for air quality improvement. Copyright © 2016 Elsevier Ltd. All rights reserved.

Arabidopsis membrane-associated acyl-CoA-binding protein ACBP1 is involved in stem cuticle formation

PubMed Central

Xue, Yan; Xiao, Shi; Kim, Juyoung; Lung, Shiu-Cheung; Chen, Liang; Tanner, Julian A.; Suh, Mi Chung; Chye, Mee-Len

2014-01-01

The membrane-anchored Arabidopsis thaliana ACYL-COA-BINDING PROTEIN1 (AtACBP1) plays important roles in embryogenesis and abiotic stress responses, and interacts with long-chain (LC) acyl-CoA esters. Here, AtACBP1 function in stem cuticle formation was investigated. Transgenic Arabidopsis transformed with an AtACBP1pro::GUS construct revealed β-glucuronidase (GUS) expression on the stem (but not leaf) surface, suggesting a specific role in stem cuticle formation. Isothermal titration calorimetry results revealed that (His)6-tagged recombinant AtACBP1 interacts with LC acyl-CoA esters (18:1-, 18:2-, and 18:3-CoAs) and very-long-chain (VLC) acyl-CoA esters (24:0-, 25:0-, and 26:0-CoAs). VLC fatty acids have been previously demonstrated to act as precursors in wax biosynthesis. Gas chromatography (GC)–flame ionization detector (FID) and GC–mass spectrometry (MS) analyses revealed that an acbp1 mutant showed a reduction in stem and leaf cuticular wax and stem cutin monomer composition in comparison with the wild type (Col-0). Consequently, the acbp1 mutant showed fewer wax crystals on the stem surface in scanning electron microscopy and an irregular stem cuticle layer in transmission electron microscopy in comparison with the wild type. Also, the mutant stems consistently showed a decline in expression of cuticular wax and cutin biosynthetic genes in comparison with the wild type, and the mutant leaves were more susceptible to infection by the necrotrophic pathogen Botrytis cinerea. Taken together, these findings suggest that AtACBP1 participates in Arabidopsis stem cuticle formation by trafficking VLC acyl-CoAs. PMID:25053648

Genetic Deletion of the Adenosine A2A Receptor Confers Postnatal Development of Relative Myopia in Mice

PubMed Central

Zhou, Xiangtian; Huang, Qinzhu; An, Jianhong; Lu, Runxia; Qin, Xiaoyi; Jiang, Liqin; Li, Yuan; Wang, Jianhua; Chen, Jiangfan; Qu, Jia

2010-01-01

Purpose. To critically evaluate whether the adenosine A2A receptor (A2AR) plays a role in postnatal refractive development in mice. Methods. Custom-built biometric systems specifically designed for mice were used to assess the development of relative myopia by examining refraction and biometrics in A2AR knockout (KO) mice and wild-type (WT) littermates between postnatal days (P)28 and P56. Ocular dimensions were measured by customized optical coherence tomography (OCT), refractive state by eccentric infrared photorefraction (EIR), and corneal radius of curvature by modified keratometry. Scleral collagen diameter and density were examined by electron microscopy on P35. The effect of A2AR activation on collagen mRNA expression and on soluble collagen production was examined in cultured human scleral fibroblasts by real-time RT-PCR and a collagen assay kit. Results. Compared with WT littermates, the A2AR KO mice displayed relative myopia (average difference, 5.1 D between P28 and P35) and associated increases in VC depth and axial length from P28 to P56. Furthermore, the myopic shift in A2AR KO mice was associated with ultrastructural changes in the sclera: Electron microscopy revealed denser collagen fibrils with reduced diameter in A2AR KO compared with WT. Last, A2AR activation induced expression of mRNAs for collagens I, III, and V and increased production of soluble collagen in cultured human scleral fibroblasts. Conclusions. Genetic deletion of the A2AR promotes development of relative myopia with increased axial length and altered scleral collagen fiber structure during postnatal development in mice. Thus, the A2AR may be important in normal refractive development. PMID:20484596

TPC2 controls pigmentation by regulating melanosome pH and size.

PubMed

Ambrosio, Andrea L; Boyle, Judith A; Aradi, Al E; Christian, Keith A; Di Pietro, Santiago M

2016-05-17

Melanin is responsible for pigmentation of skin and hair and is synthesized in a specialized organelle, the melanosome, in melanocytes. A genome-wide association study revealed that the two pore segment channel 2 (TPCN2) gene is strongly linked to pigmentation variations. TPCN2 encodes the two-pore channel 2 (TPC2) protein, a cation channel. Nevertheless, how TPC2 regulates pigmentation remains unknown. Here, we show that TPC2 is expressed in melanocytes and localizes to the melanosome-limiting membrane and, to a lesser extent, to endolysosomal compartments by confocal fluorescence and immunogold electron microscopy. Immunomagnetic isolation of TPC2-containing organelles confirmed its coresidence with melanosomal markers. TPCN2 knockout by means of clustered regularly interspaced short palindromic repeat/CRISPR-associated 9 gene editing elicited a dramatic increase in pigment content in MNT-1 melanocytic cells. This effect was rescued by transient expression of TPC2-GFP. Consistently, siRNA-mediated knockdown of TPC2 also caused a substantial increase in melanin content in both MNT-1 cells and primary human melanocytes. Using a newly developed genetically encoded pH sensor targeted to melanosomes, we determined that the melanosome lumen in TPC2-KO MNT-1 cells and primary melanocytes subjected to TPC2 knockdown is less acidic than in control cells. Fluorescence and electron microscopy analysis revealed that TPC2-KO MNT-1 cells have significantly larger melanosomes than control cells, but the number of organelles is unchanged. TPC2 likely regulates melanosomes pH and size by mediating Ca(2+) release from the organelle, which is decreased in TPC2-KO MNT-1 cells, as determined with the Ca(2+) sensor tyrosinase-GCaMP6. Thus, our data show that TPC2 regulates pigmentation through two fundamental determinants of melanosome function: pH and size.

Low RNA translation activit limits the efficacy of hydrodynamic gene transfer to pig liver in vivo.

PubMed

Sendra, Luis; Carreño, Omar; Miguel, Antonio; Montalvá, Eva; Herrero, María José; Orbis, Francisco; Noguera, Inmaculada; Barettino, Domingo; López-Andújar, Rafael; Aliño, Salvador F

2014-01-01

Hydrodynamic gene delivery has proved an efficient strategy for nonviral gene therapy in the murine liver but it has been less efficient in pigs. The reason for such inefficiency remains unclear. The present study used a surgical strategy to seal the whole pig liver in vivo. A solution of enhanced green fluorescent protein (eGFP) DNA was injected under two different venous injection conditions (anterograde and retrograde), employing flow rates of 10 and 20 ml/s in each case, with the aim of identifying the best gene transfer conditions. The gene delivery and information decoding steps were evaluated by measuring the eGFP DNA, mRNA and protein copy number 24 h after transfection. In addition, gold nanoparticles (diameters of 4 and 15 nm) were retrogradely injected (10 ml/s) to observe, by electron microscopy, the ability of the particle to access the hepatocyte. The gene delivery level was higher with anterograde injection, whereas the efficacy of gene expression was better with retrograde injection, suggesting differences in the decoding processes. Thus, retrograde injection mediates gene transcription (mRNA copy/cell) equivalent to that of intermediate expression proteins but the mRNA translation was lower than that of rare proteins. Electron microscopy showed that nanoparticles within the hepatocyte were almost exclusively 4 nm in diameter. The results suggest that the low activity of mRNA translation limits the final efficacy of the gene transfer procedure. On the other hand, the gold nanoparticles study suggests that elongated DNA conformation could offer advantages in that the access of 15-nm particles is very limited. Copyright © 2014 John Wiley & Sons, Ltd.

Glial-derived neurotrophic factor is essential for blood-nerve barrier functional recovery in an experimental murine model of traumatic peripheral neuropathy.

PubMed

Dong, Chaoling; Helton, E Scott; Zhou, Ping; Ouyang, Xuan; d'Anglemont de Tassigny, Xavier; Pascual, Alberto; López-Barneo, José; Ubogu, Eroboghene E

2018-06-18

There is emerging evidence that glial-derived neurotrophic factor (GDNF) is a potent inducer of restrictive barrier function in tight junction-forming microvascular endothelium and epithelium, including the human blood-nerve barrier (BNB) in vitro. We sought to determine the role of GDNF in restoring BNB function in vivo by evaluating sciatic nerve horseradish peroxidase (HRP) permeability in tamoxifen-inducible GDNF conditional knockout (CKO) adult mice following non-transecting crush injury via electron microscopy, with appropriate wildtype (WT) and heterozygous (HET) littermate controls. A total of 24 age-, genotype- and sex-matched mice >12 weeks of age were injected with 30 mg/kg HRP via tail vein injection 7 or 14 days following unilateral sciatic nerve crush, and both sciatic nerves were harvested 30 minutes later for morphometric assessment by light and electron microscopy. The number and percentage of HRP-permeable endoneurial microvessels were ascertained to determine the effect of GDNF in restoring barrier function in vivo. Following sciatic nerve crush, there was significant upregulation in GDNF protein expression in WT and HET mice that was abrogated in CKO mice. GDNF significantly restored sciatic nerve BNB HRP impermeability to near normal levels by day 7, with complete restoration seen by day 14 in WT and HET mice. A significant recovery lag was observed in CKO mice. This effect was independent on VE-Cadherin or claudin-5 expression on endoneurial microvessels. These results imply an important role of GDNF in restoring restrictive BNB function in vivo, suggesting a potential strategy to re-establish the restrictive endoneurial microenvironment following traumatic peripheral neuropathies.

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Pitting corrosion of titanium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Casillas, N.; Charlebois, S.; Smyrl, W.H.

1994-03-01

The breakdown of native and anodically grown oxide films on Ti electrodes is investigated by scanning electrochemical microscopy (SECM), video microscopy, transmission electron microscopy, and voltammetry. SECM is used to demonstrated that the oxidation of Br[sup [minus

Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway.

PubMed

Lin, Ruhui; Li, Zuanfang; Lin, Jiumao; Ye, Jinxia; Cai, Qiaoyan; Chen, Lidian; Peng, Jun

2015-10-01

Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis.

Hydroxyapatite promotes superior keratocyte adhesion and proliferation in comparison with current keratoprosthesis skirt materials.

PubMed

Mehta, J S; Futter, C E; Sandeman, S R; Faragher, R G A F; Hing, K A; Tanner, K E; Allan, B D S

2005-10-01

Published clinical series suggest the osteoodontokeratoprosthesis (OOKP) may have a lower extrusion rate than current synthetic keratoprostheses. The OOKP is anchored in the eye wall by autologous tooth. The authors' aim was to compare adhesion, proliferation, and morphology for telomerase transformed keratocytes seeded on calcium hydroxyapatite (the principal mineral constituent of tooth) and materials used in the anchoring elements of commercially available synthetic keratoprostheses. Test materials were hydroxyapatite, polytetrafluoroethylene (PTFE), polyhydroxyethyl methacrylate (HEMA), and glass (control). Cell adhesion and viability were quantified at 4 hours, 24 hours, and 1 week using a calcein-AM/EthD-1 viability/cytotoxicity assay. Focal contact expression and cytoskeletal organisation were studied at 24 hours by confocal microscopy with immunoflourescent labelling. Further studies of cell morphology were performed using light and scanning electron microscopy. Live cell counts were significantly greater on hydroxyapatite surfaces at each time point (p<0.04). Dead cell counts were significantly higher for PTFE at 7 days (p<0.002). ss(1) integrin expression was highest on hydroxyapatite. Adhesion structures were well expressed in flat, spread out keratocytes on both HA and glass. Keratocytes tended to be thinner and spindle shaped on PTFE. The relatively few keratocytes visible on HEMA test surfaces were rounded and poorly adherent. Keratocyte adhesion, spreading, and viability on hydroxyapatite test surfaces is superior to that seen on PTFE and HEMA. Improving the initial cell adhesion environment in the skirt element of keratoprostheses may enhance tissue integration and reduce device failure rates.

Ethanolic extract of Tulipa edulis Bak induces apoptosis in SGC-7901 human gastric carcinoma cells via the mitochondrial signaling pathway

PubMed Central

LIN, RUHUI; LI, ZUANFANG; LIN, JIUMAO; YE, JINXIA; CAI, QIAOYAN; CHEN, LIDIAN; PENG, JUN

2015-01-01

Tulipa edulis Bak (TEB) is an active ingredient in various traditional Chinese medicine compounds and is commonly used to treat swelling and redness, remove toxicity and eliminate stagnation, as well as to prevent and treat certain cancer types. However, the underlying molecular mechanism of the anticancer activity of TEB remains unclear. The aim of the current study was to investigate the effect and underlying mechanism of the ethanolic extract of TEB (EETEB) on SGC-7901 human gastric carcinoma cells. An MTT assay was performed to analyze cell viability. In addition, transmission electron microscopy, an Annexin V/fluorescein isothiocyanate assay, a JC-1 assay and laser scanning confocal microscopy with DAPI staining were used to determine the rate of apoptosis. Furthermore, reverse transcription-polymerase chain reaction and western blot analysis were used to detect the expression levels of the apoptosis gene and protein. EETEB was identified to inhibit the growth of SGC-7901 cells in a dose-dependent manner and induce changes in cell morphology. At the molecular level, EETEB induced SGC-7901 cell DNA fragmentation, loss of plasma membrane and asymmetrical collapse of the mitochondrial membrane potential, while it increased the expression of pro-apoptotic B-cell lymphoma-2 (Bcl-2)-associated X protein and reduced expression of anti-apoptotic Bcl-2. Thus, the results of the current study revealed that the application of EETEB may inhibit the growth of the SGC-7901 cells due to mitochondria-mediated apoptosis. PMID:26622854

Subcellular Localization Screening of Colletotrichum higginsianum Effector Candidates Identifies Fungal Proteins Targeted to Plant Peroxisomes, Golgi Bodies, and Microtubules.

PubMed

Robin, Guillaume P; Kleemann, Jochen; Neumann, Ulla; Cabre, Lisa; Dallery, Jean-Félix; Lapalu, Nicolas; O'Connell, Richard J

2018-01-01

The genome of the hemibiotrophic anthracnose fungus, Colletotrichum higginsianum , encodes a large inventory of putative secreted effector proteins that are sequentially expressed at different stages of plant infection, namely appressorium-mediated penetration, biotrophy and necrotrophy. However, the destinations to which these proteins are addressed inside plant cells are unknown. In the present study, we selected 61 putative effector genes that are highly induced in appressoria and/or biotrophic hyphae. We then used Agrobacterium -mediated transformation to transiently express them as N -terminal fusions with fluorescent proteins in cells of Nicotiana benthamiana for imaging by confocal microscopy. Plant compartments labeled by the fusion proteins in N. benthamiana were validated by co-localization with specific organelle markers, by transient expression of the proteins in the true host plant, Arabidopsis thaliana , and by transmission electron microscopy-immunogold labeling. Among those proteins for which specific subcellular localizations could be verified, nine were imported into plant nuclei, three were imported into the matrix of peroxisomes, three decorated cortical microtubule arrays and one labeled Golgi stacks. Two peroxisome-targeted proteins harbored canonical C -terminal tripeptide signals for peroxisome import via the PTS1 (peroxisomal targeting signal 1) pathway, and we showed that these signals are essential for their peroxisome localization. Our findings provide valuable information about which host processes are potentially manipulated by this pathogen, and also reveal plant peroxisomes, microtubules, and Golgi as novel targets for fungal effectors.

Alteration of the RANKL/RANK/OPG System in Periprosthetic Osteolysis with Septic Loosening.

PubMed

Wang, Long; Dai, Zixun; Xie, Jie; Liao, Hao; Lv, Cheng; Hu, Yihe

2016-02-01

The pathogenesis of periprosthetic osteolysis with septic loosening remains incompletely understood. The purpose of this study was to investigate whether expression of the RANKL/RANK/OPG system is altered in septic interface membranes (SIMs). Seventeen cases with a SIM, 26 cases with an aseptic interface membrane (AIM), and 12 cases with a normal synovium (NS) were assessed. Scanning and transmission electron microscopy (SEM and TEM, respectively) were used to observe the microscopic morphology of three tissue conditions. Differences in RANKL, RANK, and OPG expression at the mRNA level were assessed by real-time quantitative PCR, and differences at the protein level were assessed by immunohistochemical staining and Western blotting. SEM showed wear debris widely distributed on the AIM surface, and TEM showed Bacillus activity in the SIM. RANKL expression and the RANKL/OPG ratio were significantly increased in SIMs. Imbalance in the RANKL/RANK/OPG system is related to periprosthetic osteolysis with septic loosening but is not the only possible pathogenic mechanism.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain.

PubMed

Mochizuki, Tomofumi; Hirai, Katsuyuki; Kanda, Ayami; Ohnishi, Jun; Ohki, Takehiro; Tsuda, Shinya

2009-08-01

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the second putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.

Comparative analysis of surface wax in mature fruits between Satsuma mandarin (Citrus unshiu) and 'Newhall' navel orange (Citrus sinensis) from the perspective of crystal morphology, chemical composition and key gene expression.

PubMed

Wang, Jinqiu; Hao, Haohao; Liu, Runsheng; Ma, Qiaoli; Xu, Juan; Chen, Feng; Cheng, Yunjiang; Deng, Xiuxin

2014-06-15

Surface wax of mature Satsuma mandarin (Citrus unshiu) and 'Newhall' navel orange (Citrus sinensis) was analysed by crystal morphology, chemical composition, and gene expression levels. The epicuticular and total waxes of both citrus cultivars were mostly composed of aldehydes, alkanes, fatty acids and primary alcohols. The epicuticular wax accounted for 80% of the total wax in the Newhall fruits and was higher than that in the Satsuma fruits. Scanning electron microscopy showed that larger and more wax platelets were deposited on the surface of Newhall fruits than on the Satsuma fruits. Moreover, the expression levels of genes involved in the wax formation were consistent with the biochemical and crystal morphological analyses. These diversities of fruit wax between the two cultivars may contribute to the differences of fruit postharvest storage properties, which can provide important information for the production of synthetic wax for citrus fruits. Copyright © 2013 Elsevier Ltd. All rights reserved.

Induction of necrosis via mitochondrial targeting of Melon necrotic spot virus replication protein p29 by its second transmembrane domain

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mochizuki, Tomofumi; Hirai, Katsuyuki; Kanda, Ayami

2009-08-01

The virulence factor of Melon necrotic spot virus (MNSV), a virus that induces systemic necrotic spot disease on melon plants, was investigated. When the replication protein p29 was expressed in N. benthamiana using a Cucumber mosaic virus vector, necrotic spots appeared on the leaf tissue. Transmission electron microscopy revealed abnormal mitochondrial aggregation in these tissues. Fractionation of tissues expressing p29 and confocal imaging using GFP-tagged p29 revealed that p29 associated with the mitochondrial membrane as an integral membrane protein. Expression analysis of p29 deletion fragments and prediction of hydrophobic transmembrane domains (TMDs) in p29 showed that deletion of the secondmore » putative TMD from p29 led to deficiencies in both the mitochondrial localization and virulence of p29. Taken together, these results indicated that MNSV p29 interacts with the mitochondrial membrane and that p29 may be a virulence factor causing the observed necrosis.« less

Correlative Fluorescence and Electron Microscopy

PubMed Central

Schirra, Randall T.; Zhang, Peijun

2014-01-01

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

Bile salt tolerance of Lactococcus lactis is enhanced by expression of bile salt hydrolase thereby producing less bile acid in the cells.

PubMed

Bi, Jie; Liu, Song; Du, Guocheng; Chen, Jian

2016-04-01

Changes of bile salt tolerance, morphology and amount of bile acid within cells were studied to evaluate the exact effects of bile salt hydrolase (BSH) on bile salt tolerance of microorganism. The effect of BSHs on the bile salt tolerance of Lactococcus lactis was examined by expressing two BSHs (BSH1 and BSH2). Growth of L. lactis expressing BSH1 or BSH2 was better under bile salt stress compared to wild-type L. lactis. As indicated by transmission electron microscopy, bile acids released by the action of BSH induced the formation of micelles around the membrane surface of cells subject to conjugated bile salt stress. A similar micelle containing bile acid was observed in the cytoplasm by liquid chromatography-mass spectrometry. BSH1 produced fewer bile acid micelles in the cytoplasm and achieved better cell growth of L. lactis compared to BSH2. Expression of BSH improved bile salt tolerance of L. lactis but excessive production by BSH of bile acid micelles in the cytoplasm inhibited cell growth.

A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

PubMed

Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

2015-01-01

The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, S.-F.; Huang, Jason C.; AIDS Prevention and Research Center, School of Medicine, National Yang-Ming University, Taipei 112, Taiwan

2008-09-05

DC-SIGN, a C-type lectin receptor expressed in dendritic cells (DCs), has been identified as a receptor for human immunodeficiency virus type 1, hepatitis C virus, Ebola virus, cytomegalovirus, dengue virus, and the SARS coronavirus. We used H5N1 pseudotyped and reverse-genetics (RG) virus particles to study their ability to bind with DC-SIGN. Electronic microscopy and functional assay results indicate that pseudotyped viruses containing both HA and NA proteins express hemagglutination and are capable of infecting cells expressing {alpha}-2,3-linked sialic acid receptors. Results from a capture assay show that DC-SIGN-expressing cells (including B-THP-1/DC-SIGN and T-THP-1/DC-SIGN) and peripheral blood dendritic cells are capablemore » of transferring H5N1 pseudotyped and RG virus particles to target cells; this action can be blocked by anti-DC-SIGN monoclonal antibodies. In summary, (a) DC-SIGN acts as a capture or attachment molecule for avian H5N1 virus, and (b) DC-SIGN mediates infections in cis and in trans.« less

Low-temperature and conventional scanning electron microscopy of human urothelial neoplasms.

PubMed

Hopkins, D M; Morris, J A; Oates, K; Huddart, H; Staff, W G

1989-05-01

The appearance of neoplastic human urothelium viewed by low-temperature scanning electron microscopy (LTSEM) and conventional scanning electron microscopy (CSEM) was compared. Fixed, dehydrated neoplastic cells viewed by CSEM had well-defined, often raised cell junctions; no intercellular gaps; and varying degrees of pleomorphic surface microvilli. The frozen hydrated material viewed by LTSEM, however, was quite different. The cells had a flat or dimpled surface, but no microvilli. There were labyrinthine lateral processes which interdigitated with those of adjacent cells and outlined large intercellular gaps. The process of fixation and dehydration will inevitably distort cell contours and on theoretical grounds, the images of frozen hydrated material should more closely resemble the in vivo appearance.

Comparison of the morphology, chemical composition and microstructure of cryptocrystalline graphite and carbon black

NASA Astrophysics Data System (ADS)

Quan, Ying; Liu, Qinfu; Zhang, Shilong; Zhang, Shuai

2018-07-01

The structures of cryptocrystalline graphite (CG) and carbon black (CB) have been analyzed using scanning electron microscopy (SEM), transmission electron microscopy (TEM), organic elemental analysis (OEA), X-ray diffraction (XRD), RAMAN and high-resolution transmission electron microscopy (HRTEM). These results indicate that CG has the same elemental composition as CB, with carbon being the major element present. SL sample (CG with low graphitization degree) and CB exhibit similar microcrystalline structures. CG was shown to contain a layered graphitic structure that was significantly different to the primary spherical particles present in CB. It is proposed that these CG sheets may potentially be reduced and delaminated to afford multilayer graphene structures with improved material properties.

Formation of tetragonal gas bubble superlattice in bulk molybdenum under helium ion implantation

DOE PAGES

Sun, Cheng; Sprouster, David J.; Hattar, K.; ...

2018-02-09

In this paper, we report the formation of tetragonal gas bubble superlattice in bulk molybdenum under helium ion implantation at 573 K. The transmission electron microscopy study shows that the helium bubble lattice constant measured from the in-plane d-spacing is ~4.5 nm, while it is ~3.9 nm from the out-of-plane measurement. The results of synchrotron-based small-angle x-ray scattering agree well with the transmission electron microscopy results in terms of the measurement of bubble lattice constant and bubble size. The coupling of transmission electron microscopy and synchrotron high-energy X-ray scattering provides an effective approach to study defect superlattices in irradiated materials.

Hydrothermal Synthesis of Nanoclusters of ZnS Comprised on Nanowires

PubMed Central

Ibupoto, Zafar Hussain; Khun, Kimleang; Liu, Xianjie; Willander, Magnus

2013-01-01

Cetyltrimethyl ammonium bromide cationic (CTAB) surfactant was used as template for the synthesis of nanoclusters of ZnS composed of nanowires, by hydrothermal method. The structural and morphological studies were performed by using X-ray diffraction (XRD), scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM) techniques. The synthesized ZnS nanoclusters are composed of nanowires and high yield on the substrate was observed. The ZnS nanocrystalline consists of hexagonal phase and polycrystalline in nature. The chemical composition of ZnS nanoclusters composed of nanowires was studied by X-ray photo electron microscopy (XPS). This investigation has shown that the ZnS nanoclusters are composed of Zn and S atoms. PMID:28348350

Hydrothermal Synthesis of Nanoclusters of ZnS Comprised on Nanowires.

PubMed

Ibupoto, Zafar Hussain; Khun, Kimleang; Liu, Xianjie; Willander, Magnus

2013-09-09

Cetyltrimethyl ammonium bromide cationic (CTAB) surfactant was used as template for the synthesis of nanoclusters of ZnS composed of nanowires, by hydrothermal method. The structural and morphological studies were performed by using X-ray diffraction (XRD), scanning electron microscopy (SEM) and high resolution transmission electron microscopy (HRTEM) techniques. The synthesized ZnS nanoclusters are composed of nanowires and high yield on the substrate was observed. The ZnS nanocrystalline consists of hexagonal phase and polycrystalline in nature. The chemical composition of ZnS nanoclusters composed of nanowires was studied by X-ray photo electron microscopy (XPS). This investigation has shown that the ZnS nanoclusters are composed of Zn and S atoms.