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.

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.

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.

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.

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

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.

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

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.

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

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

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.

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.

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.

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.

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

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.

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.

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

Gold nanoparticle uptake in whole cells in liquid examined by environmental scanning electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-02-01

The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 (human lung carcinoma) cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ~80 h. Analysis of 1,041 AuNP-filled vesicles showed that the long-term AuNP storing lysosomes increased their average size by 80 nm when AuNPs with 30 nm diameter were uptaken, compared to lysosomes of cells incubated with AuNPs of 10 and 15 nm diameter.

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.

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

Live CLEM imaging to analyze nuclear structures at high resolution.

PubMed

Haraguchi, Tokuko; Osakada, Hiroko; Koujin, Takako

2015-01-01

Fluorescence microscopy (FM) and electron microscopy (EM) are powerful tools for observing molecular components in cells. FM can provide temporal information about cellular proteins and structures in living cells. EM provides nanometer resolution images of cellular structures in fixed cells. We have combined FM and EM to develop a new method of correlative light and electron microscopy (CLEM), called "Live CLEM." In this method, the dynamic behavior of specific molecules of interest is first observed in living cells using fluorescence microscopy (FM) and then cellular structures in the same cell are observed using electron microscopy (EM). Following image acquisition, FM and EM images are compared to enable the fluorescent images to be correlated with the high-resolution images of cellular structures obtained using EM. As this method enables analysis of dynamic events involving specific molecules of interest in the context of specific cellular structures at high resolution, it is useful for the study of nuclear structures including nuclear bodies. Here we describe Live CLEM that can be applied to the study of nuclear structures in mammalian cells.

Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy.

PubMed

Jaros, Josef; Petrov, Michal; Tesarova, Marketa; Hampl, Ales

2017-01-01

Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.

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.

Gold Nanoparticle Quantitation by Whole Cell Tomography.

PubMed

Sanders, Aric W; Jeerage, Kavita M; Schwartz, Cindi L; Curtin, Alexandra E; Chiaramonti, Ann N

2015-12-22

Many proposed biomedical applications for engineered gold nanoparticles require their incorporation by mammalian cells in specific numbers and locations. Here, the number of gold nanoparticles inside of individual mammalian stem cells was characterized using fast focused ion beam-scanning electron microscopy based tomography. Enhanced optical microscopy was used to provide a multiscale map of the in vitro sample, which allows cells of interest to be identified within their local environment. Cells were then serially sectioned using a gallium ion beam and imaged using a scanning electron beam. To confirm the accuracy of single cross sections, nanoparticles in similar cross sections were imaged using transmission electron microscopy and scanning helium ion microscopy. Complete tomographic series were then used to count the nanoparticles inside of each cell and measure their spatial distribution. We investigated the influence of slice thickness on counting single particles and clusters as well as nanoparticle packing within clusters. For 60 nm citrate stabilized particles, the nanoparticle cluster packing volume is 2.15 ± 0.20 times the volume of the bare gold nanoparticles.

Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy.

PubMed

Peckys, Diana B; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

2013-01-01

Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32-56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general.

Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy

PubMed Central

Peckys, Diana B.; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

2013-01-01

Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32–56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general. PMID:24022088

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.

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.

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.

Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

PubMed Central

Sun, Yuliang; Juzenas, Kevin

2017-01-01

Abstract Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types. PMID:28398585

Responses of indigenous microorganisms to soil incubation as viewed by transmission electron microscopy of cell thin sections.

NASA Technical Reports Server (NTRS)

Bae, h. C.; Casida, L. E., Jr.

1973-01-01

Indigenous soil microorganisms were cultivated in their soil habitat with 50% moisture capacity at 30 C for two weeks. Changes in microorganism cells were studied by electron microscopy during incubation, with particular attention to the dormant cell growth and to the ability of cystlike cells to germinate and reencyst. The responses of various cell species to incubation conditions are described and illustrated by photomicrographs.

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...

Studies by immune electron microscopy of hepatitis B surface antigen in PLC/PRF/5 cells.

PubMed

Shibayama, T; Watanabe, T; Kojima, H; Yoshikawa, A; Watanabe, S; Kamimura, T; Suzuki, S; Ichida, F

1984-01-01

Electron microscopic studies of the morphology of hepatitis B surface antigen (HBsAg) produced by PLC/PRF/5 cells in vitro were carried out. Aggregates of 20-nm spherical particles in 3-day culture supernatants were observed by immune electron microscopy (IEM). Aggregates of tubular structures were found with IEM in the extracts of the cells. Tubular structures 18 to 22 nm in diameter were seen by electron microscopy (EM) in the cisternae of the endoplasmic reticulum in 2-3% of the cells. The tubular structures in the cytoplasm and extracts of PLC/PRF/5 cells resembled those observed in the hepatocytes of human carriers of hepatitis B virus (HBV). Intracellular localization of HBsAg in PLC/PRF/5 cells by direct peroxidase-conjugated antibody staining was observed on the tubular structures and the cisternal wall, which contained these structures. Rotation technique analysis indicated that the tubular structures were composed of 11 or 12 subunits.

The quest for four-dimensional imaging in plant cell biology: it's just a matter of time

PubMed Central

Domozych, David S.

2012-01-01

Background Analysis of plant cell dynamics over time, or four-dimensional imaging (4-DI), represents a major goal of plant science. The ability to resolve structures in the third dimension within the cell or tissue during developmental events or in response to environmental or experimental stresses (i.e. 4-DI) is critical to our understanding of gene expression, post-expression modulations of macromolecules and sub-cellular system interactions. Scope Microscopy-based technologies have been profoundly integral to this type of investigation, and new and refined microscopy technologies now allow for the visualization of cell dynamics with unprecedented resolution, contrast and experimental versatility. However, certain realities of light and electron microscopy, choice of specimen and specimen preparation techniques limit the scope of readily attaining 4-DI. Today, the plant microscopist must use a combinatorial strategy whereby multiple microscopy-based investigations are used. Modern fluorescence, confocal laser scanning, transmission electron and scanning electron microscopy provide effective conduits for synthesizing data detailing live cell dynamics and highly resolved snapshots of specific cell structures that will ultimately lead to 4-DI. This review provides a synopsis of such technologies available. PMID:22628381

A novel cell culture technique for electron microscopy.

PubMed

Wang, F; Ledford, L B; Head, J F; Elliott, R L

1993-12-15

A simplified technique for the monolayer growth of cultured cells and their in situ embedment on the inner surface of the pyramidal portion of the Beem capsule for electron microscopy has been developed. The results demonstrated that the cell monolayers grew well on the surface of the Beem capsule and could be embedded in situ. Electron micrographs showed cells in their natural state of contact with one another. The plasma membrane and intracellular organelles were well preserved. This method minimizes many difficult steps and eliminates the disruption of cells by scraping, pelleting, or enzymatic reaction to remove them.

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.

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.

Combined time-lapse cinematography and immuno-electron microscopy.

PubMed

Balfour, B M; Goscicka, T; MacKenzie, J L; Gautam, A; Tate, M; Clark, J

1990-04-01

A method was developed to record interactions between mobile non-adherent immunocytes by time-lapse cinematography and then to study the same cells by immuno-electron microscopy, using monoclonal antibodies against surface components. For this purpose a modified stage was designed to fit an inverted microscope. The attachment included a device to cool the culture chamber with N2 gas, a micro-injector for monoclonal antibody and immuno-gold treatment, and two pairs of washing needles to change the medium without disturbance. The technique was first employed to study the formation of aggregates around the antigen-presenting cells in cultures containing cells from hyper-immunized animals. Recently peripheral blood cells from normal subjects and patients with immune deficiency syndromes were stimulated with pokeweed mitogen, cluster formation was recorded, and the cells were processed for immuno-electron microscopy.

Impact of membrane-induced particle immobilization on seeded growth monitored by in situ liquid scanning transmission electron microscopy

DOE PAGES

Weiner, Rebecca G.; Chen, Dennis P.; Unocic, Raymond R.; ...

2016-04-01

In situ liquid cell scanning transmission electron microscopy probes seeded growth in real time. The growth of Pd on Au nanocubes is monitored as a model system to compare growth within a liquid cell and traditional colloidal synthesis. Furthermore, different growth patterns are observed due to seed immobilization and the highly reducing environment within the liquid cell.

In vitro model for Campylobacter pylori adherence properties.

PubMed Central

Neman-Simha, V; Mégraud, F

1988-01-01

The adherence of 12 strains of Campylobacter pylori was studied on four cell lines. Immunofluorescence and scanning and transmission electron microscopy were used to visualize the bacteria. A heavy adherence to the epithelial cell line HEp-2 and to the intestinal cell line Int-407 was noted. By transmission electron microscopy, a close association between bacteria and cells in the form of cup-like structures was observed, but pedestals were not present. Images PMID:3182085

A simple procedure to analyze positions of interest in infectious cell cultures by correlative light and electron microscopy.

PubMed

Madela, Kazimierz; Banhart, Sebastian; Zimmermann, Anja; Piesker, Janett; Bannert, Norbert; Laue, Michael

2014-01-01

Plastic cell culture dishes that contain a thin bottom of highest optical quality including an imprinted finder grid (μ-Dish Grid-500) are optimally suited for routine correlative light and electron microscopy using chemical fixation. Such dishes allow high-resolution fluorescence and bright-field imaging using fixed and living cells and are compatible with standard protocols for scanning and transmission electron microscopy. Ease of use during cell culture and imaging, as well as a tight cover render the dishes particularly suitable for working with infectious organisms up to the highest biosafety level. Detailed protocols are provided and demonstrated by showing two examples: monitoring the production of virus-like particles of the Human Endogenous Retrovirus HERV-K(HML-2) by HeLa cells and investigation of Rab11-positive membrane-compartments of HeLa cells after infection with Chlamydia trachomatis. © 2014 Elsevier Inc. All rights reserved.

Recent developments of the in situ wet cell technology for transmission electron microscopies.

PubMed

Chen, Xin; Li, Chang; Cao, Hongling

2015-03-21

In situ wet cells for transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) allow studying structures and processes in a liquid environment with high temporal and spatial resolutions, and have been attracting increasing research interests in many fields. In this review, we highlight the structural and functional developments of the wet cells for TEM and STEM. One of the key features of the wet cells is the sealing technique used to isolate the liquid sample from the TEM/STEM vacuum environments, thus the existing in situ wet cells are grouped by different sealing methods. In this study, the advantages and shortcomings of each type of in situ wet cells are discussed, the functional developments of different wet cells are presented, and the future trends of the wet cell technology are addressed. It is suggested that in the future the in situ wet cell TEM/STEM technology will have an increasing impact on frontier nanoscale research.

Single organelle dynamics linked to 3D structure by correlative live-cell imaging and 3D electron microscopy.

PubMed

Fermie, Job; Liv, Nalan; Ten Brink, Corlinda; van Donselaar, Elly G; Müller, Wally H; Schieber, Nicole L; Schwab, Yannick; Gerritsen, Hans C; Klumperman, Judith

2018-05-01

Live-cell correlative light-electron microscopy (live-cell-CLEM) integrates live movies with the corresponding electron microscopy (EM) image, but a major challenge is to relate the dynamic characteristics of single organelles to their 3-dimensional (3D) ultrastructure. Here, we introduce focused ion beam scanning electron microscopy (FIB-SEM) in a modular live-cell-CLEM pipeline for a single organelle CLEM. We transfected cells with lysosomal-associated membrane protein 1-green fluorescent protein (LAMP-1-GFP), analyzed the dynamics of individual GFP-positive spots, and correlated these to their corresponding fine-architecture and immediate cellular environment. By FIB-SEM we quantitatively assessed morphological characteristics, like number of intraluminal vesicles and contact sites with endoplasmic reticulum and mitochondria. Hence, we present a novel way to integrate multiple parameters of subcellular dynamics and architecture onto a single organelle, which is relevant to address biological questions related to membrane trafficking, organelle biogenesis and positioning. Furthermore, by using CLEM to select regions of interest, our method allows for targeted FIB-SEM, which significantly reduces time required for image acquisition and data processing. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The three dimensionality of cell membranes: lamellar to cubic membrane transition as investigated by electron microscopy.

PubMed

Chong, Ketpin; Deng, Yuru

2012-01-01

Biological membranes are generally perceived as phospholipid bilayer structures that delineate in a lamellar form the cell surface and intracellular organelles. However, much more complex and highly convoluted membrane organizations are ubiquitously present in many cell types under certain types of stress, states of disease, or in the course of viral infections. Their occurrence under pathological conditions make such three-dimensionally (3D) folded and highly ordered membranes attractive biomarkers. They have also stimulated great biomedical interest in understanding the molecular basis of their formation. Currently, the analysis of such membrane arrangements, which include tubulo-reticular structures (TRS) or cubic membranes of various subtypes, is restricted to electron microscopic methods, including tomography. Preservation of membrane structures during sample preparation is the key to understand their true 3D nature. This chapter discusses methods for appropriate sample preparations to successfully examine and analyze well-preserved highly ordered membranes by electron microscopy. Processing methods and analysis conditions for green algae (Zygnema sp.) and amoeba (Chaos carolinense), mammalian cells in culture and primary tissue cells are described. We also discuss methods to identify cubic membranes by transmission electron microscopy (TEM) with the aid of a direct template matching method and by computer simulation. A 3D analysis of cubic cell membrane topology by electron tomography is described as well as scanning electron microscopy (SEM) to investigate surface contours of isolated mitochondria with cubic membrane arrangement. Copyright © 2012 Elsevier Inc. All rights reserved.

Interrelations between the Parasitophorous Vacuole of Toxoplasma gondii and Host Cell Organelles

NASA Astrophysics Data System (ADS)

Cardoso Magno, Rodrigo; Cobra Straker, Lorian; de Souza, Wanderley; Attias, Marcia

2005-04-01

Toxoplasma gondii, the causative agent of toxoplasmosis, is capable of actively penetrating and multiplying in any nucleated cell of warm-blooded animals. Its survival strategies include escape from fusion of the parasitophorous vacuole with host cell lysosomes and rearrangement of host cell organelles in relation to the parasitophorous vacuole. In this article we report the rearrangement of host cell organelles and elements of the cytoskeleton of LLCMK2 cells, a lineage derived from green monkey kidney epithelial cells, in response to infection by T. gondii tachyzoites. Transmission electron microscopy made on flat embedded monolayers cut horizontally to the apical side of the cells or field emission scanning electron microscopy of monolayers scraped with scotch tape before sputtering showed that association of mitochondria to the vacuole is much less frequent than previously described. On the other hand, all parasitophorous vacuoles were surrounded by elements of the endoplasmic reticulum. These data were complemented by observations by laser scanning microscopy using fluorescent probes from mitochondria and endoplasmic reticulum and reinforced by three-dimensional reconstruction from serial sections observed by transmission electron microscopy and labeling of mitochondria and endoplasmic reticulum by fluorescent probes.

Transmission electron microscopy, fluorescence microscopy, and confocal raman microscopic analysis of ultrastructural and compositional heterogeneity of Cornus alba L. wood cell wall.

PubMed

Ma, Jianfeng; Ji, Zhe; Zhou, Xia; Zhang, Zhiheng; Xu, Feng

2013-02-01

Transmission electron microscopy (TEM), fluorescence microscopy, and confocal Raman microscopy can be used to characterize ultrastructural and compositional heterogeneity of plant cell walls. In this study, TEM observations revealed the ultrastructural characterization of Cornus alba L. fiber, vessel, axial parenchyma, ray parenchyma, and pit membrane between cells, notably with the ray parenchyma consisting of two well-defined layers. Fluorescence microscopy evidenced that cell corner middle lamella was more lignified than adjacent compound middle lamella and secondary wall with variation in lignification level from cell to cell. In situ Raman images showed that the inhomogeneity in cell wall components (cellulose and lignin) among different cells and within morphologically distinct cell wall layers. As the significant precursors of lignin biosynthesis, the pattern of coniferyl alcohol and aldehyde (joint abbreviation Lignin-CAA for both structures) distribution in fiber cell wall was also identified by Raman images, with higher concentration occurring in the fiber secondary wall where there was the highest cellulose concentration. Moreover, noteworthy was the observation that higher concentration of lignin and very minor amounts of cellulose were visualized in the pit membrane areas. These complementary microanalytical methods provide more accurate and complete information with regard to ultrastructural and compositional characterization of plant cell walls.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

PubMed Central

Saitoh, Sei; Ohno, Nobuhiko; Saitoh, Yurika; Terada, Nobuo; Shimo, Satoshi; Aida, Kaoru; Fujii, Hideki; Kobayashi, Tetsuro; Ohno, Shinichi

2018-01-01

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets. PMID:29622846

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.

Detection of parvoviruses in wolf feces by electron microscopy

USGS Publications Warehouse

Muneer, M.A.; Farah, I.O.; Pomeroy, K.A.; Goyal, S.M.; Mech, L.D.

1988-01-01

One hundred fifteen wolf (Canis lupus) feces were collected between 1980 and 1984 from northeastern Minnesota and were examined for canine parvovirus by negative contrast electron microscopy. Of these, seven (6%) samples revealed the presence of parvovirus. Some of these viruses were able to grow in cell cultures forming intranuclear inclusion bodies and giant cells.

In situ flat embedding of monolayers and cell relocation in the acrylic resin LR white for comparative light and electron microscopy studies.

PubMed

Steiner, M; Schöfer, C; Mosgoeller, W

1994-12-01

A simple and reliable method has been developed for the in situ LR White embedding of cell monolayers grown on glass cover-slips. Combined with cytochemical or immunological procedures, this technique allows light and/or electron microscopy investigations of a large number of cells in the same horizontal plane within a relatively short period of time. It can be applied to cells grown on microgrid finder cover-slips which allows a distinct site of even an individual cell of a monolayer to be studied at first at the light microscope level and subsequently at the electron microscope level. Hence, it is also suitable for controlling manipulation of single cells, followed by their serial sectioning after relocation in the electron microscope.

Not all that glitters is gold-Electron microscopy study on uptake of gold nanoparticles in Daphnia magna and related artifacts.

PubMed

Jensen, Louise Helene Søgaard; Skjolding, Lars Michael; Thit, Amalie; Sørensen, Sara Nørgaard; Købler, Carsten; Mølhave, Kristian; Baun, Anders

2017-06-01

Increasing use of engineered nanoparticles has led to extensive research into their potential hazards to the environment and human health. Cellular uptake from the gut is sparsely investigated, and microscopy techniques applied for uptake studies can result in misinterpretations. Various microscopy techniques were used to investigate internalization of 10-nm gold nanoparticles in Daphnia magna gut lumen and gut epithelial cells following 24-h exposure and outline potential artifacts (i.e., high-contrast precipitates from sample preparation related to these techniques). Light sheet microscopy confirmed accumulation of gold nanoparticles in the gut lumen. Scanning transmission electron microscopy and elemental analysis revealed gold nanoparticles attached to the microvilli of gut cells. Interestingly, the peritrophic membrane appeared to act as a semipermeable barrier between the lumen and the gut epithelium, permitting only single particles through. Structures resembling nanoparticles were also observed inside gut cells. Elemental analysis could not verify these to be gold, and they were likely artifacts from the preparation, such as osmium and iron. Importantly, gold nanoparticles were found inside holocrine cells with disrupted membranes. Thus, false-positive observations of nanoparticle internalization may result from either preparation artifacts or mistaking disrupted cells for intact cells. These findings emphasize the importance of cell integrity and combining elemental analysis with the localization of internalized nanoparticles using transmission electron microscopy. Environ Toxicol Chem 2017;36:1503-1509. © 2016 SETAC. © 2016 SETAC.

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

ELECTRON MICROSCOPY OF HELA CELLS INFECTED WITH ADENOVIRUSES

PubMed Central

Harford, Carl G.; Hamlin, Alice; Parker, Esther; van Ravenswaay, Theodore

1956-01-01

HeLa cells were infected with adenoviruses (types 1–4) and sectioned for electron microscopy after intervals of 20 to 48 hours. Clusters of virus-like particles were found within the nuclei of infected cultures but not in those of uninfected controls. The particles were often arranged in rows as if in crystalline formation. Maximal diameter of particles was approximately 65 mµ, and internal bodies were demonstrated. Lesions of infected cells included target-like structures of the nuclear membrane, large nuclear vacuoles (type 2), and increased numbers of large irregular electron-dense granules in the cytoplasm 48 hours after infection. Examination of infected cultures by light microscopy, using the Feulgen reaction, showed intranuclear inclusion bodies and a cytopathogenic effect consisting of clumping of cells without pyknosis of nuclei. A lipide stain showed numerous cytoplasmic granules that were not identical with the large, irregular, electron-dense granules of the cytoplasm. Practically all the cells showed the viral cytopathogenic effect, but only a minority of cells were found to contain virus-like particles or intranuclear inclusion bodies. PMID:13357696

Rumen Bacterial Degradation of Forage Cell Walls Investigated by Electron Microscopy

PubMed Central

Akin, Danny E.; Amos, Henry E.

1975-01-01

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues. Images PMID:16350017

Correlative Light-Electron Microscopy of Lipid-Encapsulated Fluorescent Nanodiamonds for Nanometric Localization of Cell Surface Antigens.

PubMed

Hsieh, Feng-Jen; Chen, Yen-Wei; Huang, Yao-Kuan; Lee, Hsien-Ming; Lin, Chun-Hung; Chang, Huan-Cheng

2018-02-06

Containing an ensemble of nitrogen-vacancy centers in crystal matrices, fluorescent nanodiamonds (FNDs) are a new type of photostable markers that have found wide applications in light microscopy. The nanomaterial also has a dense carbon core, making it visible to electron microscopy. Here, we show that FNDs encapsulated in biotinylated lipids (bLs) are useful for subdiffraction imaging of antigens on cell surface with correlative light-electron microscopy (CLEM). The lipid encapsulation enables not only good dispersion of the particles in biological buffers but also high specific labeling of live cells. By employing the bL-encapsulated FNDs to target CD44 on HeLa cell surface through biotin-mediated immunostaining, we obtained the spatial distribution of these antigens by CLEM with a localization accuracy of ∼50 nm in routine operations. A comparative study with dual-color imaging, in which CD44 was labeled with FND and MICA/MICB was labeled with Alexa Fluor 488, demonstrated the superior performance of FNDs as fluorescent fiducial markers for CLEM of cell surface antigens.

Correlative 3D imaging of Whole Mammalian Cells with Light and Electron Microscopy

PubMed Central

Murphy, Gavin E.; Narayan, Kedar; Lowekamp, Bradley C.; Hartnell, Lisa M.; Heymann, Jurgen A. W.; Fu, Jing; Subramaniam, Sriram

2011-01-01

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA–SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ~10 to 20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA–SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues. PMID:21907806

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.

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.

Ultrastructural analysis of testicular tissue and sperm by transmission and scanning electron microscopy.

PubMed

Chemes, Hector E

2013-01-01

Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning electron microscope (SEM) extended these boundaries to the recognition of cell and organ surface features and the architectural array of cells and tissues. The merging of immunocytochemical and histochemical approaches with electron microscopy has completed a series of technical improvements that integrate structural and functional features to provide a broad understanding of cell biology in health and disease. With these advances the detailed study of the intricate structural and molecular organization as well as the chemical composition of cellular organelles is now possible. Immunocytochemistry is used to identify proteins or other components and localize them in specific cells or organelles with high specificity and sensitivity, and histochemistry can be used to understand their function (i.e., enzyme activity). When these techniques are used in conjunction with electron microscopy their resolving power is further increased to subcellular levels. In the present chapter we will describe in detail various ultrastructural techniques that are now available for basic or translational research in reproductive biology and reproductive medicine. These include TEM, ultrastructural immunocytochemistry, ultrastructural histochemistry, and SEM.

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.

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.

Transmission electron microscopy study of Listeria monocytogenes serotype 1/2a cells exposed to sublethal heat stress and carvacrol

USDA-ARS?s Scientific Manuscript database

The objective of this study was to investigate the morphological changes that occurred in Listeria monocytogenes serotype 1/2a cells as visualized by transmission electron microscopy (TEM) after exposure to sublethal heat stress at 48°C for 60 min and in combination with lethal concentration of carv...

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.

Difference of EGCg adhesion on cell surface between Staphylococcus aureus and Escherichia coli visualized by electron microscopy after novel indirect staining with cerium chloride.

PubMed

Nakayama, Motokazu; Shigemune, Naofumi; Tsugukuni, Takashi; Tokuda, Hajime; Miyamoto, Takahisa

2011-07-01

We developed a novel method using indirect staining with cerium chloride for visualization of the catechin derivative epigallocatechin gallate (EGCg) on the surface of particles, i.e., polystyrene beads and bacterial cells, by electron microscopy. The staining method is based on the fact that in an alkaline environment, EGCg produces hydrogen peroxide, and then hydrogen peroxide reacts with cerium, resulting in a cerium hydroperoxide precipitate. This precipitate subsequently reacts with EGCg to produce larger deposits. The amount of precipitate is proportional to the amount of EGCg. Highly EGCg-sensitive Staphylococcus aureus and EGCg-resistant Escherichia coli were treated with EGCg under various pH conditions. Transmission electron microscopy observation showed that the amount of deposits on S. aureus increased with an increase in EGCg concentration. After treating bacterial cells with 0.5mg/mL EGCg (pH 6.0), attachment of EGCg was significantly lower to E. coli than to S. aureus. This is the first report that shows differences in affinity of EGCg to the cell surfaces of Gram-positive and -negative bacteria by electron microscopy. Copyright © 2011 Elsevier B.V. All rights reserved.

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.

Exopolysaccharide microchannels direct bacterial motility and organize multicellular behavior

DOE PAGES

Berleman, James E.; Zemla, Marcin; Remis, Jonathan P.; ...

2016-05-06

The myxobacteria are a family of soil bacteria that form biofilms of complex architecture, aligned multilayered swarms or fruiting body structures that are simple or branched aggregates containing myxospores. Here, we examined the structural role of matrix exopolysaccharide (EPS) in the organization of these surface-dwelling bacterial cells. Using time-lapse light and fluorescence microscopy, as well as transmission electron microscopy and focused ion beam/scanning electron microscopy (FIB/SEM) electron microscopy, we found that Myxococcus xanthus cell organization in biofilms is dependent on the formation of EPS microchannels. Cells are highly organized within the three-dimensional structure of EPS microchannels that are required formore » cell alignment and advancement on surfaces. Mutants lacking EPS showed a lack of cell orientation and poor colony migration. Purified, cell-free EPS retains a channel-like structure, and can complement EPS - mutant motility defects. In addition, EPS provides the cooperative structure for fruiting body formation in both the simple mounds of M. xanthus and the complex, tree-like structures of Chondromyces crocatus. We furthermore investigated the possibility that EPS impacts community structure as a shared resource facilitating cooperative migration among closely related isolates of M. xanthus.« less

Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Tong, Yongpeng; Li, Changming; Liang, Feng; Chen, Jianmin; Zhang, Hong; Liu, Guoqing; Sun, Huibin; Luong, John H. T.

2008-12-01

Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al 2O 3 and TiO 2) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl 2) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al 2O 3 and TiO 2 nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe 2O 3 nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

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.

Microscopy techniques in flavivirus research.

PubMed

Chong, Mun Keat; Chua, Anthony Jin Shun; Tan, Terence Tze Tong; Tan, Suat Hoon; Ng, Mah Lee

2014-04-01

The Flavivirus genus is composed of many medically important viruses that cause high morbidity and mortality, which include Dengue and West Nile viruses. Various molecular and biochemical techniques have been developed in the endeavour to study flaviviruses. However, microscopy techniques still have irreplaceable roles in the identification of novel virus pathogens and characterization of morphological changes in virus-infected cells. Fluorescence microscopy contributes greatly in understanding the fundamental viral protein localizations and virus-host protein interactions during infection. Electron microscopy remains the gold standard for visualizing ultra-structural features of virus particles and infected cells. New imaging techniques and combinatory applications are continuously being developed to push the limit of resolution and extract more quantitative data. Currently, correlative live cell imaging and high resolution three-dimensional imaging have already been achieved through the tandem use of optical and electron microscopy in analyzing biological specimens. Microscopy techniques are also used to measure protein binding affinities and determine the mobility pattern of proteins in cells. This chapter will consolidate on the applications of various well-established microscopy techniques in flavivirus research, and discuss how recently developed microscopy techniques can potentially help advance our understanding in these membrane viruses. Copyright © 2013 Elsevier Ltd. All rights reserved.

Imaging interactions of metal oxide nanoparticles with macrophage cells by ultra-high resolution scanning electron microscopy techniques.

PubMed

Plascencia-Villa, Germán; Starr, Clarise R; Armstrong, Linda S; Ponce, Arturo; José-Yacamán, Miguel

2012-11-01

Use of engineered metal oxide nanoparticles in a plethora of biological applications and custom products has warned about some possible dose-dependent cytotoxic effects. Macrophages are key components of the innate immune system used to study possible toxic effects and internalization of different nanoparticulate materials. In this work, ultra-high resolution field emission scanning electron microscopy (FE-SEM) was used to offer new insights into the dynamical processes of interaction of nanomaterials with macrophage cells dosed with different concentrations of metal oxide nanoparticles (CeO(2), TiO(2) and ZnO). The versatility of FE-SEM has allowed obtaining a detailed characterization of processes of adsorption and endocytosis of nanoparticles, by using advanced analytical and imaging techniques on complete unstained uncoated cells, including secondary electron imaging, high-sensitive backscattered electron imaging, X-ray microanalysis and stereoimaging. Low voltage BF/DF-STEM confirmed nanoparticle adsorption and internalization into endosomes of CeO(2) and TiO(2), whereas ZnO develop apoptosis after 24 h of interaction caused by dissolution and invasion of cell nucleus. Ultra-high resolution scanning electron microscopy techniques provided new insights into interactions of inorganic nanoparticles with macrophage cells with high spatial resolution.

Water-clear cell adenoma of the parathyroid. A case report with immunohistochemistry and electron microscopy.

PubMed

Grenko, R T; Anderson, K M; Kauffman, G; Abt, A B

1995-11-01

We report a water-clear cell adenoma of the parathyroid gland, a lesion which to our knowledge has not been described previously. Like its rare but well-described hyperplastic counterpart, water-clear cell hyperplasia, this adenoma is composed of cells with abundant foamy-to-granular cytoplasm and mild nuclear pleomorphism. The cells form glandular structures and cell nests separated by fine fibrovascular septae. The tumor cells stain positively with anti-parathyroid hormone and show characteristic glassy and flocculate material by electron microscopy. Unlike water-clear cell hyperplasia, water-clear cell adenoma is a solitary lesion that compresses the residual nonneoplastic parathyroid gland.

Electron Microscopy of Ultrathin Sections of Sporosarcina ureae

PubMed Central

Mazanec, K.; Kocur, M.; Martinec, T.

1965-01-01

Mazanec, K. (J. E. Purkyně University, Brno, Czechoslovakia), M. Kocur, and T. Martinec. Electron microscopy of ultrathin sections of Sporosarcina ureae. J. Bacteriol. 90:808–816. 1965.—Ultrathin sections of Sporosarcina ureae cells were studied by means of electron microscopy. The cell wall consists of several layers and is 340 A thick. The cytoplasm is of globular structure and includes ribosomelike structures, occasional mesosomes, and inclusions not precisely identifiable. The nuclear area has various shapes and is formed by filaments 10 to 20 A thick which proceed in various directions. Cell division occurs similarly to that of sarcinate. Both synchronic and asynchronic cell division was observed. The spores of S. ureae consist of an outer coat having several layers, a cortex, a spore wall, and cytoplasm. The results of the present investigation substantiate our previous suggestion that S. ureae should be transferred from the family Micrococcaceae to the family Bacillaceae. Images PMID:16562085

Impact of Membrane-Induced Particle Immobilization on Seeded Growth Monitored by In Situ Liquid Scanning Transmission Electron Microscopy.

PubMed

Weiner, Rebecca G; Chen, Dennis P; Unocic, Raymond R; Skrabalak, Sara E

2016-05-01

In situ liquid cell scanning transmission electron microscopy probes seeded growth in real time. The growth of Pd on Au nanocubes is monitored as a model system to compare growth within a liquid cell and traditional colloidal synthesis. Different growth patterns are observed due to seed immobilization and the highly reducing environment within the liquid cell. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Study of the Effects of High Power Pulsed 2450 MHz Microwaves, ELF modulated Microwaves, and ELF Fields on Human Lymphocytes and Selected Cell Lines

DTIC Science & Technology

1993-01-27

Considerable effect was expended in investigating shifts in intercellular calcium of one particular cell line, Jurket, using flow cytometry methods. No...culture. The following analysis were used to characterize the immortalized cell lines: flow cytometry , electron microscopy, two-dimensional protein gel...further characterized by flow cytometry , electron microscopy, two dimensional protein electrophoresis and nuclear run-off assay. Flow cytometric analysis of

Immunogold labeling reveals subcellular localisation of silica nanoparticles in a human blood-brain barrier model

NASA Astrophysics Data System (ADS)

Ye, Dong; Anguissola, Sergio; O'Neill, Tiina; Dawson, Kenneth A.

2015-05-01

Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles.Subcellular location of nanoparticles has been widely investigated with fluorescence microscopy, via fluorescently labeled antibodies to visualise target antigens in cells. However, fluorescence microscopy, such as confocal or live cell imaging, has generally limited 3D spatial resolution. Conventional electron microscopy can be useful in bridging resolution gap, but still not ideal in resolving subcellular organelle identities. Using the pre-embedding immunogold electron microscopic imaging, we performed accurate examination of the intracellular trafficking and gathered further evidence of transport mechanisms of silica nanoparticles across a human in vitro blood-brain barrier model. Our approach can effectively immunolocalise a variety of intracellular compartments and provide new insights into the uptake and subcellular transport of nanoparticles. Electronic supplementary information (ESI) available: Nanoparticle characterisation data, preservation of cellular structures, staining controls, optimisation of size amplification via the silver enhancement, and more imaging results from anti-clathrin and anti-caveolin 1 immunolabeling. See DOI: 10.1039/c5nr01539a

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching.

PubMed

Hauwiller, Matthew R; Ondry, Justin C; Alivisatos, A Paul

2018-05-17

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in liquid environments. This manuscript describes the process for making graphene liquid cells through the example of graphene liquid cell transmission electron microscopy (TEM) experiments of gold nanocrystal etching. The protocol for making graphene liquid cells involves coating gold, holey-carbon TEM grids with chemical vapor deposition graphene and then using those graphene-coated grids to encapsulate liquid between two graphene surfaces. These pockets of liquid, with the nanomaterial of interest, are imaged in the electron microscope to see the dynamics of the nanoscale process, in this case the oxidative etching of gold nanorods. By controlling the electron beam dose rate, which modulates the etching species in the liquid cell, the underlying mechanisms of how atoms are removed from nanocrystals to form different facets and shapes can be better understood. Graphene liquid cell TEM has the advantages of high spatial resolution, compatibility with traditional TEM holders, and low start-up costs for research groups. Current limitations include delicate sample preparation, lack of flow capability, and reliance on electron beam-generated radiolysis products to induce reactions. With further development and control, graphene liquid cell may become a ubiquitous technique in nanomaterials and biology, and is already being used to study mechanisms governing growth, etching, and self-assembly processes of nanomaterials in liquid on the single particle level.

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3nm precision using aberration-corrected scanning transmission electron microscopy.

PubMed

Dukes, Madeline J; Ramachandra, Ranjan; Baudoin, Jean-Pierre; Gray Jerome, W; de Jonge, Niels

2011-06-01

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells. Copyright © 2011 Elsevier Inc. All rights reserved.

Three-dimensional locations of gold-labeled proteins in a whole mount eukaryotic cell obtained with 3 nm precision using aberration-corrected scanning transmission electron microscopy

PubMed Central

Dukes, Madeline J.; Ramachandra, Ranjan; Baudoin, Jean-Pierre; Jerome, W. Gray; de Jonge, Niels

2011-01-01

Three-dimensional (3D) maps of proteins within the context of whole cells are important for investigating cellular function. However, 3D reconstructions of whole cells are challenging to obtain using conventional transmission electron microscopy (TEM). We describe a methodology to determine the 3D locations of proteins labeled with gold nanoparticles on whole eukaryotic cells. The epidermal growth factor receptors on COS7 cells were labeled with gold nanoparticles, and critical-point dried whole-mount cell samples were prepared. 3D focal series were obtained with aberration-corrected scanning transmission electron microscopy (STEM), without tilting the specimen. The axial resolution was improved with deconvolution. The vertical locations of the nanoparticles in a whole-mount cell were determined with a precision of 3 nm. From the analysis of the variation of the axial positions of the labels we concluded that the cellular surface was ruffled. To achieve sufficient stability of the sample under the electron beam irradiation during the recording of the focal series, the sample was carbon coated. A quantitative method was developed to analyze the stability of the ultrastructure after electron beam irradiation using TEM. The results of this study demonstrate the feasibility of using aberration-corrected STEM to study the 3D nanoparticle distribution in whole cells. PMID:21440635

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2015-09-11

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e., one emitting at 655 nm and at 800 revealed similar characteristic results.

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.

Architecture of dermatophyte cell Walls: Electron microscopic and biochemical analysis

NASA Technical Reports Server (NTRS)

Nozawa, Y.; Kitajima, Y.

1984-01-01

A review with 83 references on the cell wall structure of dermatophytes is presented. Topics discussed include separation and preparation of cell walls; microstructure of cell walls by electron microscopy; chemical composition of cell walls; structural model of cell walls; and morphological structure of cell walls.

Three-Dimensional Unstained Live-Cell Imaging Using Stimulated Parametric Emission Microscopy

NASA Astrophysics Data System (ADS)

Dang, Hieu M.; Kawasumi, Takehito; Omura, Gen; Umano, Toshiyuki; Kajiyama, Shin'ichiro; Ozeki, Yasuyuki; Itoh, Kazuyoshi; Fukui, Kiichi

2009-09-01

The ability to perform high-resolution unstained live imaging is very important to in vivo study of cell structures and functions. Stimulated parametric emission (SPE) microscopy is a nonlinear-optical microscopy based on ultra-fast electronic nonlinear-optical responses. For the first time, we have successfully applied this technique to archive three-dimensional (3D) images of unstained sub-cellular structures, such as, microtubules, nuclei, nucleoli, etc. in live cells. Observation of a complete cell division confirms the ability of SPE microscopy for long time-scale imaging.

Ultrastructure and Glycoconjugate Pattern of the Foot Epithelium of the Abalone Haliotis tuberculata (Linnaeus, 1758) (Gastropoda, Haliotidae)

PubMed Central

Bravo Portela, I.; Martinez-Zorzano, V. S.; Molist- Perez, I.; Molist García, P.

2012-01-01

The foot epithelium of the gastropod Haliotis tuberculata is studied by light and electron microscopy in order to contribute to the understanding of the anatomy and functional morphology of the mollusks integument. Study of the external surface by scanning electron microscopy reveals that the side foot epithelium is characterized by a microvillus border with a very scant presence of small ciliary tufts, but the sole foot epithelium bears a dense field of long cilia. Ultrastructural examination by transmission electron microscopy of the side epithelial cells shows deeply pigmented cells with high electron-dense granular content which are not observed in the epithelial sole cells. Along the pedal epithelium, seven types of secretory cells are present; furthermore, two types of subepithelial glands are located just in the sole foot. The presence and composition of glycoconjugates in the secretory cells and subepithelial glands are analyzed by conventional and lectin histochemistry. Subepithelial glands contain mainly N-glycoproteins rich in fucose and mannose whereas secretory cells present mostly acidic sulphated glycoconjugates such as glycosaminoglycans and mucins, which are rich in galactose, N-acetyl-galactosamine, and N-acetyl-glucosamine. No sialic acid is present in the foot epithelium. PMID:22645482

Contribution of high-resolution correlative imaging techniques in the study of the liver sieve in three-dimensions.

PubMed

Braet, Filip; Wisse, Eddie; Bomans, Paul; Frederik, Peter; Geerts, Willie; Koster, Abraham; Soon, Lilian; Ringer, Simon

2007-03-01

Correlative microscopy has become increasingly important for the analysis of the structure, function, and dynamics of cells. This is largely due to the result of recent advances in light-, probe-, laser- and various electron microscopy techniques that facilitate three-dimensional studies. Furthermore, the improved understanding in the past decade of imaging cell compartments in the third dimension has resulted largely from the availability of powerful computers, fast high-resolution CCD cameras, specifically developed imaging analysis software, and various probes designed for labeling living and or fixed cells. In this paper, we review different correlative high-resolution imaging methodologies and how these microscopy techniques facilitated the accumulation of new insights in the morpho-functional and structural organization of the hepatic sieve. Various aspects of hepatic endothelial fenestrae regarding their structure, origin, dynamics, and formation will be explored throughout this paper by comparing the results of confocal laser scanning-, correlative fluorescence and scanning electron-, atomic force-, and whole-mount electron microscopy. Furthermore, the recent advances of vitrifying cells with the vitrobot in combination with the glove box for the preparation of cells for cryo-electron microscopic investigation will be discussed. Finally, the first transmission electron tomography data of the liver sieve in three-dimensions are presented. The obtained data unambiguously show the involvement of special domains in the de novo formation and disappearance of hepatic fenestrae, and focuses future research into the (supra)molecular structure of the fenestrae-forming center, defenestration center and fenestrae-, and sieve plate cytoskeleton ring by using advanced cryo-electron tomography. (c) 2007 Wiley-Liss, Inc.

Rapid microwave fixation of rat mast cells. I. Localization of granule chymase with an ultrastructural postembedding immunogold technique.

PubMed

Login, G R; Galli, S J; Morgan, E; Arizono, N; Schwartz, L B; Dvorak, A M

1987-11-01

We defined the ultrastructural localization of chymase in rat peritoneal mast cells using standard aldehyde fixation and a newly described microwave fixation method (Login GR, Dvorak AM: Microwave energy fixation for electron microscopy. Am J Pathol 120: 230, 1985; Login GR, Stavinoha WB, Dvorak AM: Ultrafast microwave energy fixation for electron microscopy. J Histochem Cytochem 34:381, 1986) and postembedding immunogold labeling. Thin sections were exposed first to goat IgG anti-rat chymase and second to gold-conjugated rabbit Ig directed against goat IgG. By transmission electron microscopy, gold particles were localized to the matrix of cytoplasmic granules. Control sections treated with nonimmune sera did not exhibit labeling of mast cells. Thin sections treated simultaneously with purified rat mast cell chymase and anti-chymase antibody in competition studies, showed a marked reduction in granule staining. These findings demonstrate that a microwave fixation method can be used to rapidly fix cell suspensions for postembedding immunocytochemical studies.

[Grape seed extract induces morphological changes of prostate cancer PC-3 cells].

PubMed

Shang, Xue-Jun; Yin, Hong-Lin; Ge, Jing-Ping; Sun, Yi; Teng, Wen-Hui; Huang, Yu-Feng

2008-12-01

To observe the morphological changes of prostate cancer PC-3 cells induced by grape seed extract (GSE). PC-3 cells were incubated with different concentrations of GSE (100, 200 and 300 microg/ml) for 24, 48 and 72 hours, and then observed for morphological changes by invert microscopy, HE staining and transmission electron microscopy. The incubated PC-3 cells appeared round, small, wrinkled and broken under the invert microscope and exhibited the classical morphological characteristics of cell death under the electron microscope, including cell atrophy, increased vacuoles, crumpled nuclear membrane, and chromosome aggregation. GSE can cause morphological changes and induce necrosis and apoptosis of PC-3 cells.

Differences in Physical and Biochemical Properties of Thermus scotoductus SA-01 Cultured with Dielectric or Convection Heating.

PubMed

Cockrell, Allison L; Fitzgerald, Lisa A; Cusick, Kathleen D; Barlow, Daniel E; Tsoi, Stanislav D; Soto, Carissa M; Baldwin, Jeffrey W; Dale, Jason R; Morris, Robert E; Little, Brenda J; Biffinger, Justin C

2015-09-01

A thermophile, Thermus scotoductus SA-01, was cultured within a constant-temperature (65°C) microwave (MW) digester to determine if MW-specific effects influenced the growth and physiology of the organism. As a control, T. scotoductus cells were also cultured using convection heating at the same temperature as the MW studies. Cell growth was analyzed by optical density (OD) measurements, and cell morphologies were characterized using electron microscopy imaging (scanning electron microscopy [SEM] and transmission electron microscopy [TEM]), dynamic light scattering (DLS), and atomic force microscopy (AFM). Biophysical properties (i.e., turgor pressure) were also calculated with AFM, and biochemical compositions (i.e., proteins, nucleic acids, fatty acids) were analyzed by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Gas chromatography-mass spectrometry (GC-MS) was used to analyze the fatty acid methyl esters extracted from cell membranes. Here we report successful cultivation of a thermophile with only dielectric heating. Under the MW conditions for growth, cell walls remained intact and there were no indications of membrane damage or cell leakage. Results from these studies also demonstrated that T. scotoductus cells grown with MW heating exhibited accelerated growth rates in addition to altered cell morphologies and biochemical compositions compared with oven-grown cells. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Electron microscopy study of antioxidant interaction with bacterial cells

NASA Astrophysics Data System (ADS)

Plotnikov, Oleg P.; Novikova, Olga V.; Konnov, Nikolai P.; Korsukov, Vladimir N.; Gunkin, Ivan F.; Volkov, Uryi P.

2000-10-01

To maintain native microorganisms genotype and phenotype features a lyophylization technique is widely used. However in this case cells are affected by influences of vacuum and low temperature that cause a part of the cells population to be destruction. Another factor reduced microorganisms vitality is formation of reactive oxygen forms that damage certain biological targets (such as DNA, membranes etc.) Recently to raise microorganism's resistance against adverse condition natural and synthetic antioxidants are used. Antioxidant- are antagonists of free radicals. Introduction of antioxidants in protective medium for lyophylization increase bacteria storage life about 2,0-4,8 fold in comparison with reference samples. In the article the main results of our investigation of antioxidants interaction with microorganism cells is described. As bacteria cells we use vaccine strain yersinia pestis EV, that were grown for 48 h at 28 degree(s)C on the Hottinger agar (pH 7,2). Antioxidants are inserted on the agar surface in specimen under test. To investigate a localization of antioxidants for electron microscopy investigation, thallium organic antioxidants were used. The thallium organic compounds have an antioxidant features if thallium is in low concentration (about 1(mu) g/ml). The localization of the thallium organic antioxidants on bacteria Y. pestis EV is visible in electron microscopy images, thallium being heavy metal with high electron density. The negatively stained bacteria and bacteria thin sections with thallium organic compounds were investigated by means of transmission electron microscopy. The localization of the thallium organic compounds is clearly visible in electron micrographs as small dark spots with size about 10-80nm. Probably mechanisms of interaction of antioxidants with bacteria cells are discussed.

Helix handedness of Leptospira interrogans as determined by scanning electron microscopy.

PubMed Central

Carleton, O; Charon, N W; Allender, P; O'Brien, S

1979-01-01

Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans. Images PMID:438122

X-ray dense cellular inclusions in the cells of the green alga Chlamydomonas reinhardtii as seen by soft-x-ray microscopy

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

Stead, A.D.; Ford, T.W.; Page, A.M.

1997-04-01

Soft x-rays, having a greater ability to penetrate biological material than electrons, have the potential for producing images of intact, living cells. In addition, by using the so-called {open_quotes}water window{close_quotes} area of the soft x-ray spectrum, a degree of natural contrast is introduced into the image due to differential absorption of the wavelengths by compounds with a high carbon content compared to those with a greater oxygen content. The variation in carbon concentration throughout a cell therefore generates an image which is dependent upon the carbon density within the specimen. Using soft x-ray contact microscopy the authors have previously examinedmore » the green alga Chlamydomonas reinhardtii, and the most prominent feature of the cells are the numerous x-ray absorbing spheres, But they were not seen by conventional transmission electron microscopy. Similar structures have also been reported by the Goettingen group using their cryo transmission x-ray microscope at BESSY. Despite the fact that these spheres appear to occupy up to 20% or more of the cell volume when seen by x-ray microscopy, they are not visible by transmission electron microscopy. Given the difficulties and criticisms associated with soft x-ray contact microscopy, the present study was aimed at confirming the existence of these cellular inclusions and learning more of their possible chemical composition.« less

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

Morphological study of the TK cholangiocarcinoma cell line with three-dimensional cell culture.

PubMed

Akiyoshi, Kohei; Kamada, Minori; Akiyama, Nobutake; Suzuki, Masafumi; Watanabe, Michiko; Fujioka, Kouki; Ikeda, Keiichi; Mizuno, Shuichi; Manome, Yoshinobu

2014-04-01

Cholangiocarcinoma is an intractable carcinoma originating from the bile duct epithelium. To gain an understanding of the cell biology of cholangiocarcinoma, in vitro cell culture is valuable. However, well‑characterized cell lines are limited. In the present study, the morphology of the TK cholangiocarcinoma cell line was analyzed by three‑dimensional culture. Dispersed TK cells were injected into a gelatin mesh scaffold and cultivated for 3‑20 days. The morphology of the TK cells was investigated by phase‑contrast microscopy, optical microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TK cells were observed to proliferate three-dimensionally in the scaffold. The cells exhibited a globoid structure and attached to the scaffold. The SEM observation demonstrated typical microvilli and plicae on the surface of the structure. Light microscopy and TEM confirmed intercellular and cell‑to‑scaffold attachment in the three‑dimensional mesh. The culture also exhibited the formation of a duct-like structure covered by structured microvilli. In conclusion, three‑dimensional culture of TK cells demonstrated the morphological characteristics of cholangiocarcinoma in vitro. Production of high levels of carbohydrate antigen (CA)19‑9, CA50 and carcinoembryonic antigen was previously confirmed in the TK cell line. As a characteristic morphology was demonstrated in the present study, the TK cholangiocarcinoma cell line may be useful as an experimental model for further study of cholangiocarcinoma.

Studying localized corrosion using liquid cell transmission electron microscopy

DOE PAGES

Chee, See Wee; Pratt, Sarah H.; Hattar, Khalid; ...

2014-11-07

Using liquid cell transmission electron microscopy (LCTEM), localized corrosion of Cu and Al thin films immersed in aqueous NaCl solutions was studied. We demonstrate that potentiostatic control can be used to initiate pitting and that local compositional changes, due to focused ion beam implantation of Au + ions, can modify the corrosion susceptibility of Al films. Likewise, a discussion on strategies to control the onset of pitting is also presented.

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.

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.

Exploring the human mesenchymal stem cell tubule communication network through electron microscopy.

PubMed

Valente, Sabrina; Rossi, Roberta; Resta, Leonardo; Pasquinelli, Gianandrea

2015-04-01

Cells use several mechanisms to transfer information to other cells. In this study, we describe micro/nanotubular connections and exosome-like tubule fragments in multipotent mesenchymal stem cells (MSCs) from human arteries. Scanning and transmission electron microscopy allowed characterization of sinusoidal microtubular projections (700 nm average size, 200 µm average length, with bulging mitochondria and actin microfilaments); short, uniform, variously shaped nanotubular projections (100 nm, bidirectional communication); and tubule fragments (50 nm). This is the first study demonstrating that MSCs from human arteries constitutively interact through an articulate and dynamic tubule network allowing long-range cell to cell communication.

Changes in neutrophil morphology and morphometry following exposure to cigarette smoke.

PubMed Central

Lannan, S.; McLean, A.; Drost, E.; Gillooly, M.; Donaldson, K.; Lamb, D.; MacNee, W.

1992-01-01

Acute cigarette smoking delays neutrophils within the pulmonary circulation in some smokers. Evidence from an in-vitro Micropore filter model of the pulmonary capillaries indicates that this may be due to a smoke induced decrease in cell deformability. In order to determine whether changes in cell shape are associated with the observed decrease in neutrophil deformability following smoke exposure, cell morphology, using scanning electron microscopy, and morphometric measurements, made using transmission electron microscopy, were performed on aliquots of neutrophils harvested from whole blood in non-smoking subjects before and after exposure in vitro to cigarette smoke. Smoke exposure increased the maximum diameter and circumference of neutrophils, without changing their area. There was also a change in the maximum to minimum cell diameter ratio, which indicated that the cells had become less spherical. Scanning electron microscopy showed that smoke exposed cells had developed blebbing of their surface membranes, suggestive of an oxidative injury to the cell membrane rather than the shape changes associated with cell activation. These changes in the morphology and morphometry of smoke exposed neutrophils may contribute to the reduction in cell deformability induced by cigarette smoke. Images Fig. 3 Fig. 4 Fig. 5 PMID:1571278

Fixation methods for electron microscopy of human and other liver

PubMed Central

Wisse, Eddie; Braet, Filip; Duimel, Hans; Vreuls, Celien; Koek, Ger; Olde Damink, Steven WM; van den Broek, Maartje AJ; De Geest, Bart; Dejong, Cees HC; Tateno, Chise; Frederik, Peter

2010-01-01

For an electron microscopic study of the liver, expertise and complicated, time-consuming processing of hepatic tissues and cells is needed. The interpretation of electron microscopy (EM) images requires knowledge of the liver fine structure and experience with the numerous artifacts in fixation, embedding, sectioning, contrast staining and microscopic imaging. Hence, the aim of this paper is to present a detailed summary of different methods for the preparation of hepatic cells and tissue, for the purpose of preserving long-standing expertise and to encourage new investigators and clinicians to include EM studies of liver cells and tissue in their projects. PMID:20556830

Discovery and characterization of iron sulfide and polyphosphate bodies coexisting in Archaeoglobus fulgidus cells

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

Toso, Daniel B.; Javed, Muhammad Mohsin; Czornyj, Elizabeth

Inorganic storage granules have long been recognized in bacterial and eukaryotic cells but were only recently identified in archaeal cells. Here, we report the cellular organization and chemical compositions of storage granules in the Euryarchaeon, Archaeoglobus fulgidusstrain VC16, a hyperthermophilic, anaerobic, and sulfate-reducing microorganism. Dense granules were apparent inA. fulgiduscells imaged by cryo electron microscopy (cryoEM) but not so by negative stain electron microscopy. Cryo electron tomography (cryoET) revealed that each cell contains one to several dense granules located near the cell membrane. Energy dispersive X-ray (EDX) spectroscopy and scanning transmission electron microscopy (STEM) show that, surprisingly, each cell containsmore » not just one but often two types of granules with different elemental compositions. One type, named iron sulfide body (ISB), is composed mainly of the elements iron and sulfur plus copper; and the other one, called polyphosphate body (PPB), is composed of phosphorus and oxygen plus magnesium, calcium, and aluminum. PPBs are likely used for energy storage and/or metal sequestration/detoxification. ISBs could result from the reduction of sulfate to sulfide via anaerobic energy harvesting pathways and may be associated with energy and/or metal storage or detoxification. The exceptional ability of these archaeal cells to sequester different elements may have novel bioengineering applications.« less

Discovery and characterization of iron sulfide and polyphosphate bodies coexisting in Archaeoglobus fulgidus cells

DOE PAGES

Toso, Daniel B.; Javed, Muhammad Mohsin; Czornyj, Elizabeth; ...

2016-01-01

Inorganic storage granules have long been recognized in bacterial and eukaryotic cells but were only recently identified in archaeal cells. Here, we report the cellular organization and chemical compositions of storage granules in the Euryarchaeon, Archaeoglobus fulgidusstrain VC16, a hyperthermophilic, anaerobic, and sulfate-reducing microorganism. Dense granules were apparent inA. fulgiduscells imaged by cryo electron microscopy (cryoEM) but not so by negative stain electron microscopy. Cryo electron tomography (cryoET) revealed that each cell contains one to several dense granules located near the cell membrane. Energy dispersive X-ray (EDX) spectroscopy and scanning transmission electron microscopy (STEM) show that, surprisingly, each cell containsmore » not just one but often two types of granules with different elemental compositions. One type, named iron sulfide body (ISB), is composed mainly of the elements iron and sulfur plus copper; and the other one, called polyphosphate body (PPB), is composed of phosphorus and oxygen plus magnesium, calcium, and aluminum. PPBs are likely used for energy storage and/or metal sequestration/detoxification. ISBs could result from the reduction of sulfate to sulfide via anaerobic energy harvesting pathways and may be associated with energy and/or metal storage or detoxification. The exceptional ability of these archaeal cells to sequester different elements may have novel bioengineering applications.« less

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.

Platinum blue staining of cells grown in electrospun scaffolds.

PubMed

Yusuf, Mohammed; Millas, Ana Luiza G; Estandarte, Ana Katrina C; Bhella, Gurdeep K; McKean, Robert; Bittencourt, Edison; Robinson, Ian K

2014-01-01

Fibroblast cells grown in electrospun polymer scaffolds were stained with platinum blue, a heavy metal stain, and imaged using scanning electron microscopy. Good contrast on the cells was achieved compared with samples that were gold sputter coated. The cell morphology could be clearly observed, and the cells could be distinguished from the scaffold fibers. Here we optimized the required concentration of platinum blue for imaging cells grown in scaffolds and show that a higher concentration causes platinum aggregation. Overall, platinum blue is a useful stain for imaging cells because of its enhanced contrast using scanning electron microscopy (SEM). In the future it would be useful to investigate cell growth and morphology using three-dimensional imaging methods.

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

Imaging interactions of metal oxide nanoparticles with macrophage cells by ultra-high resolution scanning electron microscopy techniques†

PubMed Central

Plascencia-Villa, Germán; Starr, Clarise R.; Armstrong, Linda S.; Ponce, Arturo

2016-01-01

Use of engineered metal oxide nanoparticles in a plethora of biological applications and custom products has warned about some possible dose-dependent cytotoxic effects. Macrophages are key components of the innate immune system used to study possible toxic effects and internalization of different nanoparticulate materials. In this work, ultra-high resolution field emission scanning electron microscopy (FE-SEM) was used to offer new insights into the dynamical processes of interaction of nanomaterials with macrophage cells dosed with different concentrations of metal oxide nanoparticles (CeO2, TiO2 and ZnO). The versatility of FE-SEM has allowed obtaining a detailed characterization of processes of adsorption and endocytosis of nanoparticles, by using advanced analytical and imaging techniques on complete unstained uncoated cells, including secondary electron imaging, high-sensitive backscattered electron imaging, X-ray microanalysis and stereoimaging. Low voltage BF/DF-STEM confirmed nanoparticle adsorption and internalization into endosomes of CeO2 and TiO2, whereas ZnO develop apoptosis after 24 h of interaction caused by dissolution and invasion of cell nucleus. Ultra-high resolution scanning electron microscopy techniques provided new insights into interactions of inorganic nanoparticles with macrophage cells with high spatial resolution. PMID:23023106

Investigating Antibacterial Effects of Garlic (Allium sativum) Concentrate and Garlic-Derived Organosulfur Compounds on Campylobacter jejuni by Using Fourier Transform Infrared Spectroscopy, Raman Spectroscopy, and Electron Microscopy ▿ †

PubMed Central

Lu, Xiaonan; Rasco, Barbara A.; Jabal, Jamie M. F.; Aston, D. Eric; Lin, Mengshi; Konkel, Michael E.

2011-01-01

Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy were used to study the cell injury and inactivation of Campylobacter jejuni from exposure to antioxidants from garlic. C. jejuni was treated with various concentrations of garlic concentrate and garlic-derived organosulfur compounds in growth media and saline at 4, 22, and 35°C. The antimicrobial activities of the diallyl sulfides increased with the number of sulfur atoms (diallyl sulfide < diallyl disulfide < diallyl trisulfide). FT-IR spectroscopy confirmed that organosulfur compounds are responsible for the substantial antimicrobial activity of garlic, much greater than those of garlic phenolic compounds, as indicated by changes in the spectral features of proteins, lipids, and polysaccharides in the bacterial cell membranes. Confocal Raman microscopy (532-nm-gold-particle substrate) and Raman mapping of a single bacterium confirmed the intracellular uptake of sulfur and phenolic components. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were employed to verify cell damage. Principal-component analysis (PCA), discriminant function analysis (DFA), and soft independent modeling of class analogs (SIMCA) were performed, and results were cross validated to differentiate bacteria based upon the degree of cell injury. Partial least-squares regression (PLSR) was employed to quantify and predict actual numbers of healthy and injured bacterial cells remaining following treatment. PLSR-based loading plots were investigated to further verify the changes in the cell membrane of C. jejuni treated with organosulfur compounds. We demonstrated that bacterial injury and inactivation could be accurately investigated by complementary infrared and Raman spectroscopies using a chemical-based, “whole-organism fingerprint” with the aid of chemometrics and electron microscopy. PMID:21642409

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.

Effects of tricyclazole (5-methyl-1,2,4-triazol[3,4] benzothiazole), a specific DHN-melanin inhibitor, on the morphology of Fonsecaea pedrosoi conidia and sclerotic cells.

PubMed

Franzen, Anderson J; Cunha, Marcel M L; Batista, Evander J O; Seabra, Sergio H; De Souza, Wanderley; Rozental, Sonia

2006-09-01

The influence of tricyclazole (5-methyl-1,2,4-triazol[3,4]benzothiazole), a specific DHN-melanin inhibitor, on the cell walls and intracellular structures of Fonsecaea pedrosoi conidia and sclerotic cells was analyzed by transmission electron microscopy (TEM), deep-etching, and field emission scanning electron microscopy. The treatment of the fungus with 16 microg mL(-1) of tricyclazole (TC) did not significantly affect fungal viability, but electron microscopy observations showed several important morphological differences between TC-treated and non-TC treated cells. Control sclerotic cells presented patched granules, with an average diameter of 47 nm, on the cell surface, which were absent in TC-treated cells. Also, TC-treated sclerotic cells showed an undulated relief. TC treatment leads to an accumulation of electron lucent vacuoles in the fungal cytoplasm of both conidia and sclerotic cells, and treated conidia observed by deep etching showed a relevant thickening of the fungal cell wall. Together, these observations support the previous data of our group that F. pedrosoi synthesizes melanin in intracellular organelles. In addition, we suggest that melanin is not only an extracellular constituent but could also be dispersing all over the cell walls and could have an effective role in cross-linking different cell wall compounds that help maintain the regular shape of the cell wall. (c) 2006 Wiley-Liss, Inc.

Correlative light and electron microscopic detection of GFP-labeled proteins using modular APEX.

PubMed

Ariotti, Nicholas; Hall, Thomas E; Parton, Robert G

2017-01-01

The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques. Copyright © 2017 Elsevier Inc. All rights reserved.

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

PubMed

Gadelha, Ana Paula Rocha; Benchimol, Marlene; de Souza, Wanderley

2015-06-01

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites. Copyright © 2015 Elsevier Inc. All rights reserved.

A scanning electron microscopic study of 34 cases of acute granulocytic, myelomonocytic, monoblastic and histiocytic leukemia.

PubMed

Polliack, A; McKenzie, S; Gee, T; Lampen, N; de Harven, E; Clarkson, B D

1975-09-01

This report describes the surface architecture of leukemic cells, as seen by scanning electron microscopy in 34 patients with acute nonlymphoblastic leukemia. Six patients with myeloblastic, 4 with promyelocytic, 10 with myelomonocytic, 8 with monocytic, 4 with histiocytic and 2 with undifferentiated leukemia were studied. Under the scanning electron microscope most leukemia histiocytes and monocytes appeared similar and were characterized by the presence of large, well developed broad-based ruffled membranes or prominent raised ridge-like profiles, resembling ithis respect normal monocytes. Most cells from patients with acute promyelocytic or myeloblastic leukemia exhibited narrower ridge-like profiles whereas some showed ruffles or microvilli. Patients with myelomonocytic leukemia showed mixed populations of cells with ridge-like profiles and ruffled membranes whereas cells from two patients with undifferentiated leukemia had smooth surfaces, similar to those encountered in cells from patients with acute lymphoblastic leukemia. It appears that nonlymphoblastic and lymphoblastic leukemia cells (particularly histiocytes and monocytes) can frequently be distinquished on the basis of their surface architecture. The surface features of leukemic histiocytes and monocytes are similar, suggesting that they may belong to the same cell series. The monocytes seem to have characteristic surface features recognizable with the scanning electron microscope and differ from most cells from patients with acute granulocytic leukemia. Although overlap of surface features and misidentification can occur, scanning electron microscopy is a useful adjunct to other modes of microscopy in the study and diagnosis of acute leukemia.

Rethinking cell structure.

PubMed Central

Penman, S

1995-01-01

Cell structure, emerging from behind the veil of conventional electron microscopy, appears far more complex than formerly realized. The standard plastic-embedded, ultrathin section can image only what is on the section surface and masks the elaborate networks of the cytoplasm and nucleus. Embedment-free electron microscopy gives clear, high-contrast micrographs of cell structure when combined with removal of obscuring material such as soluble proteins. The resinless ultrathin section is the technique of choice; it is simple and inexpensive, and it uses ordinary electron microscopes. The resulting pictures reveal a world of complex cell structure and function. These images necessarily change our conception of the cytoskeleton, nuclear matrix, mitosis, and the relation of membranes to cytostructure. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:7777493

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.

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.

Serial block face-scanning electron microscopy: a tool for studying embryonic development at the cell-matrix interface.

PubMed

Starborg, Tobias; Kadler, Karl E

2015-03-01

Studies of gene regulation, signaling pathways, and stem cell biology are contributing greatly to our understanding of early embryonic vertebrate development. However, much less is known about the events during the latter half of embryonic development, when tissues comprising mostly extracellular matrix (ECM) are formed. The matrix extends far beyond the boundaries of individual cells and is refractory to study by conventional biochemical and molecular techniques; thus major gaps exist in our knowledge of the formation and three-dimensional (3D) organization of the dense tissues that form the bulk of adult vertebrates. Serial block face-scanning electron microscopy (SBF-SEM) has the ability to image volumes of tissue containing numerous cells at a resolution sufficient to study the organization of the ECM. Furthermore, whereas light microscopy was once relatively straightforward and electron microscopy was performed in specialist laboratories, the tables are turned; SBF-SEM is relatively straightforward and is becoming routine in high-end resolution studies of embryonic structures in vivo. In this review, we discuss the emergence of SBF-SEM as a tool for studying embryonic vertebrate development. © 2015 Wiley Periodicals, Inc.

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.

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

Extremely thin layer plastification for focused-ion beam scanning electron microscopy: an improved method to study cell surfaces and organelles of cultured cells.

PubMed

VAN Donselaar, E G; Dorresteijn, B; Popov-Čeleketić, D; VAN DE Wetering, W J; Verrips, T C; Boekhout, T; Schneijdenberg, C T W M; Xenaki, A T; VAN DER Krift, T P; Müller, W H

2018-03-25

Since the recent boost in the usage of electron microscopy in life-science research, there is a great need for new methods. Recently minimal resin embedding methods have been successfully introduced in the sample preparation for focused-ion beam scanning electron microscopy (FIB-SEM). In these methods several possibilities are given to remove as much resin as possible from the surface of cultured cells or multicellular organisms. Here we introduce an alternative way in the minimal resin embedding method to remove excess of resin from two widely different cell types by the use of Mascotte filter paper. Our goal in correlative light and electron microscopic studies of immunogold-labelled breast cancer SKBR3 cells was to visualise gold-labelled HER2 plasma membrane proteins as well as the intracellular structures of flat and round cells. We found a significant difference (p < 0.001) in the number of gold particles of selected cells per 0.6 μm 2 cell surface: on average a flat cell contained 2.46 ± 1.98 gold particles, and a round cell 5.66 ± 2.92 gold particles. Moreover, there was a clear difference in the subcellular organisation of these two cells. The round SKBR3 cell contained many organelles, such as mitochondria, Golgi and endoplasmic reticulum, when compared with flat SKBR3 cells. Our next goal was to visualise crosswall associated organelles, septal pore caps, of Rhizoctonia solani fungal cells by the combined use of a heavy metal staining and our extremely thin layer plastification (ETLP) method. At low magnifications this resulted into easily finding septa which appeared as bright crosswalls in the back-scattered electron mode in the scanning electron microscope. Then, a septum was selected for FIB-SEM. Cross-sectioned views clearly revealed the perforate septal pore cap of R. solani next to other structures, such as mitochondria, endoplasmic reticulum, lipid bodies, dolipore septum, and the pore channel. As the ETLP method was applied on two widely different cell types, the use of the ETLP method will be beneficial to correlative studies of other cell model systems and multicellular organisms. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Preparation of curcumin-loaded pluronic F127/chitosan nanoparticles for cancer therapy

NASA Astrophysics Data System (ADS)

Phuc Le, Thi Minh; Phuc Pham, Van; Lua Dang, Thi Minh; Huyen La, Thi; Hanh Le, Thi; Huan Le, Quang

2013-06-01

Nanoparticles (NPs) have been proven to be an effective delivery system with few side effects for anticancer drugs. In this study, curcumin-loaded NPs have been prepared by an ionic gelation method using chitosan (Chi) and pluronic®F-127 (PF) as carriers to deliver curcumin to the target cancer cells. Prepared NPs were characterized using Zetasizer, fluorescence microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our results showed that the encapsulation efficiency of curcumin was approximately 50%. The average size of curcumin-loaded PF/Chi NPs was 150.9 nm, while the zeta potential was 5.09 mV. Cellular uptake of curcumin-loaded NPs into HEK293 cells was confirmed by fluorescence microscopy.

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

Microscopy and microanalysis 1996

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

Bailey, G.W.; Corbett, J.M.; Dimlich, R.V.W.

1996-12-31

The Proceedings of this Annual Meeting contain paper of members from the three societies. These proceedings emphasizes the common research interests and attempts to eliminate some unwanted overlap. Topics covered are: microscopic analysis of animals with altered gene expression and in-situ gene and antibody localizations, high-resolution elemental mapping of nucleoprofein interactions, plant biology and pathology, quantitative HREM analysis of perfect and defected materials, computational methods for TEM image analysis, high-resolution FESM in materials research, frontiers in polymer microscopy and microanalysis, oxidation and corrosion, micro XRD and XRF, molecular microspectroscopy and spectral imaging, advances in confocal and multidimensional light microscopy, analyticalmore » electron microscopy in biology, correlative microscopy in biological sciences, grain-boundary microengineering, surfaces and interfaces, telepresence microscopy in education and research, MSA educational outreach, quantitative electron probe microanalysis, frontiers of analytical electron microscopy, critical issues in ceramic microstructures, dynamic organization of the cell, pathology, microbiology, high-resolution biological and cryo SEM, and scanning-probe microscopy.« less

Cytocompatibility and uptake of halloysite clay nanotubes.

PubMed

Vergaro, Viviana; Abdullayev, Elshad; Lvov, Yuri M; Zeitoun, Andre; Cingolani, Roberto; Rinaldi, Ross; Leporatti, Stefano

2010-03-08

Halloysite is aluminosilicate clay with hollow tubular structure of 50 nm external diameter and 15 nm diameter lumen. Halloysite biocompatibility study is important for its potential applications in polymer composites, bone implants, controlled drug delivery, and for protective coating (e.g., anticorrosion or antimolding). Halloysite nanotubes were added to different cell cultures for toxicity tests. Its fluorescence functionalization by aminopropyltriethosilane (APTES) and with fluorescently labeled polyelectrolyte layers allowed following halloysite uptake by the cells with confocal laser scanning microscopy (CLSM). Quantitative Trypan blue and MTT measurements performed with two neoplastic cell lines model systems as a function of the nanotubes concentration and incubation time indicate that halloysite exhibits a high level of biocompatibility and very low cytotoxicity, rendering it a good candidate for household materials and medicine. A combination of transmission electron microscopy (TEM), scanning electron microscopy (SEM), and scanning force microscopy (SFM) imaging techniques have been employed to elucidate the structure of halloysite nanotubes.

Ultrastructural effects of silicone oil on the clear crystalline lens of the human eye.

PubMed

Soliman, Wael; Sharaf, Mohamed; Abdelazeem, Khaled; El-Gamal, Dalia; Nafady, Allam

2018-03-01

To evaluate light and electron microscopic changes of the anterior capsule and its epithelium after clear lens extraction of vitrectomized myopic eyes with silicone oil tamponade. This prospective, controlled, non-randomized, interventional study included 20 anterior lens capsular specimens that were excised during combined clear lens extraction and silicone oil removal from previously vitrectomized highly myopic patients with silicone oil tamponade for previous retinal detachment surgeries. The specimens were examined via light microscopy and electron microscopy and compared with 20 anterior capsule specimens removed during clear lens extraction of non-vitrectomized highly myopic eyes. Light microscopic examination of clear lens anterior capsule specimens of vitrectomized myopic eyes filled with silicone oil showed relatively more flat cells with irregular outline of lens' epithelial cells with wide intercellular spaces, deeply stained nuclei, and multiple intracytoplasmic vacuoles. Scanning electron microscopy revealed collagenous surfaces filled with multiple pits, depressions, and abnormal deposits. Transmission electron microscopy revealed lens epithelial cells with apoptotic changes, many cytoplasmic vacuoles, and filopodia-like protrusions between lens epithelial cells and the capsule. Epithelial proliferation and multilayering were also observed. silicone oil may play a role in the development of apoptotic and histopathological changes in clear lens epithelial cells. Clarity of the lens at the time of silicone oil removal does not indicate an absence of cataractous changes. We found justification of combined clear lens extraction and silicone oil removal or combined phacovitrectomy when silicone oil injection is planned, but further long-term studies with larger patient groups are required.

Three-dimensional bright-field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms.

PubMed

Hickey, William J; Shetty, Ameesha R; Massey, Randall J; Toso, Daniel B; Austin, Jotham

2017-01-01

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Imaging plasmodesmata with high-resolution scanning electron microscopy.

PubMed

Barton, Deborah A; Overall, Robyn L

2015-01-01

High-resolution scanning electron microscopy (HRSEM) is an effective tool to investigate the distribution of plasmodesmata within plant cell walls as well as to probe their complex, three-dimensional architecture. It is a useful alternative to traditional transmission electron microscopy (TEM) in which plasmodesmata are sectioned to reveal their internal substructures. Benefits of adopting an HRSEM approach to studies of plasmodesmata are that the specimen preparation methods are less complex and time consuming than for TEM, many plasmodesmata within a large region of tissue can be imaged in a single session, and three-dimensional information is readily available without the need for reconstructing TEM serial sections or employing transmission electron tomography, both of which are lengthy processes. Here we describe methods to prepare plant samples for HRSEM using pre- or postfixation extraction of cellular material in order to visualize plasmodesmata embedded within plant cell walls.

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

PubMed

Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

2014-01-01

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

Investigating the use of in situ liquid cell scanning transmission electron microscopy to explore DNA-mediated gold nanoparticle growth

NASA Astrophysics Data System (ADS)

Nguy, Amanda

Engineering nanoparticles with desired shape-dependent properties is the key to many applications in nanotechnology. Although many synthetic procedures exist to produce anisotropic gold nanoparticles, the dynamics of growth are typically unknown or hypothetical. In the case of seed-mediated growth in the presence of DNA into anisotropic nanoparticles, it is not known exactly how DNA directs growth into specific morphologies. A series of preliminary experiments were carried out to contribute to the investigation of the possible mechanism of DNA-mediated growth of gold nanoprisms into gold nanostars using liquid cell scanning transmission electron microscopy (STEM). Imaging in the liquid phase was achieved through the use of a liquid cell platform and liquid cell holder that allow the sample to be contained within a “chip sandwich” between two electron transparent windows. Ex situ growth experiments were performed using Au-T30 NPrisms (30-base thymine oligonucleotide-coated gold nanoprisms) that are expected to grow into gold nanostars. Growth to form these nanostars were imaged using TEM (transmission electron microscopy) and liquid cell STEM (scanning transmission electron microscopy). An attempt to perform in situ growth experiments with the same Au-T30 nanoprisms revealed challenges in obtaining desired morphology results due to the environmental differences within the liquid cell compared to the ex situ environment. Different parameters in the experimental method were explored including fluid line set up, simultaneous and alternating reagent addition, and the effect of different liquid cell volumes to ensure adequate flow of reagents into the liquid cell. Lastly, the binding affinities were compared for T30 and A30 DNA incubated with gold nanoparticles using zeta potential measurements, absorption spectroscopy, and isothermal titration calorimetry (ITC). It was previously reported thymine bases have a lower binding affinity to gold surfaces than adenine bases. However, the results reported here contradict the previously reported data. Future prospectives on this work are outlined.

Investigating the use of in situ liquid cell scanning transmission electron microscopy

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

Nguy, Amanda

2016-02-19

Engineering nanoparticles with desired shape-dependent properties is the key to many applications in nanotechnology. Although many synthetic procedures exist to produce anisotropic gold nanoparticles, the dynamics of growth are typically unknown or hypothetical. In the case of seed-mediated growth in the presence of DNA into anisotropic nanoparticles, it is not known exactly how DNA directs growth into specific morphologies. A series of preliminary experiments were carried out to contribute to the investigation of the possible mechanism of DNA-mediated growth of gold nanoprisms into gold nanostars using liquid cell scanning transmission electron microscopy (STEM). Imaging in the liquid phase was achievedmore » through the use of a liquid cell platform and liquid cell holder that allow the sample to be contained within a “chip sandwich” between two electron transparent windows. Ex situ growth experiments were performed using Au-T30 NPrisms (30-base thymine oligonucleotide-coated gold nanoprisms) that are expected to grow into gold nanostars. Growth to form these nanostars were imaged using TEM (transmission electron microscopy) and liquid cell STEM (scanning transmission electron microscopy). An attempt to perform in situ growth experiments with the same Au-T30 nanoprisms revealed challenges in obtaining desired morphology results due to the environmental differences within the liquid cell compared to the ex situ environment. Different parameters in the experimental method were explored including fluid line set up, simultaneous and alternating reagent addition, and the effect of different liquid cell volumes to ensure adequate flow of reagents into the liquid cell. Lastly, the binding affinities were compared for T30 and A30 DNA incubated with gold nanoparticles using zeta potential measurements, absorption spectroscopy, and isothermal titration calorimetry (ITC). It was previously reported thymine bases have a lower binding affinity to gold surfaces than adenine bases. However, the results reported here contradict the previously reported data. Future prospectives on this work are outlined.« less

From 2D slices to 3D volumes: image based reconstruction and morphological characterization of hippocampal cells on charged and uncharged surfaces using FIB/SEM serial sectioning.

PubMed

Schmidt, Franziska; Kühbacher, Markus; Gross, Ulrich; Kyriakopoulos, Antonius; Schubert, Helmut; Zehbe, Rolf

2011-03-01

3D imaging at a subcellular resolution is a powerful tool in the life sciences to investigate cells and their interactions with native tissues or artificial objects. While a tomographic experimental setup achieving a sufficient structural resolution can be established with either X-rays or electrons, the use of electrons is usually limited to very thin samples in transmission electron microscopy due to the poor penetration depths of electrons. The combination of a serial sectioning approach and scanning electron microscopy in state of the art dual beam experimental setups therefore offers a means to image highly resolved spatial details using a focused ion beam for slicing and an electron beam for imaging. The advantage of this technique over X-ray μCT or X-ray microscopy attributes to the fact that absorption is not a limiting factor in imaging and therefore even strong absorbing structures can be spatially reconstructed with a much higher possible resolution. This approach was used in this study to elucidate the effect of an electric potential on the morphology of cells from a hippocampal cell line (HT22) deposited on gold microelectrodes. While cells cultivated on two different controls (gold and polymer substrates) did show the expected stretched morphology, cells on both the anode and the cathode differed significantly. Cells deposited on the anode part of the electrode exhibited the most extreme deviation, being almost spherical and showed signs of chromatin condensation possibly indicating cell death. Furthermore, EDX was used as supplemental methodology for combined chemical and structural analyses. Copyright © 2010 Elsevier B.V. All rights reserved.

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.

Preparation of isolated nuclei from K 562 haemopoietic cell line for high resolution scanning electron microscopy.

PubMed

Reipert, S; Reipert, B M; Allen, T D

1994-09-01

The aim of the work is to visualise nuclear pore complexes (NPCs) in mammalian cells by high resolution scanning electron microscopy. A detergent-free isolation protocol was employed to obtain clean nuclei from the haemopoietic cell line K 562. Nuclear isolation was performed by mechanical homogenisation under hypotonic conditions followed by purification of the nuclear fraction. The isolated nuclei were attached to silicon chips, fixed, critical point dried, and sputter coated with a thin film (3-4 nm) of tantalum. Analysis of the nuclear surface by scanning electron microscopy (SEM) revealed a strong sensitivity of the outer nuclear membrane (ONM) to disruption during the isolation procedure. A significant reduction of the characteristic pattern of damage to the ONM was achieved by means of an isopicnic centrifugation on an isoosmolar balanced Percoll gradient. Analysis of the population of isolated nuclei by flow cytometry showed no signs of cell cycle specific losses of nuclei during isolation. The SEM investigations of the morphology of the nuclear envelope (NE) and of substructural details of NPCs and polyribosomes were performed using an in-lens field emission scanning electron microscope.

Visualization of Hierarchical Nanodomains in Polymer/Fullerene Bulk Heterojunction Solar Cells

DOE PAGES

Wen, Jianguo; Miller, Dean J.; Chen, Wei; ...

2014-06-20

Here, traditional electron microscopy techniques such as bright-field imaging provide poor contrast for organic films and identification of structures in amorphous material can be problematic, particularly in high-performance organic solar cells. By combining energy-filtered corrected transmission electron microscopy, together with electron energy loss and X-ray energy-dispersive hyperspectral imaging, we have imaged PTB7/ PC 61BM blended polymer optical photovoltaic films, and were able to identify domains ranging in size from several hundred nanometers to several nanometers in extent. This work verifies that microstructural domains exist in bulk heterojunctions in PTB7/PC 61BM polymeric solar cells at multiple length scales and expands ourmore » understanding of optimal device performance providing insight for the design of even higher performance cells.« less

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.

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.

Cryogenic coherent X-ray diffraction imaging of biological samples at SACLA: a correlative approach with cryo-electron and light microscopy.

PubMed

Takayama, Yuki; Yonekura, Koji

2016-03-01

Coherent X-ray diffraction imaging at cryogenic temperature (cryo-CXDI) allows the analysis of internal structures of unstained, non-crystalline, whole biological samples in micrometre to sub-micrometre dimensions. Targets include cells and cell organelles. This approach involves preparing frozen-hydrated samples under controlled humidity, transferring the samples to a cryo-stage inside a vacuum chamber of a diffractometer, and then exposing the samples to coherent X-rays. Since 2012, cryo-coherent diffraction imaging (CDI) experiments have been carried out with the X-ray free-electron laser (XFEL) at the SPring-8 Ångstrom Compact free-electron LAser (SACLA) facility in Japan. Complementary use of cryo-electron microscopy and/or light microscopy is highly beneficial for both pre-checking samples and studying the integrity or nature of the sample. This article reports the authors' experience in cryo-XFEL-CDI of biological cells and organelles at SACLA, and describes an attempt towards reliable and higher-resolution reconstructions, including signal enhancement with strong scatterers and Patterson-search phasing.

Electron Microscopy of the Infection and Subsequent Development of Soybean Nodule Cells

PubMed Central

Goodchild, D. J.; Bergersen, F. J.

1966-01-01

Goodchild, D. J. (Commonwealth Scientific and Industrial Research Organization, Canberra, Australia), and F. J. Bergersen. Electron microscopy of the infection and subsequent development of soybean nodule cells. J. Bacteriol. 92:204–213. 1966—Electron microscopy of thin sections of the developing central tissue cells of young soybean root nodules has shown that infection is initiated by a few infection threads which penetrate cells of the young central tissue. Extension growth of the threads may be a result of pressure developed from the growth of the bacteria within the threads. Release of bacteria from a thread is preceded by the development on an infection thread of a bulge with a cellulose-free membrane-bounded extension; bacteria move from this into the host cells by an endocytotic process and remain enclosed in an infection vacuole which is bounded by a membrane of host-cell origin. Multiplication of the intracellular bacteria takes place within these vacuoles. Until the host cell becomes filled with bacteria, the vacuoles separate into discrete units at each division. Later, division of the bacteria occurs within each vacuole, thus leading to the mature structure of the central tissue cells in which several bacteria are enclosed within each membrane-bounded unit. Images PMID:5949564

Electron tomography of whole cultured cells using novel transmission electron imaging technique.

PubMed

Okumura, Taiga; Shoji, Minami; Hisada, Akiko; Ominami, Yusuke; Ito, Sukehiro; Ushiki, Tatsuo; Nakajima, Masato; Ohshima, Takashi

2018-01-01

Since a three-dimensional (3D) cellular ultrastructure is significant for biological functions, it has been investigated using various electron microscopic techniques. Although transmission electron microscopy (TEM)-based techniques are traditionally used, cells must be embedded in resin and sliced into ultrathin sections in sample preparation processes. Block-face observation using a scanning electron microscope (SEM) has also been recently applied to 3D observation of cellular components, but this is a destructive inspection and does not allow re-examination. Therefore, we developed electron tomography using a transmission electron imaging technique called Plate-TEM. With Plate-TEM, the cells cultured directly on a scintillator plate are inserted into a conventional SEM equipped with a Plate-TEM observation system, and their internal structures are observed by detecting scintillation light produced by electrons passing through the cells. This technology has the following four advantages. First, the cells cultured on the plate can be observed at electron-microscopic resolution since they remain on the plate. Second, both surface and internal information can be obtained simultaneously by using electron- and photo-detectors, respectively, because a Plate-TEM detector is installed in an SEM. Third, the cells on the scintillator plate can also be inspected using light microscopy because the plate has transparent features. Finally, correlative observation with other techniques, such as conventional TEM, is possible after Plate-TEM observation because Plate-TEM is a non-destructive analysis technique. We also designed a sample stage to tilt the samples for tomography with Plate-TEM, by which 3D organization of cellular structures can be visualized as a whole cell. In the present study, Mm2T cells were investigated using our tomography system, resulting in 3D visualization of cell organelles such as mitochondria, lipid droplets, and microvilli. Correlative observations with various imaging techniques were also conducted by successive observations with light microscopy, SEM, Plate-TEM, and conventional TEM. Consequently, the Plate-TEM tomography technique encourages understanding of cellular structures at high resolution, which can contribute to cellular biological research. Copyright © 2017 Elsevier Ltd. All rights reserved.

Visualizing gold nanoparticle uptake in live cells with liquid scanning transmission electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2011-04-13

The intracellular uptake of 30 nm diameter gold nanoparticles (Au-NPs) was studied at the nanoscale in pristine eukaryotic cells. Live COS-7 cells were maintained in a microfluidic chamber and imaged using scanning transmission electron microscopy. A quantitative image analysis showed that Au-NPs bound to the membranes of vesicles, possibly lysosomes, and occupied 67% of the available surface area. The vesicles accumulated to form a micrometer-sized cluster after 24 h of incubation. Two clusters were analyzed and found to consist of 117 ± 9 and 164 ± 4 NP-filled vesicles.

Label-free imaging of the dynamics of cell-to-cell string-like structure bridging in the free-space by low-coherent quantitative phase microscopy

NASA Astrophysics Data System (ADS)

Yamauchi, Toyohiko; Iwai, Hidenao; Yamashita, Yutaka

2013-03-01

We succeeded in utilizing our low-coherent quantitative phase microscopy (LC-QPM) to achieve label-free and three-dimensional imaging of string-like structures bridging the free-space between live cells. In past studies, three dimensional morphology of the string-like structures between cells had been investigated by electron microscopies and fluorescence microscopies and these structures were called "membrane nanotubes" or "tunneling nanotubes." However, use of electron microscopy inevitably kills these cells and fluorescence microscopy is itself a potentially invasive method. To achieve noninvasive imaging of live cells, we applied our LC-QPM which is a reflection-type, phase resolved and full-field interference microscope employing a low-coherent light source. LC-QPM is able to visualize the three-dimensional morphology of live cells without labeling by means of low-coherence interferometry. The lateral (diffraction limit) and longitudinal (coherence-length) spatial resolution of LC-QPM were respectively 0.49 and 0.93 micrometers and the repeatability of the phase measurement was 0.02 radians (1.0 nm). We successfully obtained three-dimensional morphology of live cultured epithelial cells (cell type: HeLa, derived from cervix cancer) and were able to clearly observe the individual string-like structures interconnecting the cells. When we performed volumetric imaging, a 80 micrometer by 60 micrometer by 6.5 micrometer volume was scanned every 5.67 seconds and 70 frames of a three-dimensional movie were recorded for a duration of 397 seconds. Moreover, the optical phase images gave us detailed information about the three-dimensional morphology of the string-like structure at sub-wavelength resolution. We believe that our LC-QPM will be a useful tool for the study of three-dimensional morphology of live cells.

Field emission scanning electron microscopy (FE-SEM) as an approach for nanoparticle detection inside cells.

PubMed

Havrdova, M; Polakova, K; Skopalik, J; Vujtek, M; Mokdad, A; Homolkova, M; Tucek, J; Nebesarova, J; Zboril, R

2014-12-01

When developing new nanoparticles for bio-applications, it is important to fully characterize the nanoparticle's behavior in biological systems. The most common techniques employed for mapping nanoparticles inside cells include transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM). These techniques entail passing an electron beam through a thin specimen. STEM or TEM imaging is often used for the detection of nanoparticles inside cellular organelles. However, lengthy sample preparation is required (i.e., fixation, dehydration, drying, resin embedding, and cutting). In the present work, a new matrix (FTO glass) for biological samples was used and characterized by field emission scanning electron microscopy (FE-SEM) to generate images comparable to those obtained by TEM. Using FE-SEM, nanoparticle images were acquired inside endo/lysosomes without disruption of the cellular shape. Furthermore, the initial steps of nanoparticle incorporation into the cells were captured. In addition, the conductive FTO glass endowed the sample with high stability under the required accelerating voltage. Owing to these features of the sample, further analyses could be performed (material contrast and energy-dispersive X-ray spectroscopy (EDS)), which confirmed the presence of nanoparticles inside the cells. The results showed that FE-SEM can enable detailed characterization of nanoparticles in endosomes without the need for contrast staining or metal coating of the sample. Images showing the intracellular distribution of nanoparticles together with cellular morphology can give important information on the biocompatibility and demonstrate the potential of nanoparticle utilization in medicine. Copyright © 2014 Elsevier Ltd. All rights reserved.

Myofibroblasts in interstitial lung diseases show diverse electron microscopic and invasive features.

PubMed

Karvonen, Henna M; Lehtonen, Siri T; Sormunen, Raija T; Harju, Terttu H; Lappi-Blanco, Elisa; Bloigu, Risto S; Kaarteenaho, Riitta L

2012-09-01

The characteristic features of myofibroblasts in various lung disorders are poorly understood. We have evaluated the ultrastructure and invasive capacities of myofibroblasts cultured from small volumes of diagnostic bronchoalveolar lavage (BAL) fluid samples from patients with different types of lung diseases. Cells were cultured from samples of BAL fluid collected from 51 patients that had undergone bronchoscopy and BAL for diagnostic purposes. The cells were visualized by transmission electron microscopy and immunoelectron microscopy to achieve ultrastructural localization of alpha-smooth muscle actin (α-SMA) and fibronectin. The levels of α-SMA protein and mRNA and fibronectin mRNA were measured by western blot and quantitative real-time reverse transcriptase polymerase chain reaction. The invasive capacities of the cells were evaluated. The cultured cells were either fibroblasts or myofibroblasts. The structure of the fibronexus, and the amounts of intracellular actin, extracellular fibronectin and cell junctions of myofibroblasts varied in different diseases. In electron and immunoelectron microscopy, cells cultured from interstitial lung diseases (ILDs) expressed more actin filaments and α-SMA than normal lung. The invasive capacity of the cells obtained from patients with idiopathic pulmonary fibrosis was higher than that from patients with other type of ILDs. Cells expressing more actin filaments had a higher invasion capacity. It is concluded that electron and immunoelectron microscopic studies of myofibroblasts can reveal differential features in various diseases. An analysis of myofibroblasts cultured from diagnostic BAL fluid samples may represent a new kind of tool for diagnostics and research into lung diseases.

Evidence for Direct Electron Transfer by a Gram-Positive Bacterium Isolated from a Microbial Fuel Cell▿†

PubMed Central

Wrighton, K. C.; Thrash, J. C.; Melnyk, R. A.; Bigi, J. P.; Byrne-Bailey, K. G.; Remis, J. P.; Schichnes, D.; Auer, M.; Chang, C. J.; Coates, J. D.

2011-01-01

Despite their importance in iron redox cycles and bioenergy production, the underlying physiological, genetic, and biochemical mechanisms of extracellular electron transfer by Gram-positive bacteria remain insufficiently understood. In this work, we investigated respiration by Thermincola potens strain JR, a Gram-positive isolate obtained from the anode surface of a microbial fuel cell, using insoluble electron acceptors. We found no evidence that soluble redox-active components were secreted into the surrounding medium on the basis of physiological experiments and cyclic voltammetry measurements. Confocal microscopy revealed highly stratified biofilms in which cells contacting the electrode surface were disproportionately viable relative to the rest of the biofilm. Furthermore, there was no correlation between biofilm thickness and power production, suggesting that cells in contact with the electrode were primarily responsible for current generation. These data, along with cryo-electron microscopy experiments, support contact-dependent electron transfer by T. potens strain JR from the cell membrane across the 37-nm cell envelope to the cell surface. Furthermore, we present physiological and genomic evidence that c-type cytochromes play a role in charge transfer across the Gram-positive bacterial cell envelope during metal reduction. PMID:21908627

Nanoscale imaging of whole cells using a liquid enclosure and a scanning transmission electron microscope.

PubMed

Peckys, Diana B; Veith, Gabriel M; Joy, David C; de Jonge, Niels

2009-12-14

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory.

Balamuthia mandrillaris: Further morphological observations of trophozoites by light, scanning and transmission electron microscopy.

PubMed

González-Robles, Arturo; Lares-Villa, Fernando; Lares-Jiménez, Luis Fernando; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Martínez-Palomo, Adolfo

2015-10-01

Additional morphological features of Balamuthia mandrillaris observed by light and electron microscopy are reported. Trophozoites were extremely pleomorphic: their cell shapes ranged from rounded to elongated and sometimes they appeared exceptionally stretched out and branched. By transmission electron microscopy it was possible to observe two different cytoplasmic areas, the ectoplasm and the endoplasm and often sections of rough endoplasmic reticulum were found in the transition zone. The cytoplasm was very fibrogranular and most of the organelles typically found in eukaryotic cells were observed. A particular finding was the presence of numerous mitochondria with a different structure from those of other free-living amoebae. The observations reported here may reinforce the morphological knowledge of this amoeba and provide a background for further analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

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.

APT mass spectrometry and SEM data for CdTe solar cells

DOE PAGES

Li, Chen; Paudel, Naba R.; Yan, Yanfa; ...

2016-03-16

Atom probe tomography (APT) data acquired from a CAMECA LEAP 4000 XHR for the CdS/CdTe interface for a non-CdCl 2 treated CdTe solar cell as well as the mass spectrum of an APT data set including a GB in a CdCl 2-treated CdTe solar cell are presented. Scanning electron microscopy (SEM) data showing the evolution of sample preparation for APT and scanning transmission electron microscopy (STEM) electron beam induced current (EBIC) are also presented. As a result, these data show mass spectrometry peak decomposition of Cu and Te within an APT dataset, the CdS/CdTe interface of an untreated CdTe solarmore » cell, preparation of APT needles from the CdS/CdTe interface in superstrate grown CdTe solar cells, and the preparation of a cross-sectional STEM EBIC sample.« less

The origins and evolution of freeze-etch electron microscopy

PubMed Central

Heuser, John E.

2011-01-01

The introduction of the Balzers freeze-fracture machine by Moor in 1961 had a much greater impact on the advancement of electron microscopy than he could have imagined. Devised originally to circumvent the dangers of classical thin-section techniques, as well as to provide unique en face views of cell membranes, freeze-fracturing proved to be crucial for developing modern concepts of how biological membranes are organized and proved that membranes are bilayers of lipids within which proteins float and self-assemble. Later, when freeze-fracturing was combined with methods for freezing cells that avoided the fixation and cryoprotection steps that Moor still had to use to prepare the samples for his original invention, it became a means for capturing membrane dynamics on the millisecond time-scale, thus allowing a deeper understanding of the functions of biological membranes in living cells as well as their static ultrastructure. Finally, the realization that unfixed, non-cryoprotected samples could be deeply vacuum-etched or even freeze-dried after freeze-fracturing opened up a whole new way to image all the other molecular components of cells besides their membranes and also provided a powerful means to image the interactions of all the cytoplasmic components with the various membranes of the cell. The purpose of this review is to outline the history of these technical developments, to describe how they are being used in electron microscopy today and to suggest how they can be improved in order to further their utility for biological electron microscopy in the future. PMID:21844598

Virus spread and initial pathological changes in the nervous system in genital herpes simplex virus type 2 infection in mice. A correlative immunohistochemical, light and electron microscopic study.

PubMed

Georgsson, G; Martin, J R; Stoner, G L; Webster, H F

1987-01-01

Mice were infected by the vaginal route with the MS strain of herpes simplex virus type 2 (HSV-2). Serial vaginal cultures were used to confirm infection and to select mice for this study. Two mice were killed by perfusion on days 2-6 post infection (p.i.) and lumbar and sacral cord with cauda were fixed and embedded for electron microscopy. Semithin Epon-sections were stained for viral antigen using a rabbit anti-HSV-2 antiserum and the Avidin-Biotin (ABC) method. Thin sections from antigen-positive blocks were examined by electron microscopy, and the number and types of infected cells detected by these two methods were compared. A good correlation was found between detection of infected cells by these methods. Infected cells included neurons of dorsal root ganglia and spinal cord, satellite cells of dorsal root ganglia, non-myelinating Schwann cells, astrocytes, oligodendrocytes and arachnoidal cells. Infected cells were first detected in the cauda on day 3 p.i. and in the spinal cord on day 5 p.i. The temporal and spatial distribution of infected cells was consistent with neural spread to and within the CNS. The pathological lesions showed a good correlation with the distribution and number of infected cells and are probably due to a direct virus effect. The similar sensitivity of the Epon-ABC method to electron microscopy in detecting infected cells indicates that this method may have useful applications in both experimental and diagnostic work.

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

Li, Chen; Paudel, Naba R.; Yan, Yanfa

Atom probe tomography (APT) data acquired from a CAMECA LEAP 4000 XHR for the CdS/CdTe interface for a non-CdCl 2 treated CdTe solar cell as well as the mass spectrum of an APT data set including a GB in a CdCl 2-treated CdTe solar cell are presented. Scanning electron microscopy (SEM) data showing the evolution of sample preparation for APT and scanning transmission electron microscopy (STEM) electron beam induced current (EBIC) are also presented. As a result, these data show mass spectrometry peak decomposition of Cu and Te within an APT dataset, the CdS/CdTe interface of an untreated CdTe solarmore » cell, preparation of APT needles from the CdS/CdTe interface in superstrate grown CdTe solar cells, and the preparation of a cross-sectional STEM EBIC sample.« less

Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.

PubMed

Jia, Peng-Fei; Li, Hong-Ju; Yang, Wei-Cai

2017-01-01

Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).

Fine structures of embryonic discs of in vivo post-hatching porcine blastocysts at the pre-primitive streak stage.

PubMed

Xia, P; Liu, Z; Qin, P

2011-04-01

To date, reports about the ultrastructure of porcine embryonic discs have not shown details of the primitive streak. The main objective of this study was to examine the ultrastructure of interior and exterior embryonic discs in porcine in vivo blastocysts with diameters of 1, 3 and 9 mm using scanning electron microscopy and transmission electron microscopy. For the first time, we revealed the ultrastructure of the unusual group of cells in the pre-primitive streak area of embryonic discs. The cells were 1-2 μm in diameter, had high electron density and contained abundant, free ribosomes and endoplasmic reticulum. These primitive streak cells could represent original embryonic stem cells or represent a stem cell niche. The results also showed three types of cells on the exterior surface of the embryonic discs. Moreover, our results provided morphological evidence of condensed nuclei in the smooth cells on the surface of the embryonic disc. © 2010 Blackwell Verlag GmbH.

Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

PubMed

Vadivelu, Jamuna; Vellasamy, Kumutha Malar; Thimma, Jaikumar; Mariappan, Vanitha; Kang, Wen-Tyng; Choh, Leang-Chung; Shankar, Esaki M; Wong, Kum Thong

2017-01-01

During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection.

Survival and Intra-Nuclear Trafficking of Burkholderia pseudomallei: Strategies of Evasion from Immune Surveillance?

PubMed Central

Vadivelu, Jamuna; Vellasamy, Kumutha Malar; Thimma, Jaikumar; Mariappan, Vanitha; Kang, Wen-Tyng; Choh, Leang-Chung; Wong, Kum Thong

2017-01-01

Background During infection, successful bacterial clearance is achieved via the host immune system acting in conjunction with appropriate antibiotic therapy. However, it still remains a tip of the iceberg as to where persistent pathogens namely, Burkholderia pseudomallei (B. pseudomallei) reside/hide to escape from host immune sensors and antimicrobial pressure. Methods We used transmission electron microscopy (TEM) to investigate post-mortem tissue sections of patients with clinical melioidosis to identify the localisation of a recently identified gut microbiome, B. pseudomallei within host cells. The intranuclear presence of B. pseudomallei was confirmed using transmission electron microscopy (TEM) of experimentally infected guinea pig spleen tissues and Live Z-stack, and ImageJ analysis of fluorescence microscopy analysis of in vitro infection of A549 human lung epithelial cells. Results TEM investigations revealed intranuclear localization of B. pseudomallei in cells of infected human lung and guinea pig spleen tissues. We also found that B. pseudomallei induced actin polymerization following infection of A549 human lung epithelial cells. Infected A549 lung epithelial cells using 3D-Laser scanning confocal microscopy (LSCM) and immunofluorescence microscopy confirmed the intranuclear localization of B. pseudomallei. Conclusion B. pseudomallei was found within the nuclear compartment of host cells. The nucleus may play a role as an occult or transient niche for persistence of intracellular pathogens, potentially leading to recurrrent episodes or recrudescence of infection. PMID:28045926

Advantages of intermediate X-ray energies in Zernike phase contrast X-ray microscopy.

PubMed

Wang, Zhili; Gao, Kun; Chen, Jian; Hong, Youli; Ge, Xin; Wang, Dajiang; Pan, Zhiyun; Zhu, Peiping; Yun, Wenbing; Jacobsen, Chris; Wu, Ziyu

2013-01-01

Understanding the hierarchical organizations of molecules and organelles within the interior of large eukaryotic cells is a challenge of fundamental interest in cell biology. Light microscopy is a powerful tool for observations of the dynamics of live cells, its resolution attainable is limited and insufficient. While electron microscopy can produce images with astonishing resolution and clarity of ultra-thin (<1 μm thick) sections of biological specimens, many questions involve the three-dimensional organization of a cell or the interconnectivity of cells. X-ray microscopy offers superior imaging resolution compared to light microscopy, and unique capability of nondestructive three-dimensional imaging of hydrated unstained biological cells, complementary to existing light and electron microscopy. Until now, X-ray microscopes operating in the "water window" energy range between carbon and oxygen k-shell absorption edges have produced outstanding 3D images of cryo-preserved cells. The relatively low X-ray energy (<540 eV) of the water window imposes two important limitations: limited penetration (<10 μm) not suitable for imaging larger cells or tissues, and small depth of focus (DoF) for high resolution 3D imaging (e.g., ~1 μm DoF for 20 nm resolution). An X-ray microscope operating at intermediate energy around 2.5 keV using Zernike phase contrast can overcome the above limitations and reduces radiation dose to the specimen. Using a hydrated model cell with an average chemical composition reported in literature, we calculated the image contrast and the radiation dose for absorption and Zernike phase contrast, respectively. The results show that an X-ray microscope operating at ~2.5 keV using Zernike phase contrast offers substantial advantages in terms of specimen size, radiation dose and depth-of-focus. Copyright © 2012 Elsevier Inc. All rights reserved.

Click-electron microscopy for imaging metabolically tagged non-protein biomolecules

PubMed Central

Ngo, John T.; Adams, Stephen R.; Deerinck, Thomas J.; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F.; Bertozzi, Carolyn R.; Ellisman, Mark H.; Tsien, Roger Y.

2016-01-01

Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

Gonococcal attachment to eukaryotic cells

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

James, J.F.; Lammel, C.J.; Draper, D.L.

The attachment of Neisseria gonorrhoeae to eukaryotic cells grown in tissue culture was analyzed by use of light and electron microscopy and by labeling of the bacteria with (/sup 3/H)- and (/sup 14/C)adenine. Isogenic piliated and nonpiliated N. gonorrhoeae from opaque and transparent colonies were studied. The results of light microscopy studies showed that the gonococci attached to cells of human origin, including Flow 2000, HeLa 229, and HEp 2. Studies using radiolabeled gonococci gave comparable results. Piliated N. gonorrhoeae usually attached in larger numbers than nonpiliated organisms, and those from opaque colonies attached more often than isogenic variants frommore » transparent colonies. Day-to-day variation in rate of attachment was observed. Scanning electron microscopy studies showed the gonococcal attachment to be specific for microvilli of the host cells. It is concluded that more N. gonorrhoeae from opaque colonies, as compared with isogenic variants from transparent colonies, attach to eukaryotic cells grown in tissue culture.« less

Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy.

PubMed

Kreplak, Laurent; Richter, Karsten; Aebi, Ueli; Herrmann, Harald

2008-01-01

Intermediate filaments (IFs) were originally discovered and defined by electron microscopy in myoblasts. In the following it was demonstrated and confirmed that they constitute, in addition to microtubules and microfilaments, a third independent, general filament system in the cytoplasm of most metazoan cells. In contrast to the other two systems, IFs are present in cells in two principally distinct cytoskeletal forms: (i) extended and free-running filament arrays in the cytoplasm that are integrated into the cytoskeleton by associated proteins of the plakin type; and (ii) a membrane- and chromatin-bound thin 'lamina' of a more or less regular network of interconnected filaments made from nuclear IF proteins, the lamins, which differ in several important structural aspects from cytoplasmic IF proteins. In man, more than 65 genes code for distinct IF proteins that are expressed during embryogenesis in various routes of differentiation in a tightly controlled manner. IF proteins exhibit rather limited sequence identity implying that the different types of IFs have distinct biochemical properties. Hence, to characterize the structural properties of the various IFs, in vitro assembly regimes have been developed in combination with different visualization methods such as transmission electron microscopy of fixed and negatively stained samples as well as methods that do not use staining such as scanning transmission electron microscopy (STEM) and cryoelectron microscopy as well as atomic force microscopy. Moreover, with the generation of both IF-type specific antibodies and chimeras of fluorescent proteins and IF proteins, it has become possible to investigate the subcellular organization of IFs by correlative fluorescence and electron microscopic methods. The combination of these powerful methods should help to further develop our understanding of nuclear architecture, in particular how nuclear subcompartments are organized and in which way lamins are involved.

Coupling EELS/EFTEM Imaging with Environmental Fluid Cell Microscopy

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

Unocic, Raymond R; Baggetto, Loic; Veith, Gabriel M

2012-01-01

Insight into dynamically evolving electrochemical reactions and mechanisms encountered in electrical energy storage (EES) and conversion technologies (batteries, fuel cells, and supercapacitors), materials science (corrosion and oxidation), and materials synthesis (electrodeposition) remains limited due to the present lack of in situ high-resolution characterization methodologies. Electrochemical fluid cell microscopy is an emerging in-situ method that allows for the direct, real-time imaging of electrochemical processes within a fluid environment. This technique is facilitated by the use of MEMS-based biasing microchip platforms that serve the purpose of sealing the highly volatile electrolyte between two electron transparent SiNx membranes and interfacing electrodes to anmore » external potentiostat for controlled nanoscale electrochemislly experiments [!]. In order to elucidate both stmctural and chemical changes during such in situ electrochemical experiments, it is impmtant to first improve upon the spatial resolution by utilizing energy-filtered transmission electron microscopy (EFTEM) (to minimize chromatic aben ation), then to detennine the chemical changes via electron energy loss spectroscopy (EELS). This presents a formidable challenge since the overall thickness through which electrons are scattered through the multiple layers of the cell can be on the order of hundreds of nanometers to microns, scattering through which has the deleterious effect of degrading image resolution and decreasing signal-to noise for spectroscopy [2].« less

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

PubMed Central

Goode, Angela E.; Skepper, Jeremy N.; Thorley, Andrew J.; Seiffert, Joanna M.; Chung, K. Fan; Tetley, Teresa D.; Shaffer, Milo S. P.; Ryan, Mary P.

2015-01-01

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinised. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data. PMID:25606708

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

Nanoscale live cell optical imaging of the dynamics of intracellular microvesicles in neural cells.

PubMed

Lee, Sohee; Heo, Chaejeong; Suh, Minah; Lee, Young Hee

2013-11-01

Recent advances in biotechnology and imaging technology have provided great opportunities to investigate cellular dynamics. Conventional imaging methods such as transmission electron microscopy, scanning electron microscopy, and atomic force microscopy are powerful techniques for cellular imaging, even at the nanoscale level. However, these techniques have limitations applications in live cell imaging because of the experimental preparation required, namely cell fixation, and the innately small field of view. In this study, we developed a nanoscale optical imaging (NOI) system that combines a conventional optical microscope with a high resolution dark-field condenser (Cytoviva, Inc.) and halogen illuminator. The NOI system's maximum resolution for live cell imaging is around 100 nm. We utilized NOI to investigate the dynamics of intracellular microvesicles of neural cells without immunocytological analysis. In particular, we studied direct, active random, and moderate random dynamic motions of intracellular microvesicles and visualized lysosomal vesicle changes after treatment of cells with a lysosomal inhibitor (NH4Cl). Our results indicate that the NOI system is a feasible, high-resolution optical imaging system for live small organelles that does not require complicated optics or immunocytological staining processes.

Diverse protocols for correlative super-resolution fluorescence imaging and electron microscopy of chemically fixed samples

PubMed Central

Kopek, Benjamin G.; Paez-Segala, Maria G.; Shtengel, Gleb; Sochacki, Kem A.; Sun, Mei G.; Wang, Yalin; Xu, C. Shan; van Engelenburg, Schuyler B.; Taraska, Justin W.; Looger, Loren L.; Hess, Harald F.

2017-01-01

Our groups have recently developed related approaches for sample preparation for super-resolution imaging within endogenous cellular environments using correlative light and electron microscopy (CLEM). Four distinct techniques for preparing and acquiring super-resolution CLEM datasets on aldehyde-fixed specimens are provided, including Tokuyasu cryosectioning, whole-cell mount, cell unroofing and platinum replication, and resin embedding and sectioning. Choice of the best protocol for a given application depends on a number of criteria that are discussed in detail. Tokuyasu cryosectioning is relatively rapid but is limited to small, delicate specimens. Whole-cell mount has the simplest sample preparation but is restricted to surface structures. Cell unroofing and platinum replica creates high-contrast, 3-dimensional images of the cytoplasmic surface of the plasma membrane, but is more challenging than whole-cell mount. Resin embedding permits serial sectioning of large samples, but is limited to osmium-resistant probes, and is technically difficult. Expected results from these protocols include super-resolution localization (~10–50 nm) of fluorescent targets within the context of electron microscopy ultrastructure, which can help address cell biological questions. These protocols can be completed in 2–7 days, are compatible with a number of super-resolution imaging protocols, and are broadly applicable across biology. PMID:28384138

Magnetite nanoparticles functionalized with α-tocopheryl succinate (α-TOS) promote selective cervical cancer cell death

NASA Astrophysics Data System (ADS)

Angulo-Molina, Aracely; Méndez-Rojas, Miguel Ángel; Palacios-Hernández, Teresa; Contreras-López, Oscar Edel; Hirata-Flores, Gustavo Alonso; Flores-Alonso, Juan Carlos; Merino-Contreras, Saul; Valenzuela, Olivia; Hernández, Jesús; Reyes-Leyva, Julio

2014-08-01

The vitamin E analog α-tocopheryl succinate (α-TOS) selectively induces apoptosis in several cancer cells, but it is sensitive to esterases present in cervical cancer cells. Magnetite nanoparticles (Nps) were prepared by a reduction-coprecipitation method; their surface was silanized and conjugated to α-TOS to enhance its resistance. Morphology, size, and crystal structure were analyzed by scanning electron microscopy, transmission electron microscopy, and selected area electron diffraction. Chemical composition was analyzed by energy-dispersive X-ray spectroscopy; functional groups were determined by Fourier transform infrared spectroscopy; and α-TOS content was estimated by thermogravimetric analysis. The cytotoxic activity of α-TOS-Nps was evaluated in non-malignant fibroblasts and cervical cancer cells by means of the colorimetric MTT viability test. Intracellular localization was identified by confocal laser scanning microscopy. Characterization of α-TOS-Nps revealed sphere-like Nps with 15 nm average size, formed by mineral and organic constituents with high stability. α-TOS-Nps were internalized in the nucleus and selectively affected the viability of cervical cancer cells in a dose- and time-dependent manner but were biocompatible with non-malignant fibroblasts. In conclusion, functionalization of magnetite Nps protected the cytotoxic activity of α-TOS in non-sensitive cervical cancer cells.

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

PubMed Central

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; Perez-Gonzalez, Teresa; Faivre, Damien; Trubitsyn, Denis; Bazylinski, Dennis A.; Prozorov, Tanya

2014-01-01

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip window surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria. PMID:25358460

Correlative Electron and Fluorescence Microscopy of Magnetotactic Bacteria in Liquid: Toward In Vivo Imaging

DOE PAGES

Woehl, Taylor J.; Kashyap, Sanjay; Firlar, Emre; ...

2014-10-31

Magnetotactic bacteria biomineralize ordered chains of uniform, membrane-bound magnetite or greigite nanocrystals that exhibit nearly perfect crystal structures and species-specific morphologies. Transmission electron microscopy (TEM) is a critical technique for providing information regarding the organization of cellular and magnetite structures in these microorganisms. However, conventional TEM can only be used to image air-dried or vitrified bacteria removed from their natural environment. Here we present a correlative scanning TEM (STEM) and fluorescence microscopy technique for imaging viable cells of Magnetospirillum magneticum strain AMB-1 in liquid using an in situ fluid cell TEM holder. Fluorescently labeled cells were immobilized on microchip windowmore » surfaces and visualized in a fluid cell with STEM, followed by correlative fluorescence imaging to verify their membrane integrity. Notably, the post-STEM fluorescence imaging indicated that the bacterial cell wall membrane did not sustain radiation damage during STEM imaging at low electron dose conditions. We investigated the effects of radiation damage and sample preparation on the bacteria viability and found that approximately 50% of the bacterial membranes remained intact after an hour in the fluid cell, decreasing to ~30% after two hours. These results represent a first step toward in vivo studies of magnetite biomineralization in magnetotactic bacteria.« less

The role of electron irradiation history in liquid cell transmission electron microscopy.

PubMed

Moser, Trevor H; Mehta, Hardeep; Park, Chiwoo; Kelly, Ryan T; Shokuhfar, Tolou; Evans, James E

2018-04-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.

The role of electron irradiation history in liquid cell transmission electron microscopy

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

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC- TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the rolemore » of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

The role of electron irradiation history in liquid cell transmission electron microscopy

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

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

The role of electron irradiation history in liquid cell transmission electron microscopy

PubMed Central

Mehta, Hardeep

2018-01-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides. PMID:29725619

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE PAGES

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo; ...

2018-04-20

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

Nanomorphology of P3HT:PCBM-based absorber layers of organic solar cells after different processing conditions analyzed by low-energy scanning transmission electron microscopy.

PubMed

Pfaff, Marina; Klein, Michael F G; Müller, Erich; Müller, Philipp; Colsmann, Alexander; Lemmer, Uli; Gerthsen, Dagmar

2012-12-01

In this study the nanomorphology of P3HT:PC61BM absorber layers of organic solar cells was studied as a function of the processing parameters and for P3HT with different molecular weight. For this purpose we apply scanning transmission electron microscopy (STEM) at low electron energies in a scanning electron microscope. This method exhibits sensitive material contrast in the high-angle annular dark-field (HAADF) mode, which is well suited to distinguish materials with similar densities and mean atomic numbers. The images taken with low-energy HAADF STEM are compared with conventional transmission electron microscopy and atomic force microscopy images to illustrate the capabilities of the different techniques. For the interpretation of the low-energy HAADF STEM images, a semiempirical equation is used to calculate the image intensities. The experiments show that the nanomorphology of the P3HT:PC61BM blends depends strongly on the molecular weight of the P3HT. Low-molecular-weight P3HT forms rod-like domains during annealing. In contrast, only small globular features are visible in samples containing high-molecular-weight P3HT, which do not change significantly after annealing at 150°C up to 30 min.

Synthesis, Characterization, and Application of Gold Nanoparticles in Green Nanochemistry Dye-Sensitized Solar Cells

DTIC Science & Technology

2012-06-01

resolution tunneling electron microscopy (HR-TEM). 2.4 DSSC Assembly Annealed TiO2 nanoparticle photoanodes were placed into 10 mL each of the blackberry ...resolution tunneling electron microscopy, and ultraviolet-visible spectroscopy. After characterization, the NPs were found to vary in shape but had... Blackberry Anthocyanin Extraction Procedure ...............................................................3 2.3 Au Nanoparticle Synthesis

Standardized Polyalthia longifolia leaf extract (PLME) inhibits cell proliferation and promotes apoptosis: The anti-cancer study with various microscopy methods.

PubMed

Vijayarathna, Soundararajan; Chen, Yeng; Kanwar, Jagat R; Sasidharan, Sreenivasan

2017-07-01

Over the years a number of microscopy methods have been developed to assess the changes in cells. Some non-invasive techniques such as holographic digital microscopy (HDM), which although does not destroy the cells, but helps to monitor the events that leads to initiation of apoptotic cell death. In this study, the apoptogenic property and the cytotoxic effect of P. longifolia leaf methanolic extract (PLME) against the human cervical carcinoma cells (HeLa) was studied using light microscope (LM), holographic digital microscopy (HDM), scanning electron microscope (SEM) and transmission electron microscope (TEM). The average IC 50 value of PLME against HeLa cells obtained by MTT and CyQuant assay was 22.00μg/mL at 24h. However, noncancerous Vero cells tested with PLME exhibited no cytotoxicity with the IC 50 value of 51.07μg/mL at 24h by using MTT assay. Cytological observations showed nuclear condensation, cell shrinkage, multinucleation, abnormalities of mitochondrial cristae, membrane blebbing, disappearance of microvilli and filopodia, narrowing of lamellipodia, holes, formation of numerous smaller vacuoles, cytoplasmic extrusions and formation of apoptotic bodies as confirmed collectively by HDM, LM, SEM and TEM. In conclusion, PLME was able to produce distinctive morphological features of HeLa cell death that corresponds to apoptosis. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Cytotoxicity of nickel zinc ferrite nanoparticles on cancer cells of epithelial origin.

PubMed

Al-Qubaisi, Mothanna Sadiq; Rasedee, Abdullah; Flaifel, Moayad Husein; Ahmad, Sahrim H J; Hussein-Al-Ali, Samer; Hussein, Mohd Zobir; Eid, Eltayeb E M; Zainal, Zulkarnain; Saeed, Mohd; Ilowefah, Muna; Fakurazi, Sharida; Mohd Isa, Norhaszalina; El Zowalaty, Mohamed Ezzat

2013-01-01

In this study, in vitro cytotoxicity of nickel zinc (NiZn) ferrite nanoparticles against human colon cancer HT29, breast cancer MCF7, and liver cancer HepG2 cells was examined. The morphology, homogeneity, and elemental composition of NiZn ferrite nanoparticles were investigated by scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy, respectively. The exposure of cancer cells to NiZn ferrite nanoparticles (15.6-1,000 μg/mL; 72 hours) has resulted in a dose-dependent inhibition of cell growth determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The quantification of caspase-3 and -9 activities and DNA fragmentation to assess the cell death pathway of the treated cells showed that both were stimulated when exposed to NiZn ferrite nanoparticles. Light microscopy examination of the cells exposed to NiZn ferrite nanoparticles demonstrated significant changes in cellular morphology. The HepG2 cells were most prone to apoptosis among the three cells lines examined, as the result of treatment with NiZn nanoparticles. In conclusion, NiZn ferrite nanoparticles are suggested to have potential cytotoxicity against cancer cells.

Cytotoxicity of nickel zinc ferrite nanoparticles on cancer cells of epithelial origin

PubMed Central

Al-Qubaisi, Mothanna Sadiq; Rasedee, Abdullah; Flaifel, Moayad Husein; Ahmad, Sahrim HJ; Hussein-Al-Ali, Samer; Hussein, Mohd Zobir; Eid, Eltayeb EM; Zainal, Zulkarnain; Saeed, Mohd; Ilowefah, Muna; Fakurazi, Sharida; Isa, Norhaszalina Mohd; Zowalaty, Mohamed Ezzat El

2013-01-01

In this study, in vitro cytotoxicity of nickel zinc (NiZn) ferrite nanoparticles against human colon cancer HT29, breast cancer MCF7, and liver cancer HepG2 cells was examined. The morphology, homogeneity, and elemental composition of NiZn ferrite nanoparticles were investigated by scanning electron microscopy, transmission electron microscopy, and energy dispersive X-ray spectroscopy, respectively. The exposure of cancer cells to NiZn ferrite nanoparticles (15.6–1,000 μg/mL; 72 hours) has resulted in a dose-dependent inhibition of cell growth determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The quantification of caspase-3 and -9 activities and DNA fragmentation to assess the cell death pathway of the treated cells showed that both were stimulated when exposed to NiZn ferrite nanoparticles. Light microscopy examination of the cells exposed to NiZn ferrite nanoparticles demonstrated significant changes in cellular morphology. The HepG2 cells were most prone to apoptosis among the three cells lines examined, as the result of treatment with NiZn nanoparticles. In conclusion, NiZn ferrite nanoparticles are suggested to have potential cytotoxicity against cancer cells. PMID:23885175

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.

The application of polyethylene glycol (PEG) to electron microscopy

PubMed Central

1980-01-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis. PMID:7400222

The application of polyethylene glycol (PEG) to electron microscopy.

PubMed

Wolosewick, J J

1980-08-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine-coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.

Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine.

PubMed

Borba-Santos, Luana Pereira; Ishida, Kelly; Calogeropoulou, Theodora; Souza, Wanderley de; Rozental, Sonia

2016-08-01

Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells.

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

PubMed Central

Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; Shapiro, David; Kirz, Janos; Marchesini, Stefano; Neiman, Aaron M.; Turner, Joshua J.; Jacobsen, Chris

2010-01-01

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11–13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane and freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy. PMID:20368463

High-resolution x-ray diffraction microscopy of specifically labeled yeast cells

DOE PAGES

Nelson, Johanna; Huang, Xiaojing; Steinbrener, Jan; ...

2010-04-20

X-ray diffraction microscopy complements other x-ray microscopy methods by being free of lens-imposed radiation dose and resolution limits, and it allows for high-resolution imaging of biological specimens too thick to be viewed by electron microscopy. We report here the highest resolution (11-13 nm) x-ray diffraction micrograph of biological specimens, and a demonstration of molecular-specific gold labeling at different depths within cells via through-focus propagation of the reconstructed wavefield. The lectin concanavalin A conjugated to colloidal gold particles was used to label the α-mannan sugar in the cell wall of the yeast Saccharomyces cerevisiae. Cells were plunge-frozen in liquid ethane andmore » freeze-dried, after which they were imaged whole using x-ray diffraction microscopy at 750 eV photon energy.« less

[Effects of infrasound therapy on proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells].

PubMed

Bao, Yong; Fan, Jian-Zhong; Li, Ke; Li, Chuan; Yang, Jun-Feng

2008-06-01

To investigate the effect of infrasound therapy on the proliferation, apoptosis and ultrastructure of human B lymphoma Raji cells. Human B lymphoma Raji cells were exposed to infrasound treatment for 15, 30, 60, 90 and 120 min and cultured subsequently for 24 or 48 h. MTT assay, flow cytometry analysis, and electron microscopy were performed to examine the proliferative status, cell apoptosis and ultrastructural changes of the exposed cells, respectively. MTT assay revealed no significant changes in the proliferation of the cells exposed to infrasound treatment (P>0.05), nor did flow cytometry analysis identified significant variation in the cell apoptosis (P>0.05). Scanning electron microscopy, however, identified shortened or reduced cell processes and microvilli on the surface of the cells with infrasound exposure and a subsequent 24-hour culture, and the cell membrane surface became smooth. Under transmission electron microscope, the cells with infrasound treatment presented with significantly reduced microvilli, and the cell nuclei appeared homogeneous, with cytoplasmic budding and losses after a 48-hour culture. Infrasound less than 90 dB does not obviously affect the proliferation and apoptosis of Raji cells, but may directly cause cell ultrastructural changes such as reduction of the cell processes.

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.

Simulation of bone resorption-repair coupling in vitro.

PubMed

Jones, S J; Gray, C; Boyde, A

1994-10-01

In the normal adult human skeleton, new bone formation by osteoblasts restores the contours of bone surfaces following osteoclastic bone resorption, but the evidence for resorption-repair coupling remains circumstantial. To investigate whether sites of prior resorption, more than the surrounding unresorbed surface, attract osteoblasts or stimulate them to proliferate or make new matrix, we developed a simple in vitro system in which resorption-repair coupling occurs. Resorption pits were produced in mammalian dentine or bone slabs by culturing chick bone-derived cells on them for 2-3 days. The chick cells were swept off and the substrata reseeded with rat calvarial osteoblastic cells, which make bone nodules in vitro, for periods of up to 8 weeks. Cell positions and new bone formation were investigated by ordinary light microscopy, fluorescence and reflection confocal laser microscopy, and SEM, in stained and unstained samples. There was no evidence that the osteoblasts were especially attracted to, or influenced by, the sites of resorption in dentine or bone before cell confluence was reached. Bone formation was identified by light microscopy by the accumulation of matrix, staining with alizarin and calcein and by von Kossa's method, and confirmed by scanning electron microscopy (SEM) by using backscattered electron (BSE) and transmitted electron imaging of unembedded samples and BSE imaging of micro-milled embedded material. These new bone patches were located initially in the resorption pits. The model in vitro system may throw new light on the factors that control resorption-repair coupling in the mineralised tissues in vivo.

Microcapsules on Streptococcus mutans serotypes by electron microscopy.

PubMed

Grenier, E M; Gray, R H; Loesche, W J; Eveland, W C

1977-02-01

Extracellular microcapsules have been demonstrated on cells of most serotypes of Streptococcus mutans by electron microscopy, using bacterial strains of the various serotypes and peroxidase labeled or unlabeled immune serum. A correlation was noted between the amount of capsular substance on the strains of S mutans examined and degree of antigenicity as expressed by the indirect fluorescent antibody (FA) title. A serotype d strain was shown to lose both antigenicity as determined by the FA reaction and capsular material as seen by electron microscopy with repeated in vitro passage. When 10% unheated rabbit serum was added to the medium, antigenicity and capsular material were restored.

Antibacterial activity and mode of action of ε-polylysine against Escherichia coli O157:H7.

PubMed

Zhang, Xiaowei; Shi, Ce; Liu, Zuojia; Pan, Fengguang; Meng, Rizeng; Bu, Xiujuan; Xing, Heqin; Deng, Yanhong; Guo, Na; Yu, Lu

2018-04-10

Gram-negative Escherichia coli O157:H7 were chosen as model bacteria to evaluate the antimicrobial mechanism of ε-polylysine (ε-PL). The antibacterial activity of ε-PL was detected by measuring the minimum inhibitory concentration values as well as the time-kill curve. The membrane integrity was determined by ultraviolet (UV) absorption, membrane potential (MP) assay and flow cytometry (FCM) experiments. The permeability of the inner membrane was detected by β-galactosidase activity assay. Furthermore, electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)] was utilized to observe bacterial morphology. These results demonstrated that ε-PL showed its antibacterial activity by changing the integrity and permeability of cell membranes, leading to rapid cell death. The electron microscopy analysis (SEM and TEM) results indicated that the bacterial cell morphology, membrane integrity and permeability were spoiled when the E. coli O157:H7 cells were exposed to minimum inhibitory concentrations of ε-PL (16 µg ml -1 ). In addition, the bacterial membrane was damaged more severely when the concentration of ε-PL was increased. The present study investigated the antimicrobial mechanism of ε-PL by measuring the content of cytoplasmic β-galactosidase, proteins and DNA. In addition, SEM and TEM were carried out to assess the mechanism. These results show that ε-PL has the ability to decrease the content of large molecules, cellular soluble proteins and nucleic acids associated with increasing the content of cytoplasmic β-galactosidase in supernatant by causing damage to the cell membranes. Consequently, the use of ε-PL as a natural antimicrobial agent should eventually become an appealing method in the field of food preservation.

Aquaporin-0 Targets Interlocking Domains to Control the Integrity and Transparency of the Eye Lens

PubMed Central

Lo, Woo-Kuen; Biswas, Sondip K.; Brako, Lawrence; Shiels, Alan; Gu, Sumin; Jiang, Jean X.

2014-01-01

Purpose. Lens fiber cell membranes contain aquaporin-0 (AQP0), which constitutes approximately 50% of the total fiber cell membrane proteins and has a dual function as a water channel protein and an adhesion molecule. Fiber cell membranes also develop an elaborate interlocking system that is required for maintaining structural order, stability, and lens transparency. Herein, we used an AQP0-deficient mouse model to investigate an unconventional adhesion role of AQP0 in maintaining a normal structure of lens interlocking protrusions. Methods. The loss of AQP0 in AQP0−/− lens fibers was verified by Western blot and immunofluorescence analyses. Changes in membrane surface structures of wild-type and AQP0−/− lenses at age 3 to 12 weeks were examined with scanning electron microscopy. Preferential distribution of AQP0 in wild-type fiber cell membranes was analyzed with immunofluorescence and immunogold labeling using freeze-fracturing transmission electron microscopy. Results. Interlocking protrusions in young differentiating fiber cells developed normally but showed minor abnormalities at approximately 50 μm deep in the absence of AQP0 in all ages studied. Strikingly, protrusions in maturing fiber cells specifically underwent uncontrolled elongation, deformation, and fragmentation, while cells still retained their overall shape. Later in the process, these changes eventually resulted in fiber cell separation, breakdown, and cataract formation in the lens core. Immunolabeling at the light microscopy and transmission electron microscopy levels demonstrated that AQP0 was particularly enriched in interlocking protrusions in wild-type lenses. Conclusions. This study suggests that AQP0 exerts its primary adhesion or suppression role specifically to maintain the normal structure of interlocking protrusions that is critical to the integrity and transparency of the lens. PMID:24458158

Transmission and scanning electron microscopy study of the characteristics and morphology of pericytes and novel desmin-immunopositive perivascular cells before and after castration in rat anterior pituitary gland.

PubMed

Jindatip, Depicha; Fujiwara, Ken; Kouki, Tom; Yashiro, Takashi

2012-09-01

Pericytes are perivascular cells associated with microcirculation. Typically, they are localized close to the capillary wall, underneath the basement membrane, and have sparse cytoplasm and poorly developed cell organelles. However, the specific properties of pericytes vary by organ and the conditions within organs. We recently demonstrated that pericytes in rat anterior pituitary gland produce type I and III collagens. The present study attempted to determine the morphological characteristics of these pituitary pericytes. Castrated rats were used as a model of hormonal and vascular changes in the gland. Pericytes, as determined by desmin immunohistochemistry, were more numerous and stained more intensely in castrated rats. Transmission electron microscopy revealed that pituitary pericytes displayed the typical characteristics of pericytes. In pituitary sections from castrated rats, the Golgi apparatus of pericytes was well developed and the rough endoplasmic reticulum was elongated. Additionally, scanning electron microscopy revealed four pericyte shapes: oval, elongate, triangular, and multiangular. As compared with normal rats, the proportion of oval pericytes was lower, and the proportions of the other three shapes were higher, in castrated rats. These results suggest that pericytes change their fine structure and cell shape in response to hormonal and vascular changes in the anterior pituitary gland. In addition, a novel type of perivascular cell was found by desmin immunoelectron microscopy. The morphological properties of these cells were dissimilar to those of pericytes. The cells were localized in the perivascular space, had no basement membrane, and contained dilated rough endoplasmic reticulum. This new cell type will require further study of its origin and characteristics.

Nanoscale Imaging of Whole Cells Using a Liquid Enclosure and a Scanning Transmission Electron Microscope

PubMed Central

Peckys, Diana B.; Veith, Gabriel M.; Joy, David C.; de Jonge, Niels

2009-01-01

Nanoscale imaging techniques are needed to investigate cellular function at the level of individual proteins and to study the interaction of nanomaterials with biological systems. We imaged whole fixed cells in liquid state with a scanning transmission electron microscope (STEM) using a micrometer-sized liquid enclosure with electron transparent windows providing a wet specimen environment. Wet-STEM images were obtained of fixed E. coli bacteria labeled with gold nanoparticles attached to surface membrane proteins. Mammalian cells (COS7) were incubated with gold-tagged epidermal growth factor and fixed. STEM imaging of these cells resulted in a resolution of 3 nm for the gold nanoparticles. The wet-STEM method has several advantages over conventional imaging techniques. Most important is the capability to image whole fixed cells in a wet environment with nanometer resolution, which can be used, e.g., to map individual protein distributions in/on whole cells. The sample preparation is compatible with that used for fluorescent microscopy on fixed cells for experiments involving nanoparticles. Thirdly, the system is rather simple and involves only minimal new equipment in an electron microscopy (EM) laboratory. PMID:20020038

Electronic properties of conductive pili of the metal-reducing bacterium Geobacter sulfurreducens probed by scanning tunneling microscopy.

PubMed

Veazey, Joshua P; Reguera, Gemma; Tessmer, Stuart H

2011-12-01

The metal-reducing bacterium Geobacter sulfurreducens produces conductive protein appendages known as "pilus nanowires" to transfer electrons to metal oxides and to other cells. These processes can be harnessed for the bioremediation of toxic metals and the generation of electricity in bioelectrochemical cells. Key to these applications is a detailed understanding of how these nanostructures conduct electrons. However, to the best of our knowledge, their mechanism of electron transport is not known. We used the capability of scanning tunneling microscopy (STM) to probe conductive materials with higher spatial resolution than other scanning probe methods to gain insights into the transversal electronic behavior of native, cell-anchored pili. Despite the presence of insulating cellular components, the STM topography resolved electronic molecular substructures with periodicities similar to those reported for the pilus shaft. STM spectroscopy revealed electronic states near the Fermi level, consistent with a conducting material, but did not reveal electronic states expected for cytochromes. Furthermore, the transversal conductance was asymmetric, as previously reported for assemblies of helical peptides. Our results thus indicate that the Geobacter pilus shaft has an intrinsic electronic structure that could play a role in charge transport.

Application of low-energy scanning transmission electron microscopy for the study of Pt-nanoparticle uptake in human colon carcinoma cells.

PubMed

Blank, Holger; Schneider, Reinhard; Gerthsen, Dagmar; Gehrke, Helge; Jarolim, Katharina; Marko, Doris

2014-06-01

High-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) in a scanning electron microscope facilitates the acquisition of images with high chemical sensitivity and high resolution. HAADF STEM at low electron energies is particularly suited to image nanoparticles (NPs) in thin cell sections which are not subjected to poststaining procedures as demonstrated by comparison with bright-field TEM. High membrane contrast is achieved and distinction of NPs with different chemical composition is possible at first sight. Low-energy HAADF STEM was applied to systematically study the uptake of Pt-NPs with a broad size distribution in HT29 colon carcinoma cells as a function of incubation time and incubation temperature. The cellular dose was quantified, that is, the amount and number density of NPs taken up by the cells, as well as the particle-size distribution. The results show a strong dependence of the amount of incubated NPs on the exposure time which can be understood by considering size-dependent diffusion and gravitational settling of the NPs in the cell culture medium.

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.

Water without windows: Evaluating the performance of open cell transmission electron microscopy under saturated water vapor conditions, and assessing its potential for microscopy of hydrated biological specimens.

PubMed

Cassidy, Cathal; Yamashita, Masao; Cheung, Martin; Kalale, Chola; Adaniya, Hidehito; Kuwahara, Ryusuke; Shintake, Tsumoru

2017-01-01

We have performed open cell transmission electron microscopy experiments through pure water vapor in the saturation pressure regime (>0.6 kPa), in a modern microscope capable of sub-Å resolution. We have systematically studied achievable pressure levels, stability and gas purity, effective thickness of the water vapor column and associated electron scattering processes, and the effect of gas pressure on electron optical resolution and image contrast. For example, for 1.3 kPa pure water vapor and 300kV electrons, we report pressure stability of ± 20 Pa over tens of minutes, effective thickness of 0.57 inelastic mean free paths, lattice resolution of 0.14 nm on a reference Au specimen, and no significant degradation in contrast or stability of a biological specimen (M13 virus, with 6 nm body diameter). We have also done some brief experiments to confirm feasibility of loading specimens into an in situ water vapor ambient without exposure to intermediate desiccating conditions. Finally, we have also checked if water experiments had any discernible impact on the microscope performance, and report pertinent vacuum and electron optical data, for reference purposes.

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.

Electron microscopy using the genetically encoded APEX2 tag in cultured mammalian cells

PubMed Central

Martell, Jeffrey D; Deerinck, Thomas J; Lam, Stephanie S; Ellisman, Mark H; Ting, Alice Y

2018-01-01

Electron microscopy (EM) is the premiere technique for high-resolution imaging of cellular ultrastructure. Unambiguous identification of specific proteins or cellular compartments in electron micrographs, however, remains challenging because of difficulties in delivering electron-dense contrast agents to specific subcellular targets within intact cells. We recently reported enhanced ascorbate peroxidase 2 (APEX2) as a broadly applicable genetic tag that generates EM contrast on a specific protein or subcellular compartment of interest. This protocol provides guidelines for designing and validating APEX2 fusion constructs, along with detailed instructions for cell culture, transfection, fixation, heavy-metal staining, embedding in resin, and EM imaging. Although this protocol focuses on EM in cultured mammalian cells, APEX2 is applicable to many cell types and contexts, including intact tissues and organisms, and is useful for numerous applications beyond EM, including live-cell proteomic mapping. This protocol, which describes procedures for sample preparation from cell monolayers and cell pellets, can be completed in 10 d, including time for APEX2 fusion construct validation, cell growth, and solidification of embedding resins. Notably, the only additional steps required relative to a standard EM sample preparation are cell transfection and a 2- to 45-min staining period with 3,3′-diaminobenzidine (DAB) and hydrogen peroxide (H2O2). PMID:28796234

Transmission electron microscopy artifacts in characterization of the nanomaterial-cell interactions.

PubMed

Leung, Yu Hang; Guo, Mu Yao; Ma, Angel P Y; Ng, Alan M C; Djurišić, Aleksandra B; Degger, Natalie; Leung, Frederick C C

2017-07-01

We investigated transmission electron microscopy artifacts obtained using standard sample preparation protocols applied to the investigation of Escherichia coli cells exposed to common nanomaterials, such as TiO 2 , Ag, ZnO, and MgO. While the common protocols for some nanomaterials result only in known issues of nanomaterial-independent generation of anomalous deposits due to fixation and staining, for others, there are reactions between the nanomaterial and chemicals used for post-fixation or staining. Only in the case of TiO 2 do we observe only the known issues of nanomaterial-independent generation of anomalous deposits due to exceptional chemical stability of this material. For the other three nanomaterials, different artifacts are observed. For each of those, we identify causes of the observed problems and suggest alternative sample preparation protocols to avoid artifacts arising from the sample preparation, which is essential for correct interpretation of the obtained images and drawing correct conclusions on cell-nanomaterial interactions. Finally, we propose modified sample preparation and characterization protocols for comprehensive and conclusive investigations of nanomaterial-cell interactions using electron microscopy and for obtaining clear and unambiguous revelation whether the nanomaterials studied penetrate the cells or accumulate at the cell membranes. In only the case of MgO and ZnO, the unambiguous presence of Zn and Mg could be observed inside the cells.

The actin cytoskeleton in whole mount preparations and sections.

PubMed

Resch, Guenter P; Urban, Edit; Jacob, Sonja

2010-01-01

In non-muscle cells, the actin cytoskeleton plays a key role by providing a scaffold contributing to the definition of cell shape, force for driving cell motility, cytokinesis, endocytosis, and propulsion of pathogens, as well as tracks for intracellular transport. A thorough understanding of these processes requires insight into the spatial and temporal organisation of actin filaments into diverse higher-order structures, such as networks, parallel bundles, and contractile arrays. Transmission and scanning electron microscopy can be used to visualise the actin cytoskeleton, but due to the delicate nature of actin filaments, they are easily affected by standard preparation protocols, yielding variable degrees of ultrastructural preservation. In this chapter, we describe different conventional and cryo-approaches to visualise the actin cytoskeleton using transmission electron microscopy and discuss their specific advantages and drawbacks. In the first part, we present three different whole mount techniques, which allow visualisation of actin in the peripheral, thinly spread parts of cells grown in monolayers. In the second part, we describe specific issues concerning the visualisation of actin in thin sections. Techniques for three-dimensional visualisation of actin, protein localisation, and correlative light and electron microscopy are also included. Copyright © 2010 Elsevier Inc. All rights reserved.

Invasion of vascular cells in vitro by Porphyromonas endodontalis.

PubMed

Dorn, B R; Harris, L J; Wujick, C T; Vertucci, F J; Progulske-Fox, A

2002-04-01

The objective of this study was to determine whether laboratory strains and clinical isolates of microorganisms associated with root canal infections can invade primary cultures of cardiovascular cells. Quantitative levels of bacterial invasion of human coronary artery endothelial cells (HCAEC) and coronary artery smooth muscle cells (CASMC) were measured using a standard antibiotic protection assay. Transmission electron microscopy was used to confirm and visualize internalization within the vascular cells. Of the laboratory and clinical strains tested, only P. endodontalis ATCC 35406 was invasive in an antibiotic protection assay using HCAEC and CASMC. Invasion of P. endodontalis ATCC 35406 was confirmed by transmission electron microscopy. Certain microorganisms associated with endodontic infections are invasive. If bacterial invasion of the vasculature contributes to the pathogenesis of cardiovascular disease, then microorganisms in the pulp chamber represent potential pathogens.

Picoliter Drop-On-Demand Dispensing for Multiplex Liquid Cell Transmission Electron Microscopy

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

Patterson, Joseph P.; Parent, Lucas R.; Cantlon, Joshua

2016-05-03

Abstract Liquid cell transmission electron microscopy (LCTEM) provides a unique insight into the dynamics of nanomaterials in solution. Controlling the addition of multiple solutions to the liquid cell remains a key hurdle in our ability to increase throughput and to study processes dependent on solution mixing including chemical reactions. Here, we report that a piezo dispensing technique allows for mixing of multiple solutions directly within the viewing area. This technique permits deposition of 50 pL droplets of various aqueous solutions onto the liquid cell window, before assembly of the cell in a fully controlled manner. This proof-of-concept study highlights themore » great potential of picoliter dispensing in combination with LCTEM for observing nanoparticle mixing in the solution phase and the creation of chemical gradients.« less

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.

Endocytosis and interaction of poly (amidoamine) dendrimers with Caco-2 cells.

PubMed

Kitchens, Kelly M; Foraker, Amy B; Kolhatkar, Rohit B; Swaan, Peter W; Ghandehari, Hamidreza

2007-11-01

To investigate the internalization and subcellular trafficking of fluorescently labeled poly (amidoamine) (PAMAM) dendrimers in intestinal cell monolayers. PAMAM dendrimers with positive or negative surface charge were conjugated to fluorescein isothiocyanate (FITC) and visualized for colocalization with endocytosis markers using confocal microscopy. Effect of concentration, generation and charge on the morphology of microvilli was observed using transmission electron microscopy. Both cationic and anionic PAMAM dendrimers internalized within 20 min, and differentially colocalized with endocytosis markers clathrin, EEA-1, and LAMP-1. Transmission electron microscopy analysis showed a concentration-, generation- and surface charge-dependent effect on microvilli morphology. These studies provide visual evidence that endocytic mechanism(s) contribute to the internalization and subcellular trafficking of PAMAM dendrimers across the intestinal cells, and that appropriate selection of PAMAM dendrimers based on surface charge, concentration and generation number allows the application of these polymers for oral drug delivery.

Allorecognition triggers autophagy and subsequent necrosis in the cnidarian Hydractinia symbiolongicarpus.

PubMed

Buss, Leo W; Anderson, Christopher; Westerman, Erica; Kritzberger, Chad; Poudyal, Monita; Moreno, Maria A; Lakkis, Fadi G

2012-01-01

Transitory fusion is an allorecognition phenotype displayed by the colonial hydroid Hydractinia symbiolongicarpus when interacting colonies share some, but not all, loci within the allorecognition gene complex (ARC). The phenotype is characterized by an initial fusion followed by subsequent cell death resulting in separation of the two incompatible colonies. We here characterize this cell death process using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and continuous in vivo digital microscopy. These techniques reveal widespread autophagy and subsequent necrosis in both colony and grafted polyp assays. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays and ultrastructural observations revealed no evidence of apoptosis. Pharmacological inhibition of autophagy using 3-methyladenine (3-MA) completely suppressed transitory fusion in vivo in colony assays. Rapamycin did not have a significant effect in the same assays. These results establish the hydroid allorecognition system as a novel model for the study of cell death.

Electron Microscopy of Staphylococcus aureus Cell Wall Lysis

PubMed Central

Virgilio, R.; González, C.; Muñoz, Nubia; Mendoza, Silvia

1966-01-01

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018–2024. 1966.—A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents. Images PMID:5939482

Electron microscopy of Staphylococcus aureus cell wall lysis.

PubMed

Virgilio, R; González, C; Muñoz, N; Mendoza, S

1966-05-01

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.

Unraveling micro- and nanoscale degradation processes during operation of high-temperature polymer-electrolyte-membrane fuel cells

NASA Astrophysics Data System (ADS)

Hengge, K.; Heinzl, C.; Perchthaler, M.; Varley, D.; Lochner, T.; Scheu, C.

2017-10-01

The work in hand presents an electron microscopy based in-depth study of micro- and nanoscale degradation processes that take place during the operation of high-temperature polymer-electrolyte-membrane fuel cells (HT-PEMFCs). Carbon supported Pt particles were used as cathodic catalyst material and the bimetallic, carbon supported Pt/Ru system was applied as anode. As membrane, cross-linked polybenzimidazole was used. Scanning electron microscopy analysis of cross-sections of as-prepared and long-term operated membrane-electrode-assemblies revealed insight into micrometer scale degradation processes: operation-caused catalyst redistribution and thinning of the membrane and electrodes. Transmission electron microscopy investigations were performed to unravel the nanometer scale phenomena: a band of Pt and Pt/Ru nanoparticles was detected in the membrane adjacent to the cathode catalyst layer. Quantification of the elemental composition of several individual nanoparticles and the overall band area revealed that they stem from both anode and cathode catalyst layers. The results presented do not demonstrate any catastrophic failure but rather intermediate states during fuel cell operation and indications to proceed with targeted HT-PEMFC optimization.

Annual Research Report 1 October 1978-30 September 1979.

DTIC Science & Technology

1979-01-01

Roeder, R. G. and Rutter, W. J. Multiple acid polymerases in ribonucleic acid synthesis during sea urchin development. Biochemistry 9: 2543-2554...with ultrastructural transmission electron microscopy (TEM) studies and scanning electron microscopy ( SEM ) stud- ies of lateral ventricular lining and...1I alterations in animals about 100 days after Silastic implantation. SEM studies show flattening and stretching of ependymal cells in the dorsomedial

Biological phosphorus removal in wastewater treatment.

PubMed

Timmerman, M W

1984-09-01

Several commercially available systems claim to remove phosphate biologically from municipal wastewater. Techniques such as scanning electron microscopy, transmission electron microscopy and 31P nuclear magnetic resonance (NMR) have demonstrated that the phosphate removed is stored within bacterial cells as polyphosphate. Acinetobacter species are usually isolated from phosphate-removing systems although there is a great deal of evidence which casts doubt on the exclusive role of these organisms.

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy.

PubMed

Inaga, Sumire; Katsumoto, Tetsuo; Tanaka, Keiichi; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2007-04-01

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

Biomineralization of endolithic microbes in rocks from the McMurdo Dry Valleys of Antarctica: implications for microbial fossil formation and their detection.

PubMed

Wierzchos, Jacek; Sancho, Leopoldo García; Ascaso, Carmen

2005-04-01

In some zones of Antarctica's cold and dry desert, the extinction of cryptoendolithic microorganisms leaves behind inorganic traces of microbial life. In this paper, we examine the transition from live microorganisms, through their decay, to microbial fossils using in situ microscopy (transmission electron microscopy, scanning electron microscopy in back-scattered electron mode) and microanalytical (energy dispersive X-ray spectroscopy) techniques. Our results demonstrate that, after their death, endolithic microorganisms inhabiting Commonwealth Glacier sandstone from the Antarctica McMurdo Dry Valleys become mineralized. In some cases, epicellular deposition of minerals and/or simply filling up of empty moulds by minerals leads to the formation of cell-shaped structures that may be considered biomarkers. The continuous deposition of allochthonous clay minerals and sulfate-rich salts fills the sandstone pores. This process can give rise to microbial fossils with distinguishable cell wall structures. Often, fossilized cell interiors were of a different chemical composition to the mineralized cell walls. We propose that the microbial fossil formation observed was induced by mineral precipitation resulting from inorganic processes occurring after the death of cryptoendolithic microorganisms. Nevertheless, it must have been the organic template that provoked the diffusion of mineral elements and gave rise to their characteristic distribution pattern inside the fossilized cells.

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.

Some personal and historical notes on the utility of "deep-etch" electron microscopy for making cell structure/function correlations.

PubMed

Heuser, John E

2014-11-01

This brief essay talks up the advantages of metal replicas for electron microscopy and explains why they are still the best way to image frozen cells in the electron microscope. Then it explains our approach to freezing, namely the Van Harreveld trick of "slamming" living cells onto a supercold block of metal sprayed with liquid helium at -269ºC, and further talks up this slamming over the alternative of high-pressure freezing, which is much trickier but enjoys greater favor at the moment. This leads me to bemoan the fact that there are not more young investigators today who want to get their hands on electron microscopes and use our approach to get the most "true to life" views of cells out of them with a minimum of hassle. Finally, it ends with a few perspectives on my own career and concludes that, personally, I'm permanently stuck with the view of the "founding fathers" that cell ultrastructure will ultimately display and explain all of cell function, or as Palade said in his Nobel lecture,electron micrographs are "irresistible and half transparent … their meaning buried under only a few years of work," and "reasonable working hypotheses are already suggested by the ultrastructural organization itself." © 2014 Heuser.

Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review.

PubMed

Xing, Fuyong; Yang, Lin

2016-01-01

Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to interobserver variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literature. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast, fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation.

High-resolution imaging of living mammalian cells bound by nanobeads-connected antibodies in a medium using scanning electron-assisted dielectric microscopy

NASA Astrophysics Data System (ADS)

Okada, Tomoko; Ogura, Toshihiko

2017-02-01

Nanometre-scale-resolution imaging technologies for liquid-phase specimens are indispensable tools in various scientific fields. In biology, observing untreated living cells in a medium is essential for analysing cellular functions. However, nanoparticles that bind living cells in a medium are hard to detect directly using traditional optical or electron microscopy. Therefore, we previously developed a novel scanning electron-assisted dielectric microscope (SE-ADM) capable of nanoscale observations. This method enables observation of intact cells in aqueous conditions. Here, we use this SE-ADM system to clearly observe antibody-binding nanobeads in liquid-phase. We also report the successful direct detection of streptavidin-conjugated nanobeads binding to untreated cells in a medium via a biotin-conjugated anti-CD44 antibody. Our system is capable of obtaining clear images of cellular organelles and beads on the cells at the same time. The direct observation of living cells with nanoparticles in a medium allowed by our system may contribute the development of carriers for drug delivery systems (DDS).

Scanning electrochemical microscopy based evaluation of influence of pH on bioelectrochemical activity of yeast cells - Saccharomyces cerevisiae.

PubMed

Ramanavicius, A; Morkvenaite-Vilkonciene, I; Kisieliute, A; Petroniene, J; Ramanaviciene, A

2017-01-01

In this research scanning electrochemical microscopy was applied for the investigation of immobilized yeast Saccharomyces cerevisiae cells. Two redox mediators based system was applied in order to increase the efficiency of charge transfer from yeast cells. 9,10-phenanthrenequinone (PQ) was applied as a lipophilic redox mediator, which has the ability to cross the cell's membrane; another redox mediator was ferricyanide, which acted as a hydrophylic electron acceptor able to transfer electrons from the PQ to the working electrode of SECM. Hill's function was applied to determine the optimal pH for this described SECM-based system. The influence of pH on cell viability could be well described by Hill's function. It was determined that at pH 6.5 the PQ has a minimal toxic influence on yeast cells, and the kinetics of metabolic processes in cells as well as electron transfer rate achieved in consecutive action of both redox mediators were appropriate to achieve optimal current signals. Copyright © 2016 Elsevier B.V. All rights reserved.

Correlating microscopy techniques and ToF-SIMS analysis of fully grown mammalian oocytes.

PubMed

Gulin, Alexander; Nadtochenko, Victor; Astafiev, Artyom; Pogorelova, Valentina; Rtimi, Sami; Pogorelov, Alexander

2016-06-20

The 2D-molecular thin film analysis protocol for fully grown mice oocytes is described using an innovative approach. Time-of-flight secondary ion mass spectrometry (ToF-SIMS), scanning electron microscopy (SEM), atomic force microscopy (AFM) and optical microscopy imaging were applied to the same mice oocyte section on the same sample holder. A freeze-dried mice oocyte was infiltrated into embedding media, e.g. Epon, and then was cut with a microtome and 2 μm thick sections were transferred onto an ITO coated conductive glass. Mammalian oocytes can contain "nucleolus-like body" (NLB) units and ToF-SIMS analysis was used to investigate the NLB composition. The ion-spatial distribution in the cell components was identified and compared with the images acquired by SEM, AFM and optical microscopy. This study presents a significant advancement in cell embryology, cell physiology and cancer-cell biochemistry.

Ultra-structural study of insulin granules in pancreatic β-cells of db/db mouse by scanning transmission electron microscopy tomography.

PubMed

Xue, Yanhong; Zhao, Wei; Du, Wen; Zhang, Xiang; Ji, Gang; Ying, Wang; Xu, Tao

2012-07-01

Insulin granule trafficking is a key step in the secretion of glucose-stimulated insulin from pancreatic β-cells. The main feature of type 2 diabetes (T2D) is the failure of pancreatic β-cells to secrete sufficient amounts of insulin to maintain normal blood glucose levels. In this work, we developed and applied tomography based on scanning transmission electron microscopy (STEM) to image intact insulin granules in the β-cells of mouse pancreatic islets. Using three-dimensional (3D) reconstruction, we found decreases in both the number and the grey level of insulin granules in db/db mouse pancreatic β-cells. Moreover, insulin granules were closer to the plasma membrane in diabetic β-cells than in control cells. Thus, 3D ultra-structural tomography may provide new insights into the pathology of insulin secretion in T2D.

Use of scanning electron microscopy to monitor nanofibre/cell interaction in digestive epithelial cells.

PubMed

Millaku, Agron; Drobne, Damjana; Torkar, Matjaz; Novak, Sara; Remškar, Maja; Pipan-Tkalec, Živa

2013-09-15

We provide data obtained by scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) on the interaction of ingested tungsten nanofibers with epithelial cells of the digestive tubes of a test organism Porcellio scaber. Conventional toxicity endpoints including feeding behaviour, weight loss and mortality were also measured in each investigated animal. No toxicity was detected in any of exposed animals after 14 days of feeding on tungsten nanofiber dosed food, but when nanofibers enter the digestive system they can react with epithelial cells of the digestive tubes, becoming physically inserted into the cells. In this way, nanofibers can injure the epithelial cells of digestive gland tubes when they are ingested with food. Our SEM data suggest that peristaltic forces may have an important role, not predicted by in vitro experiments, in the interactions of nanomaterials with digestive intestinal cells. Copyright © 2013 Elsevier B.V. All rights reserved.

Multilamellar Structures and Filament Bundles Are Found on the Cell Surface during Bunyavirus Egress

PubMed Central

Sanz-Sánchez, Laura; Risco, Cristina

2013-01-01

Inside cells, viruses build specialized compartments for replication and morphogenesis. We observed that virus release associates with specific structures found on the surface of mammalian cells. Cultured adherent cells were infected with a bunyavirus and processed for oriented sectioning and transmission electron microscopy. Imaging of cell basal regions showed sophisticated multilamellar structures (MLS) and extracellular filament bundles with attached viruses. Correlative light and electron microscopy confirmed that both MLS and filaments proliferated during the maximum egress of new viruses. MLS dimensions and structure were reminiscent of those reported for the nanostructures on gecko fingertips, which are responsible for the extraordinary attachment capacity of these lizards. As infected cells with MLS were more resistant to detachment than control cells, we propose an adhesive function for these structures, which would compensate for the loss of adherence during release of new virus progeny. PMID:23799021

Comparative analysis of the effect of low-dimensional alumina structures on cell lines L929 and Neuro-2a

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

Fomenko, A. N., E-mail: alserova@ispms.tsc.ru; Korovin, M. S., E-mail: msk@ispms.tsc.ru

The paper presents the toxicity evaluation of nanostructures on the basis of alumina of different shape (nanofibers, nanoplates, nanosheets, nanosheet agglomerates) and with similar physical and chemical properties (particle size, specific surface area, phase composition, and zeta potential). The nanostructures were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity of nanostructures was estimated using L929 fibroblast cells and Neuro-2a tumor cells. It has been found that the L929 cells are less subject to the influence of alumina nanoparticles than the Neuro-2a tumor cells. Probably, themore » differences in the proliferation activity of normal and tumor cells in contact with the synthesized nanostructures are due to a change in the pH of the cell microenvironment.« less

Comparative analysis of the effect of low-dimensional alumina structures on cell lines L929 and Neuro-2a

NASA Astrophysics Data System (ADS)

Fomenko, A. N.; Korovin, M. S.

2016-08-01

The paper presents the toxicity evaluation of nanostructures on the basis of alumina of different shape (nanofibers, nanoplates, nanosheets, nanosheet agglomerates) and with similar physical and chemical properties (particle size, specific surface area, phase composition, and zeta potential). The nanostructures were examined by transmission electron microscopy (TEM), scanning electron microscopy (SEM), X-ray diffraction (XRD), low-temperature nitrogen adsorption, and dynamic light scattering (DLS). The cytotoxicity of nanostructures was estimated using L929 fibroblast cells and Neuro-2a tumor cells. It has been found that the L929 cells are less subject to the influence of alumina nanoparticles than the Neuro-2a tumor cells. Probably, the differences in the proliferation activity of normal and tumor cells in contact with the synthesized nanostructures are due to a change in the pH of the cell microenvironment.

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

Morphological Effect of the New Antifungal Agent ME1111 on Hyphal Growth of Trichophyton mentagrophytes, Determined by Scanning and Transmission Electron Microscopy.

PubMed

Nishiyama, Yayoi; Takahata, Sho; Abe, Shigeru

2017-01-01

The effects of ME1111, a novel antifungal agent, on the hyphal morphology and ultrastructure of Trichophyton mentagrophytes were investigated by using scanning and transmission electron microscopy. Structural changes, such as pit formation and/or depression of the cell surface, and degeneration of intracellular organelles and plasmolysis were observed after treatment with ME1111. Our results suggest that the inhibition of energy production by ME1111 affects the integrity and function of cellular membranes, leading to fungal cell death. Copyright © 2016 American Society for Microbiology.

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.

Cryo-scanning transmission electron tomography of vitrified cells.

PubMed

Wolf, Sharon Grayer; Houben, Lothar; Elbaum, Michael

2014-04-01

Cryo-electron tomography (CET) of fully hydrated, vitrified biological specimens has emerged as a vital tool for biological research. For cellular studies, the conventional imaging modality of transmission electron microscopy places stringent constraints on sample thickness because of its dependence on phase coherence for contrast generation. Here we demonstrate the feasibility of using scanning transmission electron microscopy for cryo-tomography of unstained vitrified specimens (CSTET). We compare CSTET and CET for the imaging of whole bacteria and human tissue culture cells, finding favorable contrast and detail in the CSTET reconstructions. Particularly at high sample tilts, the CSTET signals contain more informative data than energy-filtered CET phase contrast images, resulting in improved depth resolution. Careful control over dose delivery permits relatively high cumulative exposures before the onset of observable beam damage. The increase in acceptable specimen thickness broadens the applicability of electron cryo-tomography.

Ultrastructural Analysis of Different Human Mesenchymal Stem Cells After in Vitro Expansion: A Technical Review

PubMed Central

Danišovič, Ľ.; Majidi, A.; Varga, I.

2015-01-01

Transmission electron microscopy reveals ultrastructural details of cells, and it is a valuable method for studying cell organelles. That is why we used this method for detailed morphological description of different adult tissue-derived stem cells, focusing on the morphological signs of their functions (proteosynthetic activity, exchange with external environment, etc.) and their comparison. Preparing a specimen from the cell culture suitable for transmission electron microscopy is, however, much more challenging than routine tissue processing for normal histological examination. There are several issues that need to be solved while working with cell pellets instead of solid tissue. Here we describe a simple protocol for the isolation and culture of mesenchymal stem cells from different adult tissues, with applications to stem cell biology and regenerative medicine. Since we are working with population of cells that was obtained after many days of passaging, very efficient and gentle procedures are highly necessary. We demonstrated that our semi-conservative approach regarding to histological techniques and processing of cells for transmission electron microscopy is a well reproducible procedure which results in quality pictures and images of cell populations with minimum distortions and artifacts. We also commented about riskiest steps and histochemical issues (e.g., precise pH, temperature) while preparing the specimen. We bring full and detailed procedures of fixation, post-fixation, infiltration, embedding, polymerization and contrasting of cell obtained from in vitro cell and tissue cultures, with modifications according to our experience. All this steps are essential for us to know more about adult stem cells derived from different sources or about other random cell populations. The knowledge about detailed ultra-structure of adult stem cells cultured in vitro are also essential for their using in regenerative medicine and tissue engineering. PMID:26708176

THE CELL CENTERED DATABASE PROJECT: AN UPDATE ON BUILDING COMMUNITY RESOURCES FOR MANAGING AND SHARING 3D IMAGING DATA

PubMed Central

Martone, Maryann E.; Tran, Joshua; Wong, Willy W.; Sargis, Joy; Fong, Lisa; Larson, Stephen; Lamont, Stephan P.; Gupta, Amarnath; Ellisman, Mark H.

2008-01-01

Databases have become integral parts of data management, dissemination and mining in biology. At the Second Annual Conference on Electron Tomography, held in Amsterdam in 2001, we proposed that electron tomography data should be shared in a manner analogous to structural data at the protein and sequence scales. At that time, we outlined our progress in creating a database to bring together cell level imaging data across scales, The Cell Centered Database (CCDB). The CCDB was formally launched in 2002 as an on-line repository of high-resolution 3D light and electron microscopic reconstructions of cells and subcellular structures. It contains 2D, 3D and 4D structural and protein distribution information from confocal, multiphoton and electron microscopy, including correlated light and electron microscopy. Many of the data sets are derived from electron tomography of cells and tissues. In the five years since its debut, we have moved the CCDB from a prototype to a stable resource and expanded the scope of the project to include data management and knowledge engineering. Here we provide an update on the CCDB and how it is used by the scientific community. We also describe our work in developing additional knowledge tools, e.g., ontologies, for annotation and query of electron microscopic data. PMID:18054501

Biocompatibility enhancement of rare earth magnesium alloy by laser surface processing

NASA Astrophysics Data System (ADS)

Nie, Shilin; Wang, Yuqing; Liu, Haifeng; Guan, Yingchun

2018-01-01

Although magnesium and magnesium alloys are considered biocompatible and biodegradable, insufficient biocompatibility in body fluid environment is still the major drawback of magnesium alloys for their successful applications as biodegradable orthopaedic implants. In this work, magnesium alloy surface with both enhanced corrosion resistance and better cell adhesion property was directly fabricated by laser surface processing. Laser surface melting was used to improve corrosion resistance of Mg-6Gd-0.6Ca alloy. After laser surface melting, laser surface texturing was utilized on melted surface for better cell adhesion property. The corrosion resistance of laser-treated and as-received samples were evaluated using electrochemical technique. The effect of laser surface treatment on phase and microstructure evolution was evaluated using scanning electron microscopy, optical microscopy and X-ray diffraction. This work investigated the effect of laser treatment on cell distribution across the surface of magnesium alloy substrates. Osteoblast was cultured on the laser-treated surface and as-received surface. Cell morphology was observed with a scanning electron microscopy, and cell viability was evaluated by optical density measurement.

Fungus-mediated biological synthesis of gold nanoparticles: potential in detection of liver cancer

PubMed Central

Chauhan, Arun; Zubair, Swaleha; Tufail, Saba; Sherwani, Asif; Sajid, Mohammad; Raman, Suri C; Azam, Amir; Owais, Mohammad

2011-01-01

Background Nanomaterials are considered to be the pre-eminent component of the rapidly advancing field of nanotechnology. However, developments in the biologically inspired synthesis of nanoparticles are still in their infancy and consequently attracting the attention of material scientists throughout the world. Keeping in mind the fact that microorganism-assisted synthesis of nanoparticles is a safe and economically viable prospect, in the current study we report Candida albicans-mediated biological synthesis of gold nanoparticles. Methods and results Transmission electron microscopy, atomic force microscopy, and various spectrophotometric analyses were performed to characterize the gold nanoparticles. The morphology of the synthesized gold particles depended on the abundance of C. albicans cytosolic extract. Transmission electron microscopy, nanophox particle analysis, and atomic force microscopy revealed the size of spherical gold nanoparticles to be in the range of 20–40 nm and nonspherical gold particles were found to be 60–80 nm. We also evaluated the potential of biogenic gold nanoparticles to probe liver cancer cells by conjugating them with liver cancer cell surface-specific antibodies. The antibody-conjugated gold particles were found to bind specifically to the surface antigens of the cancer cells. Conclusion The antibody-conjugated gold particles synthesized in this study could successfully differentiate normal cell populations from cancerous cells. PMID:22072868

Combined Multidimensional Microscopy as a Histopathology Imaging Tool.

PubMed

Shami, Gerald J; Cheng, Delfine; Braet, Filip

2017-02-01

Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus, facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterize various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterize the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. J. Cell. Physiol. 232: 249-256, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Surface modification of tantalum pentoxide coatings deposited by magnetron sputtering and correlation with cell adhesion and proliferation in in vitro tests

NASA Astrophysics Data System (ADS)

Zykova, A.; Safonov, V.; Goltsev, A.; Dubrava, T.; Rossokha, I.; Donkov, N.; Yakovin, S.; Kolesnikov, D.; Goncharov, I.; Georgieva, V.

2016-03-01

The effect was analyzed of surface treatment by argon ions on the surface properties of tantalum pentoxide coatings deposited by reactive magnetron sputtering. The structural parameters of the as-deposited coatings were investigated by means of transmission electron microscopy, atomic force microscopy and scanning electron microscopy. X-ray diffraction profiles and X-ray photoelectron spectra were also acquired. The total surface free energy (SFE), the polar, dispersion parts and fractional polarities, were estimated by the Owens-Wendt-Rabel-Kaeble method. The adhesive and proliferative potentials of bone marrow cells were evaluated for both Ta2O5 coatings and Ta2O5 coatings deposited by simultaneous bombardment by argon ions in in vitro tests.

Seeing through walls at the nanoscale: Microwave microscopy of enclosed objects and processes in liquids

DOE PAGES

Velmurugan, Jeyavel; Kalinin, Sergei V.; Kolmakov, Andrei; ...

2016-02-11

Here, noninvasive in situ nanoscale imaging in liquid environments is a current imperative in the analysis of delicate biomedical objects and electrochemical processes at reactive liquid–solid interfaces. Microwaves of a few gigahertz frequencies offer photons with energies of ≈10 μeV, which can affect neither electronic states nor chemical bonds in condensed matter. Here, we describe an implementation of scanning near-field microwave microscopy for imaging in liquids using ultrathin molecular impermeable membranes separating scanning probes from samples enclosed in environmental cells. We imaged a model electroplating reaction as well as individual live cells. Through a side-by-side comparison of the microwave imagingmore » with scanning electron microscopy, we demonstrate the advantage of microwaves for artifact-free imaging.« less

Morphometric, quantitative, and three-dimensional analysis of the heart muscle fibers of old rats: transmission electron microscopy and high-resolution scanning electron microscopy methods.

PubMed

Cury, Diego Pulzatto; Dias, Fernando José; Sosthenes, Marcia Consentino Kronka; Dos Santos Haemmerle, Carlos Alexandre; Ogawa, Koichi; Da Silva, Marcelo Cavenaghi Pereira; Mardegan Issa, João Paulo; Iyomasa, Mamie Mizusaki; Watanabe, Ii-Sei

2013-02-01

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high-resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A-, I-, and H-bands and their M- and Z-lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm(2), a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Copyright © 2012 Wiley Periodicals, Inc.

A simple method for environmental cell depressurization for use with an electron microscope.

PubMed

Ogawa, Naoki; Mizokawa, Ryo; Saito, Minoru; Ishikawa, Akira

2017-12-01

With the aid of the environmental cell (EC) in electron microscopy, hydrated specimens have been observed at high resolutions that optical microscopy cannot attain. Due to the ultra-high vacuum conditions of the inner column of the electron microscope, the EC requires sealing films that are sufficiently thin to allow electron transmission and that are sufficiently tough to withstand the pressure difference between the inside and outside of the EC. However, most hydrated specimens can be observed at low vacuum because the saturated vapor pressure of water is known to be 0.02 atm at room temperature. These concepts have been used in the differential pumping system, but it is complicated and relatively expensive. In this work, we propose a simple method for depressurization of the EC using a 'balloon structure' and demonstrate the theoretical benefits and practical improvement for specimen observations in low-vacuum conditions. © The Author 2017. 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.

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

[Electron microscopic study of the An-750 strain of Powassan virus isolated in the Soviet Union].

PubMed

Sobolev, S G; Shestopalova, N M; Linev, M B; Rubin, S G

1978-01-01

Electron microscopic examinations of brains of white mice inoculated with the An 750 strain isolated for the first time from adult mosquitoes and with the prototype LB strain of Powassan virus were carried out. The method of combination of light and electron microscopy used in the study permitted to compare ultrastructural changes in one cell with the results of light microscopy. Sizes of virions and their localizations in the brain cells were determined. Virus particles were found in large and small neurons as well as in glial elements. Subcellular changes in neurons associated with virus multiplication are described. The causes of differences in sizes of virions measured in ultrathin sections are discussed.

Investigating the performance of nitrogen-doped graphene photoanode in dye-sensitized solar cells

NASA Astrophysics Data System (ADS)

Joseph, Easter; Singh, Balbir Singh Mahinder; Mohamed, Norani Muti; Kait, Chong Fai; Saheed, Mohamed Shuaib Mohamed; Khatani, Mehboob

2016-11-01

In this paper, the atmospheric pressure chemical vapor deposition (AP-CVD) is used to synthesize graphene on a copper substrate by utilizing methane as a precursor and N-doped graphene (NDG) in the presence of ammonia. The performance of pure titanium dioxide (TiO2), TiO2/graphene, and TiO2/NDG as photoanodes in dye-sensitized solar cell (DSSC) were compared. Field emission scanning electron microscopy (FESEM) and transmission electron microscopy (TEM) showed flakes of few layers with an interrupted layer in both graphene and NDG. DSSC consist of TiO2/NDG photoanode exhibits a better enhancement due to the high conductivity of donor N in graphene which enhances the electron transportation across nanoporous TiO2.

Microtubules in Plant Cells: Strategies and Methods for Immunofluorescence, Transmission Electron Microscopy and Live Cell Imaging

PubMed Central

Celler, Katherine; Fujita, Miki; Kawamura, Eiko; Ambrose, Chris; Herburger, Klaus; Wasteneys, Geoffrey O.

2016-01-01

Microtubules are required throughout plant development for a wide variety of processes, and different strategies have evolved to visualize and analyze them. This chapter provides specific methods that can be used to analyze microtubule organization and dynamic properties in plant systems and summarizes the advantages and limitations for each technique. We outline basic methods for preparing samples for immunofluorescence labelling, including an enzyme-based permeabilization method, and a freeze-shattering method, which generates microfractures in the cell wall to provide antibodies access to cells in cuticle-laden aerial organs such as leaves. We discuss current options for live cell imaging of MTs with fluorescently tagged proteins (FPs), and provide chemical fixation, high pressure freezing/freeze substitution, and post-fixation staining protocols for preserving MTs for transmission electron microscopy and tomography. PMID:26498784

Plastic protein microarray to investigate the molecular pathways of magnetic nanoparticle-induced nanotoxicity

NASA Astrophysics Data System (ADS)

Liu, Yingshuai; Li, Xuelian; Bao, Shujuan; Lu, Zhisong; Li, Qing; Li, Chang Ming

2013-05-01

Superparamagnetic iron oxide nanoparticles (SPIONs) (about 15 nm) were synthesized via a hydrothermal method and characterized by field emission scanning electron microscopy, transmission electron microscopy, dynamic light scattering, x-ray diffraction, and vibrating sample magnetometer. The molecular pathways of SPIONs-induced nanotoxicity was further investigated by protein microarrays on a plastic substrate from evaluation of cell viability, reactive oxygen species (ROS) generation and cell apoptosis. The experimental results reveal that 50 μg ml-1 or higher levels of SPIONs cause significant loss of cell viability, considerable generation of ROS and cell apoptosis. It is proposed that high level SPIONs could induce cell apoptosis via a mitochondria-mediated intrinsic pathway by activation of caspase 9 and caspase 3, an increase of the Bax/Bcl-2 ratio, and down-regulation of HSP70 and HSP90 survivor factors.

Germination and Outgrowth of Single Spores of Saccharomyces cerevisiae Viewed by Scanning Electron and Phase-Contrast Microscopy

PubMed Central

Rousseau, Paul; Halvorson, Harlyn O.; Bulla, Lee A.; Julian, Grant St.

1972-01-01

Single spores of Saccharomyces cerevisiae were examined during germination and outgrowth by scanning electron and phase-contrast microscopy. Also determined were changes in cell weight and light absorbance, trehalose utilization, and synthesis of protein and KOH-soluble carbohydrates. These studies reveal that development of the vegetative cell from a spore follows a definite sequence of events involving dramatic physical and chemical modifications. These changes are: initial rapid loss in cellular absorbance followed later by an abrupt gain in absorbance; reduction in cell weight and a subsequent progressive increase; modification of the spore surface with concomitant diminution in refractility; elongation of the cell and augmentation of surface irregularities; rapid decline in trehalose content of the cell accompanied by extensive formation of KOH-soluble carbohydrates; and bud formation. Images PMID:4551750

Structural, Morphological, and Electron Transport Studies of Annealing Dependent In2O3 Dye-Sensitized Solar Cell

PubMed Central

Mahalingam, S.; Abdullah, H.; Shaari, S.; Muchtar, A.; Asshari, I.

2015-01-01

Indium oxide (In2O3) thin films annealed at various annealing temperatures were prepared by using spin-coating method for dye-sensitized solar cells (DSSCs). The objective of this research is to enhance the photovoltaic conversion efficiency in In2O3 thin films by finding the optimum annealing temperature and also to study the reason for high and low performance in the annealed In2O3 thin films. The structural and morphological characteristics of In2O3 thin films were studied via XRD patterns, atomic force microscopy (AFM), field-emission scanning electron microscopy (FESEM), EDX sampling, and transmission electron microscopy (TEM). The annealing treatment modified the nanostructures of the In2O3 thin films viewed through FESEM images. The In2O3-450°C-based DSSC exhibited better photovoltaic performance than the other annealed thin films of 1.54%. The electron properties were studied by electrochemical impedance spectroscopy (EIS) unit. The In2O3-450°C thin films provide larger diffusion rate, low recombination effect, and longer electron lifetime, thus enhancing the performance of DSSC. PMID:26146652

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.

Scanning and transmission electron microscopy of the damage to small intestinal mucosa following X irradiation or hyperthermia

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

Carr, K.E.; Hume, S.P.; Marigold, J.C.

Scanning and transmission electron microscopy (S.E.M. and T.E.M.) and resin histology have been used to investigate the effects on mouse small intestinal villi of heating at 43 degrees C for 20 minutes and of irradiation with 10 Gy X-rays. Damage after irradiation included conical villi and giant cells. Damage after heating included the production of conical and rudimentary villi and the stacking of enterocytes. Individual cells showed signs of abnormalities in their cell membranes, nuclei and cytoplasmic components. The differences in the response after irradiation and hyperthermia are linked to the fact that heating has a primary effect on villousmore » structure, whereas irradiation mainly affects the proliferative pool of crypt cells.« less

Synthesis of CoOOH nanorods and application as coating materials of nickel hydroxide for high temperature Ni-MH cells.

PubMed

Hu, W K; Gao, X P; Geng, M M; Gong, Z X; Noréus, D

2005-03-31

Studies on nanoscale materials have received great interest in both fundamental and applied aspects in recent years. In this letter, we report the synthesis of CoOOH nanorods and their possible applications as coating materials on nickel hydroxide for high-temperature nickel-metal hydride (Ni-MH) cells. The morphology and structure of CoOOH nanorods and coated nickel hydroxide particles are investigated by transmission electron microscopy, X-ray diffraction, and scanning electron microscopy, respectively. The electrochemical properties in the cylindrical AA size Ni-MH cells are evaluated. Our results show that the Ni-MH cells, where the positive electrodes are composed of such nanometer sized CoOOH coatings, have a higher capacity available and good performance at elevated temperatures of >50 degrees C.

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

PubMed

Yamazawa, Toshiko; Nakamura, Naotoshi; Sato, Mari; Sato, Chikara

2016-12-01

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases. © 2016 Wiley Periodicals, Inc.

In Situ Electron Microscopy of Lactomicroselenium Particles in Probiotic Bacteria.

PubMed

Nagy, Gabor; Pinczes, Gyula; Pinter, Gabor; Pocsi, Istvan; Prokisch, Jozsef; Banfalvi, Gaspar

2016-06-30

Electron microscopy was used to test whether or not (a) in statu nascendi synthesized, and in situ measured, nanoparticle size does not differ significantly from the size of nanoparticles after their purification; and (b) the generation of selenium is detrimental to the bacterial strains that produce them. Elemental nano-sized selenium produced by probiotic latic acid bacteria was used as a lactomicroselenium (lactomicroSel) inhibitor of cell growth in the presence of lactomicroSel, and was followed by time-lapse microscopy. The size of lactomicroSel produced by probiotic bacteria was measured in situ and after isolation and purification. For these measurements the TESLA BS 540 transmission electron microscope was converted from analog (aTEM) to digital processing (dTEM), and further to remote-access internet electron microscopy (iTEM). Lactobacillus acidophilus produced fewer, but larger, lactomicroSel nanoparticles (200-350 nm) than Lactobacillus casei (L. casei), which generated many, smaller lactomicroSel particles (85-200 nm) and grains as a cloudy, less electrodense material. Streptococcus thermophilus cells generated selenoparticles (60-280 nm) in a suicidic manner. The size determined in situ in lactic acid bacteria was significantly lower than those measured by scanning electron microscopy after the isolation of lactomicroSel particles obtained from lactobacilli (100-500 nm), but higher relative to those isolated from Streptococcus thermopilus (50-100 nm). These differences indicate that smaller lactomicroSel particles could be more toxic to the producing bacteria themselves and discrepancies in size could have implications with respect to the applications of selenium nanoparticles as prebiotics.

New advances in scanning microscopy and its application to study parasitic protozoa.

PubMed

de Souza, Wanderley; Attias, Marcia

2018-07-01

Scanning electron microscopy has been used to observe and study parasitic protozoa for at least 40 years. However, field emission electron sources, as well as improvements in lenses and detectors, brought the resolution power of scanning electron microscopes (SEM) to a new level. Parallel to the refinement of instruments, protocols for preservation of the ultrastructure, immunolabeling, exposure of cytoskeleton and inner structures of parasites and host cells were developed. This review is focused on protozoan parasites of medical and veterinary relevance, e.g., Toxoplasma gondii, Tritrichomonas foetus, Giardia intestinalis, and Trypanosoma cruzi, compilating the main achievements in describing the fine ultrastructure of their surface, cytoskeleton and interaction with host cells. Two new resources, namely, Helium Ion Microscopy (HIM) and Slice and View, using either Focused Ion Beam (FIB) abrasion or Microtome Serial Sectioning (MSS) within the microscope chamber, combined to backscattered electron imaging of fixed (chemically or by quick freezing followed by freeze substitution and resin embedded samples is bringing an exponential amount of valuable information. In HIM there is no need of conductive coating and the depth of field is much higher than in any field emission SEM. As for FIB- and MSS-SEM, high resolution 3-D models of areas and volumes larger than any other technique allows can be obtained. The main results achieved with all these technological tools and some protocols for sample preparation are included in this review. In addition, we included some results obtained with environmental/low vacuum scanning microscopy and cryo-scanning electron microscopy, both promising, but not yet largely employed SEM modalities. Copyright © 2018. Published by Elsevier Inc.

Analytical electron microscopy as a powerful tool in plant cell biology: examples using electron energy loss spectroscopy and X-ray microanalysis.

PubMed

Lichtenberger, O; Neumann, D

1997-08-01

Energy filtering transmission electron microscopy in combination with energy dispersive X-ray analysis (EDX) and quantumchemical calculations opens new possibilities for elemental and bone analysis at the ultrastructural level. The possibilities and limitations of these methods, applied to botanical samples, are discussed and some examples are given. Ca-oxalate crystals in plant cell vacuoles show a specific C K-edge in the electron energy loss spectrum (EELS), which allows a more reliable identification than light microscopical or cytochemical methods. In some dicots crystalline inclusions can be observed in different cell compartments, which are identified as silicon dioxide or calcium silicate by the fine structure of the Si L2,3-edge. Their formation is discussed on the basis of EEL-spectra and quantumchemical calculations. Examples concerning heavy metal detoxification are given for some tolerant plants. In Minuartia Zn is bound as Zn-silicate in cell walls; Armeria accumulates Cu in leaf idioblasts by chelation with phenolic compounds and Cd is precipitated as CdS/phytochelatin-complexes in tomato.

Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

PubMed Central

Peddie, Christopher J.; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O׳Toole, Peter; Larijani, Banafshe; Collinson, Lucy M.

2014-01-01

Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. PMID:24637200

Morphological investigations of cells that adhered to the irregular patterned polydimethylsiloxane (PDMS) surface without reagents.

PubMed

Chung, Sung Hee; Min, Junhong

2009-07-01

Polydimethylsiloxane (PDMS) surface consisting irregular pattern was investigated to develop cell-based biochip using PDMS. PDMS surface was modified with nano- and micro-combined patterns using surface deformation technology. Hydrophobicity of nano-patterned PDMS surface was sustained. Nevertheless it has irregular patterns consisting of micro- and nano-patterns. According to atomic force microscopy (AFM), scanning electron microscopy (SEM) and confocal microscopy results by immunostaining method, human mammary epithelial cells (HMEC) adhered well on irregularly patterned surface without any reagents such as gelatin and collagen, compared to commercial culture dish. It implies PDMS material can be utilized as template for cell-based biochip without any reagents.

Adamantylidene-substituted alkylphosphocholine TCAN26 is more active against Sporothrix schenckii than miltefosine

PubMed Central

Borba-Santos, Luana Pereira; Ishida, Kelly; Calogeropoulou, Theodora; de Souza, Wanderley; Rozental, Sonia

2016-01-01

Sporotrichosis is the most frequent subcutaneous mycosis in the world and its increasing incidence has led to the search for new therapeutic options for its treatment. In this study, we demonstrated that three structural analogues of miltefosine (TCAN26, TC19, and TC70) showed inhibitory activity against Sporothrix schenckii sensu stricto and that TCAN26 was more active in vitro than miltefosine against several isolates. Scanning electron microscopy showed that S. schenckii exposure to TCAN26 resulted in cells that were slightly more elongated than untreated cells. Transmission electron microscopy showed that TCAN26 treatment induced loss of the regular cytoplasmic electron-density and altered the cell envelope (disruption of the cell membrane and cell wall, and increased cell wall thickness). Additionally, TCAN26 concentrations required to kill S. schenckii cells were lower than concentrations that were cytotoxic in mammalian cells, and TCAN26 was more selective than miltefosine. Thus, the adamantylidene-substituted alkylphosphocholine TCAN26 is a promising molecule for the development of novel antifungal compounds, although further investigations are required to elucidate the mode of action of TCAN26 in S. schenckii cells. PMID:27581121

Graphene liquid cells for multi-technique analysis of biological cells in water environment

NASA Astrophysics Data System (ADS)

Matruglio, A.; Zucchiatti, P.; Birarda, G.; Marmiroli, B.; D'Amico, F.; Kocabas, C.; Kiskinova, M.; Vaccari, L.

2018-05-01

In-cell exploration of biomolecular constituents is the new frontier of cellular biology that will allow full access to structure-activity correlation of biomolecules, overcoming the limitations imposed by dissecting the cellular milieu. However, the presence of water, which is a very strong IR absorber and incompatible with the vacuum working conditions of all analytical methods using soft x-rays and electrons, poses severe constraint to perform important imaging and spectroscopic analyses under physiological conditions. Recent advances to separate the sample compartment in liquid cell are based on electron and photon transparent but molecular-impermeable graphene membranes. This strategy has opened a unique opportunity to explore technological materials under realistic operation conditions using various types of electron microscopy. However, the widespread of the graphene liquid cell applications is still impeded by the lack of well-established approaches for their massive production. We report on the first preliminary results for the fabrication of reproducible graphene liquid cells appropriate for the analysis of biological specimens in their natural hydrated environment with several crucial analytical techniques, namely FTIR microscopy, Raman spectroscopy, AFM, SEM and TEM.

3-D Ultrastructure of O. tauri: Electron Cryotomography of an Entire Eukaryotic Cell

PubMed Central

Henderson, Gregory P.; Gan, Lu; Jensen, Grant J.

2007-01-01

The hallmark of eukaryotic cells is their segregation of key biological functions into discrete, membrane-bound organelles. Creating accurate models of their ultrastructural complexity has been difficult in part because of the limited resolution of light microscopy and the artifact-prone nature of conventional electron microscopy. Here we explored the potential of the emerging technology electron cryotomography to produce three-dimensional images of an entire eukaryotic cell in a near-native state. Ostreococcus tauri was chosen as the specimen because as a unicellular picoplankton with just one copy of each organelle, it is the smallest known eukaryote and was therefore likely to yield the highest resolution images. Whole cells were imaged at various stages of the cell cycle, yielding 3-D reconstructions of complete chloroplasts, mitochondria, endoplasmic reticula, Golgi bodies, peroxisomes, microtubules, and putative ribosome distributions in-situ. Surprisingly, the nucleus was seen to open long before mitosis, and while one microtubule (or two in some predivisional cells) was consistently present, no mitotic spindle was ever observed, prompting speculation that a single microtubule might be sufficient to segregate multiple chromosomes. PMID:17710148

Enhanced thermal stability of a polymer solar cell blend induced by electron beam irradiation in the transmission electron microscope.

PubMed

Bäcke, Olof; Lindqvist, Camilla; de Zerio Mendaza, Amaia Diaz; Gustafsson, Stefan; Wang, Ergang; Andersson, Mats R; Müller, Christian; Kristiansen, Per Magnus; Olsson, Eva

2017-05-01

We show by in situ microscopy that the effects of electron beam irradiation during transmission electron microscopy can be used to lock microstructural features and enhance the structural thermal stability of a nanostructured polymer:fullerene blend. Polymer:fullerene bulk-heterojunction thin films show great promise for use as active layers in organic solar cells but their low thermal stability is a hindrance. Lack of thermal stability complicates manufacturing and influences the lifetime of devices. To investigate how electron irradiation affects the thermal stability of polymer:fullerene films, a model bulk-heterojunction film based on a thiophene-quinoxaline copolymer and a fullerene derivative was heat-treated in-situ in a transmission electron microscope. In areas of the film that exposed to the electron beam the nanostructure of the film remained stable, while the nanostructure in areas not exposed to the electron beam underwent large phase separation and nucleation of fullerene crystals. UV-vis spectroscopy shows that the polymer:fullerene films are stable for electron doses up to 2000kGy. Copyright © 2016 Elsevier B.V. All rights reserved.

Enhanced thermal stability of a polymer solar cell blend induced by electron beam irradiation in the transmission electron microscope.

PubMed

Bäcke, Olof; Lindqvist, Camilla; de Zerio Mendaza, Amaia Diaz; Gustafsson, Stefan; Wang, Ergang; Andersson, Mats R; Müller, Christian; Kristiansen, Per Magnus; Olsson, Eva

2017-02-01

We show by in situ microscopy that the effects of electron beam irradiation during transmission electron microscopy can be used to lock microstructural features and enhance the structural thermal stability of a nanostructured polymer:fullerene blend. Polymer:fullerene bulk-heterojunction thin films show great promise for use as active layers in organic solar cells but their low thermal stability is a hindrance. Lack of thermal stability complicates manufacturing and influences the lifetime of devices. To investigate how electron irradiation affects the thermal stability of polymer:fullerene films, a model bulk-heterojunction film based on a thiophene-quinoxaline copolymer and a fullerene derivative was heat-treated in-situ in a transmission electron microscope. In areas of the film that exposed to the electron beam the nanostructure of the film remained stable, while the nanostructure in areas not exposed to the electron beam underwent large phase separation and nucleation of fullerene crystals. UV-vis spectroscopy shows that the polymer:fullerene films are stable for electron doses up to 2000kGy. Copyright © 2017 Elsevier B.V. All rights reserved.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.

PubMed

Müller, Andreas; Neukam, Martin; Ivanova, Anna; Sönmez, Anke; Münster, Carla; Kretschmar, Susanne; Kalaidzidis, Yannis; Kurth, Thomas; Verbavatz, Jean-Marc; Solimena, Michele

2017-02-02

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

A national facility for biological cryo-electron microscopy

PubMed Central

Saibil, Helen R.; Grünewald, Kay; Stuart, David I.

2015-01-01

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 of 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. PMID:25615867

Preservation of protein globules and peptidoglycan in the mineralized cell wall of nitrate-reducing, iron(II)-oxidizing bacteria: a cryo-electron microscopy study.

PubMed

Miot, J; Maclellan, K; Benzerara, K; Boisset, N

2011-11-01

Iron-oxidizing bacteria are important actors of the geochemical cycle of iron in modern environments and may have played a key role all over Earth's history. However, in order to better assess that role on the modern and the past Earth, there is a need for better understanding the mechanisms of bacterial iron oxidation and for defining potential biosignatures to be looked for in the geologic record. In this study, we investigated experimentally and at the nanometre scale the mineralization of iron-oxidizing bacteria with a combination of synchrotron-based scanning transmission X-ray microscopy (STXM), scanning transmission electron microscopy (STEM) and cryo-transmission electron microscopy (cryo-TEM). We show that the use of cryo-TEM instead of conventional microscopy provides detailed information of the successive iron biomineralization stages in anaerobic nitrate-reducing iron-oxidizing bacteria. These results suggest the existence of preferential Fe-binding and Fe-oxidizing sites on the outer face of the plasma membrane leading to the nucleation and growth of Fe minerals within the periplasm of these cells that eventually become completely encrusted. In contrast, the septa of dividing cells remain nonmineralized. In addition, the use of cryo-TEM offers a detailed view of the exceptional preservation of protein globules and the peptidoglycan within the Fe-mineralized cell walls of these bacteria. These organic molecules and ultrastructural details might be protected from further degradation by entrapment in the mineral matrix down to the nanometre scale. This is discussed in the light of previous studies on the properties of Fe-organic interactions and more generally on the fossilization of mineral-organic assemblies. © 2011 Blackwell Publishing Ltd.

Water without windows: Evaluating the performance of open cell transmission electron microscopy under saturated water vapor conditions, and assessing its potential for microscopy of hydrated biological specimens

PubMed Central

Yamashita, Masao; Cheung, Martin; Kalale, Chola; Adaniya, Hidehito; Kuwahara, Ryusuke; Shintake, Tsumoru

2017-01-01

We have performed open cell transmission electron microscopy experiments through pure water vapor in the saturation pressure regime (>0.6 kPa), in a modern microscope capable of sub-Å resolution. We have systematically studied achievable pressure levels, stability and gas purity, effective thickness of the water vapor column and associated electron scattering processes, and the effect of gas pressure on electron optical resolution and image contrast. For example, for 1.3 kPa pure water vapor and 300kV electrons, we report pressure stability of ± 20 Pa over tens of minutes, effective thickness of 0.57 inelastic mean free paths, lattice resolution of 0.14 nm on a reference Au specimen, and no significant degradation in contrast or stability of a biological specimen (M13 virus, with 6 nm body diameter). We have also done some brief experiments to confirm feasibility of loading specimens into an in situ water vapor ambient without exposure to intermediate desiccating conditions. Finally, we have also checked if water experiments had any discernible impact on the microscope performance, and report pertinent vacuum and electron optical data, for reference purposes. PMID:29099843

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.

A novel immuno-gold labeling protocol for nanobody-based detection of HER2 in breast cancer cells using immuno-electron microscopy.

PubMed

Kijanka, M; van Donselaar, E G; Müller, W H; Dorresteijn, B; Popov-Čeleketić, D; El Khattabi, M; Verrips, C T; van Bergen En Henegouwen, P M P; Post, J A

2017-07-01

Immuno-electron microscopy is commonly performed with the use of antibodies. In the last decade the antibody fragment indicated as nanobody (VHH or single domain antibody) has found its way to different applications previously done with conventional antibodies. Nanobodies can be selected to bind with high affinity and specificity to different antigens. They are small (molecular weight ca. 15kDa) and are usually easy to produce in microorganisms. Here we have evaluated the feasibility of a nanobody binding to HER2 for application in immuno-electron microscopy. To obtain highest labeling efficiency combined with optimal specificity, different labeling conditions were analysed, which included nanobody concentration, fixation and blocking conditions. The obtained optimal protocol was applied for post-embedment labeling of Tokuyasu cryosections and for pre-embedment labeling of HER2 for fluorescence microscopy and both transmission and scanning electron microscopy. We show that formaldehyde fixation after incubation with the anti-HER2 nanobody, improves labeling intensity. Among all tested blocking agents the best results were obtained with a mixture of cold water fish gelatine and acetylated bovine serum albumin, which prevented a-specific interactions causing background labeling while preserving specific interactions at the same time. In conclusion, we have developed a nanobody-based protocol for immuno-gold labeling of HER2 for Tokuyasu cryosections in TEM as well as for pre-embedment gold labeling of cells for both TEM and SEM. Copyright © 2017. Published by Elsevier Inc.

Adipose‑derived stem cells and hyaluronic acid based gel compatibility, studied in vitro.

PubMed

Guo, Jiayan; Guo, Shu; Wang, Yuxin; Yu, Yanqiu

2017-10-01

Minimally invasive aesthetic and cosmetic procedures have increased in popularity. Injectable dermal fillers provide soft tissue augmentation, improve facial rejuvenation and wrinkles, and correct tissue defects. To investigate the use of adipose‑derived stem cells integrated with a hyaluronic acid based gel as a dermal filler, the present study used cytotoxicity studies, proliferation studies, adipogenic and osteogenic differentiation, apoptosis assays and scanning electron microscopy. Although hyaluronic acid induced low levels of apoptosis in adipose‑derived stem cells, its significantly promoted proliferation of adipose‑derived stem cells. Hyaluronic acid demonstrates little toxicity against adipose‑derived stem cells. Adipose‑derived stem cells were able to differentiate into adipocytes and osteoblasts. Furthermore, scanning electron microscopy revealed that adipose‑derived stem cells maintained intact structures on the surface of hyaluronic acid as well as in it, and demonstrated abundant cell attachments. The present study demonstrated the compatibility of adipose‑derived stem cells and hyaluronic acid based gels in vitro.

Interfacial Ferromagnetism and Exchange Bias in CaRuO3/CaMnO3 Superlattices

DTIC Science & Technology

2012-11-07

microscopy and electron energy loss spectroscopy indicate that the difference in magnitude of the Mn valence states between the center of the CaMnO3 layer...CaMnO3 thickness dependence of the exchange bias field together indicate that the interfacial 1. REPORT DATE (DD-MM-YYYY) 4. TITLE AND SUBTITLE 13...superlattices of CaRuO3/CaMnO3 that arises in one unit cell at the interface. Scanning transmission electron microscopy and electron energy loss

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

PubMed Central

Romero-Brey, Inés; Bartenschlager, Ralf

2015-01-01

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications. PMID:26633469

Precise and economic FIB/SEM for CLEM: with 2 nm voxels through mitosis.

PubMed

Luckner, Manja; Wanner, Gerhard

2018-05-23

A portfolio is presented documenting economic, high-resolution correlative focused ion beam scanning electron microscopy (FIB/SEM) in routine, comprising: (i) the use of custom-labeled slides and coverslips, (ii) embedding of cells in thin, or ultra-thin resin layers for correlative light and electron microscopy (CLEM) and (iii) the claim to reach the highest resolution possible with FIB/SEM in xyz. Regions of interest (ROIs) defined in light microscope (LM), can be relocated quickly and precisely in SEM. As proof of principle, HeLa cells were investigated in 3D context at all stages of the cell cycle, documenting ultrastructural changes during mitosis: nuclear envelope breakdown and reassembly, Golgi degradation and reconstitution and the formation of the midzone and midbody.

Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

PubMed

Romero-Brey, Inés; Bartenschlager, Ralf

2015-12-03

As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

Protective effects of edaravone against cisplatin-induced hair cell damage in zebrafish.

PubMed

Hong, Seok Jin; Im, Gi Jung; Chang, Jiwon; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon Young; Jung, Hak Hyun; Chung, Ah Young; Park, Hae Chul; Choi, June

2013-06-01

Edaravone is known to have a potent free radical scavenging effect. The objective of the present study was to evaluate the effects of edaravone on cisplatin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five day post-fertilization zebrafish larvae were exposed to 1000 μM cisplatin and 50 μM, 100 μM, 250 μM, 500 μM, 750 μM, and 1000 μM concentrations of edaravone for 4h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of the hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Edaravone protected cisplatin-induced hair cell loss of neuromasts (edaravone 750 μM: 8.7 ± 1.5 cells, cisplatin 1000 μM only: 3.7 ± 0.9 cells; n=10, p<0.0001) and decreased the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. Structures of mitochondria and hair cell within neuromasts in ultrastructural analysis were preserved in zebrafish exposed to 1000 μM cisplatin and 750 μM edaravone for 4h. Edaravone attenuated cisplatin-induced hair cell damage in zebrafish. The results of the current study suggest that cisplatin induces apoptosis, and the apoptotic cell death can be prevented by treatment with edaravone in zebrafish. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

Morphological classification of bioaerosols from composting using scanning electron microscopy

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

Tamer Vestlund, A.; FIRA International Ltd., Maxwell Road, Stevenage, Herts SG1 2EW; Al-Ashaab, R.

2014-07-15

Highlights: • Bioaerosols were captured using the filter method. • Bioaerosols were analysed using scanning electron microscope. • Bioaerosols were classified on the basis of morphology. • Single small cells were found more frequently than aggregates and larger cells. • Smaller cells may disperse further than heavier aggregate structures. - Abstract: This research classifies the physical morphology (form and structure) of bioaerosols emitted from open windrow composting. Aggregation state, shape and size of the particles captured are reported alongside the implications for bioaerosol dispersal after release. Bioaerosol sampling took place at a composting facility using personal air filter samplers. Samplesmore » were analysed using scanning electron microscopy. Particles were released mainly as small (<1 μm) single, spherical cells, followed by larger (>1 μm) single cells, with aggregates occurring in smaller proportions. Most aggregates consisted of clusters of 2–3 particles as opposed to chains, and were <10 μm in size. No cells were attached to soil debris or wood particles. These small single cells or small aggregates are more likely to disperse further downwind from source, and cell viability may be reduced due to increased exposure to environmental factors.« less

Chondroptosis in Alkaptonuric Cartilage

PubMed Central

Millucci, Lia; Giorgetti, Giovanna; Viti, Cecilia; Ghezzi, Lorenzo; Gambassi, Silvia; Braconi, Daniela; Marzocchi, Barbara; Paffetti, Alessandro; Lupetti, Pietro; Bernardini, Giulia; Orlandini, Maurizio

2015-01-01

Alkaptonuria (AKU) is a rare genetic disease that affects the entire joint. Current standard of treatment is palliative and little is known about AKU physiopathology. Chondroptosis, a peculiar type of cell death in cartilage, has been so far reported to occur in osteoarthritis, a rheumatic disease that shares some features with AKU. In the present work, we wanted to assess if chondroptosis might also occur in AKU. Electron microscopy was used to detect the morphological changes of chondrocytes in damaged cartilage distinguishing apoptosis from its variant termed chondroptosis. We adopted histological observation together with Scanning Electron Microscopy and Transmission Electron Microscopy to evaluate morphological cell changes in AKU chondrocytes. Lipid peroxidation in AKU cartilage was detected by fluorescence microscopy. Using the above‐mentioned techniques, we performed a morphological analysis and assessed that AKU chondrocytes undergo phenotypic changes and lipid oxidation, resulting in a progressive loss of articular cartilage structure and function, showing typical features of chondroptosis. To the best of our knowledge, AKU is the second chronic pathology, following osteoarthritis, where chondroptosis has been documented. Our results indicate that Golgi complex plays an important role in the apoptotic process of AKU chondrocytes and suggest a contribution of chondroptosis in AKU pathogenesis. These findings also confirm a similarity between osteoarthritis and AKU. J. Cell. Physiol. 230: 1148–1157, 2015. © 2014 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. PMID:25336110

Liquid-phase electron microscopy of molecular drug response in breast cancer cells reveals irresponsive cell subpopulations related to lack of HER2 homodimers.

PubMed

Peckys, Diana B; Korf, Ulrike; Wiemann, Stefan; de Jonge, Niels

2017-08-09

The development of drug resistance in cancer poses a major clinical problem. An example is human epidermal growth factor receptor 2 (HER2) overexpressing breast cancer often treated with anti-HER2 antibody therapies, such as trastuzumab. Since drug resistance is rooted mainly in tumor cell heterogeneity, we examined the drug effect in different subpopulations of SKBR3 breast cancer cells, and compared the results with a drug resistant cell line, HCC1954. Correlative light microscopy and liquid-phase scanning transmission electron microscopy (STEM) were used to quantitatively analyze HER2 responses upon drug binding, whereby many tens of whole cells were imaged. Trastuzumab was found to selectively cross-link and down regulate HER2 homodimers from the plasma membranes of bulk cancer cells. In contrast, HER2 resided mainly as monomers in rare subpopulations of resting- and cancer stem cells (CSCs), and these monomers were not internalized after drug binding. The HER2 distribution was hardly influenced by trastuzumab for the HCC1954 cells. These findings show that resting cells and CSCs are irresponsive to the drug, and thus point towards a molecular explanation behind the origin of drug resistance. This analytical method is broadly applicable to study membrane protein interactions in the intact plasma membrane, while accounting for cell heterogeneity. © 2017 by The American Society for Cell Biology.

Garlic (Allium sativum) feeding impairs Sertoli cell junctional proteins in male Wistar rat testis: microscopy study.

PubMed

Hammami, I; Nahdi, A; Atig, F; El May, A; El May, M V

2016-12-01

Sertoli cell junctions, such as adhesion junction (AJ), gap junction (GJ) and tight junction (TJ), are important for maintaining spermatogenesis. In previous studies, we showed the inhibitory effect of crude garlic (Allium sativum, As) on spermatogenesis and steroidogenesis. The aim of this work was to complete our investigation on the impact of this plant, especially on Sertoli cell junctional proteins (SCJPs). During 1 month, 24 male rats were divided into groups: group control (0% of As) and treated groups fed 5%, 10% and 15% of As. Light and electron microscopy observations were performed to localise junctional proteins: connexin-43, Zona Occluding-1 and N-cadherin (immunohistochemistry) and to describe junctions. We showed that the specific cells involved in the localisation of the SCJP were similar in both control and treated groups, but with different immunoreactivity intensity between them. The electron microscopy observation focused on TJs between Sertoli cells, constituting the blood-testis barrier, showed ultrastructural changes such as fragmentation of TJs between adjacent Sertoli cell membranes and dilatation of rough endoplasmic reticulum saccules giving an aspect of scale to these junctions. We concluded that crude garlic consumption during 1 month induces perturbations on Sertoli cell junctions. These alterations can explain apoptosis in testicular germ cells previously showed. © 2016 Blackwell Verlag GmbH.

Microgravity effects during fertilization, cell division, development, and calcium metabolism in sea urchins

NASA Technical Reports Server (NTRS)

Schatten, Heide

1996-01-01

The overall objectives of this project are to explore the role of microgravity during fertilization, early development, cytoskeletal organization, and skeletal calcium deposition in a model development system: the sea urchin eggs and embryos. While pursuing these objectives, we have also helped to develop, test, and fly the Aquatic Research Facility (ARF) system. Cells were fixed at preselected time points to preserve the structures and organelles of interest with regards to cell biology events during development. The protocols used for the analysis of the results had been developed during the earlier part of this research and were applied for post-flight analysis using light and (immuno)fluorescence microscopy, scanning electron microscopy, and transmission electron microscopy. The structures of interest are: microtubules during fertilization, cell division, and cilia movement; microfilaments during cell surface restructuring and cell division; centrosomes and centrioles during cell division, cell differentiation, and cilia formation and movement; membranes, Golgi, endoplasmic reticulum, mitochondria, and chromosomes at all stages of development; and calcium deposits during spicule formation in late-stage embryos. In addition to further explore aspects important or living in space, several aspects of this research are also aimed at understanding diseases that affect humans on Earth which may be accelerated in space.

Golgi, electron-microscopic and combined Golgi-electron-microscopic studies of the mitral cells in the goldfish olfactory bulb.

PubMed

Oka, Y

1983-04-01

The local neuronal circuitry of goldfish olfactory bulb was analyzed in Golgi preparations combining light- and electron-microscopy, as well as in routinely prepared ultrastructural preparations. Mitral cells were identified with the light-microscope in Golgi-impregnated thick sections according to the following criteria: (1) cell bodies were distributed irregularly in a wide layer between 100 and 200 micrometer from the surface, (2) cell bodies were larger than other neurons (10-20 micrometer in diameter), and (3) the dendrites were directed toward the superficially-located olfactory nerve layer where they ended as highly branched glomerular tufts. These impregnated cells were examined by electron-microscopy in serial section. The results demonstrate synaptic organization in relation to the mitral cells. (1) Glomerular tufts received afferent input from primary olfactory axons which made Gray's Type I synaptic contacts. These dendrites also had reciprocal dendrodendritic synapses with dendrites of certain non-mitral cells. (2) Dendritic shafts of mitral cells made reciprocal dendritic synapses with dendrites of certain non-mitral cells. (3) Cell bodies and their initial axon segments had reciprocal synapses with certain dendrites but occurred infrequently. In reciprocal synapses, the direction of the Gray Type I (asymmetrical) is away from the mitral cell while those with Gray Type II synapses (symmetrical) are toward the mitral cell. Assuming that the type I synapse is excitatory and Type II is inhibitory, these findings explain the electrophysiological demonstration of self-inhibition discharge found in mitral cells.

[A STUDY OF THE ISOLATED BACTERIOPHAGE ΦAB-SP7 ADSORPTION ON THE CELL SURFACE OF THE AZOSPIRILLUM BRASILENSE SP7].

PubMed

Guliy, O I; Karavaeva, O A; Velikov, V A; Sokolov, O I; Pavily, S A; Larionova, O S; Burov, A M; Ignatov, O V

2016-01-01

The bacteriophage ΦAb-Sp7 was isolated from the cells of the Azospirillum brasilense Sp7. The morphology, size of the gram-negative colonies, and range of lytic activity against other strains and species of the genus Azospirillum was tested. The isolated phage DNA was examined using electrophoretic and restriction analysis, and the size of the genome were established. The electron microscopy. resuIts show that the phage (capsid) has a strand-like form. The electron microscopy study of the bacteriophage ΦAb-Sp7 adsorption on the A. brasilense Sp7 bacterial surface was performed.

ELECTRON MICROSCOPIC STUDY OF THE ATPASE ACTIVITY OF THE BAI STRAIN A (MYELOBLASTOSIS) AVIAN TUMOR VIRUS

PubMed Central

Novikoff, Alex B.; de Thé, Guy; Beard, D.; Beard, J. W.

1962-01-01

Thymus glands of chicks with leukemia induced by BAI strain A (myeloblastosis) virus were fixed in cold 4 per cent formaldehyde-sucrose. Frozen sections were incubated in the ATPase medium of Wachstein and Meisel and studied by light microscopy and electron microscopy. The ATPase activity of the virus is localized to the outermost membrane of the virus. The membrane of the blast-like cells of the thymus cortex from which the virus emerges, by budding, also possesses such activity. It appears likely that the outermost membrane of the virus is derived from the plasma membrane of these cells. PMID:13939125

The inhibitory potential of Thai mango seed kernel extract against methicillin-resistant Staphylococcus aureus.

PubMed

Jiamboonsri, Pimsumon; Pithayanukul, Pimolpan; Bavovada, Rapepol; Chomnawang, Mullika T

2011-07-25

Plant extracts are a valuable source of novel antibacterial compounds to combat pathogenic isolates of methicillin-resistant Staphylococcus aureus (MRSA), a global nosocomial infection. In this study, the alcoholic extract from Thai mango (Mangifera indica L. cv. 'Fahlun') seed kernel extract (MSKE) and its phenolic principles (gallic acid, methyl gallate and pentagalloylglucopyranose) demonstrated potent in vitro antibacterial activity against Staphylococcus aureus and 19 clinical MRSA isolates in studies of disc diffusion, broth microdilution and time-kill assays. Electron microscopy studies using scanning electron microscopy and transmission electron microscopy revealed impaired cell division and ultra-structural changes in bacterial cell morphology, including the thickening of cell walls, of microorganisms treated with MSKE; these damaging effects were increased with increasing concentrations of MSKE. MSKE and its phenolic principles enhanced and intensified the antibacterial activity of penicillin G against 19 clinical MRSA isolates by lowering the minimum inhibitory concentration by at least 5-fold. The major phenolic principle, pentagalloylglucopyranose, was demonstrated to be the major contributor to the antibacterial activity of MSKE. These results suggest that MSKE may potentially be useful as an alternative therapeutic agent or an adjunctive therapy along with penicillin G in the treatment of MRSA infections.

Electron microscopy investigations of nanoparticles for cancer diagnostic applications

NASA Astrophysics Data System (ADS)

Koh, Ai Leen

This dissertation concerns electron microscopy characterization of magnetic (MNP) and surface enhanced Raman scattering (SERS) nanoparticles for in-vitro cancer diagnostic applications. Electron microscopy is an essential characterization tool owing to its (sub) nanometer spatial resolution. Structural information about the nanoparticles can be obtained using transmission electron microscopy (TEM), which can in turn be correlated to their physical characteristics. The scanning electron microscope (SEM) has excellent depth of field and can be effectively utilized to obtain high resolution information about nanoparticles binding onto cell surfaces. Part One of this thesis focuses on MNPs for bio-sensing and detection applications. As a preliminary study, chemically-synthesized, commercially-available iron oxide nanoparticles were compared against their laboratory-synthesized counterparts to assess their suitability for this application. The motivation for this initial study came about due to the lack of published data on commercially available iron oxide nanoparticles. TEM studies show that the latter are "beads" composed of multiple iron oxide cores encapsulated by a polymer shell, with large standard deviations in core diameter. Laboratory-synthesized iron oxide nanoparticles, on the other hand, are single core particles with small variations in diameter and therefore are expected to be better candidates for the required application. A key limitation in iron oxide nanoparticles is their relatively weak magnetic signals. The development of high moment Synthetic Anti-Ferromagnetic (SAF) nanoparticles aims to overcome this issue. SAFs are a novel class of MNPs fabricated using nanoimprint lithography, direct deposition of multilayer structure and final suspension into liquid medium (water). TEM analyses of cross-section specimens reveal that the SAFs possess characteristics similar to those of sputtered magnetic multilayer thin films. Their layered structure is preserved after a chemical etch. Magnetic measurements show a slight decrease in magnetic moment after ion milling. From TEM characterization, the introduction of oxygen into the copper release layer, prior the film deposition process, can effectively control the topography of the oxidized-copper grains and, consequently, lead to the production of SAF nanoparticles with flatter layers. Size distribution studies performed on SAFs fabricated using self-assembled stamps show that it is possible to produce monodisperse nanoparticles with diameters from 70 nm up. Part Two of the dissertation describes structural characterization experiments performed on Composite Organic-Inorganic Nanoparticles (COINs), which are a novel type of SERS nanoclusters formed by aggregating silver nanoparticles with Raman molecules, and then encapsulating them with an organic coating that stabilizes the aggregates and promotes subsequent functionalization with antibodies. Part Three of this dissertation focuses on the development and application of electron microscopy-based techniques to characterize the nanomaterial-biology interactions, to assess how, or indeed whether, nanoparticles are attaching to the cancer cells. The technique of negative staining was applied to simultaneously visualize inorganic nanoparticles and their biofunctionalized entities under the TEM and to verify the successful functionalization of nanoparticles with antibodies. The interpretation of the negatively-stained COINs was consistent with the EFTEM data. Next, the localization and characterization of CD54-functionalized COINs on the apicolateral portions of U937 leukemia cell lines was determined using TEM, SEM and Scanning Auger Microscopy. The analyses show that CD54 antigens are localized at a specific region on U937 leukemia cell surfaces. SEM imaging and SER spectroscopy correlation studies of different antibody-conjugated COINs attached onto different cancer cell lines show a direct correlation between the number of COINs binding to cells and the corresponding SER intensity. Finally, TEM was used to locate intra-cellularly labeled COINs and to trace the phospho-stat6 signaling pathway in U937 leukemia cells, demonstrating that COINs can be used to detect intracellular phosphorylation signaling events. These experiments demonstrate the importance of electron microscopy for analyzing the material-biology interface and for validating the attachment of nanoparticles on and in cells. Thus, electron microscope provides complementary imaging and spectroscopic information to current magnetic and SERS bio-detection technologies. (Abstract shortened by UMI.)

Enhancing microparticle internalization by nonphagocytic cells through the use of noncovalently conjugated polyethyleneimine

PubMed Central

Patiño, Tania; Nogués, Carme; Ibáñez, Elena; Barrios, Leonardo

2012-01-01

Development of micro- and nanotechnology for the study of living cells, especially in the field of drug delivery, has gained interest in recent years. Although several studies have reported successful results in the internalization of micro- and nanoparticles in phagocytic cells, when nonphagocytic cells are used, the low internalization efficiency represents a limitation that needs to be overcome. It has been reported that covalent surface modification of micro- and nanoparticles increases their internalization rate. However, this surface modification represents an obstacle for their use as drug-delivery carriers. For this reason, the aim of the present study was to increase the capability for microparticle internalization of HeLa cells through the use of noncovalently bound transfection reagents: polyethyleneimine (PEI) Lipofectamine™ 2000 and FuGENE 6®. Both confocal microscopy and flow cytometry techniques allowed us to precisely quantify the efficiency of microparticle internalization by HeLa cells, yielding similar results. In addition, intracellular location of microparticles was analyzed through transmission electron microscopy and confocal microscopy procedures. Our results showed that free PEI at a concentration of 0.05 mM significantly increased microparticle uptake by cells, with a low cytotoxic effect. As determined by transmission electron and confocal microscopy analyses, microparticles were engulfed by plasma-membrane projections during internalization, and 24 hours later they were trapped in a lysosomal compartment. These results show the potential use of noncovalently conjugated PEI in microparticle internalization assays. PMID:23152683

Some personal and historical notes on the utility of “deep-etch” electron microscopy for making cell structure/function correlations

PubMed Central

Heuser, John E.

2014-01-01

This brief essay talks up the advantages of metal replicas for electron microscopy and explains why they are still the best way to image frozen cells in the electron microscope. Then it explains our approach to freezing, namely the Van Harreveld trick of “slamming” living cells onto a supercold block of metal sprayed with liquid helium at −269ºC, and further talks up this slamming over the alternative of high-pressure freezing, which is much trickier but enjoys greater favor at the moment. This leads me to bemoan the fact that there are not more young investigators today who want to get their hands on electron microscopes and use our approach to get the most “true to life” views of cells out of them with a minimum of hassle. Finally, it ends with a few perspectives on my own career and concludes that, personally, I'm permanently stuck with the view of the “founding fathers” that cell ultrastructure will ultimately display and explain all of cell function, or as Palade said in his Nobel lecture,electron micrographs are “irresistible and half transparent … their meaning buried under only a few years of work,” and “reasonable working hypotheses are already suggested by the ultrastructural organization itself.” PMID:25360049

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.

Confocal laser scanning microscopic photoconversion: a new method to stabilize fluorescently labeled cellular elements for electron microscopic analysis.

PubMed

Colello, Raymond J; Tozer, Jordan; Henderson, Scott C

2012-01-01

Photoconversion, the method by which a fluorescent dye is transformed into a stable, osmiophilic product that can be visualized by electron microscopy, is the most widely used method to enable the ultrastructural analysis of fluorescently labeled cellular structures. Nevertheless, the conventional method of photoconversion using widefield fluorescence microscopy requires long reaction times and results in low-resolution cell targeting. Accordingly, we have developed a photoconversion method that ameliorates these limitations by adapting confocal laser scanning microscopy to the procedure. We have found that this method greatly reduces photoconversion times, as compared to conventional wide field microscopy. Moreover, region-of-interest scanning capabilities of a confocal microscope facilitate the targeting of the photoconversion process to individual cellular or subcellular elements within a fluorescent field. This reduces the area of the cell exposed to light energy, thereby reducing the ultrastructural damage common to this process when widefield microscopes are employed. © 2012 by John Wiley & Sons, Inc.

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

PubMed Central

Barrasa, J. M.; Gutiérrez, A.; Escaso, V.; Guillén, F.; Martínez, M. J.; Martínez, A. T.

1998-01-01

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H2O2-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw. PMID:9435085

Differentiating Pseudomonas sp. strain ADP cells in suspensions and biofilms using Raman spectroscopy and scanning electron microscopy.

PubMed

Henry, Victoria A; Jessop, Julie L P; Peeples, Tonya L

2017-02-01

High quality spectra of Pseudomonas sp. strain ADP in the planktonic and biofilm state were obtained using Raman microspectroscopy. These spectra enabled the identification of key differences between free and biofilm cells in the fingerprint region of Raman spectra in the nucleic acid, carbohydrate, and protein regions. Scanning electron microscopy (SEM) enabled detailed visualization of ADP biofilm with confirmation of associated extracellular matrix structure. Following extraction and Raman analysis of extracellular polymeric substances, Raman spectral differences between free and biofilm cells were largely attributed to the contribution of extracellular matrix components produced in mature biofilms. Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species. Graphical Abstract Raman spectroscopy complemented with SEM proves to be useful in distinguishing physiological properties among cells of the same species.

Preparation of herpes simplex virus-infected primary neurons for transmission electron microscopy.

PubMed

Miranda-Saksena, Monica; Boadle, Ross; Cunningham, Anthony L

2014-01-01

Transmission electron microscopy (TEM) provides the resolution necessary to identify both viruses and subcellular components of cells infected with many types of viruses, including herpes simplex virus. Recognized as a powerful tool in both diagnostic and research-based virology laboratories, TEM has made possible the identification of new viruses and has contributed to the elucidation of virus life cycle and virus-host cell interaction. Whilst there are many sample preparation techniques for TEM, conventional processing using chemical fixation and resin embedding remains a useful technique, available in virtually all EM laboratories, for studying virus/cell ultrastructure. In this chapter, we describe the preparation of herpes simplex virus-infected primary neurons, grown on plastic cover slips, to allow sectioning of neurons and axons in their growth plane. This technique allows TEM examination of cell bodies, axons, growth cones, and varicosities, providing powerful insights into virus-cell interaction.

Ultrananocrystalline diamond-coated nanoporous membranes support SK-N-SH neuroblastoma endothelial cell attachment.

PubMed

Yang, Kai-Hung; Nguyen, Alexander K; Goering, Peter L; Sumant, Anirudha V; Narayan, Roger J

2018-06-06

Ultrananocrystalline diamond (UNCD) has been demonstrated to have attractive features for biomedical applications and can be combined with nanoporous membranes for applications in drug delivery systems, biosensing, immunoisolation and single molecule analysis. In this study, free-standing nanoporous UNCD membranes with pore sizes of 100 or 400 nm were fabricated by directly depositing ultrathin UNCD films on nanoporous silicon nitride membranes and then etching away silicon nitride using reactive ion etching. Successful deposition of UNCD on the substrate with a novel process was confirmed with Raman spectroscopy, X-ray photoelectron spectroscopy, cross-section scanning electron microscopy (SEM) and transmission electron microscopy. Both sample types exhibited uniform geometry and maintained a clear hexagonal pore arrangement. Cellular attachment of SK-N-SH neuroblastoma endothelial cells was examined using confocal microscopy and SEM. Attachment of SK-N-SH cells onto UNCD membranes on both porous regions and solid surfaces was shown, indicating the potential use of UNCD membranes in biomedical applications such as biosensors and tissue engineering scaffolds.

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.

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

Telocytes and putative stem cells in the lungs: electron microscopy, electron tomography and laser scanning microscopy.

PubMed

Popescu, Laurentiu M; Gherghiceanu, Mihaela; Suciu, Laura C; Manole, Catalin G; Hinescu, Mihail E

2011-09-01

This study describes a novel type of interstitial (stromal) cell - telocytes (TCs) - in the human and mouse respiratory tree (terminal and respiratory bronchioles, as well as alveolar ducts). TCs have recently been described in pleura, epicardium, myocardium, endocardium, intestine, uterus, pancreas, mammary gland, etc. (see www.telocytes.com ). TCs are cells with specific prolongations called telopodes (Tp), frequently two to three per cell. Tp are very long prolongations (tens up to hundreds of μm) built of alternating thin segments known as podomers (≤ 200 nm, below the resolving power of light microscope) and dilated segments called podoms, which accommodate mitochondria, rough endoplasmic reticulum and caveolae. Tp ramify dichotomously, making a 3-dimensional network with complex homo- and heterocellular junctions. Confocal microscopy reveals that TCs are c-kit- and CD34-positive. Tp release shed vesicles or exosomes, sending macromolecular signals to neighboring cells and eventually modifying their transcriptional activity. At bronchoalveolar junctions, TCs have been observed in close association with putative stem cells (SCs) in the subepithelial stroma. SCs are recognized by their ultrastructure and Sca-1 positivity. Tp surround SCs, forming complex TC-SC niches (TC-SCNs). Electron tomography allows the identification of bridging nanostructures, which connect Tp with SCs. In conclusion, this study shows the presence of TCs in lungs and identifies a TC-SC tandem in subepithelial niches of the bronchiolar tree. In TC-SCNs, the synergy of TCs and SCs may be based on nanocontacts and shed vesicles.

Imaging mammalian cells with soft x rays: The importance of specimen preparation

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

Brown, J.T.; Meyer-Ilse, W.

1997-04-01

Studies of mammalian cell structure and spatial organization are a very prominent part of modern cell biology. The interest in them as well as their size make them very accommodating subject specimens for imaging with soft x-rays using the XM-1 transmission microscope built and operated by The Center for X-ray Optics on Beam Line 6.1 at the Advanced Light Source. The purpose of these experiments was to determine if the fixative protocols normally used in electron or visible light microscopy were adequate to allow imaging cells, either fibroblasts or neurons, with minimal visible radiation damage due to imaging with softmore » x-rays at 2.4 nm. Two cell types were selected. Fibroblasts are easily cultured but fragile cells which are commonly used as models for the detailed study of cell physiology. Neurons are complex and sensitive cells which are difficult to prepare and to culture for study in isolation from their connections with surrounding cells. These cell types pose problems in their preparation for any microscopy. To improve the contrast and to prevent postmortem alteration of the chemistry and hence the structure of cells extracted from culture or from living organisms, fixation and staining techniques are employed in electron and visible light microscopy. It has been accepted by biologists for years that these treatments create artifacts and false structure. The authors have begun to develop protocols for specimens of each of these two cell types for soft x-ray microscopy which will preserve them in as near normal state as possible using minimal fixation, and make it possible to image them in either a hydrated or dried state free of secondary addition of stains or other labels.« less

Robust Nucleus/Cell Detection and Segmentation in Digital Pathology and Microscopy Images: A Comprehensive Review

PubMed Central

Xing, Fuyong; Yang, Lin

2016-01-01

Digital pathology and microscopy image analysis is widely used for comprehensive studies of cell morphology or tissue structure. Manual assessment is labor intensive and prone to inter-observer variations. Computer-aided methods, which can significantly improve the objectivity and reproducibility, have attracted a great deal of interest in recent literatures. Among the pipeline of building a computer-aided diagnosis system, nucleus or cell detection and segmentation play a very important role to describe the molecular morphological information. In the past few decades, many efforts have been devoted to automated nucleus/cell detection and segmentation. In this review, we provide a comprehensive summary of the recent state-of-the-art nucleus/cell segmentation approaches on different types of microscopy images including bright-field, phase-contrast, differential interference contrast (DIC), fluorescence, and electron microscopies. In addition, we discuss the challenges for the current methods and the potential future work of nucleus/cell detection and segmentation. PMID:26742143

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

Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium.

PubMed

Zhu, Wei; Teel, George; O'Brien, Christopher M; Zhuang, Taisen; Keidar, Michael; Zhang, Lijie Grace

2015-01-01

Surface modification of titanium for use in orthopedics has been explored for years; however, an ideal method of integrating titanium with native bone is still required to this day. Since human bone cells directly interact with nanostructured extracellular matrices, one of the most promising methods of improving titanium's osseointegration involves inducing bio-mimetic nanotopography to enhance cell-implant interaction. In this regard, we explored an approach to functionalize the surface of titanium by depositing a thin film of textured titanium nanoparticles via a cathodic arc discharge plasma. The aim is to improve human bone marrow mesenchymal stem cell (MSC) attachment and differentiation and to reduce deleterious effects of more complex surface modification methods. Surface functionalization was analyzed by scanning electron microscopy, atomic force microscopy, contact angle testing, and specific protein adsorption. Scanning electron microscopy and atomic force microscopy examination demonstrate the deposition of titanium nanoparticles and the surface roughness change after coating. The specific fibronectin adsorption was enhanced on the modified titanium surface that associates with the improved hydrophilicity. MSC adhesion and proliferation were significantly promoted on the nanocoated surface. More importantly, compared to bare titanium, greater production of total protein, deposition of calcium mineral, and synthesis of alkaline phosphatase were observed from MSCs on nanocoated titanium after 21 days. The method described herein presents a promising alternative method for inducing more cell favorable nanosurface for improved orthopedic applications.

In vitro inhibitory activity of probiotic products against oral Candida species.

PubMed

Zhao, C; Lv, X; Fu, J; He, C; Hua, H; Yan, Z

2016-07-01

To evaluate the inhibitory activity of probiotics against oral Candida species. Four commercial probiotic products were screened. Bacillus subtilis R0179 was found to have a significant antifungal effect. Bacillus subtilis-Candida interactions were evaluated using disc diffusion tests, confocal laser scanning microscopy, scanning electron microscopy and interaction with engineered human oral mucosa tissue. Bacillus subtilis exhibited clear zones of inhibition for Candida albicans and Candida parapsilosis but not for Candida krusei. A remarkable reduction in the number of Candida cells and abundant Candida cell death were visualized with confocal laser scanning microscopy. Shrinkage and deformation of Candida cells was observed using scanning electron microscopy. Culture of C. albicans on engineered human oral mucosa tissues resulted in the presence of a large number of yeast cells on the tissue surface and the development of large-scale tissue damage. However, comparatively fewer Candida cells were observed on B. subtilis-treated tissues. We also use ultra performance liquid chromatography/time of flight mass spectrometry (UPLC/TOF MS) to explore the preliminary antifungal mechanism of B. subtilis R0179 and to detect that whether it can secrete an antifungal agent, Iturin A. Bacillus subtilis R0179 exhibits a significant inhibitory effect on the growth of Candida species. Bacillus subtilis has the potential to be used in the prevention or treatment of oral candidiasis. © 2016 The Society for Applied Microbiology.

Visualization of early influenza A virus trafficking in human dendritic cells using STED microscopy.

PubMed

Baharom, Faezzah; Thomas, Oliver S; Lepzien, Rico; Mellman, Ira; Chalouni, Cécile; Smed-Sörensen, Anna

2017-01-01

Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.

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

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.

Biocompatibility of tungsten disulfide inorganic nanotubes and fullerene-like nanoparticles with salivary gland cells.

PubMed

Goldman, Elisheva B; Zak, Alla; Tenne, Reshef; Kartvelishvily, Elena; Levin-Zaidman, Smadar; Neumann, Yoav; Stiubea-Cohen, Raluca; Palmon, Aaron; Hovav, Avi-Hai; Aframian, Doron J

2015-03-01

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles.

Biocompatibility of Tungsten Disulfide Inorganic Nanotubes and Fullerene-Like Nanoparticles with Salivary Gland Cells

PubMed Central

Goldman, Elisheva B.; Zak, Alla; Tenne, Reshef; Kartvelishvily, Elena; Levin-Zaidman, Smadar; Neumann, Yoav; Stiubea-Cohen, Raluca; Palmon, Aaron; Hovav, Avi-Hai

2015-01-01

Impaired salivary gland (SG) function leading to oral diseases is relatively common with no adequate solution. Previously, tissue engineering of SG had been proposed to overcome this morbidity, however, not yet clinically available. Multiwall inorganic (tungsten disulfide [WS2]) nanotubes (INT-WS2) and fullerene-like nanoparticles (IF-WS2) have many potential medical applications. A yet unexplored venue application is their interaction with SG, and therefore, our aim was to test the biocompatibility of INT/IF-WS2 with the A5 and rat submandibular cells (RSC) SG cells. The cells were cultured and subjected after 1 day to different concentrations of INT-WS2 and were compared to control groups. Growth curves, trypan blue viability test, and carboxyfluorescein succinimidyl ester (CFSE) proliferation assay were obtained. Furthermore, cells morphology and interaction with the nanoparticles were observed by light microscopy, scanning electron microscopy and transmission electron microscopy (TEM), and energy dispersive X-ray spectroscopy. The results showed no significant differences in growth curves, proliferation kinetics, and viability between the groups compared. Moreover, no alterations were observed in the cell morphology. Interestingly, TEM images indicated that the nanoparticles are uptaken by the cells and accumulate in cytoplasmic vesicles. These results suggest promising future medical applications for these nanoparticles. PMID:25366879

Quantification of endocytosis using a folate functionalized silica hollow nanoshell platform

PubMed Central

Sandoval, Sergio; Mendez, Natalie; Alfaro, Jesus G.; Yang, Jian; Aschemeyer, Sharraya; Liberman, Alex; Trogler, William C.; Kummel, Andrew C.

2015-01-01

Abstract. A quantification method to measure endocytosis was designed to assess cellular uptake and specificity of a targeting nanoparticle platform. A simple N-hydroxysuccinimide ester conjugation technique to functionalize 100-nm hollow silica nanoshell particles with fluorescent reporter fluorescein isothiocyanate and folate or polyethylene glycol (PEG) was developed. Functionalized nanoshells were characterized using scanning electron microscopy and transmission electron microscopy and the maximum amount of folate functionalized on nanoshell surfaces was quantified with UV-Vis spectroscopy. The extent of endocytosis by HeLa cervical cancer cells and human foreskin fibroblast (HFF-1) cells was investigated in vitro using fluorescence and confocal microscopy. A simple fluorescence ratio analysis was developed to quantify endocytosis versus surface adhesion. Nanoshells functionalized with folate showed enhanced endocytosis by cancer cells when compared to PEG functionalized nanoshells. Fluorescence ratio analyses showed that 95% of folate functionalized silica nanoshells which adhered to cancer cells were endocytosed, while only 27% of PEG functionalized nanoshells adhered to the cell surface and underwent endocytosis when functionalized with 200 and 900  μg, respectively. Additionally, the endocytosis of folate functionalized nanoshells proved to be cancer cell selective while sparing normal cells. The developed fluorescence ratio analysis is a simple and rapid verification/validation method to quantify cellular uptake between datasets by using an internal control for normalization. PMID:26315280

Persistent measles virus infection of the intestine: confirmation by immunogold electron microscopy.

PubMed Central

Lewin, J; Dhillon, A P; Sim, R; Mazure, G; Pounder, R E; Wakefield, A J

1995-01-01

This study sought to investigate persistent measles virus infection of the intestine: a novel protocol for immunogold electron microscopy was developed using a polyclonal anti-measles nucleoprotein antibody on reprocessed, formalin fixed paraffin wax embedded tissue sections. Antibody binding was detected using both immunoperoxidase and light microscopy on tissue sections, and 10 nm gold conjugated secondary antibody and electron microscopy on ultrathin sections. The techniques were validated using both measles infected vero cells and human tissues with established measles infection: these included brain affected by subacute sclerosing panencephalitis and acute measles appendicitis. The technique was applied subsequently to six untreated cases of granulomatous Crohn's disease, and two cases of ileocaecal tuberculosis, a granulomatous control. Mumps primary antibody--applied to both mumps infected vero cells, and measles infected vero cells and tissues studied by immunoperoxidase, and measles antibody on mumps infected cells studied by immunoperoxidase and immunogold--were used as specificity controls: the primary antibodies identified their respective target antigen and there was no antibody cross reactivity. Measles virus nucleocapsids labelled with gold conjugated antibody in both infected cells and tissues, including foci of granulomatous inflammation in five of six cases of Crohn's disease: in the fifth case, the granuloma could not be identified in ultrathin section. In one of the tuberculosis cases, a low level of signal was noted while the second case was negative. Labelling adopted a characteristic pattern in all infected tissues, strengthening the specificity of these findings. This study provides the first direct confirmation of persistent measles virus infection of the intestine. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:7737565

A cellular uptake and cytotoxicity properties study of gallic acid-loaded mesoporous silica nanoparticles on Caco-2 cells

NASA Astrophysics Data System (ADS)

Rashidi, Ladan; Vasheghani-Farahani, Ebrahim; Soleimani, Masoud; Atashi, Amir; Rostami, Khosrow; Gangi, Fariba; Fallahpour, Masoud; Tahouri, Mohammad Taher

2014-03-01

In this study, the effects of intracellular delivery of various concentrations of gallic acid (GA) as a semistable antioxidant, gallic acid-loaded mesoporous silica nanoparticles (MSNs-GA), and cellular uptake of nanoparticles into Caco-2 cells were investigated. MSNs were synthesized and loaded with GA, then characterized using transmission electron microscopy (TEM), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy, N2 adsorption isotherms, X-ray diffraction, and thermal gravimetric analysis. The cytotoxicity of MSNs and MSNs-GA at low and high concentrations were studied by means of 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) test and flow cytometry. MSNs did not show significant toxicity in various concentrations (0-500 μg/ml) on Caco-2 cells. For MSNs-GA, cell viability was reduced as a function of incubation time and different concentrations of nanoparticles. The in vitro GA release from MSNs-GA exhibited the same antitumor properties as free GA on Caco-2 cells. Flow cytometry results confirmed those obtained using MTT assay. TEM and fluorescent microscopy confirmed the internalization of MSNs by Caco-2 cells through nonspecific cellular uptake. MSNs can easily internalize into Caco-2 cells without deleterious effects on cell viability. The cell viability of Caco-2 cells was affected during MSNs-GA uptake. MSNs could be designed as suitable nanocarriers for antioxidants delivery.

Serial Section Scanning Electron Microscopy (S3EM) on Silicon Wafers for Ultra-Structural Volume Imaging of Cells and Tissues

PubMed Central

Horstmann, Heinz; Körber, Christoph; Sätzler, Kurt; Aydin, Daniel; Kuner, Thomas

2012-01-01

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S3EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm3 volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S3EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S3EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation. PMID:22523574

Serial section scanning electron microscopy (S3EM) on silicon wafers for ultra-structural volume imaging of cells and tissues.

PubMed

Horstmann, Heinz; Körber, Christoph; Sätzler, Kurt; Aydin, Daniel; Kuner, Thomas

2012-01-01

High resolution, three-dimensional (3D) representations of cellular ultrastructure are essential for structure function studies in all areas of cell biology. While limited subcellular volumes have been routinely examined using serial section transmission electron microscopy (ssTEM), complete ultrastructural reconstructions of large volumes, entire cells or even tissue are difficult to achieve using ssTEM. Here, we introduce a novel approach combining serial sectioning of tissue with scanning electron microscopy (SEM) using a conductive silicon wafer as a support. Ribbons containing hundreds of 35 nm thick sections can be generated and imaged on the wafer at a lateral pixel resolution of 3.7 nm by recording the backscattered electrons with the in-lens detector of the SEM. The resulting electron micrographs are qualitatively comparable to those obtained by conventional TEM. S(3)EM images of the same region of interest in consecutive sections can be used for 3D reconstructions of large structures. We demonstrate the potential of this approach by reconstructing a 31.7 µm(3) volume of a calyx of Held presynaptic terminal. The approach introduced here, Serial Section SEM (S(3)EM), for the first time provides the possibility to obtain 3D ultrastructure of large volumes with high resolution and to selectively and repetitively home in on structures of interest. S(3)EM accelerates process duration, is amenable to full automation and can be implemented with standard instrumentation.

Interactions of Condensed Tannins with Saccharomyces cerevisiae Yeast Cells and Cell Walls: Tannin Location by Microscopy.

PubMed

Mekoue Nguela, Julie; Vernhet, Aude; Sieczkowski, Nathalie; Brillouet, Jean-Marc

2015-09-02

Interactions between grape tannins/red wine polyphenols and yeast cells/cell walls was previously studied within the framework of red wine aging and the use of yeast-derived products as an alternative to aging on lees. Results evidenced a quite different behavior between whole cells (biomass grown to elaborate yeast-derived products, inactivated yeast, and yeast inactivated after autolysis) and yeast cell walls (obtained from mechanical disruption of the biomass). Briefly, whole cells exhibited a high capacity to irreversibly adsorb grape and wine tannins, whereas only weak interactions were observed for cell walls. This last point was quite unexpected considering the literature and called into question the real role of cell walls in yeasts' ability to fix tannins. In the present work, tannin location after interactions between grape and wine tannins and yeast cells and cell walls was studied by means of transmission electron microscopy, light epifluorescence, and confocal microscopy. Microscopy observations evidenced that if tannins interact with cell walls, and especially cell wall mannoproteins, they also diffuse freely through the walls of dead cells to interact with their plasma membrane and cytoplasmic components.

Staining of Tissue Sections for Electron Microscopy with Heavy Metals

PubMed Central

Watson, Michael L.

1958-01-01

Descriptions of three heavy metal stains and methods of application to tissue sections for electron microscopy are presented. Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles. Barium hydroxide emphasizes certain bodies within vesicles of the Golgi region of hepatic cells. Alkalized lead acetate is useful as a general stain, as are also lead and barium hydroxides. PMID:13610936

Folate-grafted boron nitride nanotubes: possible exploitation in cancer therapy.

PubMed

Ferreira, Tiago H; Marino, Attilio; Rocca, Antonella; Liakos, Ioannis; Nitti, Simone; Athanassiou, Athanassia; Mattoli, Virgilio; Mazzolai, Barbara; de Sousa, Edesia M B; Ciofani, Gianni

2015-03-15

Boron nitride nanotubes (BNNTs) have generated considerable interest among the scientific community because of their unique physical and chemical properties. They present good chemical inertness, high thermal stability, and optimal resistance to oxidation, that make them ideal candidates for biomedical applications, in particular as nanovectors for drug, gene and protein delivery into the cells. In this study, BNNTs were prepared through a synthesis based on a chemical vapor deposition (CVD) method, and thereafter chemically functionalized with folic acid. The obtained nanostructures have been characterized by Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The characterization showed efficiently functionalized BNNTs of length of about 1 μm. Furthermore, confocal laser microscopy demonstrated that our nanotubes can be fluorescently-traced under appropriate excitation. Thanks to this property, it has been possible to investigate their internalization by HeLa cells through confocal microscopy, demonstrating that the BNNT up-take clearly increases after the functionalization with folate, a result confirmed by inductively coupled plasma (ICP) assessment of boron content inside the treated cell cultures. Copyright © 2015 Elsevier B.V. All rights reserved.

X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning

DOE PAGES

Deng, Junjing; Vine, David J.; Chen, Si; ...

2017-03-27

X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. Here, by working with cells that have been rapidly frozen without the use of chemicalmore » fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.« less

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

Prolymphocytic leukaemia: surface morphology in 21 cases as seen by scanning electron microscopy and comparison with B-type CLL and CLL in 'prolymphocytoid' transformation.

PubMed

Polliack, A; Leizerowitz, R; Berrebi, A; Gamliel, H; Galili, N; Gurfel, D; Catovsky, D

1984-08-01

The surface architecture of leukaemic cells obtained from 21 cases of proven prolymphocytic leukaemia (PLL) and eight cases of chronic lymphocytic leukaemia (CLL) with 'prolymphocytoid' transformation (PL-CLL) was compared with the cell surface morphology of leukaemic cells obtained from 46 cases of B-type CLL, using the scanning electron microscope (SEM). All cases were defined by cytochemistry, immunological markers and transmission electron microscopy prior to SEM examination. B-CLL cells showed the well-recognized spectrum of surface architecture described in earlier studies. The majority of cells had moderate numbers of short microvilli, although in a minority, cells with relatively smooth surfaces predominated. In seven of the eight cases of PL-CLL, cells were villous in nature and in this respect similar to CLL cells; however, more cells with dense microvilli were seen. The prolymphocytic cells were recognized by their larger size and in 18 of the 19 cases of B-derived PLL, villous cells predominated. Two cases of T-derived PLL showed variable cell surface morphology ranging from smooth to moderately villous. It appears that B-PLL cells are most frequently villous and display more surface microvilli than B-CLL cells. B-prolymphocytes display the surface features regarded as characteristic for neoplastic B-cells as seen in patients with B-type lymphoma and leukaemia.

Rapid fibroblast activation in mammalian cells induced by silicon nanowire arrays

NASA Astrophysics Data System (ADS)

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

2014-06-01

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.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. Electronic supplementary information (ESI) available: (1) ESEM cross-sectional view images of the flat silicon and SiNW substrates. (2) Bright field morphology images of fibroblasts cultured in Petri dishes. (3) FIB/SEM 52° tilt images of fibroblasts cultured on SiNW 2 and SiNW 3. (4) Immunofluorescence images of FAP expression in fibroblasts re-cultured in Petri dishes after detachment from flat silicon and a series of SiNW substrates. (5) ESEM images of cells re-cultured in Petri dishes after detachment from each group. See DOI: 10.1039/c4nr01415d

Electron tomography of HEK293T cells using scanning electron microscope-based scanning transmission electron microscopy.

PubMed

You, Yun-Wen; Chang, Hsun-Yun; Liao, Hua-Yang; Kao, Wei-Lun; Yen, Guo-Ji; Chang, Chi-Jen; Tsai, Meng-Hung; Shyue, Jing-Jong

2012-10-01

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

Determination of redox reaction rates and orders by in situ liquid cell electron microscopy of Pd and Au solution growth.

PubMed

Sutter, Eli A; Sutter, Peter W

2014-12-03

In-situ liquid cell transmission and scanning transmission electron microscopy (TEM/STEM) experiments are important, as they provide direct insight into processes in liquids, such as solution growth of nanoparticles, among others. In liquid cell TEM/STEM redox reaction experiments, the hydrated electrons e(-)aq created by the electron beam are responsible for the reduction of metal-ion complexes. Here we investigate the rate equation of redox reactions involving reduction by e(-)aq generated by the electron beam during in situ liquid TEM/STEM. Specifically we consider the growth of Pd on Au seeds in aqueous solutions containing Pd-chloro complexes. From the quantification of the rate of Pd deposition at different electron beam currents and as a function of distance from a stationary, nanometer-sized exciting beam, we determine that the reaction is first order with respect to the concentration of hydrated electrons, [e(-)aq]. By comparing Pd- and Au-deposition, we further demonstrate that measurements of the local deposition rate on nanoparticles in the solution via real-time imaging can be used to measure not only [e(-)aq] but also the rate of reduction of a metal-ion complex to zerovalent metal atoms in solution.

Determination of redox reaction rates and orders by in situ liquid cell electron microscopy of Pd and Au solution growth

DOE PAGES

Sutter, Eli A.; Sutter, Peter W.

2014-11-19

In-situ liquid cell transmission and scanning transmission electron microscopy (TEM/STEM) experiments are important as they provide direct insight into processes in liquids, such as solution growth of nanoparticles among others. In liquid cell TEM/STEM redox reaction experiments the hydrated electrons e⁻ aq created by the electron beam are responsible for the reduction of metal-ion complexes. Here we investigate the rate equation of redox reactions involving reduction by e⁻ aq generated by the electron beam during in-situ liquid TEM/STEM. Specifically we consider the growth of Pd on Au seeds in aqueous solutions containing Pd-chloro complexes. From the quantification of the ratemore » of Pd deposition at different electron beam currents and as a function of distance from a stationary, nanometer-sized exciting beam, we determine that the reaction is first order with respect to the concentration of hydrated electrons, [e⁻ aq]. In addition, by comparing Pd- and Au-deposition, we further demonstrate that measurements of the local deposition rate on nanoparticles in the solution via real-time imaging can be used to measure not only [e⁻ aq] but also the rate of reduction of a metal-ion complex to zero-valent metal atoms in solution.« less

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

Resinless section electron microscopy reveals the yeast cytoskeleton.

PubMed

Penman, J; Penman, S

1997-04-15

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or "soluble" proteins are distinct from the retained or "structural" proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters-5 nm and 15-20 nm-which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300-500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.

Resinless section electron microscopy reveals the yeast cytoskeleton

PubMed Central

Penman, Joshua; Penman, Sheldon

1997-01-01

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or “soluble” proteins are distinct from the retained or “structural” proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters—5 nm and 15–20 nm—which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300–500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture. PMID:9108046

Scanning superlens microscopy for non-invasive large field-of-view visible light nanoscale imaging

NASA Astrophysics Data System (ADS)

Wang, Feifei; Liu, Lianqing; Yu, Haibo; Wen, Yangdong; Yu, Peng; Liu, Zhu; Wang, Yuechao; Li, Wen Jung

2016-12-01

Nanoscale correlation of structural information acquisition with specific-molecule identification provides new insight for studying rare subcellular events. To achieve this correlation, scanning electron microscopy has been combined with super-resolution fluorescent microscopy, despite its destructivity when acquiring biological structure information. Here we propose time-efficient non-invasive microsphere-based scanning superlens microscopy that enables the large-area observation of live-cell morphology or sub-membrane structures with sub-diffraction-limited resolution and is demonstrated by observing biological and non-biological objects. This microscopy operates in both non-invasive and contact modes with ~200 times the acquisition efficiency of atomic force microscopy, which is achieved by replacing the point of an atomic force microscope tip with an imaging area of microspheres and stitching the areas recorded during scanning, enabling sub-diffraction-limited resolution. Our method marks a possible path to non-invasive cell imaging and simultaneous tracking of specific molecules with nanoscale resolution, facilitating the study of subcellular events over a total cell period.

Long helical filaments are not seen encircling cells in electron cryotomograms of rod-shaped bacteria

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

Swulius, Matthew T.; Chen, Songye; Jane Ding, H.

2011-04-22

Highlights: {yields} No long helical filaments are seen near or along rod-shaped bacterial inner membranes by electron cryo-tomography. {yields} Electron cryo-tomography has the resolution to detect single filaments in vivo. -- Abstract: How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (>80 nm)more » helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.« less

Effect of ultrasound sonication on clonogenic survival and mitochondria of ovarian cancer cells in the presence of methylene blue.

PubMed

Xiang, Junyan; Leung, Albert Wingnang; Xu, Chuanshan

2014-10-01

This study aimed to investigate the effect of ultrasound sonication in the presence of methylene blue on clonogenic survival and mitochondria of ovarian cancer cells. Human ovarian cancer HO-8910 cells, which were incubated with different concentrations of methylene blue for 1 hour, were exposed to an ultrasonic wave for 5 seconds with intensity of 0.46 W/cm(2). Clonogenic survival of HO-8910 cells after ultrasound sonication was measured by a colony-forming unit assay. Mitochondrial structural changes were observed on transmission electron microscopy, and the mitochondrial membrane potential was evaluated by confocal laser-scanning microscopy with rhodamine 123 staining. The colony-forming units of HO-8910 cells decreased considerably after ultrasound sonication in the presence of methylene blue. Transmission electron microscopy showed slightly enlarged mitochondria in the ultrasound-treated cells in the absence of methylene blue; however, seriously damaged mitochondria, even with almost complete disappearance of cristae, were found in the cells treated by ultrasound sonication in the presence of methylene blue. The mitochondrial membrane potential collapsed significantly when HO-8910 cells were treated by ultrasound sonication in the presence of methylene blue (P < .05). Ultrasound sonication in the presence of methylene blue markedly damaged mitochondrial structure and function and decreased clonogenic survival of HO-8910 cells. © 2014 by the American Institute of Ultrasound in Medicine.

Fluorescence Microscopy Methods for Determining the Viability of Bacteria in Association with Mammalian Cells

PubMed Central

Johnson, M. Brittany; Criss, Alison K.

2013-01-01

Central to the field of bacterial pathogenesis is the ability to define if and how microbes survive after exposure to eukaryotic cells. Current protocols to address these questions include colony count assays, gentamicin protection assays, and electron microscopy. Colony count and gentamicin protection assays only assess the viability of the entire bacterial population and are unable to determine individual bacterial viability. Electron microscopy can be used to determine the viability of individual bacteria and provide information regarding their localization in host cells. However, bacteria often display a range of electron densities, making assessment of viability difficult. This article outlines protocols for the use of fluorescent dyes that reveal the viability of individual bacteria inside and associated with host cells. These assays were developed originally to assess survival of Neisseria gonorrhoeae in primary human neutrophils, but should be applicable to any bacterium-host cell interaction. These protocols combine membrane-permeable fluorescent dyes (SYTO9 and 4',6-diamidino-2-phenylindole [DAPI]), which stain all bacteria, with membrane-impermeable fluorescent dyes (propidium iodide and SYTOX Green), which are only accessible to nonviable bacteria. Prior to eukaryotic cell permeabilization, an antibody or fluorescent reagent is added to identify extracellular bacteria. Thus these assays discriminate the viability of bacteria adherent to and inside eukaryotic cells. A protocol is also provided for using the viability dyes in combination with fluorescent antibodies to eukaryotic cell markers, in order to determine the subcellular localization of individual bacteria. The bacterial viability dyes discussed in this article are a sensitive complement and/or alternative to traditional microbiology techniques to evaluate the viability of individual bacteria and provide information regarding where bacteria survive in host cells. PMID:24056524

Effects of Piper cubeba L. essential oil on methicillin-resistant Staphylococcus aureus: an AFM and TEM study.

PubMed

Alharbi, Naiyf S; Khaled, Jamal M; Alzaharni, Khalid E; Mothana, Ramzi A; Alsaid, Mansour S; Alhoshan, Mansour; Dass, Lawrence Arockiasamy; Kadaikunnan, Shine; Alobaidi, Ahmed S

2017-01-01

The increasing prevalence of antibiotic-resistant bacteria is creating a real challenge for health care systems worldwide, making the development of novel antibiotics a necessity. In addition to the development of new antibiotics, there is an urgent need for in-depth characterization of the mechanisms of bacterial resistance toward new drugs. Here, we used essential oils extracted in our laboratory from Piper cubeba against methicillin-resistant Staphylococcus aureus ATCC 43300, one of the most prominent antibiotic-resistant bacteria. Effects of the essential oils extracted from P cubeba on bacteria were mainly evaluated using 2 powerful microscopy techniques: atomic force microscopy and transmission electron microscopy. High-resolution atomic force microscopy images of the cells were obtained close to their native environment by immobilizing the cells on porous Polyether sulfone membranes, which were prepared in our laboratory with a wide range and distribution of pore sizes and depth. Inhibition zones (mm) and minimum inhibitory concentrations were determined. Two different concentrations of the oil were used to treat the cells: 50 μg/mL minimum inhibitory concentration and 25 μg/mL. The 50 μg/mL oil solution caused severe damage to the bacterial cells at microscopic levels while the 25 μg/mL solution showed no effects compared to the control. However, at nanoscopic levels, the 25 μg/mL oil solution caused significant changes in the cell wall, which could potentially impair bacterial activities. These results were also confirmed by transmission electron microscopy micrographs. Our results indicate that the extract has a good biological activity against methicillin- and oxacillin-resistant S aureus and that it acts on the cell wall and plasma (cytoplasmic) membrane. Copyright © 2016 John Wiley & Sons, Ltd.

High-pressure freezing and freeze substitution of Arabidopsis for electron microscopy.

PubMed

Austin, Jotham R

2014-01-01

The objectives of electron microscopy ultrastructural studies are to examine cellular architecture and relate the cell's structural machinery to dynamic functional roles. This aspiration is difficult to achieve if specimens have not been adequately preserved in a "living state"; hence specimen preparation is of the utmost importance for the success of any electron micrographic study. High-pressure freezing (HPF)/freeze substitution (FS) has long been recognized as the primer technique for the preservation of ultrastructure in biological samples. In most cases a basic HPF/freeze substitution protocol is sufficient to obtain superior ultrastructural preservation and structural contrast, which allows one to use more advanced microscopy techniques such as 3D electron tomography. However, for plant tissues, which have a thick cell wall, large water-filled vacuoles, and air spaces (all of which are detrimental to cryopreservation), these basic HPF/FS protocols often yield undesirable results. In particular, ice crystal artifacts and the staining of membrane systems are often poorly or negatively stained, which make 3D segmentation of a tomogram difficult. To overcome these problems, various aspects of the HPF/FS protocol can be altered, including the cryo-filler(s) used, freeze substitution cocktail, and the resin infiltration process. This chapter will describe these modifications for the preparation of plant tissues for routine electron microscopic studies, immunocytochemistry, and 3D tomographic electron imaging.

Association of intracellular and synaptic organization in cochlear inner hair cells revealed by 3D electron microscopy.

PubMed

Bullen, Anwen; West, Timothy; Moores, Carolyn; Ashmore, Jonathan; Fleck, Roland A; MacLellan-Gibson, Kirsty; Forge, Andrew

2015-07-15

The ways in which cell architecture is modelled to meet cell function is a poorly understood facet of cell biology. To address this question, we have studied the cytoarchitecture of a cell with highly specialised organisation, the cochlear inner hair cell (IHC), using multiple hierarchies of three-dimensional (3D) electron microscopy analyses. We show that synaptic terminal distribution on the IHC surface correlates with cell shape, and the distribution of a highly organised network of membranes and mitochondria encompassing the infranuclear region of the cell. This network is juxtaposed to a population of small vesicles, which represents a potential new source of neurotransmitter vesicles for replenishment of the synapses. Structural linkages between organelles that underlie this organisation were identified by high-resolution imaging. Taken together, these results describe a cell-encompassing network of membranes and mitochondria present in IHCs that support efficient coding and transmission of auditory signals. Such techniques also have the potential for clarifying functionally specialised cytoarchitecture of other cell types. © 2015. Published by The Company of Biologists Ltd.

Cytotoxic and antimicrobial effect of biosynthesized silver nanoparticles using the fruit extract of Ribes nigrum

NASA Astrophysics Data System (ADS)

Dobrucka, Renata; Kaczmarek, Mariusz; Dlugaszewska, Jolanta

2018-06-01

The present study reveals the efficiency of the fruit extract of Ribes nigrum in the green synthesis of silver nanoparticles (Ag-NPs). Biosynthesized Ag-NPs were characterized by UV-vis, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The nanoparticles were found to be 5–10 nm. In some places, the particles were agglomerated. The nanoparticles showed strong bactericidal activity and fungicidal activity against dermatophytes Trichophyton rubrum ATCC 28188. Moreover, the A549 and CCD39Lu cells under the influence of the highest concentration of nanoparticles synthesized using the fruit extract of Ribes nigrum showed the maximum mortality. Also, the results indicate that Ag-NPs synthesized using the fruit extract of Ribes nigrum exhibit efficiency in therapy of human non-small cell lung cancer A549.

Electron microscopy of whole cells in liquid with nanometer resolution

PubMed Central

de Jonge, N.; Peckys, D. B.; Kremers, G. J.; Piston, D. W.

2009-01-01

Single gold-tagged epidermal growth factor (EGF) molecules bound to cellular EGF receptors of fixed fibroblast cells were imaged in liquid with a scanning transmission electron microscope (STEM). The cells were placed in buffer solution in a microfluidic device with electron transparent windows inside the vacuum of the electron microscope. A spatial resolution of 4 nm and a pixel dwell time of 20 μs were obtained. The liquid layer was sufficiently thick to contain the cells with a thickness of 7 ± 1 μm. The experimental findings are consistent with a theoretical calculation. Liquid STEM is a unique approach for imaging single molecules in whole cells with significantly improved resolution and imaging speed over existing methods. PMID:19164524

Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by Widefield Epifluorescence, super-resolution light, and immunoelectron microscopy.

PubMed

Killingsworth, Murray C; Lai, Ken; Wu, Xiaojuan; Yong, Jim L C; Lee, C Soon

2012-11-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy.

Quantum Dot Immunocytochemical Localization of Somatostatin in Somatostatinoma by Widefield Epifluorescence, Super-resolution Light, and Immunoelectron Microscopy

PubMed Central

Lai, Ken; Wu, Xiaojuan; Yong, Jim L. C.; Lee, C. Soon

2012-01-01

Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy. PMID:22899862

Probing battery chemistry with liquid cell electron energy loss spectroscopy

DOE PAGES

Unocic, Raymond R.; Baggetto, Loic; Veith, Gabriel M.; ...

2015-09-15

Electron energy loss spectroscopy (EELS) was used to determine the chemistry and oxidation state of LiMn 2O 4 and Li 4Ti 5O 12 thin film battery electrodes in liquid cells for in situ scanning/transmission electron microscopy (S/TEM). Using the L2,3 white line intensity ratio method we determine the oxidation state of Mn and Ti in a liquid electrolyte solvent and discuss experimental parameters that influence measurement sensitivity.

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.

Scanning electron microscopy coupled with energy-dispersive X-ray spectrometry for quick detection of sulfur-oxidizing bacteria in environmental water samples

NASA Astrophysics Data System (ADS)

Sun, Chengjun; Jiang, Fenghua; Gao, Wei; Li, Xiaoyun; Yu, Yanzhen; Yin, Xiaofei; Wang, Yong; Ding, Haibing

2017-01-01

Detection of sulfur-oxidizing bacteria has largely been dependent on targeted gene sequencing technology or traditional cell cultivation, which usually takes from days to months to carry out. This clearly does not meet the requirements of analysis for time-sensitive samples and/or complicated environmental samples. Since energy-dispersive X-ray spectrometry (EDS) can be used to simultaneously detect multiple elements in a sample, including sulfur, with minimal sample treatment, this technology was applied to detect sulfur-oxidizing bacteria using their high sulfur content within the cell. This article describes the application of scanning electron microscopy imaging coupled with EDS mapping for quick detection of sulfur oxidizers in contaminated environmental water samples, with minimal sample handling. Scanning electron microscopy imaging revealed the existence of dense granules within the bacterial cells, while EDS identified large amounts of sulfur within them. EDS mapping localized the sulfur to these granules. Subsequent 16S rRNA gene sequencing showed that the bacteria detected in our samples belonged to the genus Chromatium, which are sulfur oxidizers. Thus, EDS mapping made it possible to identify sulfur oxidizers in environmental samples based on localized sulfur within their cells, within a short time (within 24 h of sampling). This technique has wide ranging applications for detection of sulfur bacteria in environmental water samples.

Effects of spaceflight in the adductor longus muscle of rats flown in the Soviet Biosatellite COSMOS 2044. A study employing neural cell adhesion molecule (N-CAM) immunocytochemistry and conventional morphological techniques (light and electron microscopy)

NASA Technical Reports Server (NTRS)

D'Amelio, F.; Daunton, N. G.

1992-01-01

The effects of spaceflight upon the "slow" muscle adductor longus were examined in rats flown in the Soviet Biosatellite COSMOS 2044. The techniques employed included standard methods for light microscopy, neural cell adhesion molecule (N-CAM) immunocytochemistry and electron microscopy. Light microscopic observations revealed myofiber atrophy and segmental necrosis accompanied by cellular infiltrates composed of macrophages, leukocytes and mononuclear cells. Neural cell adhesion molecule immunoreactivity (N-CAM-IR) was seen on the myofiber surface and in regenerating myofibers. Ultrastructural alterations included Z band streaming, disorganization of myofibrillar architecture, sarcoplasmic degradation, extensive segmental necrosis with apparent preservation of the basement membrane, degenerative phenomena of the capillary endothelium and cellular invasion of necrotic areas. Regenerating myofibers were identified by the presence of increased amounts of ribosomal aggregates and chains of polyribosomes associated with myofilaments. The principal electron microscopic changes of the neuromuscular junctions showed axon terminals with a decrease or absence of synaptic vesicles replaced by microtubules and neurofilaments, degeneration of axon terminals, vacant axonal spaces and changes suggestive of axonal sprouting. The present observations suggest that alterations such as myofibrillar disruption and necrosis, muscle regeneration and denervation and synaptic remodeling at the level of the neuromuscular junction may take place during spaceflight.

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

Surface characteristics of isopod digestive gland epithelium studied by SEM.

PubMed

Millaku, Agron; Leser, Vladka; Drobne, Damjana; Godec, Matjaz; Torkar, Matjaz; Jenko, Monika; Milani, Marziale; Tatti, Francesco

2010-05-01

The structure of the digestive gland epithelium of a terrestrial isopod Porcellio scaber has been investigated by conventional scanning electron microscopy (SEM), focused ion beam-scanning electron microscopy (FIB/SEM), and light microscopy in order to provide evidence on morphology of the gland epithelial surface in animals from a stock culture. We investigated the shape of cells, extrusion of lipid droplets, shape and distribution of microvilli, and the presence of bacteria on the cell surface. A total of 22 animals were investigated and we found some variability in the appearance of the gland epithelial surface. Seventeen of the animals had dome-shaped digestive gland "normal" epithelial cells, which were densely and homogeneously covered by microvilli and varying proportions of which extruded lipid droplets. On the surface of microvilli we routinely observed sparsely distributed bacteria of different shapes. Five of the 22 animals had "abnormal" epithelial cells with a significantly altered shape. In three of these animals, the cells were much smaller, partly or completely flat or sometimes pyramid-like. A thick layer of bacteria was detected on the microvillous border, and in places, the shape and size of microvilli were altered. In two animals, hypertrophic cells containing large vacuoles were observed indicating a characteristic intracellular infection. The potential of SEM in morphological investigations of epithelial surfaces is discussed.

Chemo-spectroscopic sensor for carboxyl terminus overexpressed in carcinoma cell membrane.

PubMed

Stanca, Sarmiza E; Matthäus, Christian; Neugebauer, Ute; Nietzsche, Sandor; Fritzsche, Wolfgang; Dellith, Jan; Heintzmann, Rainer; Weber, Karina; Deckert, Volker; Krafft, Christoph; Popp, Jürgen

2015-10-01

Certain carboxyl groups of the plasma membrane are involved in tumorgenesis processes. A gold core-hydroxyapatite shell (AuHA) nanocomposite is introduced as chemo-spectroscopic sensor to monitor these carboxyl groups of the cell membrane. Hydroxyapatite (HA) plays the role both of a chemical detector and of a biocompatible Raman marker. The principle of detection is based on chemical interaction between the hydroxyl groups of the HA and the carboxyl terminus of the proteins. The AuHA exhibits a surface enhanced Raman scattering (SERS) signal at 954 cm(-1) which can be used for its localization. The bio-sensing capacity of AuHA towards human skin epidermoid carcinoma (A431) and Chinese hamster ovary (CHO) cell lines is investigated using Raman microspectroscopic imaging. The localization of AuHA on cells is correlated with scanning electron microscopy, transmission electron microscopy and structured illumination fluorescence microscopy. This qualitative approach is a step towards a quantitative study of the proteins terminus. This method would enable further studies on the molecular profiling of the plasma membrane, in an attempt to provide accurate cell identification. Using a gold core-hydroxyapatite shell (AuHA) nanocomposite, the authors in this paper showed the feasibility of detecting and differentiating cell surface molecules by surface enhanced Raman scattering. Copyright © 2015 Elsevier Inc. All rights reserved.

Extrusion of amyloid fibrils to the extracellular space in experimental mesangial AL-amyloidosis: transmission and scanning electron microscopy studies and correlation with renal biopsy observations.

PubMed

Teng, Jiamin; Turbat-Herrera, Elba A; Herrera, Guillermo A

2014-04-01

In vitro studies have provided much information regarding the process of glomerular AL-amyloidogenesis. Research efforts have been successful in deciphering how glomerulopathic light chains interact with mesangial cells. The sequential steps involved in the genesis of amyloid fibrils include interactions with surface caveolae in mesangial cells and internalization of the monoclonal light chains through a clathrin-mediated process followed by trafficking in the mesangial cells to the mature lysosomal compartment where fibrils are formed. This manuscript focuses on how mesangial cells, once amyloid has been formed, deliver the fibrils to the extracellular matrix. The delivery of amyloid fibrils to the outside of the cells is carried out by lysosomes, which abut the mesangial cell membranes and extrude their contents into the extracellular space. This final step responsible for the fibrils to be present predominantly in the extracellular space is well demonstrated with scanning electron microscopy.

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.

Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

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

Mader, Jamie S.; Richardson, Angela; Salsman, Jayme

2007-07-15

Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused themore » death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB.« less

A transmission electron microscopy study of the diversity of Candida albicans cells induced by Euphorbia hirta L. leaf extract in vitro

PubMed Central

Basma, Abu Arra; Zuraini, Zakaria; Sasidharan, Sreenivasan

2011-01-01

Objective To determine the major changes in the microstructure of Candida albicans (C. albicans) after treatment with Euphorbia hirta (E. hirta) L. leaf extract. Methods Transmission electron microscopy was used to study the ultrastructural changes caused by E. hirta extract on C. albicans cells at various exposure time. Results It was found that the main abnormalities were the alterations in morphology, lysis and complete collapse of the yeast cells after 36 h of exposure to the extract. Whereas the control cultures showed a typical morphology of Candida with a uniform central density, typically structured nucleus, and a cytoplasm with several elements of endomembrane system and enveloped by a regular, intact cell wall. Conclusions The significant antifungal activity shown by this methanol extract of E. hirta L. suggests its potential against infections caused by C. albicans. The extract may be developed as an anticandidal agent. PMID:23569719

Three-Dimensional Human Bronchial-Tracheal Epithelial Tissue-Like Assemblies (TLAs) as Hosts for Severe Acute Respiratory Syndrome (SARS)-CoV Infection

NASA Technical Reports Server (NTRS)

Suderman, M. T.; McCarthy, M.; Mossell, E.; Watts, D. M.; Peters, C. J.; Shope, R.; Goodwin, T. J.

2006-01-01

A three-dimensional (3-D) tissue-like assembly (TLA) of human bronchial-tracheal mesenchymal (HBTC) cells with an overlay of human bronchial epithelial (BEAS-2B) cells was constructed using a NASA Bioreactor to survey the infectivity of SARS-CoV. This TLA was inoculated with a low passage number Urbani strain of SARS-CoV. At selected intervals over a 10-day period, media and cell aliquots of the 3-D TLA were harvested for viral titer assay and for light and electron microscopy examination. All viral titer assays were negative in both BEAS-2B two-dimensional monolayer and TLA. Light microscopy immunohistochemistry demonstrated antigen-antibody reactivity with anti-SARS-CoV polyclonal antibody to spike and nuclear proteins on cell membranes and cytoplasm. Coronavirus Group 2 cross-reactivity was demonstrated by positive reaction to anti-FIPV 1 and anti-FIPV 1 and 2 antibodies. TLA examination by transmission electron microscopy indicated increasing cytoplasmic vacuolation with numerous electron-dense bodies measuring 45 to 270 nm from days 4 through 10. There was no evidence of membrane blebbing, membrane duplication, or fragmentation of organelles in the TLAs. However, progressive disruption of endoplasmic reticulum was observed throughout the cells. Antibody response to SARS-CoV specific spike and nucleocapsid glycoproteins, cross-reactivity with FIPV antibodies, and the cytoplasmic pathology suggests this HBTE TLA model is permissive to SARS-CoV infection.

How do oocytes disappear?

PubMed

Bonilla-Musoles, F; Renau, J; Hernandez-Yago, J; Torres, J

1975-07-29

It has been study using transmission and scanner electron microscopy the mean procedures of dessaparence of the oocytes. On described three methods: 1. The necrosis of the oocytes. 2. The autolysis and fagocitosis by granulosa cells. 3. The migration of those to the superphicie and fall into the peritoneal cavity. Using the scanner electron microscopy in ovaries of fetus and newborn it seems the latest method to bee the most important during the intrauterine life. After the birth, this last phenomenon seems to disappear.

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.

Shedding light on cell compartmentation in the candidate phylum Poribacteria by high resolution visualisation and transcriptional profiling

NASA Astrophysics Data System (ADS)

Jahn, Martin T.; Markert, Sebastian M.; Ryu, Taewoo; Ravasi, Timothy; Stigloher, Christian; Hentschel, Ute; Moitinho-Silva, Lucas

2016-10-01

Assigning functions to uncultivated environmental microorganisms continues to be a challenging endeavour. Here, we present a new microscopy protocol for fluorescence in situ hybridisation-correlative light and electron microscopy (FISH-CLEM) that enabled, to our knowledge for the first time, the identification of single cells within their complex microenvironment at electron microscopy resolution. Members of the candidate phylum Poribacteria, common and uncultivated symbionts of marine sponges, were used towards this goal. Cellular 3D reconstructions revealed bipolar, spherical granules of low electron density, which likely represent carbon reserves. Poribacterial activity profiles were retrieved from prokaryotic enriched sponge metatranscriptomes using simulation-based optimised mapping. We observed high transcriptional activity for proteins related to bacterial microcompartments (BMC) and we resolved their subcellular localisation by combining FISH-CLEM with immunohistochemistry (IHC) on ultra-thin sponge tissue sections. In terms of functional relevance, we propose that the BMC-A region may be involved in 1,2-propanediol degradation. The FISH-IHC-CLEM approach was proven an effective toolkit to combine -omics approaches with functional studies and it should be widely applicable in environmental microbiology.

Spontaneous cytotoxic earthworm leukocytes kill K562 tumor cells.

PubMed

Suzuki, M M; Cooper, E L

1995-08-01

Earthworm coelomocytes may act as effector cells which destroy targets in vitro. In a 51Cr release assay, Lumbricus coelomocyte effectors showed lytic activities of 3-14% against K562 human tumor cells when incubated 1-4 hr at 23 degrees C or 37 degrees C. Cytotoxicity was correlated with effector: target ratio. However, targets were not killed by incubating them in cell-free, 0.2 micron filtered coelomic fluid. The supernatant from coelomocytes cultured alone failed to kill K562 targets but coelomocyte lysates were toxic to target cells in a concentration-dependent manner. Coelomocytes were examined using transmission electron microscopy (TEM) and scanning electron microscopy (SEM). When effectors and targets were examined under TEM, we found close apposition of effector granulocytic coelomocytes and target cell membranes but not with coelomocytes nor eleocytes at up to 15 min incubation. By SEM, effector cells appeared not only to be in close contact with targets, but instances of target lysis were observed. These results suggest that effector cell/target cell contact is essential for cytotoxicity to occur.

The role of 3D microenvironmental organization in MCF-7 epithelial–mesenchymal transition after 7 culture days

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

Foroni, Laura; Vasuri, Francesco, E-mail: vasurifrancesco@libero.it; Chair of Vascular Surgery, Department of Specialistic Surgery and Anaesthesiological Sciences, S. Orsola-Malpighi Hospital, Bologna University

2013-06-10

We present a multi-technique study on in vitro epithelial–mesenchymal transition (EMT) in human MCF-7 cells cultured on electrospun scaffolds of poly(L-lactic acid) (PLA), with random and aligned fiber orientations. Our aim is to investigate the morphological and genetic characteristics induced by extracellular matrix in tumor cells cultured in different 3D environments, and at different time points. Cell vitality was assessed with AlamarBlue at days 1, 3, 5 and 7. Scanning electron microscopy was performed at culture days 3 and 7. Immunohistochemistry (for E-cadherin, β-catenin, cytokeratins, nucleophosmin, tubulin, Ki-67 and vimentin), immunofluorescence (for F-actin) western blot (for E-cadherin, β-catenin and vimentin)more » and transmission electron microscopy were carried out at day 7. An EMT gene array followed by PCR analysis confirmed the regulation of selected genes. At day 7, scanning electron microscopy on aligned-PLA revealed spindle-shaped cells gathered in buds and ribbon-like structures, with a higher nucleolar/nuclear ratio and a loss in E-cadherin and β-catenin at immunohistochemistry and western blot. An up-regulation of SMAD2, TGF-β2, TFPI2 and SOX10 was found in aligned-PLA compared to random-PLA cultured cells. The topography of the extracellular matrix has a role in tumor EMT, and a more aggressive phenotype characterizes MCF-7 cells cultured on aligned-PLA scaffold. -- Highlights: • After 7 culture days an aligned-PLA scaffold induces a spindle shape to MCF-7 cells. • Despite these changes, the aligned MCF-7 cells keep an epithelial phenotype. • The extracellular environment alone influences the E-cadherin/β-catenin axis. • The extracellular environment can promote the epithelial–mesenchymal transition.« less

Reversible Cryopreservation of Living Cells Using an Electron Microscopy Cryo-Fixation Method.

PubMed

Huebinger, Jan; Han, Hong-Mei; Grabenbauer, Markus

2016-01-01

Rapid cooling of aqueous solutions is a useful approach for two important biological applications: (I) cryopreservation of cells and tissues for long-term storage, and (II) cryofixation for ultrastructural investigations by electron and cryo-electron microscopy. Usually, both approaches are very different in methodology. Here we show that a novel, fast and easy to use cryofixation technique called self-pressurized rapid freezing (SPRF) is-after some adaptations-also a useful and versatile technique for cryopreservation. Sealed metal tubes with high thermal diffusivity containing the samples are plunged into liquid cryogen. Internal pressure builds up reducing ice crystal formation and therefore supporting reversible cryopreservation through vitrification of cells. After rapid rewarming of pressurized samples, viability rates of > 90% can be reached, comparable to best-performing of the established rapid cooling devices tested. In addition, the small SPRF tubes allow for space-saving sample storage and the sealed containers prevent contamination from or into the cryogen during freezing, storage, or thawing.

Aldehyde dehydrogenase, Ald4p, is a major component of mitochondrial fluorescent inclusion bodies in the yeast Saccharomyces cerevisiae

PubMed Central

Misonou, Yoshiko; Kikuchi, Maiko; Sato, Hiroshi; Inai, Tomomi; Kuroiwa, Tsuneyoshi; Tanaka, Kenji; Miyakawa, Isamu

2014-01-01

ABSTRACT When Saccharomyces cerevisiae strain 3626 was cultured to the stationary phase in a medium that contained glucose, needle-like structures that emitted autofluorescence were observed in almost all cells by fluorescence microscopy under UV excitation. The needle-like structures completely overlapped with the profile of straight elongated mitochondria. Therefore, these structures were designated as mitochondrial fluorescent inclusion bodies (MFIBs). The MFIB-enriched mitochondrial fractions were successfully isolated and 2D-gel electrophoresis revealed that a protein of 54 kDa was only highly concentrated in the fractions. Determination of the N-terminal amino acid sequence of the 54-kDa protein identified it as a mitochondrial aldehyde dehydrogenase, Ald4p. Immunofluorescence microscopy showed that anti-Ald4p antibody specifically stained MFIBs. Freeze-substitution electron microscopy demonstrated that cells that retained MFIBs had electron-dense filamentous structures with a diameter of 10 nm in straight elongated mitochondria. Immunoelectron microscopy showed that Ald4p was localized to the electron-dense filamentous structures in mitochondria. These results together showed that a major component of MFIBs is Ald4p. In addition, we demonstrate that MFIBs are common features that appear in mitochondria of many species of yeast. PMID:24771619

Iron- and 4-hydroxy-2-alkylquinoline-containing periplasmic inclusion bodies of Pseudomonas aeruginosa: A chemical analysis

USGS Publications Warehouse

Royt, P.W.; Honeychuck, R.V.; Pant, R.R.; Rogers, M.L.; Asher, L.V.; Lloyd, J.R.; Carlos, W.E.; Belkin, H.E.; Patwardhan, S.

2007-01-01

Dark aggregated particles were seen on pellets of iron-rich, mid-logarithmic phase Pseudomonas aeruginosa. Transmission electron microscopy of these cells showed inclusion bodies in periplasmic vacuoles. Aggregated particles isolated from the spent medium of these cells contained iron as indicated by atomic absorption spectroscopy and by electron paramagnetic resonance spectroscopy that revealed Fe3+. Scanning electron microscopy/energy dispersive X-ray analysis of whole cells revealed the presence of iron-containing particles beneath the surface of the cell, indicating that the isolated aggregates were the intracellular inclusion bodies. Collectively, mass spectroscopy and nuclear magnetic resonance spectroscopy of the isolated inclusion bodies revealed the presence of 3,4-dihydroxy-2-heptylquinoline which is the Pseudomonas quinolone signaling compound (PQS) and an iron chelator; 4-hydroxy-2-heptylquinoline (pseudan VII), which is an iron chelator, antibacterial compound and precursor of PQS; 4-hydroxy-2-nonylquinoline (pseudan IX) which is an iron chelator and antibacterial compound; 4-hydroxy-2-methylquinoline (pseudan I), and 4-hydroxy-2-nonylquinoline N-oxide. ?? 2006 Elsevier Inc. All rights reserved.

Note on in situ (scanning) transmission electron microscopy study of liquid samples.

PubMed

Jiang, Nan

2017-08-01

Liquid cell (scanning) transmission electron microscopy has been developed rapidly, using amorphous SiN x membranes as electron transparent windows. The current interpretations of electron beam effects are mainly based on radiolytic processes. In this note, additional effects of the electric field due to electron-beam irradiation are discussed. The electric field can be produced by the charge accumulation due to the emission of secondary and Auger electrons. Besides various beam-induced phenomena, such as nanoparticle precipitation and gas bubble formation and motion, two other effects need to be considered; one is the change of Gibbs free energy of nucleation and the other is the violation of Brownian motion due to ion drifting driven by the electric field. Copyright © 2017 Elsevier B.V. All rights reserved.

Three-dimensional reconstruction of root cells and interdental cells in the rat inner ear by serial section scanning electron microscopy.

PubMed

Shodo, Ryusuke; Hayatsu, Manabu; Koga, Daisuke; Horii, Arata; Ushiki, Tatsuo

2017-01-01

In the cochlea, a high K + environment in the endolymph is essential for the maintenance of normal hearing function, and the transport of K + ions through gap junctions of the cochlear epithelium is thought to play an important role in endolymphatic homeostasis. The aim of the present study was to demonstrate the three-dimensional (3D) ultrastructure of spiral ligament root cells and interdental cells, which are located at both ends of the gap junction system of the cochlea epithelium. Serial semi-thin sections of plastic-embedded rat cochlea were mounted on glass slides, stained with uranyl acetate and lead citrate, and observed by scanning electron microscopy (SEM) using the backscattered electron (BSE) mode. 3D reconstruction of BSE images of serial sections revealed that the root cells were linked together to form a branched structure like an elaborate "tree root" in the spiral ligament. The interdental cells were also connected to each other, forming a comb-shaped cellular network with a number of cellular strands in the spiral limbus. Furthermore, TEM studies of ultra-thin sections revealed the rich presence of gap junctions in both root cells and interdental cells. These findings suggest the possibility that both root cells and interdental cells contribute to K + circulation as the end portion of the epithelial cell gap junction system of the cochlea.

Application of a quick-freezing and deep-etching method to pathological diagnosis: a case of elastofibroma.

PubMed

Hemmi, Akihiro; Tabata, Masahiko; Homma, Taku; Ohno, Nobuhiko; Terada, Nobuo; Fujii, Yasuhisa; Ohno, Shinichi; Nemoto, Norimichi

2006-04-01

A case of elastofibroma in a middle-aged Japanese woman was examined by the quick-freezing and deep-etching (QF-DE) method, as well as by immunohistochemistry and conventional electron microscopy. The slowly growing tumor developed at the right scapular region and was composed of fibrous connective tissue with unique elastic materials called elastofibroma fibers. A normal elastic fiber consists of a central core and peripheral zone, in which the latter has small aggregates of 10 nm microfibrils. By the QF-DE method, globular structures consisting of numerous fibrils (5-20 nm in width) were observed between the collagen bundles. We could confirm that they were microfibril-rich peripheral zones of elastofibroma fibers by comparing the replica membrane and conventional electron microscopy. One of the characteristics of elastofibroma fibers is that they are assumed to contain numerous microfibrils. Immunohistochemically, spindle tumor cells showed positive immunoreaction for vimentin, whereas alpha-smooth muscle actin, desmin, S-100 protein and CD34 showed negative immunoreaction. By conventional electron microscopy, the tumor cell had thin cytoplasmic processes, pinocytotic vesicles and prominent rough endoplasmic reticulum. Abundant intracytoplasmic filaments were observed in some tumor cells. Thick lamina-like structures along with their inner nuclear membrane were often observed in the tumor cell nuclei. The whole image of the tumor cell was considered to be a periosteal-derived cell, which would produce numerous microfibrils in the peripheral zone of elastofibroma fibers. This study indicated that the QF-DE method could be applied to the pathological diagnosis and analysis of pathomechanism, even for surgical specimens obtained from a patient.

Biosynthesis of lead nanoparticles by the aquatic water fern, Salvinia minima Baker, when exposed to high lead concentration.

PubMed

Castro-Longoria, E; Trejo-Guillén, K; Vilchis-Nestor, A R; Avalos-Borja, M; Andrade-Canto, S B; Leal-Alvarado, D A; Santamaría, J M

2014-02-01

Salvinia minima Baker is a small floating aquatic fern that is efficient for the removal and storage of heavy metals such as lead and cadmium. In this study, we report that lead removal by S. minima causes large accumulation of lead inside the cells in the form of nanoparticles (PbNPs). The accumulation pattern of lead was analyzed in both, submerged root-like modified fronds (here named "roots"), and in its aerial leaf-like fronds ("leaves"). Analysis by scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS), transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM) confirmed the biosynthesis of PbNPs by the plant. In both, roots and leaves, PbNPs were found to accumulate almost exclusively at the cell wall and closely associated to the cell membrane. Two types of PbNPs shapes were found in cells of both tissues, those associated to the cell wall were quasi-spherical with 17.2±4.2 nm of diameter, while those associated to the cell membrane/cytoplasm were elongated. Elongated particles were 53.7±29.6 nm in length and 11.1±2.4 nm wide. Infrared spectroscopy (IR) results indicate that cellulose, lignin and pectin are the major components that may be acting as the reducing agents for lead ions; these findings strongly suggest the potential use of this fern to further explore the bio-assisted synthesis of heavy metal nanostructures. Copyright © 2013 Elsevier B.V. All rights reserved.

Evaluation of Ultrasound-Induced Damage to Escherichia coli and Staphylococcus aureus by Flow Cytometry and Transmission Electron Microscopy

PubMed Central

Li, Jiao; Ahn, Juhee; Liu, Donghong; Chen, Shiguo; Ye, Xingqian

2016-01-01

As a nonthermal sterilization technique, ultrasound has attracted great interest in the field of food preservation. In this study, flow cytometry and transmission electron microscopy were employed to investigate ultrasound-induced damage to Escherichia coli and Staphylococcus aureus. For flow cytometry studies, single staining with propidium iodide (PI) or carboxyfluorescein diacetate (cFDA) revealed that ultrasound treatment caused cell death by compromising membrane integrity, inactivating intracellular esterases, and inhibiting metabolic performance. The results showed that ultrasound damage was independent of initial bacterial concentrations, while the mechanism of cellular damage differed according to the bacterial species. For the Gram-negative bacterium E. coli, ultrasound worked first on the outer membrane rather than the cytoplasmic membrane. Based on the double-staining results, we inferred that ultrasound treatment might be an all-or-nothing process: cells ruptured and disintegrated by ultrasound cannot be revived, which can be considered an advantage of ultrasound over other nonthermal techniques. Transmission electron microscopy studies revealed that the mechanism of ultrasound-induced damage was multitarget inactivation, involving the cell wall, cytoplasmic membrane, and inner structure. Understanding of the irreversible antibacterial action of ultrasound has great significance for its further utilization in the food industry. PMID:26746712

Brain Tissue Compartment Density Estimated Using Diffusion-Weighted MRI Yields Tissue Parameters Consistent With Histology

PubMed Central

Sepehrband, Farshid; Clark, Kristi A.; Ullmann, Jeremy F.P.; Kurniawan, Nyoman D.; Leanage, Gayeshika; Reutens, David C.; Yang, Zhengyi

2015-01-01

We examined whether quantitative density measures of cerebral tissue consistent with histology can be obtained from diffusion magnetic resonance imaging (MRI). By incorporating prior knowledge of myelin and cell membrane densities, absolute tissue density values were estimated from relative intra-cellular and intra-neurite density values obtained from diffusion MRI. The NODDI (neurite orientation distribution and density imaging) technique, which can be applied clinically, was used. Myelin density estimates were compared with the results of electron and light microscopy in ex vivo mouse brain and with published density estimates in a healthy human brain. In ex vivo mouse brain, estimated myelin densities in different sub-regions of the mouse corpus callosum were almost identical to values obtained from electron microscopy (Diffusion MRI: 42±6%, 36±4% and 43±5%; electron microscopy: 41±10%, 36±8% and 44±12% in genu, body and splenium, respectively). In the human brain, good agreement was observed between estimated fiber density measurements and previously reported values based on electron microscopy. Estimated density values were unaffected by crossing fibers. PMID:26096639

Enhanced human bone marrow mesenchymal stem cell functions on cathodic arc plasma-treated titanium

PubMed Central

Zhu, Wei; Teel, George; O’Brien, Christopher M; Zhuang, Taisen; Keidar, Michael; Zhang, Lijie Grace

2015-01-01

Surface modification of titanium for use in orthopedics has been explored for years; however, an ideal method of integrating titanium with native bone is still required to this day. Since human bone cells directly interact with nanostructured extracellular matrices, one of the most promising methods of improving titanium’s osseointegration involves inducing bio-mimetic nanotopography to enhance cell–implant interaction. In this regard, we explored an approach to functionalize the surface of titanium by depositing a thin film of textured titanium nanoparticles via a cathodic arc discharge plasma. The aim is to improve human bone marrow mesenchymal stem cell (MSC) attachment and differentiation and to reduce deleterious effects of more complex surface modification methods. Surface functionalization was analyzed by scanning electron microscopy, atomic force microscopy, contact angle testing, and specific protein adsorption. Scanning electron microscopy and atomic force microscopy examination demonstrate the deposition of titanium nanoparticles and the surface roughness change after coating. The specific fibronectin adsorption was enhanced on the modified titanium surface that associates with the improved hydrophilicity. MSC adhesion and proliferation were significantly promoted on the nanocoated surface. More importantly, compared to bare titanium, greater production of total protein, deposition of calcium mineral, and synthesis of alkaline phosphatase were observed from MSCs on nanocoated titanium after 21 days. The method described herein presents a promising alternative method for inducing more cell favorable nanosurface for improved orthopedic applications. PMID:26677327

Initial stem cell adhesion on porous silicon surface: molecular architecture of actin cytoskeleton and filopodial growth

PubMed Central

2014-01-01

The way cells explore their surrounding extracellular matrix (ECM) during development and migration is mediated by lamellipodia at their leading edge, acting as an actual motor pulling the cell forward. Lamellipodia are the primary area within the cell of actin microfilaments (filopodia) formation. In this work, we report on the use of porous silicon (pSi) scaffolds to mimic the ECM of mesenchymal stem cells from the dental pulp (DPSC) and breast cancer (MCF-7) cells. Our atomic force microscopy (AFM), fluorescence microscopy, and scanning electron microscopy (SEM) results show that pSi promoted the appearance of lateral filopodia protruding from the DPSC cell body and not only in the lamellipodia area. The formation of elongated lateral actin filaments suggests that pores provided the necessary anchorage points for protrusion growth. Although MCF-7 cells displayed a lower presence of organized actin network on both pSi and nonporous silicon, pSi stimulated the formation of extended cell protrusions. PMID:25386101

Membrane protein stoichiometry studied in intact mammalian cells using liquid-phase electron microscopy.

PubMed

DE Jonge, N

2018-02-01

Receptor membrane proteins in the plasma membranes of cells respond to extracellular chemical signals by conformational changes, spatial redistribution, and (re-)assembly into protein complexes, for example, into homodimers (pairs of the same protein type). The functional state of the proteins can be determined from information about how subunits are assembled into protein complexes. Stoichiometric information about the protein complex subunits, however, is generally not obtained from intact cells but from pooled material extracted from many cells, resulting in a lack of fundamental knowledge about the functioning of membrane proteins. First, functional states may dramatically differ from cell to cell on account of cell heterogeneity. Second, extracting the membrane proteins from the plasma membrane may lead to many artefacts. Liquid-phase scanning transmission electron microscopy (STEM), in short liquid STEM, is a new technique capable of determining the locations of individual membrane proteins within the intact plasma membranes of cells in liquid. Many tens of whole cells can readily be imaged. It is possible to analyse the stoichiometry of membrane proteins in single cells while accounting for heterogenic cell populations. Liquid STEM was used to image epidermal growth factor receptors in whole COS7 cells. A study of the dimerisation of the HER2 protein in breast cancer cells revealed the presence of rare cancer cells in which HER2 was in a different functional state than in the bulk cells. Stoichiometric information about receptors is essential not only for basic science but also for biomedical application because they present many important pharmaceutical targets. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

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.

Resistance in mango against infection by Ceratocystis fimbriata.

PubMed

Araujo, Leonardo; Bispo, Wilka Messner Silva; Cacique, Isaías Severino; Moreira, Wiler Ribas; Rodrigues, Fabrício Ávila

2014-08-01

This study was designed to characterize and describe host cell responses of stem tissue to mango wilt disease caused by the fungus Ceratocystis fimbriata in Brazil. Disease progress was followed, through time, in inoculated stems for two cultivars, 'Ubá' (field resistant) and 'Haden' (field susceptible). Stem sections from inoculated areas were examined using fluorescence light microscopy and transmission and scanning electron microscopy, coupled with energy-dispersive X-ray microanalysis. Tissues from Ubá colonized by C. fimbriata had stronger autofluorescence than those from Haden. The X-ray microanalysis revealed that the tissues of Ubá had higher levels of insoluble sulfur and calcium than those of Haden. Scanning electron microscopy revealed that fungal hyphae, chlamydospores (aleurioconidia), and perithecia-like structures of C. fimbriata were more abundant in Haden relative to Ubá. At the ultrastructural level, pathogen hyphae had grown into the degraded walls of parenchyma, fiber cells, and xylem vessels in the tissue of Haden. However, in Ubá, plant cell walls were rarely degraded and hyphae were often surrounded by dense, amorphous granular materials and hyphae appeared to have died. Taken together, the results of this study characterize the susceptible and resistant basal cell responses of mango stem tissue to infection by C. fimbriata.

The chromoplasts of Or mutants of cauliflower (Brassica oleracea L. var. botrytis).

PubMed

Paolillo, D J; Garvin, D F; Parthasarathy, M V

2004-12-01

The Or mutation in cauliflower (Brassica oleracea L. var. botrytis) leads to abnormal accumulations of beta-carotene in orange chromoplasts, in tissues in which leucoplasts are characteristic of wild-type plants. Or chromoplasts were investigated by light microscopy of fresh materials and electron microscopy of glutaraldehyde- and potassium permanganate-fixed materials. Carotenoid inclusions in Or chromoplasts resemble those found in carrot root chromoplasts in their optical activity and angular shape. Electron microscopy revealed that the inclusions are made up of parallel, membrane-bound compartments. These stacks of membranes are variously rolled and folded into three-dimensional objects. We classify Or chromoplasts as "membranous" chromoplasts. The Or mutation also limits plastid replication so that a single chromoplast constitutes the plastidome in most of the affected cells. There are one to two chromoplasts in each cell of a shoot apex. The ability of differentiated chromoplasts to divide in the apical meristems of Or mutant plants resembles the ability of proplastids to maintain plastid continuity from cell to cell in meristems of Arabidopsis thaliana mutants in which plastid replication is drastically limited. The findings are used to discuss the number of levels of regulation involved in plastid replication.

Influence of dichloromethylene bisphosphonate on the in vitro phagocytosis of hydroxyapatite particles by rat peritoneal exudate cells: an electron microscopic and chemiluminescence study.

PubMed Central

Hyvönen, P M; Kowolik, M J

1992-01-01

Transmission electron microscopy and standard chemiluminescence assays were used to investigate the in vivo effect of dichloromethylene bisphosphonate (clodronate) on the phagocytosis of pure hydroxyapatite particles by rat peritoneal macrophages and the production of chemiluminescence by the peritoneal exudate cells. Hydroxyapatite (control) and a hydroxyapatite/clodronate suspension (28 mumol clodronate per gram of hydroxyapatite, experimental) were injected into the peritoneum of rats, the clodronate dose being 10 micrograms/kg. Macrophages were harvested at 12, 24, 48, and 96 hours after injection and the particle phagocytosis was assessed by transmission electron microscopy. Hydroxyapatite alone was completely phagocytosed by 24 hours and hydroxyapatite reacted with clodronate was completely phagocytosed by 48 hours. From 48 hours onwards hydroxyapatite particle dissolution was observed in the phagosomes of cells in the two groups. At 48 hours the chemiluminescence produced by the peritoneal exudate cells was also measured. Clodronate and clodronate/hydroxyapatite enhanced cell activity on subsequent challenge with phorbol myristate acetate or zymosan. Clodronate seemed to exhibit an inhibitory effect on the phagocytic activity and an enhancement of the chemiluminescence production by the cells in this model, indicating that it was modifying the inflammatory cell response. Images PMID:1532298

Respiratory adenovirus-like infection in a rose-ringed parakeet (Psittacula krameri).

PubMed

Desmidt, M; Ducatelle, R; Uyttebroek, E; Charlier, G; Hoorens, J

1991-01-01

Intranuclear inclusions were observed under light microscopy in the bronchial epithelial cells of a recently purchased female rose-ringed parakeet that died of chlamydiosis. Transmission electron microscopy revealed the presence of numerous particles of adenovirus morphology. A latent adenovirus infection may have become more severe following chlamydiosis and the stress of handling.

Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.

PubMed

Bharat, Tanmay A M; Hoffmann, Patrick C; Kukulski, Wanda

2018-04-10

Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation. Copyright © 2018 MRC Laboratory of Molecular Biology. Published by Elsevier Ltd.. All rights reserved.

Morphological Analysis of Human Induced Pluripotent Stem Cells During Induced Differentiation and Reverse Programming

PubMed Central

Magniez, Aurélie; Oudrhiri, Noufissa; Féraud, Olivier; Bacci, Josette; Gobbo, Emilie; Proust, Stéphanie; Turhan, Ali G.

2014-01-01

Abstract The fine analysis of cell components during the generation of pluripotent cells and their comparison to bone fide human embryonic stem cells (hESCs) are valuable tools to understand their biological behavior. In this report, human mesenchymal cells (hMSCs) generated from the human ES cell line H9, were reprogrammed back to induced pluripotent state using Oct-4, Sox2, Nanog, and Lin28 transgenes. Human induced pluripotent stem cells (hIPSCs) were analyzed using electron microscopy and compared with regard to the original hESCs and the hMSCs from which they were derived. This analysis shows that hIPSCs and the original hESCs are morphologically undistinguishable but differ from the hMSCs with respect to the presence of several morphological features of undifferentiated cells at both the cytoplasmic (ribosomes, lipid droplets, glycogen, scarce reticulum) and nuclear levels (features of nuclear plasticity, presence of euchromatin, reticulated nucleoli). We show that hIPSC colonies generated this way presented epithelial aspects with specialized junctions highlighting morphological criteria of the mesenchymal–epithelial transition in cells engaged in a successful reprogramming process. Electron microscopic analysis revealed also specific morphological aspects of partially reprogrammed cells. These results highlight the valuable use of electron microscopy for a better knowledge of the morphological aspects of IPSC and cellular reprogramming. PMID:25371857

Surface topography of hairy cell leukemia cells compared to other leukemias as seen by scanning electron microscopy.

PubMed

Polliack, Aaron; Tadmor, Tamar

2011-06-01

This short review deals with the ultrastructural surface architecture of hairy cell leukemia (HCL) compared to other leukemic cells, as seen by scanning electron microscopy (SEM). The development of improved techniques for preparing blood cells for SEM in the 1970s readily enabled these features to be visualized more accurately. This review returns us to the earlier history of SEM, when the surface topography of normal and neoplastic cells was visualized and reported for the first time, in an era before the emergence and use of monoclonal antibodies and flow cytometry, now used routinely to define cells by their immunophenotype. Surface microvilli are characteristic for normal and leukemic lymphoid cells, myelo-monocytic cells lack microvilli and show surface ruffles, while leukemic plasma and myeloma cells and megakaryocytes display large surface blebs. HCL cell surfaces are complex and typically 'hybrid' in nature, displaying both lymphoid and monocytic features with florid ruffles of varying sizes interspersed with clumps of short microvilli cytoplasm. The surface features of other leukemic cells and photomicrographs of immuno-SEM labeling of cells employing antibodies and colloidal gold, reported more than 20 years ago, are shown.

Electron microscopy of vesicular-arbuscular mycorrhizae of yellow poplar. II. Intracellular hyphae and vesicles.

PubMed

Kinden, D A; Brown, M F

1975-11-01

Intracellular hyphae and vesicles in mycorrhizal roots of yellow poplar were examined by electron microscopy. An investing layer of host wall material and cytoplasm enclosed the endophyte within the cells. Young developing hyphae contained abundant cytoplasm and few vacuoles. As hyphae matured, they became highly vacuolated and accumulated carbohydrate (glycogen) and lipid reserves. Mature vesicles were engorged with lipid droplets, possessed a trilaminate wall and were also enclosed by host wall material and cytoplasm. Compared with uninfected cells, infected cortical cells showed an increase in cytoplasmic volume, enlarged nuclei, and a reduction of starch reserves. Host nuclei were always proximal to the hyphae during hyphal development and deterioration. While other cytoplasmic components of infected and uninfected cells were comparable large electron-dense bodies occurred in vacuoles of most cells containing hyphae. Deterioration of intracellular hyphae occurred throughout the samples examined. Septa separated functional and degenerating portions of the hyphae. Hyphal deterioration involved degeneration and ultimate disappearance of fungal cytoplasm as well as collapse of hyphal walls. Based on these observations, the authors hypothesize that deterioration of the endophyte may release significant quantities of mineral nutrients, via hyphal contents, which are absorbed by the host.

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.

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.

Protein-accumulating cells and dilated cisternae of the endoplasmic reticulum in three glucosinolate-containing genera: Armoracia, Capparis, Drypetes.

PubMed

Jørgensen, L B; Behnke, H D; Mabry, T J

1977-01-01

Three glucosinolate-containing species, Armoracia rusticana Gaertner, Meyer et Scherbius (Brassicaceae), Capparis cynophallophora L. (Capparaceae) and Drypetes roxburghii (Wall.) Hurusawa (Euphorbiaceae), are shown by both light and electron microscopy to contain protein-accumulating cells (PAC). The PAC of Armoracia and Copparis (former "myrosin cells") occur as idioblasts. The PAC of Drypetes are usual members among axial phloem parenchyma cells rather than idioblasts. In Drypetes the vacuoles of the PAC are shown ultrastructurally to contain finely fibrillar material and to originate from local dilatations of the endoplasmic reticulum. The vacuoles in PAC of Armoracia and Capparis seem to originate in the same way; but ultrastructurally, their content is finely granular. In addition, Armoracia and Capparis are shown by both light and electron microscopy to contain dilated cisternae (DC) of the endoplasmic reticulum in normal parenchyma cells, in accord with previous findings for several species within Brassicaceae. The relationship of PAC and DC to glucosinolates and the enzyme myrosinase is discussed.

Immunomicrospheres - Reagents for cell labeling and separation

NASA Technical Reports Server (NTRS)

Rembaum, A.; Dreyer, W. J.

1980-01-01

Immunomicrospheres are specially designed microscopic particles that have antibodies or similar molecules chemically bound to their surfaces. The antibody-coated microspheres react in a highly specific way with target cells, viruses, or other antigenic agents. Immunomicrospheres may be synthesized so that they incorporate compounds that are highly radioactive, intensely fluorescent, magnetic, electron opaque, highly colored, or pharmacologically active. These various types of microspheres may be coated with pure, highly specific monoclonal antibodies obtained by the new hybridoma cell cloning techniques or with conventional antibody preparations. Some of the many present and potential applications for these new reagents are (1) new types of radioimmune or immunofluorescent assays, (2) improved fluorescence microscopy, (3) separation of cells on the basis of the fluorescent, electrophoretic, or magnetic properties of bound immunomicrospheres, (4) markers for use in several types of electron or standard light microscopy, and (5) delivery of lethal compouds to specific undesirable living cells. The combination of the various new types of synthetic microspheres and the newly available homogeneous antibodies offers new opportunities in research, diagnosis, and therapy.

Neural differentiation of transplanted neural stem cells in a rat model of striatal lacunar infarction: light and electron microscopic observations

PubMed Central

Muñetón-Gómez, Vilma C.; Doncel-Pérez, Ernesto; Fernandez, Ana P.; Serrano, Julia; Pozo-Rodrigálvarez, Andrea; Vellosillo-Huerta, Lara; Taylor, Julian S.; Cardona-Gómez, Gloria P.; Nieto-Sampedro, Manuel; Martínez-Murillo, Ricardo

2012-01-01

The increased risk and prevalence of lacunar stroke and Parkinson's disease (PD) makes the search for better experimental models an important requirement for translational research. In this study we assess ischemic damage of the nigrostriatal pathway in a model of lacunar stroke evoked by damaging the perforating arteries in the territory of the substantia nigra (SN) of the rat after stereotaxic administration of endothelin-1 (ET-1), a potent vasoconstrictor peptide. We hypothesized that transplantation of neural stem cells (NSCs) with the capacity of differentiating into diverse cell types such as neurons and glia, but with limited proliferation potential, would constitute an alternative and/or adjuvant therapy for lacunar stroke. These cells showed neuritogenic activity in vitro and a high potential for neural differentiation. Light and electron microscopy immunocytochemistry was used to characterize GFP-positive neurons derived from the transplants. 48 h after ET-1 injection, we characterized an area of selective degeneration of dopaminergic neurons within the nigrostriatal pathway characterized with tissue necrosis and glial scar formation, with subsequent behavioral signs of Parkinsonism. Light microscopy showed that grafted cells within the striatal infarction zone differentiated with a high yield into mature glial cells (GFAP-positive) and neuron types present in the normal striatum. Electron microscopy revealed that NSCs-derived neurons integrated into the host circuitry establishing synaptic contacts, mostly of the asymmetric type. Astrocytes were closely associated with normal small-sized blood vessels in the area of infarct, suggesting a possible role in the regulation of the blood brain barrier and angiogenesis. Our results encourage the use of NSCs as a cell-replacement therapy for the treatment of human vascular Parkinsonism. PMID:22876219

The comparative safety of genipin versus UVA-riboflavin crosslinking of rabbit corneas

PubMed Central

Song, Wenjing; Tang, Yun; Qiao, Jing; Li, Haili; Rong, Bei; Yang, Songlin; Wu, Yuan

2017-01-01

Purpose To investigate, after 24 h, the safety of genipin or ultraviolet A (UVA)-riboflavin crosslinking of keratocytes and endothelial cells. Methods Fifteen New Zealand white rabbits were selected and divided into a PBS group (five rabbits), a 0.2% genipin crosslinking (GP-CXL) group (five rabbits), and a UVA-riboflavin crosslinking (UVA-CXL) group (five rabbits). In the GP-CXL and PBS groups, 0.2% genipin or PBS was applied to the corneal surface of the right eyes. In the UVA-CXL group, a clinical crosslinking procedure was used. Before and after surgery, the operated eyes of each group were characterized with confocal microscopy, and the corneal buttons were excised for endothelium staining and electron microscopy. Results The corneal endothelial cell density of the GP-CXL, UVA-CLX, and PBS groups changed. There was a statistically significant difference in thickness and changes in corneal endothelial cell density between the UVA-CXL group and the PBS group (p<0.05), and between the UVA-CXL group and the GP-CXL group (p<0.05), but no statistically significant difference between the GP-CXL group and the PBS group. Confocal microscopy, transmission electron microscopy, and hematoxylin and eosin staining showed that there was keratocyte apoptosis in the anterior and middle stroma and endothelial cell damage in the UVA-CXL group. In the GP-CXL group, only active keratocytes were found and minimal endothelial cell damage. Conclusions Treatment of rabbit corneas with 0.2% genipin showed minimal toxicity toward keratocytes and endothelial cells. Genipin is safer than UVA-CXL for crosslinking of thin corneas. PMID:28761323

Transmission and Scanning Electron Microscopy of the Accessory Cells and Chorion During Development of Ciona intestinalis Type B Embryos and the Impact of Their Removal on Cell Morphology.

PubMed

Thompson, Helen; Shimeld, Sebastian M

2015-06-01

Spawned ascidian oocytes are surrounded by a membrane called the chorion (or vitelline coat) and associated with two populations of maternally-supplied cells. Outside the chorion are follicle cells, which may affect the buoyancy of eggs. Inside the chorion are test cells, which during oogenesis provision the egg and which after fertilisation contribute to the larval tunic. The structure of maternal cells may vary between species. The model ascidian Ciona intestinalis has been recently split into two species, currently named type A and type B. The ultrastructure of extraembryonic cells and structures from type A embryos has been reported. Here we describe the ultrastructure of follicle and test cells from C. intestinalis type B embryos. Test cells are about 5 µm in diameter and line the inside of the chorion of developing embryos in a dense sheet. Follicle cells are large (> 100 µm long) and spike-shaped, with many large vesicles. Terminal electron dense granules are found towards the tips of spikes, adjacent to cytoplasm containing numerous small electron dense bodies connected by filaments. These are probably vesicles containing material for the terminal granules. Removal of maternal structures and cells just after fertilisation, as commonly used in many experiments manipulating C. intestinalis development, has been reported to affect embryonic patterning. We examined the impact of this on embryonic ectoderm cells by scanning electron microscopy. Cells of embryos that developed without maternal structures still developed cilia, but had indistinct cell boundaries and a more flattened appearance than those that developed within the chorion.

Osteoblast-like cell response to macro- and micro-patterned carbon scaffolds obtained from the sea rush Juncus maritimus.

PubMed

López-Álvarez, M; Pereiro, I; Serra, J; de Carlos, A; González, P

2011-08-01

Carbon scaffolds with a directional patterned surface were obtained by pyrolysis of the sea rush Juncus maritimus. The structure of the scaffolds was investigated using scanning electron microscopy, mercury porosimetry and interferometric profilometry. X-ray diffraction and x-ray fluorescence were the techniques used for their chemical characterization. The alignment and differentiation of pre-osteoblasts (MC3T3-E1 cell line) incubated on the patterned scaffolds were evaluated by scanning electron microscopy, confocal laser scanning microscopy and by the quantification of the phosphatase alkaline activity and the osteocalcin synthesis. It was found that pyrolysis at 500 °C preserved and even enhanced the natural macro- and micro-patterning of the plant. The results obtained for porosity and chemical composition validated these structures as viable scaffolds for tissue engineering applications. Finally, the patterned surface was confirmed to promote the oriented growth of the pre-osteoblasts MC3T3-E1, not only after short periods of incubation (hours) but also after longer ones (several weeks). The quantification of the cell differentiation markers together with the evaluation of the cell layer morphology up to 28 days of incubation confirmed the differentiation of MC3T3-E1 cells to osteoblasts. © 2011 IOP Publishing Ltd

Absorption and Effect of Azaspiracid-1 Over the Human Intestinal Barrier.

PubMed

Abal, Paula; Louzao, M Carmen; Fraga, María; Vilariño, Natalia; Ferreiro, Sara; Vieytes, Mercedes R; Botana, Luis M

2017-01-01

Azaspiracids (AZAs) are marine biotoxins produced by the dinoflagellates genera Azadinium and Amphidoma. These toxins cause azaspiracid poisoning (AZP), characterized by severe gastrointestinal illness in humans after the consumption of bivalve molluscs contaminated with AZAs. The main aim of the present study was to examine the consequences of human exposure to AZA1 by the study of absorption and effects of the toxin on Caco-2 cells, a reliable model of the human intestine. The ability of AZA1 to cross the human intestinal epithelium has been evaluated by the Caco-2 transepithelial permeability assay. The toxin has been detected and quantified using a microsphere-based immunoassay. Cell alterations and ultrastructural effects has been observed with confocal and transmission electron microscopy Results: AZA1 was absorbed by Caco-2 cells in a dose-dependent way without affecting cell viability. However, modifications on occludin distribution detected by confocal microscopy imaging indicated a possible monolayer integrity disruption. Nevertheless, transmission electron microscopy imaging revealed ultrastructural damages at the nucleus and mitochondria with autophagosomes in the cytoplasm, however, tight junctions and microvilli remained unaffected. After the ingestion of molluscs with the AZA1, the toxin will be transported through the human intestinal barrier to blood causing damage on epithelial cells. © 2017 The Author(s). Published by S. Karger AG, Basel.

Reflections on the value of electron microscopy in the study of heterogeneous catalysts

PubMed Central

2017-01-01

Electron microscopy (EM) is arguably the single most powerful method of characterizing heterogeneous catalysts. Irrespective of whether they are bulk and multiphasic, or monophasic and monocrystalline, or nanocluster and even single-atom and on a support, their structures in atomic detail can be visualized in two or three dimensions, thanks to high-resolution instruments, with sub-Ångstrom spatial resolutions. Their topography, tomography, phase-purity, composition, as well as the bonding, and valence-states of their constituent atoms and ions and, in favourable circumstances, the short-range and long-range atomic order and dynamics of the catalytically active sites, can all be retrieved by the panoply of variants of modern EM. The latter embrace electron crystallography, rotation and precession electron diffraction, X-ray emission and high-resolution electron energy-loss spectra (EELS). Aberration-corrected (AC) transmission (TEM) and scanning transmission electron microscopy (STEM) have led to a revolution in structure determination. Environmental EM is already playing an increasing role in catalyst characterization, and new advances, involving special cells for the study of solid catalysts in contact with liquid reactants, have recently been deployed. PMID:28265196

Ultrastructural Characterization of the Glomerulopathy in Alport Mice by Helium Ion Scanning Microscopy (HIM).

PubMed

Tsuji, Kenji; Suleiman, Hani; Miner, Jeffrey H; Daley, James M; Capen, Diane E; Păunescu, Teodor G; Lu, Hua A Jenny

2017-09-15

The glomerulus exercises its filtration barrier function by establishing a complex filtration apparatus consisting of podocyte foot processes, glomerular basement membrane and endothelial cells. Disruption of any component of the glomerular filtration barrier leads to glomerular dysfunction, frequently manifested as proteinuria. Ultrastructural studies of the glomerulus by transmission electron microscopy (TEM) and conventional scanning electron microscopy (SEM) have been routinely used to identify and classify various glomerular diseases. Here we report the application of newly developed helium ion scanning microscopy (HIM) to examine the glomerulopathy in a Col4a3 mutant/Alport syndrome mouse model. Our study revealed unprecedented details of glomerular abnormalities in Col4a3 mutants including distorted podocyte cell bodies and disorganized primary processes. Strikingly, we observed abundant filamentous microprojections arising from podocyte cell bodies and processes, and presence of unique bridging processes that connect the primary processes and foot processes in Alport mice. Furthermore, we detected an altered glomerular endothelium with disrupted sub-endothelial integrity. More importantly, we were able to clearly visualize the complex, three-dimensional podocyte and endothelial interface by HIM. Our study demonstrates that HIM provides nanometer resolution to uncover and rediscover critical ultrastructural characteristics of the glomerulopathy in Col4a3 mutant mice.

Confocal laser scanning microscopy coupled to a spectrofluorometric detector as a rapid tool for determining the in vivo effect of metals on phototrophic bacteria.

PubMed

Burnat, Mireia; Diestra, Elia; Esteve, Isabel; Solé, Antonio

2010-01-01

In this paper, we determine for the first time the in vivo effect of heavy metals in a phototrophic bacterium. We used Confocal Laser Scanning Microscopy coupled to a spectrofluorometric detector as a rapid technique to measure pigment response to heavy-metal exposure. To this end, we selected lead and copper (toxic and essential metals) and Microcoleus sp. as the phototrophic bacterium because it would be feasible to see this cyanobacterium as a good biomarker, since it covers large extensions of coastal sediments. The results obtained demonstrate that, while cells are still viable, pigment peak decreases whereas metal concentration increases (from 0.1 to 1 mM Pb). Pigments are totally degraded when cultures were polluted with lead and copper at the maximum doses used (25 mM Pb(NO(3))(2) and 10 mM CuSO(4)). The aim of this study was also to identify the place of metal accumulation in Microcoleus cells. Element analysis of this cyanobacterium in the above mentioned conditions determined by Energy Dispersive X-ray microanalysis (EDX) coupled to Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM), shows that Pb (but not Cu) accumulates externally and internally in cells.

Development of flange and reticulate wall ingrowths in maize (Zea mays L.) endosperm transfer cells.

PubMed

Monjardino, Paulo; Rocha, Sara; Tavares, Ana C; Fernandes, Rui; Sampaio, Paula; Salema, Roberto; da Câmara Machado, Artur

2013-04-01

Maize (Zea mays L.) endosperm transfer cells are essential for kernel growth and development so they have a significant impact on grain yield. Although structural and ultrastructural studies have been published, little is known about the development of these cells, and prior to this study, there was a general consensus that they contain only flange ingrowths. We characterized the development of maize endosperm transfer cells by bright field microscopy, transmission electron microscopy, and confocal laser scanning microscopy. The most basal endosperm transfer cells (MBETC) have flange and reticulate ingrowths, whereas inner transfer cells only have flange ingrowths. Reticulate and flange ingrowths are mostly formed in different locations of the MBETC as early as 5 days after pollination, and they are distinguishable from each other at all stages of development. Ingrowth structure and ultrastructure and cellulose microfibril compaction and orientation patterns are discussed during transfer cell development. This study provides important insights into how both types of ingrowths are formed in maize endosperm transfer cells.

Morphological and ultrastructural characterization of ionoregulatory cells in the teleost Oreochromis niloticus following salinity challenge combining complementary confocal scanning laser microscopy and transmission electron microscopy using a novel prefixation immunogold labeling technique.

PubMed

Fridman, Sophie; Rana, Krishen J; Bron, James E

2013-10-01

Aspects of ionoregulatory or mitochondria-rich cell (MRC) differentiation and adaptation in Nile tilapia yolk-sac larvae following transfer from freshwater to elevated salinities, that is, 12.5 and 20 ppt are described. Investigations using immunohistochemistry on whole-mount Nile tilapia larvae using anti- Na⁺/K⁺-ATPase as a primary antibody and Fluoronanogold™ (Nanoprobes) as a secondary immunoprobe allowed fluorescent labeling with the high resolution of confocal scanning laser microscopy combined with the detection of immunolabeled target molecules at an ultrastructural level using transmission electron microscopy (TEM). It reports, for the first time, various developmental stages of MRCs within the epithelial layer of the tail of yolk-sac larvae, corresponding to immature, developing, and mature MRCs, identifiable by their own characteristic ultrastructure and form. Following transfer to hyperosmotic salinities the density of immunogold particles and well as the intricacy of the tubular system appeared to increase. In addition, complementary confocal scanning laser microscopy allowed identification of immunopositive ramifying extensions that appeared to emanate from the basolateral portion of the cell that appeared to be correlated with the localization of subsurface tubular areas displaying immunogold labeled Na⁺/K⁺-ATPase. This integrated approach describes a reliable and repeatable prefixation immunogold labeling technique allowing precise visualization of NaK within target cells combined with a 3D imaging that offers valuable insights into MRC dynamics at an ultrastructural level. Copyright © 2013 Wiley Periodicals, Inc.

Light and electron microscopic observations of the reproductive swarmer cells of nassellarian and spumellarian polycystines (Radiolaria).

PubMed

Yuasa, Tomoko; Takahashi, Osamu

2016-06-01

We observed reproductive swarmer cells of the nassellarian and spumellarian polycystine radiolarians Didymocyrtis ceratospyris, Pterocanium praetextum, Tetrapyle sp., and Triastrum aurivillii using light, scanning and transmission electron microscopy. The swarmer cells had subspherical to ovoid or spindle shapes with two unequal flagella tapered to whip-like ends. The cell size was approximately 2.5-5.5μm long and 1.6-2.2μm wide, which is significantly smaller than that of the collodarian (colonial or naked) polycystine radiolarians. Transmission electron microscopy revealed that the swarmer cells possessed a nucleus, mitochondria with tubular cristae, Golgi body, and small lipid droplets in the cytoplasm; they also had a large vacuole in which a single crystalline inclusion (approx. 1.0-1.5μm) that was probably celestite (SrSO4) was enclosed. The swarmer cells were released directly from the parent cells. At that time, morphological change such as encystment was not observed in the parent cells, and the axopodia remained extended in a period of swarmer reproduction for floating existence. This may have prevented the polycystine swarmers from rapidly sinking down to great depths. Thus, we concluded that the polycystine radiolarians release the swarmer cells into the photic layer in the same way as the symbiotic acantharians. Copyright © 2016 Elsevier GmbH. All rights reserved.

Calcium phosphate nanoparticles as versatile carrier for small and large molecules across cell membranes

NASA Astrophysics Data System (ADS)

Sokolova, Viktoriya; Rotan, Olga; Klesing, Jan; Nalbant, Perihan; Buer, Jan; Knuschke, Torben; Westendorf, Astrid M.; Epple, Matthias

2012-06-01

The successful transport of molecules across the cell membrane is a key point in biology and medicine. In most cases, molecules alone cannot penetrate the cell membrane, therefore an efficient carrier is needed. Calcium phosphate nanoparticles (diameter: 100-250 nm, depending on the functionalization) were loaded with fluorescent oligonucleotides, peptide, proteins, antibodies, polymers or porphyrins and characterized by dynamic light scattering, nanoparticle tracking analysis and scanning electron microscopy. Any excess of molecules was removed by ultracentrifugation, and the dissolved molecules at the same concentration were used as control. The uptake of such fluorescence-labeled nanoparticles into HeLa cells was monitored by fluorescence microscopy and confocal laser scanning microscopy. Calcium phosphate nanoparticles were able to transport all molecules across the cell membrane, whereas the dissolved molecules alone were taken up only to a very small extent or even not at all.

Synthesis and study of photovoltaic performance on various photoelectrode materials for DSSCs: Optimization of compact layer on nanometer thickness

NASA Astrophysics Data System (ADS)

Surya, Subramanian; Thangamuthu, Rangasamy; Senthil Kumar, Sakkarapalayam Murugesan; Murugadoss, Govindhasamy

2017-02-01

Dye-sensitized solar cells (DSSCs) have gained widespread attention in recent years because of their low production costs, ease of fabrication process and tuneable optical properties, such as colour and transparency. In this work, we explored a strategy wherein nanoparticles of pure TiO2, TiO2sbnd SnO2 nanocomposite, Sn (10%) doped TiO2 and SnO2 synthesized by the simple chemical precipitation method were employed as photoelectrodes to enhance the photovoltaic conversion efficiency of solar cells. The nanoparticles were characterized by different characterization techniques such as X-ray diffraction (XRD), scanning electron microscopy (SEM with EDX), transmission electron microscopy (TEM), high resolution electron microscopy (HR-TEM), UV-Visible absorbance (UV-vis), photoluminescence (PL), thermal gravimetric analysis (TGA) and X-ray photoelectron spectroscopy (XPS) measurements. Moreover, we also demonstrated the effect of thin compact layer in DSSCs by architecture with various precursor materials of different concentrations. We found that the optimized compact layer material TDIP (titanium diisopropoxide) with a concentration of 0.3 M % is produced the highest efficiency of 2.25% for Sn (10%) doped TiO2 electron transport material (ETM) and 4.38% was achieved for pure TiO2 ETM using SnCl2 compact layer with 0.1 M concentrations.

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.

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.

METABOLIC AND MORPHOLOGICAL OBSERVATIONS ON THE EFFECT OF SURFACE-ACTIVE AGENTS ON LEUKOCYTES

PubMed Central

Graham, R. C.; Karnovsky, M. J.; Shafer, A. W.; Glass, E. A.; Karnovsky, Manfred L.

1967-01-01

Morphological and metabolic observations have been made on the effects of endotoxin, deoxycholate, and digitonin (at less than 50 µg/ml) on polymorphonuclear leukocytes and mononuclear cells. The agents stimulate the respiration and glucose oxidation of these cells in a manner similar to that seen during phagocytosis. Electron microscopy revealed no morphological changes with the first two agents, but dramatic membrane changes were seen in the case of digitonin. Here tubular projections of characteristic size and shape formed on and split off the membrane. All the agents stimulated uptake of inulin, but efforts to demonstrate increased pinocytosis by electron microscopy have not so far succeeded, probably due to limitations in present experimental techniques. PMID:6034482

Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

NASA Astrophysics Data System (ADS)

Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

2016-03-01

Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

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

Suggestive evidence of a vesicle-mediated mode of cell degranulation in chromaffin cells. A high-resolution scanning electron microscopy investigation

PubMed Central

Crivellato, Enrico; Solinas, Paola; Isola, Raffaella; Ribatti, Domenico; Riva, Alessandro

2010-01-01

In this study we used a modified osmium maceration method for high-resolution scanning electron microscopy to study some ultrastructural details fitting the schema of piecemeal degranulation in chromaffin cells. Piecemeal degranulation refers to a particulate pattern of cell secretion that is accomplished by vesicle-mediated extracellular transport of granule-stored material. We investigated adrenal samples from control and angiotensin II-treated rats, and identified a variable proportion of smooth, 30–60-nm-diameter vesicles in the cytoplasm of chromaffin cells. A percentage of these vesicles were interspersed in the cytosol among chromaffin granules but the majority appeared to be attached to granules. Remarkably, the number of unattached cytoplasmic vesicles was greatly increased in chromaffin cells from angiotensin II-treated animals. Vesicles of the same structure and dimension were detected close to or attached to the cytoplasmic face of the plasma membrane; these, too, were increased in number in chromaffin cells from rats stimulated with angiotensin II. In specimens shaken with a rotating agitator during maceration, the cytoplasmic organelles could be partially removed and the fine structure of the vesicular interaction with the inner side of the plasma membrane emerged most clearly. A proportion of chromaffin granules showed protrusions that we interpreted as vesicular structures budding from the granular envelope. In some instances, the transection plane intersected granules with putative vesicles emerging from the surfaces. In these cases, the protrusions of budding vesicles could be observed from the internal side. This study provides high-resolution scanning electron microscopy images compatible with a vesicle-mediated degranulation mode of cell secretion in adrenal chromaffin cells. The data indicating an increase in the number of vesicles observed in chromaffin cells after stimulation with the chromaffin cell secretagogue angiotensin II suggests that this secretory process may be susceptible to fine regulation. PMID:20136671

Extending the knowledge in histochemistry and cell biology.

PubMed

Heupel, Wolfgang-Moritz; Drenckhahn, Detlev

2010-01-01

Central to modern Histochemistry and Cell Biology stands the need for visualization of cellular and molecular processes. In the past several years, a variety of techniques has been achieved bridging traditional light microscopy, fluorescence microscopy and electron microscopy with powerful software-based post-processing and computer modeling. Researchers now have various tools available to investigate problems of interest from bird's- up to worm's-eye of view, focusing on tissues, cells, proteins or finally single molecules. Applications of new approaches in combination with well-established traditional techniques of mRNA, DNA or protein analysis have led to enlightening and prudent studies which have paved the way toward a better understanding of not only physiological but also pathological processes in the field of cell biology. This review is intended to summarize articles standing for the progress made in "histo-biochemical" techniques and their manifold applications.

Mast cell dynamics in the house rat (Rattus rattus) ovary during estrus cycle, pregnancy and lactation.

PubMed

Batth, B K; Parshad, R K

2000-02-01

The distribution of mast cells in various ovarian compartments was studied during different stages of the reproductive cycles in Rattus rattus. Two types of mast cell populations were recognized with light microscopy i.e., light purple and deep purple, the latter also includes deeply stained cells with extruded granules. Mast cells identified by electron microscopy showed the ultrastructural features during granule formation and release of their content. Significantly higher numbers of mast cells per unit area of ovary were seen at estrus and diestrus. Numbers of mast cells also remained high during pregnancy with possible involvement of mast cell products in vascularization of corpora lutea. A positive correlation existed between mast cell counts and embryo number during pregnancy. However, numbers of mast cells declined significantly after parturition.

Inhibition effects of protein-conjugated amorphous zinc sulfide nanoparticles on tumor cells growth

NASA Astrophysics Data System (ADS)

Cao, Ying; Wang, Hua-Jie; Cao, Cui; Sun, Yuan-Yuan; Yang, Lin; Wang, Bao-Qing; Zhou, Jian-Guo

2011-07-01

In this article, a facile and environmentally friendly method was applied to fabricate BSA-conjugated amorphous zinc sulfide (ZnS) nanoparticles using bovine serum albumin (BSA) as the matrix. Transmission electron microscopy analysis indicated that the stable and well-dispersed nanoparticles with the diameter of 15.9 ± 2.1 nm were successfully prepared. The energy dispersive X-ray, X-ray powder diffraction, Fourier transform infrared spectrograph, high resolution transmission electron microscope, and selected area electron diffraction measurements showed that the obtained nanoparticles had the amorphous structure and the coordination occurred between zinc sulfide surfaces and BSA in the nanoparticles. In addition, the inhibition effects of BSA-conjugated amorphous zinc sulfide nanoparticles on tumor cells growth were described in detail by cell viability analysis, optical and electron microscopy methods. The results showed that BSA-conjugated amorphous zinc sulfide nanoparticles could inhibit the metabolism and proliferation of human hepatocellular carcinoma cells, and the inhibition was dose dependent. The half maximal inhibitory concentration (IC50) was 0.36 mg/mL. Overall, this study suggested that BSA-conjugated amorphous zinc sulfide nanoparticles had the application potential as cytostatic agents and BSA in the nanoparticles could provide the modifiable site for the nanoparticles to improve their bioactivity or to endow them with the target function.

Scanning and transmission electron microscopic analysis of ampullary segment of oviduct during estrous cycle in caprines.

PubMed

Sharma, R K; Singh, R; Bhardwaj, J K

2015-01-01

The ampullary segment of the mammalian oviduct provides suitable milieu for fertilization and development of zygote before implantation into uterus. It is, therefore, in the present study, the cyclic changes in the morphology of ampullary segment of goat oviduct were studied during follicular and luteal phases using scanning and transmission electron microscopy techniques. Topographical analysis revealed the presence of uniformly ciliated ampullary epithelia, concealing apical processes of non-ciliated cells along with bulbous secretory cells during follicular phase. The luteal phase was marked with decline in number of ciliated cells with increased occurrence of secretory cells. The ultrastructure analysis has demonstrated the presence of indented nuclear membrane, supranuclear cytoplasm, secretory granules, rough endoplasmic reticulum, large lipid droplets, apically located glycogen masses, oval shaped mitochondria in the secretory cells. The ciliated cells were characterized by the presence of elongated nuclei, abundant smooth endoplasmic reticulum, oval or spherical shaped mitochondria with crecentric cristae during follicular phase. However, in the luteal phase, secretory cells were possessing highly indented nucleus with diffused electron dense chromatin, hyaline nucleosol, increased number of lipid droplets. The ciliated cells had numerous fibrous granules and basal bodies. The parallel use of scanning and transmission electron microscopy techniques has enabled us to examine the cyclic and hormone dependent changes occurring in the topography and fine structure of epithelium of ampullary segment and its cells during different reproductive phases that will be great help in understanding major bottle neck that limits success rate in vitro fertilization and embryo transfer technology. © Wiley Periodicals, Inc.

Ultrastructural analysis of oral exfoliated epithelial cells of tobacco smokers and betel nut chewers: A scanning electron microscopy study.

PubMed

Khan, Sameera Shamim; Shreedhar, Balasundari; Kamboj, Mala

2016-01-01

The study was undertaken to correlate epithelial surface pattern changes of oral exfoliated cells of tobacco smokers and betel nut chewers and also to compare them with patients of oral squamous cell carcinoma (OSCC) and healthy individuals. In this cross-sectional study, a total of fifty persons were included in the study, out of which thirty formed the study group (15 each tobacco smokers and betel nut chewers) and twenty formed the control group (ten each of OSCC patients - positive control and ten normal buccal mucosa - negative control). Their oral exfoliated cells were scraped, fixed, and studied under scanning electron microscope (SEM). The statistical analysis was determined using ANOVA, Tukey honestly significant difference, Chi-square test, and statistical SPASS software, P < 0.05. OSCC, Individual cell modifications, intercellular relationships and surface characteristics observed by scanning electron microscopy between OSCC, tobacco smokers, betel nut chewers compared to normal oral mucosa have been tabulated. In normal oral mucosa, cell surface morphology depends on the state of keratinization of the tissue. Thus, it could prove helpful in detecting any carcinomatous change at its incipient stage and also give an insight into the ultra-structural details of cellular differentiations in epithelial tissues.

Ultrastructure and Pathology of Microsporidium phytoseiuli n. sp. Infecting the Predatory Mite, Phytoseiulus persimilis Athias-Henriot (Acari: Phytoseiidae)

PubMed

Bjørnson; Steiner; Keddie

1996-11-01

Ultrastructure and pathology of Microsporidium phytoseiuli n. sp. infecting the predatory mite Phytoseiulus persimilis Athias-Henriot is described using light and transmission electron microscopy. Infected mites showed no gross, external symptoms. All observed stages of the parasite had unpaired nuclei. Schizonts were commonly observed within nuclei of digestive cells of the ventriculus and within the cytoplasm of cells lining the cecal wall and in muscle tissue underlying it. Sporoblasts and spores occurred in the nuclei and cytoplasm of digestive cells within the ventriculus, in cortical regions of the sub- and supraesophageal ganglia, within the cecal wall and muscle tissue, and in parenchyma cells underlying the cuticle. Mature spores were also observed in developing eggs within gravid females. These were broad- to elongate-ovoid, measured 4.33 ± 0.35 x 1.27 ± 0.15 &mgr;m (electron micrographs), 5.37 ± 0.46 x 2.22 ± 0.17 &mgr;m (fixed and stained), and 5.88 ± 0.34 x 2.22 ± 0.19 &mgr;m (fresh) and had an isolfilar polar filament coiled 12 to 15 times within the posterior two-thirds. Within cells, individual spores appeared to be in direct contact with host cytoplasm, while groups of spores were infrequently observed within interfacial envelopes. Groups of 4, 8, to more than 16 spores were observed by light microscopy, while 8 was the maximum observed by electron microscopy. No spores were observed in Tetranychus urticae, a mite used as food during this study.

Wide-range high-resolution transmission electron microscopy reveals morphological and distributional changes of endomembrane compartments during log to stationary transition of growth phase in tobacco BY-2 cells.

PubMed

Toyooka, Kiminori; Sato, Mayuko; Kutsuna, Natsumaro; Higaki, Takumi; Sawaki, Fumie; Wakazaki, Mayumi; Goto, Yumi; Hasezawa, Seiichiro; Nagata, Noriko; Matsuoka, Ken

2014-09-01

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth. © The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Ultrastructure of Pericystic or Intracystic Blood Vessels in Epidermoid Cysts-A Transmission Electron Microscopy Study: Laboratory Investigation.

PubMed

Ren, Xiao-Hui; Ma, Jun; Zeng, Chun; Sun, Yi-Lin; Lin, Song

2017-07-01

Recently, we reported a tendency toward spontaneous hemorrhage in both the preoperative and postoperative periods in patients with intracranial epidermoid cyst (EC). According to our experience, this tendency for spontaneous hemorrhage was partly caused by the pathologic blood vessels adjacent to the EC. This study was designed to testify this hypothesis. Twenty-three removable pericystic or intracystic blood vessels from 17 patients with EC were collected during surgery and were then examined by transmission electron microscopy. The microvascular structure in gliomas was chosen as the control. Under electron microscopy, variant pathologic changes of vessels were found in all patients with EC. In the tunicae intima, we found vacuolization, apoptosis, necrosis, and intralumenal protrusion of endothelial cells, as well as swollen basement and highly flexed and discontinued elastic plate. In the tunicae media, vacuolization and swollen mitochondria were found in muscular cells. In the tunicae adventitia, extravascular erythrocytes, edema or apoptosis of pericytes, collagen predominance, and inflammatory cell infiltration and destruction were found. Neuron denature and necrosis were found in the peripheral brain tissue. In the microvascular structure of 5 glioma specimens, we found enlargement and hyperplasia of endothelial cells, swollen basement membrane, swollen pericytes, and astrocytic hyperplasia and neuron denature in adjacent brain tissues. Our findings provide strong evidence for the hypothesis that intracystic or pericystic vascular degeneration or destruction accounts for the spontaneous hemorrhage tendency before and after surgical resection of ECs. Copyright © 2017 Elsevier Inc. All rights reserved.

Structural study of Purkinje cell axonal torpedoes in essential tremor.

PubMed

Louis, Elan D; Yi, Hong; Erickson-Davis, Cordelia; Vonsattel, Jean-Paul G; Faust, Phyllis L

2009-02-06

Essential tremor (ET) is one of the most common neurological diseases. A basic understanding of its neuropathology is now emerging. Aside from Purkinje cell loss, a prominent finding is an abundance of torpedoes (rounded swellings of Purkinje cell axons). Such swellings often result from the mis-accumulation of cell constituents. Identifying the basic nature of these accumulations is an important step in understanding the underlying disease process. Torpedoes, only recently identified in ET, have not yet been characterized ultrastructurally. Light and electron microscopy were used to characterize the structural constituents of torpedoes in ET. Formalin-fixed cerebellar cortical tissue from four prospectively collected ET brains was sectioned and immunostained with a monoclonal phosphorylated neurofilament antibody (SMI-31, Covance, Emeryville, CA). Using additional sections from three ET brains, torpedoes were assessed using electron microscopy. Immunoreactivity for phosphorylated neurofilament protein revealed clear labeling of torpedoes in each case. Torpedoes were strongly immunoreactive; in many instances, two or more torpedoes were noted in close proximity to one another. On electron microscopy, torpedoes were packed with randomly arranged 10-12nm neurofilaments. Mitochondria and smooth endoplasmic reticulum were abundant as well, particularly at the periphery of the torpedo. We demonstrated that the torpedoes in ET represent the mis-accumulation of disorganized neurofilaments and other organelles. It is not known where in the pathogenic cascade these accumulations occur (i.e., whether these accumulations are the primary event or a secondary/downstream event) and this deserves further study.

Ultrastructural investigations for reducing endothelial cell damage of vein grafts during CABG-operation and practical consequences.

PubMed

Hickethier, T; Dämmrich, J; Silber, R E; Finster, S; Elert, O

1999-02-01

In the present study the influence of different storage solutions on endothelial integrity or damage was investigated with direct methods particularly with transmission electron microscopy (TEM), scanning electron microscopy (SEM) and immunohistochemistry. Saphenous vein segments of 10 cm in length were taken surgically from 6 male CABG-patients (aged 60-70) under standardized conditions. Each vein segment was cut into rings, which were incubated at room temperature for 45 minutes in different storage solutions, particularly in 0.9% sodium chloride solution and in buffered solution (M 199) with 5% human serum albumin respectively. Then, the vein segments were fixed in 3.5% glutaraldehyde and prepared for scanning and transmission electron microscopy to evaluate the endothelial damage. In addition, immunohistochemical staining (CD34, PECAM and Factor VIII) was performed. When using 0.9% sodium chloride solution, the SEM-examination revealed that 55% of the cell population was destroyed. In comparison to these findings only 26% of the endothelial cell population was damaged when the venous segment was stored in buffered solution with 5% albumin (p<0.01). In immunohistochemistry (CD34, PECAM, Factor VIII) these findings were supported. This study demonstrates the importance of storage solutions in regard to endothelial integrity. For best preservation of endothelium it is necessary to modify conventional storage methods. So, storage in buffered solution with albumin has shown much better endothelial cell preservation compared with physiological saline which might reduce the obliteration rate of CABG in future.

Whole-Cell Analysis of Low-Density Lipoprotein Uptake by Macrophages Using STEM Tomography

PubMed Central

Baudoin, Jean-Pierre; Jerome, W. Gray; Kübel, Christian; de Jonge, Niels

2013-01-01

Nanoparticles of heavy materials such as gold can be used as markers in quantitative electron microscopic studies of protein distributions in cells with nanometer spatial resolution. Studying nanoparticles within the context of cells is also relevant for nanotoxicological research. Here, we report a method to quantify the locations and the number of nanoparticles, and of clusters of nanoparticles inside whole eukaryotic cells in three dimensions using scanning transmission electron microscopy (STEM) tomography. Whole-mount fixed cellular samples were prepared, avoiding sectioning or slicing. The level of membrane staining was kept much lower than is common practice in transmission electron microscopy (TEM), such that the nanoparticles could be detected throughout the entire cellular thickness. Tilt-series were recorded with a limited tilt-range of 80° thereby preventing excessive beam broadening occurring at higher tilt angles. The 3D locations of the nanoparticles were nevertheless determined with high precision using computation. The obtained information differed from that obtained with conventional TEM tomography data since the nanoparticles were highlighted while only faint contrast was obtained on the cellular material. Similar as in fluorescence microscopy, a particular set of labels can be studied. This method was applied to study the fate of sequentially up-taken low-density lipoprotein (LDL) conjugated to gold nanoparticles in macrophages. Analysis of a 3D reconstruction revealed that newly up-taken LDL-gold was delivered to lysosomes containing previously up-taken LDL-gold thereby forming onion-like clusters. PMID:23383042

Scanning electron microscopy of the collodion membrane from a self-healing collodion baby*

PubMed Central

de Almeida Jr., Hiram Larangeira; Isaacsson, Henrique; Guarenti, Isabelle Maffei; Silva, Ricardo Marques e; de Castro, Luis Antônio Suita

2015-01-01

Abstract Self-healing collodion baby is a well-established subtype of this condition. We examined a male newborn, who was covered by a collodion membrane. The shed membrane was examined with scanning electron microscopy. The outer surface showed a very compact keratin without the normal elimination of corneocytes. The lateral view of the specimen revealed a very thick, horny layer. The inner surface showed the structure of lower corneocytes with polygonal contour. With higher magnifications villous projections were seen in the cell membrane. PMID:26375232

Neuron cells uptake of polymeric microcapsules and subsequent intracellular release.

PubMed

Pavlov, Anton M; Sapelkin, Andrei V; Huang, Xinyue; P'ng, Ken M Y; Bushby, Andy J; Sukhorukov, Gleb B; Skirtach, André G

2011-06-14

Neuron cells uptake of biodegradable and synthetic polymeric microcapsules functionalized with aggregates of gold nanoparticles incorporated into their shells is demonstrated in situ. In addition to traditionally used optical microscopy, electron microscopy is used both for higher-resolution imaging and for confirming the uptake by focused ion beam cross-sectioning of specific cells in situ. Subsequently, physical methods of release are compared to chemical methods wherein laser-induced intracellular release of dextran molecules into the cytosol of hippocampal neuron cells is studied in comparison to biodegradation. Implications of this work for neuroscience, bio-medicine and single cell studies are discussed. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structural protein 4.1 is located in mammalian centrosomes

PubMed Central

Krauss, Sharon Wald; Chasis, Joel Anne; Rogers, Catherine; Mohandas, Narla; Krockmalnic, Gabriela; Penman, Sheldon

1997-01-01

Structural protein 4.1 was first characterized as an important 80-kDa protein in the mature red cell membrane skeleton. It is now known to be a member of a family of protein isoforms detected at diverse intracellular sites in many nucleated mammalian cells. We recently reported that protein 4.1 isoforms are present at interphase in nuclear matrix and are rearranged during the cell cycle. Here we report that protein 4.1 epitopes are present in centrosomes of human and murine cells and are detected by using affinity-purified antibodies specific for 80-kDa red cell 4.1 and for 4.1 peptides. Immunofluorescence, by both conventional and confocal microscopy, showed that protein 4.1 epitopes localized in the pericentriolar region. Protein 4.1 epitopes remained in centrosomes after extraction of cells with detergent, salt, and DNase. Higher resolution electron microscopy of detergent-extracted cell whole mounts showed centrosomal protein 4.1 epitopes distributed along centriolar cylinders and on pericentriolar fibers, at least some of which constitute the filamentous network surrounding each centriole. Double-label electron microscopy showed that protein 4.1 epitopes were predominately localized in regions also occupied by epitopes for centrosome-specific autoimmune serum 5051 but were not found on microtubules. Our results suggest that protein 4.1 is an integral component of centrosome structure, in which it may play an important role in centrosome function during cell division and organization of cellular architecture. PMID:9207085

Improvement of pentathiophene/fullerene planar heterojunction photovoltaic cells by improving the organic films morphology through the anode buffer bilayer

NASA Astrophysics Data System (ADS)

El Jouad, Zouhair; Cattin, Linda; Martinez, Francisco; Neculqueo, Gloria; Louarn, Guy; Addou, Mohammed; Predeep, Padmanabhan; Manuvel, Jayan; Bernède, Jean-Christian

2016-05-01

Organic photovoltaic cells (OPVCs) are based on a heterojunction electron donor (ED)/electron acceptor (EA). In the present work, the electron donor which is also the absorber of light is pentathiophene. The typical cells were ITO/HTL/pentathiophene/fullerene/Alq3/Al with HTL (hole transport layer) = MoO3, CuI, MoO3/CuI. After optimisation of the pentathiophene thickness, 70 nm, the highest efficiency, 0.81%, is obtained with the bilayer MoO3/CuI as HTL. In order to understand these results the pentathiophene films deposited onto the different HTLs were characterized by scanning electron microscopy, atomic force microscopy, X-rays diffraction, optical absorption and electrical characterization. It is shown that CuI improves the conductivity of the pentathiophene layer through the modification of the film structure, while MoO3 decreases the leakage current. Using the bilayer MoO3/CuI allows cumulating the advantages of each layer. Contribution to the topical issue "Materials for Energy Harvesting, Conversion and Storage (ICOME 2015) - Elected submissions", edited by Jean-Michel Nunzi, Rachid Bennacer and Mohammed El Ganaoui

Imaging the Dynamics of Cell Wall Polymer Deposition in the Unicellular Model Plant, Penium margaritaceum.

PubMed

Domozych, David; Lietz, Anna; Patten, Molly; Singer, Emily; Tinaz, Berke; Raimundo, Sandra C

2017-01-01

The unicellular green alga, Penium margaritaceum, represents a novel and valuable model organism for elucidating cell wall dynamics in plants. This organism's cell wall contains several polymers that are highly similar to those found in the primary cell walls of land plants. Penium is easily grown in laboratory culture and is effectively manipulated in various experimental protocols including microplate assays and correlative microscopy. Most importantly, Penium can be live labeled with cell wall-specific antibodies or other probes and returned to culture where specific cell wall developmental events can be monitored. Additionally, live cells can be rapidly cryo-fixed and cell wall surface microarchitecture can be observed with variable pressure scanning electron microscopy. Here, we describe the methodology for maintaining Penium for experimental cell wall enzyme studies.

Lulo cell line derived from Lutzomyia longipalpis (Diptera: Psychodidae): a novel model to assay Leishmania spp. and vector interaction.

PubMed

Côrtes, Luzia Mc; Silva, Roger Mm; Pereira, Bernardo As; Guerra, Camila; Zapata, Angela C; Bello, Felio J; Finkelstein, Léa C; Madeira, Maria F; Brazil, Reginaldo P; Côrte-Real, Suzana; Alves, Carlos R

2011-11-14

Leishmania (Vianna) braziliensis, Leishmania (Leishmania) amazonensis and Leishmania (Leishmania) chagasi are important parasites in the scenario of leishmaniasis in Brazil. During the life cycle of these parasites, the promastigote forms adhere to the midgut epithelial microvillii of phlebotomine insects to avoid being secreted along with digestive products. Lulo cells are a potential model that will help to understand the features of this adhesion phenomenon. Here, we analyze the interaction between Leishmania spp. promastigotes and Lulo cells in vitro, specifically focusing on adhesion events occurring between three Leishmania species and this cell line. Confluent monolayers of Lulo cells were incubated with promastigotes and adhesion was assessed using both light microscopy and scanning electron microscopy. The results indicate that species from the subgenera Leishmania and Viannia have great potential to adhere to Lulo cells. The highest adherence rate was observed for L. (L.) chagasi after 24 h of incubation with Lulo cells (27.3 ± 1.8% of cells with adhered promastigotes), followed by L. (L.) amazonensis (16.0 ± 0.7%) and L. (V.) braziliensis (3.0 ± 0.7%), both after 48 h. In the ultrastructural analysis, promastigote adherence was also assessed by scanning electron microscopy, showing that, for parasites from both subgenera, adhesion occurs by both the body and the flagellum. The interaction of Lulo cells with Leishmania (L.) chagasi showed the participation of cytoplasmic projections from the former closely associating the parasites with the cells. We present evidence that Lulo cells can be useful in studies of insect-parasite interactions for Leishmania species.

Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography.

PubMed

Murata, Kazuyoshi; Esaki, Masatoshi; Ogura, Teru; Arai, Shigeo; Yamamoto, Yuta; Tanaka, Nobuo

2014-11-01

Electron tomography using a high-voltage electron microscope (HVEM) provides three-dimensional information about cellular components in sections thicker than 1 μm, although in bright-field mode image degradation caused by multiple inelastic scattering of transmitted electrons limit the attainable resolution. Scanning transmission electron microscopy (STEM) is believed to give enhanced contrast and resolution compared to conventional transmission electron microscopy (CTEM). Samples up to 1 μm in thickness have been analyzed with an intermediate-voltage electron microscope because inelastic scattering is not a critical limitation, and probe broadening can be minimized. Here, we employed STEM at 1 MeV high-voltage to extend the useful specimen thickness for electron tomography, which we demonstrate by a seamless tomographic reconstruction of a whole, budding Saccharomyces cerevisiae yeast cell, which is ~3 μm in thickness. High-voltage STEM tomography, especially in the bright-field mode, demonstrated sufficiently enhanced contrast and intensity, compared to CTEM tomography, to permit segmentation of major organelles in the whole cell. STEM imaging also reduced specimen shrinkage during tilt-series acquisition. The fidelity of structural preservation was limited by cytoplasmic extraction, and the spatial resolution was limited by the relatively large convergence angle of the scanning probe. However, the new technique has potential to solve longstanding problems of image blurring in biological specimens beyond 1 μm in thickness, and may facilitate new research in cellular structural biology. Copyright © 2014 Elsevier B.V. All rights reserved.

The Development of M Cells in Peyer’s Patches Is Restricted to Specialized Dome-Associated Crypts

PubMed Central

Gebert, Andreas; Fassbender, Susanne; Werner, Kerstin; Weissferdt, Annikka

1999-01-01

It is controversial whether the membranous (M) cells of the Peyer’s patches represent a separate cell line or develop from enterocytes under the influence of lymphocytes on the domes. To answer this question, the crypts that produce the dome epithelial cells were studied and the distribution of M cells over the domes was determined in mice. The Ulex europaeus agglutinin was used to detect M cells in mouse Peyer’s patches. Confocal microscopy with lectin-gold labeling on ultrathin sections, scanning electron microscopy, and laminin immuno-histochemistry were combined to characterize the cellular composition and the structure of the dome-associated crypts and the dome epithelium. In addition, the sites of lymphocyte invasion into the dome epithelium were studied after removal of the epithelium using scanning electron microscopy. The domes of Peyer’s patches were supplied with epithelial cells that derived from two types of crypt: specialized dome-associated crypts and ordinary crypts differing not only in shape, size, and cellular composition but also in the presence of M cell precursors. When epithelial cells derived from ordinary crypts entered the domes, they formed converging radial strips devoid of M cells. In contrast to the M cells, the sites where lymphocytes invaded the dome epithelium were not arranged in radial strips, but randomly distributed over the domes. M cell development is restricted to specialized dome-associated crypts. Only dome epithelial cells that derive from these specialized crypts differentiate into M cells. It is concluded that M cells represent a separate cell line that is induced in the dome-associated crypts by still unknown, probably diffusible lymphoid factors. PMID:10329609

Correlated Fluorescence-Atomic Force Microscopy Studies of the Clathrin Mediated Endocytosis in SKMEL Cells

NASA Astrophysics Data System (ADS)

Smith, Steve; Hor, Amy; Luu, Anh; Kang, Lin; Scott, Brandon; Bailey, Elizabeth; Hoppe, Adam

Clathrin-mediated endocytosis is one of the central pathways for cargo transport into cells, and plays a major role in the maintenance of cellular functions, such as intercellular signaling, nutrient intake, and turnover of plasma membrane in cells. The clathrin-mediated endocytosis process involves invagination and formation of clathrin-coated vesicles. However, the biophysical mechanisms of vesicle formation are still debated. We investigate clathrin vesicle formation mechanisms through the utilization of tapping-mode atomic force microscopy for high resolution topographical imaging in neutral buffer solution of unroofed cells exposing the inner membrane, combined with fluorescence imaging to definitively label intracellular constituents with specific fluorescent fusion proteins (actin filaments labeled with green phalloidin-antibody and clathrin coated vesicles with the fusion protein Tq2) in SKMEL (Human Melanoma) cells. Results from our work are compared against dynamical polarized total internal fluorescence (TIRF), super-resolution photo-activated localization microscopy (PALM) and transmission electron microscopy (TEM) to draw conclusions regarding the prominent model of vesicle formation in clathrin-mediated endocytosis. Funding provided by NSF MPS/DMR/BMAT award # 1206908.

Midbody Accumulation in Breast Cancer Stem Cells

DTIC Science & Technology

2011-08-01

transit amplifying or differentiating cells. These results suggest that MBds are in almost exclusively in stem cells and putative breast cancer stem...confer tumor-like properties to these cells. We were unable to establish GFP-MKLP1 breast cancer cell lines for this analysis for some reason that we...and nonpolarized cells (Fig. 1c, d). Immuno- electron microscopy confirmed this localization and revealed ultrastructural features characteristic of

New approaches in renal microscopy: volumetric imaging and superresolution microscopy.

PubMed

Kim, Alfred H J; Suleiman, Hani; Shaw, Andrey S

2016-05-01

Histologic and electron microscopic analysis of the kidney has provided tremendous insight into structures such as the glomerulus and nephron. Recent advances in imaging, such as deep volumetric approaches and superresolution microscopy, have the capacity to dramatically enhance our current understanding of the structure and function of the kidney. Volumetric imaging can generate images millimeters below the surface of the intact kidney. Superresolution microscopy breaks the diffraction barrier inherent in traditional light microscopy, enabling the visualization of fine structures. Here, we describe new approaches to deep volumetric and superresolution microscopy of the kidney. Rapid advances in lasers, microscopic objectives, and tissue preparation have transformed our ability to deep volumetric image the kidney. Innovations in sample preparation have allowed for superresolution imaging with electron microscopy correlation, providing unprecedented insight into the structures within the glomerulus. Technological advances in imaging have revolutionized our capacity to image both large volumes of tissue and the finest structural details of a cell. These new advances have the potential to provide additional profound observations into the normal and pathologic functions of the kidney.

Triple retinal infection with human immunodeficiency virus type 1, cytomegalovirus, and herpes simplex virus type 1. Light and electron microscopy, immunohistochemistry, and in situ hybridization.

PubMed

Rummelt, V; Rummelt, C; Jahn, G; Wenkel, H; Sinzger, C; Mayer, U M; Naumann, G O

1994-02-01

This report describes the histopathologic and virologic findings of the retina from a 55-year-old bisexual patient with the acquired immune deficiency syndrome (AIDS), who had concurrent human immunodeficiency virus type 1 (HIV-1), cytomegalovirus (CMV), and herpes simplex virus type 1 (HSV-1) retinitis, and was treated with ganciclovir. The eyes were obtained at autopsy and processed for light microscopy and transmission electron microscopy. Immunohistochemical stains for HSV-1, CMV, HIV-1, varicella zoster virus, and glial fibrillary acidic protein were carried out using the peroxidase-antiperoxidase and streptavidin-biotin-alkaline phosphatase techniques. For in situ hybridization, a radiolabeled CMV DNA probe (Eco-RI-Y fragment of strain AD 169) was used. Results of histopathologic examination showed a full-thickness necrotizing retinitis with cytomegalic and herpes viral intranuclear inclusions in cells of the neurosensory retina, retinal vascular endothelium, and the retinal pigment epithelium. Some areas of the retina were replaced by glial tissue. The choroid contained only a few chronic inflammatory cells. Immunoperoxidase studies disclosed CMV antigens diffusely distributed throughout all layers of the retina and the retinal pigment epithelium. Herpes simplex virus type 1 antigens were present in retinal cells and the retinal vascular endothelium. Human immunodeficiency virus type 1 antigens were found in mononuclear cells in all layers of the sensory retina. Dual infections with HIV-1 and CMV of individual multinucleated giant cells of glial origin were demonstrated immunohistochemically. Transmission electron microscopy showed herpes viral particles in the vascular endothelium of the retinal vessels and the choriocapillaris. Human immunodeficiency virus particles were identified in the endothelium of the choriocapillaris. The possibility of multiple viral infections of the retina, mimicking classic CMV retinitis, should be considered in the clinical and histologic differential diagnosis of necrotizing retinitis in patients with AIDS.

Polymeric capsule-cushioned leukocyte cell membrane vesicles as a biomimetic delivery platform

NASA Astrophysics Data System (ADS)

Gao, Changyong; Wu, Zhiguang; Lin, Zhihua; Lin, Xiankun; He, Qiang

2016-02-01

We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy.We report a biomimetic delivery of microsized capsule-cushioned leukocyte membrane vesicles (CLMVs) through the conversion of freshly reassembled leukocyte membrane vesicles (LMVs), including membrane lipids and membrane-bound proteins onto the surface of layer-by-layer assembled polymeric multilayer microcapsules. The leukocyte membrane coating was verified by using electron microscopy, a quartz crystal microbalance, dynamic light scattering, and confocal laser scanning microscopy. The resulting CLMVs have the ability to effectively evade clearance by the immune system and thus prolong the circulation time in mice. Moreover, we also show that the right-side-out leukocyte membrane coating can distinctly improve the accumulation of capsules in tumor sites through the molecular recognition of membrane-bound proteins of CLMVs with those of tumor cells in vitro and in vivo. The natural cell membrane camouflaged polymeric multilayer capsules with the immunosuppressive and tumor-recognition functionalities of natural leukocytes provide a new biomimetic delivery platform for disease therapy. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08407e

Novel of core-shell AlOOH/Cu nanostructures: Synthesis, characterization, antimicrobial activity and in vitro toxicity in Neuro-2a cells

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

Bakina, O. V., E-mail: ovbakina@ispms.tsc.ru; Fomenko, A. N., E-mail: alserova@ispms.tsc.ru; Korovin, M. S., E-mail: msk@ispms.tsc.ru

Core-shell micro/nanostructures were fabricated by the reaction of Al/Cu bimetallic nanoparticles with water. Al/Cu nanoparticles have been obtained using the method of simultaneous electrical explosion of a pair of the corresponding metal wires in an argon atmosphere. The nanoparticles are chemically active and interact with water at 60°C to form core-shell micro/nanostructures. The obtained products were characterized by means of X-ray diffraction, scanning electron microscopy, transmission electron microscopy and dynamic light scattering and the nitrogen adsorption method. The antibacterial activity of the synthesized structures was investigated against E. coli and St. aureus. The toxic effect of these nanostructures against themore » Neuro-2a neuroblastoma cell line was investigated. AlOOH/Cu nanostructures are shown to inhibit cell proliferation. The AlOOH/Cu nanostructures are good candidates for medical applications.« less

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

Bio-camouflage of anatase nanoparticles explored by in situ high-resolution electron microscopy.

PubMed

Ribeiro, Ana R; Mukherjee, Arijita; Hu, Xuan; Shafien, Shayan; Ghodsi, Reza; He, Kun; Gemini-Piperni, Sara; Wang, Canhui; Klie, Robert F; Shokuhfar, Tolou; Shahbazian-Yassar, Reza; Borojevic, Radovan; Rocha, Luis A; Granjeiro, José M

2017-08-03

While titanium is the metal of choice for most prosthetics and inner body devices due to its superior biocompatibility, the discovery of Ti-containing species in the adjacent tissue as a result of wear and corrosion has been associated with autoimmune diseases and premature implant failures. Here, we utilize the in situ liquid cell transmission electron microscopy (TEM) in a liquid flow holder and graphene liquid cells (GLCs) to investigate, for the first time, the in situ nano-bio interactions between titanium dioxide nanoparticles and biological medium. This imaging and spectroscopy methodology showed the process of formation of an ionic and proteic bio-camouflage surrounding Ti dioxide (anatase) nanoparticles that facilitates their internalization by bone cells. The in situ understanding of the mechanisms of the formation of the bio-camouflage of anatase nanoparticles may contribute to the definition of strategies aimed at the manipulation of these NPs for bone regenerative purposes.

Gold and iodine diffusion in large area perovskite solar cells under illumination.

PubMed

Cacovich, S; Ciná, L; Matteocci, F; Divitini, G; Midgley, P A; Di Carlo, A; Ducati, C

2017-04-06

Operational stability is the main issue hindering the commercialisation of perovskite solar cells. Here, a long term light soaking test was performed on large area hybrid halide perovskite solar cells to investigate the morphological and chemical changes associated with the degradation of photovoltaic performance occurring within the devices. Using Scanning Transmission Electron Microscopy (STEM) in conjunction with EDX analysis on device cross sections, we observe the formation of gold clusters in the perovskite active layer as well as in the TiO 2 mesoporous layer, and a severe degradation of the perovskite due to iodine migration into the hole transporter. All these phenomena are associated with a drastic drop of all the photovoltaic parameters. The use of advanced electron microscopy techniques and data processing provides new insights on the degradation pathways, directly correlating the nanoscale structure and chemistry to the macroscopic properties of hybrid perovskite devices.

Observations on the antibody-dependent cytotoxic cell by scanning electron microscopy.

PubMed Central

Inglis, J R; Penhale, W J; Farmer, A; Irvine, W J; Williams, A E

1975-01-01

The cytotoxic effect of human peripheral blood leucocytes on antibody-coated sheep erythrocyte monolayers has been investigated using scanning electron microscopy. Only a small proportion of leucocytes were found to adhere to the monolayers. A progressive destruction was observed beginning as small plaque-like areas of erythrocyte clearing which later became confluent. Three distinct cell types were found to be associated with the areas of lysis. No destruction was observed in control monolayers incubated for a similar period in the absence of either antibody of leucocytes. Surface changes in the erthrocytes adjacent to the leucocytes suggest that mechanical factors may be involved in erythrocyte lysis in this system. It is concluded that more than one leucocyte type may damage antibody-coated erythrocytes, possibly by a mechanism involving attachment to and mechanical disruption of the red cell membrane. Images FIG. 5 FIG. 2 FIG. 3 FIG. 1 FIG. 2 FIG. 4 PMID:1191386

The (2 × 2) tunnels structured manganese dioxide nanorods with α phase for lithium air batteries

NASA Astrophysics Data System (ADS)

Ghouri, Zafar Khan; Zahoor, Awan; Barakat, Nasser A. M.; Alsoufi, Mohammad S.; Bawazeer, Tahani M.; Mohamed, Ahmed F.; Kim, Hak Yong

2016-02-01

The (2 × 2) tunnels structured manganese dioxide nanorods with α phase (α-MnO2) are synthesized via simplistic hydrothermal method at low temperature. The obtained tunnels structured α-MnO2 nanorods are characterized by, Transmission electron microscopy, Scanning electron microscopy, and X-ray diffraction techniques. The oxygen reduction reaction (ORR) activity was studied by cyclic voltammetry and rotating ring-disc electrode voltammetry techniques in alkaline media. Moreover; the highly electrocatalytic tunnels structured α-MnO2 nanorods were then also applied as cathode in rechargeable Li-O2 cells. The Li-O2 cells exhibited initial discharge capacity as high as ∼4000 mAh/g with the tunnels structured α-MnO2 nanorods which was double the original capacity of the cells without any catalyst. Also we obtained 100% round trip efficiency upon cycling with limited capacity for more than 50 cycles.

Novel of core-shell AlOOH/Cu nanostructures: Synthesis, characterization, antimicrobial activity and in vitro toxicity in Neuro-2a cells

NASA Astrophysics Data System (ADS)

Bakina, O. V.; Fomenko, A. N.; Korovin, M. S.; Glazkova, E. A.; Svarovskaya, N. V.

2016-08-01

Core-shell micro/nanostructures were fabricated by the reaction of Al/Cu bimetallic nanoparticles with water. Al/Cu nanoparticles have been obtained using the method of simultaneous electrical explosion of a pair of the corresponding metal wires in an argon atmosphere. The nanoparticles are chemically active and interact with water at 60°C to form core-shell micro/nanostructures. The obtained products were characterized by means of X-ray diffraction, scanning electron microscopy, transmission electron microscopy and dynamic light scattering and the nitrogen adsorption method. The antibacterial activity of the synthesized structures was investigated against E. coli and St. aureus. The toxic effect of these nanostructures against the Neuro-2a neuroblastoma cell line was investigated. AlOOH/Cu nanostructures are shown to inhibit cell proliferation. The AlOOH/Cu nanostructures are good candidates for medical applications.

Revealing the correlation between real-space structure and chiral magnetic order at the atomic scale

NASA Astrophysics Data System (ADS)

Hauptmann, Nadine; Dupé, Melanie; Hung, Tzu-Chao; Lemmens, Alexander K.; Wegner, Daniel; Dupé, Bertrand; Khajetoorians, Alexander A.

2018-03-01

We image simultaneously the geometric, the electronic, and the magnetic structures of a buckled iron bilayer film that exhibits chiral magnetic order. We achieve this by combining spin-polarized scanning tunneling microscopy and magnetic exchange force microscopy (SPEX) to independently characterize the geometric as well as the electronic and magnetic structures of nonflat surfaces. This new SPEX imaging technique reveals the geometric height corrugation of the reconstruction lines resulting from strong strain relaxation in the bilayer, enabling the decomposition of the real-space from the electronic structure at the atomic level and the correlation with the resultant spin-spiral ground state. By additionally utilizing adatom manipulation, we reveal the chiral magnetic ground state of portions of the unit cell that were not previously imaged with spin-polarized scanning tunneling microscopy alone. Using density functional theory, we investigate the structural and electronic properties of the reconstructed bilayer and identify the favorable stoichiometry regime in agreement with our experimental result.

Formation of newly synthesized adeno-associated virus capsids in the cell nucleus.

PubMed

Bell, Peter; Vandenberghe, Luk H; Wilson, James M

2014-06-01

Adeno-associated virus (AAV) particles inside the nucleus of a HEK 293 cell are shown by electron microscopy. Cells have been triple-transfected for vector production and were analyzed for capsid formation three days later. Newly assembled particle are visible as seemingly unstructured conglomerates or crystal-like arrays.

Ultra-fine Pt nanoparticles on graphene aerogel as a porous electrode with high stability for microfluidic methanol fuel cell

NASA Astrophysics Data System (ADS)

Kwok, Y. H.; Tsang, Alpha C. H.; Wang, Yifei; Leung, Dennis Y. C.

2017-05-01

Platinum-decorated graphene aerogel as a porous electrode for flow-through direct methanol microfluidic fuel cell is introduced. Ultra-fine platinum nanoparticles with size ranged from diameter 1.5 nm-3 nm are evenly anchored on the graphene nanosheets without agglomeration. The electrode is characterized by scanning electron microscopy, transmission electron microscopy and energy-dispersive X-ray spectroscopy. Catalytic activity is confirmed by cyclic voltammetry. The electroactive surface area and catalytic activity of platinum on graphene oxide (Pt/GO) are much larger than commercial platinum on carbon black (Pt/C). A counterflow microfluidic fuel cell is designed for contrasting the cell performance between flow-over type and flow-through type electrodes using Pt/C on carbon paper and Pt/GO, respectively. The Pt/GO electrode shows 358% increment in specific power compared with Pt/C anode. Apart from catalytic activity, the effect of porous electrode conductivity to cell performance is also studied. The conductivity of the porous electrode should be further enhanced to achieve higher cell performance.

Phagocytosis of PLGA Microparticles in Rat Peritoneal Exudate Cells: A Time-Dependent Study

NASA Astrophysics Data System (ADS)

Gomes, Anderson De Jesus; Nain Lunardi, Claure; Henrique Caetano, Flávio; Orive Lunardi, Laurelúcia; da Hora Machado, Antonio Eduardo

2006-07-01

With the purpose of enhancing the efficacy of microparticle-encapsulated therapeutic agents, in this study we evaluated the phagocytic ability of rat peritoneal exudate cells and the preferential location of poly(D,L-lactide-co-glycolic acid) (PLGA) microparticles inside these cells. The microparticles used were produced by a solvent evaporation method and were characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Size distribution analysis using DLS and SEM showed that the particles were spherical, with diameters falling between 0.5 and 1.5 [mu]m. Results from cell adhesion by SEM assay, indicated that the PLGA microparticles are not toxic to cells and do not cause any distinct damage to them as confirmed by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Among the large variety of cell populations found in the peritoneal exudates (neutrophils, eosinophils, monocytes, and macrophages), TEM showed that only the latter phagocytosed PLGA microparticles, in a time-dependent manner. The results obtained indicate that the microparticles studied show merits as possible carriers of drugs for intracellular delivery.

The Structure of Resting Bacterial Populations in Soil and Subsoil Permafrost

NASA Astrophysics Data System (ADS)

Soina, Vera S.; Mulyukin, Andrei L.; Demkina, Elena V.; Vorobyova, Elena A.; El-Registan, Galina I.

2004-09-01

The structure of individual cells in microbial populations in situ of the Arctic and Antarctic permafrost was studied by scanning and transmission electron microscopy methods and compared with that of cyst-like resting forms generated under special conditions by the non-sporeforming bacteria Arthrobacter and Micrococcus isolated from the permafrost. Electron microscopy examination of microorganisms in situ revealed several types of bacterial cells having no signs of damage, including "dwarf" curved forms similar to nanoforms. Intact bacterial cells in situ and frozen cultures of the permafrost isolates differed from vegetative cells by thickened cell walls, the altered structure of cytoplasm, and the compact nucleoid, and were similar in these features to cyst-like resting forms of non-spore-forming "permafrost" bacterial strains of Arthrobacter and Micrococcus spp. Cyst-like cells, being resistant to adverse external factors, are regarded as being responsible for survival of the non-spore-formers under prolonged exposure to subzero temperatures and can be a target to search for living microorganisms in natural environments both on the Earth and on extraterrestrial bodies.

Solution-Processible Crystalline NiO Nanoparticles for High-Performance Planar Perovskite Photovoltaic Cells.

PubMed

Kwon, Uisik; Kim, Bong-Gi; Nguyen, Duc Cuong; Park, Jong-Hyeon; Ha, Na Young; Kim, Seung-Joo; Ko, Seung Hwan; Lee, Soonil; Lee, Daeho; Park, Hui Joon

2016-07-28

In this work, we report on solution-based p-i-n-type planar-structured CH3NH3PbI3 perovskite photovoltaic (PV) cells, in which precrystallized NiO nanoparticles (NPs) without post-treatment are used to form a hole transport layer (HTL). X-ray diffraction and high-resolution transmission electron microscopy showed the crystallinity of the NPs, and atomic force microscopy and scanning electron microscopy confirmed the uniform surfaces of the resultant NiO thin film and the subsequent perovskite photoactive layer. Compared to the conventional poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) ( PSS) HTL, the NiO HTL had excellent energy-level alignment with that of CH3NH3PbI3 and improved electron-blocking capability, as analyzed by photoelectron spectroscopy and diode modeling, resulting in Voc ~0.13 V higher than conventional PSS-based devices. Consequently, a power conversion efficiency (PCE) of 15.4% with a high fill factor (FF, 0.74), short-circuit current density (Jsc, 20.2 mA·cm(-2)), and open circuit voltage (Voc, 1.04 V) having negligible hysteresis and superior air stability has been achieved.

Solution-Processible Crystalline NiO Nanoparticles for High-Performance Planar Perovskite Photovoltaic Cells

PubMed Central

Kwon, Uisik; Kim, Bong-Gi; Nguyen, Duc Cuong; Park, Jong-Hyeon; Ha, Na Young; Kim, Seung-Joo; Ko, Seung Hwan; Lee, Soonil; Lee, Daeho; Park, Hui Joon

2016-01-01

In this work, we report on solution-based p-i-n-type planar-structured CH3NH3PbI3 perovskite photovoltaic (PV) cells, in which precrystallized NiO nanoparticles (NPs) without post-treatment are used to form a hole transport layer (HTL). X-ray diffraction and high-resolution transmission electron microscopy showed the crystallinity of the NPs, and atomic force microscopy and scanning electron microscopy confirmed the uniform surfaces of the resultant NiO thin film and the subsequent perovskite photoactive layer. Compared to the conventional poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS) HTL, the NiO HTL had excellent energy-level alignment with that of CH3NH3PbI3 and improved electron-blocking capability, as analyzed by photoelectron spectroscopy and diode modeling, resulting in Voc ~0.13 V higher than conventional PEDOT:PSS-based devices. Consequently, a power conversion efficiency (PCE) of 15.4% with a high fill factor (FF, 0.74), short-circuit current density (Jsc, 20.2 mA·cm−2), and open circuit voltage (Voc, 1.04 V) having negligible hysteresis and superior air stability has been achieved. PMID:27465263

Ultrastructural pathogenesis of lesions produced by exposure to oxygen difluoride with correlative light microscopy

NASA Technical Reports Server (NTRS)

Harrison, G.; Mackenzie, W.

1973-01-01

The lungs of rats exposed to OF2 were examined by light and electron microscopy. The exposures were for 30 to 60 minutes to an average of 4.5 ppm OF2, the minimal lethal dose. Animals were sacrificed after 30 (group 1) and 60 minutes (group 2) exposure and 1 (group 3) and 2 (group 4) hours following 60 minutes exposure. Mild gross changes were observed in groups 3 and 4, but no light microscopic lesions were found. Alterations were noted in all four groups using electron microscopy. These were mostly indicative of fluid change and consisted of blebbing of the endothelial and epithelial layers of the alveolocapillary wall and rarification of the cytoplasm of these cells. The lamellar bodies of the Type II cells showed an increasing and consistent loss of matrix structure and density. These fine structural changes increased in quantity and severity as time of exposure or post-exposure period increased. (Modified author abstract)

Evaluation of hemocompatibility and in vitro immersion on microwave-assisted hydroxyapatite-alumina nanocomposites.

PubMed

Radha, G; Balakumar, S; Venkatesan, Balaji; Vellaichamy, Elangovan

2015-05-01

This study reports the microwave-assisted synthesis and characterization of nHAp (nano-hydroxyapatite)-alumina composites. The crystalline phase and interaction of alumina with nHAp was analyzed using X-ray diffraction (XRD) and Raman microscopy analysis, respectively. High resolution transmission electron microscopy (HRTEM) micrographs exhibit morphological changes of nHAp composites with increasing alumina concentrations. Microhardness studies reveal the enhanced mechanical strength of nHAp10 and nHAp20 nanocomposites than pure nHAp. In vitro bioactivity of the nanocomposites was studied by immersing samples in simulated body fluid (Hank's solution) for 21 days. The surface of biomineralized samples were analyzed using field emission scanning electron microscopy (FESEM) and energy dispersive X-ray spectroscopy (EDX). Hemolytic assay revealed acceptable compatibility for varying concentrations of all the samples. Cell proliferation assay was systematically investigated for 1 day and 3 days on Saos-2 osteoblast-like cell lines and it was found that nHAp nanocomposites improved the proliferation. Copyright © 2015 Elsevier B.V. All rights reserved.

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.

Identification of Foreign Particles in Human Tissues using Raman Microscopy.

PubMed

Campion, Alan; Smith, Kenneth J; Fedulov, Alexey V; Gregory, David; Fan, Yuwei; Godleski, John J

2018-06-12

The precise identification of foreign particles in tissue for patient care and research has been studied using polarized light microscopy, electron microscopy with X-ray analysis, and electron diffraction. The goal of this study was to unambiguously identify particles in tissues using a combina-tion of polarized light microscopy and Raman microscopy, which provides chemical composition and microstructural characterization of complex materials with submicron spatial resolution. We designed a model system of stained and unstained cells that contained birefringent talc particles, and systematically investigated the influence of slide and coverslip materials, laser wavelengths, and mounting media on the Raman spectra ob-tained. Hematoxylin and eosin stained slides did not produce useful results because of fluorescence interference from the stains. Unstained cell samples prepared with standard slides and coverslips produce high quality Raman spectra when excited at 532 nm; the spectra are uniquely as-signed to talc. We also obtain high quality Raman spectra specific for talc in unstained tissue samples (pleural tissue following talc pleurodesis and ovarian tissue following long-term perineal talc exposure). Raman microscopy is sufficiently sensitive and compositionally selective to identify particles as small as one micron in diameter. Among commonly used coverslip mounting media, Cytoseal 60 is recommended; Permount was unacceptable due to intense background interference. Raman spectra have been catalogued for thousands of substances, which suggests that this approach is likely to be successful in identifying other particles of interest in tissues, potentially making Raman microscopy a powerful new tool in pathology.

New techniques enable comparative analysis of microtubule orientation, wall texture, and growth rate in intact roots of Arabidopsis.

PubMed

Sugimoto, K; Williamson, R E; Wasteneys, G O

2000-12-01

This article explores root epidermal cell elongation and its dependence on two structural elements of cells, cortical microtubules and cellulose microfibrils. The recent identification of Arabidopsis morphology mutants with putative cell wall or cytoskeletal defects demands a procedure for examining and comparing wall architecture and microtubule organization patterns in this species. We developed methods to examine cellulose microfibrils by field emission scanning electron microscopy and microtubules by immunofluorescence in essentially intact roots. We were able to compare cellulose microfibril and microtubule alignment patterns at equivalent stages of cell expansion. Field emission scanning electron microscopy revealed that Arabidopsis root epidermal cells have typical dicot primary cell wall structure with prominent transverse cellulose microfibrils embedded in pectic substances. Our analysis showed that microtubules and microfibrils have similar orientation only during the initial phase of elongation growth. Microtubule patterns deviate from a predominantly transverse orientation while cells are still expanding, whereas cellulose microfibrils remain transverse until well after expansion finishes. We also observed microtubule-microfibril alignment discord before cells enter their elongation phase. This study and the new technology it presents provide a starting point for further investigations on the physical properties of cell walls and their mechanisms of assembly.

The structure of the desiccated Richtersius coronifer (Richters, 1903).

PubMed

Czerneková, Michaela; Jönsson, K Ingemar; Chajec, Lukasz; Student, Sebastian; Poprawa, Izabela

2017-05-01

Tun formation is an essential morphological adaptation for entering the anhydrobiotic state in tardigrades, but its internal structure has rarely been investigated. We present the structure and ultrastructure of organs and cells in desiccated Richtersius coronifer by transmission and scanning electron microscopy, confocal microscopy, and histochemical methods. A 3D reconstruction of the body organization of the tun stage is also presented. The tun formation during anhydrobiosis of tardigrades is a process of anterior-posterior body contraction, which relocates some organs such as the pharyngeal bulb. The cuticle is composed of epicuticle, intracuticle and procuticle; flocculent coat; and trilaminate layer. Moulting does not seem to restrict the tun formation, as evidenced from tardigrade tuns that were in the process of moulting. The storage cells of desiccated specimens filled up the free inner space and surrounded internal organs, such as the ovary and digestive system, which were contracted. All cells (epidermal cells, storage cells, ovary cells, cells of the digestive system) underwent shrinkage, and their cytoplasm was electron dense. Lipids and polysaccharides dominated among reserve material of storage cells, while the amount of protein was small. The basic morphology of specific cell types and organelles did not differ between active and anhydrobiotic R. coronifer.

Cell Uptake and Validation of Novel PECs for Biomedical Applications.

PubMed

Palamà, Ilaria E; Musarò, Mariarosaria; Coluccia, Addolorata M L; D'Amone, Stefania; Gigli, Giuseppe

2011-01-01

This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability.

Cell Uptake and Validation of Novel PECs for Biomedical Applications

PubMed Central

Palamà, Ilaria E.; Musarò, Mariarosaria; Coluccia, Addolorata M. L.; D'Amone, Stefania; Gigli, Giuseppe

2011-01-01

This pilot study provides the proof of principle for biomedical application of novel polyelectrolyte complexes (PECs) obtained via electrostatic interactions between dextran sulphate (DXS) and poly(allylamine hydrochloride) (PAH). Scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed that DXS/PAH polyelectrolyte complexes were Monodispersed with regular rounded-shape features and average diameters of 250 nm at 2 : 1 weight ratios of DXS/PAH. Fluorescently labelled DXS and fluorescein-isothiocyanate- (FITC-)conjugate DXS were used to follow cell uptake efficiency of PECs and biodegradability of their enzymatically degradable DXS-layers by using confocal laser scanning microscopy (CLSM). Moreover, quantitative MTT and Trypan Blue assays were employed to validate PECs as feasible and safe nanoscaled carriers at single-cell level without adverse effects on metabolism and viability. PMID:21876815

Sensing of silver nanoparticles on/in endothelial cells using atomic force spectroscopy.

PubMed

Kolodziejczyk, Agnieszka; Jakubowska, Aleksandra; Kucinska, Magdalena; Wasiak, Tomasz; Komorowski, Piotr; Makowski, Krzysztof; Walkowiak, Bogdan

2018-05-10

Endothelial cells, due to their location, are interesting objects for atomic force spectroscopy study. They constitute a barrier between blood and vessel tissues located deeper, and therefore they are the first line of contact with various substances present in blood, eg, drugs or nanoparticles. This work intends to verify whether the mechanical response of immortalized human umbilical vein endothelial cells (EA.hy926), when exposed to silver nanoparticles, as measured using force spectroscopy, could be effectively used as a bio-indicator of the physiological state of the cells. Silver nanoparticles were characterized with transmission electron microscopy and dynamic light scattering techniques. Tetrazolium salt reduction test was used to determine cell viability after treatment with silver nanoparticles. An elasticity of native cells was examined in the Hanks' buffer whereas fixed cells were softly fixed with formaldehyde. Additional aspect of the work is the comparative force spectroscopy utilizing AFM probes of ball-shape and conical geometries, in order to understand what changes in cell elasticity, caused by SNPs, were detectable with each probe. As a supplement to elasticity studies, cell morphology observation by atomic force microscopy and detection of silver nanoparticles inside cells using transmission electron microscopy were also performed. Cells exposed to silver nanoparticles at the highest selected concentrations (3.6 μg/mL, 16 μg/mL) are less elastic. It may be associated with the reorganization of the cellular cytoskeleton and the "strengthening" of the cell cortex caused by presence of silver nanoparticles. This observation does not depend on cell fixation. Agglomerates of silver nanoparticles were observed on the cell membrane as well as inside the cells. Copyright © 2018 John Wiley & Sons, Ltd.

An Enteroendocrine Cell – Enteric Glia Connection Revealed by 3D Electron Microscopy

PubMed Central

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. PMID:24587096

Engulfment of ceramic particles by fibroblasts does not alter cell behavior.

PubMed

Faye, Pierre-Antoine; Roualdes, Olivier; Rossignol, Fabrice; Hartmann, Daniel Jean; Desmoulière, Alexis

2017-02-17

Despite many studies, the impact of ceramic particles on cell behavior remains unclear. The aim of the present study was to investigate the effects of nano-sized ceramic particles on fibroblastic cells. Fibroblasts (dermal fibroblasts freshly isolated from skin samples and WI26 fibroblastic cells) were cultured in a monolayer in the presence of alumina or cerium-zirconia particles (≈50 nm diameter) at two concentrations (100 or 500 μg ml -1 ). Fluorescent alumina particles were also used. The following properties were analyzed: cell morphology, cytoplasmic ceramic incorporation (using confocal and transmission electron microscopy) and migration (using a silicon insert). Sedimentation field-flow fractionation (SdFFF) was also used to evaluate the rate of incorporation of ceramic particles into the cells. Finally, after treatment with various concentrations of ceramic particles, fibroblasts were also included in a collagen type I lattice constituting a dermal equivalent (DE), and the collagen lattice retraction and cell proliferation were evaluated. In monolayer conditions, the presence of both alumina and cerium-zirconia ceramic particles did not cause any deleterious effects on cultured cells (dermal fibroblast and WI26 cells) and cell fate was not affected in any way by the presence of ceramic particles in the cytoplasm. Confocal (using fluorescent alumina particles) and electron microscopy (using both alumina and cerium-zirconia particles) showed that ceramic particles were internalized in the WI26 cells. Using fluorescent membrane labeling and fluorescent alumina particles, a membrane was observed around the particle-containing vesicles present in the cytoplasm. Electron microscopy on WI26 cells showed the presence of a classical bilayer membrane around the ceramic particles. Interestingly, SdFFF confirmed that some dermal fibroblasts contained many alumina ceramic particles while others contained very few; in WI26 cells, the uptake of alumina ceramic was more homogeneous. In DE, collagen lattice retraction and cell proliferation were unchanged when WI26 fibroblastic cells contained alumina or cerium-zirconia ceramic particles. Our data suggest that ceramic particles are internalized in the cells by endocytosis. The presence of ceramic particles in the cytoplasm has no affect on cell behavior, confirming the excellent biocompatibility of this material and anticipating a minimal harmful effect of potential wear debris.

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

Bioaccumulation and distribution of selenium in Enterococcus durans.

PubMed

Pieniz, Simone; Andreazza, Robson; Mann, Michele Bertoni; Camargo, Flávio; Brandelli, Adriano

2017-03-01

Selenium is an essential nutrient for all living organisms. Under appropriate conditions lactic acid bacteria (LAB) are capable for accumulating large amounts of trace elements, such as selenium, and incorporating them into organic compounds. In this study, the capacity of selenium bioaccumulation by Enterococcus durans LAB18s was evaluated. The distribution of organic selenium in selenium-enriched E. durans LAB18s biomass was analyzed, and the highest percentage of organic selenium was found in the fraction of total protein, followed by the fractions of polysaccharides and nucleic acids. When the protein fraction was obtained by different extractions (water, NaCl, ethanol and NaOH) it was demonstrated that alkali-soluble protein showed the higher Selenium content. Analysis of protein fractions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that selenium was present in the proteins ranging from 23 to 100kDa. The cells were analyzed by scanning electron microscopy (SEM); scanning electron microscopy/energy dispersive spectrometry (SEM/EDS) and transmission electron microscopy (TEM). SEM, TEM and SEM/EDS showed the morphology, the selenium particles bioaccumulated into and on the cells and the amounts of selenium present into the cells, respectively. Thus, the isolate E. durans LAB18s can be a promising probiotic to be used as selenium-enriched biomass in feed trials. Copyright © 2016 Elsevier GmbH. All rights reserved.

Anterior lens epithelium in intumescent white cataracts - scanning and transmission electron microscopy study.

PubMed

Andjelic, Sofija; Drašlar, Kazimir; Hvala, Anastazija; Hawlina, Marko

2016-02-01

Our purpose was to study the structure of the lens epithelial cells (LECs) of intumescent white cataracts (IC) in comparison with nuclear cataracts (NC) in order to investigate possible structural reasons for development of IC. The anterior lens capsule (aLC: basement membrane and associated LECs) were obtained from cataract surgery and prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM). We observed by SEM that in IC, LEC swelling was pronounced with the clefts surrounding the groups of LECs. Another structural feature was spherical formations, that were observed on the apical side of LEC's, towards the fibre cell layer, both by SEM and TEM. Development of these structures, bulging out from the apical cell membrane of the LEC's and disrupting it, could be followed in steps towards the sphere formation. The degeneration of the lens epithelium and the structures of the aLC in IC similar to Morgagnian globules were also observed. None of these structural changes were observed in NC. We show by SEM and TEM that, in IC, LECs have pronounced structural features not observed in NC. This supports the hypothesis that the disturbed structure of LECs plays a role in water accumulation in the IC lens. We also suggest that, in IC, LECs produce bulging spheres that represent unique structures of degenerated material, extruded from the LEC.

[Histologic and ultrastructural studies of the patient died of highly pathogenic H5N1 avian influenza virus infection in China].

PubMed

Li, Ning; Zhu, Qing-Yu; Yu, Qi; Wang, Wei; Wang, Yi-Ping

2008-03-01

To explore histopathologic and ultrastructural characteristics of human avian influenza (AI) infection and related etiological pathogenesis. Postmortem lung and heart samples were collected from the patient who died of avian influenza virus infection on November 29, 2003 in China. Light and electron microscopy, immunohistochemistry and histochemistry were used to investigate the pathological changes. The main pathological findings included extensive pulmonary consolidation, hemorrhage, pulmonary edema and local hemorrhagic infarct. The lamina of alveoli and bronchioles were abundantly filled with protein-rich fluid, erythrocytes, fibrin and cell debris admixed with many neutrophilis, macrophages, lymphocytes and a few of monokaryon and multinuclear giant cells. Hyaline membranes were formed. Local pulmonary tissues were heavily damaged by hemorrhage and necrosis. Alveolar septum was disintegrated. Mesenchymal edema with a few of macrophages infiltration of heart was found. Electron microscopy showed the avian influenza A virus-like particles (type C and type A) of 80 - 120 nm diameter and envelopes in the cytoplasm of pneumocytes and endothelial cells. Fatal pneumonia associated with highly pathogenic avian influenza A virus (H5N1) infection leads to extensive pulmonary consolidation, edema and marked hemorrhagic necrosis and inflammation. Electron microscopy can identify avian influenza A virus-like particles. The findings may offer an important theoretical basis for clinical diagnosis and treatment.

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.

High-angle annular dark field scanning transmission electron microscopy on carbon-based functional polymer systems.

PubMed

Sourty, Erwan; van Bavel, Svetlana; Lu, Kangbo; Guerra, Ralph; Bar, Georg; Loos, Joachim

2009-06-01

Two purely carbon-based functional polymer systems were investigated by bright-field conventional transmission electron microscopy (CTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM). For a carbon black (CB) filled polymer system, HAADF-STEM provides high contrast between the CB agglomerates and the polymer matrix so that details of the interface organization easily can be revealed and assignment of the CB phase is straightforward. For a second system, the functional polymer blend representing the photoactive layer of a polymer solar cell, details of its nanoscale organization could be observed that were not accessible with CTEM. By varying the camera length in HAADF-STEM imaging, the contrast can be enhanced between crystalline and amorphous compounds due to diffraction contrast so that nanoscale interconnections between domains are identified. In general, due to its incoherent imaging characteristics HAADF-STEM allows for reliable interpretation of the data obtained.

In-situ spectro-microscopy on organic films: Mn-Phthalocyanine on Ag(100)

NASA Astrophysics Data System (ADS)

Al-Mahboob, Abdullah; Sadowski, Jerzy T.; Vescovo, Elio

2013-03-01

Metal phthalocyanines are attracting significant attention, owing to their potential for applications in chemical sensors, solar cells and organic magnets. As the electronic properties of molecular films are determined by their crystallinity and molecular packing, the optimization of film quality is important for improving the performance of organic devices. Here, we present the results of in situ low-energy electron microscopy / photoemission electron microscopy (LEEM/PEEM) studies of incorporation-limited growth of manganese-phthalocyanine (MnPc) on Ag(100) surfaces. MnPc thin films were grown on both, bulk Ag(100) surface and thin Ag(100)/Fe(100) films, where substrate spin-polarized electronic states can be modified through tuning the thickness of the Ag film. We also discuss the electronic structure and magnetic ordering in MnPc thin films, investigated by angle- and spin-resolved photoemission spectroscopy. Research carried out at the Center for Functional Nanomaterials and National Synchrotron Light Source, Brookhaven National Laboratory, which are supported by the U.S. Dept. of Energy, Office of Basic Energy Sciences, under Contract No. DE-AC02-98CH10886.

A large-sized bubbling appearance of the glomerular basement membrane in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis.

PubMed

Suga, Norihiro; Miura, Naoto; Uemura, Yuko; Nakamura, Toshinobu; Morita, Hiroyuki; Banno, Shogo; Imai, Hirokazu

2011-12-01

We report an unusual pathological finding, a large-sized bubbling appearance of the glomerular basement membrane (GBM), in a patient with pulmonary limited AL amyloidosis and a past history of lupus nephritis. The first renal biopsy specimen from 10 years ago, when systemic lupus erythematosus was diagnosed, demonstrated mild mesangial proliferation and subepithelial deposits (WHO classification: III + V). Light microscopy of the current biopsy using periodic acid methenamine silver (PAMS) stain demonstrated a large-sized bubbling appearance of the GBM; however, very weak immunoglobulin and complement deposition was observed in immunofluorescence studies. Routine electron microscopy demonstrated partial subendothelial expansion with electron-lucent materials, but no electron-dense deposits or amyloid fibrils. Electron microscopy with PAMS stain revealed electron-lucent endothelial scalloping, including some cellular components and microspheres in the GBM; however, it is not clear if these materials are derived from endothelial cells. One possibility is that these unique findings represent a recovery phase of lupus membranous nephritis; another is that these findings correspond to a new disease entity.

Automated detection of fluorescent cells in in-resin fluorescence sections for integrated light and electron microscopy.

PubMed

Delpiano, J; Pizarro, L; Peddie, C J; Jones, M L; Griffin, L D; Collinson, L M

2018-04-26

Integrated array tomography combines fluorescence and electron imaging of ultrathin sections in one microscope, and enables accurate high-resolution correlation of fluorescent proteins to cell organelles and membranes. Large numbers of serial sections can be imaged sequentially to produce aligned volumes from both imaging modalities, thus producing enormous amounts of data that must be handled and processed using novel techniques. Here, we present a scheme for automated detection of fluorescent cells within thin resin sections, which could then be used to drive automated electron image acquisition from target regions via 'smart tracking'. The aim of this work is to aid in optimization of the data acquisition process through automation, freeing the operator to work on other tasks and speeding up the process, while reducing data rates by only acquiring images from regions of interest. This new method is shown to be robust against noise and able to deal with regions of low fluorescence. © 2018 The Authors. Journal of Microscopy published by JohnWiley & Sons Ltd on behalf of Royal Microscopical Society.

Evidence of specialized tissue in human interatrial septum: histological, immunohistochemical and ultrastructural findings.

PubMed

Mitrofanova, Lubov B; Gorshkov, Andrey N; Lebedev, Dmitry S; Mikhaylov, Evgeny N

2014-01-01

There is a paucity of information on structural organization of muscular bundles in the interatrial septum (IAS). The aim was to investigate histologic and ultrastructural organization of muscular bundles in human IAS, including fossa ovalis (FO) and flap valve. Macroscopic and light microscopy evaluations of IAS were performed from postmortem studies of 40 patients. Twenty three IAS specimens underwent serial transverse sectioning, and 17--longitudinal sectioning. The transverse sections from 10 patients were immunolabeled for HCN4, Caveolin3 and Connexin43. IAS specimens from 6 other patients underwent electron microscopy. In all IAS specimens sections the FO, its rims and the flap valve had muscle fibers consisting of working cardiac myocytes. Besides the typical cardiomyocytes there were unusual cells: tortuous and horseshoe-shaped intertangled myocytes, small and large rounded myocytes with pale cytoplasm. The cells were aggregated in a definite structure in 38 (95%) cases, which was surrounded by fibro-fatty tissue. The height of the structure on transverse sections positively correlated with age (P = 0.03) and AF history (P = 0.045). Immunohistochemistry showed positive staining of the cells for HCN4 and Caveolin3. Electron microscopy identified cells with characteristics similar to electrical conduction cells. Specialized conduction cells in human IAS have been identified, specifically in the FO and its flap valve. The cells are aggregated in a structure, which is surrounded by fibrous and fatty tissue. Further investigations are warranted to explore electrophysiological characteristics of this structure.

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.

Silver nanoparticles synthesized with Rumex hymenosepalus extracts: effective broad-spectrum microbicidal agents and cytotoxicity study.

PubMed

Rodríguez-León, Ericka; Íñiguez-Palomares, Ramón A; Navarro, Rosa Elena; Rodríguez-Beas, César; Larios-Rodríguez, Eduardo; Alvarez-Cirerol, Francisco J; Íñiguez-Palomares, Claudia; Ramírez-Saldaña, Maricela; Hernández Martínez, Javier; Martínez-Higuera, Aarón; Galván-Moroyoqui, José Manuel; Martínez-Soto, Juan Manuel

2017-08-21

We synthesized silver nanoparticles using Rumex hymenosepalus root extract (Rh). Nanoparticles were subjected to a purification process and final product is a composite of Rh and silver nanoparticles (AgNPsC). Transmission electron microscopy (TEM), high-resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD), and X-ray photoelectron spectroscopy (XPS) were used to perform a microstructure study. Additionally, two fractions (RhA and RhB) were obtained from the original extract by filtration with tetrahydrofuran (THF); both fractions were analyzed using UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FT-IR), and 2,2-diphenyl-1-picrylhydrazyl (DPPH); total polyphenol content was also determined. Separate inhibition tests for AgNPsC and RhA and RhB were applied to Gram-positive bacteria, Gram-negative bacteria, and yeast (Candida albicans) using the well diffusion method. Extract fractions were found to have inhibitory effects only over Gram-positive bacteria, and silver nanoparticles showed inhibitory effects over all the evaluated microorganisms. Cytotoxicity was evaluated using the tetrazolium dye (MTT) assay in mononuclear peripheral blood cells. In addition, we assessment AgNPsC in THP-1 monocyte cell line, using the cell viability estimation by trypan blue dye exclusion test (TB) and Live/Dead (LD) cell viability assays by confocal microscopy.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells.

PubMed

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa E; Cuervo, Patricia; Jesus, José Batista de

2017-10-01

Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis.

Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane

PubMed Central

2005-01-01

The effects of antimicrobial peptides on artificial membranes have been well-documented; however, reports on the ultrastructural effects on the membranes of micro-organisms are relatively scarce. We compared the effects of histatin 5 and LL-37, two antimicrobial peptides present in human saliva, on the functional and morphological properties of the Candida albicans cell membrane. Fluorescence microscopy and immunogold transmission electron microscopy revealed that LL-37 remained associated with the cell wall and cell membrane, whereas histatin 5 transmigrated over the membrane and accumulated intracellularly. Freeze-fracture electron microscopy revealed that LL-37 severely affected the membrane morphology, resulting in the disintegration of the membrane bilayer into discrete vesicles, and an instantaneous efflux of small molecules such as ATP as well as larger molecules such as proteins with molecular masses up to 40 kDa. The effects of histatin 5 on the membrane morphology were less pronounced, but still resulted in the efflux of nucleotides. As the morphological defects induced by histatin 5 are much smaller than those induced by LL-37, but the efflux of nucleotides is similar at comparable candidacidal concentrations, we suggest that the loss of nucleotides plays an important role in the killing process. PMID:15707390

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

Impact of fipronil on the mushroom bodies of the stingless bee Scaptotrigona postica.

PubMed

Jacob, Cynthia R O; Soares, Hellen M; Nocelli, Roberta C F; Malaspina, Osmar

2015-01-01

Studies on stingless bees are scarce, and little is known about these insects, especially regarding the effects of contamination by neurotoxic insecticides, which can cause damage to important structures of the insect brain. This study evaluated the morphological changes in the intrinsic neurons of the protocerebral mushroom bodies (Kenyon cells) of the stingless bee Scaptotrigona postica after exposure to different doses of fipronil, using light microscopy and transmission electron microscopy. This region of the brain was selected for analysis because of its importance as a sensory integration centre. In both oral and topical treatments, Kenyon cells presented pyknotic profiles, suggesting cell death. Statistical analysis showed significant differences among doses and exposure times. Transmission electron microscopy revealed changes in the nucleus and cellular organelles. Depending on the dose, the characteristics observed suggested apoptotosis or necrosis. This study demonstrates the toxic effects of fipronil. An increase in the number of pyknotic profiles of Kenyon cells of mushroom bodies was observed even at the sublethal doses of 0.27 ng AI bee(-1) and 0.24 ng AI µL(-1) in the topical and oral treatments respectively. Also, differences in the number of pyknotic profiles were dose and time dependent. © 2014 Society of Chemical Industry.

Fine structures and ion images on fresh frozen dried ultrathin sections by transmission electron and scanning ion microscopy

NASA Astrophysics Data System (ADS)

Takaya, K.; Okabe, M.; Sawataishi, M.; Takashima, H.; Yoshida, T.

2003-01-01

Ion microscopy (IM) of air-dried or freeze-dried cryostat and semi-thin cryosections has provided ion images of elements and organic substances in wide areas of the tissue. For reproducible ion images by a shorter time of exposure to the primary ion beam, fresh frozen dried ultrathin sections were prepared by freezing the tissue in propane chilled with liquid nitrogen, cryocut at 60 nm, mounted on grids and silicon wafer pieces, and freeze-dried. Rat Cowper gland and sciatic nerve, bone marrow of the rat administered of lithium carbonate, tree frog and African toad spleen and buffy coat of atopic dermatitis patients were examined. Fine structures and ion images of the corresponding areas in the same or neighboring sections were observed by transmission electron microscopy (TEM) followed by sector type and time-of-flight type IM. Cells in the buffy coat contained larger amounts of potassium and magnesium while plasma had larger amounts of sodium and calcium. However, in the tissues, lithium, sodium, magnesium, calcium and potassium were distributed in the cell and calcium showed a granular appearance. A granular cell of the tree frog spleen contained sodium and potassium over the cell and magnesium and calcium were confined to granules.

Morphologic study of the effect of iron on pseudocyst formation in Trichomonas vaginalis and its interaction with human epithelial cells

PubMed Central

Dias-Lopes, Geovane; Saboia-Vahia, Leonardo; Margotti, Eliane Trindade; Fernandes, Nilma de Souza; Castro, Cássia Luana de Faria; Oliveira, Francisco Odencio; Peixoto, Juliana Figueiredo; Britto, Constança; Silva, Fernando Costa e; Cuervo, Patricia; de Jesus, José Batista

2017-01-01

BACKGROUND Trichomonas vaginalis is the aetiological agent of human trichomoniasis, which is one of the most prevalent sexually transmitted diseases in humans. Iron is an important element for the survival of this parasite and the colonisation of the host urogenital tract. OBJECTIVES In this study, we investigated the effects of iron on parasite proliferation in the dynamics of pseudocyst formation and morphologically characterised iron depletion-induced pseudocysts. METHODS We performed structural and ultrastructural analyses using light microscopy, scanning electron microscopy and transmission electron microscopy. FINDINGS It was observed that iron depletion (i) interrupts the proliferation of T. vaginalis, (ii) induces morphological changes in typical multiplicative trophozoites to spherical non-proliferative, non-motile pseudocysts, and (iii) induces the arrest of cell division at different stages of the cell cycle; (iv) iron is the fundamental element for the maintenance of typical trophozoite morphology; (v) pseudocysts induced by iron depletion are viable and reversible forms; and, finally, (vi) we demonstrated that pseudocysts induced by iron depletion are able to interact with human epithelial cells maintaining their spherical forms. MAIN CONCLUSIONS Together, these data suggest that pseudocysts could be induced as a response to iron nutritional stress and could have a potential role in the transmission and infection of T. vaginalis. PMID:28953994

[Screening antihydatid drugs using cultivated germinal cells of Echinococcus granulosus].

PubMed

Feng, J J; Xiao, S H; Guo, H F; Shen, B G; Jiao, W

1993-01-01

Germinal cells isolated from Echinococcus granulosus cysts harbored in mice have been maintained in an in vitro culture system containing RPMI 1640 supplemented by 20% calf serum, and used as a model for screening anti-hydatid drugs. When the germinal cells were maintained in the medium for 6 days, the cell proliferation rate was rather high in the first four days but declined in the last two days. In screening drugs, 1.4 x 10(6) germinal cells were exposed to known effective drugs against metacestodes of E. granulosus in mice, such as mebendazole (Meb), albendazole (Alb) or praziquantel (Pra) at various concentrations. One to three days after exposure, cell counts were made daily in 3 samples of each drug concentration. The mean cell number of each group was compared with that of the control and the inhibition rate of the cell was then calculated. The results showed that the minimal effective concentrations of Meb, Alb and Pra, were 1.0 (48 h), 2.5 (24 h) and 10.0 (72 h) micrograms/ml, respectively, while the inhibition rates of the cell were 34.1, 55.7 and 18.5%. Interestingly, the in vitro effects of Meb, Alb and Pra were consistent to those obtained from the in vivo tests, ie Meb > Alb > Pra. Nevertheless, after exposure of germinal cells to Meb at 2.5 micrograms/ml for 24 h, the cells appeared in roughness, indistinction, shrunk or swelling, collapse, deformation and hole-like feature detected by light microscopy and scanning electron-microscopy, while the ultrastructure alterations of the cells noted by transmission electron-microscopy were lysis in cytoplasm, disruption or disappearance of nucleus and even darkness of the whole cell.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Peptide nanoparticles (PNPs) modified disposable platform for sensitive electrochemical cytosensing of DLD-1 cancer cells.

PubMed

Yaman, Yesim Tugce; Akbal, Öznur; Bolat, Gulcin; Bozdogan, Betul; Denkbas, Emir Baki; Abaci, Serdar

2018-05-01

A novel diphenylalaninamid (FFA) based peptide nanoparticles (PNPs) modified pencil graphite electrodes (PGEs) for construction of electrochemical cytosensor was demonstrated for the first time in this study. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images revealed the spherical nanostructure of the synthesized FFA based PNPs while attenuated total reflectance-fourier transform infrared (ATR-FTIR) spectra provided information about the structure and conformation of proteins in their structure. Self-assembly of PNPs on PGE surface and adhesion of DLD-1 cancer cells on this surface was also characterized by electrochemical measurements. PNP/PGEs acted as a sensitive platform for simple and rapid quantification of low concentration of DLD-1 cancer cells in early diagnosis using the electrochemical impedance method (EIS). The offered cytosensor demonstrated outstanding performance for the detection of DLD-1 cells by the EIS method. The impedance of electronic transduction was associated with the amount of the immobilized cells ranging from 2 × 10 2 to 2.0 × 10 5 cellsmL -1 with a limit of detection of 100 cellsmL -1 . The efficient performance of the cytosensor was attributed to the well-defined nanostructure and biocompability of PNPs on the substrate. Copyright © 2017 Elsevier B.V. All rights reserved.

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.

Characteristic thickened cell walls of the bracts of the 'eternal flower' Helichrysum bracteatum.

PubMed

Nishikawa, Kuniko; Ito, Hiroaki; Awano, Tatsuya; Hosokawa, Munetaka; Yazawa, Susumu

2008-07-01

Helichrysum bracteatum is called an 'eternal flower' and has large, coloured, scarious bracts. These maintain their aesthetic value without wilting or discoloration for many years. There have been no research studies of cell death or cell morphology of the scarious bract, and hence the aim of this work was to elucidate these characteristics for the bract of H. bracteatum. DAPI (4'6-diamidino-2-phenylindol dihydrochloride) staining and fluorescence microscopy were used for observation of cell nuclei. Light microscopy (LM), transmission electron microscopy (TEM) and polarized light microscopy were used for observation of cells, including cell wall morphology. Cell death occurred at the bract tip during the early stage of flower development. The cell wall was the most prominent characteristic of H. bracteatum bract cells. Characteristic thickened secondary cell walls on the inside of the primary cell walls were observed in both epidermal and inner cells. In addition, the walls of all cells exhibited birefringence. Characteristic thickened secondary cell walls have orientated cellulose microfibrils as well as general secondary cell walls of the tracheary elements. For comparison, these characters were not observed in the petal and bract tissues of Chrysanthemum morifolium. Bracts at anthesis are composed of dead cells. Helichrysum bracteatum bracts have characteristic thickened secondary cell walls that have not been observed in the parenchyma of any other flowers or leaves. The cells of the H. bracteatum bract differ from other tissues with secondary cell walls, suggesting that they may be a new cell type.

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

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

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney

PubMed Central

Vega, Israel A.; Castro-Vazquez, Alfredo

2015-01-01

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail. PMID:25893243

Immune Defenses of the Invasive Apple Snail Pomacea canaliculata (Caenogastropoda, Ampullariidae): Phagocytic Hemocytes in the Circulation and the Kidney.

PubMed

Cueto, Juan A; Rodriguez, Cristian; Vega, Israel A; Castro-Vazquez, Alfredo

2015-01-01

Hemocytes in the circulation and kidney islets, as well as their phagocytic responses to microorganisms and fluorescent beads, have been studied in Pomacea canaliculata, using flow cytometry, light microscopy (including confocal laser scanning microscopy) and transmission electron microscopy (TEM). Three circulating hemocyte types (hyalinocytes, agranulocytes and granulocytes) were distinguished by phase contrast microscopy of living cells and after light and electron microscopy of fixed material. Also, three different populations of circulating hemocytes were separated by flow cytometry, which corresponded to the three hemocyte types. Hyalinocytes showed a low nucleus/cytoplasm ratio, and no apparent granules in stained material, but showed granules of moderate electron density under TEM (L granules) and at least some L granules appear acidic when labeled with LysoTracker Red. Both phagocytic and non-phagocytic hyalinocytes lose most (if not all) L granules when exposed to microorganisms in vitro. The phagosomes formed differed whether hyalinocytes were exposed to yeasts or to Gram positive or Gram negative bacteria. Agranulocytes showed a large nucleus/cytoplasm ratio and few or no granules. Granulocytes showed a low nucleus/cytoplasm ratio and numerous eosinophilic granules after staining. These granules are electron dense and rod-shaped under TEM (R granules). Granulocytes may show merging of R granules into gigantic ones, particularly when exposed to microorganisms. Fluorescent bead exposure of sorted hemocytes showed phagocytic activity in hyalinocytes, agranulocytes and granulocytes, but the phagocytic index was significantly higher in hyalinocytes. Extensive hemocyte aggregates ('islets') occupy most renal hemocoelic spaces and hyalinocyte-like cells are the most frequent component in them. Presumptive glycogen deposits were observed in most hyalinocytes in renal islets (they also occur in the circulation but less frequently) and may mean that hyalinocytes participate in the storage and circulation of this compound. Injection of microorganisms in the foot results in phagocytosis by hemocytes in the islets, and the different phagosomes formed are similar to those in circulating hyalinocytes. Dispersed hemocytes were obtained after kidney collagenase digestion and cell sorting, and they were able to phagocytize fluorescent beads. A role for the kidney as an immune barrier is proposed for this snail.

Mechanoreceptor Cells on the Tertiary Pulvini of Mimosa pudica L.

PubMed Central

Világi, Ildikó; Varró, Petra; Kristóf, Zoltán

2007-01-01

Special red cells were found on the adaxial surface of tertiary pulvini of Mimosa pudica and experiments performed to determine the origin and function of these cells. Using anatomical (light, scanning electron and transmission electron microscopy) and electrophysiological techniques, we have demonstrated that these red cells are real mechanoreceptor cells. They can generate receptor potential following mechanical stimuli and they are in connection with excitable motor cells (through plasmodesmata). We also provide evidence that these red cells are derived from stomatal subsidiary cells and not guard cells. As histochemical studies show red cells contain tannin, which is important in development of action potentials and movements of plants. These cells could be one of unidentified mechanoreceptors of mimosa. PMID:19517007

Measuring the Autocorrelation Function of Nanoscale Three-Dimensional Density Distribution in Individual Cells Using Scanning Transmission Electron Microscopy, Atomic Force Microscopy, and a New Deconvolution Algorithm.

PubMed

Li, Yue; Zhang, Di; Capoglu, Ilker; Hujsak, Karl A; Damania, Dhwanil; Cherkezyan, Lusik; Roth, Eric; Bleher, Reiner; Wu, Jinsong S; Subramanian, Hariharan; Dravid, Vinayak P; Backman, Vadim

2017-06-01

Essentially all biological processes are highly dependent on the nanoscale architecture of the cellular components where these processes take place. Statistical measures, such as the autocorrelation function (ACF) of the three-dimensional (3D) mass-density distribution, are widely used to characterize cellular nanostructure. However, conventional methods of reconstruction of the deterministic 3D mass-density distribution, from which these statistical measures can be calculated, have been inadequate for thick biological structures, such as whole cells, due to the conflict between the need for nanoscale resolution and its inverse relationship with thickness after conventional tomographic reconstruction. To tackle the problem, we have developed a robust method to calculate the ACF of the 3D mass-density distribution without tomography. Assuming the biological mass distribution is isotropic, our method allows for accurate statistical characterization of the 3D mass-density distribution by ACF with two data sets: a single projection image by scanning transmission electron microscopy and a thickness map by atomic force microscopy. Here we present validation of the ACF reconstruction algorithm, as well as its application to calculate the statistics of the 3D distribution of mass-density in a region containing the nucleus of an entire mammalian cell. This method may provide important insights into architectural changes that accompany cellular processes.

Measuring the Autocorrelation Function of Nanoscale Three-Dimensional Density Distribution in Individual Cells Using Scanning Transmission Electron Microscopy, Atomic Force Microscopy, and a New Deconvolution Algorithm

PubMed Central

Li, Yue; Zhang, Di; Capoglu, Ilker; Hujsak, Karl A.; Damania, Dhwanil; Cherkezyan, Lusik; Roth, Eric; Bleher, Reiner; Wu, Jinsong S.; Subramanian, Hariharan; Dravid, Vinayak P.; Backman, Vadim

2018-01-01

Essentially all biological processes are highly dependent on the nanoscale architecture of the cellular components where these processes take place. Statistical measures, such as the autocorrelation function (ACF) of the three-dimensional (3D) mass–density distribution, are widely used to characterize cellular nanostructure. However, conventional methods of reconstruction of the deterministic 3D mass–density distribution, from which these statistical measures can be calculated, have been inadequate for thick biological structures, such as whole cells, due to the conflict between the need for nanoscale resolution and its inverse relationship with thickness after conventional tomographic reconstruction. To tackle the problem, we have developed a robust method to calculate the ACF of the 3D mass–density distribution without tomography. Assuming the biological mass distribution is isotropic, our method allows for accurate statistical characterization of the 3D mass–density distribution by ACF with two data sets: a single projection image by scanning transmission electron microscopy and a thickness map by atomic force microscopy. Here we present validation of the ACF reconstruction algorithm, as well as its application to calculate the statistics of the 3D distribution of mass–density in a region containing the nucleus of an entire mammalian cell. This method may provide important insights into architectural changes that accompany cellular processes. PMID:28416035

Internalization of Carbon Nano-onions by Hippocampal Cells Preserves Neuronal Circuit Function and Recognition Memory.

PubMed

Trusel, Massimo; Baldrighi, Michele; Marotta, Roberto; Gatto, Francesca; Pesce, Mattia; Frasconi, Marco; Catelani, Tiziano; Papaleo, Francesco; Pompa, Pier Paolo; Tonini, Raffaella; Giordani, Silvia

2018-05-23

One area where nanomedicine may offer superior performances and efficacy compared to current strategies is in the diagnosis and treatment of central nervous system (CNS) diseases. However, the application of nanomaterials in such complex arenas is still in its infancy and an optimal vector for the therapy of CNS diseases has not been identified. Graphitic carbon nano-onions (CNOs) represent a class of carbon nanomaterials that shows promising potential for biomedical purposes. To probe the possible applications of graphitic CNOs as a platform for therapeutic and diagnostic interventions on CNS diseases, fluorescently labeled CNOs were stereotaxically injected in vivo in mice hippocampus. Their diffusion within brain tissues and their cellular localization were analyzed ex vivo by confocal microscopy, electron microscopy, and correlative light-electron microscopy techniques. The subsequent fluorescent staining of hippocampal cells populations indicates they efficiently internalize the nanomaterial. Furthermore, the inflammatory potential of the CNOs injection was found comparable to sterile vehicle infusion, and it did not result in manifest neurophysiological and behavioral alterations of hippocampal-mediated functions. These results clearly demonstrate that CNOs can interface effectively with several cell types, which encourages further their development as possible brain disease-targeted diagnostics or therapeutics nanocarriers.

An Unroofing Method to Observe the Cytoskeleton Directly at Molecular Resolution Using Atomic Force Microscopy

PubMed Central

Usukura, Eiji; Narita, Akihiro; Yagi, Akira; Ito, Shuichi; Usukura, Jiro

2016-01-01

An improved unroofing method enabled the cantilever of an atomic force microscope (AFM) to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in phosphate-buffered saline (PBS) at a higher resolution than conventional electron microscopy. All of the actin filaments clearly exhibited a short periodicity of approximately 5–6 nm, which was derived from globular actins linked to each other to form filaments, as well as a long helical periodicity. The polarity of the actin filaments appeared to be determined by the shape of the periodic striations. Microtubules were identified based on their thickness. Clathrin coats and caveolae were observed on the cytoplasmic surface of cell membranes. The area containing clathrin molecules and their terminal domains was directly visualized. Characteristic ridge structures located at the surface of the caveolae were observed at high resolution, similar to those observed with electron microscopy (EM). Overall, unroofing allowed intracellular AFM imaging in a liquid environment with a level of quality equivalent or superior to that of EM. Thus, AFMs are anticipated to provide cutting-edge findings in cell biology and histology. PMID:27273367

Microstructural observation of fuel cell catalyst inks by Cryo-SEM and Cryo-TEM.

PubMed

Shimanuki, Junichi; Takahashi, Shinichi; Tohma, Hajime; Ohma, Atsushi; Ishihara, Ayumi; Ito, Yoshiko; Nishino, Yuri; Miyazawa, Atsuo

2017-06-01

In order to improve the electricity generation performance of fuel cell electric vehicles, it is necessary to optimize the microstructure of the catalyst layer of a polymer electrolyte fuel cell. The catalyst layer is formed by a wet coating process using catalyst inks. Therefore, it is very important to observe the microstructure of the catalyst ink. In this study, the morphology of carbon-supported platinum (Pt/C) particles in catalyst inks with a different solvent composition was investigated by cryogenic scanning electron microscopy (cryo-SEM). In addition, the morphology of the ionomer, which presumably influences the formation of agglomerated Pt/C particles in a catalyst ink, was investigated by cryogenic transmission electron microscopy (cryo-TEM). The results of a cryo-SEM observation revealed that the agglomerated Pt/C particles tended to become coarser with a higher 1-propanol (NPA) weight fraction. The results of a cryo-TEM observation indicated that the actual ionomer dispersion in a catalyst ink formed a network structure different from that of the ionomer in the solvent. © The Author 2017. 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.

Design of a bioresorbable polymeric scaffold for osteoblast culture

NASA Astrophysics Data System (ADS)

Ditaranto, Vincent M., Jr.

Bioresorbable polymeric scaffolds were designed for the purpose of growing rat osteosarcoma cells (ROS 17/2.8) using the compression molding method. The material used in the construction of the scaffolds was a mixture of polycaprolactone (PCL), Hydroxyapatite (HA), Glycerin (GL) and salt (NaCl) for porosity. The concentration of the several materials utilized, was determined by volume. Past research at the University of Massachusetts Lowell (UML) has successfully utilized the compression molding method for the construction of scaffolds, but was unable to accomplish the goal of long term cell survival and complete cellular proliferation throughout a three dimensional scaffold. This research investigated various concentrations of the materials and molding temperatures used for the manufacture of scaffolds in order to improve the scaffold design and address those issues. The design of the scaffold using the compression molding process is detailed in the Method and Materials section of this thesis. The porogen (salt) used for porosity was suspected as a possible source of contamination causing cell apoptosis in past studies. This research addressed the issues for cell survival and proliferation throughout a three dimensional scaffold. The leaching of the salt was one major design modification. This research successfully used ultrasonic leaching in addition to the passive method. Prior to cell culture, the scaffolds were irradiated to 2.75 Mrad, with cobalt-60 gamma radionuclide. The tissue culture consisted of two trials: (1) cell culture in scaffolds cleaned with passive leaching; (2) cell culture with scaffolds cleaned with ultrasonic leaching. Cell survival and proliferation was accomplished only with the addition of ultrasonic leaching of the scaffolds. Analysis of the scaffolds included Scanning Electron Microscopy (SEM), Nikon light microscopy and x-ray mapping of the calcium, sodium and chloride ion distribution. The cells were analyzed by Environmental Scanning Electron Microscopy (ESEM) and Nikon light microscopy. The high magnification of ESEM up to 60,000 x revealed an unexpected discovery. The osteoblasts appeared to be remodeling the PCL scaffold shown in the last two figures of this research.

The response of single human cells to zero gravity

NASA Technical Reports Server (NTRS)

Montgomery, P. O., Jr.; Cook, J. E.; Reynolds, R. C.; Paul, J. S.; Hayflick, L.; Stock, D.; Schulz, W. W.; Kimzey, S. L.; Thirolf, R. G.; Rogers, T.

1974-01-01

The SO15 experiment was designed to extend observations of the effects of zero-gravity to living human cells during and subsequent to a 59-day flight on Skylab 3. A strain of diploid human embryonic lung cells, WI-38, was chosen for this purpose. The studies were concerned with observations designed to detect the effects of zero-gravity on cell growth rates and on cell structure as observed by light microscopy, transmission and scanning electron microscopy and histochemistry. Studies of the effects of zero-gravity on the cell function and the cell cycle were performed by time lapse motion picture photography and microspectrophotometry. Subsequent study of the returned living cells included karotyping, G- and C-banding, and analyses of the culture media used. Some of the living cells returned were banked by deep freeze techniques for possible future experiments.

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.

The Relationship between Fenestrations, Sieve Plates and Rafts in Liver Sinusoidal Endothelial Cells

PubMed Central

McNerney, Gregory P.; Owen, Dylan M.; Zencak, Dusan; Zykova, Svetlana N.; Crane, Harry; Huser, Thomas; Quinn, Ronald J.; Smedsrød, Bård; Le Couteur, David G.; Cogger, Victoria C.

2012-01-01

Fenestrations are transcellular pores in endothelial cells that facilitate transfer of substrates between blood and the extravascular compartment. In order to understand the regulation and formation of fenestrations, the relationship between membrane rafts and fenestrations was investigated in liver sinusoidal endothelial cells where fenestrations are grouped into sieve plates. Three dimensional structured illumination microscopy, scanning electron microscopy, internal reflectance fluorescence microscopy and two-photon fluorescence microscopy were used to study liver sinusoidal endothelial cells isolated from mice. There was an inverse distribution between sieve plates and membrane rafts visualized by structured illumination microscopy and the fluorescent raft stain, Bodipy FL C5 ganglioside GM1. 7-ketocholesterol and/or cytochalasin D increased both fenestrations and lipid-disordered membrane, while Triton X-100 decreased both fenestrations and lipid-disordered membrane. The effects of cytochalasin D on fenestrations were abrogated by co-administration of Triton X-100, suggesting that actin disruption increases fenestrations by its effects on membrane rafts. Vascular endothelial growth factor (VEGF) depleted lipid-ordered membrane and increased fenestrations. The results are consistent with a sieve-raft interaction, where fenestrations form in non-raft lipid-disordered regions of endothelial cells once the membrane-stabilizing effects of actin cytoskeleton and membrane rafts are diminished. PMID:23029409

Foundation laid for understanding essentials of cell division | Center for Cancer Research

Cancer.gov

NCI Center for Cancer Research (CCR) scientists reported new molecular insights into understanding a critical aspect of cell division through a cross-disciplinary effort that combines cryo-electron microscopy (cryo-EM), biochemical and cell biological approaches. Errors in segregation of chromosomes during mitosis can lead to an aberrant number of chromosomes, a condition

As-prepared MoS2 quantum dot as a facile fluorescent probe for long-term tracing of live cells

NASA Astrophysics Data System (ADS)

Zhou, Kai; Zhang, Yue; Xia, Zhining; Wei, Weili

2016-07-01

Recently, the newly emerged two-dimensional nanomaterials, layered transition metal dichalcogenide (e.g. MoS2) nanosheets, have drawn tremendous attentions due to their extraordinary electronic and optical properties, and MoS2 quantum dots (MoS2 QDs) with lateral sizes less than 10 nm have been found to be highly luminescent. In the present study, a facile approach for large-scale preparation of MoS2 QDs by Na intercalation reaction without using any toxic organic reagents is proposed. MoS2 QDs were carefully characterized by various techniques including transmission electron microscopy, atomic force microscopy, dynamic light scattering, spectroscopy, in vitro cytotoxicology, and capillary electrophoresis. The as-prepared MoS2 QDs were strongly fluorescent, highly photo-stable, low in cytotoxicity, and readily reactive to thiols. These inherent properties of MoS2 QDs make them excellent fluorescent probes for long-term live cell tracing. The results of live cells imaging indicated that MoS2 QD stained cells remained highly fluorescent after long-term culture, and could be easily traced from other co-cultured cell lines.

Biocompatibility of MG-63 cells on collagen, poly-L-lactic acid, hydroxyapatite scaffolds with different parameters.

PubMed

Cecen, Berivan; Kozaci, Didem; Yuksel, Mithat; Erdemli, Diler; Bagriyanik, Alper; Havitcioglu, Hasan

2015-03-18

In this study, osteoblast-like MG-63 cells were cultured on 3 different scaffold types composed of (a) collagen + poly-L-lactic acid (PLLA), (b) collagen + hydroxyapatite (HA; 30ºC) or (c) collagen + hydroxyapatite (HA; 37ºC) and produced with different porosities. Biomechanical properties of the scaffolds were characterized by tensile strength measurements. Properties of the cell-seeded scaffolds were evaluated with scanning electron microscopy (SEM). Cell adhesion and proliferation capacities were evaluated. Alkaline phosphatase (ALP) levels in media were measured. Transmission electron microscopy (TEM) and histological analyses were used to assess morphological characteristics. Our results showed that collagen-based PLLA and HA scaffolds have good cell biocompatibility. MTT test showed that the scaffolds exhibited no cytotoxicity. According to the force and displacement data, collagen + HA at 37ºC showed the highest mechanical strength and displacement. The results suggest that collagen-based PLLA and HA scaffolds might improve osteoblastic growth in vitro and have biomaterial integration potential in possible therapeutic approaches for future clinical studies.