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Sample records for biological electron microscopy

  1. Scanning transmission electron microscopy of biological structures.

    PubMed

    Colliex, C; Mory, C

    1994-01-01

    The design of the scanning transmission electron microscope (STEM) has been conceived to optimize its detection efficiency of the different elastic and inelastic signals resulting from the interaction of the high energy primary electrons with the specimen. Its potential use to visualize and measure biological objects was recognized from the first studies by Crewe and coworkers in the seventies. Later the real applications have not followed the initial hopes. The purpose of the present paper is to describe how the instrument has practically evolved and recently begun to demonstrate all its potentialities for quantitative electron microscopy of a wide range of biological specimens, from freeze-dried isolated macromolecules to unstained cryosections. Emphasis will be put on the mass-mapping, multi-signal and elemental mapping modes which are unique features of the STEM instruments.

  2. Application of Electron Diffraction to Biological Electron Microscopy

    PubMed Central

    Glaeser, Robert M.; Thomas, Gareth

    1969-01-01

    Three methods by which electron diffraction may be applied to problems in electron microscopy are discussed from a fundamental point of view, and experimental applications with biological specimens are demonstrated for each case. It is shown that wide-angle electron diffraction provides valuable information for evaluating specimen damage that can occur either during specimen preparation or while in the electron beam. Dark-field electron microscopy can be used both to enhance the image contrast and to provide highly restricted and therefore highly specific information about the object. Low-angle electron diffraction provides quantitative information about the object structure in the range from 20 A to ∼ 1000 A. Lowangle electron diffraction also demonstrates the important role of Fourier contrast with biological specimens, which are usually characterized by structural features with dimensions of 20 A or larger. ImagesFigure 1Figure 2Figure 5Figure 6Figure 7Figure 8Figure 9Figure 10Figure 11Figure 13 PMID:4896898

  3. 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.

  4. Electron Microscopy.

    ERIC Educational Resources Information Center

    Beer, Michael

    1980-01-01

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

  5. 3D electron microscopy of biological nanomachines: principles and applications.

    PubMed

    Sorzano, C O S; Jonic, S; Cottevieille, M; Larquet, E; Boisset, N; Marco, S

    2007-11-01

    Transmission electron microscopy is a powerful technique for studying the three-dimensional (3D) structure of a wide range of biological specimens. Knowledge of this structure is crucial for fully understanding complex relationships among macromolecular complexes and organelles in living cells. In this paper, we present the principles and main application domains of 3D transmission electron microscopy in structural biology. Moreover, we survey current developments needed in this field, and discuss the close relationship of 3D transmission electron microscopy with other experimental techniques aimed at obtaining structural and dynamical information from the scale of whole living cells to atomic structure of macromolecular complexes.

  6. Electron Microscopy of Biological Materials at the Nanometer Scale

    NASA Astrophysics Data System (ADS)

    Kourkoutis, Lena Fitting; Plitzko, Jürgen M.; Baumeister, Wolfgang

    2012-08-01

    Electron microscopy of biological matter uses three different imaging modalities: (a) electron crystallography, (b) single-particle analysis, and (c) electron tomography. Ideally, these imaging modalities are applied to frozen-hydrated samples to ensure an optimal preservation of the structures under scrutiny. Cryo-electron microscopy of biological matter has made important advances in the past decades. It has become a research tool that further expands the scope of structural research into unique areas of cell and molecular biology, and it could augment the materials research portfolio in the study of soft and hybrid materials. This review addresses how researchers using transmission electron microscopy can derive structural information at high spatial resolution from fully hydrated specimens, despite their sensitivity to ionizing radiation, despite the adverse conditions of high vacuum for samples that have to be kept in aqueous environments, and despite their low contrast resulting from weakly scattering building blocks.

  7. Unravelling biological macromolecules with cryo-electron microscopy.

    PubMed

    Fernandez-Leiro, Rafael; Scheres, Sjors H W

    2016-09-14

    Knowledge of the three-dimensional structures of proteins and other biological macromolecules often aids understanding of how they perform complicated tasks in the cell. Because many such tasks involve the cleavage or formation of chemical bonds, structural characterization at the atomic level is most useful. Developments in the electron microscopy of frozen hydrated samples (cryo-electron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules. This is resulting in a wave of information about processes in the cell that were impossible to characterize with existing techniques in structural biology.

  8. Unravelling biological macromolecules with cryo-electron microscopy.

    PubMed

    Fernandez-Leiro, Rafael; Scheres, Sjors H W

    2016-01-01

    Knowledge of the three-dimensional structures of proteins and other biological macromolecules often aids understanding of how they perform complicated tasks in the cell. Because many such tasks involve the cleavage or formation of chemical bonds, structural characterization at the atomic level is most useful. Developments in the electron microscopy of frozen hydrated samples (cryo-electron microscopy) are providing unprecedented opportunities for the structural characterization of biological macromolecules. This is resulting in a wave of information about processes in the cell that were impossible to characterize with existing techniques in structural biology. PMID:27629640

  9. Another 60 years in electron microscopy: development of phase-plate electron microscopy and biological applications.

    PubMed

    Nagayama, Kuniaki

    2011-01-01

    It has been six decades since the concept of phase-plate electron microscopy was first reported by Boersch, but an experimental report on a phase plate with a theoretically rational performance has only recently been released by a group including the present author. Currently, many laboratories around the world are attempting to develop a wide range of phase plates to enhance the capabilities of transmission electron microscopy. They are reporting not only advantages of their own developments but also a fundamental problem inherent to electron beam devices, namely charging, i.e. the accumulation of electrostatic charge. In this report, we review the 60-year history of phase-plate development, with a particular focus on the fundamental issue of phase-plate charging. Next, we review biological applications of qualified phase plates, which have been successful in avoiding charging to some extent. Finally, we compare and discuss electron microscopic images, taken with or without phase plates, of biological targets such as proteins (GroEL and TRPV4), protein complexes (flagellar motor), viruses (T4 phage, ε-15 phage and herpes simplex virus), bacterial (cyanobacteria) and mammalian (PtK2) cells. PMID:21844600

  10. A national facility for biological cryo-electron microscopy

    SciTech Connect

    Saibil, Helen R.; 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 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.

  11. Transmission electron microscopy in molecular structural biology: A historical survey.

    PubMed

    Harris, J Robin

    2015-09-01

    In this personal, historic account of macromolecular transmission electron microscopy (TEM), published data from the 1940s through to recent times is surveyed, within the context of the remarkable progress that has been achieved during this time period. The evolution of present day molecular structural biology is described in relation to the associated biological disciplines. The contribution of numerous electron microscope pioneers to the development of the subject is discussed. The principal techniques for TEM specimen preparation, thin sectioning, metal shadowing, negative staining and plunge-freezing (vitrification) of thin aqueous samples are described, with a selection of published images to emphasise the virtues of each method. The development of digital image analysis and 3D reconstruction is described in detail as applied to electron crystallography and reconstructions from helical structures, 2D membrane crystals as well as single particle 3D reconstruction of icosahedral viruses and macromolecules. The on-going development of new software, algorithms and approaches is highlighted before specific examples of the historical progress of the structural biology of proteins and viruses are presented.

  12. Transmission electron microscopy in molecular structural biology: A historical survey.

    PubMed

    Harris, J Robin

    2015-09-01

    In this personal, historic account of macromolecular transmission electron microscopy (TEM), published data from the 1940s through to recent times is surveyed, within the context of the remarkable progress that has been achieved during this time period. The evolution of present day molecular structural biology is described in relation to the associated biological disciplines. The contribution of numerous electron microscope pioneers to the development of the subject is discussed. The principal techniques for TEM specimen preparation, thin sectioning, metal shadowing, negative staining and plunge-freezing (vitrification) of thin aqueous samples are described, with a selection of published images to emphasise the virtues of each method. The development of digital image analysis and 3D reconstruction is described in detail as applied to electron crystallography and reconstructions from helical structures, 2D membrane crystals as well as single particle 3D reconstruction of icosahedral viruses and macromolecules. The on-going development of new software, algorithms and approaches is highlighted before specific examples of the historical progress of the structural biology of proteins and viruses are presented. PMID:25475529

  13. 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

  14. A national facility for biological cryo-electron microscopy.

    PubMed

    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

  15. Scanning tunneling and scanning transmission electron microscopy of biological membranes

    NASA Astrophysics Data System (ADS)

    Stemmer, A.; Reichelt, R.; Engel, A.; Rosenbusch, J. P.; Ringger, M.; Hidber, H. R.; Güntherodt, H. J.

    1987-03-01

    The feasibility of imaging porin membrane, which is a reconstituted biological membrane consisting of phospholipid and protein, was studied by scanning tunneling microscopy (STM). Due to detailed knowledge of its composition from biochemical and its three-dimensional (3D) structure from electron microscopical analysis, porin vesicles seem to be a suitable model specimen for exploring the application of STM in biology. Unstained vesicles adsorbed onto a thin amorphous carbon film supported by a finder grid were localized using a scanning transmission electron microscope (STEM) at low irradiation doses ( < 100 {e -}/{nm 2}). Suitable areas of the sample were then positioned in the STM by a light optical telescope. STM images taken under ambient pressure from empty amorphous carbon films exhibited corrugations in the range of ⩽ 1 nm, whereas steps having a height of 5 nm were reproducibly observed on grids with porin vesicles. Since this value is in good agreement with that obtained from air-dried metal shadowed vesicles, we interpret these steps as the edges of porin membranes.

  16. Three-dimensional scanning transmission electron microscopy of biological specimens.

    PubMed

    de Jonge, Niels; Sougrat, Rachid; Northan, Brian M; Pennycook, Stephen J

    2010-02-01

    A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2-3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

  17. Three-dimensional scanning transmission electron microscopy of biological specimens

    SciTech Connect

    De Jonge, Niels; Sougrat, Rachid; Northan, Brian; Pennycook, Stephen J

    2010-01-01

    A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2 - 3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original data set. The precision of the height determination was 0.2 nm. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy (TEM). However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved data set.

  18. Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

    EPA Science Inventory

    Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum "cast" intended for examination by transmission electron microscopy. Specimens are subjected to u...

  19. Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.

    PubMed

    Lucas, Miriam S; Günthert, Maja; Gasser, Philippe; Lucas, Falk; Wepf, Roger

    2012-01-01

    The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

  20. Use of fluorescence and scanning electron microscopy as tools in teaching biology

    NASA Astrophysics Data System (ADS)

    Ghosh, Nabarun; Silva, Jessica; Vazquez, Aracely; Das, A. B.; Smith, Don W.

    2011-06-01

    Recent nationwide surveys reveal significant decline in students' interest in Math and Sciences. The objective of this project was to inspire young minds in using various techniques involved in Sciences including Scanning Electron Microscopy. We used Scanning Electron Microscope in demonstrating various types of Biological samples. An SEM Tabletop model in the past decade has revolutionized the use of Scanning Electron Microscopes. Using SEM Tabletop model TM 1000 we studied biological specimens of fungal spores, pollen grains, diatoms, plant fibers, dust mites, insect parts and leaf surfaces. We also used fluorescence microscopy to view, to record and analyze various specimens with an Olympus BX40 microscope equipped with FITC and TRITC fluorescent filters, a mercury lamp source, DP-70 digital camera with Image Pro 6.0 software. Micrographs were captured using bright field microscopy, the fluoresceinisothiocyanate (FITC) filter, and the tetramethylrhodamine (TRITC) filter settings at 40X. A high pressure mercury lamp or UV source was used to excite the storage molecules or proteins which exhibited autofluorescence. We used fluorescent microscopy to confirm the localization of sugar beet viruses in plant organs by viewing the vascular bundles in the thin sections of the leaves and other tissues. We worked with the REU summer students on sample preparation and observation on various samples utilizing the SEM. Critical Point Drying (CPD) and metal coating with the sputter coater was followed before observing some cultured specimen and the samples that were soft in textures with high water content. SEM Top allowed investigating the detailed morphological features that can be used for classroom teaching. Undergraduate and graduate researchers studied biological samples of Arthropods, pollen grains and teeth collected from four species of snakes using SEM. This project inspired the research students to pursue their career in higher studies in science and 45% of the

  1. A new method of scanning electron microscopy for imaging biological tissues.

    PubMed

    Boyde, A; Reid, S A

    1983-04-01

    The scanning electron microscope (SEM) has proved of little value in the examination of material prepared for light microscopic histology. One of the chief reasons for this is that the secondary electron signal used for image formation in routine scanning microscopy derives from the surface of the specimen. In the case of histological material this surface is one which has been severely distorted by processing and cutting procedures. Light microscopy sections can be usefully studied in te SEM if the signal used to form the image derives from a considerable portion of the thickness of the section. Thus the backscattered electron (BSE) image has been successfully used in studying the distribution of dense material or densely staining components several micrometres deep to the surface of dried sections. Such sections are, however, usually mounted on low density (poorly BSE reflecting) non-transparent substrates such as beryllium or carbon, so that matching light microscopy of the same samples is not possible. We report here a method by which histological sections mounted on glass slides can be imaged in the SEM at a resolution higher than that obtained using conventional light microscopy. The method exploits the facts that the ordinary, cheap light microscope slide is strongly cathodoluminescent, yet the standard histological (7 micrometers) section is of such a mass thickness that it absorbs a significant proportion of electrons which energies (5-20 keV) usually used in biological SEM. Thus the measure of the glass cathodoluminescence signal is the measure of the electron flux passing through the specimen.

  2. Investigation of resins suitable for the preparation of biological sample for 3-D electron microscopy.

    PubMed

    Kizilyaprak, Caroline; Longo, Giovanni; Daraspe, Jean; Humbel, Bruno M

    2015-02-01

    In the last two decades, the third-dimension has become a focus of attention in electron microscopy to better understand the interactions within subcellular compartments. Initially, transmission electron tomography (TEM tomography) was introduced to image the cell volume in semi-thin sections (∼ 500 nm). With the introduction of the focused ion beam scanning electron microscope, a new tool, FIB-SEM tomography, became available to image much larger volumes. During TEM tomography and FIB-SEM tomography, the resin section is exposed to a high electron/ion dose such that the stability of the resin embedded biological sample becomes an important issue. The shrinkage of a resin section in each dimension, especially in depth, is a well-known phenomenon. To ensure the dimensional integrity of the final volume of the cell, it is important to assess the properties of the different resins and determine the formulation which has the best stability in the electron/ion beam. Here, eight different resin formulations were examined. The effects of radiation damage were evaluated after different times of TEM irradiation. To get additional information on mass-loss and the physical properties of the resins (stiffness and adhesion), the topography of the irradiated areas was analysed with atomic force microscopy (AFM). Further, the behaviour of the resins was analysed after ion milling of the surface of the sample with different ion currents. In conclusion, two resin formulations, Hard Plus and the mixture of Durcupan/Epon, emerged that were considerably less affected and reasonably stable in the electron/ion beam and thus suitable for the 3-D investigation of biological samples. PMID:25433274

  3. Biological Applications and Transmission Electron Microscopy Investigations of Mesoporous Silica Nanoparticles

    SciTech Connect

    Trewyn, Brian G.

    2006-01-01

    The research presented and discussed within involves the development of novel biological applications of mesoporous silica nanoparticles (MSN) and an investigation of mesoporous material by transmission electron microscopy (TEM). Mesoporous silica nanoparticles organically functionalized shown to undergo endocytosis in cancer cells and drug release from the pores was controlled intracellularly and intercellularly. Transmission electron microscopy investigations demonstrated the variety of morphologies produced in this field of mesoporous silica nanomaterial synthesis. A series of room-temperature ionic liquid (RTIL) containing mesoporous silica nanoparticle (MSN) materials with various particle morphologies, including spheres, ellipsoids, rods, and tubes, were synthesized. By changing the RTIL template, the pore morphology was tuned from the MCM-41 type of hexagonal mesopores to rotational moire type of helical channels, and to wormhole-like porous structures. These materials were used as controlled release delivery nanodevices to deliver antibacterial ionic liquids against Escherichia coli K12. The involvement of a specific organosiloxane function group, covalently attached to the exterior of fluorescein doped mesoporous silica nanoparticles (FITC-MSN), on the degree and kinetics of endocytosis in cancer and plant cells was investigated. The kinetics of endocystosis of TEG coated FITC-MSN is significantly quicker than FITC-MSN as determined by flow cytometry experiments. The fluorescence confocal microscopy investigation showed the endocytosis of TEG coated-FITC MSN triethylene glycol grafted fluorescein doped MSN (TEG coated-FITC MSN) into both KeLa cells and Tobacco root protoplasts. Once the synthesis of a controlled-release delivery system based on MCM-41-type mesoporous silica nanorods capped by disulfide bonds with superparamagnetic iron oxide nanoparticles was completed. The material was characterized by general methods and the dosage and kinetics of the

  4. Scanning ultrafast electron microscopy

    PubMed Central

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

    2010-01-01

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

  5. Biological applications and transmission electron microscopy investigation of mesoporous silica nanoparticles

    NASA Astrophysics Data System (ADS)

    Trewyn, Brian G.

    The research presented and discussed within involves the development of novel biological applications of mesoporous silica nanoparticles (MSN) and an investigation of mesoporous material by transmission electron microscopy (TEM). A series of room-temperature ionic liquid (RTIL) containing mesoporous silica nanoparticle (MSN) materials with various particle morphologies, including spheres, ellipsoids, rods, and tubes, were synthesized. By changing the RTIL template, the pore morphology was tuned from the MCM-41 type of hexagonal mesopores to rotational moire type of helical channels, and to wormhole-like porous structures. These materials were used as controlled release delivery nanodevices to deliver antibacterial ionic liquids against Escherichia coli K12. The involvement of a specific organosiloxane function group, covalently attached to the exterior of fluorescein doped mesoporous silica nanoparticles (FITC-MSN), on the degree and kinetics of endocytosis in cancer and plant cells was investigated. The kinetics of endocystosis of TEG coated FITC-MSN is significantly quicker than FITC-MSN as determined by flow cytometry experiments. The fluorescence confocal microscopy investigation showed the endocytosis of TEG coated-FITC MSN triethylene glycol grafted fluorescein doped MSN (TEG coated-FITC MSN) into both HeLa cells and Tobacco root protoplasts. Once the synthesis of a controlled-release delivery system based on MCM-41-type mesoporous silica nanorods capped by disulfide bonds with superparamagnetic iron oxide nanoparticles was completed. The material was characterized by general methods and the dosage and kinetics of the antioxidant dependent release was measured. Finally, the biological interaction of the material was determined along with TEM measurements. An electron microscopy investigation proved that the pore openings of the MSN were indeed blocked by the Fe 3O4 nanoparticles. The biological interaction investigation demonstrated Fe3O4-capped MSN

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

    PubMed

    Chen, S; Goode, A E; Skepper, J N; Thorley, A J; Seiffert, J M; Chung, K F; Tetley, T D; Shaffer, M S P; Ryan, M P; Porter, A E

    2016-02-01

    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 scrutinized. 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.

  7. Silver stain for electron microscopy

    NASA Technical Reports Server (NTRS)

    Corbett, R. L.

    1972-01-01

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

  8. An introduction to sample preparation and imaging by cryo-electron microscopy for structural biology

    PubMed Central

    Thompson, Rebecca F.; Walker, Matt; Siebert, C. Alistair; Muench, Stephen P.; Ranson, Neil A.

    2016-01-01

    Transmission electron microscopy (EM) is a versatile technique that can be used to image biological specimens ranging from intact eukaryotic cells to individual proteins >150 kDa. There are several strategies for preparing samples for imaging by EM, including negative staining and cryogenic freezing. In the last few years, cryo-EM has undergone a ‘resolution revolution’, owing to both advances in imaging hardware, image processing software, and improvements in sample preparation, leading to growing number of researchers using cryo-EM as a research tool. However, cryo-EM is still a rapidly growing field, with unique challenges. Here, we summarise considerations for imaging of a range of specimens from macromolecular complexes to cells using EM. PMID:26931652

  9. Multi-resolution correlative focused ion beam scanning electron microscopy: applications to cell biology.

    PubMed

    Narayan, Kedar; Danielson, Cindy M; Lagarec, Ken; Lowekamp, Bradley C; Coffman, Phil; Laquerre, Alexandre; Phaneuf, Michael W; Hope, Thomas J; Subramaniam, Sriram

    2014-03-01

    Efficient correlative imaging of small targets within large fields is a central problem in cell biology. Here, we demonstrate a series of technical advances in focused ion beam scanning electron microscopy (FIB-SEM) to address this issue. We report increases in the speed, robustness and automation of the process, and achieve consistent z slice thickness of ∼3 nm. We introduce "keyframe imaging" as a new approach to simultaneously image large fields of view and obtain high-resolution 3D images of targeted sub-volumes. We demonstrate application of these advances to image post-fusion cytoplasmic intermediates of the HIV core. Using fluorescently labeled cell membranes, proteins and HIV cores, we first produce a "target map" of an HIV infected cell by fluorescence microscopy. We then generate a correlated 3D EM volume of the entire cell as well as high-resolution 3D images of individual HIV cores, achieving correlative imaging across a volume scale of 10(9) in a single automated experimental run.

  10. Fundamental technical elements of freeze-fracture/freeze-etch in biological electron microscopy.

    PubMed

    Carson, Johnny L

    2014-01-01

    Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum "cast" intended for examination by transmission electron microscopy. Specimens are subjected to ultrarapid freezing rates, often in the presence of cryoprotective agents to limit ice crystal formation, with subsequent fracturing of the specimen at liquid nitrogen cooled temperatures under high vacuum. The resultant fractured surface is replicated and stabilized by evaporation of carbon and platinum from an angle that confers surface three-dimensional detail to the cast. This technique has proved particularly enlightening for the investigation of cell membranes and their specializations and has contributed considerably to the understanding of cellular form to related cell function. In this report, we survey the instrument requirements and technical protocol for performing freeze-fracture, the associated nomenclature and characteristics of fracture planes, variations on the conventional procedure, and criteria for interpretation of freeze-fracture images. This technique has been widely used for ultrastructural investigation in many areas of cell biology and holds promise as an emerging imaging technique for molecular, nanotechnology, and materials science studies. PMID:25285532

  11. Scanning transmission electron microscopy through-focal tilt-series on biological specimens.

    PubMed

    Trepout, Sylvain; Messaoudi, Cédric; Perrot, Sylvie; Bastin, Philippe; Marco, Sergio

    2015-10-01

    Since scanning transmission electron microscopy can produce high signal-to-noise ratio bright-field images of thick (≥500 nm) specimens, this tool is emerging as the method of choice to study thick biological samples via tomographic approaches. However, in a convergent-beam configuration, the depth of field is limited because only a thin portion of the specimen (from a few nanometres to tens of nanometres depending on the convergence angle) can be imaged in focus. A method known as through-focal imaging enables recovery of the full depth of information by combining images acquired at different levels of focus. In this work, we compare tomographic reconstruction with the through-focal tilt-series approach (a multifocal series of images per tilt angle) with reconstruction with the classic tilt-series acquisition scheme (one single-focus image per tilt angle). We visualised the base of the flagellum in the protist Trypanosoma brucei via an acquisition and image-processing method tailored to obtain quantitative and qualitative descriptors of reconstruction volumes. Reconstructions using through-focal imaging contained more contrast and more details for thick (≥500 nm) biological samples.

  12. Perspectives on low voltage transmission electron microscopy as applied to cell biology.

    PubMed

    Bendayan, Moise; Paransky, Eugene

    2014-12-01

    Low voltage transmission electron microscopy (LVTEM) with accelerating voltages as low as 5 kV was applied to cell biology. To take advantage of the increased contrast given by LVTEM, tissue preparation was modified omitting all heavy metals such as osmium, uranium, and lead from the fixation, on block staining and counterstaining. Nonstained ultra-thin tissue sections (40 nm thick) generated highly contrasted images. While the aspect of the cells remains similar to that obtained by conventional TEM, some new substructures were revealed. The pancreatic acinar cells granules present a heterogeneous matrix with partitions corresponding to segregation of their different secretory proteins. Microvilli display their core of microfilaments anchored to the dense top membrane. Mitochondria revealed the presence of distinct particles along their cristea membranes that may correspond to the ATP synthase complexes or oxysomes. The dense nuclear chromatin displays a honey-comb appearance while distinct beads aligned along thin threads were seen in the dispersed chromatin. These new features revealed by LVTEM correlate with structures described or predicted through other approaches. Masking effects due to thickness of the tissue sections and to the presence of heavy metals must have prevented their observation by conventional TEM. Furthermore, the immunogold was adapted to LVTEM revealing nuclear lamin-A at the edge of the dense chromatin ribbons. Combining cytochemistry with LVTEM brings additional advantages to this new approach in cell biology.

  13. The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases.

    PubMed

    Hehl, A B; Hemphill, A

    2006-09-01

    Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host. PMID:17024976

  14. Workshop on the validation and modeling of electron cryo-microscopy structures of biological nanomachines.

    PubMed

    Ludtke, Steven J; Lawson, Catherine L; Kleywegt, Gerard J; Berman, Helen M; Chiu, Wah

    2011-01-01

    Electron cryo-microscopy (cryoEM) is a rapidly maturing methodology in structural biology, which now enables the determination of 3D structures of molecules, macromolecular complexes and cellular components at resolutions as high as 3.5Å, bridging the gap between light microscopy and X-ray crystallography/NMR. In recent years structures of many complex molecular machines have been visualized using this method. Single particle reconstruction, the most widely used technique in cryoEM, has recently demonstrated the capability of producing structures at resolutions approaching those of X-ray crystallography, with over a dozen structures at better than 5 Å resolution published to date. This method represents a significant new source of experimental data for molecular modeling and simulation studies. CryoEM derived maps and models are archived through EMDataBank.org joint deposition services to the EM Data Bank (EMDB) and Protein Data Bank (PDB), respectively. CryoEM maps are now being routinely produced over the 3 - 30 Å resolution range, and a number of computational groups are developing software for building coordinate models based on this data and developing validation techniques to better assess map and model accuracy. In this workshop we will present the results of the first cryoEM modeling challenge, in which computational groups were asked to apply their tools to a selected set of published cryoEM structures. We will also compare the results of the various applied methods, and discuss the current state of the art and how we can most productively move forward.

  15. Dynamic Transmission Electron Microscopy

    SciTech Connect

    Evans, James E.; Jungjohann, K. L.; Browning, Nigel D.

    2012-10-12

    Dynamic transmission electron microscopy (DTEM) combines the benefits of high spatial resolution electron microscopy with the high temporal resolution of ultrafast lasers. The incorporation of these two components into a single instrument provides a perfect platform for in situ observations of material processes. However, previous DTEM applications have focused on observing structural changes occurring in samples exposed to high vacuum. Therefore, in order to expand the pump-probe experimental regime to more natural environmental conditions, in situ gas and liquid chambers must be coupled with Dynamic TEM. This chapter describes the current and future applications of in situ liquid DTEM to permit time-resolved atomic scale observations in an aqueous environment, Although this chapter focuses mostly on in situ liquid imaging, the same research potential exists for in situ gas experiments and the successful integration of these techniques promises new insights for understanding nanoparticle, catalyst and biological protein dynamics with unprecedented spatiotemporal resolution.

  16. 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. PMID:25392009

  17. High sensitivity electron spin magnetic resonance force microscopy for labeled biological samples

    NASA Astrophysics Data System (ADS)

    Moore, Eric W.; Lee, Sanggap; Hickman, Steven A.; Wright, Sarah J.; Harrell, Lee E.; Longenecker, Jonilyn G.; Borbat, Peter P.; Freed, Jack H.; Marohn, John A.

    2010-03-01

    Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of organic materials due to its high sensitivity and isotopic specificity. Labeling of proteins, DNA and biomolecular assemblies with free radical labels for inductive detection are well established techniques, although many of these radical's relaxation times are too short to support previously demonstrated techniques for single electron detection by magnetic resonance force microscopy. We report on our efforts toward sub-single electron sensitivity on organic radicals using batch fabricated 100 nm nickel nanorod tipped ultrasensitive cantilevers.

  18. New and unconventional approaches for advancing resolution in biological transmission electron microscopy by improving macromolecular specimen preparation and preservation.

    SciTech Connect

    Massover, W.; Materials Science Division

    2011-02-01

    Resolution in transmission electron microscopy (TEM) now is limited by the properties of specimens, rather than by those of instrumentation. The long-standing difficulties in obtaining truly high-resolution structure from biological macromolecules with TEM demand the development, testing, and application of new ideas and unconventional approaches. This review concisely describes some new concepts and innovative methodologies for TEM that deal with unsolved problems in the preparation and preservation of macromolecular specimens. The selected topics include use of better support films, a more protective multi-component matrix surrounding specimens for cryo-TEM and negative staining, and, several quite different changes in microscopy and micrography that should decrease the effects of electron radiation damage; all these practical approaches are non-traditional, but have promise to advance resolution for specimens of biological macromolecules beyond its present level of 3-10 {angstrom} (0.3-1.0 nm). The result of achieving truly high resolution will be a fulfillment of the still unrealized potential of transmission electron microscopy for directly revealing the structure of biological macromolecules down to the atomic level.

  19. Dynamic Processes in Biology, Chemistry, and Materials Science: Opportunities for UltraFast Transmission Electron Microscopy - Workshop Summary Report

    SciTech Connect

    Kabius, Bernd C.; Browning, Nigel D.; Thevuthasan, Suntharampillai; Diehl, Barbara L.; Stach, Eric A.

    2012-07-25

    This report summarizes a 2011 workshop that addressed the potential role of rapid, time-resolved electron microscopy measurements in accelerating the solution of important scientific and technical problems. A series of U.S. Department of Energy (DOE) and National Academy of Science workshops have highlighted the critical role advanced research tools play in addressing scientific challenges relevant to biology, sustainable energy, and technologies that will fuel economic development without degrading our environment. Among the specific capability needs for advancing science and technology are tools that extract more detailed information in realistic environments (in situ or operando) at extreme conditions (pressure and temperature) and as a function of time (dynamic and time-dependent). One of the DOE workshops, Future Science Needs and Opportunities for Electron Scattering: Next Generation Instrumentation and Beyond, specifically addressed the importance of electron-based characterization methods for a wide range of energy-relevant Grand Scientific Challenges. Boosted by the electron optical advancement in the last decade, a diversity of in situ capabilities already is available in many laboratories. The obvious remaining major capability gap in electron microscopy is in the ability to make these direct in situ observations over a broad spectrum of fast (µs) to ultrafast (picosecond [ps] and faster) temporal regimes. In an effort to address current capability gaps, EMSL, the Environmental Molecular Sciences Laboratory, organized an Ultrafast Electron Microscopy Workshop, held June 14-15, 2011, with the primary goal to identify the scientific needs that could be met by creating a facility capable of a strongly improved time resolution with integrated in situ capabilities. The workshop brought together more than 40 leading scientists involved in applying and/or advancing electron microscopy to address important scientific problems of relevance to DOE’s research

  20. Single-particle reconstruction of biological macromolecules in electron microscopy – 30 years

    PubMed Central

    Frank, Joachim

    2010-01-01

    This essay gives the author’s personal account on the development of concepts underlying single-particle reconstruction, a technique in electron microscopy of macromolecular assemblies with a remarkable record of achievements as of late. The ribosome proved to be an ideal testing ground for the development of specimen preparation methods, cryo-EM techniques, and algorithms, with discoveries along the way as a rich reward. Increasingly, cryo-EM and single-particle reconstruction, in combination with classification techniques, is revealing dynamic information on functional molecular machines uninhibited by molecular contacts. PMID:20025794

  1. Closer to the native state. Critical evaluation of cryo-techniques for Transmission Electron Microscopy: preparation of biological samples.

    PubMed

    Mielanczyk, Lukasz; Matysiak, Natalia; Michalski, Marek; Buldak, Rafal; Wojnicz, Romuald

    2014-01-01

    Over the years Transmission Electron Microscopy (TEM) has evolved into a powerful technique for the structural analysis of cells and tissues at various levels of resolution. However, optimal sample preservation is required to achieve results consistent with reality. During the last few decades, conventional preparation methods have provided most of the knowledge about the ultrastructure of organelles, cells and tissues. Nevertheless, some artefacts can be introduced at all stagesofstandard electron microscopy preparation technique. Instead, rapid freezing techniques preserve biological specimens as close as possible to the native state. Our review focuses on different cryo-preparation approaches, starting from vitrification methods dependent on sample size. Afterwards, we discuss Cryo-Electron Microscopy Of VItreous Sections (CEMOVIS) and the main difficulties associated with this technique. Cryo-Focused Ion Beam (cryo-FIB) is described as a potential alternative for CEMOVIS. Another post-processing route for vitrified samples is freeze substitution and embedding in resin for structural analysis or immunolocalization analysis. Cryo-sectioning according to Tokuyasu is a technique dedicated to high efficiency immunogold labelling. Finally, we introduce hybrid techniques, which combine advantages of primary techniques originally dedicated to different approaches. Hybrid approaches permit to perform the study of difficult-to-fix samples and antigens or help optimize the sample preparation protocol for the integrated Laser and Electron Microscopy (iLEM) technique.

  2. Graphene as a transparent conductive support for studying biological molecules by transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Nair, R. R.; Blake, P.; Blake, J. R.; Zan, R.; Anissimova, S.; Bangert, U.; Golovanov, A. P.; Morozov, S. V.; Geim, A. K.; Novoselov, K. S.; Latychevskaia, T.

    2010-10-01

    We demonstrate the application of graphene as a support for imaging individual biological molecules in transmission electron microscope (TEM). A simple procedure to produce free-standing graphene membranes has been designed. Such membranes are extremely robust and can support practically any submicrometer object. Tobacco mosaic virus has been deposited on graphene samples and observed in a TEM. High contrast has been achieved even though no staining has been applied.

  3. Graphene as a transparent conductive support for studying biological molecules by transmission electron microscopy

    SciTech Connect

    Nair, R. R.; Anissimova, S.; Novoselov, K. S.; Blake, P.; Blake, J. R.; Geim, A. K.; Zan, R.; Bangert, U.; Golovanov, A. P.; Morozov, S. V.; Latychevskaia, T.

    2010-10-11

    We demonstrate the application of graphene as a support for imaging individual biological molecules in transmission electron microscope (TEM). A simple procedure to produce free-standing graphene membranes has been designed. Such membranes are extremely robust and can support practically any submicrometer object. Tobacco mosaic virus has been deposited on graphene samples and observed in a TEM. High contrast has been achieved even though no staining has been applied.

  4. Electron microscopy of frozen biological objects: a study using cryosectioning and cryosubstitution.

    PubMed

    Erk, I; Nicolas, G; Caroff, A; Lepault, J

    1998-03-01

    Freezing of bulk biological objects was investigated by X-ray cryodiffraction. Freezing at atmospheric pressure of most microscopic biological samples gives rise to large hexagonal crystals and leads to poor structural preservation of these specimens. High-pressure freezing induces the formation of different ices (hexagonal, cubic and a high-pressure form) consisting of crystals having sizes smaller than those formed at atmospheric pressure. With both freezing methods, a cryoprotectant has to be added to the biological object to avoid the formation of ice crystals. However, special cases can be encountered: some biological objects contain large amounts of natural cryoprotectant or have a low water content. In these cases, vitrification can be achieved, especially using high-pressure freezing. Cryo-sectioning can be performed on vitrified samples, and the sections studied by electron cryomicroscopy. Images and electron diffraction patterns having a resolution better than 2 and 0.2 nm, respectively, can be obtained with such sections. Because samples containing crystalline ices cannot be cryosectioned, their structure has to be studied using cryosubstitution and resin embedding. We show that bacteria, yeast, and ciliate and marine worm elytrum have cellular compartments with an organization that has not been described by classical techniques relying on chemical fixation of the tissues. A high-pressure artefact affecting the Paramecium trichocysts is described. Such artefacts are not general; for example, we show that 70% of high-pressure frozen yeast cells survive successive high-pressure freezing and thawing steps.

  5. Direct observation of unstained wet biological samples by scanning-electron generation X-ray microscopy.

    PubMed

    Ogura, Toshihiko

    2010-01-01

    Analytical tools of nanometre-scale resolution are indispensable in the fields of biology, physics and chemistry. One suitable tool, the soft X-ray microscope, provides high spatial resolution of visible light for wet specimens. For biological specimens, X-rays of water-window wavelength between carbon (284 eV; 4.3 nm) and oxygen (540 eV; 2.3 nm) absorption edges provide high-contrast imaging of biological samples in water. Among types of X-ray microscope, the transmission X-ray microscope using a synchrotron radiation source with diffractive zone plates offers the highest spatial resolution, approaching 15-10nm. However, even higher resolution is required to measure proteins and protein complexes in biological specimens; therefore, a new type of X-ray microscope with higher resolution that uses a simple light source is desirable. Here we report a novel scanning-electron generation X-ray microscope (SGXM) that demonstrates direct imaging of unstained wet biological specimens. We deposited wet yeasts in the space between two silicon nitride (Si(3)N(4)) films. A scanning electron beam of accelerating voltage 5 keV and current 1.6 nA irradiates the titanium (Ti)-coated Si(3)N(4) film, and the soft X-ray signal from it is detected by an X-ray photodiode (PD) placed below the sample. The SGXM can theoretically achieve better than 5 nm resolution. Our method can be utilized easily for various wet biological samples of bacteria, viruses, and protein complexes.

  6. A New Approach to Studying Biological and Soft Materials Using Focused Ion Beam Scanning Electron Microscopy (FIB SEM)

    NASA Astrophysics Data System (ADS)

    Stokes, D. J.; Morrissey, F.; Lich, B. H.

    2006-02-01

    Over the last decade techniques such as confocal light microscopy, in combination with fluorescent labelling, have helped biologists and life scientists to study biological architectures at tissue and cell level in great detail. Meanwhile, obtaining information at very small length scales is possible with the combination of sample preparation techniques and transmission electron microscopy (TEM) or scanning transmission electron microscopy (STEM). Scanning electron microscopy (SEM) is well known for the determination of surface characteristics and morphology. However, the desire to understand the three dimensional relationships of meso-scale hierarchies has led to the development of advanced microscopy techniques, to give a further complementary approach. A focused ion beam (FIB) can be used as a nano-scalpel and hence allows us to reveal internal microstructure in a site-specific manner. Whilst FIB instruments have been used to study and verify the three-dimensional architecture of man made materials, SEM and FIB technologies have now been brought together in a single instrument representing a powerful combination for the study of biological specimens and soft materials. We demonstrate the use of FIB SEM to study three-dimensional relationships for a range of length scales and materials, from small-scale cellular structures to the larger scale interactions between biomedical materials and tissues. FIB cutting of heterogeneous mixtures of hard and soft materials, resulting in a uniform cross-section, has proved to be of particular value since classical preparation methods tend to introduce artefacts. Furthermore, by appropriate selection, we can sequentially cross-section to create a series of 'slices' at specific intervals. 3D reconstruction software can then be used to volume-render information from the 2D slices, enabling us to immediately see the spatial relationships between microstructural components.

  7. 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

  8. Computation in electron microscopy.

    PubMed

    Kirkland, Earl J

    2016-01-01

    Some uses of the computer and computation in high-resolution transmission electron microscopy are reviewed. The theory of image calculation using Bloch wave and multislice methods with and without aberration correction is reviewed and some applications are discussed. The inverse problem of reconstructing the specimen structure from an experimentally measured electron microscope image is discussed. Some future directions of software development are given. PMID:26697863

  9. Preparation, theory, and biological applications of highly luminescent cadmium selenide/zinc sulfide quantum dots in optical and electron microscopy

    NASA Astrophysics Data System (ADS)

    Bouwer, James Christopher

    This dissertation describes the preparation, theory, and applications of ZnS overcoated CdSe (core) quantum dots for applications as fluorescent probes in optical microscopy and as electron energy loss spectroscopy (EELS) probes in electron microscopy, with applications to the biological sciences. The dissertation begins with a brief overview of quantum dots and their history. Next, a brief overview of the necessary semiconductor theory is discussed including the origin of the band gap, the origin of holes, the concepts of phonons, and trap states. Then, the role of the confinement potential in the quantum dot fluorescent spectrum is discussed in the context of the 3-dimensional spherical well. Included in this discussion is the role of excitonic electron-hole bound states. To provide a complete document useful to anyone who wishes to continue work along these lines, included is a methods section which describes the complete process of synthesis of the CdSe cores, overcoating the cores with ZnS, size selection of nanocrystals, water solubilization, and protein conjugation. The methods used in live cell labeling are included as well. In the section that follows, a discussion of the mathematical methods of image correlation spectroscopy (ICS) for extracting dynamic constants such as flow rates and diffusion constants from time lapse optical image data is discussed in the context of quantum dot fluorescent probes. Dynamic constants were obtained using live NIH3T3 mouse fibroblast cells labeled with IgG-anti-EGF conjugated quantum dots. These same cells were then fixed, imbedded in resin, sectioned to 100nm thick sections and imaged under the electron microscope. The electron dense cadmium selinide provides the contrast necessary to perform direct imaging of EGF receptor sites. In order to improve the data and move toward multi-channel imaging in the electron microscope, EELS spectroscopy and elemental mapping of quantum dots was performed. The theory along with a

  10. Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules.

    PubMed

    Han, Bong-Gyoon; Watson, Zoe; Kang, Hannah; Pulk, Arto; Downing, Kenneth H; Cate, Jamie; Glaeser, Robert M

    2016-08-01

    We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

  11. Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules.

    PubMed

    Han, Bong-Gyoon; Watson, Zoe; Kang, Hannah; Pulk, Arto; Downing, Kenneth H; Cate, Jamie; Glaeser, Robert M

    2016-08-01

    We describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2μm holes of the support films. Randomly biotinylated 70S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure. PMID:27320699

  12. Electron microscopy of biological macromolecules: Bridging the gapbetween what physics allows and what we currently can get

    SciTech Connect

    Typke, Dieter; Downing, Kenneth H.; Glaeser, Robert M.

    2003-04-30

    The resolution achieved in low-dose electron microscopy of biological macromolecules is significantly worse than what can be obtained on the same microscopes with more robust specimens. When two-dimensional crystals are used, it is also apparent that the high-resolution image contrast is much less than what it could be if the images were perfect. Since specimen charging is one factor that might limit the contrast and resolution achieved with biological specimens, we have investigated the use of holey support films that have been coated with a metallic film before depositing specimens onto a thin carbon film that is suspended over the holes. Monolayer crystals of paraffin (C44H90) are used as a test specimen for this work because of the relative ease in imaging Bragg spacings at {approx}0.4 nm resolution, the relative ease of measuring the contrast in these images, and the similar degree of radiation sensitivity of these crystals when compared to biological macromolecules. A metallic coating on the surrounding support film does, indeed, produce a significant improvement in the high-resolution contrast for a small fraction of the images. The majority of images show little obvious improvement, however, and even the coated area of the support film continues to show a significant amount of beam-induced movement under low-dose conditions. The fact that the contrast in the best images can be as much as 25 percent-35 percent of what it would be in a perfect image is nevertheless encouraging, demonstrating that it should be possible, in principle, to achieve the same performance for every image. Routine data collection of this quality would make it possible to determine the structure of large, macromolecular complexes without the need to grow crystals of these difficult specimen materials.

  13. Liquid Cell Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-01

    Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM.

  14. Liquid Cell Transmission Electron Microscopy.

    PubMed

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-27

    Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM.

  15. Spectroscopic imaging in electron microscopy

    SciTech Connect

    Pennycook, Stephen J; Colliex, C.

    2012-01-01

    In the scanning transmission electron microscope, multiple signals can be simultaneously collected, including the transmitted and scattered electron signals (bright field and annular dark field or Z-contrast images), along with spectroscopic signals such as inelastically scattered electrons and emitted photons. In the last few years, the successful development of aberration correctors for the electron microscope has transformed the field of electron microscopy, opening up new possibilities for correlating structure to functionality. Aberration correction not only allows for enhanced structural resolution with incident probes into the sub-angstrom range, but can also provide greater probe currents to facilitate mapping of intrinsically weak spectroscopic signals at the nanoscale or even the atomic level. In this issue of MRS Bulletin, we illustrate the power of the new generation of electron microscopes with a combination of imaging and spectroscopy. We show the mapping of elemental distributions at atomic resolution and also the mapping of electronic and optical properties at unprecedented spatial resolution, with applications ranging from graphene to plasmonic nanostructures, and oxide interfaces to biology.

  16. Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells

    PubMed Central

    2012-01-01

    Background Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130

  17. Electron microscopy and forensic practice

    NASA Astrophysics Data System (ADS)

    Kotrlý, Marek; Turková, Ivana

    2013-05-01

    Electron microanalysis in forensic practice ranks among basic applications used in investigation of traces (latents, stains, etc.) from crime scenes. Applying electron microscope allows for rapid screening and receiving initial information for a wide range of traces. SEM with EDS/WDS makes it possible to observe topography surface and morphology samples and examination of chemical components. Physical laboratory of the Institute of Criminalistics Prague use SEM especially for examination of inorganic samples, rarely for biology and other material. Recently, possibilities of electron microscopy have been extended considerably using dual systems with focused ion beam. These systems are applied mainly in study of inner micro and nanoparticles , thin layers (intersecting lines in graphical forensic examinations, analysis of layers of functional glass, etc.), study of alloys microdefects, creating 3D particles and aggregates models, etc. Automated mineralogical analyses are a great asset to analysis of mineral phases, particularly soils, similarly it holds for cathode luminescence, predominantly colour one and precise quantitative measurement of their spectral characteristics. Among latest innovations that are becoming to appear also at ordinary laboratories are TOF - SIMS systems and micro Raman spectroscopy with a resolution comparable to EDS/WDS analysis (capable of achieving similar level as through EDS/WDS analysis).

  18. Soil microstructure and electron microscopy

    NASA Technical Reports Server (NTRS)

    Smart, P.; Fryer, J. R.

    1988-01-01

    As part of the process of comparing Martian soils with terrestial soils, high resolution electron microscopy and associated techniques should be used to examine the finer soil particles, and various techniques of electron and optical microscopy should be used to examine the undisturbed structure of Martian soils. To examine the structure of fine grained portions of the soil, transmission electron microscopy may be required. A striking feature of many Martian soils is their red color. Although the present-day Martian climate appears to be cold, this color is reminiscent of terrestial tropical red clays. Their chemical contents are broadly similar.

  19. Electronic Blending in Virtual Microscopy

    ERIC Educational Resources Information Center

    Maybury, Terrence S.; Farah, Camile S.

    2010-01-01

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

  20. High-resolution low-temperature scanning electron microscopy for observing intracellular structures of quick frozen biological specimens.

    PubMed

    Inoué, T; Koike, H

    1989-11-01

    Intracellular structures of rapidly frozen biological tissues were observed in 3-D under a low-temperature scanning electron microscope using a newly developed side-entry type cryo-holder. The present low-temperature SEM is simple, easy to operate and effective for observing biological materials at high magnification. Biological tissues (the pancreas, small intestine, brown adipose tissue and Harderian gland) freshly removed from the mouse were immediately frozen in liquid propane cooled with liquid nitrogen, and their surfaces were manually fractured using a precooled razor blade in liquid nitrogen before introducing the cryo-holder into the SEM. When intracellular structures were revealed after appropriate sublimation, the specimens were coated with gold using a metal evaporator fitted to the side of the microscope column at one of the specimen chamber ports. The cryo-holder was connected to a copper braid coming from a liquid nitrogen reservoir to maintain a low temperature. Using this method, intracellular structures such as the mitochondria and endoplasmic reticulum were demonstrated at high magnifications. Ribosomal granules were discerned on the rough endoplasmic reticulum of the pancreatic acinar cells. Granular substances, presumably elementary particles, were also recognized on the mitochondrial cristae of the brown adipose tissue. The method was particularly effective for studying the 3-D configuration of lipid droplets which had been difficult to preserve by chemical fixation. PMID:2593147

  1. 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. PMID:26919369

  2. 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.

  3. Electron microscopy of electromagnetic waveforms.

    PubMed

    Ryabov, A; Baum, P

    2016-07-22

    Rapidly changing electromagnetic fields are the basis of almost any photonic or electronic device operation. We report how electron microscopy can measure collective carrier motion and fields with subcycle and subwavelength resolution. A collimated beam of femtosecond electron pulses passes through a metamaterial resonator that is previously excited with a single-cycle electromagnetic pulse. If the probing electrons are shorter in duration than half a field cycle, then time-frozen Lorentz forces distort the images quasi-classically and with subcycle time resolution. A pump-probe sequence reveals in a movie the sample's oscillating electromagnetic field vectors with time, phase, amplitude, and polarization information. This waveform electron microscopy can be used to visualize electrodynamic phenomena in devices as small and fast as available. PMID:27463670

  4. Electron microscopy of electromagnetic waveforms

    NASA Astrophysics Data System (ADS)

    Ryabov, A.; Baum, P.

    2016-07-01

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

  5. Electron microscopy of electromagnetic waveforms.

    PubMed

    Ryabov, A; Baum, P

    2016-07-22

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

  6. Sample preparation of biological macromolecular assemblies for the determination of high-resolution structures by cryo-electron microscopy.

    PubMed

    Stark, Holger; Chari, Ashwin

    2016-02-01

    Single particle cryo-EM has recently developed into a powerful tool to determine the 3D structure of macromolecular complexes at near-atomic resolution, which allows structural biologists to build atomic models of proteins. All technical aspects of cryo-EM technology have been considerably improved over the last two decades, including electron microscopic hardware, image processing software and the ever growing speed of computers. This leads to a more widespread use of the technique, and it can be anticipated that further automation of electron microscopes and image processing tools will soon fully shift the focus away from the technological aspects, onto biological questions that can be answered. In single particle cryo-EM, no crystals of a macromolecule are required. In contrast to X-ray crystallography, this significantly facilitates structure determination by cryo-EM. Nevertheless, a relatively high level of biochemical control is still essential to obtain high-resolution structures by cryo-EM, and it can be anticipated that the success of the cryo-EM technology goes hand in hand with further developments of sample purification and preparation techniques. This will allow routine high-resolution structure determination of the many macromolecular complexes of the cell that until now represent evasive targets for X-ray crystallographers. Here we discuss the various biochemical tools that are currently available and the existing sample purification and preparation techniques for cryo-EM grid preparation that are needed to obtain high-resolution images for structure determination. PMID:26671943

  7. Sample preparation of biological macromolecular assemblies for the determination of high-resolution structures by cryo-electron microscopy.

    PubMed

    Stark, Holger; Chari, Ashwin

    2016-02-01

    Single particle cryo-EM has recently developed into a powerful tool to determine the 3D structure of macromolecular complexes at near-atomic resolution, which allows structural biologists to build atomic models of proteins. All technical aspects of cryo-EM technology have been considerably improved over the last two decades, including electron microscopic hardware, image processing software and the ever growing speed of computers. This leads to a more widespread use of the technique, and it can be anticipated that further automation of electron microscopes and image processing tools will soon fully shift the focus away from the technological aspects, onto biological questions that can be answered. In single particle cryo-EM, no crystals of a macromolecule are required. In contrast to X-ray crystallography, this significantly facilitates structure determination by cryo-EM. Nevertheless, a relatively high level of biochemical control is still essential to obtain high-resolution structures by cryo-EM, and it can be anticipated that the success of the cryo-EM technology goes hand in hand with further developments of sample purification and preparation techniques. This will allow routine high-resolution structure determination of the many macromolecular complexes of the cell that until now represent evasive targets for X-ray crystallographers. Here we discuss the various biochemical tools that are currently available and the existing sample purification and preparation techniques for cryo-EM grid preparation that are needed to obtain high-resolution images for structure determination.

  8. Electron Diffraction Using Transmission Electron Microscopy

    PubMed Central

    Bendersky, Leonid A.; Gayle, Frank W.

    2001-01-01

    Electron diffraction via the transmission electron microscope is a powerful method for characterizing the structure of materials, including perfect crystals and defect structures. The advantages of electron diffraction over other methods, e.g., x-ray or neutron, arise from the extremely short wavelength (≈2 pm), the strong atomic scattering, and the ability to examine tiny volumes of matter (≈10 nm3). The NIST Materials Science and Engineering Laboratory has a history of discovery and characterization of new structures through electron diffraction, alone or in combination with other diffraction methods. This paper provides a survey of some of this work enabled through electron microscopy. PMID:27500060

  9. Nuclear microscopy of biological specimens

    NASA Astrophysics Data System (ADS)

    Watt, F.; Grime, G. W.; Brook, A. J.; Gadd, G. M.; Perry, C. C.; Pearce, R. B.; Turnau, K.; Watkinson, S. C.

    1991-03-01

    Recent developments in technology have enabled the scanning proton microprobe to scan at submicron spatial resolution on a routine basis. The use of the powerful combination of techniques PIXE (proton induced X-ray emission), nuclear (or Rutherford) backscattering (RBS), and secondary electron detection operating at this resolution will open up new areas in many scientific disciplines. This paper describes some of the work carried out in the biological sciences over the last year, using the Oxford SPM facility. Collaborations with biological scientists have drawn attention to the wealth of information that can be derived when these techniques are applied to micro-organisms, cells and plant tissue. Briefly described here are investigations into the uptake of heavy metals by the alga Pandorina morum, the structure of the diatom Stephanopyxis turris, the presence of various types of crystal structures within the cells of Spirogyra, the heavy metal uptake of a mycorrhizal fungus present in the bracken ( Pteridium aquilinum) root, the role of sphagnum moss in the absorption of inorganic elements, the measurement of heavy metals in environmentally-adapted cells of the yeast Saccharomyces cerevisiae, and the elemental distribution in the growing tip of a spore from the plant Equisetum arvense, with special emphasis placed on the visual interpretation of the elemental and secondary-electron maps provided by the nuclear microscopical techniques.

  10. Dynamic imaging with electron microscopy

    ScienceCinema

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2016-07-12

    Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

  11. Dynamic imaging with electron microscopy

    SciTech Connect

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2014-02-20

    Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

  12. The future of electron microscopy

    SciTech Connect

    Zhu, Yimei; Durr, Hermann

    2015-04-01

    Seeing is believing. So goes the old adage and seen evidence is undoubtedly satisfying because it can be interpreted easily, though not always correctly. For centuries, humans have developed such instruments as telescopes that observe the heavens and microscopes that reveal bacteria and viruses. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their foundational work on superresolution fluorescence microscopy in which they overcame the Abbe diffraction limit for the resolving power of conventional light microscopes. (See Physics Today, December 2014, page 18.) That breakthrough enabled discoveries in biological research and testifies to the importance of modern microscopy.

  13. The future of electron microscopy

    DOE PAGES

    Zhu, Yimei; Durr, Hermann

    2015-04-01

    Seeing is believing. So goes the old adage and seen evidence is undoubtedly satisfying because it can be interpreted easily, though not always correctly. For centuries, humans have developed such instruments as telescopes that observe the heavens and microscopes that reveal bacteria and viruses. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their foundational work on superresolution fluorescence microscopy in which they overcame the Abbe diffraction limit for the resolving power of conventional light microscopes. (See Physics Today, December 2014, page 18.) That breakthrough enabled discoveries in biological research and testifiesmore » to the importance of modern microscopy.« less

  14. Low-temperature electron microscopy: techniques and protocols.

    PubMed

    Fleck, Roland A

    2015-01-01

    Low-temperature electron microscopy endeavors to provide "solidification of a biological specimen by cooling with the aim of minimal displacement of its components through the use of low temperature as a physical fixation strategy" (Steinbrecht and Zierold, Cryotechniques in biological electron microscopy. Springer-Verlag, Berlin, p 293, 1987). The intention is to maintain confidence that the tissue observed retains the morphology and dimensions of the living material while also ensuring soluble cellular components are not displaced. As applied to both scanning and transmission electron microscopy, cryo-electron microscopy is a strategy whereby the application of low-temperature techniques are used to reduce or remove processing artifacts which are commonly encountered in more conventional room temperature electron microscopy techniques which rely heavily on chemical fixation and heavy metal staining. Often, cryo-electron microscopy allows direct observation of specimens, which have not been stained or chemically fixed.

  15. Electron Microscopy Methods and Protocols (Volume 17 of the Series on Methods in Molecular Biology), Edited by M.A. Nasser Hajibagheri 1999. Humana Press, Totowa, NJ. xi + 281 pages. ($89.50)

    NASA Astrophysics Data System (ADS)

    Bendayan, Moise

    1999-10-01

    At first glance, this book seems to come up short with regard to the application of electron microscopy in biological sciences. However, one has to consider that it is part of a series on molecular biology. The purpose here was not to collect a large variety of techniques related to electron microscopy but rather to focus on methods in molecular morphology. In this context, the book offers 15 short (some very short) chapters dealing with morphological approaches related to and used in molecular biology.

  16. Direct Detectors for Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Clough, R. N.; Moldovan, G.; Kirkland, A. I.

    2014-06-01

    There is interest in improving the detectors used to capture images in transmission electron microscopy. Detectors with an improved modulation transfer function at high spatial frequencies allow for higher resolution in images at lower magnification, which leads to an increased effective field of view. Detectors with improved detective quantum efficiency are important for low dose applications. One way in which these performance enhancements can be achieved is through direct detection, where primary electrons are converted directly into suitable electrical signals by the detector rather than relying on an indirect electron to photon conversion before detection. In this paper we present the characterisation of detector performance for a number of different direct detection technologies, and compare these technologies to traditional indirect detectors. Overall our results show that direct detection enables a significant improvement in all aspects of detector performance.

  17. Synthetic incoherence for electron microscopy.

    PubMed

    Levine, Zachary H; Dunstan, Robyn M

    2007-08-01

    Tomographic studies of submicrometer samples in materials science using electron microscopy have been inhibited by diffraction effects. In the present work, we describe a practical method for ameliorating these effects. First, we find an analytic expression for the mutual coherence function for hollow-cone illumination. Then, we use this analytic expression to propose a Gaussian weighting of hollow-cone illumination, which we name tapered solid-cone illumination, and present an analytic expression for its mutual coherence function. Finally, we investigate numerically an n-ring approximation to tapered solid-cone illumination. The results suggest a method for removing diffraction effects and hence enabling tomography.

  18. Electron microscopy of pharmaceutical systems.

    PubMed

    Klang, Victoria; Valenta, Claudia; Matsko, Nadejda B

    2013-01-01

    During the last decades, the focus of research in pharmaceutical technology has steadily shifted towards the development and optimisation of nano-scale drug delivery systems. As a result, electron microscopic methods are increasingly employed for the characterisation of pharmaceutical systems such as nanoparticles and microparticles, nanoemulsions, microemulsions, solid lipid nanoparticles, different types of vesicles, nanofibres and many more. Knowledge of the basic properties of these systems is essential for an adequate microscopic analysis. Classical transmission and scanning electron microscopic techniques frequently have to be adapted for an accurate analysis of formulation morphology, especially in case of hydrated colloidal systems. Specific techniques such as environmental scanning microscopy or cryo preparation are required for their investigation. Analytical electron microscopic techniques such as electron energy-loss spectroscopy or energy-dispersive X-ray spectroscopy are additional assets to determine the elemental composition of the systems, but are not yet standard tools in pharmaceutical research. This review provides an overview of pharmaceutical systems of interest in current research and strategies for their successful electron microscopic analysis. Advantages and limitations of the different methodological approaches are discussed and recent findings of interest are presented. PMID:22921788

  19. ESR Microscopy for Biological and Biomedical Applications.

    PubMed

    Shin, C S; Dunnam, C R; Borbat, P P; Dzikovski, B; Barth, E D; Halpern, H J; Freed, J H

    2011-08-01

    We report on electron-spin resonance microscopy (ESRM) providing sub-micron resolution (~700nm) with a high spin concentration sample, i.e. lithium phthalocyanine (LiPc) crystal. For biomedical applications of our ESRM, we have imaged samples containing rat basophilic leukemia (RBL) cells as well as cancerous tissue samples with a resolution of several microns using a water soluble spin probe, Trityl_OX063_d24. Phantom samples with the nitroxide spin label, (15)N PDT, were also imaged to demonstrate that nitroxides, which are commonly used as spin labels, may also be used for ESRM applications. ESRM tissue imaging would therefore be valuable for diagnostic or therapeutic purposes. Also, ESRM can be used to study the motility or the metabolism of cells in various environments. With further modification and/or improvement of imaging probe and spectrometer instrumentation sub-micron biological images should be obtainable, thereby providing a useful tool for various biomedical applications.

  20. Light and Electron Microscopy of the European Beaver (Castor fiber) Stomach Reveal Unique Morphological Features with Possible General Biological Significance

    PubMed Central

    Petryński, Wojciech; Palkowska, Katarzyna; Prusik, Magdalena; Targońska, Krystyna; Giżejewski, Zygmunt; Przybylska-Gornowicz, Barbara

    2014-01-01

    Anatomical, histological, and ultrastructural studies of the European beaver stomach revealed several unique morphological features. The prominent attribute of its gross morphology was the cardiogastric gland (CGG), located near the oesophageal entrance. Light microscopy showed that the CGG was formed by invaginations of the mucosa into the submucosa, which contained densely packed proper gastric glands comprised primarily of parietal and chief cells. Mucous neck cells represented <0.1% of cells in the CGG gastric glands and 22–32% of cells in the proper gastric glands of the mucosa lining the stomach lumen. These data suggest that chief cells in the CGG develop from undifferentiated cells that migrate through the gastric gland neck rather than from mucous neck cells. Classical chief cell formation (i.e., arising from mucous neck cells) occurred in the mucosa lining the stomach lumen, however. The muscularis around the CGG consisted primarily of skeletal muscle tissue. The cardiac region was rudimentary while the fundus/corpus and pyloric regions were equally developed. Another unusual feature of the beaver stomach was the presence of specific mucus with a thickness up to 950 µm (in frozen, unfixed sections) that coated the mucosa. Our observations suggest that the formation of this mucus is complex and includes the secretory granule accumulation in the cytoplasm of pit cells, the granule aggregation inside cells, and the incorporation of degenerating cells into the mucus. PMID:24727802

  1. High resolution scanning electron microscopy in biology: artefacts caused by the nature and mode of application of the coating material.

    PubMed

    Bråten, T

    1978-05-01

    Results of high resolution secondary electron observations of the haemocyanin molecule from the marine whelk Buccinum undatum are given. The choice of metal used for surface coating of the sample (gold, gold--palladium or carbon/gold--palldium) and the mode of application of this metal (thermal vacuum evaporation or diode sputtering) were both found to be of utmost importance when working at high magnifications in the scanning electron microscope. Sputter coating with gold gives poor results because of a granular and cracked appearance of the surface film. The haemocyanin molecules were difficult to recognize in these preparations. Best results were obtained by a thermal vacuum evaporation of gold--palladium. It is suggested that the inferior results seen after sputter coating may be due to the high temperatures wihch the specimen experiences during the sputtering, even when cooling of the specimen stage is carried out. Other specimens (diatom valves and latex spheres) were also studied at high magnification giving the same results as the haemocyanin molecules.

  2. Electron transfer in biology

    NASA Astrophysics Data System (ADS)

    Williams, R. J. P.

    Electron transfer is one of the key reactions of biology not just in catalysis of oxidation/reduction reactions but in the conversion of sources of energy such as light to usable form for chemical transformations. There are then two intriguing problems. What is the nature of the matrix in which electrons flow in a biological cell after the initial charge separation due for example to the absorption of light. Here we are examining biological structures similar to man's electronic wires and the construction must be of low resistance in what are apparently insulators - organic polymers. It has been found that the electronic conduction system is largely made from metallo-proteins associated with lipid membranes. We understand much about these biological wires today. The second problem concerns the conversion of the energy captured from the light into usable chemical form. The major synthetic step in the production of biological polymers, including proteins, DNA, RNA, polysaccharides and fats, is condensation, i.e. the removal of water in the formation of amides, esters and so on. Now these condensation reactions are driven in biology by using a drying agent in water, namely the anhydride, pyrophosphate, in a special compound ATP, adenosine triphosphate. The central problem is to discover exactly how the flow of electrons can be related to the synthesis of (bound) pyrophosphate. (In a thermodynamic sense pyrophosphate is a water soluble kinetically stable drying agent comparable with solid P2O5.) In the biological systems the connection between these different classes of reaction, electron transfer and condensation, is known to be via the production of an energized gradient of protons across the biological membrane which arises from the flow of electrons across the same membrane in the electron transport wires of biology. However we do not understand thoroughly the steps which lead from electron flow in a membrane to proton gradients in that membrane, i.e. electron

  3. 4D electron microscopy: principles and applications.

    PubMed

    Flannigan, David J; Zewail, Ahmed H

    2012-10-16

    The transmission electron microscope (TEM) is a powerful tool enabling the visualization of atoms with length scales smaller than the Bohr radius at a factor of only 20 larger than the relativistic electron wavelength of 2.5 pm at 200 keV. The ability to visualize matter at these scales in a TEM is largely due to the efforts made in correcting for the imperfections in the lens systems which introduce aberrations and ultimately limit the achievable spatial resolution. In addition to the progress made in increasing the spatial resolution, the TEM has become an all-in-one characterization tool. Indeed, most of the properties of a material can be directly mapped in the TEM, including the composition, structure, bonding, morphology, and defects. The scope of applications spans essentially all of the physical sciences and includes biology. Until recently, however, high resolution visualization of structural changes occurring on sub-millisecond time scales was not possible. In order to reach the ultrashort temporal domain within which fundamental atomic motions take place, while simultaneously retaining high spatial resolution, an entirely new approach from that of millisecond-limited TEM cameras had to be conceived. As shown below, the approach is also different from that of nanosecond-limited TEM, whose resolution cannot offer the ultrafast regimes of dynamics. For this reason "ultrafast electron microscopy" is reserved for the field which is concerned with femtosecond to picosecond resolution capability of structural dynamics. In conventional TEMs, electrons are produced by heating a source or by applying a strong extraction field. Both methods result in the stochastic emission of electrons, with no control over temporal spacing or relative arrival time at the specimen. The timing issue can be overcome by exploiting the photoelectric effect and using pulsed lasers to generate precisely timed electron packets of ultrashort duration. The spatial and temporal resolutions

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

    PubMed

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

    2016-08-01

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

  5. Electron microscopy of atmospheric particles

    NASA Astrophysics Data System (ADS)

    Huang, Po-Fu

    Electron microscopy coupled with energy dispersive spectrometry (EM/EDS) is a powerful tool for single particle analysis. However, the accuracy with which atmospheric particle compositions can be quantitatively determined by EDS is often hampered by substrate-particle interactions, volatilization losses in the low pressure microscope chamber, electron beam irradiation and use of inaccurate quantitation factors. A pseudo-analytical solution was derived to calculate the temperature rise due to the dissipation of the electron energy on a particle-substrate system. Evaporative mass loss for a spherical cap-shaped sulfuric acid particle resting on a thin film supported by a TEM grid during electron beam impingement has been studied. Measured volatilization rates were found to be in very good agreement with theoretical predictions. The method proposed can also be used to estimate the vapor pressure of a species by measuring the decay of X-ray intensities. Several types of substrates were studied. We found that silver-coated silicon monoxide substrates give carbon detection limits comparable to commercially available substrates. An advantage of these substrates is that the high thermal conductivity of the silver reduces heating due to electron beam impingement. In addition, exposure of sulfuric acid samples to ammonia overnight substantially reduces sulfur loss in the electron beam. Use of size-dependent k-factors determined from particles of known compositions shows promise for improving the accuracy of atmospheric particle compositions measured by EM/EDS. Knowledge accumulated during the course of this thesis has been used to analyze atmospheric particles (Minneapolis, MN) selected by the TDMA and collected by an aerodynamic focusing impactor. 'Less' hygroscopic particles, which do not grow to any measurable extent when humidified to ~90% relative humidity, included chain agglomerates, spheres, flakes, and irregular shapes. Carbon was the predominant element detected in

  6. Tomographic phase microscopy and its biological applications

    NASA Astrophysics Data System (ADS)

    Choi, Wonshik

    2012-12-01

    Conventional interferometric microscopy techniques such as digital holographic microscopy and quantitative phase microscopy are often classified as 3D imaging techniques because a recorded complex field image can be numerically propagated to a different depth. In a strict sense, however, a single complex field image contains only 2D information on a specimen. The measured 2D image is only a subset of the 3D structure. For the 3D mapping of an object, multiple independent 2D images are to be taken, for example at multiple incident angles or wavelengths, and then combined by the so-called optical diffraction tomography (ODT). In this Letter, tomographic phase microscopy (TPM) is reviewed that experimentally realizes the concept of the ODT for the 3D mapping of biological cells in their native state, and some of its interesting biological and biomedical applications are introduced. [Figure not available: see fulltext.

  7. PLS photoemission electron microscopy beamline

    NASA Astrophysics Data System (ADS)

    Kang, Tai-Hee; Kim, Ki-jeong; Hwang, C. C.; Rah, S.; Park, C. Y.; Kim, Bongsoo

    2001-07-01

    The performance of a recently commissioned beamline at the Pohang Light Source (PLS) is described. The beamline, which is located at 4B1 at PLS, is a Varied Line Spacing (VLS) Plane Grating Monochromator (PGM) beamline. VLS PGM has become very popular because of the simple scanning mechanism and the fixed exit slit. The beamline which takes 3 mrad horizontal beam fan from bending magnet, covers the energy range 200-1000 eV for Photoemission Electron Microscopy (PEEM), X-ray Photoelectron Spectroscopy (XPS) and Magnetic Circular Dichroism (MCD) experiments. Simplicity of the optics and high flux with medium resolution were the design goals for these applications. The beamline consists of a horizontal focusing mirror, a vertical focusing mirror, VLS plane grating and exit slit. The source of PLS could be used as a virtual entrance slit because of its small size and stability. The flux and the resolution of the beamline at the experimental station have been measured using an ion chamber and a calibrated photodiode. Test images of PEEM from a standard sample were taken to illustrate the further performance of the beamline and PEEM station.

  8. Atomic force microscopy of biological samples

    SciTech Connect

    Doktycz, Mitchel John

    2010-01-01

    The ability to evaluate structural-functional relationships in real time has allowed scanning probe microscopy (SPM) to assume a prominent role in post genomic biological research. In this mini-review, we highlight the development of imaging and ancillary techniques that have allowed SPM to permeate many key areas of contemporary research. We begin by examining the invention of the scanning tunneling microscope (STM) by Binnig and Rohrer in 1982 and discuss how it served to team biologists with physicists to integrate high-resolution microscopy into biological science. We point to the problems of imaging nonconductive biological samples with the STM and relate how this led to the evolution of the atomic force microscope (AFM) developed by Binnig, Quate, and Gerber, in 1986. Commercialization in the late 1980s established SPM as a powerful research tool in the biological research community. Contact mode AFM imaging was soon complemented by the development of non-contact imaging modes. These non-contact modes eventually became the primary focus for further new applications including the development of fast scanning methods. The extreme sensitivity of the AFM cantilever was recognized and has been developed into applications for measuring forces required for indenting biological surfaces and breaking bonds between biomolecules. Further functional augmentation to the cantilever tip allowed development of new and emerging techniques including scanning ion-conductance microscopy (SICM), scanning electrochemical microscope (SECM), Kelvin force microscopy (KFM) and scanning near field ultrasonic holography (SNFUH).

  9. Immunogold Labeling for Scanning Electron Microscopy.

    PubMed

    Goldberg, Martin W; Fišerová, Jindřiška

    2016-01-01

    Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components, and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo, or real 3D at magnifications ranging from about 10× to 1,000,000×. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualized. In order to identify that protein at high resolution, or to see and quantify its distribution at lower magnifications, samples can be labeled with antibodies. Any surface that can be exposed can potentially be studied in this way. Presented here is a generic method for immunogold labeling for scanning electron microscopy, using two examples of specimens: isolated nuclear envelopes and the cytoskeleton of mammalian culture cells. Various parameters for sample preparation, fixation, immunogold labeling, drying, metal coating, and imaging are discussed so that the best immunogold scanning electron microscopy results can be obtained from different types of specimens. PMID:27515090

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

    PubMed

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

    2015-06-11

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

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

    PubMed Central

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

    2015-01-01

    The application of electron microscopy to hydrated biological samples has been limited by high-vacuum operating conditions. Traditional methods utilize harsh and laborious sample dehydration procedures, often leading to structural artefacts and creating difficulties for correlating results with high-resolution fluorescence microscopy. Here, we utilize graphene, a single-atom-thick carbon meshwork, as the thinnest possible impermeable and conductive membrane to protect animal cells from vacuum, thus enabling high-resolution electron microscopy of wet and untreated whole cells with exceptional ease. Our approach further allows for facile correlative super-resolution and electron microscopy of wet cells directly on the culturing substrate. In particular, individual cytoskeletal actin filaments are resolved in hydrated samples through electron microscopy and well correlated with super-resolution results. PMID:26066680

  12. Silicon Nitride Windows for Electron Microscopy of Whole Cells

    PubMed Central

    Ring, E. A.; Peckys, D. B.; Dukes, M. J.; Baudoin, J. P.; de Jonge, N.

    2012-01-01

    Summary Silicon microchips with thin electron transparent silicon nitride windows provide a sample support that accommodates both light-, and electron microscopy of whole eukaryotic cells in vacuum or liquid, with minimum sample preparation steps. The windows are robust enough that cellular samples can be cultured directly onto them, with no addition of a supporting film, and no need to embed or section the sample, as is typically required in electron microscopy. By combining two microchips, a microfluidic chamber can be constructed for the imaging of samples in liquid in the electron microscope. We provide microchip design specifications, a fabrication outline, instructions on how to prepare them for biological samples, and examples of images obtained using different light-, and electron microscopy modalities. The use of these microchips is particularly advantageous for correlative light-, and electron microscopy. PMID:21770941

  13. Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function

    PubMed Central

    Begemann, Isabell; Galic, Milos

    2016-01-01

    Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in microscopy techniques, labeling tools, and fixation or preparation procedures have fueled the development of a series of hybrid approaches that allow correlating functional fluorescence microscopy data and ultrastructural information from electron micrographs from a singular biological event. As correlative light electron microscopy (CLEM) approaches become increasingly accessible, long-standing neurobiological questions regarding structure-function relation are being revisited. In this review, we will survey what developments in electron and light microscopy have spurred the advent of correlative approaches, highlight the most relevant CLEM techniques that are currently available, and discuss its potential and limitations with respect to neuronal and synapse-specific applications.

  14. Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function.

    PubMed

    Begemann, Isabell; Galic, Milos

    2016-01-01

    Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in microscopy techniques, labeling tools, and fixation or preparation procedures have fueled the development of a series of hybrid approaches that allow correlating functional fluorescence microscopy data and ultrastructural information from electron micrographs from a singular biological event. As correlative light electron microscopy (CLEM) approaches become increasingly accessible, long-standing neurobiological questions regarding structure-function relation are being revisited. In this review, we will survey what developments in electron and light microscopy have spurred the advent of correlative approaches, highlight the most relevant CLEM techniques that are currently available, and discuss its potential and limitations with respect to neuronal and synapse-specific applications. PMID:27601992

  15. Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function

    PubMed Central

    Begemann, Isabell; Galic, Milos

    2016-01-01

    Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in microscopy techniques, labeling tools, and fixation or preparation procedures have fueled the development of a series of hybrid approaches that allow correlating functional fluorescence microscopy data and ultrastructural information from electron micrographs from a singular biological event. As correlative light electron microscopy (CLEM) approaches become increasingly accessible, long-standing neurobiological questions regarding structure-function relation are being revisited. In this review, we will survey what developments in electron and light microscopy have spurred the advent of correlative approaches, highlight the most relevant CLEM techniques that are currently available, and discuss its potential and limitations with respect to neuronal and synapse-specific applications. PMID:27601992

  16. Correlative Light Electron Microscopy: Connecting Synaptic Structure and Function.

    PubMed

    Begemann, Isabell; Galic, Milos

    2016-01-01

    Many core paradigms of contemporary neuroscience are based on information obtained by electron or light microscopy. Intriguingly, these two imaging techniques are often viewed as complementary, yet separate entities. Recent technological advancements in microscopy techniques, labeling tools, and fixation or preparation procedures have fueled the development of a series of hybrid approaches that allow correlating functional fluorescence microscopy data and ultrastructural information from electron micrographs from a singular biological event. As correlative light electron microscopy (CLEM) approaches become increasingly accessible, long-standing neurobiological questions regarding structure-function relation are being revisited. In this review, we will survey what developments in electron and light microscopy have spurred the advent of correlative approaches, highlight the most relevant CLEM techniques that are currently available, and discuss its potential and limitations with respect to neuronal and synapse-specific applications.

  17. Analytical transmission electron microscopy in materials science

    SciTech Connect

    Fraser, H.L.

    1980-01-01

    Microcharacterization of materials on a scale of less than 10 nm has been afforded by recent advances in analytical transmission electron microscopy. The factors limiting accurate analysis at the limit of spatial resolution for the case of a combination of scanning transmission electron microscopy and energy dispersive x-ray spectroscopy are examined in this paper.

  18. Fast electron microscopy via compressive sensing

    SciTech Connect

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

    2014-12-09

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

  19. Multimodal dyes: toward correlative two-photon and electron microscopy

    NASA Astrophysics Data System (ADS)

    Bolze, Frédéric; Ftouni, Hussein; Nicoud, Jean-François; Leoni, Piero; Schwab, Yannick; Rehspringer, Jean-Luc; Mafouana, Rodrigues R.

    2013-03-01

    Nowadays, many crucial biological questions involve the observation of biological samples at different scales. Thus, optical microscopy can be associated to magnetic nuclear imaging allowing access to data from the cellular to the organ level, or can be associated to electron microscopy to reach the sub cellular level. We will describe here the design, synthesis and characterization of new bimodal probes, which can be used as dye in two-photon excited microscopy (TPEM) and electron dense markers in scanning and transmission electron microscopy (EM). In a first part, we will describe new molecular dyes with small organic systems grafted on metal atoms (Pt, Au). Such systems show good twophoton induced fluorescence and two-photon images of HeLa cells will be presented. In a second part, we will present hybrid organic-inorganic fluorescent systems with diketopyrrolopyrole-based dye grafted on iron oxide-silica core shell nanoparticles by peptide bond. Such systems present high two-photon absorption cross sections and good fluorescence quantum yields. These nanoparticles are rapidly internalized in HeLa cells and high quality two-photon images were performed with low laser power. Then we will present our results on correlative light-electron microscopy were twophoton and electron microscopy (both scanning and transmission) images were obtained on the same biological sample.

  20. The CryoCapsule: Simplifying correlative light to electron microscopy

    PubMed Central

    Heiligenstein, Xavier; Heiligenstein, Jérôme; Delevoye, Cédric; Hurbain, Ilse; Bardin, Sabine; Paul-Gilloteaux, Perrine; Sengmanivong, Lucie; Régnier, Gilles; Salamero, Jean; Antony, Claude; Raposo, Graca

    2014-01-01

    Correlating complementary multiple scale images of the same object is a straightforward means to decipher biological processes. Light and electron microscopy are the most commonly used imaging techniques, yet despite their complementarity, the experimental procedures available to correlate them are technically complex. We designed and manufactured a new device adapted to many biological specimens, the CryoCapsule, that simplifies the multiple sample preparation steps, which at present separate live cell fluorescence imaging from contextual high-resolution electron microscopy, thus opening new strategies for full correlative light to electron microscopy. We tested the biological application of this highly optimized tool on three different specimens: the in-vitro Xenopus laevis mitotic spindle, melanoma cells over-expressing YFP-langerin sequestered in organized membranous subcellular organelles and a pigmented melanocytic cell in which the endosomal system was labeled with internalized fluorescent transferrin. PMID:24533564

  1. Entanglement-assisted electron microscopy based on a flux qubit

    SciTech Connect

    Okamoto, Hiroshi; Nagatani, Yukinori

    2014-02-10

    A notorious problem in high-resolution biological electron microscopy is radiation damage caused by probe electrons. Hence, acquisition of data with minimal number of electrons is of critical importance. Quantum approaches may represent the only way to improve the resolution in this context, but all proposed schemes to date demand delicate control of the electron beam in highly unconventional electron optics. Here we propose a scheme that involves a flux qubit based on a radio-frequency superconducting quantum interference device, inserted in a transmission electron microscope. The scheme significantly improves the prospect of realizing a quantum-enhanced electron microscope for radiation-sensitive specimens.

  2. Study of nanoscale structural biology using advanced particle beam microscopy

    NASA Astrophysics Data System (ADS)

    Boseman, Adam J.

    This work investigates developmental and structural biology at the nanoscale using current advancements in particle beam microscopy. Typically the examination of micro- and nanoscale features is performed using scanning electron microscopy (SEM), but in order to decrease surface charging, and increase resolution, an obscuring conductive layer is applied to the sample surface. As magnification increases, this layer begins to limit the ability to identify nanoscale surface structures. A new technology, Helium Ion Microscopy (HIM), is used to examine uncoated surface structures on the cuticle of wild type and mutant fruit flies. Corneal nanostructures observed with HIM are further investigated by FIB/SEM to provide detailed three dimensional information about internal events occurring during early structural development. These techniques are also used to reconstruct a mosquito germarium in order to characterize unknown events in early oogenesis. Findings from these studies, and many more like them, will soon unravel many of the mysteries surrounding the world of developmental biology.

  3. Advanced electron microscopy for advanced materials.

    PubMed

    Van Tendeloo, Gustaaf; Bals, Sara; Van Aert, Sandra; Verbeeck, Jo; Van Dyck, Dirk

    2012-11-01

    The idea of this Review is to introduce newly developed possibilities of advanced electron microscopy to the materials science community. Over the last decade, electron microscopy has evolved into a full analytical tool, able to provide atomic scale information on the position, nature, and even the valency atoms. This information is classically obtained in two dimensions (2D), but can now also be obtained in 3D. We show examples of applications in the field of nanoparticles and interfaces.

  4. Digital detectors for electron microscopy

    NASA Astrophysics Data System (ADS)

    Faruqi, A. R.; Cattermole, D. M.

    2002-02-01

    Film has traditionally been used for recording images in transmission electron microscopes but there is an essential need for computer-interfaced electronic detectors. Cooled-CCD detectors, developed over the past few years, though not ideal, are increasingly used as the preferred detection system in a number of applications. We describe briefly the design of CCD-based detectors, along with their main properties, which have been used in electron crystallography. A newer detector design with a much bigger sensitive area, incorporating a 2×2 tiled array of CCDs with tapered fibre optics will overcome some of the limitations of existing CCD detectors. We also describe some preliminary results for 8 keV imaging, from (direct detection) silicon hybrid pixel detectors, which offer advantages over CCDs in terms of better spatial resolution, faster readout with minimal noise.

  5. Low voltage transmission electron microscopy of graphene.

    PubMed

    Bachmatiuk, Alicja; Zhao, Jiong; Gorantla, Sandeep Madhukar; Martinez, Ignacio Guillermo Gonzalez; Wiedermann, Jerzy; Lee, Changgu; Eckert, Juergen; Rummeli, Mark Hermann

    2015-02-01

    The initial isolation of graphene in 2004 spawned massive interest in this two-dimensional pure sp(2) carbon structure due to its incredible electrical, optical, mechanical, and thermal effects. This in turn led to the rapid development of various characterization tools for graphene. Examples include Raman spectroscopy and scanning tunneling microscopy. However, the one tool with the greatest prowess for characterizing and studying graphene is the transmission electron microscope. State-of-the-art (scanning) transmission electron microscopes enable one to image graphene with atomic resolution, and also to conduct various other characterizations simultaneously. The advent of aberration correctors was timely in that it allowed transmission electron microscopes to operate with reduced acceleration voltages, so that damage to graphene is avoided while still providing atomic resolution. In this comprehensive review, a brief introduction is provided to the technical aspects of transmission electron microscopes relevant to graphene. The reader is then introduced to different specimen preparation techniques for graphene. The different characterization approaches in both transmission electron microscopy and scanning transmission electron microscopy are then discussed, along with the different aspects of electron diffraction and electron energy loss spectroscopy. The use of graphene for other electron microscopy approaches such as in-situ investigations is also presented.

  6. Electron microscopy at atomic resolution

    SciTech Connect

    Gronsky, R.

    1983-11-01

    The direct imaging of atomic structure in solids has become increasingly easier to accomplish with modern transmission electron microscopes, many of which have an information retrieval limit near 0.2 nm point resolution. Achieving better resolution, particularly with any useful range of specimen tilting, requires a major design effort. This presentation describes the new Atomic Resolution Microscope (ARM), recently put into operation at the Lawrence Berkeley Laboratory. Capable of 0.18 nm or better interpretable resolution over a voltage range of 400 kV to 1000 kV with +- 40/sup 0/ biaxial specimen tilting, the ARM features a number of new electron-optical and microprocessor-control designs. These are highlighted, and its atomic resolution performance demonstrated for a selection of inorganic crystals.

  7. Correlated light and electron microscopy: ultrastructure lights up!

    PubMed

    de Boer, Pascal; Hoogenboom, Jacob P; Giepmans, Ben N G

    2015-06-01

    Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development-including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy-now paves the way for broad CLEM implementation in biology.

  8. Fibreoptic fluorescent microscopy in studying biological objects

    SciTech Connect

    Morozov, A N; Turchin, Il'ya V; Kamenskii, V A; Fiks, I I; Lazutkin, A A; Bezryadkov, D V; Ivanova, A A; Toptunov, D M; Anokhin, K V

    2010-11-13

    The method of fluorescent microscopy is developed based on employment of a single-mode fibreoptic channel to provide high spatial resolution 3D images of large cleared biological specimens using the 488-nm excitation laser line. The transverse and axial resolution of the setup is 5 and 13 {mu}m, respectively. The transversal sample size under investigation is up to 10 mm. The in-depth scanning range depends on the sample transparency and reaches 4 mm in the experiment. The 3D images of whole mouse organs (heart, lungs, brain) and mouse embryos obtained using autofluorescence or fluorescence of exogenous markers demonstrate a high contrast and cellular-level resolution.

  9. Electron microscopy of Paramecium (Ciliata).

    PubMed

    Hausmann, Klaus; Allen, Richard D

    2010-01-01

    Paramecium may be the best known single-celled organism in existence (Hausmann et al., 2003). Today its image often appears on television programs where the producers use it to illustrate a stereotypic microorganism, be it pathogenic or nonpathogenic, prokaryotic or eukaryotic. Paramecium was probably one of the first single-celled organisms observed with a light microscope by the Dutch cloth vendor and amateur lens maker Antoni van Leuwenhoek (1632-1723) (Dobell, 1932), and it is still being investigated in the 21st century in the days of the modern electron microscopes.

  10. Electron Microscopy of Natural and Epitaxial Diamond

    NASA Technical Reports Server (NTRS)

    Posthill, J. B.; George, T.; Malta, D. P.; Humphreys, T. P.; Rudder, R. A.; Hudson, G. C.; Thomas, R. E.; Markunas, R. J.

    1993-01-01

    Semiconducting diamond films have the potential for use as a material in which to build active electronic devices capable of operating at high temperatures or in high radiation environments. Ultimately, it is preferable to use low-defect-density single crystal diamond for device fabrication. We have previously investigated polycrystalline diamond films with transmission electron microscopy (TEM) and scanning electron microscopy (SEM), and homoepitaxial films with SEM-based techniques. This contribution describes some of our most recent observations of the microstructure of natural diamond single crystals and homoepitaxial diamond thin films using TEM.

  11. Electron Microscopy of the Cell

    PubMed Central

    Leeson, T. S.

    1965-01-01

    The use of the electron microscope has added much to our knowledge of the cell. The fine structure of the component parts of the nucleus and the cytoplasm is described, and their functions are indicated. The nature and structural modifications of the plasma membrane are illustrated with particular reference to function. To illustrate the interrelationships of the nucleus and cytoplasm, the theory of protein secretion is discussed, the secretion of a particular protein or polypeptide being determined by a particular nucleotide sequence in the desoxyribonucleic acid of a chromosome, that is, by a gene. This information is transferred from nucleus to cytoplasm. It is in the cytoplasm that the majority of the work is performed while the nucleus directs the work of the cell. ImagesFig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11Fig. 12Fig. 13Fig. 14Fig. 15Fig. 16Fig. 17Fig. 18Fig. 19Fig. 20Fig. 21Fig. 22Fig. 23Fig. 24Fig. 25Fig. 26 PMID:5829410

  12. Electron Microscopy of Retinal Photoreceptors

    PubMed Central

    Lasansky, Arnaldo; de Robertis, Eduardo

    1960-01-01

    The fine structure of the cone and rod outer segments of the toad was studied under the electron microscope after fixation in osmium tetroxide and fixation in formaldehyde followed by chromation. In the OsO4-fixed specimens, the rod outer segment appears to be built of a stack of lobulated flattened sacs, each of which is made of two membranes of about 40 A separated by an innerspace of about 30 A. The distance between the rod sacs is about 50 A. The sacs in the cone outer segment are originated by the folding of a continuous membrane. The thickness of the membranes and width of the spaces between the cone sacs is the same as in rod, but the sac innerspace is slightly narrower in the cone (∼ 20 A). After fixation in formaldehyde and chromation, two different dense lines (l1 and l2) separated by spaces of less density appear. One of the lines, l1, has a thickness of 70 A and is less dense than the other, l2, which is 30 A thick. The correlation of the patterns obtained with both fixatives is considered and two possible interpretations are given. The possibility that l2 is related to a soluble phospholipid component is discussed. It is suggested that the outer segments have a paracrystallin organization similar to that found in myelin. PMID:14414323

  13. Advanced Electron Microscopy in Materials Physics

    SciTech Connect

    Zhu, Y.; Jarausch, K.

    2009-06-01

    Aberration correction has opened a new frontier in electron microscopy by overcoming the limitations of conventional round lenses, providing sub-angstrom-sized probes and extending information limits. The imaging and analytical performance of these corrector-equipped microscopes affords an unprecedented opportunity to study structure-property relationships of matter at the atomic scale. This new generation of microscopes is able to retrieve high-quality structural information comparable to neutron and synchrotron x-ray experiments, but with local atomic resolution. These advances in instrumentation are accelerating the research and development of various functional materials ranging from those for energy generation, conversion, transportation and storage to those for catalysis and nano-device applications. The dramatic improvements in electron-beam illumination and detection also present a host of new challenges for the interpretation and optimization of experiments. During 7-9 November 2007, a workshop, entitled 'Aberration Corrected Electron Microscopy in Material Physics', was convened at the Center for Functional Nanomaterials, Brookhaven National Laboratories (BNL) to address these opportunities and challenges. The workshop was co-sponsored by Hitachi High Technologies, a leader in electron microscopy instrumentation, and BNL's Institute of Advanced Electron Microscopy, a leader in materials physics research using electron microscopy. The workshop featured presentations by internationally prominent scientists working at the frontiers of electron microscopy, both on developing instrumentation and applying it in materials physics. The meeting, structured to stimulate scientific exchanges and explore new capabilities, brought together {approx}100 people from over 10 countries. This special issue complies many of the advances in instrument performance and materials physics reported by the invited speakers and attendees at the workshop.

  14. Molecular Electron Microscopy: State of the Art and Current Challenges

    PubMed Central

    2008-01-01

    The objective of molecular electron microscopy (EM) is to use electron microscopes to visualize the structure of biological molecules. This Review provides a brief overview of the methods used in molecular EM, their respective strengths and successes, and current developments that promise an even more exciting future for molecular EM in the structural investigation of proteins and macromolecular complexes, studied in isolation or in the context of cells and tissues. PMID:18484707

  15. Correlative video-light-electron microscopy: development, impact and perspectives.

    PubMed

    Rizzo, Riccardo; Parashuraman, Seetharaman; Luini, Alberto

    2014-08-01

    Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the 'history,' or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light-electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular 'context' to the fluorescent dynamic structures of interest. CLEM 'multiplies' the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging. PMID:25030356

  16. Integrated fluorescence and transmission electron microscopy.

    PubMed

    Agronskaia, Alexandra V; Valentijn, Jack A; van Driel, Linda F; Schneijdenberg, Chris T W M; Humbel, Bruno M; van Bergen en Henegouwen, Paul M P; Verkleij, Arie J; Koster, Abraham J; Gerritsen, Hans C

    2008-11-01

    Correlative microscopy is a powerful technique that combines the strengths of fluorescence microscopy and electron microscopy. The first enables rapid searching for regions of interest in large fields of view while the latter exhibits superior resolution over a narrow field of view. Routine use of correlative microscopy is seriously hampered by the cumbersome and elaborate experimental procedures. This is partly due to the use of two separate microscopes for fluorescence and electron microscopy. Here, an integrated approach to correlative microscopy is presented based on a laser scanning fluorescence microscope integrated in a transmission electron microscope. Using this approach the search for features in the specimen is greatly simplified and the time to carry out the experiment is strongly reduced. The potential of the integrated approach is demonstrated at room temperature on specimens of rat intestine cells labeled with AlexaFluor488 conjugated to wheat germ agglutinin and on rat liver peroxisomes immunolabeled with anti-catalase antibodies and secondary AlexaFluor488 antibodies and 10nm protein A-gold.

  17. Optical microscopy versus scanning electron microscopy in urolithiasis.

    PubMed

    Marickar, Y M Fazil; Lekshmi, P R; Varma, Luxmi; Koshy, Peter

    2009-10-01

    Stone analysis is incompletely done in many clinical centers. Identification of the stone component is essential for deciding future prophylaxis. X-ray diffraction, Fourier transform infrared spectroscopy, and scanning electron microscopy (SEM) still remains a distant dream for routine hospital work. It is in this context that optical microscopy is suggested as an alternate procedure. The objective of this article was to assess the utility of an optical microscope which gives magnification of up to 40x and gives clear picture of the surface of the stones. In order to authenticate the morphological analysis of urinary stones, SEM and elemental distribution analysis were performed. A total of 250 urinary stones of different compositions were collected from stone clinic, photographed, observed under an optical microscope, and optical photographs were taken at different angles. Twenty-five representative samples among these were gold sputtered to make them conductive and were fed into the SEM machine. Photographs of the samples were taken at different angles at magnifications up to 4,000. Elemental distribution analysis (EDAX) was done to confirm the composition. The observations of the two studies were compared. The different appearances of the stones under optical illuminated microscopy were mostly standardized appearances, namely bosselations of pure whewellite, spiculations of weddellite, bright yellow colored appearance of uric acid, and dirty white amorphous appearance of phosphates. SEM and EDAX gave clearer pictures and gave added confirmation of the stone composition. From the references thus obtained, it was possible to confirm the composition by studying the optical microscopic pictures. Higher magnification capacity of the SEM and the EDAX patterns are useful to give reference support for performing optical microscopy work. After standardization, routine analysis can be performed with optical microscopy. The advantage of the optical microscope is that, it

  18. Active Pixel Sensors for electron microscopy

    NASA Astrophysics Data System (ADS)

    Denes, P.; Bussat, J.-M.; Lee, Z.; Radmillovic, V.

    2007-09-01

    The technology used for monolithic CMOS imagers, popular for cell phone cameras and other photographic applications, has been explored for charged particle tracking by the high-energy physics community for several years. This technology also lends itself to certain imaging detector applications in electron microscopy. We have been developing such detectors for several years at Lawrence Berkeley National Laboratory, and we and others have shown that this technology can offer excellent point-spread function, direct detection and high readout speed. In this paper, we describe some of the design constraints peculiar to electron microscopy and summarize where such detectors could play a useful role.

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

    SciTech Connect

    Timmermans, F. J.; Otto, C.

    2015-01-15

    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.

  20. 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.

  1. [Pili annulati. A scanning electron microscopy study].

    PubMed

    Lalević-Vasić, B; Polić, D

    1988-01-01

    A case of ringed hair studied by light and electron microscopy is reported. The patient, a 20-year old girl, had been presenting with the hair abnormality since birth. At naked eye examination the hairs were dry, 6 to 7 cm long, and they showed dull and shining areas giving the scalp hair a scintillating appearance (fig. 1). Several samples of hair were taken and examined by light microscopy under white and polarized light. Hair shafts and cryo-fractured surfaces were examined by scanning electron microscopy. RESULTS. 1. Light microscopy. Lesions were found in every hair examined. There were abnormal, opaque and fusiform areas alternating with normal areas all along the hair shaft (fig. 2). The abnormal areas resulted from intracortical air-filled cavities. Fractures similar to those of trichorrhexis nodosa were found in the opaque areas of the distal parts of the hairs. 2. Scanning electron microscopy. A. Hair shaft surface. The abnormal areas showed a longitudinal, "curtain-like" folding of the cuticular cells which had punctiform depressions on their surface and worn free edges (fig. 4, 5, 6); trichorrhexis-type fractures were seen in the distal parts of the hair shafts (fig. 7, 8). Normal areas regularly presented with longitudinal, superficial, short and non-systematized depressions (fig. 9); the cuticular cells were worn, and there were places where the denuded cortex showed dissociated cortical fibres (fig. 10).(ABSTRACT TRUNCATED AT 250 WORDS)

  2. Wet electron microscopy with quantum dots.

    PubMed

    Timp, Winston; Watson, Nicki; Sabban, Alon; Zik, Ory; Matsudaira, Paul

    2006-09-01

    Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM. PMID:16989089

  3. Wet electron microscopy with quantum dots.

    PubMed

    Timp, Winston; Watson, Nicki; Sabban, Alon; Zik, Ory; Matsudaira, Paul

    2006-09-01

    Wet electron microscopy (EM) is a new imaging method with the potential to allow higher spatial resolution of samples. In contrast to most EM methods, it requires little time to perform and does not require complicated equipment or difficult steps. We used this method on a common murine macrophage cell line, IC-21, in combination with various stains and preparations, to collect high resolution images of the actin cytoskeleton. Most importantly, we demonstrated the use of quantum dots in conjunction with this technique to perform light/electron correlation microscopy. We found that wet EM is a useful tool that fits into a niche between the simplicity of light microscopy and the high spatial resolution of EM.

  4. The rapidly changing face of electron microscopy

    NASA Astrophysics Data System (ADS)

    Thomas, John Meurig; Leary, Rowan K.; Eggeman, Alexander S.; Midgley, Paul A.

    2015-07-01

    This short but wide-ranging review is intended to convey to chemical physicists and others engaged in the interfaces between solid-state chemistry and solid-state physics the growing power and extensive applicability of multiple facets of the technique of electron microscopy.

  5. Photon gating in four-dimensional ultrafast electron microscopy

    PubMed Central

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

    2015-01-01

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

  6. Photon-induced near field electron microscopy

    NASA Astrophysics Data System (ADS)

    Park, Sang Tae; Zewail, Ahmed H.

    2013-09-01

    Ultrafast electron microscopy in the space and time domains utilizes a pulsed electron probe to directly map structural dynamics of nanomaterials initiated by an optical pump pulse, in imaging, di raction, spectroscopy, and their combinations. It has demonstrated its capability in the studies of phase transitions, mechanical vibrations, and chemical reactions. Moreover, electrons can directly interact with photons via the near eld component of light scattering by nanostructures, and either gain or lose light quanta discretely in energy. By energetically selecting those electrons that exchanged photon energies, we can map this photon-electron interaction, and the technique is termed photon-induced near eld electron microscopy (PINEM). Here, we give an account of the theoretical understanding of PINEM. Experimentally, nanostructures such as a sphere, cylinder, strip, and triangle have been investigated. Theoretically, time-dependent Schrodinger and Dirac equations for an electron under light are directly solved to obtain analytical solutions. The interaction probability is expressed by the mechanical work done by an optical wave on a traveling electron, which can be evaluated analytically by the near eld components of the Rayleigh scattering for small spheres and thin cylinders, and numerically by the discrete dipole approximation for other geometries. Application in visualization of plasmon elds is discussed.

  7. Analytical scanning electron microscopy for solid surface.

    PubMed

    Ichinokawa, T

    1989-07-01

    A scanning electron microscope of ultra-high-vacuum (UHV-SEM) with a field emission gun (FEG) is operated at the primary electron energies of from 100 eV to 3 keV. The instrument can form the images that contain information on surface chemical composition, chemical bonding state (electronic structure), and surface crystal structure in a microscopic resolution of several hundred angstroms (A) using the techniques of scanning Auger electron microscope, scanning electron energy loss microscope, and scanning low-energy electron diffraction (LEED) microscope. A scanning tunneling microscope (STM) also has been combined with the SEM in order to obtain the atomic resolution for the solid surface. The instrumentation and examples of their applications are presented both for scanning LEED microscopy and STM.

  8. Photoemission electron microscopy and scanning electron microscopy of Magnetospirillum magnetotacticum's magnetosome chains.

    PubMed

    Keutner, Christoph; von Bohlen, Alex; Berges, Ulf; Espeter, Philipp; Schneider, Claus M; Westphal, Carsten

    2014-10-01

    Magnetotactic bacteria are of great interdisciplinary interest, since a vast field of applications from magnetic recording media to medical nanorobots is conceivable. A key feature for a further understanding is the detailed knowledge about the magnetosome chain within the bacteria. We report on two preparation procedures suitable for UHV experiments in reflective geometry. Further, we present the results of scanning electron microscopy, as well as the first photoemission electron microscopy experiments, both accessing the magnetosomes within intact magnetotactic bacteria and compare these to scanning electron microscopy data from the literature. From the images, we can clearly identify individual magnetosomes within their chains.

  9. Aberration corrected Lorentz scanning transmission electron microscopy.

    PubMed

    McVitie, S; McGrouther, D; McFadzean, S; MacLaren, D A; O'Shea, K J; Benitez, M J

    2015-05-01

    We present results from an aberration corrected scanning transmission electron microscope which has been customised for high resolution quantitative Lorentz microscopy with the sample located in a magnetic field free or low field environment. We discuss the innovations in microscope instrumentation and additional hardware that underpin the imaging improvements in resolution and detection with a focus on developments in differential phase contrast microscopy. Examples from materials possessing nanometre scale variations in magnetisation illustrate the potential for aberration corrected Lorentz imaging as a tool to further our understanding of magnetism on this lengthscale.

  10. High-resolution low-dose scanning transmission electron microscopy

    PubMed Central

    Buban, James P.; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D.; Stahlberg, Henning

    2010-01-01

    During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM. PMID:19915208

  11. High-resolution low-dose scanning transmission electron microscopy.

    PubMed

    Buban, James P; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D; Stahlberg, Henning

    2010-01-01

    During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

  12. Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

    PubMed Central

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

    2013-01-01

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

  13. Frontiers of in situ electron microscopy

    DOE PAGES

    Zheng, Haimei; Zhu, Yimei; Meng, Shirley Ying

    2015-01-01

    In situ transmission electron microscopy (TEM) has become an increasingly important tool for materials characterization. It provides key information on the structural dynamics of a material during transformations and the correlation between structure and properties of materials. With the recent advances in instrumentation, including aberration corrected optics, sample environment control, the sample stage, and fast and sensitive data acquisition, in situ TEM characterization has become more and more powerful. In this article, a brief review of the current status and future opportunities of in situ TEM is included. It also provides an introduction to the six articles covered by inmore » this issue of MRS Bulletin explore the frontiers of in situ electron microscopy, including liquid and gas environmental TEM, dynamic four-dimensional TEM, nanomechanics, ferroelectric domain switching studied by in situ TEM, and state-of-the-art atomic imaging of light elements (i.e., carbon atoms) and individual defects.« less

  14. Experiments in electron microscopy: from metals to nerves

    NASA Astrophysics Data System (ADS)

    Unwin, Nigel

    2015-04-01

    Electron microscopy has advanced remarkably as a tool for biological structure research since the development of methods to examine radiation-sensitive unstained specimens and the introduction of cryo-techniques. Structures of biological molecules at near-atomic resolution can now be obtained from images of single particles as well as crystalline arrays. It has also become possible to analyze structures of molecules in their functional context, i.e. in their natural membrane or cellular setting, and in an ionic environment like that in living tissue. Electron microscopy is thus opening ways to answer definitively questions about physiological mechanisms. Here I recall a number of experiments contributing to, and benefiting from the technical advances that have taken place. I begin—in the spirit of this crystallography series—with some biographical background, and then sketch the path to an analysis by time-resolved microscopy of the opening mechanism of an ion channel (nicotinic acetylcholine receptor). This analysis illustrates how electron imaging can be combined with freeze-trapping to illuminate a transient biological event: in our case, chemical-to-electrical transduction at the nerve-muscle synapse.

  15. Scanning electron microscopy of superficial white onychomycosis*

    PubMed Central

    de Almeida Jr., Hiram Larangeira; Boabaid, Roberta Oliveira; Timm, Vitor; Silva, Ricardo Marques e; de Castro, Luis Antonio Suita

    2015-01-01

    Superficial white onychomycosis is characterized by opaque, friable, whitish superficial spots on the nail plate. We examined an affected halux nail of a 20-year-old male patient with scanning electron microscopy. The mycological examination isolated Trichophyton mentagrophytes. Abundant hyphae with the formation of arthrospores were found on the nail's surface, forming small fungal colonies. These findings showed the great capacity for dissemination of this form of onychomycosis. PMID:26560225

  16. Analytical transmission electron microscopy in minerals processing

    SciTech Connect

    Fraser, H.L.; Hsieh, K.C.; Twigg, M.E.

    1981-01-01

    A review of the possibilities of performing microchemical analysis in thin sections using a combination of scanning transmission electron microscopy and energy dispersive spectroscopy of x-rays is given. Particular attention is paid to the factors that limit accurate analysis at the highest spatial resolution. As an example of the use of these techniques applied to a potential problem in minerals processing, the identification of pyrite and pyrrhotite particles in Illinois, Herrin number 6 coal is presented.

  17. Electron Microscopy Study of Tin Whisker Growth

    SciTech Connect

    Norton, Murray G.; Lebret, Joel

    2003-03-30

    The growth of tin whiskers formed on sputtered tin layers deposited on brass was studied using electron microscopy. The occurrence of whiskers appeared to be largely independent of the macroscopic stress state in the film; rather it was microscopic compressive stresses arising from the formation of an intermetallic phase that appeared to be the necessary precursor. Whisker morphology was a result of whether nucleation had occurred on single grains or on multiple grains. In the latter case, the whiskers had a fluted or striated surface. The formation of whiskers on electron transparent samples was demonstrated. These samples showed the whiskers were monocrystalline and defect free, and that the growth direction could be determined.

  18. Self-labelling enzymes as universal tags for fluorescence microscopy, super-resolution microscopy and electron microscopy.

    PubMed

    Liss, Viktoria; Barlag, Britta; Nietschke, Monika; Hensel, Michael

    2015-01-01

    Research in cell biology demands advanced microscopy techniques such as confocal fluorescence microscopy (FM), super-resolution microscopy (SRM) and transmission electron microscopy (TEM). Correlative light and electron microscopy (CLEM) is an approach to combine data on the dynamics of proteins or protein complexes in living cells with the ultrastructural details in the low nanometre scale. To correlate both data sets, markers functional in FM, SRM and TEM are required. Genetically encoded markers such as fluorescent proteins or self-labelling enzyme tags allow observations in living cells. Various genetically encoded tags are available for FM and SRM, but only few tags are suitable for CLEM. Here, we describe the red fluorescent dye tetramethylrhodamine (TMR) as a multimodal marker for CLEM. TMR is used as fluorochrome coupled to ligands of genetically encoded self-labelling enzyme tags HaloTag, SNAP-tag and CLIP-tag in FM and SRM. We demonstrate that TMR can additionally photooxidize diaminobenzidine (DAB) to an osmiophilic polymer visible on TEM sections, thus being a marker suitable for FM, SRM and TEM. We evaluated various organelle markers with enzymatic tags in mammalian cells labelled with TMR-coupled ligands and demonstrate the use as efficient and versatile DAB photooxidizer for CLEM approaches. PMID:26643905

  19. Opportunities and challenges in liquid cell electron microscopy.

    PubMed

    Ross, Frances M

    2015-12-18

    Transmission electron microscopy offers structural and compositional information with atomic resolution, but its use is restricted to thin, solid samples. Liquid samples, particularly those involving water, have been challenging because of the need to form a thin liquid layer that is stable within the microscope vacuum. Liquid cell electron microscopy is a developing technique that allows us to apply the powerful capabilities of the electron microscope to imaging and analysis of liquid specimens. We describe its impact in materials science and biology. We discuss how its applications have expanded via improvements in equipment and experimental techniques, enabling new capabilities and stimuli for samples in liquids, and offering the potential to solve grand challenge problems.

  20. Metallothioneins for correlative light and electron microscopy.

    PubMed

    Fernández de Castro, Isabel; Sanz-Sánchez, Laura; Risco, Cristina

    2014-01-01

    Structural biologists have been working for decades on new strategies to identify proteins in cells unambiguously. We recently explored the possibilities of using the small metal-binding protein, metallothionein (MT), as a tag to detect proteins in transmission electron microscopy. It had been reported that, when fused with a protein of interest and treated in vitro with gold salts, a single MT tag will build an electron-dense gold cluster ~1 nm in diameter; we provided proof of this principle by demonstrating that MT can be used to detect intracellular proteins in bacteria and eukaryotic cells. The method, which is compatible with a variety of sample processing techniques, allows specific detection of proteins in cells with exceptional sensitivity. We illustrated the applicability of the technique in a series of studies to visualize the intracellular distribution of bacterial and viral proteins. Immunogold labeling was fundamental to confirm the specificity of the MT-gold method. When proteins were double-tagged with green fluorescent protein and MT, direct correlative light and electron microscopy allowed visualization of the same macromolecular complexes with different spatial resolutions. MT-gold tagging might also become a useful tool for mapping proteins into the 3D-density maps produced by (cryo)-electron tomography. New protocols will be needed for double or multiple labeling of proteins, using different versions of MT with fluorophores of different colors. Further research is also necessary to render the MT-gold labeling procedure compatible with immunogold labeling on Tokuyasu cryosections and with cryo-electron microscopy of vitreous sections.

  1. The Electron Microscopy eXchange (EMX) initiative

    PubMed Central

    Marabini, Roberto; Ludtke, Steven J.; Murray, Stephen C.; Chiu, Wah; de la Rosa-Trevín, Jose M.; Patwardhan, Ardan; Heymann, J. Bernard; Carazo, Jose M.

    2016-01-01

    Three-dimensional electron microscopy (3DEM) of ice-embedded samples allows the structural analysis of large biological macromolecules close to their native state. Different techniques have been developed during the last forty years to process cryo-electron microscopy (cryo-EM) data. Not surprisingly, success in analysis and interpretation is highly correlated with the continuous development of image processing packages. The field has matured to the point where further progress in data and methods sharing depends on an agreement between the packages on how to describe common image processing tasks. Such standardization will facilitate the use of software as well as seamless collaboration, allowing the sharing of rich information between different platforms. Our aim here is to describe the Electron Microscopy eXchange (EMX) initiative, launched at the 2012 Instruct Image Processing Center Developer Workshop, with the intention of developing a first set of standard conventions for the interchange of information for single-particle analysis (EMX version 1.0). These conventions cover the specification of the metadata for micrograph and particle images, including contrast transfer function (CTF) parameters and particle orientations. EMX v1.0 has already been implemented in the Bsoft, EMAN, Xmipp and Scipion image processing packages. It has been and will be used in the CTF and EMDataBank Validation Challenges respectively. It is also being used in EMPIAR, the Electron Microscopy Pilot Image Archive, which stores raw image data related to the 3DEM reconstructions in EMDB. PMID:26873784

  2. The Electron Microscopy eXchange (EMX) initiative.

    PubMed

    Marabini, Roberto; Ludtke, Steven J; Murray, Stephen C; Chiu, Wah; de la Rosa-Trevín, Jose M; Patwardhan, Ardan; Heymann, J Bernard; Carazo, Jose M

    2016-05-01

    Three-dimensional electron microscopy (3DEM) of ice-embedded samples allows the structural analysis of large biological macromolecules close to their native state. Different techniques have been developed during the last forty years to process cryo-electron microscopy (cryo-EM) data. Not surprisingly, success in analysis and interpretation is highly correlated with the continuous development of image processing packages. The field has matured to the point where further progress in data and methods sharing depends on an agreement between the packages on how to describe common image processing tasks. Such standardization will facilitate the use of software as well as seamless collaboration, allowing the sharing of rich information between different platforms. Our aim here is to describe the Electron Microscopy eXchange (EMX) initiative, launched at the 2012 Instruct Image Processing Center Developer Workshop, with the intention of developing a first set of standard conventions for the interchange of information for single-particle analysis (EMX version 1.0). These conventions cover the specification of the metadata for micrograph and particle images, including contrast transfer function (CTF) parameters and particle orientations. EMX v1.0 has already been implemented in the Bsoft, EMAN, Xmipp and Scipion image processing packages. It has been and will be used in the CTF and EMDataBank Validation Challenges respectively. It is also being used in EMPIAR, the Electron Microscopy Pilot Image Archive, which stores raw image data related to the 3DEM reconstructions in EMDB. PMID:26873784

  3. A quick guide to light microscopy in cell biology

    PubMed Central

    Thorn, Kurt

    2016-01-01

    Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments. PMID:26768859

  4. Biological Electron Transport Systems

    PubMed Central

    Cowan, Dwaine O.; Pasternak, Gavril; Kaufman, Frank

    1970-01-01

    The solid-state electrical conductivities of a number of ferredoxin model compounds are reported. For one of these compounds, (KFeS2)n, an electron transfer rate for a 25 Å unit is shown to be at least 1 × 108 electrons sec-1. The rate becomes proportionally larger for smaller molecular units. This rapid rate is consistant with a short pipe model for electron transport between two reaction sites. Some of the factors leading to this rapid transfer rate are considered. PMID:5269247

  5. 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...

  6. Phase-contrast scanning transmission electron microscopy.

    PubMed

    Minoda, Hiroki; Tamai, Takayuki; Iijima, Hirofumi; Hosokawa, Fumio; Kondo, Yukihito

    2015-06-01

    This report introduces the first results obtained using phase-contrast scanning transmission electron microscopy (P-STEM). A carbon-film phase plate (PP) with a small center hole is placed in the condenser aperture plane so that a phase shift is introduced in the incident electron waves except those passing through the center hole. A cosine-type phase-contrast transfer function emerges when the phase-shifted scattered waves interfere with the non-phase-shifted unscattered waves, which passed through the center hole before incidence onto the specimen. The phase contrast resulting in P-STEM is optically identical to that in phase-contrast transmission electron microscopy that is used to provide high contrast for weak phase objects. Therefore, the use of PPs can enhance the phase contrast of the STEM images of specimens in principle. The phase shift resulting from the PP, whose thickness corresponds to a phase shift of π, has been confirmed using interference fringes displayed in the Ronchigram of a silicon single crystal specimen. The interference fringes were found to abruptly shift at the edge of the PP hole by π.

  7. The intermediate size direct detection detector for electron microscopy

    NASA Astrophysics Data System (ADS)

    Jin, Liang; Milazzo, Anna-Clare; Kleinfelder, Stuart; Li, Shengdong; Leblanc, Philippe; Duttweiler, Fred; Bouwer, James C.; Peltier, Steve T.; Ellisman, Mark; Xuong, Nguyen-Huu

    2007-02-01

    In a longstanding effort to overcome limits of film and the charge coupled device (CCD) systems in electron microscopy, we have developed a radiation-tolerant system that can withstand direct electron bombardment. A prototype Direct Detection Device (DDD) detector based on an Active Pixel Sensor (APS) has delivered unprecedented performance with an excellent signal-to-noise ratio (approximately 5/1 for a single incident electron in the range of 200-400 keV) and a very high spatial resolution. This intermediate size prototype features a 512×550 pixel format of 5μm pitch. The detector response to uniform beam illumination and to single electron hits is reported. Radiation tolerance with high-energy electron exposure is also impressive, especially with cooling to -15 °C. Stable performance has been demonstrated, even after a total dose of 3.3×10 6 electrons/pixel. The characteristics of this new detector have exciting implications for transmission electron microscopy, especially for cryo-EM as applied to biological macromolecules.

  8. Feature Adaptive Sampling for Scanning Electron Microscopy

    PubMed Central

    Dahmen, Tim; Engstler, Michael; Pauly, Christoph; Trampert, Patrick; de Jonge, Niels; Mücklich, Frank; Slusallek, Philipp

    2016-01-01

    A new method for the image acquisition in scanning electron microscopy (SEM) was introduced. The method used adaptively increased pixel-dwell times to improve the signal-to-noise ratio (SNR) in areas of high detail. In areas of low detail, the electron dose was reduced on a per pixel basis, and a-posteriori image processing techniques were applied to remove the resulting noise. The technique was realized by scanning the sample twice. The first, quick scan used small pixel-dwell times to generate a first, noisy image using a low electron dose. This image was analyzed automatically, and a software algorithm generated a sparse pattern of regions of the image that require additional sampling. A second scan generated a sparse image of only these regions, but using a highly increased electron dose. By applying a selective low-pass filter and combining both datasets, a single image was generated. The resulting image exhibited a factor of ≈3 better SNR than an image acquired with uniform sampling on a Cartesian grid and the same total acquisition time. This result implies that the required electron dose (or acquisition time) for the adaptive scanning method is a factor of ten lower than for uniform scanning. PMID:27150131

  9. Low energy electron microscopy and photoemission electron microscopy investigation of graphene

    NASA Astrophysics Data System (ADS)

    Man, K. L.; Altman, M. S.

    2012-08-01

    Low energy electron microscopy (LEEM) and photoemission electron microscopy (PEEM) are two powerful techniques for the investigation of surfaces, thin films and surface supported nanostructures. In this review, we examine the contributions of these microscopy techniques to our understanding of graphene in recent years. These contributions have been made in studies of graphene on various metal and SiC surfaces and free-standing graphene. We discuss how the real-time imaging capability of LEEM facilitates a deeper understanding of the mechanisms of dynamic processes, such as growth and intercalation. Numerous examples also demonstrate how imaging and the various available complementary measurement capabilities, such as selected area or micro low energy electron diffraction (μLEED) and micro angle resolved photoelectron spectroscopy (μARPES), allow the investigation of local properties in spatially inhomogeneous graphene samples.

  10. Introduction to Atomic Force Microscopy (AFM) in Biology.

    PubMed

    Kreplak, Laurent

    2016-01-01

    The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution over a wide range of time scales from milliseconds to hours. In addition to providing topographical images of surfaces with nanometer- to angstrom-scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated from the nano-scale to the micro-scale. Importantly, the measurements are made in buffer solutions, allowing biological samples to "stay alive" within a physiological-like environment while temporal changes in structure are measured-e.g., before and after addition of chemical reagents. These qualities distinguish AFM from conventional imaging techniques of comparable resolution, e.g., electron microscopy (EM). This unit provides an introduction to AFM on biological systems and describes specific examples of AFM on proteins, cells, and tissues. The physical principles of the technique and methodological aspects of its practical use and applications are also described. © 2016 by John Wiley & Sons, Inc. PMID:27479503

  11. Nanometric crystal defects in transmission electron microscopy.

    PubMed

    Schäublin, Robin

    2006-05-01

    Transmission electron microscopy (TEM) is revisited in order to define methods for the identification of nanometric defects. Nanometric crystal defects play an important role as they influence, generally in a detrimental way, physical properties. For instance, radiation-induced damage in metals strongly degrades mechanical properties, rendering the material stronger but brittle. The difficulty in using TEM to identify the nature and size of such defects resides in their small size. TEM image simulations are deployed to explore limits and possible ways to improve on spatial resolution and contrast. The contrast of dislocation loops, cavities, and a stacking fault tetrahedra (SFT) are simulated in weak beam, interfering reflections (HRTEM), and scanned condensed electron probe (STEM) mode. Results indicate that STEM is a possible way to image small defects. In addition, a new objective aperture is proposed to improve resolution in diffraction contrast. It is investigated by simulations of the weak beam imaging of SFT and successfully applied in experimental observations.

  12. High resolution scanning electron microscopy of plasmodesmata.

    PubMed

    Brecknock, Sarah; Dibbayawan, Teresa P; Vesk, Maret; Vesk, Peter A; Faulkner, Christine; Barton, Deborah A; Overall, Robyn L

    2011-10-01

    Symplastic transport occurs between neighbouring plant cells through functionally and structurally dynamic channels called plasmodesmata (PD). Relatively little is known about the composition of PD or the mechanisms that facilitate molecular transport into neighbouring cells. While transmission electron microscopy (TEM) provides 2-dimensional information about the structural components of PD, 3-dimensional information is difficult to extract from ultrathin sections. This study has exploited high-resolution scanning electron microscopy (HRSEM) to reveal the 3-dimensional morphology of PD in the cell walls of algae, ferns and higher plants. Varied patterns of PD were observed in the walls, ranging from uniformly distributed individual PD to discrete clusters. Occasionally the thick walls of the giant alga Chara were fractured, revealing the surface morphology of PD within. External structures such as spokes, spirals and mesh were observed surrounding the PD. Enzymatic digestions of cell wall components indicate that cellulose or pectin either compose or stabilise the extracellular spokes. Occasionally, the PD were fractured open and desmotubule-like structures and other particles were observed in their central regions. Our observations add weight to the argument that Chara PD contain desmotubules and are morphologically similar to higher plant PD.

  13. Characterization of hydroxyapatite by electron microscopy.

    PubMed

    Rodríguez-Lugo, V; Hernández, J Sanchez; Arellano-Jimenez, Ma J; Hernández-Tejeda, P H; Recillas-Gispert, S

    2005-12-01

    The obtention of hydroxyapatite (HAp) is reported using brushite (CaHPO4.2H2O) and the skeleton of a starfish (Mellita eduardobarrosoi sp. nov.), primarily composed of magnesian calcite ((Ca,Mg)CO3) as precursors. Stoichiometric amounts of both were reacted under hydrothermal conditions: a pressure of 5.8 MPa and a temperature of 200 degrees C for 2, 4, 6, 8, 10, and 20 h of reaction times. The samples obtained were characterized by means of scanning electron microscopy, X-ray diffraction, infrared spectroscopy, and transmission electron microscopy. Two defined populations of HAp fibers were found: A bundle of fibers 75 mum in length and 1-13 mum in diameter, and a second bundle of fibers 5 mum in length and less than 0.5 mum in diameter. Furthermore, an increase in HAp formation and a Ca/P ratio as a function of reaction time were observed. The growth mechanism of HAp is also discussed. PMID:17481330

  14. Characterization of Hydroxyapatite by Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Rodríguez-Lugo, V.; Sanchez Hernández, J.; Arellano-Jimenez, Ma. J.; Hernández-Tejeda, P. H.; Recillas-Gispert, S.

    2005-12-01

    The obtention of hydroxyapatite (HAp) is reported using brushite (CaHPO4·2H2O) and the skeleton of a starfish (Mellita eduardobarrosoi sp. nov.), primarily composed of magnesian calcite ((Ca,Mg)CO3) as precursors. Stoichiometric amounts of both were reacted under hydrothermal conditions: a pressure of 5.8 MPa and a temperature of 200°C for 2, 4, 6, 8, 10, and 20 h of reaction times. The samples obtained were characterized by means of scanning electron microscopy, X-ray diffraction, infrared spectroscopy, and transmission electron microscopy. Two defined populations of HAp fibers were found: A bundle of fibers 75 [mu]m in length and 1 13 [mu]m in diameter, and a second bundle of fibers 5 [mu]m in length and less than 0.5 [mu]m in diameter. Furthermore, an increase in HAp formation and a Ca/P ratio as a function of reaction time were observed. The growth mechanism of HAp is also discussed.

  15. Determination of surface topography of biological specimens at high resolution by scanning tunnelling microscopy.

    PubMed

    Baró, A M; Miranda, R; Alamán, J; García, N; Binnig, G; Rohrer, H; Gerber, C; Carrascosa, J L

    Although techniques are available for the determination of the three-dimensional structure of biological specimens, for example scanning electron microscopy, they all have some serious drawback, such as low resolution, the requirement for crystals or for the sample to be analysed in a high vacuum. In an attempt to develop a technique for high-resolution three-dimensional structure analysis of non-crystalline biological material, we have tested the applicability of scanning tunnelling microscopy (STM), a method that has been used successfully in the analysis of metal and semiconductor surface structures. We report here that scanning tunnelling electron microscopy can be used to determine the surface topography of biological specimens at atmospheric pressure and room temperature, giving a vertical resolution of the order of 1 A. Our results show that quantum mechanical tunnelling of electrons through biological material is possible provided that the specimen is deposited on a conducting surface.

  16. Cryo electron microscopy to determine the structure of macromolecular complexes.

    PubMed

    Carroni, Marta; Saibil, Helen R

    2016-02-15

    Cryo-electron microscopy (cryo-EM) is a structural molecular and cellular biology technique that has experienced major advances in recent years. Technological developments in image recording as well as in processing software make it possible to obtain three-dimensional reconstructions of macromolecular assemblies at near-atomic resolution that were formerly obtained only by X-ray crystallography or NMR spectroscopy. In parallel, cryo-electron tomography has also benefitted from these technological advances, so that visualization of irregular complexes, organelles or whole cells with their molecular machines in situ has reached subnanometre resolution. Cryo-EM can therefore address a broad range of biological questions. The aim of this review is to provide a brief overview of the principles and current state of the cryo-EM field.

  17. High-Resolution Secondary Electron Microscopy and Scanning Reflection Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Liu, Jingyue

    1990-01-01

    High resolution secondary electron microscopy (HRSEM) utilizes the low energy electrons emitted from the sample to form images of the surface. By using a very small incident electron probe subnanometer resolution images of solid surfaces can be obtained by collecting secondary electrons. Surfaces of both electron beam transparent samples and bulk samples can be investigated by high resolution secondary electron (SE) imaging technique. The emission of secondary electrons is determined by three different processes: (1) the generation of secondary electrons inside the sample; (2) the transport of the excited electrons to the vacuum-sample interface and (3) the escape of secondary electrons over the surface potential barrier into vacuum. The total yield of the emitted secondary electrons is sensitive to sample surface conditions. Surface electronic and geometric modifications will influence the total yield of secondary electrons. The contrast in a SE image is determined by the change of the total SE yield. Therefore the knowledge of the origin of SE emission is essential for interpreting the experimental high resolution secondary electron images. The first part of this dissertation is to discuss the origins of the collected secondary electrons, to develop the theory of surface imaging by secondary electrons and to investigate the contrast mechanisms of high resolution SE images. By combining HRSEM with secondary electron spectroscopy information about the surface topographic and, to some extent, surface electronic structures can be obtained. Experimental results obtained in the ultra-high vacuum (UHV) scanning transmission electron microscope have yielded fruitful information about the electron emission processes. Scanning reflection electron microscopy (SREM) utilizes the high energy electrons reflected from a bulk crystal to form images of the crystal surface. At glancing incident angle specularlly Bragg diffracted beam satisfying surface resonance conditions can

  18. Characterization of nanomaterials with transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Anjum, D. H.

    2016-08-01

    The field of nanotechnology is about research and development on materials whose at least one dimension is in the range of 1 to 100 nanometers. In recent years, the research activity for developing nano-materials has grown exponentially owing to the fact that they offer better solutions to the challenges faced by various fields such as energy, food, and environment. In this paper, the importance of transmission electron microscopy (TEM) based techniques is demonstrated for investigating the properties of nano-materials. Specifically the nano-materials that are investigated in this report include gold nano-particles (Au-NPs), silver atom-clusters (Ag-ACs), tantalum single-atoms (Ta-SAs), carbon materials functionalized with iron cobalt (Fe-Co) NPs and titania (TiO2) NPs, and platinum loaded Ceria (Pt-CeO2) Nano composite. TEM techniques that are employed to investigate nano-materials include aberration corrected bright-field TEM (BF-TEM), high-angle dark-field scanning TEM (HAADF-STEM), electron energy-loss spectroscopy (EELS), and BF-TEM electron tomography (ET). With the help presented of results in this report, it is proved herein that as many TEM techniques as available in a given instrument are essential for a comprehensive nano-scale analysis of nanomaterials.

  19. 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

  20. The origins and evolution of freeze-etch electron microscopy.

    PubMed

    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.

  1. From high symmetry to high resolution in biological electron microscopy: a commentary on Crowther (1971) 'Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs'.

    PubMed

    Rosenthal, Peter B

    2015-04-19

    Elucidation of the structure of biological macromolecules and larger assemblies has been essential to understanding the roles they play in living processes. Methods for three-dimensional structure determination of biological assemblies from images recorded in the electron microscope were therefore a key development. In his paper published in Philosophical Transactions B in 1971, Crowther described new computational procedures applied to the first three-dimensional reconstruction of an icosahedral virus from images of virus particles preserved in negative stain. The method for determining the relative orientation of randomly oriented particles and combining their images for reconstruction exploited the high symmetry of the virus particle. Computational methods for image analysis have since been extended to include biological assemblies without symmetry. Further experimental advances, combined with image analysis, have led to the method of cryomicroscopy, which is now used by structural biologists to study the structure and dynamics of biological machines and assemblies in atomic detail. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society. PMID:25750240

  2. From high symmetry to high resolution in biological electron microscopy: a commentary on Crowther (1971) ‘Procedures for three-dimensional reconstruction of spherical viruses by Fourier synthesis from electron micrographs’

    PubMed Central

    Rosenthal, Peter B.

    2015-01-01

    Elucidation of the structure of biological macromolecules and larger assemblies has been essential to understanding the roles they play in living processes. Methods for three-dimensional structure determination of biological assemblies from images recorded in the electron microscope were therefore a key development. In his paper published in Philosophical Transactions B in 1971, Crowther described new computational procedures applied to the first three-dimensional reconstruction of an icosahedral virus from images of virus particles preserved in negative stain. The method for determining the relative orientation of randomly oriented particles and combining their images for reconstruction exploited the high symmetry of the virus particle. Computational methods for image analysis have since been extended to include biological assemblies without symmetry. Further experimental advances, combined with image analysis, have led to the method of cryomicroscopy, which is now used by structural biologists to study the structure and dynamics of biological machines and assemblies in atomic detail. This commentary was written to celebrate the 350th anniversary of the journal Philosophical Transactions of the Royal Society. PMID:25750240

  3. ELECTRON MICROSCOPY OF PLASMOLYSIS IN ESCHERICHIA COLI.

    PubMed

    COTA-ROBLES, E H

    1963-03-01

    Cota-Robles, Eugene H. (University of California, Riverside). Electron microscopy of plasmolysis in Escherichia coli. J. Bacteriol. 85:499-503. 1963.-Escherichia coli cells plasmolyzed in 0.35 m sucrose reveal plasmolysis at one tip of a cell or in the center of dividing cells in which protoplast partition has been complete. Central plasmolysis reveals that protoplast separation can be completed before the invagination of the cell wall is complete. These studies support the concept that these cells divide by constriction. The strength of the union between cell wall and cytoplasm is not uniform around the entire cell. It is strongest along the sides of these rod-shaped cells and weakest at one tip of the single cell. Thus, a single cell generally forms one cup-shaped vacuole in which the cytoplasm has collapsed away from one tip of the cell.

  4. Scanning electron microscopy of tinea nigra.

    PubMed

    Guarenti, Isabelle Maffei; Almeida, Hiram Larangeira de; Leitão, Aline Hatzenberger; Rocha, Nara Moreira; Silva, Ricardo Marques E

    2014-01-01

    Tinea nigra is a rare superficial mycosis caused by Hortaea werneckii. This infection presents as asymptomatic brown to black maculae mostly in palmo-plantar regions. We performed scanning electron microscopy of a superficial shaving of a tinea nigra lesion. The examination of the outer surface of the sample showed the epidermis with corneocytes and hyphae and elimination of fungal filaments. The inner surface of the sample showed important aggregation of hyphae among keratinocytes, which formed small fungal colonies. The ultrastructural findings correlated with those of dermoscopic examination - the small fungal aggregations may be the dark spicules seen on dermoscopy - and also allowed to document the mode of dissemination of tinea nigra, showing how hyphae are eliminated on the surface of the lesion.

  5. Improved methods for high resolution electron microscopy

    SciTech Connect

    Taylor, J.R.

    1987-04-01

    Existing methods of making support films for high resolution transmission electron microscopy are investigated and novel methods are developed. Existing methods of fabricating fenestrated, metal reinforced specimen supports (microgrids) are evaluated for their potential to reduce beam induced movement of monolamellar crystals of C/sub 44/H/sub 90/ paraffin supported on thin carbon films. Improved methods of producing hydrophobic carbon films by vacuum evaporation, and improved methods of depositing well ordered monolamellar paraffin crystals on carbon films are developed. A novel technique for vacuum evaporation of metals is described which is used to reinforce microgrids. A technique is also developed to bond thin carbon films to microgrids with a polymer bonding agent. Unique biochemical methods are described to accomplish site specific covalent modification of membrane proteins. Protocols are given which covalently convert the carboxy terminus of papain cleaved bacteriorhodopsin to a free thiol. 53 refs., 19 figs., 1 tab.

  6. Improved methods for high resolution electron microscopy

    NASA Astrophysics Data System (ADS)

    Taylor, J. R.

    1987-04-01

    Existing methods of making support films for high resolution transmission electron microscopy are investigated and novel methods are developed. Existing methods of fabricating fenestrated, metal reinforced specimen supports (microgrids) are evaluated for their potential to reduce beam induced movement of monolamellar crystals of C44H90 paraffin supported on thin carbon films. Improved methods of producing hydrophobic carbon films by vacuum evaporation, and improved methods of depositing well ordered monolamellar paraffin crystals on carbon films are developed. A novel technique for vacuum evaporation of metals is described which is used to reinforce microgrids. A technique is also developed to bond thin carbon films to microgrids with a polymer bonding agent. Unique biochemical methods are described to accomplish site specific covalent modification of membrane proteins. Protocols are given which covalently convert the carboxy terminus of papain cleaved bacteriorhodopsin to a free thiol.

  7. Hexamethyldisilazane for scanning electron microscopy of Gastrotricha.

    PubMed

    Hochberg, R; Litvaitis, M K

    2000-01-01

    We evaluated treatment with hexamethyldisilazane (HMDS) as an alternative to critical-point drying (CPD) for preparing microscopic Gastrotricha for scanning electron microscopy (SEM). We prepared large marine (2 mm) and small freshwater (100 microm) gastrotrichs using HMDS as the primary dehydration solvent and compared the results to earlier investigations using CPD. The results of HMDS dehydration are similar to or better than CPD for resolution of two important taxonomic features: cuticular ornamentation and patterns of ciliation. The body wall of both sculpted (Lepidodermella) and smooth (Dolichodasys) gastrotrichs retained excellent morphology as did the delicate sensory and locomotory cilia. The only unfavorable result of HMDS dehydration was an occasional coagulation of gold residue when the solvent had not fully evaporated before sputter-coating. We consider HMDS an effective alternative for preparing of gastrotrichs for SEM because it saves time and expense compared to CPD. PMID:10810982

  8. Electron microscopy of frozen hydrated eukaryotic flagella.

    PubMed

    Murray, J M

    1986-01-01

    Resting and active sea urchin sperm flagella have been examined by low-dose electron microscopy of frozen hydrated specimens. The flagella are unfixed, unstained, completely intact, and able to swim vigorously after going through the entire preparative procedure. The most prominent features of the image arise from the edges of the axonemal doublets and central-pair microtubules seen in projection. By comparison with these longitudinal markings, transverse features are less easy to discern, being camouflaged by superposition. However, Fourier transforms of digitized micrographs reveal a remarkable degree of crystalline order in quiescent flagella. Filtered images derived from these Fourier transforms show clearly features arising from the central-pair complex and radial spokes that were obscured in the original data. Potentially complicating effects of specimen thickness are shown to be quantitatively insignificant in the formation of images of unstained frozen hydrated flagella. Determination of native flagellar structure by 3-D reconstruction from multiple-tilted views appears to be feasible.

  9. Hexamethyldisilazane for scanning electron microscopy of Gastrotricha.

    PubMed

    Hochberg, R; Litvaitis, M K

    2000-01-01

    We evaluated treatment with hexamethyldisilazane (HMDS) as an alternative to critical-point drying (CPD) for preparing microscopic Gastrotricha for scanning electron microscopy (SEM). We prepared large marine (2 mm) and small freshwater (100 microm) gastrotrichs using HMDS as the primary dehydration solvent and compared the results to earlier investigations using CPD. The results of HMDS dehydration are similar to or better than CPD for resolution of two important taxonomic features: cuticular ornamentation and patterns of ciliation. The body wall of both sculpted (Lepidodermella) and smooth (Dolichodasys) gastrotrichs retained excellent morphology as did the delicate sensory and locomotory cilia. The only unfavorable result of HMDS dehydration was an occasional coagulation of gold residue when the solvent had not fully evaporated before sputter-coating. We consider HMDS an effective alternative for preparing of gastrotrichs for SEM because it saves time and expense compared to CPD.

  10. Electric fields in Scanning Electron Microscopy simulations

    NASA Astrophysics Data System (ADS)

    Arat, K. T.; Bolten, J.; Klimpel, T.; Unal, N.

    2016-03-01

    The electric field distribution and charging effects in Scanning Electron Microscopy (SEM) were studied by extending a Monte-Carlo based SEM simulator by a fast and accurate multigrid (MG) based 3D electric field solver. The main focus is on enabling short simulation times with maintaining sufficient accuracy, so that SEM simulation can be used in practical applications. The implementation demonstrates a gain in computation speed, when compared to a Gauss-Seidel based reference solver is roughly factor of 40, with negligible differences in the result (~10-6 𝑉). In addition, the simulations were compared with experimental SEM measurements using also complex 3D sample, showing that i) the modelling of e-fields improves the simulation accuracy, and ii) multigrid method provide a significant benefit in terms of simulation time.

  11. Biological oscillations: Fluorescence monitoring by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Chattoraj, Shyamtanu; Bhattacharyya, Kankan

    2016-09-01

    Fluctuations play a vital role in biological systems. Single molecule spectroscopy has recently revealed many new kinds of fluctuations in biological molecules. In this account, we focus on structural fluctuations of an antigen-antibody complex, conformational dynamics of a DNA quadruplex, effects of taxol on dynamics of microtubules, intermittent red-ox oscillations at different organelles in a live cell (mitochondria, lipid droplets, endoplasmic reticulum and cell membrane) and stochastic resonance in gene silencing. We show that there are major differences in these dynamics between a cancer cell and the corresponding non-cancer cell.

  12. High pressure freezing, electron microscopy, and immuno-electron microscopy of Tetrahymena thermophila basal bodies.

    PubMed

    Meehl, Janet B; Giddings, Thomas H; Winey, Mark

    2009-01-01

    Preservation of Tetrahymena thermophila basal body ultrastructure for visualization by transmission electron microscopy is improved by a combination of high pressure freezing (HPF) and freeze substitution (FS). These methods also reliably retain the antigenicity of cellular proteins for immuno-electron microscopy, which enables the precise localization of green fluorescent protein (GFP)-tagged and native basal body proteins. The plastic-embedded samples generated by these methods take full advantage of higher resolution visualization techniques such as electron tomography. We describe protocols for cryofixation, FS, immunolabeling, and staining. Suggestions for trouble shooting and evaluation of specimen quality are discussed. In combination with identification and manipulation of a rapidly expanding list of basal body-associated gene products, these methods are being used to increase our understanding of basal body composition, assembly, and function.

  13. Digital position determination system for electron microscopy.

    PubMed

    Hohmann-Marriott, Martin F; Sharp, William P; Roberson, Robert W; Blankenship, Robert E

    2005-06-01

    The precise determination of object positions within a specimen grid is important for many applications in electron microscopy. For example, real-time position determination is necessary for current statistical approaches and the efficient mapping and relocation of objects. Unfortunately, precise real-time position determination is not available on many older electron microscopes with manual stage controls. This report demonstrates the cost-effective and flexible implementation of a digital position determination system that can be adapted to many hand-operated electron microscopes. A customized solution that includes the hardware and software to accomplish position determination is presented. Lists of required parts, instructions for building the hardware, and descriptions of the developed programs are included. Two LED-photodiode assemblies detect x and y movements via an optical wheel that is in physical contact with the mechanical x and y stage control elements. These detector assemblies are interfaced with an integrated circuit that converts movement information into serial port-compatible signals, which are interpreted by a computer with specialized software. Two electron microscopes, a Philips CM12 (S)TEM and a Philips 201 TEM, were equipped with the described digital position determination system. The position fidelity and position fidelity after reloading of grids were determined for both microscopes. The determined position deviation was 1.06 microm in the x axis and 0.565 microm in the y axis for the Philips CM12 (S)TEM, and 0.303 microm in the x axis and 0.545 microm in the y axis for the Philips 201 TEM. After reloading and computational realigning, the determined average position variation was 2.66 microm in the x axis and 2.61 microm in the y axis for the Philips CM12 (S)TEM, and 1.13 microm in the x axis and 1.27 microm in the y axis for the Philips 201 TEM.

  14. Development and biological applications of high-resolution ion beam induced fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Zhaohong, Mi

    High-resolution fluorescence microscopy has become an essential tool in both biological and biomedical sciences, to directly visualize biological processes at the cellular and subcellular levels through specific fluorescence labeling. Among the fluorescence microscopy techniques, mega-electron-volt (MeV) ion-induced fluorescence microscopy has unique advantages because MeV ions can penetrate through biological cells with little deflection in their trajectories. The state-of-the-art bioimaging facility in the Centre for Ion Beam Applications, National University of Singapore can achieve sub-30 nm spatial resolutions for structural imaging of biological cells, which is well below the diffraction limits imposed by optical microscopy. Our aim is to achieve similar spatial resolutions for Ion Beam Induced Fluorescence Imaging. (Abstract shortened by UMI.).

  15. Development and testing of hyperbaric atomic force microscopy (AFM) and fluorescence microscopy for biological applications.

    PubMed

    D'Agostino, D P; McNally, H A; Dean, J B

    2012-05-01

    A commercially available atomic force microscopy and fluorescence microscope were installed and tested inside a custom-designed hyperbaric chamber to provide the capability to study the effects of hyperbaric gases on biological preparations, including cellular mechanism of oxidative stress. In this report, we list details of installing and testing atomic force microscopy and fluorescence microscopy inside a hyperbaric chamber. The pressure vessel was designed to accommodate a variety of imaging equipment and ensures full functionality at ambient and hyperbaric conditions (≤85 psi). Electrical, gas and fluid lines were installed to enable remote operation of instrumentation under hyperbaric conditions, and to maintain viable biological samples with gas-equilibrated superfusate and/or drugs. Systems were installed for vibration isolation and temperature regulation to maintain atomic force microscopy performance during compression and decompression. Results of atomic force microscopy testing demonstrate sub-nanometre resolution at hyperbaric pressure in dry scans and fluid scans, in both contact mode and tapping mode. Noise levels were less when measurements were taken under hyperbaric pressure with air, helium (He) and nitrogen (N(2) ). Atomic force microscopy and fluorescence microscopy measurements were made on a variety of living cell cultures exposed to hyperbaric gases (He, N(2) , O(2) , air). In summary, atomic force microscopy and fluorescence microscopy were installed and tested for use at hyperbaric pressures and enables the study of cellular and molecular effects of hyperbaric gases and pressure per se in biological preparations.

  16. [Morton's disease: optic and electron microscopy observations].

    PubMed

    De Palma, L; Tulli, A

    1991-01-01

    The authors performed an optic and electron-microscope investigation above the common digital nerve of the foot, whose fragments had been surgically removed from patients suffering from "Morton metatarsalgia" (neuroma). Histological sections were taken from pre-stenotic swelling in patients with clinical symptoms persisting for one year; perineural thickening without evidence of fibroblastic proliferation could be demonstrated, together with an intraneural deposition of an amorphous substance. In other patients suffering from Morton's disease for a longer time, a more pronounced epineural thickening in the pre-stenotic zone could be shown, with partial replacement of nerve fibers by amorphous substance. In the same patients endoneural fibrositis was seen at the level of the stenosis. Electron-microscopy in patients after one year showed an increase in collagenous endoneural fibers and microfibrils. These histopathological findings suggest a compressive mechanism in the pathogenesis of the damage to the common interdigital nerve in Morton's disease, caused by the extrinsic anatomical structures surrounding the nerve. The so-called "neuroma" can be identified with the pre-stenotic swelling.

  17. Quantitative Phase Microscopy of Live Biological Cell Dynamics

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.; Wax, Adam

    2010-04-01

    Interferometric phase microscopy of biological cell dynamics has the potential to provide a label-free quantitative tool for cell biology, as well as for medical diagnosis and monitoring. The current state of the art of this field, the open questions, and specific solutions developed in our laboratory will be presented.

  18. 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.

  19. Advanced electron microscopy characterization of multimetallic nanoparticles

    NASA Astrophysics Data System (ADS)

    Khanal, Subarna Raj

    Research in noble metal nanoparticles has led to exciting progress in a versatile array of applications. For the purpose of better tailoring of nanoparticles activities and understanding the correlation between their structures and properties, control over the composition, shape, size and architecture of bimetallic and multimetallic nanomaterials plays an important role on revealing their new or enhanced functions for potentials application. Advance electron microscopy techniques were used to provide atomic scale insights into the structure-properties of different materials: PtPd, Au-Au3Cu, Cu-Pt, AgPd/Pt and AuCu/Pt nanoparticles. The objective of this work is to understand the physical and chemical properties of nanomaterials and describe synthesis, characterization, surface properties and growth mechanism of various bimetallic and multimetallic nanoparticles. The findings have provided us with novel and significant insights into the physical and chemical properties of noble metal nanoparticles. Different synthesis routes allowed us to synthesize bimetallic: Pt-Pd, Au-Au3Cu, Cu-Pt and trimetallic: AgPd/Pt, AuCu/Pt, core-shell and alloyed nanoparticles with monodispersed sizes, controlled shapes and tunable surface properties. For example, we have synthesized the polyhedral PtPd core-shell nanoparticles with octahedral, decahedral, and triangular plates. Decahedral PtPd core-shell structures are novel morphologies for this system. For the first time we fabricated that the Au core and Au3Cu alloyed shell nanoparticles passivated with CuS2 surface layers and characterized by Cs-corrected scanning transmission electron microscopy. The analysis of the high-resolution micrographs reveals that these nanoparticles have decahedral structure with shell periodicity, and that each of the particles is composed by Au core and Au3Cu ordered superlattice alloyed shell surrounded by CuS 2 surface layer. Additionally, we have described both experimental and theoretical methods of

  20. Scanning electron microscopy of macrophages: a bibliography.

    PubMed

    Carr, K E; Toner, P G

    1979-01-01

    In this bibliography an attempt has been made to gather together as much as possible of the widely spread literature on the scanning electron microscopic appearances of macrophages. The primary source for these references was a medline search, supplemented by any secondary references obtained. The listed references have all been examined in detail by the compilers. In the subject index, an attempt has been made to draw the reference together into broad categories of biologic interest, as well as indexing for specific anatomic sites, pathologic conditions, toxic agents and so on. We hope that this compilation may be of some help to those seeking information on the surface morphology of the mononuclear phagocytes. PMID:392725

  1. Single-particle cryo-electron microscopy of macromolecular complexes.

    PubMed

    Skiniotis, Georgios; Southworth, Daniel R

    2016-02-01

    Recent technological breakthroughs in image acquisition have enabled single-particle cryo-electron microscopy (cryo-EM) to achieve near-atomic resolution structural information for biological complexes. The improvements in image quality coupled with powerful computational methods for sorting distinct particle populations now also allow the determination of compositional and conformational ensembles, thereby providing key insights into macromolecular function. However, the inherent instability and dynamic nature of biological assemblies remain a tremendous challenge that often requires tailored approaches for successful implementation of the methodology. Here, we briefly describe the fundamentals of single-particle cryo-EM with an emphasis on covering the breadth of techniques and approaches, including low- and high-resolution methods, aiming to illustrate specific steps that are crucial for obtaining structural information by this method.

  2. Electron microscopy imaging of proteins on gallium phosphide semiconductor nanowires

    NASA Astrophysics Data System (ADS)

    Hjort, Martin; Bauer, Mikael; Gunnarsson, Stefan; Mårsell, Erik; Zakharov, Alexei A.; Karlsson, Gunnel; Sanfins, Elodie; Prinz, Christelle N.; Wallenberg, Reine; Cedervall, Tommy; Mikkelsen, Anders

    2016-02-01

    We have imaged GaP nanowires (NWs) incubated with human laminin, serum albumin (HSA), and blood plasma using both cryo-transmission electron microscopy and synchrotron based X-ray photoemission electron microscopy. This extensive imaging methodology simultaneously reveals structural, chemical and morphological details of individual nanowires and the adsorbed proteins. We found that the proteins bind to NWs, forming coronas with thicknesses close to the proteins' hydrodynamic diameters. We could directly image how laminin is extending from the NWs, maximizing the number of proteins bound to the NWs. NWs incubated with both laminin and HSA show protein coronas with a similar appearance to NWs incubated with laminin alone, indicating that the presence of HSA does not affect the laminin conformation on the NWs. In blood plasma, an intermediate sized corona around the NWs indicates a corona with a mixture of plasma proteins. The ability to directly visualize proteins on nanostructures in situ holds great promise for assessing the conformation and thickness of the protein corona, which is key to understanding and predicting the properties of engineered nanomaterials in a biological environment.We have imaged GaP nanowires (NWs) incubated with human laminin, serum albumin (HSA), and blood plasma using both cryo-transmission electron microscopy and synchrotron based X-ray photoemission electron microscopy. This extensive imaging methodology simultaneously reveals structural, chemical and morphological details of individual nanowires and the adsorbed proteins. We found that the proteins bind to NWs, forming coronas with thicknesses close to the proteins' hydrodynamic diameters. We could directly image how laminin is extending from the NWs, maximizing the number of proteins bound to the NWs. NWs incubated with both laminin and HSA show protein coronas with a similar appearance to NWs incubated with laminin alone, indicating that the presence of HSA does not affect the

  3. Quantitative characterization of electron detectors for transmission electron microscopy.

    PubMed

    Ruskin, Rachel S; Yu, Zhiheng; Grigorieff, Nikolaus

    2013-12-01

    A new generation of direct electron detectors for transmission electron microscopy (TEM) promises significant improvement over previous detectors in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE). However, the performance of these new detectors needs to be carefully monitored in order to optimize imaging conditions and check for degradation over time. We have developed an easy-to-use software tool, FindDQE, to measure MTF and DQE of electron detectors using images of a microscope's built-in beam stop. Using this software, we have determined the DQE curves of four direct electron detectors currently available: the Gatan K2 Summit, the FEI Falcon I and II, and the Direct Electron DE-12, under a variety of total dose and dose rate conditions. We have additionally measured the curves for the Gatan US4000 and TVIPS TemCam-F416 scintillator-based cameras. We compare the results from our new method with published curves. PMID:24189638

  4. Imaging Cytoskeleton Components by Electron Microscopy

    PubMed Central

    Svitkina, Tatyana

    2016-01-01

    The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers—actin filaments, microtubules, and intermediate filaments—are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell. PMID:26498781

  5. Scanning electron microscopy of rabbit corneal scars.

    PubMed

    Cintron, C; Szamier, R B; Hassinger, L C; Kublin, C L

    1982-07-01

    Central full-thickness perforating excision wounds were made in rabbit corneas and were examined by light and scanning electron microscopy at various times after wounding to study the three-dimensional morphologic changes in the tissue during healing and remodeling. Formation of a fibrin clot soon after wounding seals the hole and functions as a substrate for the healing epithelium. Changes in the histologic appearance of the fibrin lot immediately below the new epithelium are followed by migration of adjacent stromal cells under the epithelium, parallel to the basal surface of this tissue. Further healing is characterized by the organization of stromal fibroblasts into several layers parallel to the corneal surface and the deposition of collagen as a matted meshwork of fibrils tangential to the cell surface. Although remodeling of the collagenous matrix of corneal scar is evident and the scar eventually appears less opaque, the lamellae of the scar are narrower and shorter than normal. Evidence from this and other studies suggests that the orientation of the fibroblasts in healing tissues is determined by the organization of the newly formed epithelium. Furthermore, our observations are consistent with the hypothesis that collagen fibrils are deposited parallel to the flat surface of the fibroblasts during scar formation. Subsequent reorganization of this collagenous matrix approaches the normal lamellar appearance, but the matrix fails to regenerate even after 2 years.

  6. Imaging Cytoskeleton Components by Electron Microscopy

    PubMed Central

    Svitkina, Tatyana

    2010-01-01

    Summary The cytoskeleton is a complex of detergent-insoluble components of the cytoplasm playing critical roles in cell motility, shape generation, and mechanical properties of a cell. Fibrillar polymers-actin filaments, microtubules, and intermediate filaments- are major constituents of the cytoskeleton, which constantly change their organization during cellular activities. The actin cytoskeleton is especially polymorphic, as actin filaments can form multiple higher order assemblies performing different functions. Structural information about cytoskeleton organization is critical for understanding its functions and mechanisms underlying various forms of cellular activity. Because of the nanometer-scale thickness of cytoskeletal fibers, electron microscopy (EM) is a key tool to determine the structure of the cytoskeleton. This article describes application of rotary shadowing (or metal replica) EM for visualization of the cytoskeleton. The procedure is applicable to thin cultured cells growing on glass coverslips and consists of detergent extraction of cells to expose their cytoskeleton, chemical fixation to provide stability, ethanol dehydration and critical point drying to preserve three-dimensionality, rotary shadowing with platinum to create contrast, and carbon coating to stabilize replicas. This technique provides easily interpretable three-dimensional images, in which individual cytoskeletal fibers are clearly resolved, and individual proteins can be identified by immunogold labeling. More importantly, replica EM is easily compatible with live cell imaging, so that one can correlate the dynamics of a cell or its components, e.g., expressed fluorescent proteins, with high resolution structural organization of the cytoskeleton in the same cell. PMID:19768431

  7. Electron microscopy and theoretical modeling of cochleates.

    PubMed

    Nagarsekar, Kalpa; Ashtikar, Mukul; Thamm, Jana; Steiniger, Frank; Schacher, Felix; Fahr, Alfred; May, Sylvio

    2014-11-11

    Cochleates are self-assembled cylindrical condensates that consist of large rolled-up lipid bilayer sheets and represent a novel platform for oral and systemic delivery of therapeutically active medicinal agents. With few preceding investigations, the physical basis of cochleate formation has remained largely unexplored. We address the structure and stability of cochleates in a combined experimental/theoretical approach. Employing different electron microscopy methods, we provide evidence for cochleates consisting of phosphatidylserine and calcium to be hollow tubelike structures with a well-defined constant lamellar repeat distance and statistically varying inner and outer radii. To rationalize the relation between inner and outer radii, we propose a theoretical model. Based on the minimization of a phenomenological free energy expression containing a bending, adhesion, and frustration contribution, we predict the optimal tube dimensions of a cochleate and estimate ratios of material constants for cochleates consisting of phosphatidylserines with varied hydrocarbon chain structures. Knowing and understanding these ratios will ultimately benefit the successful formulation of cochleates for drug delivery applications.

  8. Biomechanics of DNA structures visualized by 4D electron microscopy

    PubMed Central

    Lorenz, Ulrich J.; Zewail, Ahmed H.

    2013-01-01

    We present a technique for in situ visualization of the biomechanics of DNA structural networks using 4D electron microscopy. Vibrational oscillations of the DNA structure are excited mechanically through a short burst of substrate vibrations triggered by a laser pulse. Subsequently, the motion is probed with electron pulses to observe the impulse response of the specimen in space and time. From the frequency and amplitude of the observed oscillations, we determine the normal modes and eigenfrequencies of the structures involved. Moreover, by selective “nano-cutting” at a given point in the network, it was possible to obtain Young’s modulus, and hence the stiffness, of the DNA filament at that position. This experimental approach enables nanoscale mechanics studies of macromolecules and should find applications in other domains of biological networks such as origamis. PMID:23382239

  9. Biological applications of confocal fluorescence polarization microscopy

    NASA Astrophysics Data System (ADS)

    Bigelow, Chad E.

    Fluorescence polarization microscopy is a powerful modality capable of sensing changes in the physical properties and local environment of fluorophores. In this thesis we present new applications for the technique in cancer diagnosis and treatment and explore the limits of the modality in scattering media. We describe modifications to our custom-built confocal fluorescence microscope that enable dual-color imaging, optical fiber-based confocal spectroscopy and fluorescence polarization imaging. Experiments are presented that indicate the performance of the instrument for all three modalities. The limits of confocal fluorescence polarization imaging in scattering media are explored and the microscope parameters necessary for accurate polarization images in this regime are determined. A Monte Carlo routine is developed to model the effect of scattering on images. Included in it are routines to track the polarization state of light using the Mueller-Stokes formalism and a model for fluorescence generation that includes sampling the excitation light polarization ellipse, Brownian motion of excited-state fluorophores in solution, and dipole fluorophore emission. Results from this model are compared to experiments performed on a fluorophore-embedded polymer rod in a turbid medium consisting of polystyrene microspheres in aqueous suspension. We demonstrate the utility of the fluorescence polarization imaging technique for removal of contaminating autofluorescence and for imaging photodynamic therapy drugs in cell monolayers. Images of cells expressing green fluorescent protein are extracted from contaminating fluorescein emission. The distribution of meta-tetrahydroxypheny1chlorin in an EMT6 cell monolayer is also presented. A new technique for imaging enzyme activity is presented that is based on observing changes in the anisotropy of fluorescently-labeled substrates. Proof-of-principle studies are performed in a model system consisting of fluorescently labeled bovine

  10. Direct imaging electron microscopy (EM) methods in modern structural biology: overview and comparison with X-ray crystallography and single-particle cryo-EM reconstruction in the studies of large macromolecules.

    PubMed

    Miyaguchi, Katsuyuki

    2014-10-01

    Determining the structure of macromolecules is important for understanding their function. The fine structure of large macromolecules is currently studied primarily by X-ray crystallography and single-particle cryo-electron microscopy (EM) reconstruction. Before the development of these techniques, macromolecular structure was often examined by negative-staining, rotary-shadowing and freeze-etching EM, which are categorised here as 'direct imaging EM methods'. In this review, the results are summarised by each of the above techniques and compared with respect to four macromolecules: the ryanodine receptor, cadherin, rhodopsin and the ribosome-translocon complex (RTC). The results of structural analysis of the ryanodine receptor and cadherin are consistent between each technique. The results obtained for rhodopsin vary to some extent within each technique and between the different techniques. Finally, the results for RTC are inconsistent between direct imaging EM and other analytical techniques, especially with respect to the space within RTC, the reasons for which are discussed. Then, the role of direct imaging EM methods in modern structural biology is discussed. Direct imaging methods should support and verify the results obtained by other analytical methods capable of solving three-dimensional molecular architecture, and they should still be used as a primary tool for studying macromolecule structure in vivo.

  11. Soft x-ray holography and microscopy of biological cells

    NASA Astrophysics Data System (ADS)

    Chen, Jianwen; Gao, Hongyi; Xie, Honglan; Li, Ruxin; Xu, Zhizhan

    2003-10-01

    Some experimental results on soft X-ray microscopy and holography imaging of biological specimens are presented in the paper. As we know, due to diffraction effects, there exists a resolution limit determined by wavelength λ and numerical aperture NA in conventional optical microscopy. In order to improve resolution, the num erical aperture should be made as large as possible and the wavelength as short as possible. Owing to the shorter wavelength, X-rays provide the potential of higher resolution in X-ray microscopy, holography image and allow for exam ination the interior structures of thicker specimens. In the experiments, we used synchrotron radiation source in Hefei as light source. Soft X-rays come from a bending magnet in 800 M eV electron storage ring with characteristic wavelength of 2.4 nm. The continuous X-ray spectrums are monochromatized by a zone-plate and a pinhole with 300 m diameter. The experimental set-up is typical contact microscopic system, its main advantage is simplicity and no special optical element is needed. The specimens used in the experiments of microscopic imaging are the colibacillus, the gingko vascular hundle and the fritillaries ovary karyon. The specimen for holographic imaging is the spider filam ents. The basic structures of plant cells such as the cell walls, the cytoplasm and the karyon especially the joint structures between the cells are observed clearly. An experimental study on a thick biological specimen that is a whole sporule w ith the thickness of about 30 μm is performed. In the holographic experiments, the experimental setup is typical Gabor in-line holography. The specimen is placed in line with X-ray source, which provides both the reference w aves and specimen illum ination. The specimen is some spider filament, which adhere to a Si3N4 film. The recording medium is PM M A, which is placed at recording distance of about 400 μm from the specimen. The hologram s were reconstructed by digital method with 300 nm

  12. Probing Structural and Electronic Dynamics with Ultrafast Electron Microscopy

    SciTech Connect

    Plemmons, DA; Suri, PK; Flannigan, DJ

    2015-05-12

    In this Perspective, we provide an overview,of the field of ultrafast electron microscopy (UEM). We begin by briefly discussing the emergence of methods for probing ultrafast structural dynamics and the information that can be obtained. Distinctions are drawn between the two main types a probes for femtosecond (fs) dynamics fast electrons and X-ray photons and emphasis is placed on hour the nature of charged particles is exploited in ultrafast electron-based' experiments:. Following this, we describe the versatility enabled by the ease with which electron trajectories and velocities can be manipulated with transmission electron microscopy (TEM): hardware configurations, and we emphasize how this is translated to the ability to measure scattering intensities in real, reciprocal, and energy space from presurveyed and selected rianoscale volumes. Owing to decades of ongoing research and development into TEM instrumentation combined with advances in specimen holder technology, comprehensive experiments can be conducted on a wide range of materials in various phases via in situ methods. Next, we describe the basic operating concepts, of UEM, and we emphasize that its development has led to extension of several of the formidable capabilities of TEM into the fs domain, dins increasing the accessible temporal parameter spade by several orders of magnitude. We then divide UEM studies into those conducted in real (imaging), reciprocal (diffraction), and energy (spectroscopy) spate. We begin each of these sections by providing a brief description of the basic operating principles and the types of information that can be gathered followed by descriptions of how these approaches are applied in UM, the type of specimen parameter space that can be probed, and an example of the types of dynamics that can be resolved. We conclude with an Outlook section, wherein we share our perspective on some future directions of the field pertaining to continued instrument development and

  13. Visualizing Macromolecular Complexes with In Situ Liquid Scanning Transmission Electron Microscopy

    SciTech Connect

    Evans, James E.; Jungjohann, K. L.; Wong, Peony C. K.; Chiu, Po-Lin; Dutrow, Gavin H.; Arslan, Ilke; Browning, Nigel D.

    2012-11-01

    A central focus of biological research is understanding the structure/function relationship of macromolecular protein complexes. Yet conventional transmission electron microscopy techniques are limited to static observations. Here we present the first direct images of purified macromolecular protein complexes using in situ liquid scanning transmission electron microscopy. Our results establish the capability of this technique for visualizing the interface between biology and nanotechnology with high fidelity while also probing the interactions of biomolecules within solution. This method represents an important advancement towards allowing future high-resolution observations of biological processes and conformational dynamics in real-time.

  14. Visualizing macromolecular complexes with in situ liquid scanning transmission electron microscopy.

    PubMed

    Evans, James E; Jungjohann, Katherine L; Wong, Peony C K; Chiu, Po-Lin; Dutrow, Gavin H; Arslan, Ilke; Browning, Nigel D

    2012-11-01

    A central focus of biological research is understanding the structure/function relationship of macromolecular protein complexes. Yet conventional transmission electron microscopy techniques are limited to static observations. Here we present the first direct images of purified macromolecular protein complexes using in situ liquid scanning transmission electron microscopy. Our results establish the capability of this technique for visualizing the interface between biology and nanotechnology with high fidelity while also probing the interactions of biomolecules within solution. This method represents an important advancement towards allowing future high-resolution observations of biological processes and conformational dynamics in real-time.

  15. Coherent Raman Scattering Microscopy in Biology and Medicine.

    PubMed

    Zhang, Chi; Zhang, Delong; Cheng, Ji-Xin

    2015-01-01

    Advancements in coherent Raman scattering (CRS) microscopy have enabled label-free visualization and analysis of functional, endogenous biomolecules in living systems. When compared with spontaneous Raman microscopy, a key advantage of CRS microscopy is the dramatic improvement in imaging speed, which gives rise to real-time vibrational imaging of live biological samples. Using molecular vibrational signatures, recently developed hyperspectral CRS microscopy has improved the readout of chemical information available from CRS images. In this article, we review recent achievements in CRS microscopy, focusing on the theory of the CRS signal-to-noise ratio, imaging speed, technical developments, and applications of CRS imaging in bioscience and clinical settings. In addition, we present possible future directions that the use of this technology may take. PMID:26514285

  16. Coherent Raman Scattering Microscopy in Biology and Medicine

    PubMed Central

    Zhang, Chi; Zhang, Delong; Cheng, Ji-Xin

    2016-01-01

    Advancements in coherent Raman scattering (CRS) microscopy have enabled label-free visualization and analysis of functional, endogenous biomolecules in living systems. When compared with spontaneous Raman microscopy, a key advantage of CRS microscopy is the dramatic improvement in imaging speed, which gives rise to real-time vibrational imaging of live biological samples. Using molecular vibrational signatures, recently developed hyperspectral CRS microscopy has improved the readout of chemical information available from CRS images. In this article, we review recent achievements in CRS microscopy, focusing on the theory of the CRS signal-to-noise ratio, imaging speed, technical developments, and applications of CRS imaging in bioscience and clinical settings. In addition, we present possible future directions that the use of this technology may take. PMID:26514285

  17. Surface Biology of DNA by Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Hansma, Helen G.

    2001-10-01

    The atomic force microscope operates on surfaces. Since surfaces occupy much of the space in living organisms, surface biology is a valid and valuable form of biology that has been difficult to investigate in the past owing to a lack of good technology. Atomic force microscopy (AFM) of DNA has been used to investigate DNA condensation for gene therapy, DNA mapping and sizing, and a few applications to cancer research and to nanotechnology. Some of the most exciting new applications for atomic force microscopy of DNA involve pulling on single DNA molecules to obtain measurements of single-molecule mechanics and thermodynamics.

  18. Ultrastructure of Candida albicans pleomorphic forms: phase-contrast microscopy, scanning and transmission electron microscopy.

    PubMed

    Staniszewska, Monika; Bondaryk, Małgorzata; Siennicka, Katarzyna; Kurzatkowski, Wiesław

    2012-01-01

    A modified method of glutaraldeyde-osmium tetroxide fixation was adjusted to characterize the ultrastructure of Candida albicans pleomorphic forms, using phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy. The discovered morphological criteria defining the individual morphotypes are discussed in terms of mycological and histopathological diagnostics of candidiasis. The relations are discussed between fungal pleomorphism, virulence and susceptibility of different morphotypes to fungicides.

  19. Light microscopy applications in systems biology: opportunities and challenges

    PubMed Central

    2013-01-01

    Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology. PMID:23578051

  20. Fully Hydrated Yeast Cells Imaged with Electron Microscopy

    PubMed Central

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

    2011-01-01

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

  1. Fully hydrated yeast cells imaged with electron microscopy.

    PubMed

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

    2011-05-18

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

  2. X-ray microscopy of live biological micro-organisms

    NASA Astrophysics Data System (ADS)

    Raja Al-Ani, Ma'an Nassar

    Real-time, compact x-ray microscopy has the potential to benefit many scientific fields, including microbiology, pharmacology, organic chemistry, and physics. Single frame x-ray micro-radiography, produced by a compact, solid-state laser plasma source, allows scientists to use x-ray emission for elemental analysis, and to observe biological specimens in their natural state. In this study, x-ray images of mouse kidney tissue, live bacteria, Pseudomonas aeruginosa and Burkholderia cepacia, and the bacteria's interaction with the antibiotic gentamicin, are examined using x-ray microscopy. For the purposes of comparing between confocal microscopy and x-ray microscopy, we introduced to our work the technique of gold labeling. Indirect immunofluorescence staining and immuno-gold labeling were applied on human lymphocytes and human tumor cells. Differential interference contrast microscopy (DIC) showed the lymphocyte body and nucleus, as did x-ray microscopy. However, the high resolution of x-ray microscopy allows us to differentiate between the gold particles bound to the antibodies and the free gold. A compact, tabletop Nd: glass laser is used in this study to produce x-rays from an Yttrium target. An atomic force microscope is used to scan the x-ray images from the developed photo-resist. The use of compact, tabletop laser plasma sources, in conjunction with x-ray microscopy, is a new technique that has great potential as a flexible, user-friendly scientific research tool.

  3. Collaborative Computational Project for Electron cryo-Microscopy

    SciTech Connect

    Wood, Chris; Burnley, Tom; Patwardhan, Ardan; Scheres, Sjors; Topf, Maya; Roseman, Alan; Winn, Martyn

    2015-01-01

    The Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM) is a new initiative for the structural biology community, following the success of CCP4 for macromolecular crystallography. Progress in supporting the users and developers of cryoEM software is reported. The Collaborative Computational Project for Electron cryo-Microscopy (CCP-EM) has recently been established. The aims of the project are threefold: to build a coherent cryoEM community which will provide support for individual scientists and will act as a focal point for liaising with other communities, to support practising scientists in their use of cryoEM software and finally to support software developers in producing and disseminating robust and user-friendly programs. The project is closely modelled on CCP4 for macromolecular crystallography, and areas of common interest such as model fitting, underlying software libraries and tools for building program packages are being exploited. Nevertheless, cryoEM includes a number of techniques covering a large range of resolutions and a distinct project is required. In this article, progress so far is reported and future plans are discussed.

  4. High-Contrast Observation of Unstained Proteins and Viruses by Scanning Electron Microscopy

    PubMed Central

    Ogura, Toshihiko

    2012-01-01

    Scanning electron microscopy (SEM) is an important tool for the nanometre-scale analysis of the various samples. Imaging of biological specimens can be difficult for two reasons: (1) Samples must often be left unstained to observe detail of the biological structures; however, lack of staining significantly decreases image contrast. (2) Samples are prone to serious radiation damage from electron beam. Herein we report a novel method for sample preparation involving placement on a new metal-coated insulator film. This method enables obtaining high-contrast images from unstained proteins and viruses by scanning electron microscopy with minimal electron radiation damage. These images are similar to those obtained by transmission electron microscopy. In addition, the method can be easily used to observe specimens of proteins, viruses and other organic samples by using SEM. PMID:23056522

  5. Value of electron microscopy in the diagnosis of glomerular diseases.

    PubMed

    Darouich, Sihem; Goucha, Rym Louzir; Jaafoura, Mohamed Habib; Moussa, Fatma Ben; Zekri, Semy; Maiz, Hédi Ben

    2010-04-01

    To evaluate the contribution of electron microscopy to the final diagnosis of glomerulopathies, the authors established a prospective study during the first semester of 2006. A total of 52 kidney biopsies were performed with 3 samples for light microscopy, immunofluorescence, and electron microscopy. Among these renal biopsies, only 20 were examined with electron microscopy because the diagnosis made on the basis of conventional methods had remained unclear or doubtful. In 18 cases, electron microscopy was undertaken for the investigation of primary kidney disease. The 2 remaining cases were transplant biopsies. In this series of 20 patients, there were 3 children with an average age of 9 years and 17 adults with an average age of 35.5 years. Fifteen patients (75%) were nephrotic. The study revealed that electron microscopy was essential for diagnosis in 8 cases (40%) and was helpful in 12 cases (60%). In conclusion, the results showed that the ultrastructural study provides essential or helpful information in many cases of glomerular diseases, and therefore electron microscopy should be considered an important tool of diagnostic renal pathology. As was recommended, it is important to reserve renal tissue for ultrastructural study unless electron microscopy can be routinely used in all biopsies. Thus, this technique could be performed wherever a renal biopsy has to be ultrastructurally evaluated.

  6. Quantitative Scanning Transmission Electron Microscopy of Electronic and Nanostructured Materials

    NASA Astrophysics Data System (ADS)

    Yankovich, Andrew B.

    Electronic and nanostructured materials have been investigated using advanced scanning transmission electron microscopy (STEM) techniques. The first topic is the microstructure of Ga and Sb-doped ZnO. Ga-doped ZnO is a candidate transparent conducting oxide material. The microstructure of GZO thin films grown by MBE under different growth conditions and different substrates were examined using various electron microscopy (EM) techniques. The microstructure, prevalent defects, and polarity in these films strongly depend on the growth conditions and substrate. Sb-doped ZnO nanowires have been shown to be the first route to stable p-type ZnO. Using Z-contrast STEM, I have showed that an unusual microstructure of Sb-decorated head-to-head inversion domain boundaries and internal voids contain all the Sb in the nanowires and cause the p-type conduction. InGaN thin films and InGaN / GaN quantum wells (QW) for light emitting diodes are the second topic. Low-dose Z-contrast STEM, PACBED, and EDS on InGaN QW LED structures grown by MOCVD show no evidence for nanoscale composition variations, contradicting previous reports. In addition, a new extended defect in GaN and InGaN was discovered. The defect consists of a faceted pyramid-shaped void that produces a threading dislocation along the [0001] growth direction, and is likely caused by carbon contamination during growth. Non-rigid registration (NRR) and high-precision STEM of nanoparticles is the final topic. NRR is a new image processing technique that corrects distortions arising from the serial nature of STEM acquisition that previously limited the precision of locating atomic columns and counting the number of atoms in images. NRR was used to demonstrate sub-picometer precision in STEM images of single crystal Si and GaN, the best achieved in EM. NRR was used to measure the atomic surface structure of Pt nanoacatalysts and Au nanoparticles, which revealed new bond length variation phenomenon of surface atoms. In

  7. Annular dark field transmission electron microscopy for protein structure determination.

    PubMed

    Koeck, Philip J B

    2016-02-01

    Recently annular dark field (ADF) transmission electron microscopy (TEM) has been advocated as a means of recording images of biological specimens with better signal to noise ratio (SNR) than regular bright field images. I investigate whether and how such images could be used to determine the three-dimensional structure of proteins given that an ADF aperture with a suitable pass-band can be manufactured and used in practice. I develop an approximate theory of ADF-TEM image formation for weak amplitude and phase objects and test this theory using computer simulations. I also test whether these simulated images can be used to calculate a three-dimensional model of the protein using standard software and discuss problems and possible ways to overcome these. PMID:26656466

  8. High Resolution Scanning Electron Microscopy of Cells Using Dielectrophoresis

    PubMed Central

    Tang, Shi-Yang; Zhang, Wei; Soffe, Rebecca; Nahavandi, Sofia; Shukla, Ravi; Khoshmanesh, Khashayar

    2014-01-01

    Ultrastructural analysis of cells can reveal valuable information about their morphological, physiological, and biochemical characteristics. Scanning electron microscopy (SEM) has been widely used to provide high-resolution images from the surface of biological samples. However, samples need to be dehydrated and coated with conductive materials for SEM imaging. Besides, immobilizing non-adherent cells during processing and analysis is challenging and requires complex fixation protocols. In this work, we developed a novel dielectrophoresis based microfluidic platform for interfacing non-adherent cells with high-resolution SEM at low vacuum mode. The system enables rapid immobilization and dehydration of samples without deposition of chemical residues over the cell surface. Moreover, it enables the on-chip chemical stimulation and fixation of immobilized cells with minimum dislodgement. These advantages were demonstrated for comparing the morphological changes of non-budding and budding yeast cells following Lyticase treatment. PMID:25089528

  9. Cryogenic transmission electron microscopy: aqueous suspensions of nanoscale objects.

    PubMed

    Burrows, Nathan D; Penn, R Lee

    2013-12-01

    Direct imaging of nanoscale objects suspended in liquid media can be accomplished using cryogenic transmission electron microscopy (cryo-TEM). Cryo-TEM has been used with particular success in microbiology and other biological fields. Samples are prepared by plunging a thin film of sample into an appropriate cryogen, which essentially produces a snapshot of the suspended objects in their liquid medium. With successful sample preparation, cryo-TEM images can facilitate elucidation of aggregation and self-assembly, as well as provide detailed information about cells and viruses. This work provides an explanation of sample preparation, detailed examples of the many artifacts found in cryo-TEM of aqueous samples, and other key considerations for successful cryo-TEM imaging.

  10. High voltage electron microscopy and low voltage scanning electron microscopy of human neoplastic cell culture.

    PubMed

    Malecki, M

    1991-01-01

    Improved procedures were developed to correlate cell culture data with the images provided by advanced ultrastructural technologies. These procedures were compatible with the two main types of cellular behavior: adherent, spreading (melanomas, rhabdomyosarcomas) and non-adherent in suspension (leukemias). The ultrastructure and function of spreading neoplastic cells primarily depend on surface properties of the attaching substrates. Therefore, the films used for cultured cell whole-mount ultrastructural analysis must have adherence features identical to those of standard cell culture vessels. Improved procedures were developed to produce the polystyrene films of required qualities. These films allowed processing of cells for electron microscopy including chemical fixation, cryo-immobilization, and immunolabelling. Furthermore, these polystyrene films permitted observations of the same cell in the high voltage electron microscope to reveal the internal organization and in the low voltage scanning electron microscope to reveal the surface topography. Neoplastic cells in suspension may dramatically change their ultrastructure as a result of interactions with substrates or other cells. Therefore, immobilization of cellular processes must occur rapidly while cells remain in suspension. These processes were cryo-immobilized by high pressure freezing through the use of the newly designed specimen carrier. Procedures allowing high yield attachment of cryo-fixed neoplastic cells to amino-propyl-derived glass carriers enabled observations of cell surface topography. Furthermore, freeze-substitution and drying of freeze-fractured cells revealed their three-dimensional internal organization in the low voltage scanning electron microscope. PMID:1822024

  11. Aberration-Coreected Electron Microscopy at Brookhaven National Laboratory

    SciTech Connect

    Zhu,Y.; Wall, J.

    2008-04-01

    The last decade witnessed the rapid development and implementation of aberration correction in electron optics, realizing a more-than-70-year-old dream of aberration-free electron microscopy with a spatial resolution below one angstrom [1-9]. With sophisticated aberration correctors, modern electron microscopes now can reveal local structural information unavailable with neutrons and x-rays, such as the local arrangement of atoms, order/disorder, electronic inhomogeneity, bonding states, spin configuration, quantum confinement, and symmetry breaking [10-17]. Aberration correction through multipole-based correctors, as well as the associated improved stability in accelerating voltage, lens supplies, and goniometers in electron microscopes now enables medium-voltage (200-300kV) microscopes to achieve image resolution at or below 0.1nm. Aberration correction not only improves the instrument's spatial resolution but, equally importantly, allows larger objective lens pole-piece gaps to be employed thus realizing the potential of the instrument as a nanoscale property-measurement tool. That is, while retaining high spatial resolution, we can use various sample stages to observe the materials response under various temperature, electric- and magnetic- fields, and atmospheric environments. Such capabilities afford tremendous opportunities to tackle challenging science and technology issues in physics, chemistry, materials science, and biology. The research goal of the electron microscopy group at the Dept. of Condensed Matter Physics and Materials Science and the Center for Functional Nanomaterials, as well as the Institute for Advanced Electron Microscopy, Brookhaven National Laboratory (BNL), is to elucidate the microscopic origin of the physical- and chemical-behavior of materials, and the role of individual, or groups of atoms, especially in their native functional environments. We plan to accomplish this by developing and implementing various quantitative electron

  12. Cryomesh: a new substrate for cryo-electron microscopy.

    PubMed

    Yoshioka, Craig; Carragher, Bridget; Potter, Clinton S

    2010-02-01

    Here we evaluate a new grid substrate developed by ProtoChips Inc. (Raleigh, NC) for cryo-transmission electron microscopy. The new grids are fabricated from doped silicon carbide using processes adapted from the semiconductor industry. A major motivating purpose in the development of these grids was to increase the low-temperature conductivity of the substrate, a characteristic that is thought to affect the appearance of beam-induced movement (BIM) in transmission electron microscope (TEM) images of biological specimens. BIM degrades the quality of data and is especially severe when frozen biological specimens are tilted in the microscope. Our results show that this new substrate does indeed have a significant impact on reducing the appearance and severity of beam-induced movement in TEM images of tilted cryo-preserved samples. Furthermore, while we have not been able to ascertain the exact causes underlying the BIM phenomenon, we have evidence that the rigidity and flatness of these grids may play a major role in its reduction. This improvement in the reliability of imaging at tilt has a significant impact on using data collection methods such as random conical tilt or orthogonal tilt reconstruction with cryo-preserved samples. Reduction in BIM also has the potential for improving the resolution of three-dimensional cryo-reconstructions in general.

  13. Near-infrared branding efficiently correlates light and electron microscopy.

    PubMed

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

    2011-06-05

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

  14. Ion-induced electron emission microscopy

    DOEpatents

    Doyle, Barney L.; Vizkelethy, Gyorgy; Weller, Robert A.

    2001-01-01

    An ion beam analysis system that creates multidimensional maps of the effects of high energy ions from an unfocussed source upon a sample by correlating the exact entry point of an ion into a sample by projection imaging of the secondary electrons emitted at that point with a signal from a detector that measures the interaction of that ion within the sample. The emitted secondary electrons are collected in a strong electric field perpendicular to the sample surface and (optionally) projected and refocused by the electron lenses found in a photon emission electron microscope, amplified by microchannel plates and then their exact position is sensed by a very sensitive X Y position detector. Position signals from this secondary electron detector are then correlated in time with nuclear, atomic or electrical effects, including the malfunction of digital circuits, detected within the sample that were caused by the individual ion that created these secondary electrons in the fit place.

  15. Image Resolution in Scanning Transmission Electron Microscopy

    SciTech Connect

    Pennycook, S. J.; Lupini, A.R.

    2008-06-26

    Digital images captured with electron microscopes are corrupted by two fundamental effects: shot noise resulting from electron counting statistics and blur resulting from the nonzero width of the focused electron beam. The generic problem of computationally undoing these effects is called image reconstruction and for decades has proved to be one of the most challenging and important problems in imaging science. This proposal concerned the application of the Pixon method, the highest-performance image-reconstruction algorithm yet devised, to the enhancement of images obtained from the highest-resolution electron microscopes in the world, now in operation at Oak Ridge National Laboratory.

  16. Cryo-scanning electron microscopy and light microscopy for the study of fungi interactions.

    PubMed

    Sempere, F; Santamarina, M P

    2011-03-01

    The application of the cryo-scanning electron microscopy and light microscopy for the study of the interactions at different environmental conditions between Penicillium oxalicum and Fusarium verticillioides is described. A dual microculture was developed for the light microscopy analysis of the interaction. The microscope and macroscopic examinations were compared. Analysis of Petri plates revealed that F. verticillioides was a competitor for space and nutrients while P. oxalicum was a mycoparasite under the microscopic observations.

  17. Phase contrast in high resolution electron microscopy

    DOEpatents

    Rose, H.H.

    1975-09-23

    This patent relates to a device for developing a phase contrast signal for a scanning transmission electron microscope. The lens system of the microscope is operated in a condition of defocus so that predictable alternate concentric regions of high and low electron density exist in the cone of illumination. Two phase detectors are placed beneath the object inside the cone of illumination, with the first detector having the form of a zone plate, each of its rings covering alternate regions of either higher or lower electron density. The second detector is so configured that it covers the regions of electron density not covered by the first detector. Each detector measures the number of electrons incident thereon and the signal developed by the first detector is subtracted from the signal developed by the record detector to provide a phase contrast signal. (auth)

  18. Gold nanocluster formation using metallothionein: mass spectrometry and electron microscopy.

    PubMed

    Mercogliano, Christopher P; DeRosier, David J

    2006-01-13

    Clonable contrasting agents for light microscopy, such as green fluorescent protein, have revolutionized biology, but few such agents have been developed for transmission electron microscopy (TEM). As an attempt to develop a novel clonable contrasting agent for TEM, we have evaluated metallothionein, a small metal-binding protein, reacted with aurothiomalate, an anti-arthritic gold compound. Electro spray ionization and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry measurements show a distribution of gold atoms bound to individual metallothionein molecules. Unlike previous reports, these data show gold binding occurred as the addition of single atoms without retention of additional ligands. Moreover, under certain conditions, MALDI spectra show gold binding ratios of greater than 1:1 with the cysteine residues of metallothionein. Together, this may hint at a gold-binding mechanism similar to gold nanocluster formation. Finally, metallothionein-gold complexes visualized in the TEM show a range of sizes similar to those used as current TEM labels, and show the potential of the protein as a clonable TEM label in which the gold cluster is grown on the label, thereby circumventing the problems associated with attaching gold clusters.

  19. Scanning transmission electron microscopy methods for the analysis of nanoparticles.

    PubMed

    Ponce, Arturo; Mejía-Rosales, Sergio; José-Yacamán, Miguel

    2012-01-01

    Here we review the scanning transmission electron microscopy (STEM) characterization technique and STEM imaging methods. We describe applications of STEM for studying inorganic nanoparticles, and other uses of STEM in biological and health sciences and discuss how to interpret STEM results. The STEM imaging mode has certain benefits compared with the broad-beam illumination mode; the main advantage is the collection of the information about the specimen using a high angular annular dark field (HAADF) detector, in which the images registered have different levels of contrast related to the chemical composition of the sample. Another advantage of its use in the analysis of biological samples is its contrast for thick stained sections, since HAADF images of samples with thickness of 100-120 nm have notoriously better contrast than those obtained by other techniques. Combining the HAADF-STEM imaging with the new aberration correction era, the STEM technique reaches a direct way to imaging the atomistic structure and composition of nanostructures at a sub-angstrom resolution. Thus, alloying in metallic nanoparticles is directly resolved at atomic scale by the HAADF-STEM imaging, and the comparison of the STEM images with results from simulations gives a very powerful way of analysis of structure and composition. The use of X-ray energy dispersive spectroscopy attached to the electron microscope for STEM mode is also described. In issues where characterization at the atomic scale of the interaction between metallic nanoparticles and biological systems is needed, all the associated techniques to STEM become powerful tools for the best understanding on how to use these particles in biomedical applications. PMID:22791456

  20. Scanning transmission electron microscopy methods for the analysis of nanoparticles.

    PubMed

    Ponce, Arturo; Mejía-Rosales, Sergio; José-Yacamán, Miguel

    2012-01-01

    Here we review the scanning transmission electron microscopy (STEM) characterization technique and STEM imaging methods. We describe applications of STEM for studying inorganic nanoparticles, and other uses of STEM in biological and health sciences and discuss how to interpret STEM results. The STEM imaging mode has certain benefits compared with the broad-beam illumination mode; the main advantage is the collection of the information about the specimen using a high angular annular dark field (HAADF) detector, in which the images registered have different levels of contrast related to the chemical composition of the sample. Another advantage of its use in the analysis of biological samples is its contrast for thick stained sections, since HAADF images of samples with thickness of 100-120 nm have notoriously better contrast than those obtained by other techniques. Combining the HAADF-STEM imaging with the new aberration correction era, the STEM technique reaches a direct way to imaging the atomistic structure and composition of nanostructures at a sub-angstrom resolution. Thus, alloying in metallic nanoparticles is directly resolved at atomic scale by the HAADF-STEM imaging, and the comparison of the STEM images with results from simulations gives a very powerful way of analysis of structure and composition. The use of X-ray energy dispersive spectroscopy attached to the electron microscope for STEM mode is also described. In issues where characterization at the atomic scale of the interaction between metallic nanoparticles and biological systems is needed, all the associated techniques to STEM become powerful tools for the best understanding on how to use these particles in biomedical applications.

  1. Invited review article: Advanced light microscopy for biological space research.

    PubMed

    De Vos, Winnok H; Beghuin, Didier; Schwarz, Christian J; Jones, David B; van Loon, Jack J W A; Bereiter-Hahn, Juergen; Stelzer, Ernst H K

    2014-10-01

    As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy. PMID:25362364

  2. Invited Review Article: Advanced light microscopy for biological space research

    SciTech Connect

    De Vos, Winnok H.; Beghuin, Didier; Schwarz, Christian J.; Jones, David B.; Loon, Jack J. W. A. van

    2014-10-15

    As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

  3. Invited review article: Advanced light microscopy for biological space research.

    PubMed

    De Vos, Winnok H; Beghuin, Didier; Schwarz, Christian J; Jones, David B; van Loon, Jack J W A; Bereiter-Hahn, Juergen; Stelzer, Ernst H K

    2014-10-01

    As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

  4. Invited Review Article: Advanced light microscopy for biological space research

    NASA Astrophysics Data System (ADS)

    De Vos, Winnok H.; Beghuin, Didier; Schwarz, Christian J.; Jones, David B.; van Loon, Jack J. W. A.; Bereiter-Hahn, Juergen; Stelzer, Ernst H. K.

    2014-10-01

    As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

  5. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

    PubMed

    Faas, F G A; Bárcena, M; Agronskaia, A V; Gerritsen, H C; Moscicka, K B; Diebolder, C A; van Driel, L F; Limpens, R W A L; Bos, E; Ravelli, R B G; Koning, R I; Koster, A J

    2013-03-01

    Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells. PMID:23261400

  6. Transmission electron microscopy: Visualizing fullerene chemistry

    NASA Astrophysics Data System (ADS)

    Terrones, Mauricio

    2010-02-01

    Chemical reactions of fullerenes and metallofullerenes lined up inside single-walled carbon nanotubes can be monitored at the atomic scale inside an aberration-corrected transmission electron microscope.

  7. Structure of Wet Specimens in Electron Microscopy

    ERIC Educational Resources Information Center

    Parsons, D. F.

    1974-01-01

    Discussed are past work and recent advances in the use of electron microscopes for viewing structures immersed in gas and liquid. Improved environmental chambers make it possible to examine wet specimens easily. (Author/RH)

  8. Nanowire growth kinetics in aberration corrected environmental transmission electron microscopy

    DOE PAGES

    Chou, Yi -Chia; Panciera, Federico; Reuter, Mark C.; Stach, Eric A.; Ross, Frances M.

    2016-03-15

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

  9. 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

    2015-02-01

    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.

  10. Photoemission electron microscopy and scanning electron microscopy of Magnetospirillum magnetotacticum’s magnetosome chains

    SciTech Connect

    Keutner, Christoph; von Bohlen, Alex; Berges, Ulf; Espeter, Philipp; Schneider, Claus M.; Westphal, Carsten

    2014-10-07

    Magnetotactic bacteria are of great interdisciplinary interest, since a vast field of applications from magnetic recording media to medical nanorobots is conceivable. A key feature for a further understanding is the detailed knowledge about the magnetosome chain within the bacteria. We report on two preparation procedures suitable for UHV experiments in reflective geometry. Further, we present the results of scanning electron microscopy, as well as the first photoemission electron microscopy experiments, both accessing the magnetosomes within intact magnetotactic bacteria and compare these to scanning electron microscopy data from the literature. From the images, we can clearly identify individual magnetosomes within their chains.

  11. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy.

    PubMed Central

    Xu, C; Zipfel, W; Shear, J B; Williams, R M; Webb, W W

    1996-01-01

    Intrinsic, three-dimensionally resolved, microscopic imaging of dynamical structures and biochemical processes in living preparations has been realized by nonlinear laser scanning fluorescence microscopy. The search for useful two-photon and three-photon excitation spectra, motivated by the emergence of nonlinear microscopy as a powerful biophysical instrument, has now discovered a virtual artist's palette of chemical indicators, fluorescent markers, and native biological fluorophores, including NADH, flavins, and green fluorescent proteins, that are applicable to living biological preparations. More than 25 two-photon excitation spectra of ultraviolet and visible absorbing molecules reveal useful cross sections, some conveniently blue-shifted, for near-infrared absorption. Measurements of three-photon fluorophore excitation spectra now define alternative windows at relatively benign wavelengths to excite deeper ultraviolet fluorophores. The inherent optical sectioning capability of nonlinear excitation provides three-dimensional resolution for imaging and avoids out-of-focus background and photodamage. Here, the measured nonlinear excitation spectra and their photophysical characteristics that empower nonlinear laser microscopy for biological imaging are described. Images Fig. 1 Fig. 5 Fig. 7 PMID:8855254

  12. Atmospheric pressure scanning transmission electron microscopy.

    PubMed

    de Jonge, Niels; Bigelow, Wilbur C; Veith, Gabriel M

    2010-03-10

    Scanning transmission electron microscope (STEM) images of gold nanoparticles at atmospheric pressure have been recorded through a 0.36 mm thick mixture of CO, O2, and He. This was accomplished using a reaction cell consisting of two electron-transparent silicon nitride membranes. Gold nanoparticles of a full width at half-maximum diameter of 1.0 nm were visible above the background noise, and the achieved edge resolution was 0.4 nm in accordance with calculations of the beam broadening.

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

    PubMed

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

    2015-12-01

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

  14. Cellulose Acetate Membranes: Electron Microscopy of Structure.

    PubMed

    Riley, R; Gardner, J O; Merten, U

    1964-02-21

    Electron photomicrographs of cellulose acetate membranes used in the reverse osmosis processof water desalination reveal a dense surface layer with a porous substructure. The high rate oftransmission for water can be correlated with the thickness of the dense layer on the air-driedsurface of the membrane.

  15. Microstructure of Mixed Surfactant Solutions by Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Naranjo, Edward

    1995-01-01

    Surfactant mixtures add a new dimension to the design of complex fluid microstructure. By combining different surfactants it is not only possible to modify aggregate morphology and control the macrascopic properties of colloidal dispersions but also to produce a variety of novel synergistic phases. Mixed systems produce new microstructures by altering the intermolecular and interaggregate forces in ways impossible for single component systems. In this dissertation, we report on the phase behavior and microstructure of several synthetic and biological surfactant mixtures as elucidated by freeze-fracture and cryo-transmission electron microscopy. We have discovered that stable, spontaneous unilamellar vesicles can be prepared from aqueous mixtures of commercially available single-tailed cationic and anionic surfactants. Vesicle stability is determined by the length and volume of the hydrocarbon chains of the "catanionic" pairs. Mixtures containing bulky or branched surfactant pairs (C _{16}/C_{12 -14}) in water produce defect-free fairly monodisperse equilibrium vesicles at high dilution. In contrast, mixtures of catanionic surfactants with highly asymmetric tails (C_{16}/C_8 ) form phases of porous vesicles, dilute lamellar L_{alpha}, and anomalous isotropic L_3 phases. Images of the microstructure by freeze-fracture microscopy show that the L_3 phase consists of multiconnected self-avoiding bilayers with saddle shaped curvature. The forces between bilayers of vesicle-forming cationic and anionic surfactant mixtures were also measured using the Surface Force Apparatus (SFA). We find that the vesicles are stabilized by a long range electrostatic repulsion at large separations (>20 A) and an additional salt-independent repulsive force below 20 A. The measured forces correlate very well with the ternary phase diagram and the vesicle microstructures observed by electron microscopy. In addition to studying ionic surfactants, we have also done original work with

  16. 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.

  17. Quantitative analytical electron microscopy of multiphase alloys.

    PubMed

    Prybylowski, J; Ballinger, R; Elliott, C

    1989-02-01

    In this paper, we present a technique for analysis of composition gradients, using an analytical electron microscope, within the primary phase of a two-phase alloy for the case where the second-phase particle size is similar to the size of the irradiated volume. If the composition difference between the two phases is large, the detected compositional fluctuations associated with varying phase fractions may mask any underlying composition gradient of the primary phase. The analysis technique was used to determine grain boundary chromium concentration gradients in a nickel-base superalloy, alloy X-750. The technique may also be of use in other alloy systems. PMID:2709131

  18. Biological imaging by soft x-ray diffraction microscopy

    SciTech Connect

    Shapiro, D.; Thibault, P.; Beetz, T.; Elser, V.; Howells, M.; Jacobsen, C.; Kirz, J.; Lima, E.; Miao, H.; Neiman, A. M.; Sayre, D.

    2005-10-25

    We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffraction microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.

  19. Biological imaging by soft x-ray diffraction microscopy

    DOE PAGES

    Shapiro, D.; Thibault, P.; Beetz, T.; Elser, V.; Howells, M.; Jacobsen, C.; Kirz, J.; Lima, E.; Miao, H.; Neiman, A. M.; et al

    2005-10-25

    We have used the method of x-ray diffraction microscopy to image the complex-valued exit wave of an intact and unstained yeast cell. The images of the freeze-dried cell, obtained by using 750-eV x-rays from different angular orientations, portray several of the cell's major internal components to 30-nm resolution. The good agreement among the independently recovered structures demonstrates the accuracy of the imaging technique. To obtain the best possible reconstructions, we have implemented procedures for handling noisy and incomplete diffraction data, and we propose a method for determining the reconstructed resolution. This work represents a previously uncharacterized application of x-ray diffractionmore » microscopy to a specimen of this complexity and provides confidence in the feasibility of the ultimate goal of imaging biological specimens at 10-nm resolution in three dimensions.« less

  20. A practical guide to evaluating colocalization in biological microscopy

    PubMed Central

    Kamocka, Malgorzata M.; McDonald, John H.

    2011-01-01

    Fluorescence microscopy is one of the most powerful tools for elucidating the cellular functions of proteins and other molecules. In many cases, the function of a molecule can be inferred from its association with specific intracellular compartments or molecular complexes, which is typically determined by comparing the distribution of a fluorescently labeled version of the molecule with that of a second, complementarily labeled probe. Although arguably the most common application of fluorescence microscopy in biomedical research, studies evaluating the “colocalization” of two probes are seldom quantified, despite a diversity of image analysis tools that have been specifically developed for that purpose. Here we provide a guide to analyzing colocalization in cell biology studies, emphasizing practical application of quantitative tools that are now widely available in commercial and free image analysis software. PMID:21209361

  1. Collective electronic effects in scanning probe microscopy

    NASA Astrophysics Data System (ADS)

    Passian, Ali

    The surface plasmon dispersion relations are calculated for a metal coated dielectric probe above a dielectric half space with and without metal coating. Employing prolate spheroidal coordinate system this configuration was modeled as confocal single-sheeted hyperboloids of revolution superimposed on planar domains. The involved media are characterized by frequency dependent, spatially local dielectric functions. Due to subwavelength dimensions of the region of interest, nonretarded electrodynamics is utilized to derive exact analytical expressions describing the resonant surface modes. The dispersion relations are studied as functions of the parameter that defines the hyperboloidal boundaries of the tip and the corresponding coating, and as functions of the involved coating thicknesses. Both parallel and perpendicular polarizations are considered. The results are simulated numerically and limiting cases are discussed with comparison to the Cartesian thin foil case. Using this new type of probe-substrate configuration, the surface plasmon coupling mechanism is investigated experimentally utilizing a scanning probe microscope, and the signal strength acquired by the probe is measured as a function of the distance between the probe and the sample. This is repeated at three different wavelengths of the incident p-polarized photons used to stimulate surface plasmons in the thin metal foil. The results are compared with the theory. Utilizing the prolate spheroidal coordinate system, the related and relevant problem of the Coulomb interaction of a dielectric probe tip with a uniform field existing above a semiinfinite, homogeneous dielectric substrate was studied. This is of interest in atomic force microscopy when the sample surface is electrically charged. The induced polarization surface charge density and the field distribution at the bounding surface of the dielectric medium with the geometry of a single-sheeted hyperboloid of revolution located above the dielectric

  2. Sad State of Phage Electron Microscopy. Please Shoot the Messenger

    PubMed Central

    Ackermann, Hans-W.

    2013-01-01

    Two hundred and sixty publications from 2007 to 2012 were classified according to the quality of electron micrographs; namely as good (71); mediocre (21); or poor (168). Publications were from 37 countries; appeared in 77 journals; and included micrographs produced with about 60 models of electron microscopes. The quality of the micrographs was not linked to any country; journal; or electron microscope. Main problems were poor contrast; positive staining; low magnification; and small image size. Unsharp images were frequent. Many phage descriptions were silent on virus purification; magnification control; even the type of electron microscope and stain used. The deterioration in phage electron microscopy can be attributed to the absence of working instructions and electron microscopy courses; incompetent authors and reviewers; and lenient journals. All these factors are able to cause a gradual lowering of standards.

  3. Scanning electron microscopy of cells and tissues under fully hydrated conditions.

    PubMed

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

    2004-03-01

    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 approximately 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.

  4. Chemical Force Microscopy of Chemical and Biological Interactions

    SciTech Connect

    Noy, A

    2006-01-02

    Interactions between chemical functionalities define outcomes of the vast majority of important events in chemistry, biology and materials science. Chemical Force Microscopy (CFM)--a technique that uses direct chemical functionalization of AFM probes with specific functionalities--allows researchers to investigate these important interactions directly. We review the basic principles of CFM, some examples of its application, and theoretical models that provide the basis for understanding the experimental results. We also emphasize application of modern kinetic theory of non-covalent interactions strength to the analysis of CFM data.

  5. Cryogenic X-ray Diffraction Microscopy for Biological Samples

    SciTech Connect

    E Lima; L Wiegart; P Pernot; M Howells; J Timmins; F Zontone; A Madsen

    2011-12-31

    X-ray diffraction microscopy (XDM) is well suited for nondestructive, high-resolution biological imaging, especially for thick samples, with the high penetration power of x rays and without limitations imposed by a lens. We developed nonvacuum, cryogenic (cryo-) XDM with hard x rays at 8 keV and report the first frozen-hydrated imaging by XDM. By preserving samples in amorphous ice, the risk of artifacts associated with dehydration or chemical fixation is avoided, ensuring the imaging condition closest to their natural state. The reconstruction shows internal structures of intact D. radiodurans bacteria in their natural contrast.

  6. Transmission Electron Microscopy of Itokawa Regolith Grains

    NASA Technical Reports Server (NTRS)

    Keller, Lindsay P.; Berger, E. L.

    2013-01-01

    Introduction: In a remarkable engineering achievement, the JAXA space agency successfully recovered the Hayabusa space-craft in June 2010, following a non-optimal encounter and sur-face sampling mission to asteroid 25143 Itokawa. These are the first direct samples ever obtained and returned from the surface of an asteroid. The Hayabusa samples thus present a special op-portunity to directly investigate the evolution of asteroidal sur-faces, from the development of the regolith to the study of the effects of space weathering. Here we report on our preliminary TEM measurements on two Itokawa samples. Methods: We were allocated particles RA-QD02-0125 and RA-QD02-0211. Both particles were embedded in low viscosity epoxy and thin sections were prepared using ultramicrotomy. High resolution images and electron diffraction data were ob-tained using a JEOL 2500SE 200 kV field-emission scanning-transmission electron microscope. Quantitative maps and anal-yses were obtained using a Thermo thin-window energy-dispersive x-ray (EDX) spectrometer. Results: Both particles are olivine-rich (Fo70) with µm-sized inclusions of FeS and have microstructurally complex rims. Par-ticle RA-QD02-0125 is rounded and has numerous sub-µm grains attached to its surface including FeS, albite, olivine, and rare melt droplets. Solar flare tracks have not been observed, but the particle is surrounded by a continuous 50 nm thick, stuctur-ally disordered rim that is compositionally similar to the core of the grain. One of the surface adhering grains is pyrrhotite show-ing a S-depleted rim (8-10 nm thick) with nanophase Fe metal grains (<5 nm) decorating the outermost surface. The pyrrhotite displays a complex superstructure in its core that is absent in the S-depleted rim. Particle RA-QD02-0211 contains solar flare particle tracks (2x109 cm-2) and shows a structurally disordered rim 100 nm thick. The track density corresponds to a surface exposure of 103-104 years based on the track production rate

  7. Atomic resolution imaging of graphene by transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Robertson, Alex W.; Warner, Jamie H.

    2013-05-01

    The atomic structure of a material influences its electronic, chemical, magnetic and mechanical properties. Characterising carbon nanomaterials, such as fullerenes, nanotubes and graphene, at the atomic level is challenging due to their chemical reactivity and low atomic mass. Transmission electron microscopy and scanning probe microscopy are two of the leading methods for imaging graphene at the atomic level. Here, we report on recent advances in atomic resolution imaging of graphene using aberration-corrected high resolution transmission electron microscopy and how it has revealed many of the structural deviations from the pristine monolayer form. Structures in graphene such as vacancy defects, edges, grain boundaries, linear chains, impurity dopants, layer number, layer stacking and bond rotations are explored.

  8. Imaging hydrated microbial extracellular polymers: Comparative analysis by electron microscopy

    SciTech Connect

    Dohnalkova, A.C.; Marshall, M. J.; Arey, B. W.; Williams, K. H.; Buck, E. C.; Fredrickson, J. K.

    2011-01-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigating microscale associations. Electron microscopy has been used extensively for geomicrobial investigations and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions using conventional electron microscopy approaches of imaging at room temperature and a suite of cryogenic electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of the hydrated bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in their collapse into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding nature of interactions between microbial extracellular polymers and their environment.

  9. Imaging Hydrated Microbial Extracellular Polymers: Comparative Analysis by Electron Microscopy

    SciTech Connect

    Dohnalkova, Alice; Marshall, Matthew J.; Arey, Bruce W.; Williams, Kenneth H.; Buck, Edgar C.; Fredrickson, Jim K.

    2011-02-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigating microscale associations. Electron microscopy has been used extensively for geomicrobial investigations and although used bona fide, the traditional methods of sample preparation do not preserve the native morphology of microbiological components, especially extracellular polymers. Herein, we present a direct comparative analysis of microbial interactions using conventional electron microscopy approaches of imaging at room temperature and a suite of cryo-electron microscopy methods providing imaging in the close-to-natural hydrated state. In situ, we observed an irreversible transformation of bacterial extracellular polymers during the traditional dehydration-based sample preparation that resulted in the collapse of hydrated gel-like EPS into filamentous structures. Dehydration-induced polymer collapse can lead to inaccurate spatial relationships and hence could subsequently affect conclusions regarding nature of interactions between microbial extracellular polymers and their environment.

  10. Laboratory design for high-performance electron microscopy

    SciTech Connect

    O'Keefe, Michael A.; Turner, John H.; Hetherington, Crispin J.D.; Cullis, A.G.; Carragher, Bridget; Jenkins, Ron; Milgrim, Julie; Milligan,Ronald A.; Potter, Clinton S.; Allard, Lawrence F.; Blom, Douglas A.; Degenhardt, Lynn; Sides, William H.

    2004-04-23

    Proliferation of electron microscopes with field emission guns, imaging filters and hardware spherical aberration correctors (giving higher spatial and energy resolution) has resulted in the need to construct special laboratories. As resolutions improve, transmission electron microscopes (TEMs) and scanning transmission electron microscopes (STEMs) become more sensitive to ambient conditions. State-of-the-art electron microscopes require state-of-the-art environments, and this means careful design and implementation of microscope sites, from the microscope room to the building that surrounds it. Laboratories have been constructed to house high-sensitive instruments with resolutions ranging down to sub-Angstrom levels; we present the various design philosophies used for some of these laboratories and our experiences with them. Four facilities are described: the National Center for Electron Microscopy OAM Laboratory at LBNL; the FEGTEM Facility at the University of Sheffield; the Center for Integrative Molecular Biosciences at TSRI; and the Advanced Microscopy Laboratory at ORNL.

  11. Microscopy with slow electrons: from LEEM to XPEEM

    ScienceCinema

    Bauer, Ernst [Arizona State University, Phoenix, Arizona, United States

    2016-07-12

    The short penetration and escape depth of electrons with energies below 1 keV make them ideally suited for the study of surfaces and ultrathin films. The combination of the low energy electrons and the high lateral resolution of a microscope produces a powerful method for the characterization of nanostructures on bulk samples, in particular if the microscope is equipped with an imaging energy filter and connected to a synchrotron radiation source. Comprehensive characterization by imaging, diffraction, and spectroscope of the structural, chemical, and magnetic properties is then possible. The Talk will describe the various imaging techniques in using reflected and emitted electrons in low-energy electron microscopy (LEEM) and x-ray photoemission electron microscopy (XPEEM), with an emphasis on magnetic materials with spin-polarized LEEM and x-ray magnetic circular dichroism PEEM. The talk with end with an outlook on future possibilities.

  12. Extended focus Fourier domain optical coherence microscopy assists developmental biology

    NASA Astrophysics Data System (ADS)

    Villiger, Martin L.; Beleut, Manfred; Brisken, Cathrin; Lasser, Theo; Leitgeb, Rainer A.

    2007-07-01

    We present a novel detection scheme for Fourier domain optical coherence microscopy (FDOCM). A Bessel-like interference pattern with a strong central lobe was created with an axicon lens. This pattern was then imaged by a telescopic system into the sample space to obtain a laterally highly confined illumination needle, extending over a long axial range. For increased efficiency, the detection occurs decoupled from the illumination, avoiding a double pass through the axicon. Nearly constant transverse resolution of ~1.5μm along a focal range of 200μm with a maximum sensitivity of 105dB was obtained. A broad bandwidth Ti:Sapphire laser allowed for an axial resolution of 3μm in air, providing the nearly isotropic resolution necessary to access the microstructure of biological tissues. Together with the speed- and sensitivity-advantage of FDOCT, this system can perform in vivo measurements in a minimally invasive way. Tomograms of the mouse mammary gland and the mouse follicle, recorded in vitro, revealed biologically relevant structural details. Images acquired with classical microscopy techniques, involving stained and fluorescent samples, validate these structures and emphasize the high contrast of the tomograms. It is comparable to the contrast achieved with classical techniques, but employing neither staining, labeling nor slicing of the samples, stressing the high potential of FDOCM for minimally invasive in vivo small animal imaging.

  13. Exploring lipids with nonlinear optical microscopy in multiple biological systems

    NASA Astrophysics Data System (ADS)

    Alfonso-Garcia, Alba

    Lipids are crucial biomolecules for the well being of humans. Altered lipid metabolism may give rise to a variety of diseases that affect organs from the cardiovascular to the central nervous system. A deeper understanding of lipid metabolic processes would spur medical research towards developing precise diagnostic tools, treatment methods, and preventive strategies for reducing the impact of lipid diseases. Lipid visualization remains a complex task because of the perturbative effect exerted by traditional biochemical assays and most fluorescence markers. Coherent Raman scattering (CRS) microscopy enables interrogation of biological samples with minimum disturbance, and is particularly well suited for label-free visualization of lipids, providing chemical specificity without compromising on spatial resolution. Hyperspectral imaging yields large datasets that benefit from tailored multivariate analysis. In this thesis, CRS microscopy was combined with Raman spectroscopy and other label-free nonlinear optical techniques to analyze lipid metabolism in multiple biological systems. We used nonlinear Raman techniques to characterize Meibum secretions in the progression of dry eye disease, where the lipid and protein contributions change in ratio and phase segregation. We employed similar tools to examine lipid droplets in mice livers aboard a spaceflight mission, which lose their retinol content contributing to the onset of nonalcoholic fatty-liver disease. We also focused on atherosclerosis, a disease that revolves around lipid-rich plaques in arterial walls. We examined the lipid content of macrophages, whose variable phenotype gives rise to contrasting healing and inflammatory activities. We also proposed new label-free markers, based on lifetime imaging, for macrophage phenotype, and to detect products of lipid oxidation. Cholesterol was also detected in hepatitis C virus infected cells, and in specific strains of age-related macular degeneration diseased cells by

  14. Ultrastructural Analysis of Drosophila Ovaries by Electron Microscopy

    PubMed Central

    Hurd, Thomas R.; Sanchez, Carlos G.; Teixeira, Felipe K.; Petzold, Chris; Dancel-Manning, Kristen; Wang, Ju-Yu S.; Lehmann, Ruth; Liang, Feng-Xia A.

    2016-01-01

    i. Summary The Drosophila melanogaster ovary is a powerful, genetically tractable system through which one can elucidate the principles underlying cellular function and organogenesis in vivo. In order to understand the intricate process of oogenesis at the subcellular level, microscopic analysis with the highest possible resolution is required. In this chapter, we describe the preparation of ovaries for ultrastructural analysis using transmission electron microscopy and focused ion beam scanning electron microscopy. We discuss and provide protocols for chemical fixation of Drosophila ovaries that facilitate optimal imaging with particular attention paid to preserving and resolving mitochondrial membrane morphology and structure. PMID:26324436

  15. Scanning electron microscopy of biosynthetic wound dressings Biocol.

    PubMed

    Pogorelov, A G; Gavriluk, V B; Pogorelova, V N; Gavriluk, B K

    2012-11-01

    The surface of wound dressing Biocol was studied by scanning electron microscopy. This composite system consists of latex matrix with incorporated water-soluble polysaccharide. The peculiarities of the surface are important for manufacturing of the dressing and for modification of its surface upon contact with fluids, e.g. during de novo tissue reconstruction. The method for studying the fine structure of the polymeric film surface was developed. The relief of the wound dressing changes during interaction with the fluid and nanopores appear on the surface. Thus, scanning electron microscopy is an informative method for studying the surface of biosynthetic films. PMID:23330116

  16. Surface morphology of Trichinella spiralis by scanning electron microscopy

    SciTech Connect

    Kim, C.W.; Ledbetter, M.C.

    1980-02-01

    The surface morphology of larval and adult Trichinella spiralis was studied by scanning electron microscopy (SEM) of fixed, dried, and metal-coated specimens. The results are compared with those found earlier by various investigators using light and transmission electron microscopy. Some morphological features reported here are revealed uniquely by SEM. These include the pores of the cephalic sense organs, the character of secondary cuticular folds, variations of the hypodermal gland cell openings or pores, and the presence of particles on the copulatory bell.

  17. Adaptive quantum measurement for low-dose electron microscopy

    SciTech Connect

    Okamoto, Hiroshi

    2010-04-15

    The resolution of current cryoelectron microscopy of radiation-sensitive specimens is ultimately limited by statistical noise because of the necessarily small number of electrons. To bypass this resolution barrier, we propose an extended in-focus phase-contrast microscopy scheme. The scheme incorporates a pixelwise deformable electrostatic mirror at a plane conjugate to the back focal plane of the objective lens. This setup would extract structural information more efficiently than otherwise and naturally enable generation of phase contrast. We present key concepts regarding microscope operations and estimate the degree of electron dose reduction that should in turn enable a resolution improvement.

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

    PubMed Central

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

    2013-01-01

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

  19. Scanning electron microscopy and transmission electron microscopy study of hot-deformed gamma-TiAl-based alloy microstructure.

    PubMed

    Chrapoński, J; Rodak, K

    2006-09-01

    The aim of this work was to assess the changes in the microstructure of hot-deformed specimens made of alloys containing 46-50 at.% Al, 2 at.% Cr and 2 at.% Nb (and alloying additions such as carbon and boron) with the aid of scanning electron microscopy and transmission electron microscopy techniques. After homogenization and heat treatment performed in order to make diverse lamellae thickness, the specimens were compressed at 1000 degrees C. Transmission electron microscopy examinations of specimens after the compression test revealed the presence of heavily deformed areas with a high density of dislocation. Deformation twins were also observed. Dynamically recrystallized grains were revealed. For alloys no. 2 and no. 3, the recovery and recrystallization processes were more extensive than for alloy no. 1.

  20. Standardless atom counting in scanning transmission electron microscopy.

    PubMed

    LeBeau, James M; Findlay, Scott D; Allen, Leslie J; Stemmer, Susanne

    2010-11-10

    We demonstrate that high-angle annular dark-field imaging in scanning transmission electron microscopy allows for quantification of the number and location of all atoms in a three-dimensional, crystalline, arbitrarily shaped specimen without the need for a calibration standard. We show that the method also provides for an approach to directly measure the finite effective source size of a scanning transmission electron microscope.

  1. Improved Imaging in Low Energy Electron Microscopy and Photo Emission Electron Microscopy Using MEDIPIX2 Pixel Detector

    NASA Astrophysics Data System (ADS)

    Sikharulidze, I.; van Gastel, R.; Schramm, S.; Abrahams, J. P.; Poelsema, B.; Trom, R. M.; van der Molen, S. J.

    2010-04-01

    The application of the Medipix2 hybrid pixel detector in Low Energy Electron Microscopy (LEEM) and Photo Emission Electron Microscopy (PEEM) led to an improvement of the recorded image quality compared to the original setup based on microchannel plate (MCP), phosphor screen and CCD. The measurements were performed on an Elmitec LEEM III instrument without energy filter using an Ir(111) sample with graphene islands grown on the surface. The Medipix2 images exhibited better resolution and higher contrast compared to the MCP data. The results suggest that Medipix2 has potential to become the detector of choice for LEEM/PEEM instruments.

  2. Light and electron microscopy of classical Ehlers-Danlos syndrome.

    PubMed

    de Almeida, Hiram L; Bicca, Eduardo; Rocha, Nara M; de Castro, Luis A S

    2013-02-01

    A 12-year-old boy with difficulty in wound healing and abnormal scars since early childhood was examined. Light microscopy showed loose and disperse dermal collagen with rare bundles, and fibroblasts show an irregular morphology. The fibrous sheath of hair presented a normal parallel distribution of the collagen fibers with normal spindle-shaped fibroblasts. Transmission electron microscopy also found disorganized collagen fibers, which were seen in a same field in longitudinal and cross sections. With high magnifications, an amorphous substance was seen near to loose collagen fibers, which showed variable diameters in cross sections. Scanning electron microscopy of the dermis showed disorganized collagen fibers and with higher magnification, important collagen disarrangement was observed with isolated and crossed-over fibers.

  3. Quantifying Nanoscale Order in Amorphous Materials via Fluctuation Electron Microscopy

    ERIC Educational Resources Information Center

    Bogle, Stephanie Nicole

    2009-01-01

    Fluctuation electron microscopy (FEM) has been used to study the nanoscale order in various amorphous materials. The method is explicitly sensitive to 3- and 4-body atomic correlation functions in amorphous materials; this is sufficient to establish the existence of structural order on the nanoscale, even when the radial distribution function…

  4. Multiple reaction pathways of metallofullerenes investigated by transmission electron microscopy.

    PubMed

    Koshino, Masanori

    2014-05-28

    Recent advances in molecule-by-molecule transmission electron microscopy (TEM) have provided time-series structural information of individual molecules supported by nano-carbon materials, enabling researchers to trace their motions and reactions. In this paper, the chemical reactions of fullerenes and metallofullerene derivatives, focusing on their deformation process, are reviewed and discussed based on the single-molecule-resolved TEM analysis.

  5. Electron microscopy of Lednice virus in chick embryo cells.

    PubMed

    Jelínková, A; Málková, D; Holubová, J; Novák, M

    1980-01-01

    Replication of Lednice virus in chick embryo cells was studied for 72 hr after inoculation by infectivity titration and the indirect immunofluorescence technique. At 24 and 48 hr after inoculation, electron microscopy revealed spherical virions of uniform morphology, 80-105 nm in diameter, which were localized mostly extracellularly.

  6. 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.

  7. Preparation of zeolites for TEM (Transmission Electron Microscopy) using microtomy

    SciTech Connect

    Csencsits, R.; Gronsky, R.

    1987-12-01

    The application of microtomy to Transmission Electron Microscopy (TEM) specimen preparation of zeolite catalysts is explained. Using a new acrylic resin (LR White) thin sections (less than or equal to60 nm) may be cut with relative ease. Although the details described are specific to catalysts, microtomy and the use of the acrylic resin are applicable to any hard ceramic powder sample. 3 figs.

  8. Collaboration at the Nanoscale: Exploring Viral Genetics with Electron Microscopy

    ERIC Educational Resources Information Center

    Duboise, S. Monroe; Moulton, Karen D.; Jamison, Jennifer L.

    2009-01-01

    The Maine Science Corps is a project sponsored by the National Science Foundation's (NSF) Graduate Teaching Fellows in K-12 Education (GK-12 ) program. Through this program, the University of Southern Maine's (USM) virology and transmission electron microscopy (TEM) research group provides high school teachers and students in rural areas with…

  9. Preparation of Articular Cartilage Specimens for Scanning Electron Microscopy.

    PubMed

    Stupina, T A

    2016-08-01

    We developed and adapted a technology for preparation of articular cartilage specimens for scanning electron microscopy. The method includes prefixation processing, fixation, washing, and dehydration of articular cartilage specimens with subsequent treatment in camphene and air-drying. The technological result consists in prevention of deformation of the articular cartilage structures. The method is simpler and cheaper than the known technologies. PMID:27591865

  10. Scanning electron microscopy of Serratia marcescens producing prodigiosin.

    PubMed

    Geron, M; Botershvili, I; Rokem, J S

    1988-01-01

    Production of high concentrations of prodigiosin by growing cells of Serratia marcescens was accompanied by the formation of extracellular protrusions as was revealed by scanning electron microscopy. Prodigiosin extracted from the bacterium was compared with the extracellular material. Bacteria which did not produce prodigiosin showed no extracellular protrusions.

  11. Generation and application of bessel beams in electron microscopy.

    PubMed

    Grillo, Vincenzo; Harris, Jérémie; Gazzadi, Gian Carlo; Balboni, Roberto; Mafakheri, Erfan; Dennis, Mark R; Frabboni, Stefano; Boyd, Robert W; Karimi, Ebrahim

    2016-07-01

    We report a systematic treatment of the holographic generation of electron Bessel beams, with a view to applications in electron microscopy. We describe in detail the theory underlying hologram patterning, as well as the actual electron-optical configuration used experimentally. We show that by optimizing our nanofabrication recipe, electron Bessel beams can be generated with relative efficiencies reaching 37±3%. We also demonstrate by tuning various hologram parameters that electron Bessel beams can be produced with many visible rings, making them ideal for interferometric applications, or in more highly localized forms with fewer rings, more suitable for imaging. We describe the settings required to tune beam localization in this way, and explore beam and hologram configurations that allow the convergences and topological charges of electron Bessel beams to be controlled. We also characterize the phase structure of the Bessel beams generated with our technique, using a simulation procedure that accounts for imperfections in the hologram manufacturing process. PMID:27203186

  12. 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.

  13. 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.

  14. Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

    PubMed

    Killingsworth, Murray C; Bobryshev, Yuri V

    2016-01-01

    A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region. PMID:27584907

  15. Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

    NASA Astrophysics Data System (ADS)

    Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R.; Chess, Jordan; McMorran, Benjamin J.; Czarnik, Cory; Rose, Harald H.; Ercius, Peter

    2016-02-01

    The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.

  16. Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry.

    PubMed

    Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R; Chess, Jordan; McMorran, Benjamin J; Czarnik, Cory; Rose, Harald H; Ercius, Peter

    2016-01-01

    The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals. PMID:26923483

  17. Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

    PubMed Central

    Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R.; Chess, Jordan; McMorran, Benjamin J.; Czarnik, Cory; Rose, Harald H.; Ercius, Peter

    2016-01-01

    The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals. PMID:26923483

  18. Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry.

    PubMed

    Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R; Chess, Jordan; McMorran, Benjamin J; Czarnik, Cory; Rose, Harald H; Ercius, Peter

    2016-02-29

    The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.

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

    PubMed Central

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

    2009-01-01

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

  20. Atomic force microscopy application in biological research: a review study.

    PubMed

    Vahabi, Surena; Nazemi Salman, Bahareh; Javanmard, Anahita

    2013-06-01

    Atomic force microscopy (AFM) is a three-dimensional topographic technique with a high atomic resolution to measure surface roughness. AFM is a kind of scanning probe microscope, and its near-field technique is based on the interaction between a sharp tip and the atoms of the sample surface. There are several methods and many ways to modify the tip of the AFM to investigate surface properties, including measuring friction, adhesion forces and viscoelastic properties as well as determining the Young modulus and imaging magnetic or electrostatic properties. The AFM technique can analyze any kind of samples such as polymers, adsorbed molecules, films or fibers, and powders in the air whether in a controlled atmosphere or in a liquid medium. In the past decade, the AFM has emerged as a powerful tool to obtain the nanostructural details and biomechanical properties of biological samples, including biomolecules and cells. The AFM applications, techniques, and -in particular- its ability to measure forces, are not still familiar to most clinicians. This paper reviews the literature on the main principles of the AFM modality and highlights the advantages of this technique in biology, medicine, and- especially- dentistry. This literature review was performed through E-resources, including Science Direct, PubMed, Blackwell Synergy, Embase, Elsevier, and Scholar Google for the references published between 1985 and 2010.

  1. Use of Atomic Force Microscopy and Transmission Electron Microscopy for Correlative Studies of Bacterial Capsules▿ †

    PubMed Central

    Stukalov, Oleg; Korenevsky, Anton; Beveridge, Terry J.; Dutcher, John R.

    2008-01-01

    Bacteria can possess an outermost assembly of polysaccharide molecules, a capsule, which is attached to their cell wall. We have used two complementary, high-resolution microscopy techniques, atomic force microscopy (AFM) and transmission electron microscopy (TEM), to study bacterial capsules of four different gram-negative bacterial strains: Escherichia coli K30, Pseudomonas aeruginosa FRD1, Shewanella oneidensis MR-4, and Geobacter sulfurreducens PCA. TEM analysis of bacterial cells using different preparative techniques (whole-cell mounts, conventional embeddings, and freeze-substitution) revealed capsules for some but not all of the strains. In contrast, the use of AFM allowed the unambiguous identification of the presence of capsules on all strains used in the present study, including those that were shown by TEM to be not encapsulated. In addition, the use of AFM phase imaging allowed the visualization of the bacterial cell within the capsule, with a depth sensitivity that decreased with increasing tapping frequency. PMID:18606791

  2. Studying Atomic Structures by Aberration-Corrected Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Urban, Knut W.

    2008-07-01

    Seventy-five years after its invention, transmission electron microscopy has taken a great step forward with the introduction of aberration-corrected electron optics. An entirely new generation of instruments enables studies in condensed-matter physics and materials science to be performed at atomic-scale resolution. These new possibilities are meeting the growing demand of nanosciences and nanotechnology for the atomic-scale characterization of materials, nanosynthesized products and devices, and the validation of expected functions. Equipped with electron-energy filters and electron-energy loss spectrometers, the new instruments allow studies not only of structure but also of elemental composition and chemical bonding. The energy resolution is about 100 milli electron volts, and the accuracy of spatial measurements has reached a few picometers. However, understanding the results is generally not straightforward and only possible with extensive quantum-mechanical computer calculations.

  3. Studying atomic structures by aberration-corrected transmission electron microscopy.

    PubMed

    Urban, Knut W

    2008-07-25

    Seventy-five years after its invention, transmission electron microscopy has taken a great step forward with the introduction of aberration-corrected electron optics. An entirely new generation of instruments enables studies in condensed-matter physics and materials science to be performed at atomic-scale resolution. These new possibilities are meeting the growing demand of nanosciences and nanotechnology for the atomic-scale characterization of materials, nanosynthesized products and devices, and the validation of expected functions. Equipped with electron-energy filters and electron-energy-loss spectrometers, the new instruments allow studies not only of structure but also of elemental composition and chemical bonding. The energy resolution is about 100 milli-electron volts, and the accuracy of spatial measurements has reached a few picometers. However, understanding the results is generally not straightforward and only possible with extensive quantum-mechanical computer calculations. PMID:18653874

  4. Reporting methods for processing and analysis of data from serial block face scanning electron microscopy.

    PubMed

    Borrett, S; Hughes, L

    2016-07-01

    Serial block face scanning electron microscopy is rapidly becoming a popular tool for collecting large three-dimensional data sets of cells and tissues, filling the resolution and volume gap between fluorescence microscopy and high-resolution electron microscopy. The automated collection of data within the instrument occupies the smallest proportion of the time required to prepare and analyse biological samples. It is the processing of data once it has been collected that proves the greatest challenge. In this review we discuss different methods that are used to process data. We suggest potential workflows that can be used to facilitate the transfer of raw image stacks into quantifiable data as well as propose a set of criteria for reporting methods for data analysis to enable replication of work. PMID:26800017

  5. Human enamel structure studied by high resolution electron microscopy

    SciTech Connect

    Wen, S.L. )

    1989-01-01

    Human enamel structural features are characterized by high resolution electron microscopy. The human enamel consists of polycrystals with a structure similar to Ca10(PO4)6(OH)2. This article describes the structural features of human enamel crystal at atomic and nanometer level. Besides the structural description, a great number of high resolution images are included. Research into the carious process in human enamel is very important for human beings. This article firstly describes the initiation of caries in enamel crystal at atomic and unit-cell level and secondly describes the further steps of caries with structural and chemical demineralization. The demineralization in fact, is the origin of caries in human enamel. The remineralization of carious areas in human enamel has drawn more and more attention as its potential application is realized. This process has been revealed by high resolution electron microscopy in detail in this article. On the other hand, the radiation effects on the structure of human enamel are also characterized by high resolution electron microscopy. In order to reveal this phenomenon clearly, a great number of electron micrographs have been shown, and a physical mechanism is proposed. 26 references.

  6. Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique

    PubMed Central

    2008-01-01

    Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried, the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique, samples are frozen, fractured and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed. PMID:18385807

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

    PubMed

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

    2014-01-01

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

  8. Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution

    PubMed Central

    Joens, Matthew S.; Huynh, Chuong; Kasuboski, James M.; Ferranti, David; Sigal, Yury J.; Zeitvogel, Fabian; Obst, Martin; Burkhardt, Claus J.; Curran, Kevin P.; Chalasani, Sreekanth H.; Stern, Lewis A.; Goetze, Bernhard; Fitzpatrick, James A. J.

    2013-01-01

    Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure. PMID:24343236

  9. Helium Ion Microscopy (HIM) for the imaging of biological samples at sub-nanometer resolution.

    PubMed

    Joens, Matthew S; Huynh, Chuong; Kasuboski, James M; Ferranti, David; Sigal, Yury J; Zeitvogel, Fabian; Obst, Martin; Burkhardt, Claus J; Curran, Kevin P; Chalasani, Sreekanth H; Stern, Lewis A; Goetze, Bernhard; Fitzpatrick, James A J

    2013-12-17

    Scanning Electron Microscopy (SEM) has long been the standard in imaging the sub-micrometer surface ultrastructure of both hard and soft materials. In the case of biological samples, it has provided great insights into their physical architecture. However, three of the fundamental challenges in the SEM imaging of soft materials are that of limited imaging resolution at high magnification, charging caused by the insulating properties of most biological samples and the loss of subtle surface features by heavy metal coating. These challenges have recently been overcome with the development of the Helium Ion Microscope (HIM), which boasts advances in charge reduction, minimized sample damage, high surface contrast without the need for metal coating, increased depth of field, and 5 angstrom imaging resolution. We demonstrate the advantages of HIM for imaging biological surfaces as well as compare and contrast the effects of sample preparation techniques and their consequences on sub-nanometer ultrastructure.

  10. Immuno-electron microscopy of primary cell cultures from genetically modified animals in liquid by atmospheric scanning electron microscopy.

    PubMed

    Kinoshita, Takaaki; Mori, Yosio; Hirano, Kazumi; Sugimoto, Shinya; Okuda, Ken-ichi; Matsumoto, Shunsuke; Namiki, Takeshi; Ebihara, Tatsuhiko; Kawata, Masaaki; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Higashiyama, Kenichi; Sonomoto, Kenji; Mizunoe, Yoshimitsu; Nishihara, Shoko; Sato, Chikara

    2014-04-01

    High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future. PMID:24564988

  11. Scanning electron microscopy of pulmonary alveolar capillary vessels

    PubMed Central

    Alexander, I. G. S.; Ritchie, B. C.; Maloney, J. E.

    1973-01-01

    The pattern of subepithelial vessels in pulmonary alveoli of rabbits has been studied using scanning electron microscopy. Alveolar capillaries form a network of interconnecting vascular rings, most of which surround the periphery of type II cells of the alveolar epithelium. Individual capillaries contributing to the formation of adjacent rings follow a corrugated course with angulations located on the sites of junction with other capillaries completing the rings; the capillaries are covered by type I epithelial cells which also extend into and form the alveolar lining at the peripheral area of the interstices of the capillary network. Single type II cells form the alveolar lining at the centre of vascular rings. The pattern of pulmonary alveolar capillaries revealed by scanning electron microscopy is thus similar to that postulated by Weibel (1963) on the basis of transmission microscopic studies. Images PMID:4731118

  12. Microfabricated high-bandpass foucault aperture for electron microscopy

    DOEpatents

    Glaeser, Robert; Cambie, Rossana; Jin, Jian

    2014-08-26

    A variant of the Foucault (knife-edge) aperture is disclosed that is designed to provide single-sideband (SSB) contrast at low spatial frequencies but retain conventional double-sideband (DSB) contrast at high spatial frequencies in transmission electron microscopy. The aperture includes a plate with an inner open area, a support extending from the plate at an edge of the open area, a half-circle feature mounted on the support and located at the center of the aperture open area. The radius of the half-circle portion of reciprocal space that is blocked by the aperture can be varied to suit the needs of electron microscopy investigation. The aperture is fabricated from conductive material which is preferably non-oxidizing, such as gold, for example.

  13. Electron microscopy of legionella and legionella-infected cells.

    PubMed

    Faulkner, Gary; Garduño, Rafael A

    2013-01-01

    Those investigators who study the morphology of Legionella and Legionella-infected cells have greatly benefited from the superior resolution afforded by electron microscopy (EM). It can also be said with confidence that EM will continue to reveal as yet to be discovered features of this fascinating intracellular pathogen. In this chapter we detail our practical experience in the application of three transmission electron microscopy (TEM) techniques to the study of Legionella: conventional ultrastructural analysis, immuno-gold labeling, and negative staining. Each of these techniques has particular, well-defined applications, which are discussed in the context of our in-house developed methods. We invite researchers to try the methods given here in the study of Legionella, and adopt TEM as part of their research tools arsenal. PMID:23150403

  14. WHOLE CELL TOMOGRAPHY/MOLECULAR BIOLOGY/STRUCTURAL BIOLOGY: Affordable x-ray microscopy with nanoscale resolution

    SciTech Connect

    Evans, James E.; Blackborow, Paul; Horne, Stephen J.; Gelb, Jeff

    2013-03-01

    Biological research spans 10 orders of magnitude from angstroms to meters. While electron microscopy can reveal structural details at most of these spatial length scales, transmission electron tomography only reliably reconstructs three-dimensional (3-D) volumes of cellular material with a spatial resolution between 1-5 nm from samples less than 500 nm thick1. Most biological cells are 2-30 times thicker than this threshold, which means that a cell must be cut into consecutive slices with each slice reconstructed individually in order to approximate the contextual information of the entire cell. Fortunately, due to a larger penetration depth2, X-ray computed tomography bypasses the need to physically section a cell and enables imaging of intact cells and tissues on the micrometer or larger scale with tens to hundreds of nanometer spatial resolution. While the technique of soft x-ray microscopy has been extensively developed in synchrotron facilities, advancements in laboratory x-ray source designs now increase its accessibility by supporting commercial systems suitable for a standard laboratory. In this paper, we highlight a new commercial compact cryogenic soft x-ray microscope designed for a standard laboratory setting and explore its capabilities for mesoscopic investigations of intact prokaryotic and eukaryotic cells.

  15. Blotting protein complexes from native gels to electron microscopy grids.

    PubMed

    Knispel, Roland Wilhelm; Kofler, Christine; Boicu, Marius; Baumeister, Wolfgang; Nickell, Stephan

    2012-01-08

    We report a simple and generic method for the direct transfer of protein complexes separated by native gel electrophoresis to electron microscopy grids. After transfer, sufficient material remains in the gel for identification and characterization by mass spectrometry. The method should facilitate higher-throughput single-particle analysis by substantially reducing the time needed for protein purification, as demonstrated for three complexes from Thermoplasma acidophilum.

  16. In Situ Electron Microscopy of Lactomicroselenium Particles in Probiotic Bacteria.

    PubMed

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

    2016-01-01

    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. PMID:27376279

  17. Quantitative electron microscopy of InN-GaN alloys

    NASA Astrophysics Data System (ADS)

    Bartel, T.; Jinschek, J. R.; Freitag, B.; Specht, P.; Kisielowski, C.

    2006-01-01

    The local element distribution in quantum wells largely affects physical properties of devices made from such materials. In the past, quantitative electron microscopy was developed to access the stoichiometry on an atomic scale as shown on the cover page of this issue's Editor's Choice [1] for the GaN/InxGa1-xN/GaN and the GaAs/AlxGa1-xAs/GaAs system by the application of QUANTITEM and Chemical Imaging, respectively. In case of GaN/InxGa1-xN/GaN local strain mapping allows for extracting similar data and an unusual large indium fluctuation can be observed if compared with the aluminum distribution in GaAs/AlxGa1-xAs/GaAs quantum well structures. However, radiation damage, sample preparation and microscope stability affect the data analyses and it is of essence to monitor and control such effects as outlined in the related paper.The first author Til Bartel is a PhD candidate in physics at the Technical University of Berlin, currently visiting LBNL in California to apply transmission electron microscopy to III-nitride semiconductors. Christian Kisielowski is Staff Scientist and Principle Investigator at the National Center for Electron Microscopy (NCEM) and is responsible for the development and application of high resolution electron microscopy.The present special issue of physica status solidi (a) is a compilation of presentations from the recent symposium on Indium Nitride and Indium Rich Related Alloys at the E-MRS 2005 Fall Meeting in Warsaw.

  18. Ultra-High-Resolution Electron Microscopy of Carbon Nanotube Walls

    SciTech Connect

    Cowley, J. M.; Winterton, Jamie

    2001-07-02

    The resolution in scanning transmission electron microscopy may be enhanced by taking advantage of the information contained in the nanodiffraction patterns recorded for each position of the scanning incident beam. We have demonstrated the first production of ultrahigh resolution, better than 0.1nm, by this method, in the imaging of an essentially one-dimensional object, the wall of a multiwalled carbon nanotube, using an instrument for which the resolution for normal imaging is about 0.3nm.

  19. Cross-sectional transmission electron microscopy of semiconductors

    SciTech Connect

    Sadana, D.K.

    1982-10-01

    A method to prepare cross-sectional (X) semiconductor specimens for transmission electron microscopy (TEM) has been described. The power and utility of XTEM has been demonstrated. It has been shown that accuracy and interpretation of indirect structural-defects profiling techniques, namely, MeV He/sup +/ channeling and secondary ion mass spectrometry (SIMS) can be greatly enhanced by comparing their results with those obtained by XTEM from the same set of samples.

  20. Studying localized corrosion using liquid cell transmission electron microscopy

    DOE PAGES

    Chee, See Wee; Pratt, Sarah H.; Hattar, Khalid; Duquette, David; Ross, Frances M.; Hull, Robert

    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.

  1. In Situ Electron Microscopy of Lactomicroselenium Particles in Probiotic Bacteria

    PubMed Central

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

    2016-01-01

    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. PMID:27376279

  2. Studying localized corrosion using liquid cell transmission electron microscopy

    SciTech Connect

    Chee, See Wee; Pratt, Sarah H.; Hattar, Khalid; Duquette, David; Ross, Frances M.; Hull, Robert

    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.

  3. Transmission electron microscopy of a model crystalline organic, theophylline

    NASA Astrophysics Data System (ADS)

    Cattle, J.; S'ari, M.; Hondow, N.; Abellán, P.; Brown, A. P.; Brydson, R. M. D.

    2015-10-01

    We report on the use of transmission electron microscopy (TEM) to analyse the diffraction patterns of the model crystalline organic theophylline to investigate beam damage in relation to changing accelerating voltage, sample temperature and TEM grid support films. We find that samples deposited on graphene film grids have the longest lifetimes when also held at -190 °C and imaged at 200 kV accelerating voltage. Finally, atomic lattice images are obtained in bright field STEM by working close to the estimated critical electron dose for theophylline.

  4. Cellular structural biology as revealed by cryo-electron tomography.

    PubMed

    Irobalieva, Rossitza N; Martins, Bruno; Medalia, Ohad

    2016-02-01

    Understanding the function of cellular machines requires a thorough analysis of the structural elements that underline their function. Electron microscopy (EM) has been pivotal in providing information about cellular ultrastructure, as well as macromolecular organization. Biological materials can be physically fixed by vitrification and imaged with cryo-electron tomography (cryo-ET) in a close-to-native condition. Using this technique, one can acquire three-dimensional (3D) information about the macromolecular architecture of cells, depict unique cellular states and reconstruct molecular networks. Technical advances over the last few years, such as improved sample preparation and electron detection methods, have been instrumental in obtaining data with unprecedented structural details. This presents an exciting opportunity to explore the molecular architecture of both individual cells and multicellular organisms at nanometer to subnanometer resolution. In this Commentary, we focus on the recent developments and in situ applications of cryo-ET to cell and structural biology.

  5. Evaluation of collagen gel microstructure by scanning electron microscopy.

    PubMed

    Pogorelov, A G; Selezneva, I I

    2010-12-01

    We performed qualitative comparison of freeze drying and chemical drying as methods of preparing 3D wet specimens for scanning electron microscopy. Human fibroblasts immobilized in collagen gel were used as a model system. Specimens fixed with glutaraldehyde were frozen in liquid nitrogen and freeze-dried at low temperature in high vacuum. In parallel experiments, glutaraldehyde-fixed samples were dehydrated in ascending ethanol solutions, absolute ethanol, and 100% hexamethyldisilazane and then dried at room temperature. Scanning electron microscopy microphotographs of collagen fibers and cells were characterized by high resolution and the absence of collapsed or deformed structures even at high magnification (×50,000) for both chemical drying and high-vacuum freeze drying. However, high-vacuum freeze drying is superior to chemical drying for the investigation of the internal space of 3D scaffolds, because sample fracture can be prepared directly in liquid nitrogen. These techniques are a part of the sample preparation process for scanning electron microscopy and can also be used for studies of cell adhesion, morphology, and arrangement in wet specimens (3D gels and flexible tissue engineering scaffolds). PMID:21161075

  6. System and method for compressive scanning electron microscopy

    DOEpatents

    Reed, Bryan W

    2015-01-13

    A scanning transmission electron microscopy (STEM) system is disclosed. The system may make use of an electron beam scanning system configured to generate a plurality of electron beam scans over substantially an entire sample, with each scan varying in electron-illumination intensity over a course of the scan. A signal acquisition system may be used for obtaining at least one of an image, a diffraction pattern, or a spectrum from the scans, the image, diffraction pattern, or spectrum representing only information from at least one of a select subplurality or linear combination of all pixel locations comprising the image. A dataset may be produced from the information. A subsystem may be used for mathematically analyzing the dataset to predict actual information that would have been produced by each pixel location of the image.

  7. Visualization of Microbial Biomarkers by Scanning Electron Microscopy

    NASA Technical Reports Server (NTRS)

    Wainwright, Norman R.; Allen, Carlton C.; Child, Alice

    2001-01-01

    . Fortunately, many antimicrobial defense systems of higher organisms require sensitive detection to combat microbial pathogens. We employ here the primitive immune system of the evolutionarily ancient horseshoe crab, Limulus polyphemus. This species relies on multi-enzyme signal amplification detection of cell wall molecules and they can be applied to the development of useful detectors of life. An extension of this work includes the visualization of microbial signatures by labeling LAL components with chromogenic or electron dense markers. The protein Limulus Anti-LPS Factor (LALF) has an extremely high affinity for LPS. By coupling LALF binding with colloidal gold labels we demonstrate a correlation of the structures visible by electron microscopy with biochemical evidence of microbial cell wall materials. Pure silica particles were mixed with cultures of E. coli (10(exp 6) cfu/mL). Samples were washed sequentially with buffered saline, LALF, antibody to LALF and finally colloidal gold-labeled Protein A. Negative controls were not exposed to E. coli but received identical treatment otherwise. Samples were coated with carbon and imaged on a JEOL JSM-840 scanning electron microscope with LaB6 source in the back scatter mode with the JEOL annular back scatter detector. 20 nm-scale black spots in this contrast-reversed image originate from electrons back-scattered by gold atoms. Negative controls did not give any signal. Future work will expand application of this technique to soil simulants and mineralized rock samples.

  8. Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography.

    PubMed

    Schröder, Rasmus R

    2015-09-01

    Macromolecular imaging and tomography of ice embedded samples has developed into a mature imaging technology, in structural biology today widely referred to simply as cryo electron microscopy.(1) While the pioneers of the technique struggled with ill-suited instruments, state-of-the-art cryo microscopes are now readily available and an increasing number of groups are producing excellent high-resolution structural data of macromolecular complexes, of cellular organelles, or the morphology of whole cells. Instrumentation developers, however, are offering yet more novel electron optical devices, such as energy filters and monochromators, aberration correctors or physical phase plates. Here we discuss how current instrumentation has already changed cryo EM, and how newly available instrumentation - often developed in other fields of electron microscopy - may further develop the use and applicability of cryo EM to the imaging of single isolated macromolecules of smaller size or molecules embedded in a crowded cellular environment.

  9. Imaging liver biology in vivo using conventional confocal microscopy.

    PubMed

    Marques, Pedro E; Antunes, Maísa M; David, Bruna A; Pereira, Rafaela V; Teixeira, Mauro M; Menezes, Gustavo B

    2015-02-01

    Imaging of live animals using intravital microscopy (IVM) has provided a substantial advance in our understanding of cell biology. Here we describe how to adapt a conventional, relatively low-cost laser-scanning microscope to operate as a versatile imaging station. We present the surgical procedures needed to perform liver confocal IVM in mice, thereby allowing one to image different cells in their native environment, including hepatocytes, endothelial cells and leukocytes, as well as to analyze their morphology and function under physiological or pathological conditions. In addition, we propose a plethora of working doses of antibodies and probes to stain multiple cells and molecules simultaneously in vivo. Considering the central role of the liver in metabolism and immunity and the growing interest in the relationship between immune and parenchymal cells, this protocol, in which 20 min of preparation yields up to 4 h of imaging, provides useful insights for various research fields. In addition, the protocol can be easily adapted to investigate adipose tissue, mesentery, intestines, spleen and virtually any abdominal organ.

  10. 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. PMID:25173605

  11. 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.

  12. Peptide π-Electron Conjugates: Organic Electronics for Biology?

    PubMed

    Ardoña, Herdeline Ann M; Tovar, John D

    2015-12-16

    Highly ordered arrays of π-conjugated molecules are often viewed as a prerequisite for effective charge-transporting materials. Studies involving these materials have traditionally focused on organic electronic devices, with more recent emphasis on biological systems. In order to facilitate the transition to biological environments, biomolecules that can promote hierarchical ordering and water solubility are often covalently appended to the π-electron unit. This review highlights recent work on π-conjugated systems bound to peptide moieties that exhibit self-assembly and aims to provide an overview on the development and emerging applications of peptide-based supramolecular π-electron systems.

  13. Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

    NASA Astrophysics Data System (ADS)

    Seacor, Taylor; Howell, Carina

    2013-03-01

    Nematode worms account for the vast majority of the animals in the biosphere. They are colossally important to global public health as parasites, and to agriculture both as pests and as beneficial inhabitants of healthy soil. Amphid neurons are the anterior chemosensory neurons in nematodes, mediating critical behaviors including chemotaxis and mating. We are examining the cellular morphology and external anatomy of amphid neurons, using fluorescence microscopy and scanning electron microscopy, respectively, of a wide range of soil nematodes isolated in the wild. We use both classical systematics (e.g. diagnostic keys) and molecular markers (e.g. ribosomal RNA) to classify these wild isolates. Our ultimate aim is to build a detailed anatomical database in order to dissect genetic pathways of neuronal development and function across phylogeny and ecology. Research supported by NSF grants 092304, 0806660, 1058829 and Lock Haven University FPDC grants

  14. Probing structures of nanomaterials using advanced electron microscopy methods, including aberration-corrected electron microscopy at the Angstrom scale.

    PubMed

    Gai, Pratibha L; Yoshida, Kenta; Shute, Carla; Jia, Xiaoting; Walsh, Michael; Ward, Michael; Dresselhaus, Mildred S; Weertman, Julia R; Boyes, Edward D

    2011-07-01

    Structural and compositional studies of nanomaterials of technological importance have been carried out using advanced electron microscopy methods, including aberration-corrected transmission electron microscopy (AC-TEM), AC-high angle annular dark field scanning TEM (AC-HAADF-STEM), AC-energy filtered TEM, electron-stimulated energy dispersive spectroscopy in the AC-(S)TEM and high-resolution TEM (HRTEM) with scanning tunneling microscopy (STM) holder. The AC-EM data reveal improvements in resolution and minimization in image delocalization. A JEOL 2200FS double-AC field emission gun TEM/STEM operating at 200 kV in the Nanocentre at the University of York has been used to image single metal atoms on crystalline supports in catalysts, grain boundaries in nanotwinned metals, and nanostructures of tetrapods. Joule heating studies using HRTEM integrated with an STM holder reveal in situ crystallization and edge reconstruction in graphene. Real-time in situ AC-HAADF-STEM studies at elevated temperatures are described. Dynamic in-column energy filtering in an AC environment provides an integral new approach to perform dynamic in situ studies with aberration correction. The new results presented here open up striking new opportunities for atomic scale studies of nanomaterials and indicate future development directions.

  15. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    NASA Astrophysics Data System (ADS)

    Jesse, S.; Chi, M.; Belianinov, A.; Beekman, C.; Kalinin, S. V.; Borisevich, A. Y.; Lupini, A. R.

    2016-05-01

    Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. Here, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. However, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.

  16. High-Pressure Freezing Electron Microscopy of Zebrafish Oocytes.

    PubMed

    Kanagaraj, Palsamy; Riedel, Dietmar; Dosch, Roland

    2016-01-01

    Oogenesis is an essential cellular and developmental process to prepare the oocyte for propagation of a species after fertilization. Oocytes of oviparous animals are enormous cells endowed with many, big cellular compartments, which are interconnected through active intracellular transport. The dynamic transport pathways and the big organelles of the oocyte provide the opportunity to study cellular trafficking with outstanding resolution. Hence, oocytes were classically used to investigate cellular compartments. Though many novel regulators of vesicle trafficking have been discovered in yeast, tissue culture cells and invertebrates, recent forward genetic screens in invertebrate and vertebrate oocytes isolated novel control proteins specific to multicellular organisms. Zebrafish is a widely used vertebrate model to study cellular and developmental processes in an entire animal. The transparency of zebrafish embryos allows following cellular events during early development with in vivo imaging. Unfortunately, the active endocytosis of the oocyte also represents a drawback for imaging. The massive amounts of yolk globules prevent the penetration of light-beams and currently make in vivo microscopy a challenge. As a consequence, electron microscopy (EM) still provides the highest resolution to analyze the ultra-structural details of compartments and organelles and the mechanisms controlling many cellular pathways of the oocyte. Among different fixation approaches for EM, High Pressure Freezing (HPF) in combination with freeze substitution significantly improves the samples preservation closest to their natural status. Here, we describe the HPF with freeze substitution embedding method for analyzing cellular processes in zebrafish oocytes using electron microscopy. PMID:27557580

  17. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    DOE PAGES

    Jesse, S.; Chi, M.; Belianinov, A.; Beekman, C.; Kalinin, S. V.; Borisevich, A. Y.; Lupini, A. R.

    2016-05-23

    Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. In this paper, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature andmore » does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. Finally, however, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.« less

  18. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography.

    PubMed

    Jesse, S; Chi, M; Belianinov, A; Beekman, C; Kalinin, S V; Borisevich, A Y; Lupini, A R

    2016-05-23

    Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. Here, we discuss the application of so-called "big-data" methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. However, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.

  19. Electron Microscopy Study of Exotic Nanostructures of Cadmium Sulfide

    NASA Astrophysics Data System (ADS)

    Dong, Lifeng; Jiao, Jun

    2005-04-01

    In this article, two simple methods, evaporation-condensation and catalytic thermal evaporation, were used to investigate the synthesis of CdS nanostructures for nanoscale optoelectronic applications. To understand their growth mechanisms, various electron microscopy and microanalysis techniques were utilized in characterizing their morphologies, internal structures, growth directions and elemental compositions. The electron microscopy study reveals that when using the evaporation-condensation method, branched CdS nanorods and self-assembled arrays of CdS nanorods were synthesized at 800°C and 1000°C, respectively. Instead of morphological differences, both types of CdS nanorods grew along the [0001] direction. However, when using the catalytic thermal evaporation method (Au as the catalyst), patterned CdS nanowires and nanobelts were formed at the temperature region of 500 600°C and 600 750°C, respectively. Their growth direction was along the direction [1010] instead of [0001]. Based on the microscopy and microanalysis results, we propose some growth mechanisms in relation to the growth processes of those exotic CdS nanostructures.

  20. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    PubMed Central

    Jesse, S.; Chi, M.; Belianinov, A.; Beekman, C.; Kalinin, S. V.; Borisevich, A. Y.; Lupini, A. R.

    2016-01-01

    Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. Here, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. However, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy. PMID:27211523

  1. Ultrastructural and elemental imaging of biological specimens by soft x-ray contact microscopy

    SciTech Connect

    Panessa, B.J.; Hoffman, P. . Dept. of Orthopedics); Warren, J.B. ); Feder, R.; Sayre, D. . Thomas J. Watson Research Center)

    1980-01-01

    Soft X-ray contact microscopy offers a means of visualizing unstained as well as stained biological materials at better than 6 nm resolution. Soft X-ray imaging depends on differential absorption of incident soft (1--10nm wavelength) X-rays by the endogenous elements within a specimen. The advantages of using soft X-rays for imaging are: (1) reduced specimen damage during exposure; (2) ability to image hydrated specimens at atmospheric pressure; (3) ability to image specimens ranging in thickness from less than 40 nm to as much as 10{mu}m; and (4) ability to map the elemental composition of the specimen through observation of the differential absorption of properly chosen incident x-ray wavelengths. This paper explains the principles of image formation and demonstrates the use of soft X-ray contact microscopy with biological samples which could not readily be imaged in their natural form using conventional electron microscopy methods. Data are also presented on the recognition of compositional features in histochemically treated articular joint tissues. 30 refs., 15 figs.

  2. Time resolved electron microscopy for in situ experiments

    SciTech Connect

    Campbell, Geoffrey H. McKeown, Joseph T.; Santala, Melissa K.

    2014-12-15

    Transmission electron microscopy has functioned for decades as a platform for in situ observation of materials and processes with high spatial resolution. Yet, the dynamics often remain elusive, as they unfold too fast to discern at these small spatial scales under traditional imaging conditions. Simply shortening the exposure time in hopes of capturing the action has limitations, as the number of electrons will eventually be reduced to the point where noise overtakes the signal in the image. Pulsed electron sources with high instantaneous current have successfully shortened exposure times (thus increasing the temporal resolution) by about six orders of magnitude over conventional sources while providing the necessary signal-to-noise ratio for dynamic imaging. We describe here the development of this new class of microscope and the principles of its operation, with examples of its application to problems in materials science.

  3. Effects of instrument imperfections on quantitative scanning transmission electron microscopy.

    PubMed

    Krause, Florian F; Schowalter, Marco; Grieb, Tim; Müller-Caspary, Knut; Mehrtens, Thorsten; Rosenauer, Andreas

    2016-02-01

    Several instrumental imperfections of transmission electron microscopes are characterized and their effects on the results of quantitative scanning electron microscopy (STEM) are investigated and quantified using simulations. Methods to either avoid influences of these imperfections during acquisition or to include them in reference calculations are proposed. Particularly, distortions inflicted on the diffraction pattern by an image-aberration corrector can cause severe errors of more than 20% if not accounted for. A procedure for their measurement is proposed here. Furthermore, afterglow phenomena and nonlinear behavior of the detector itself can lead to incorrect normalization of measured intensities. Single electrons accidentally impinging on the detector are another source of error but can also be exploited for threshold-less calibration of STEM images to absolute dose, incident beam current determination and measurement of the detector sensitivity.

  4. Electron microscopy: essentials for viral structure, morphogenesis and rapid diagnosis.

    PubMed

    Zhang, Ying; Hung, Tao; Song, Jingdong; He, Jinsheng

    2013-05-01

    Electron microscopy (EM) should be used in the front line for detection of agents in emergencies and bioterrorism, on accounts of its speed and accuracy. However, the number of EM diagnostic laboratories has decreased considerably and an increasing number of people encounter difficulties with EM results. Therefore, the research on viral structure and morphologyant in EM diagnostic practice. EM has several technological advantages, and should be a fundamental tool in clinical diagnosis of viruses, particularly when agents are unknown or unsuspected. In this article, we review the historical contribution of EM to virology, and its use in virus differentiation, localization of specific virus antigens, virus-cell interaction, and viral morphogenesis. It is essential that EM investigations are based on clinical and comprehensive pathogenesis data from light or confocal microscopy. Furthermore, avoidance of artifacts or false results is necessary to exploit fully the advantages while minimizing its limitations.

  5. 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.

  6. High-resolution electron microscopy of advanced materials

    SciTech Connect

    Mitchell, T.E.; Kung, H.H.; Sickafus, K.E.; Gray, G.T. III; Field, R.D.; Smith, J.F.

    1997-11-01

    This final report chronicles a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The High-Resolution Electron Microscopy Facility has doubled in size and tripled in quality since the beginning of the three-year period. The facility now includes a field-emission scanning electron microscope, a 100 kV field-emission scanning transmission electron microscope (FE-STEM), a 300 kV field-emission high-resolution transmission electron microscope (FE-HRTEM), and a 300 kV analytical transmission electron microscope. A new orientation imaging microscope is being installed. X-ray energy dispersive spectrometers for chemical analysis are available on all four microscopes; parallel electron energy loss spectrometers are operational on the FE-STEM and FE-HRTEM. These systems enable evaluation of local atomic bonding, as well as chemical composition in nanometer-scale regions. The FE-HRTEM has a point-to-point resolution of 1.6 {angstrom}, but the resolution can be pushed to its information limit of 1 {angstrom} by computer reconstruction of a focal series of images. HRTEM has been used to image the atomic structure of defects such as dislocations, grain boundaries, and interfaces in a variety of materials from superconductors and ferroelectrics to structural ceramics and intermetallics.

  7. Molecular ferroelectrics: where electronics meet biology

    PubMed Central

    Li, Jiangyu; Liu, Yuanming; Zhang, Yanhang; Cai, Hong-Ling; Xiong, Ren-Gen

    2013-01-01

    In the last several years, we have witnessed significant advances in molecular ferroelectrics, with ferroelectric properties of molecular crystals approaching those of barium titanate. In addition, ferroelectricity has been observed in biological systems, filling an important missing link in bioelectric phenomena. In this perspective, we will present short historical notes on ferroelectrics, followed by overview on the fundamentals of ferroelectricity. Latest development in molecular ferroelectrics and biological ferroelectricity will then be highlighted, and their implications and potential applications will be discussed. We close by noting molecular ferroelectric as an exciting frontier between electronics and biology, and a number of challenges ahead are also noted. PMID:24018952

  8. Molecular ferroelectrics: where electronics meet biology.

    PubMed

    Li, Jiangyu; Liu, Yuanming; Zhang, Yanhang; Cai, Hong-Ling; Xiong, Ren-Gen

    2013-12-28

    In the last several years, we have witnessed significant advances in molecular ferroelectrics, with the ferroelectric properties of molecular crystals approaching those of barium titanate. In addition, ferroelectricity has been observed in biological systems, filling an important missing link in bioelectric phenomena. In this perspective, we will present short historical notes on ferroelectrics, followed by an overview of the fundamentals of ferroelectricity. The latest developments in molecular ferroelectrics and biological ferroelectricity will then be highlighted, and their implications and potential applications will be discussed. We close by noting molecular ferroelectric as an exciting frontier between electronics and biology, and a number of challenges ahead are also described.

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

    PubMed

    Kubota, Yoshiyuki

    2015-02-01

    Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed.

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

    PubMed

    Kubota, Yoshiyuki

    2015-02-01

    Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed. PMID:25564566

  11. Nanocrystal size distribution analysis from transmission electron microscopy images

    NASA Astrophysics Data System (ADS)

    van Sebille, Martijn; van der Maaten, Laurens J. P.; Xie, Ling; Jarolimek, Karol; Santbergen, Rudi; van Swaaij, René A. C. M. M.; Leifer, Klaus; Zeman, Miro

    2015-12-01

    We propose a method, with minimal bias caused by user input, to quickly detect and measure the nanocrystal size distribution from transmission electron microscopy (TEM) images using a combination of Laplacian of Gaussian filters and non-maximum suppression. We demonstrate the proposed method on bright-field TEM images of an a-SiC:H sample containing embedded silicon nanocrystals with varying magnifications and we compare the accuracy and speed with size distributions obtained by manual measurements, a thresholding method and PEBBLES. Finally, we analytically consider the error induced by slicing nanocrystals during TEM sample preparation on the measured nanocrystal size distribution and formulate an equation to correct this effect.We propose a method, with minimal bias caused by user input, to quickly detect and measure the nanocrystal size distribution from transmission electron microscopy (TEM) images using a combination of Laplacian of Gaussian filters and non-maximum suppression. We demonstrate the proposed method on bright-field TEM images of an a-SiC:H sample containing embedded silicon nanocrystals with varying magnifications and we compare the accuracy and speed with size distributions obtained by manual measurements, a thresholding method and PEBBLES. Finally, we analytically consider the error induced by slicing nanocrystals during TEM sample preparation on the measured nanocrystal size distribution and formulate an equation to correct this effect. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06292f

  12. 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

  13. Electron microscopy study of direct laser deposited IN718

    SciTech Connect

    Ding, R.G.; Huang, Z.W.; Li, H.Y.; Mitchell, I.; Baxter, G.; Bowen, P.

    2015-08-15

    The microstructure of direct laser deposited (DLD) IN718 has been investigated in detail using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results confirm that the dendrite core microstructure can be linked to the cooling rate experienced during the deposition. A ~ 100 μm wide δ partially dissolved region in the IN718 substrate was observed close to the substrate/deposit boundary. In the deposited IN718, γ/Laves eutectic constituent is the predominant minor microconstituent. Irregular and regular (small) (Nb,Ti)C carbides and a mixture of the carbides and Laves were observed. Most M{sub 3}B{sub 2} borides were nucleated around a (Nb,Ti)C carbide. Needles of δ phase precipitated from the Laves phase were also observed. A complex constituent (of Laves, δ, α-Cr, γ″, and γ matrix) is reported in IN718 for the first time. The formation of α-Cr particles could be related to Cr rejection during the formation and growth of Cr-depleted δ phase. - Highlights: • Secondary phases in IN718 deposits were identified using electron diffraction and EDS. • MC, M{sub 3}B{sub 2}, γ/Laves eutectic and γ/NbC/Laves eutectic were observed. • Needle-like δ phases were precipitated from the Laves phase. • A complex constituent (Laves, δ, α-Cr, γ″ and γ) was reported for the first time.

  14. 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.

  15. Exploiting desktop supercomputing for three-dimensional electron microscopy reconstructions using ART with blobs.

    PubMed

    Bilbao-Castro, J R; Marabini, R; Sorzano, C O S; García, I; Carazo, J M; Fernández, J J

    2009-01-01

    Three-dimensional electron microscopy allows direct visualization of biological macromolecules close to their native state. The high impact of this technique in the structural biology field is highly correlated with the development of new image processing algorithms. In order to achieve subnanometer resolution, the size and number of images involved in a three-dimensional reconstruction increase and so do computer requirements. New chips integrating multiple processors are hitting the market at a reduced cost. This high-integration, low-cost trend has just begun and is expected to bring real supercomputers to our laboratory desktops in the coming years. This paper proposes a parallel implementation of a computation-intensive algorithm for three-dimensional reconstruction, ART, that takes advantage of the computational power in modern multicore platforms. ART is a sophisticated iterative reconstruction algorithm that has turned out to be well suited for the conditions found in three-dimensional electron microscopy. In view of the performance obtained in this work, these modern platforms are expected to play an important role to face the future challenges in three-dimensional electron microscopy.

  16. Exploiting desktop supercomputing for three-dimensional electron microscopy reconstructions using ART with blobs.

    PubMed

    Bilbao-Castro, J R; Marabini, R; Sorzano, C O S; García, I; Carazo, J M; Fernández, J J

    2009-01-01

    Three-dimensional electron microscopy allows direct visualization of biological macromolecules close to their native state. The high impact of this technique in the structural biology field is highly correlated with the development of new image processing algorithms. In order to achieve subnanometer resolution, the size and number of images involved in a three-dimensional reconstruction increase and so do computer requirements. New chips integrating multiple processors are hitting the market at a reduced cost. This high-integration, low-cost trend has just begun and is expected to bring real supercomputers to our laboratory desktops in the coming years. This paper proposes a parallel implementation of a computation-intensive algorithm for three-dimensional reconstruction, ART, that takes advantage of the computational power in modern multicore platforms. ART is a sophisticated iterative reconstruction algorithm that has turned out to be well suited for the conditions found in three-dimensional electron microscopy. In view of the performance obtained in this work, these modern platforms are expected to play an important role to face the future challenges in three-dimensional electron microscopy. PMID:18940260

  17. Modular electron transfer circuits for synthetic biology

    PubMed Central

    Agapakis, Christina M

    2010-01-01

    Electron transfer is central to a wide range of essential metabolic pathways, from photosynthesis to fermentation. The evolutionary diversity and conservation of proteins that transfer electrons makes these pathways a valuable platform for engineered metabolic circuits in synthetic biology. Rational engineering of electron transfer pathways containing hydrogenases has the potential to lead to industrial scale production of hydrogen as an alternative source of clean fuel and experimental assays for understanding the complex interactions of multiple electron transfer proteins in vivo. We designed and implemented a synthetic hydrogen metabolism circuit in Escherichia coli that creates an electron transfer pathway both orthogonal to and integrated within existing metabolism. The design of such modular electron transfer circuits allows for facile characterization of in vivo system parameters with applications toward further engineering for alternative energy production. PMID:21468209

  18. Electron microscopy of primary cell cultures in solution and correlative optical microscopy using ASEM.

    PubMed

    Hirano, Kazumi; Kinoshita, Takaaki; Uemura, Takeshi; Motohashi, Hozumi; Watanabe, Yohei; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Sato, Mari; Suga, Mitsuo; Maruyama, Yuusuke; Tsuji, Noriko M; Yamamoto, Masayuki; Nishihara, Shoko; Sato, Chikara

    2014-08-01

    Correlative light-electron microscopy of cells in a natural environment of aqueous liquid facilitates high-throughput observation of protein complex formation. ASEM allows the inverted SEM to observe the wet sample from below, while an optical microscope observes it from above quasi-simultaneously. The disposable ASEM dish with a silicon nitride (SiN) film window can be coated variously to realize the primary-culture of substrate-sensitive cells in a few milliliters of culture medium in a stable incubator environment. Neuron differentiation, neural networking, proplatelet-formation and phagocytosis were captured by optical or fluorescence microscopy, and imaged at high resolution by gold-labeled immuno-ASEM with/without metal staining. Fas expression on the cell surface was visualized, correlated to the spatial distribution of F-actin. Axonal partitioning was studied using primary-culture neurons, and presynaptic induction by GluRδ2-N-terminus-linked fluorescent magnetic beads was correlated to the presynaptic-marker Bassoon. Further, megakaryocytes secreting proplatelets were captured, and P-selectins with adherence activity were localized to some of the granules present by immuno-ASEM. The phagocytosis of lactic acid bacteria by dendritic cells was also imaged. Based on these studies, ASEM correlative microscopy promises to allow the study of various mesoscopic-scale dynamics in the near future. PMID:24216127

  19. Analytical electron microscopy study of radioactive ceramic waste form

    SciTech Connect

    O'Holleran, T. P.; Sinkler, W.; Moschetti, T. L.; Johnson, S. G.; Goff, K. M.

    1999-11-11

    A ceramic waste form has been developed to immobilize the halide high-level waste stream from electrometallurgical treatment of spent nuclear fuel. Analytical electron microscopy studies, using both scanning and transmission instruments, have been performed to characterize the microstructure of this material. The microstructure consists primarily of sodalite granules (containing the bulk of the halides) bonded together with glass. The results of these studies are discussed in detail. Insight into the waste form fabrication process developed as a result of these studies is also discussed.

  20. Microstructural studies of dental amalgams using analytical transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Hooghan, Tejpal Kaur

    Dental amalgams have been used for centuries as major restorative materials for decaying teeth. Amalgams are prepared by mixing alloy particles which contain Ag, Sn, and Cu as the major constituent elements with liquid Hg. The study of microstructure is essential in understanding the setting reactions and improving the properties of amalgams. Until the work reported in this dissertation, optical microscopy (OM), scanning electron microscopy (SEM), and x-ray diffractometry (XRD) were used commonly to analyze amalgam microstructures. No previous systematic transmission electron microscopy (TEM) study has been performed due to sample preparation difficulties and composite structure of dental amalgams. The goal of this research was to carry out detailed microstructural and compositional studies of dental amalgams. This was accomplished using the enhanced spatial resolution of the TEM and its associated microanalytical techniques, namely, scanning transmission electron microscopy (STEM), x-ray energy dispersive spectroscopy (XEDS) and micro-microdiffraction (mumuD). A new method was developed for thinning amalgam samples to electron transparency using the "wedge technique." Velvalloy, a low-Cu amalgam, and Tytin, a high-Cu amalgam, were the two amalgams characterized. Velvalloy is composed of a Agsb2Hgsb3\\ (gammasb1)/HgSnsb{7-9}\\ (gammasb2) matrix surrounding unreacted Agsb3Sn (gamma) particles. In addition, hitherto uncharacterized reaction layers between Agsb3Sn(gamma)/Agsb2Hgsb3\\ (gammasb2)\\ and\\ Agsb2Hgsb3\\ (gammasb1)/HgSnsb{7-9}\\ (gammasb2) were observed and analyzed. An Ag-Hg-Sn (betasb1) phase was clearly identified for the first time. In Tytin, the matrix consists of Agsb2Hgsb3\\ (gammasb1) grains. Fine precipitates of Cusb6Snsb5\\ (etasp') are embedded inside the gammasb1 and at the grain boundaries. These precipitates are responsible for the improved creep resistance of Tytin compared to Velvalloy. The additional Cu has completely eliminated the gammasb

  1. Simultaneous orientation and thickness mapping in transmission electron microscopy

    SciTech Connect

    Tyutyunnikov, Dmitry; Özdöl, V. Burak; Koch, Christoph T.

    2014-12-04

    In this paper we introduce an approach for simultaneous thickness and orientation mapping of crystalline samples by means of transmission electron microscopy. We show that local thickness and orientation values can be extracted from experimental dark-field (DF) image data acquired at different specimen tilts. The method has been implemented to automatically acquire the necessary data and then map thickness and crystal orientation for a given region of interest. We have applied this technique to a specimen prepared from a commercial semiconductor device, containing multiple 22 nm technology transistor structures. The performance and limitations of our method are discussed and compared to those of other techniques available.

  2. Bulk electronic structure of metals resolved with scanning tunneling microscopy.

    PubMed

    Pascual, J I; Dick, A; Hansmann, M; Rust, H-P; Neugebauer, J; Horn, K

    2006-02-01

    We demonstrate that bulk band structure can have a strong influence in scanning tunneling microscopy measurements by resolving electronic interference patterns associated with scattering phenomena of bulk states at a metal surface and reconstructing the bulk band topology. Our data reveal that bulk information can be detected because states at the edge of the surface-projected bulk band have a predominant role on the scattering patterns. With the aid of density functional calculations, we associate this effect with an intrinsic increase in the projected density of states of edge states. This enhancement is characteristic of the three-dimensional bulk band curvature, a phenomenon analog to a van Hove singularity.

  3. Correlated cryogenic photoactivated localization microscopy and cryo-electron tomography.

    PubMed

    Chang, Yi-Wei; Chen, Songye; Tocheva, Elitza I; Treuner-Lange, Anke; Löbach, Stephanie; Søgaard-Andersen, Lotte; Jensen, Grant J

    2014-07-01

    Cryo-electron tomography (CET) produces three-dimensional images of cells in a near-native state at macromolecular resolution, but identifying structures of interest can be challenging. Here we describe a correlated cryo-PALM (photoactivated localization microscopy)-CET method for localizing objects within cryo-tomograms to beyond the diffraction limit of the light microscope. Using cryo-PALM-CET, we identified multiple and new conformations of the dynamic type VI secretion system in the crowded interior of Myxococcus xanthus.

  4. Electron microscopy of a Gd-Ba-Cu-O superconductor

    NASA Technical Reports Server (NTRS)

    Ramesh, R.; Thomas, G.; Meng, R. L.; Hor, P. H.; Chu, C. W.

    1989-01-01

    An electron microscopy study has been carried out to characterize the microstructure of a sintered Gd-Ba-Cu-O superconductor alloy. The GdBa2Cu3O(7-x) phase in the oxygen annealed sample is orthorhombic, while in the vacuum annealed sample it is tetragonal. It is shown that the details of the fine structure in the 001-line zone axis convergent beam patterns can be used to distinguish between the orthorhombic form and the tetragonal form. In addition to this matrix phase, an amorphous phase is frequently observed at the triple grain junctions. Gd-rich inclusions have been observed inside the matrix phase.

  5. Photoemission Electron Microscopy of a Plasmonic Silver Nanoparticle Trimer

    SciTech Connect

    Peppernick, Samuel J.; Joly, Alan G.; Beck, Kenneth M.; Hess, Wayne P.; Wang, Jinyong; Wang, Yi-Chung; Wei, Wei

    2013-07-01

    We present a combined experimental and theoretical study to investigate the spatial distribution of photoelectrons emitted from core-shell silver (Ag) nanoparticles. We use two-photon photoemission microscopy (2P-PEEM) to spatially resolve electron emission from a trimeric core-shell aggregate of triangular symmetry. Finite difference time domain (FDTD) simulations are performed to model the intensity distributions of the electromagnetic near-fields resulting from femtosecond (fs) laser excitation of localized surface plasmon oscillations in the triangular core-shell structure. We demonstrate that the predicted FDTD near-field intensity distribution reproduces the 2P-PEEM photoemission pattern.

  6. Segmentation of virus particle candidates in transmission electron microscopy images.

    PubMed

    Kylberg, G; Uppström, M; Hedlund, K-O; Borgefors, G; Sintorn, I-M

    2012-02-01

    In this paper, we present an automatic segmentation method that detects virus particles of various shapes in transmission electron microscopy images. The method is based on a statistical analysis of local neighbourhoods of all the pixels in the image followed by an object width discrimination and finally, for elongated objects, a border refinement step. It requires only one input parameter, the approximate width of the virus particles searched for. The proposed method is evaluated on a large number of viruses. It successfully segments viruses regardless of shape, from polyhedral to highly pleomorphic.

  7. Scanning electron microscopy of adult Gongylonema pulchrum (Nematoda: Spirurida).

    PubMed

    Naem, S; Seifi, H; Simon, G T

    2000-05-01

    Scanning electron microscopy (SEM) was used to study the surface ultrastructure of adult worms of Gongylonema pulchrum. The anterior end in both sexes was covered by numerous cuticular platelets. There was a pair of lateral cervical papillac. The buccal opening was small and extended in the dorsoventral direction. Around the mouth a cuticular elevation enclosed the labia, and eight papillae were located laterodorsally and lateroventrally. Two large lateral amphids were seen. On the lateral sides of the female's tail, phasmidal apertures were observed. The caudal end of the male was asymmetrically alate and bore 10 pairs of papillae and two phasmidal apertures.

  8. Visualizing aquatic bacteria by light and transmission electron microscopy.

    PubMed

    Silva, Thiago P; Noyma, Natália P; Duque, Thabata L A; Gamalier, Juliana P; Vidal, Luciana O; Lobão, Lúcia M; Chiarini-Garcia, Hélio; Roland, Fábio; Melo, Rossana C N

    2014-01-01

    The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.

  9. Cryogenic electron microscopy and single-particle analysis.

    PubMed

    Elmlund, Dominika; Elmlund, Hans

    2015-01-01

    About 20 years ago, the first three-dimensional (3D) reconstructions at subnanometer (<10-Å) resolution of an icosahedral virus assembly were obtained by cryogenic electron microscopy (cryo-EM) and single-particle analysis. Since then, thousands of structures have been determined to resolutions ranging from 30 Å to near atomic (<4 Å). Almost overnight, the recent development of direct electron detectors and the attendant improvement in analysis software have advanced the technology considerably. Near-atomic-resolution reconstructions can now be obtained, not only for megadalton macromolecular complexes or highly symmetrical assemblies but also for proteins of only a few hundred kilodaltons. We discuss the developments that led to this breakthrough in high-resolution structure determination by cryo-EM and point to challenges that lie ahead.

  10. Theory and application of scanning electron acoustic microscopy

    NASA Technical Reports Server (NTRS)

    Cantrell, John H.; Qian, Menglu; Chen, Ruiyi; Yost, William T.

    1992-01-01

    A three-dimensional theoretical model based on the application of the thermal conduction and Navier equations to a chopped electron beam incident on a disk specimen is used to obtain the particle displacement field in the specimen. The results lead to a consideration of the signal generation, spatial resolution, and contrast mechanisms in scanning electron acoustic microscopy (SEAM). The model suggests that the time-variant heat source produced by the beam chopping generates driving source, thermal wave, and acoustic wave displacements simultaneously in the specimen. Evidence of the correctness of the prediction is obtained from the mathematically similar problem of pulsed laser light injection into a tank of water. High speed Schlieren photographs taken following laser injection show the simultaneous evolution of thermal and acoustic waveforms. Examples of contrast reversal, stress-induced contrast, and acoustic zone contrast and resolution with SEAM are presented and explained in terms of the model features.

  11. Combined Scanning Transmission Electron Microscopy Tilt- and Focal Series

    SciTech Connect

    Dahmen, Tim; Baudoin, Jean-Pierre G; Lupini, Andrew R; Kubel, Christian; Slusallek, Phillip; De Jonge, Niels

    2014-01-01

    In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller missing wedge artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.

  12. Four-dimensional ultrafast electron microscopy of phase transitions.

    PubMed

    Grinolds, Michael S; Lobastov, Vladimir A; Weissenrieder, Jonas; Zewail, Ahmed H

    2006-12-01

    Reported here is direct imaging (and diffraction) by using 4D ultrafast electron microscopy (UEM) with combined spatial and temporal resolutions. In the first phase of UEM, it was possible to obtain snapshot images by using timed, single-electron packets; each packet is free of space-charge effects. Here, we demonstrate the ability to obtain sequences of snapshots ("movies") with atomic-scale spatial resolution and ultrashort temporal resolution. Specifically, it is shown that ultrafast metal-insulator phase transitions can be studied with these achieved spatial and temporal resolutions. The diffraction (atomic scale) and images (nanometer scale) we obtained manifest the structural phase transition with its characteristic hysteresis, and the time scale involved (100 fs) is now studied by directly monitoring coordinates of the atoms themselves. PMID:17130445

  13. Modeling atomic-resolution scanning transmission electron microscopy images.

    PubMed

    Findlay, Scott D; Oxley, Mark P; Allen, Leslie J

    2008-02-01

    A real-space description of inelastic scattering in scanning transmission electron microscopy is derived with particular attention given to the implementation of the projected potential approximation. A hierarchy of approximations to expressions for inelastic images is presented. Emphasis is placed on the conditions that must hold in each case. The expressions that justify the most direct, visual interpretation of experimental data are also the most approximate. Therefore, caution must be exercised in selecting experimental parameters that validate the approximations needed for the analysis technique used. To make the most direct, visual interpretation of electron-energy-loss spectroscopic images from core-shell excitations requires detector improvements commensurate with those that aberration correction provides for the probe-forming lens. Such conditions can be relaxed when detailed simulations are performed as part of the analysis of experimental data. PMID:18096101

  14. Modelling atomic resolution scanning transmission electron microscopy images

    SciTech Connect

    Findlay, Scott D.; Oxley, Mark P; Allen, L. J.

    2008-01-01

    A real-space description of inelastic scattering in scanning transmission electron microscopy is derived with particular attention given to the implementation of the projected potential approximation. A hierarchy of approximations to expressions for inelastic images is presented. Emphasis is placed on the conditions that must hold in each case. The expressions that justify the most direct, visual interpretation of experimental data are also the most approximate. Therefore, caution must be exercised in selecting experimental parameters that validate the approximations needed for the analysis technique used. To make the most direct, visual interpretation of electron-energy-loss spectroscopic images from core-shell excitations requires detector improvements commensurate with those that aberration correction provides for the probe-forming lens. Such conditions can be relaxed when detailed simulations are performed as part of the analysis of experimental data.

  15. Sample heating system for spin-polarized scanning electron microscopy.

    PubMed

    Kohashi, Teruo; Motai, Kumi

    2013-08-01

    A sample-heating system for spin-polarized scanning electron microscopy (spin SEM) has been developed and used for microscopic magnetization analysis at temperatures up to 500°C. In this system, a compact ceramic heater and a preheating operation keep the ultra-high vacuum conditions while the sample is heated during spin SEM measurement. Moreover, the secondary-electron collector, which is arranged close to the sample, was modified so that it is not damaged at high temperatures. The system was used to heat a Co(1000) single-crystal sample from room temperature up to 500°C, and the magnetic-domain structures were observed. Changes of the domain structures were observed around 220 and 400°C, and these changes are considered to be due to phase transitions of this sample.

  16. Single sideband imaging in high-resolution electron microscopy

    NASA Astrophysics Data System (ADS)

    Hohenstein, M.

    1992-06-01

    More then 20 years ago, Hanßen and Morgenstern [1] described the case of single sideband imaging in electron microscopy. Single sideband imaging allows to correct artifacts in the imaging process due to spherical aberration and defocus and to reconstruct the electron wave function at the exit surface of the sample from experimental micrographs. In the present work, optimized imaging parameters allowed us to obtain new experimental results, thus confirming the resolution limit of single sideband imaging (0.13 nm) to be close to the information limit of a JEOL 4000EX microscope. Furthermore, the reconstructed exit surface wave functions were throuroughly checked by using them to calculate a focus series, which was compared with an experimental focus series.

  17. Study of Supported Particle Catalysts by Electron Microscopy Methods.

    NASA Astrophysics Data System (ADS)

    Yao, Ming-Hui

    1994-01-01

    The imaging conditions for electron microscopy study of supported ultrafine particle catalysts were investigated both theoretically and experimentally. Particles supported on crystalline supports were simulated and compared in high resolution electron microscopy (HREM) plan view and profile view as a function of defocus, voltage, aperture size, and support thickness. Possibilities and techniques for improving particle visibility and resolution by selecting objective lens defocus, Fourier filtering, and profile imaging were discussed. Various microscopy techniques, including HREM, high resolution scanning electron microscopy (HRSEM) and high-angle annular dark field imaging (HAADF) were used in parallel to study supported metal particle catalysts, and relative merits and shortcomings of each method were evaluated. It was pointed out that HREM profile imaging was the most effective technique for direct observation of microstructure, especially the surface structure of supported particles, whereas HRSEM and HAADF, respectively, were preferred for characterizing the surface topology of catalyst supports and the size distribution of supported particles. The HREM profile imaging method was used to study the strong metal-support interaction on various temperature treated Pt/CeO_2, and Pt/TiO _2 samples. Ti oxide monolayer on Pt/TiO _2 was observed, and related to the suppressed hydrogenolysis activity observed after high temperature reduction. No similar surface layer was observed on Pt/CeO_2 after high temperature treatment even though the hydrogenolysis activity was also strongly suppressed. It is proposed that decoration model is the main mechanism responsible for the SMSI for Pt/TiO _2, while morphological change and epitaxial relation is the major cause for the metal-support interaction for Pt/CeO_2. As well as surface structure, surface area was also studied in detail. A procedure for measuring surface area of supported particles by TEM was developed and applied to

  18. Characterization and Detection of Biological Weapons with Atomic Force Microscopy

    SciTech Connect

    Malkin, A J; Plomp, M; Leighton, T J; McPherson, A

    2006-09-25

    Critical gaps exist in our capabilities to rapidly characterize threat agents which could be used in attacks on facilities and military forces. DNA-based PCR and immunoassay-based techniques provide unique identification of species, strains and protein signatures of pathogens. However, differentiation between naturally occurring and weaponized bioagents and the identification of formulation signatures are beyond current technologies. One of the most effective and often the only definitive means to identify a threat agent is by its direct visualization. Atomic force microscopy (AFM) is a rapid imaging technique that covers the size range of most biothreat agents (several nanometers to tens of microns), is capable of resolving pathogen morphology and structure, and could be developed into a portable device for biological weapons (BW) field characterization. AFM can detect pathogens in aerosol, liquid, surface and soil samples while concomitantly acquiring their weaponization and threat agent digital signatures. BW morphological and structural signatures, including modifications to pathogen microstructural architecture and topology that occur during formulation and weaponization, provide the means for their differentiation from crude or purified unformulated agent, processing signatures, as well as assessment of their potential for dispersion, inhalation and environmental persistence. AFM visualization of pathogen morphology and architecture often provides valuable digital signatures and allows direct detection and identification of threat agents. We have demonstrated that pathogens, spanning the size range from several nanometers for small agricultural satellite viruses to almost half micron for pox viruses, and to several microns for bacteria and bacterial spores, can be visualized by AFM under physiological conditions to a resolution of {approx}20-30 {angstrom}. We have also demonstrated that viruses from closely related families could be differentiated by AFM on

  19. Advantages of environmental scanning electron microscopy in studies of microorganisms.

    PubMed

    Collins, S P; Pope, R K; Scheetz, R W; Ray, R I; Wagner, P A; Little, B J

    1993-08-01

    Microorganisms, including bacteria, fungi, protozoa, and microalgae, are composed predominantly of water which prohibits direct observation in a traditional scanning electron microscope (SEM). Preparation for SEM requires that microorganisms be fixed, frozen or dehydrated, and coated with a conductive film before observation in a high vacuum environment. Sample preparation may mechanically disturb delicate samples, compromise morphological information, and introduce other artifacts. The environmental scanning electron microscope (ESEM) provides a technology for imaging hydrated or dehydrated biological samples with minimal manipulation and without the need for conductive coatings. Sporulating cultures of three fungi, Aspergillus sp., Cunninghamella sp., and Mucor sp., were imaged in the ESEM to assess usefulness of the instrument in the direct observation of delicate, uncoated, biological specimens. Asexual sporophores showed no evidence of conidial displacement or disruption of sporangia. Uncoated algal cells of Euglena gracilis and Spirogyra sp. were examined using the backscatter electron detector (BSE) and the environmental secondary electron detector (ESD) of the ESEM. BSE images had more clearly defined intracellular structures, whereas ESD gave a clearer view of the surface E. gracilis cells fixed with potassium permanganate, Spirogyra sp. stained with Lugol's solution, and Saprolegnia sp. fixed with osmium tetroxide were compared using BSE and ESD to demonstrate that cellular details could be enhanced by the introduction of heavy metals. The effect of cellular water on signal quality was evaluated by comparing hydrated to critical point dried specimens.

  20. Advantages of environmental scanning electron microscopy in studies of microorganisms.

    PubMed

    Collins, S P; Pope, R K; Scheetz, R W; Ray, R I; Wagner, P A; Little, B J

    1993-08-01

    Microorganisms, including bacteria, fungi, protozoa, and microalgae, are composed predominantly of water which prohibits direct observation in a traditional scanning electron microscope (SEM). Preparation for SEM requires that microorganisms be fixed, frozen or dehydrated, and coated with a conductive film before observation in a high vacuum environment. Sample preparation may mechanically disturb delicate samples, compromise morphological information, and introduce other artifacts. The environmental scanning electron microscope (ESEM) provides a technology for imaging hydrated or dehydrated biological samples with minimal manipulation and without the need for conductive coatings. Sporulating cultures of three fungi, Aspergillus sp., Cunninghamella sp., and Mucor sp., were imaged in the ESEM to assess usefulness of the instrument in the direct observation of delicate, uncoated, biological specimens. Asexual sporophores showed no evidence of conidial displacement or disruption of sporangia. Uncoated algal cells of Euglena gracilis and Spirogyra sp. were examined using the backscatter electron detector (BSE) and the environmental secondary electron detector (ESD) of the ESEM. BSE images had more clearly defined intracellular structures, whereas ESD gave a clearer view of the surface E. gracilis cells fixed with potassium permanganate, Spirogyra sp. stained with Lugol's solution, and Saprolegnia sp. fixed with osmium tetroxide were compared using BSE and ESD to demonstrate that cellular details could be enhanced by the introduction of heavy metals. The effect of cellular water on signal quality was evaluated by comparing hydrated to critical point dried specimens. PMID:8400431

  1. Spiral phase plate contrast in optical and electron microscopy

    NASA Astrophysics Data System (ADS)

    Juchtmans, Roeland; Clark, Laura; Lubk, Axel; Verbeeck, Jo

    2016-08-01

    The use of phase plates in the back focal plane of a microscope is a well-established technique in optical microscopy to increase the contrast of weakly interacting samples and is gaining interest in electron microscopy as well. In this paper we study the spiral phase plate (SPP), also called helical, vortex, or two-dimensional Hilbert phase plate, which adds an angularly dependent phase of the form ei ℓ ϕk to the exit wave in Fourier space. In the limit of large collection angles, we analytically calculate that the average of a pair of ℓ =±1 SPP filtered images is directly proportional to the gradient squared of the exit wave, explaining the edge contrast previously seen in optical SPP work. We discuss the difference between a clockwise-anticlockwise pair of SPP filtered images and derive conditions under which the modulus of the wave's gradient can be seen directly from one SPP filtered image. This work provides the theoretical background to interpret images obtained with a SPP, thereby opening new perspectives for new experiments to study, for example, magnetic materials in an electron microscope.

  2. Frontiers of in situ electron microscopy

    SciTech Connect

    Zheng, Haimei; Zhu, Yimei; Meng, Shirley Ying

    2015-01-01

    In situ transmission electron microscopy (TEM) has become an increasingly important tool for materials characterization. It provides key information on the structural dynamics of a material during transformations and the correlation between structure and properties of materials. With the recent advances in instrumentation, including aberration corrected optics, sample environment control, the sample stage, and fast and sensitive data acquisition, in situ TEM characterization has become more and more powerful. In this article, a brief review of the current status and future opportunities of in situ TEM is included. It also provides an introduction to the six articles covered by in this issue of MRS Bulletin explore the frontiers of in situ electron microscopy, including liquid and gas environmental TEM, dynamic four-dimensional TEM, nanomechanics, ferroelectric domain switching studied by in situ TEM, and state-of-the-art atomic imaging of light elements (i.e., carbon atoms) and individual defects.

  3. Amyloid structure and assembly: insights from scanning transmission electron microscopy.

    PubMed

    Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N; Steven, Alasdair C; Engel, Andreas; Wall, Joseph S; Aebi, Ueli; Müller, Shirley A

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

  4. Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

    SciTech Connect

    Goldsbury, C.; Wall, J.; Baxa, U.; Simon, M. N.; Steven, A. C.; Engel, A.; Aebi, U.; Muller, S. A.

    2011-01-01

    Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

  5. In-Situ Transmission Electron Microscopy with Nanosecond Temporal Resolution

    NASA Astrophysics Data System (ADS)

    Browning, Nigel

    2012-02-01

    The dynamic transmission electron microscope (DTEM) can obtain both high spatial (˜1nm or better) and high temporal (˜1μs or faster) resolution. The high temporal resolution is achieved by using a short pulse laser to create the pulse of electrons through photo-emission. This pulse of electrons is propagated down the microscope column in the same way as in a conventional high-resolution TEM. The only difference is that the spatial resolution is limited by the electron-electron interactions in the pulse (a typical 10ns pulse contains ˜10^9 electrons). To synchronize this pulse of electrons with a particular dynamic event, a second laser is used to ``drive'' the sample a defined time interval prior to the arrival of the laser pulse. The important aspect of the DTEM is that one pulse of electrons is used to form the whole image, allowing irreversible transitions and cumulative phenomena such as nucleation and growth, to be studied directly in the microscope. The use of the drive laser for fast heating of the specimen presents differences and several advantages over conventional resistive heating in-situ TEM -- such as the ability to drive the sample into non-equilibrium states. So far, the drive laser has been used for in-situ processing of nanoscale materials, rapid and high temperature phase transformations, and controlled thermal activation of materials. In this presentation, a summary of the development of the DTEM and in-situ stages to control the environment around the specimen will be described. Particular attention will be paid to the potential for gas stages to study catalytic processes and liquid stages to study biological specimens in their live hydrated states. The future potential improvements in spatial and temporal resolution that can be expected through the implementation of upgrades to the lasers, electron optics and detectors will also be discussed.

  6. Carbon Nanotube Atomic Force Microscopy for Proteomics and Biological Forensics

    SciTech Connect

    Noy, A; De Yoreo, J J; Malkin, A J

    2002-01-01

    The Human Genome Project was focused on mapping the complete genome. Yet, understanding the structure and function of the proteins expressed by the genome is the real end game. But there are approximately 100,000 proteins in the human body and the atomic structure has been determined for less than 1% of them. Given the current rate at which structures are being solved, it will take more than one hundred years to complete this task. The rate-limiting step in protein structure determination is the growth of high-quality single crystals for X-ray diffraction. Synthesis of the protein stock solution as well as X-ray diffraction and analysis can now often be done in a matter of weeks, but developing a recipe for crystallization can take years and, especially in the case of membrane proteins, is often completely unsuccessful. Consequently, techniques that can either help to elucidate the factors controlling macromolecular crystallization, increase the amount of structural information obtained from crystallized macromolecules or eliminate the need for crystallization altogether are of enormous importance. In addition, potential applications for those techniques extend well beyond the challenges of proteomics. The global spread of modern technology has brought with it an increasing threat from biological agents such as viruses. As a result, developing techniques for identifying and understanding the operation of such agents is becoming a major area of forensic research for DOE. Previous to this project, we have shown that we can use in situ atomic force microscopy (AFM) to image the surfaces of growing macromolecular crystals with molecular resolution (1-5) In addition to providing unprecedented information about macromolecular nucleation, growth and defect structure, these results allowed us to obtain low-resolution phase information for a number of macromolecules, providing structural information that was not obtainable from X-ray diffraction(3). For some virus systems

  7. Dimensional comparison between amplitude-modulation atomic force microscopy and scanning ion conductance microscopy of biological samples

    NASA Astrophysics Data System (ADS)

    Kim, Joonhui; Choi, MyungHoon; Jung, Goo-Eun; Rahim Ferhan, Abdul; Cho, Nam-Joon; Cho, Sang-Joon

    2016-08-01

    The range of scanning probe microscopy (SPM) applications for atomic force microscopy (AFM) is expanding in the biological sciences field, reflecting an increasing demand for tools that can improve our fundamental understanding of the physics behind biological systems. However, the complexity associated with applying SPM techniques in biomedical research hampers the full exploitation of its capabilities. Recently, the development of scanning ion conductance microscopy (SICM) has overcome these limitations and enabled contact-free, high resolution imaging of live biological specimens. In this work, we demonstrate the limitation of AFM for imaging biological samples in liquid due to artifacts arising from AFM tip-sample interaction, and how SICM imaging is able to overcome those limitations with contact-free scanning. We also demonstrate that SICM measurements, when compared to AFM, show better fit to the actual dimensions of the biological samples. Our results highlight the superiority of SICM imaging, enabling it to be widely adopted as a general and versatile research tool for biological studies in the nanoscale.

  8. Dimensional comparison between amplitude-modulation atomic force microscopy and scanning ion conductance microscopy of biological samples

    NASA Astrophysics Data System (ADS)

    Kim, Joonhui; Choi, MyungHoon; Jung, Goo-Eun; Rahim Ferhan, Abdul; Cho, Nam-Joon; Cho, Sang-Joon

    2016-08-01

    The range of scanning probe microscopy (SPM) applications for atomic force microscopy (AFM) is expanding in the biological sciences field, reflecting an increasing demand for tools that can improve our fundamental understanding of the physics behind biological systems. However, the complexity associated with applying SPM techniques in biomedical research hampers the full exploitation of its capabilities. Recently, the development of scanning ion conductance microscopy (SICM) has overcome these limitations and enabled contact-free, high resolution imaging of live biological specimens. In this work, we demonstrate the limitation of AFM for imaging biological samples in liquid due to artifacts arising from AFM tip–sample interaction, and how SICM imaging is able to overcome those limitations with contact-free scanning. We also demonstrate that SICM measurements, when compared to AFM, show better fit to the actual dimensions of the biological samples. Our results highlight the superiority of SICM imaging, enabling it to be widely adopted as a general and versatile research tool for biological studies in the nanoscale.

  9. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells.

    PubMed

    Moreno-Azanza, Miguel; Bauluz, Blanca; Canudo, José Ignacio; Gasca, José Manuel; Torcida Fernández-Baldor, Fidel

    2016-01-01

    Abnormalities in the histo- and ultrastructure of the amniote eggshell are often related to diverse factors, such as ambient stress during egg formation, pathologies altering the physiology of the egg-laying females, or evolutionarily selected modifications of the eggshell structure that vary the physical properties of the egg, for example increasing its strength so as to avoid fracture during incubation. When dealing with fossil materials, all the above hypotheses are plausible, but a detailed taphonomical study has to be performed to rule out the possibility that secondary processes of recrystallization have occurred during fossilization. Traditional analyses, such as optical microscopy inspection and cathodoluminescence, have proven not to be enough to understand the taphonomic story of some eggshells. Recently, electron backscatter diffraction has been used, in combination with other techniques, to better understand the alteration of fossil eggshells. Here we present a combined study using scanning electron microscopy, optical microscopy, cathodoluminescence and electron backscatter diffraction of eggshell fragments assigned to Megaloolithus cf. siruguei from the Upper Cretaceous outcrops of the Cameros Basin. We focus our study on the presence of secondary shell units that mimic most aspects of the ultrastructure of the eggshell mammillae, but grow far from the inner surface of the eggshell. We call these structures extra-spherulites, describe their crystal structure and demonstrate their secondary origin. Our study has important implications for the interpretation of secondary shell units as biological or pathological structures. Thus, electron backscatter diffraction complements other microscope techniques as a useful tool for understanding taphonomical alterations in fossil eggshells.

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

    PubMed Central

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

    2010-01-01

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

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

    PubMed

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

    2010-07-27

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

  12. Scanning electron microscopy to probe working nanowire gas sensors

    NASA Astrophysics Data System (ADS)

    Liu, Yangmingyue

    This study is dedicated to the implementing of Electron-Beam-Induced Current (EBIC) microscopy to study the behavior of metal oxide semiconducting (MOS) nanowire (NW) gas sensor in situ under exposure to different environment. First, we reported the development of a single nanowire gas sensor compatible with an environmental cell. The major component of the device we use in this study is a single SnO2 nanowire attached to an electron transparent SiN membrane (50-100 nm thick), which was used for mounting nanowire working electrodes and surface imaging of NW. First the NW's conductivity is investigated in different temperatures. Higher temperature is proved to cause higher conductivity of NW. We also found that often the Schottky barrier is formed at the nanowire's contacts with Au and Au/Cr electrodes. Then NW's responses to gas and electron beam (from SEM) are analyzed quantitatively by current measurement. Electron-Beam-Induced Current technique was introduced for the first time to characterize the conductivity behavior of the nanowire during the gas sensing process. Resistive contrast was observed in the EBIC image.

  13. Contamination mitigation strategies for scanning transmission electron microscopy.

    PubMed

    Mitchell, D R G

    2015-06-01

    Modern scanning transmission electron microscopy (STEM) enables imaging and microanalysis at very high magnification. In the case of aberration-corrected STEM, atomic resolution is readily achieved. However, the electron fluxes used may be up to three orders of magnitude greater than those typically employed in conventional STEM. Since specimen contamination often increases with electron flux, specimen cleanliness is a critical factor in obtaining meaningful data when carrying out high magnification STEM. A range of different specimen cleaning methods have been applied to a variety of specimen types. The contamination rate has been measured quantitatively to assess the effectiveness of cleaning. The methods studied include: baking, cooling, plasma cleaning, beam showering and UV/ozone exposure. Of the methods tested, beam showering is rapid, experimentally convenient and very effective on a wide range of specimens. Oxidative plasma cleaning is also very effective and can be applied to specimens on carbon support films, albeit with some care. For electron beam-sensitive materials, cooling may be the method of choice. In most cases, preliminary removal of the bulk of the contamination by methods such as baking or plasma cleaning, followed by beam showering, where necessary, can result in a contamination-free specimen suitable for extended atomic scale imaging and analysis.

  14. High Speed, Radiation Hard CMOS Pixel Sensors for Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Contarato, Devis; Denes, Peter; Doering, Dionisio; Joseph, John; Krieger, Brad

    CMOS monolithic active pixel sensors are currently being established as the technology of choice for new generation digital imaging systems in Transmission Electron Microscopy (TEM). A careful sensor design that couples μm-level pixel pitches with high frame rate readout and radiation hardness to very high electron doses enables the fabrication of direct electron detectors that are quickly revolutionizing high-resolution TEM imaging in material science and molecular biology. This paper will review the principal characteristics of this novel technology and its advantages over conventional, optically-coupled cameras, and retrace the sensor development driven by the Transmission Electron Aberration corrected Microscope (TEAM) project at the LBNL National Center for Electron Microscopy (NCEM), illustrating in particular the imaging capabilities enabled by single electron detection at high frame rate. Further, the presentation will report on the translation of the TEAM technology to a finer feature size process, resulting in a sensor with higher spatial resolution and superior radiation tolerance currently serving as the baseline for a commercial camera system.

  15. A large area cooled-CCD detector for electron microscopy

    NASA Astrophysics Data System (ADS)

    Faruqi, A. R.; Andrews, H. N.; Raeburn, C.

    1994-09-01

    Large area cooled-CCDs are an excellent medium for (indirectly) recording electron images and electron diffraction patterns in real time and for use in electron tomography; real-time imaging is extremely useful in making rapid adjustments in the electron microscope. CCDs provide high sensitivity (useful for minimising dosage to radiation-sensitive biological specimen), good resolution, stable performance, excellent dynamic range and linearity and a reasonably fast readout. We have built an electron imaging device based on the EEV 1152 by 814 pixel CCD which is controlled from a unix based SUN Sparestation operating under X-Windows. The incident 100 kV electrons are converted to visible light in a 0.5 mm thick YAG single crystal which is imaged through a lens on to the CCD. The CCD electronics is designed to be as flexible as possible and allows a wide variation in the readout speed to cater for the relatively fast application where readout noise is less critical and low readout noise applications where the extra few seconds of readout time are not significant. The CCD electronics is built in VME format which is controlled through a S-bus to VME driver. With two parallel channels of readout the whole image can be read out in ˜ 1 s (using the faster readout speed) with 16 bit precision and the image is displayed under X-Windows in a few seconds. The present readout works at 500 kHz and has a noise of ˜ 30 e rms per pixel. With a Peltier cooling device we can operate the CCD at ˜ -40°C which reduces the dark current adequately to allow exposures of up to several minutes. Several examples of patterns collected with the system on a Philips CM12 microscope will be presented.

  16. Towards correlative super-resolution fluorescence and electron cryo-microscopy.

    PubMed

    Wolff, Georg; Hagen, Christoph; Grünewald, Kay; Kaufmann, Rainer

    2016-09-01

    Correlative light and electron microscopy (CLEM) has become a powerful tool in life sciences. Particularly cryo-CLEM, the combination of fluorescence cryo-microscopy (cryo-FM) permitting for non-invasive specific multi-colour labelling, with electron cryo-microscopy (cryo-EM) providing the undisturbed structural context at a resolution down to the Ångstrom range, has enabled a broad range of new biological applications. Imaging rare structures or events in crowded environments, such as inside a cell, requires specific fluorescence-based information for guiding cryo-EM data acquisition and/or to verify the identity of the structure of interest. Furthermore, cryo-CLEM can provide information about the arrangement of specific proteins in the wider structural context of their native nano-environment. However, a major obstacle of cryo-CLEM currently hindering many biological applications is the large resolution gap between cryo-FM (typically in the range of ∼400 nm) and cryo-EM (single nanometre to the Ångstrom range). Very recently, first proof of concept experiments demonstrated the feasibility of super-resolution cryo-FM imaging and the correlation with cryo-EM. This opened the door towards super-resolution cryo-CLEM, and thus towards direct correlation of structural details from both imaging modalities.

  17. 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.

  18. Waveguide evanescent field fluorescence microscopy & its application in cell biology

    NASA Astrophysics Data System (ADS)

    Hassanzadeh, Abdollah

    There are many powerful microscopy technologies available for the investigation of bulk materials as well as for thin film samples. Nevertheless, for imaging an interface, especially live cells on a substrate and ultra thin-films, only Total Internal Reflection Fluorescence (TIRF) microscopy is available. This TIRF microscopy allows imaging without interference of the bulk. Various approaches are employed in fluorescence microscopy applications to restrict the excitation and detection of fluorophores to a thin region of the specimen. Elimination of background fluorescence from outside the focal plane can dramatically improve the signal-to-noise ratio, and consequently, the spatial resolution of the features or events of interest. TIRF microscopy is an evanescent field based microscopy. In this method, fluorescent dyes are only excited within an evanescent field: roughly within 100 nm above a glass coverslip. This will allow imaging surface and interfacial issues of the glass coverslip and an adjacent material. Waveguide evanescent field fluorescence (WEFF) microscopy is a new development for imaging cell-substrate interactions in real time and in vitro. It is an alternative to TIRF microscopy. In this method the light is coupled into a waveguide via an optical grating. The coupled light propagates as a waveguide mode and exhibits an evanescent field on top of the waveguide. This can be used as a surface-bound illumination source to excite fluorophores. This evanescent field serves as an extremely powerful tool for quality control of thin films, to study cell-substrate contacts, and investigating the effect of external agents and drugs on the cell-substrate interaction in real time and in vitro. This new method has been established and optimized to minimize non-uniformity, scattering and photo bleaching issues. Visualizing and quantifying of the cell-substrates and solid thin films have been carried out by WEFF microscopy. The images of the cell-substrate interface

  19. Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

    PubMed Central

    Carlson, David B.; Evans, James E.

    2011-01-01

    The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples. PMID:21673645

  20. The dento-gingival junction as seen with light microscopy and scanning electron microscopy.

    PubMed

    Garnick, J J; Ringle, R D

    1988-06-01

    The purpose of this paper is to review the anatomical relationship of the Dento-Gingival Junction as seen in the human dentition. The junction is described under light microscopy and then reviewed as seen in the SEM with the author's unpublished findings. The authors' material was derived from extracted human teeth with remaining marginal gingival tissue. The specimens were fixed with 2% glutaraldehyde in 0.15M sodium cacodylate buffer (pH 7.2) for 24 h. The specimens were then washed and freeze-fractured in Freon 113 using liquid nitrogen. Afterwards they were processed by freeze-drying or CPD methods, coated with gold, and placed in the scanning electron microscope (SEM) for viewing. These specimens demonstrated the presence of numerous Sharpey's fibers at the cemental surface. A large number of fibrils intermingled with the fibers to produce a dense mass of tissue. Junctional epithelium, with the adjacent homogeneous dental cuticle was demonstrated. Plaque deposits on the tooth surface extended to a cell-free zone. Morphological detail viewed with SEM and light microscopy are compared. PMID:3041569

  1. Light microscopy and scanning electron microscopy study on microstructure of gallbladder mucosa in pig.

    PubMed

    Prozorowska, Ewelina; Jackowiak, Hanna

    2015-03-01

    The present light microscopy (LM) and scanning electron microscopy (SEM) studies on porcine gallbladder mucosa provide a description of the microstructures of great functional importance such as mucosal folds, the epithelium, glands, and lymphatic nodules. The results showed the regional structural differences of the porcine gallbladder wall. Depending on the part of the gallbladder, three types of mucosal structures were described: simple and branched folds and mucosal crypts. An important structural feature found in the mucosa is connected with the structural variety of type of mucosal folds, which change from simple located in the neck, to most composed, i.e., branched or joined, in the polygonal crypts toward the fundus of the gallbladder. The morphometric analysis showed statistically significantly differences in the form and size of the folds and between the fundus, body, and neck of the gallbladder. Differences in the size of mucosal epithelium are discussed in terms of processes of synthesis and secretion of glycoproteins. Regional, species-specific differences in morphology of mucosal subepithelial glands, i.e., their secretory units and openings, and intensity of mucus secretion were described. Our results on the pig gallbladder show adaptation and/or specialization in particular areas of the mucosa for (1) secretion of mucus in the neck or body of gallbladder and (2) for cyclic volume changes, especially in the fundus of gallbladder. The description of the microstructures of mucosa in the porcine gallbladder could be useful as reference data for numerous experiments on the bile tract in the pig.

  2. Robert Feulgen Prize Lecture 1995. Electronic light microscopy: present capabilities and future prospects.

    PubMed

    Shotton, D M

    1995-08-01

    Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy--video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy--considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical

  3. Robert Feulgen Prize Lecture 1995. Electronic light microscopy: present capabilities and future prospects.

    PubMed

    Shotton, D M

    1995-08-01

    Electronic light microscopy involves the combination of microscopic techniques with electronic imaging and digital image processing, resulting in dramatic improvements in image quality and ease of quantitative analysis. In this review, after a brief definition of digital images and a discussion of the sampling requirements for the accurate digital recording of optical images, I discuss the three most important imaging modalities in electronic light microscopy--video-enhanced contrast microscopy, digital fluorescence microscopy and confocal scanning microscopy--considering their capabilities, their applications, and recent developments that will increase their potential. Video-enhanced contrast microscopy permits the clear visualisation and real-time dynamic recording of minute objects such as microtubules, vesicles and colloidal gold particles, an order of magnitude smaller than the resolution limit of the light microscope. It has revolutionised the study of cellular motility, and permits the quantitative tracking of organelles and gold-labelled membrane bound proteins. In combination with the technique of optical trapping (optical tweezers), it permits exquisitely sensitive force and distance measurements to be made on motor proteins. Digital fluorescence microscopy enables low-light-level imaging of fluorescently labelled specimens. Recent progress has involved improvements in cameras, fluorescent probes and fluorescent filter sets, particularly multiple bandpass dichroic mirrors, and developments in multiparameter imaging, which is becoming particularly important for in situ hybridisation studies and automated image cytometry, fluorescence ratio imaging, and time-resolved fluorescence. As software improves and small computers become more powerful, computational techniques for out-of-focus blur deconvolution and image restoration are becoming increasingly important. Confocal microscopy permits convenient, high-resolution, non-invasive, blur-free optical

  4. A perspective of biological supramolecular electron transfer.

    PubMed

    Ramasarma, T

    1999-12-01

    Electron transfer is an essential activity in biological systems. The migrating electron originates from water-oxygen in photosynthesis and reverts to dioxygen in respiration. In this cycle two metal porphyrin complexes possessing circular conjugated system and macrocyclic pi-clouds, chlorophyll and heme, play a decisive role in mobilising electrons for travel over biological structures as extraneous electrons. Transport of electrons within proteins (as in cytochromes) and within DNA (during oxidative damage and repair) is known to occur. Initial evaluations did not favour formation of semiconducting pathways of delocalized electrons of the peptide bonds in proteins and of the bases in nucleic acids. Direct measurement of conductivity of bulk material and quantum chemical calculations of their polymeric structures also did not support electron transfer in both proteins and nucleic acids. New experimental approaches have revived interest in the process of charge transfer through DNA duplex. The fluorescence on photo-excitation of Ru-complex was found to be quenched by Rh-complex, when both were tethered to DNA and intercalated in the base stack. Similar experiments showed that damage to G-bases and repair of T-T dimers in DNA can occur by possible long range electron transfer through the base stack. The novelty of this phenomenon prompted the apt name, "chemistry at a distance". Based on experiments with ruthenium modified proteins, intramolecular electron transfer in proteins is now proposed to use pathways that include C-C sigma-bonds and surprisingly hydrogen bonds which remained out of favour for a long time. In support of this, some experimental evidence is now available showing that hydrogen bond-bridges facilitate transfer of electrons between metal-porphyrin complexes. By molecular orbital calculations over 20 years ago we found that "delocalization of an extraneous electron is pronounced when it enters low-lying virtual orbitals of the electronic structures

  5. In Situ Analytical Electron Microscopy for Probing Nanoscale Electrochemistry

    SciTech Connect

    Graetz J.; Meng, Y.S.; McGilvray, T.; Yang, M.-C.; Gostovic, D.; Wang, F.; Zeng, D.; Zhu, Y.

    2011-10-31

    Oxides and their tailored structures are at the heart of electrochemical energy storage technologies and advances in understanding and controlling the dynamic behaviors in the complex oxides, particularly at the interfaces, during electrochemical processes will catalyze creative design concepts for new materials with enhanced and better-understood properties. Such knowledge is not accessible without new analytical tools. New innovative experimental techniques are needed for understanding the chemistry and structure of the bulk and interfaces, more importantly how they change with electrochemical processes in situ. Analytical Transmission Electron Microscopy (TEM) is used extensively to study electrode materials ex situ and is one of the most powerful tools to obtain structural, morphological, and compositional information at nanometer scale by combining imaging, diffraction and spectroscopy, e.g., EDS (energy dispersive X-ray spectrometry) and Electron Energy Loss Spectrometry (EELS). Determining the composition/structure evolution upon electrochemical cycling at the bulk and interfaces can be addressed by new electron microscopy technique with which one can observe, at the nanometer scale and in situ, the dynamic phenomena in the electrode materials. In electrochemical systems, for instance in a lithium ion battery (LIB), materials operate under conditions that are far from equilibrium, so that the materials studied ex situ may not capture the processes that occur in situ in a working battery. In situ electrochemical operation in the ultra-high vacuum column of a TEM has been pursued by two major strategies. In one strategy, a 'nano-battery' can be fabricated from an all-solid-state thin film battery using a focused ion beam (FIB). The electrolyte is either polymer based or ceramic based without any liquid component. As shown in Fig. 1a, the interfaces between the active electrode material/electrolyte can be clearly observed with TEM imaging, in contrast to the

  6. Fluctuation Electron Microscopy of Amorphous and Polycrystalline Materials

    NASA Astrophysics Data System (ADS)

    Rezikyan, Aram

    Fluctuation Electron Microscopy (FEM) has become an effective materials' structure characterization technique, capable of probing medium-range order (MRO) that may be present in amorphous materials. Although its sensitivity to MRO has been exercised in numerous studies, FEM is not yet a quantitative technique. The holdup has been the discrepancy between the computed kinematical variance and the experimental variance, which previously was attributed to source incoherence. Although high-brightness, high coherence, electron guns are now routinely available in modern electron microscopes, they have not eliminated this discrepancy between theory and experiment. The main objective of this thesis was to explore, and to reveal, the reasons behind this conundrum. The study was started with an analysis of the speckle statistics of tilted dark-field TEM images obtained from an amorphous carbon sample, which confirmed that the structural ordering is sensitively detected by FEM. This analysis also revealed the inconsistency between predictions of the source incoherence model and the experimentally observed variance. FEM of amorphous carbon, amorphous silicon and ultra nanocrystalline diamond samples was carried out in an attempt to explore the conundrum. Electron probe and sample parameters were varied to observe the scattering intensity variance behavior. Results were compared to models of probe incoherence, diffuse scattering, atom displacement damage, energy loss events and multiple scattering. Models of displacement decoherence matched the experimental results best. Decoherence was also explored by an interferometric diffraction method using bilayer amorphous samples, and results are consistent with strong displacement decoherence in addition to temporal decoherence arising from the electron source energy spread and energy loss events in thick samples. It is clear that decoherence plays an important role in the long-standing discrepancy between experimental FEM and its

  7. Visualization and quantitative analysis of nanoparticles in the respiratory tract by transmission electron microscopy

    PubMed Central

    Mühlfeld, Christian; Rothen-Rutishauser, Barbara; Vanhecke, Dimitri; Blank, Fabian; Gehr, Peter; Ochs, Matthias

    2007-01-01

    Nanotechnology in its widest sense seeks to exploit the special biophysical and chemical properties of materials at the nanoscale. While the potential technological, diagnostic or therapeutic applications are promising there is a growing body of evidence that the special technological features of nanoparticulate material are associated with biological effects formerly not attributed to the same materials at a larger particle scale. Therefore, studies that address the potential hazards of nanoparticles on biological systems including human health are required. Due to its large surface area the lung is one of the major sites of interaction with inhaled nanoparticles. One of the great challenges of studying particle-lung interactions is the microscopic visualization of nanoparticles within tissues or single cells both in vivo and in vitro. Once a certain type of nanoparticle can be identified unambiguously using microscopic methods it is desirable to quantify the particle distribution within a cell, an organ or the whole organism. Transmission electron microscopy provides an ideal tool to perform qualitative and quantitative analyses of particle-related structural changes of the respiratory tract, to reveal the localization of nanoparticles within tissues and cells and to investigate the 3D nature of nanoparticle-lung interactions. This article provides information on the applicability, advantages and disadvantages of electron microscopic preparation techniques and several advanced transmission electron microscopic methods including conventional, immuno and energy-filtered electron microscopy as well as electron tomography for the visualization of both model nanoparticles (e.g. polystyrene) and technologically relevant nanoparticles (e.g. titanium dioxide). Furthermore, we highlight possibilities to combine light and electron microscopic techniques in a correlative approach. Finally, we demonstrate a formal quantitative, i.e. stereological approach to analyze the

  8. Image formation modeling in cryo-electron microscopy.

    PubMed

    Vulović, Miloš; Ravelli, Raimond B G; van Vliet, Lucas J; Koster, Abraham J; Lazić, Ivan; Lücken, Uwe; Rullgård, Hans; Öktem, Ozan; Rieger, Bernd

    2013-07-01

    Accurate modeling of image formation in cryo-electron microscopy is an important requirement for quantitative image interpretation and optimization of the data acquisition strategy. Here we present a forward model that accounts for the specimen's scattering properties, microscope optics, and detector response. The specimen interaction potential is calculated with the isolated atom superposition approximation (IASA) and extended with the influences of solvent's dielectric and ionic properties as well as the molecular electrostatic distribution. We account for an effective charge redistribution via the Poisson-Boltzmann approach and find that the IASA-based potential forms the dominant part of the interaction potential, as the contribution of the redistribution is less than 10%. The electron wave is propagated through the specimen by a multislice approach and the influence of the optics is included via the contrast transfer function. We incorporate the detective quantum efficiency of the camera due to the difference between signal and noise transfer characteristics, instead of using only the modulation transfer function. The full model was validated against experimental images of 20S proteasome, hemoglobin, and GroEL. The simulations adequately predict the effects of phase contrast, changes due to the integrated electron flux, thickness, inelastic scattering, detective quantum efficiency and acceleration voltage. We suggest that beam-induced specimen movements are relevant in the experiments whereas the influence of the solvent amorphousness can be neglected. All simulation parameters are based on physical principles and, when necessary, experimentally determined.

  9. On mapping subangstrom electron clouds with force microscopy.

    PubMed

    Wright, C Alan; Solares, Santiago D

    2011-11-01

    In 2004 Hembacher et al. (Science 2004, 305, 380-383) reported simultaneous higher-harmonics atomic force mocroscopy (AFM)/scanning tunneling microscopy (STM) images acquired while scanning a graphite surface with a tungsten tip. They interpreted the observed subatomic features in the AFM images as the signature of lobes of increased electron density at the tungsten tip apex. Although these intriguing images have stirred controversy, an in-depth theoretical feasibility study has not yet been produced. Here we report on the development of a method for simulating higher harmonics AFM images and its application to the same system. Our calculations suggest that four lobes of increased electron density are expected to be present at a W(001) tip apex atom and that the corresponding higher harmonics AFM images of graphite can exhibit 4-fold symmetry features. Despite these promising results, open questions remain since the calculated amplitudes of the higher harmonics generated by the short-range forces are on the order of hundredths of picometers, leading to very small corrugations in the theoretical images. Additionally, the complex, intermittent nature of the tip-sample interaction, which causes constant readjustment of the tip and sample orbitals as the tip approaches and retracts from the surface, prevents a direct quantitative connection between the electron density and the AFM image features.

  10. High-Resolution Transmission Electron Microscopy Using Negative Spherical Aberration

    NASA Astrophysics Data System (ADS)

    Jia, Chun-Lin; Lentzen, Markus

    2004-04-01

    A novel imaging mode for high-resolution transmission electron microscopy is described. It is based on the adjustment of a negative value of the spherical aberration CS of the objective lens of a transmission electron microscope equipped with a multipole aberration corrector system. Negative spherical aberration applied together with an overfocus yields high-resolution images with bright-atom contrast. Compared to all kinds of images taken in conventional transmission electron microscopes, where the then unavoidable positive spherical aberration is combined with an underfocus, the contrast is dramatically increased. This effect can only be understood on the basis of a full nonlinear imaging theory. Calculations show that the nonlinear contrast contributions diminish the image contrast relative to the linear image for a positive-CS setting whereas they reinforce the image contrast relative to the linear image for a negative-CS setting. The application of the new mode to the imaging of oxygen in SrTiO3 and YBa2Cu3O7 demonstrates the benefit to materials science investigations. It allows us to image directly, without further image processing, strongly scattering heavy-atom columns together with weakly scattering light-atom columns.

  11. Image formation modeling in cryo-electron microscopy.

    PubMed

    Vulović, Miloš; Ravelli, Raimond B G; van Vliet, Lucas J; Koster, Abraham J; Lazić, Ivan; Lücken, Uwe; Rullgård, Hans; Öktem, Ozan; Rieger, Bernd

    2013-07-01

    Accurate modeling of image formation in cryo-electron microscopy is an important requirement for quantitative image interpretation and optimization of the data acquisition strategy. Here we present a forward model that accounts for the specimen's scattering properties, microscope optics, and detector response. The specimen interaction potential is calculated with the isolated atom superposition approximation (IASA) and extended with the influences of solvent's dielectric and ionic properties as well as the molecular electrostatic distribution. We account for an effective charge redistribution via the Poisson-Boltzmann approach and find that the IASA-based potential forms the dominant part of the interaction potential, as the contribution of the redistribution is less than 10%. The electron wave is propagated through the specimen by a multislice approach and the influence of the optics is included via the contrast transfer function. We incorporate the detective quantum efficiency of the camera due to the difference between signal and noise transfer characteristics, instead of using only the modulation transfer function. The full model was validated against experimental images of 20S proteasome, hemoglobin, and GroEL. The simulations adequately predict the effects of phase contrast, changes due to the integrated electron flux, thickness, inelastic scattering, detective quantum efficiency and acceleration voltage. We suggest that beam-induced specimen movements are relevant in the experiments whereas the influence of the solvent amorphousness can be neglected. All simulation parameters are based on physical principles and, when necessary, experimentally determined. PMID:23711417

  12. An electron microscopy study of wear in polysilicon microelectromechanical systems.

    SciTech Connect

    Dugger, Michael Thomas; Enachescu, M.; Stach, Eric A.; Alsem, Daan Hein; Ritchie, Robert O.

    2005-02-01

    Wear is a critical factor in determining the durability of microelectromechanical systems (MEMS). While the reliability of polysilicon MEMS has received extensive attention, the mechanisms responsible for this failure mode at the microscale have yet to be conclusively determined. We have used on-chip polycrystalline silicon side-wall friction MEMS specimens to study active mechanisms during sliding wear in ambient air. Worn parts were examined by analytical scanning and transmission electron microscopy, while local temperature changes were monitored using advanced infrared microscopy. Observations show that small amorphous debris particles ({approx}50-100 nm) are removed by fracture through the silicon grains ({approx}500 nm) and are oxidized during this process. Agglomeration of such debris particles into larger clusters also occurs. Some of these debris particles/clusters create plowing tracks on the beam surface. A nano-crystalline surface layer ({approx}20-200 nm), with higher oxygen content, forms during wear at and below regions of the worn surface; its formation is likely aided by high local stresses. No evidence of dislocation plasticity or of extreme local temperature increases was found, ruling out the possibility of high temperature-assisted wear mechanisms.

  13. Simultaneous orientation and thickness mapping in transmission electron microscopy

    DOE PAGES

    Tyutyunnikov, Dmitry; Özdöl, V. Burak; Koch, Christoph T.

    2014-12-04

    In this paper we introduce an approach for simultaneous thickness and orientation mapping of crystalline samples by means of transmission electron microscopy. We show that local thickness and orientation values can be extracted from experimental dark-field (DF) image data acquired at different specimen tilts. The method has been implemented to automatically acquire the necessary data and then map thickness and crystal orientation for a given region of interest. We have applied this technique to a specimen prepared from a commercial semiconductor device, containing multiple 22 nm technology transistor structures. The performance and limitations of our method are discussed and comparedmore » to those of other techniques available.« less

  14. Temperature Calibration for In Situ Environmental Transmission Electron Microscopy Experiments

    PubMed Central

    Winterstein, JP; Lin, PA; Sharma, R

    2016-01-01

    In situ environmental transmission electron microscopy (ETEM) experiments require specimen heating holders to study material behavior in gaseous environments at elevated temperatures. In order to extract meaningful kinetic parameters, such as activation energies, it is essential to have a direct and accurate measurement of local sample temperature. This is particularly important if the sample temperature might fluctuate, for example when room temperature gases are introduced to the sample area. Using selected-area diffraction (SAD) in an ETEM, the lattice parameter of Ag nanoparticles was measured as a function of the temperature and pressure of hydrogen gas to provide a calibration of the local sample temperature. SAD permits measurement of temperature to an accuracy of ± 30 °C using Ag lattice expansion. Gas introduction can cause sample cooling of several hundred degrees celsius for gas pressures achievable in the ETEM. PMID:26441334

  15. Scanning SQUID microscopy with single electron spin sensitivity

    NASA Astrophysics Data System (ADS)

    Vasyukov, Denis

    2014-03-01

    Superconducting interference devices (SQUIDs) have been traditionally used for studying fundamental properties of magnetic materials and superconductors. Although widely used in scanning magnetic microscopy, their progress towards detection of small magnetic moments was stagnating of late due to limitations imposed by conventional designs of planar SQUIDs and contemporary lithography techniques, restricting sample-to-sensor distance smaller than ~ 0.5 micron and SQUIDs diameters smaller than ~ 200 nm. These limitations were overcome by the invention of a SQUID-on-tip device, subsequent realization of a SQUID-on-tip microscope, and by creation of an ultra-small sensor with spatial resolution of 20 nm and sensitivity to a single electron spin per 1 Hz bandwidth. In this talk I will describe the principles of scanning SQUID magnetometry, its applications to study superconductors and its potential for magnetic nano-scale imaging of novel materials.

  16. Scanning electron microscopy fractography analysis of fractured hollow implants.

    PubMed

    Sbordone, Ludovico; Traini, Tonino; Caputi, Sergio; Scarano, Antonio; Bortolaia, Claudia; Piattelli, Adriano

    2010-01-01

    Fracture of the implant is one of the possible complications affecting dental implants; it is a rare event but of great clinical relevance. The aim of the present study was to perform a scanning electron microscopy (SEM) fractography evaluation of 7 International Team for oral Implantology (ITI) hollow implants removed because of fracture. The most common clinical risk factors, such as malocclusion, bruxism, and cantilevers on the prosthesis, were absent. Seven fractured ITI hollow implants were retrieved from 5 patients and were analyzed with the use of SEM. SEM analysis showed typical signs of a cleavage-type fracture. Fractures could be due to an association of multiple factors such as fatigue, inner defects, material electrochemical problems, and tensocorrosion. PMID:20426587

  17. Electron microscopy of life-tested semiconductor laser diodes

    PubMed

    Vanzi; Bonfiglio; Magistrali; Salmini

    2000-06-01

    Electron Microscopy on life-tested 980 nm SL SQW InGaAs/AlGaAs laser diodes is able to find and analyze lattice defects responsible for the detected failures. Anyway, the origin and evolution of those defects remains questionable. Only the comparative analysis of life-test measurements, EBIC-FIB/TEM images, and charge-transport physics is able to point out a coherent framework for complete decoding of the failure kinetics. Minority-carrier diffusion and their enhanced recombination at defective lattice points are indicated, as the energy supply required for defect reaction and growth. The rules of charge diffusion drive both the reaction model, the interpretation of EBIC images and the expected electrical and optical effects. Strain release at the ultimate propagation of defects into the strained InGaAs quantum layer is then easily related to the final state of the failed devices.

  18. Morphological classification of bioaerosols from composting using scanning electron microscopy.

    PubMed

    Tamer Vestlund, A; Al-Ashaab, R; Tyrrel, S F; Longhurst, P J; Pollard, S J T; Drew, G H

    2014-07-01

    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. Samples 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. PMID:24565805

  19. Electron Microscopy Analysis of the Nucleolus of Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

    López-Velázquez, Gabriel; Hernández, Roberto; López-Villaseñor, Imelda; Reyes-Vivas, Horacio; Segura-Valdez, María De L.; Jiménez-García, Luis F.

    2005-08-01

    The nucleolus is the main site for synthesis and processing of ribosomal RNA in eukaryotes. In mammals, plants, and yeast the nucleolus has been extensively characterized by electron microscopy, but in the majority of the unicellular eukaryotes no such studies have been performed. Here we used ultrastructural cytochemical and immunocytochemical techniques as well as three-dimensional reconstruction to analyze the nucleolus of Trypanosoma cruzi, which is an early divergent eukaryote of medical importance. In T. cruzi epimastigotes the nucleolus is a spherical intranuclear ribonucleoprotein organelle localized in a relatively central position within the nucleus. Dense fibrillar and granular components but not fibrillar centers were observed. In addition, nuclear bodies resembling Cajal bodies were observed associated to the nucleolus in the surrounding nucleoplasm. Our results provide additional morphological data to better understand the synthesis and processing of the ribosomal RNA in kinetoplastids.

  20. Advanced analytical electron microscopy for alkali-ion batteries

    DOE PAGES

    Qian, Danna; Ma, Cheng; Meng, Ying Shirley; More, Karren; Chi, Miaofang

    2015-01-01

    Lithium-ion batteries are a leading candidate for electric vehicle and smart grid applications. However, further optimizations of the energy/power density, coulombic efficiency and cycle life are still needed, and this requires a thorough understanding of the dynamic evolution of each component and their synergistic behaviors during battery operation. With the capability of resolving the structure and chemistry at an atomic resolution, advanced analytical transmission electron microscopy (AEM) is an ideal technique for this task. The present review paper focuses on recent contributions of this important technique to the fundamental understanding of the electrochemical processes of battery materials. A detailed reviewmore » of both static (ex situ) and real-time (in situ) studies will be given, and issues that still need to be addressed will be discussed.« less

  1. Advanced analytical electron microscopy for alkali-ion batteries

    SciTech Connect

    Qian, Danna; Ma, Cheng; Meng, Ying Shirley; More, Karren; Chi, Miaofang

    2015-01-01

    Lithium-ion batteries are a leading candidate for electric vehicle and smart grid applications. However, further optimizations of the energy/power density, coulombic efficiency and cycle life are still needed, and this requires a thorough understanding of the dynamic evolution of each component and their synergistic behaviors during battery operation. With the capability of resolving the structure and chemistry at an atomic resolution, advanced analytical transmission electron microscopy (AEM) is an ideal technique for this task. The present review paper focuses on recent contributions of this important technique to the fundamental understanding of the electrochemical processes of battery materials. A detailed review of both static (ex situ) and real-time (in situ) studies will be given, and issues that still need to be addressed will be discussed.

  2. Watershed Merge Tree Classification for Electron Microscopy Image Segmentation

    SciTech Connect

    Liu, TIng; Jurrus, Elizabeth R.; Seyedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2012-11-11

    Automated segmentation of electron microscopy (EM) images is a challenging problem. In this paper, we present a novel method that utilizes a hierarchical structure and boundary classification for 2D neuron segmentation. With a membrane detection probability map, a watershed merge tree is built for the representation of hierarchical region merging from the watershed algorithm. A boundary classifier is learned with non-local image features to predict each potential merge in the tree, upon which merge decisions are made with consistency constraints in the sense of optimization to acquire the final segmentation. Independent of classifiers and decision strategies, our approach proposes a general framework for efficient hierarchical segmentation with statistical learning. We demonstrate that our method leads to a substantial improvement in segmentation accuracy.

  3. Characterization of protein immobilization on nanoporous gold using atomic force microscopy and scanning electron microscopy

    NASA Astrophysics Data System (ADS)

    Tan, Yih Horng; Schallom, John R.; Ganesh, N. Vijaya; Fujikawa, Kohki; Demchenko, Alexei V.; Stine, Keith J.

    2011-08-01

    Nanoporous gold (NPG), made by dealloying low carat gold alloys, is a relatively new nanomaterial finding application in catalysis, sensing, and as a support for biomolecules. NPG has attracted considerable interest due to its open bicontinuous structure, high surface-to-volume ratio, tunable porosity, chemical stability and biocompatibility. NPG also has the attractive feature of being able to be modified by self-assembled monolayers. Here we use scanning electron microscopy (SEM) and atomic force microscopy (AFM) to characterize a highly efficient approach for protein immobilization on NPG using N-hydroxysuccinimide (NHS) ester functionalized self-assembled monolayers on NPG with pore sizes in the range of tens of nanometres. Comparison of coupling under static versus flow conditions suggests that BSA (Bovine Serum Albumin) and IgG (Immunoglobulin G) can only be immobilized onto the interior surfaces of free standing NPG monoliths with good coverage under flow conditions. AFM is used to examine protein coverage on both the exterior and interior of protein modified NPG. Access to the interior surface of NPG for AFM imaging is achieved using a special procedure for cleaving NPG. AFM is also used to examine BSA immobilized on rough gold surfaces as a comparative study. In principle, the general approach described should be applicable to many enzymes, proteins and protein complexes since both pore sizes and functional groups present on the NPG surfaces are controllable.Nanoporous gold (NPG), made by dealloying low carat gold alloys, is a relatively new nanomaterial finding application in catalysis, sensing, and as a support for biomolecules. NPG has attracted considerable interest due to its open bicontinuous structure, high surface-to-volume ratio, tunable porosity, chemical stability and biocompatibility. NPG also has the attractive feature of being able to be modified by self-assembled monolayers. Here we use scanning electron microscopy (SEM) and atomic force

  4. Liquid scanning transmission electron microscopy: Nanoscale imaging in micrometers-thick liquids

    NASA Astrophysics Data System (ADS)

    Schuh, Tobias; de Jonge, Niels

    2014-02-01

    Scanning transmission electron microscopy (STEM) of specimens in liquid is possible using a microfluidic chamber with thin silicon nitride windows. This paper includes an analytic equation of the resolution as a function of the sample thickness and the vertical position of an object in the liquid. The equipment for STEM of liquid specimen is briefly described. STEM provides nanometer resolution in micrometer-thick liquid layers with relevance for both biological research and materials science. Using this technique, we investigated tagged proteins in whole eukaryotic cells, and gold nanoparticles in liquid with time-lapse image series. Possibly future applications are discussed.

  5. High resolution electron microscopy and spectroscopy of ferritin in thin window liquid cells

    NASA Astrophysics Data System (ADS)

    Wang, Canhui; Qiao, Qiao; Shokuhfar, Tolou; Klie, Robert

    2014-03-01

    In-situ transmission electron microscopy (TEM) has seen a dramatic increase in interest in recent years with the commercial development of liquid and gas stages. High-resolution TEM characterization of samples in a liquid environment remains limited by radiation damage and loss of resolution due to the thick window-layers required by the in-situ stages. We introduce thin-window static-liquid cells that enable sample imaging with atomic resolution and electron energy-loss (EEL) spectroscopy with 1.3 nm resolution. Using this approach, atomic and electronic structures of biological samples such as ferritin is studied via in-situ transmission electron microscopy experiments. Ferritin in solution is encapsulated using the static liquid cells with reduced window thickness. The integrity of the thin window liquid cell is maintained by controlling the electron dose rate. Radiation damage of samples, such as liquid water and protein, is quantitatively studied to allow precision control of radiation damage level within the liquid cells. Biochemical reactions, such as valence change of the iron in a functioning ferritin, is observed and will be quantified. Relevant biochemical activity: the release and uptake of Fe atoms through the channels of ferritin protein shell is also imaged at atomic resolution. This work is funded by Michigan Technological University. The UIC JEOL JEM-ARM200CF is supported by an MRI-R2 grant from the National Science Foundation (Grant No. DMR-0959470).

  6. 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.

  7. Scanning and three-dimensional electron microscopy methods for the study of Trypanosoma brucei and Leishmania mexicana flagella

    PubMed Central

    Gluenz, Eva; Wheeler, Richard John; Hughes, Louise; Vaughan, Sue

    2015-01-01

    Three-dimensional electron microscopy tools have revolutionized our understanding of cell structure and molecular complexes in biology. Here, we describe methods for studying flagellar ultrastructure and biogenesis in two unicellular parasites—Trypanosoma brucei and Leishmania mexicana. We describe methods for the preparation of these parasites for scanning electron microscopy cellular electron tomography, and serial block face scanning electron microscopy (SBFSEM). These parasites have a highly ordered cell shape and form, with a defined positioning of internal cytoskeletal structures and organelles. We show how knowledge of these can be used to dissect cell cycles in both parasites and identify the old flagellum from the new in T. brucei. Finally, we demonstrate the use of SBFSEM three-dimensional models for analysis of individual whole cells, demonstrating the excellent potential this technique has for future studies of mutant cell lines. PMID:25837406

  8. Sea Spray Aerosol Structure and Composition Using Cryogenic Transmission Electron Microscopy

    PubMed Central

    2016-01-01

    The composition and surface properties of atmospheric aerosol particles largely control their impact on climate by affecting their ability to uptake water, react heterogeneously, and nucleate ice in clouds. However, in the vacuum of a conventional electron microscope, the native surface and internal structure often undergo physicochemical rearrangement resulting in surfaces that are quite different from their atmospheric configurations. Herein, we report the development of cryogenic transmission electron microscopy where laboratory generated sea spray aerosol particles are flash frozen in their native state with iterative and controlled thermal and/or pressure exposures and then probed by electron microscopy. This unique approach allows for the detection of not only mixed salts, but also soft materials including whole hydrated bacteria, diatoms, virus particles, marine vesicles, as well as gel networks within hydrated salt droplets—all of which will have distinct biological, chemical, and physical processes. We anticipate this method will open up a new avenue of analysis for aerosol particles, not only for ocean-derived aerosols, but for those produced from other sources where there is interest in the transfer of organic or biological species from the biosphere to the atmosphere. PMID:26878061

  9. Immuno EM-OM correlative microscopy in solution by atmospheric scanning electron microscopy (ASEM).

    PubMed

    Maruyama, Yuusuke; Ebihara, Tatsuhiko; Nishiyama, Hidetoshi; Suga, Mitsuo; Sato, Chikara

    2012-11-01

    In the atmospheric scanning electron microscope (ASEM), an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized the cytoskeletons formed by actin and tubulin, the chaperone PDI that catalyses native disulfide bond formation of proteins in the endoplasmic reticulum (ER) and the calcium sensor STIM1 that is integrated in ER membranes, using established cell lines. In particular, a dynamic string-like gathering of STIM1 was observed on the ER in Jurkat T cells in response to Ca(2+) store depletion. We have also visualized filamentous actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca(2+) signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework. Based on this work, ASEM correlative microscopy promises to allow the dynamics of various protein complexes to be investigated in the near future. PMID:22959994

  10. Combined scanning transmission electron microscopy tilt- and focal series.

    PubMed

    Dahmen, Tim; Baudoin, Jean-Pierre; Lupini, Andrew R; Kübel, Christian; Slusallek, Philipp; de Jonge, Niels

    2014-04-01

    In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt-focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller "missing wedge" artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.

  11. Morphological classification of bioaerosols from composting using scanning electron microscopy

    SciTech Connect

    Tamer Vestlund, A.; Al-Ashaab, R.; Tyrrel, S.F.; Longhurst, P.J.; Pollard, S.J.T.; Drew, G.H.

    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. Samples 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.

  12. [Light and electron microscopy of rhinoscleroma (author's transl)].

    PubMed

    Balázs, M; Elö, J; Juhász, J

    1975-02-01

    The authors report the case of a 50 year old male patient whose rhinoscleroma, localized to the upper respiratory tract, was demonstrated by the isolation of Klebsiella bacilli and histologically. Electron microscopically the Mikulicz cells were characterized by fused vacuoles occupying the largest portion of the cytoplasm and displacing the damaged cytoplasmic organelles. Phagosomes and dense bodies reminiscent of Russel bodies also occurred in the Mikulicz cells, in the vacuoles of which formations representing Klebsiella rhinoscleromatis were demonstrated. A light halo was visible around some of these formations. It could not be, however, decided whether these halos represented the mucous sheath of the bacillus or an artifact only. In the plasmacells the authors observed the bag-like dilatation of the ergastoplasm and the presence of Russel bodies. Transitory forms were not seen among the plasma and Mikulicz cells. As a result of the treatment, Klebsiella disappeared from the nasal mucosa of the patient. The authors wish to follow by means of electron microscopy the changes of the granulation tissue and pathogens following antibiotic therapy. PMID:1225879

  13. The characterization of nanoparticles using analytical electron microscopy

    NASA Astrophysics Data System (ADS)

    Hill, Whitney B.

    2011-06-01

    Nanoparticles are often overlooked during routine trace evidence analyses because of their small size and the degree of difficulty needed to efficiently characterize them. However, analytical electron microscopy (AEM) enables the characterization and/or identification of nanoparticles because of its high magnification capability, the ability to gather elemental data and also the ability to determine the internal structure of a single nanoparticles(1). There is a wide variety of natural and manufactured nanoparticles that are prominent within the environment and their presence becomes very valuable in the absence of larger particles. The combustion of materials produces by-products such as nano-sized carbon soot, fumes, fly ash and gun-shot residue (GSR). Using AEM, nano-sized carbon soot, fumes, fly ash and GSR can not only be distinguished from other nanoparticles within the environment but can also be distinguished from each other because of differences in morphology, elemental composition, and internal structure. The elemental information gathered from combustion by-products during AEM analysis can also give an indication of the original source material. Other nanoparticles such as paint pigments and fillers can also be characterized by AEM using morphology, electron diffraction and elemental composition.

  14. Thin Dielectric Film Thickness Determination by Advanced Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Diebold, A. C.; Foran, B.; Kisielowski, C.; Muller, D. A.; Pennycook, S. J.; Principe, E.; Stemmer, S.

    2003-12-01

    High-resolution transmission electron microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by nonspecialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark-field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods has been steadily improved reaching now into the sub-Ångstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this article, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this article is the proposal of a reproducible method for film thickness determination.

  15. Seeing Inside Materials by Aberration-Corrected Electron Microscopy

    SciTech Connect

    Pennycook, Stephen J

    2011-01-01

    The recent successful correction of lens aberrations in the electron microscope has improved resolution by more than a factor of two in just a few years, bringing many benefits for the study of materials. These benefits extend significantly beyond enhanced resolution alone. Aberration correction gives higher resolution by allowing the objective lens to have a wider aperture, which also results in a reduced depth of field. This effect can be used to only focus specific sections inside materials for the first time. In this contribution we describe recent results exploiting this capability. Additionally, we show how combining the microscopy data with first-principles theory gives new insights into materials properties. We cover two applications, both involving heavy atoms in a lighter host. The first shows how single Hf atoms can be mapped in three dimensions inside the 1 nm-wide SiO2 region of a high dielectric constant device structure, and how a link to macroscopic device properties results through theoretical calculations. The second example is from the field of nanoscience, where individual Au atoms are imaged inside Si nanowires grown by a vapor-liquid-solid mechanism. The majority of Au atoms are probably injected by the highly energetic electron beam. However, their observed sites and atomic configurations represent at least meta-stable configurations and match well to results from density functional calculations.

  16. Analytical electron microscopy in mineralogy; exsolved phases in pyroxenes

    USGS Publications Warehouse

    Nord, G.L.

    1982-01-01

    Analytical scanning transmission electron microscopy has been successfully used to characterize the structure and composition of lamellar exsolution products in pyroxenes. At operating voltages of 100 and 200 keV, microanalytical techniques of x-ray energy analysis, convergent-beam electron diffraction, and lattice imaging have been used to chemically and structurally characterize exsolution lamellae only a few unit cells wide. Quantitative X-ray energy analysis using ratios of peak intensities has been adopted for the U.S. Geological Survey AEM in order to study the compositions of exsolved phases and changes in compositional profiles as a function of time and temperature. The quantitative analysis procedure involves 1) removal of instrument-induced background, 2) reduction of contamination, and 3) measurement of correction factors obtained from a wide range of standard compositions. The peak-ratio technique requires that the specimen thickness at the point of analysis be thin enough to make absorption corrections unnecessary (i.e., to satisfy the "thin-foil criteria"). In pyroxenes, the calculated "maximum thicknesses" range from 130 to 1400 nm for the ratios Mg/Si, Fe/Si, and Ca/Si; these "maximum thicknesses" have been contoured in pyroxene composition space as a guide during analysis. Analytical spatial resolutions of 50-100 nm have been achieved in AEM at 200 keV from the composition-profile studies, and analytical reproducibility in AEM from homogeneous pyroxene standards is ?? 1.5 mol% endmember. ?? 1982.

  17. Thin dielectric film thickness determination by advanced transmission electron microscopy

    SciTech Connect

    Diebold, A.C.; Foran, B.; Kisielowski, C.; Muller, D.; Pennycook, S.; Principe, E.; Stemmer, S.

    2003-09-01

    High Resolution Transmission Electron Microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by non-specialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods was steadily improved reaching now into the sub Angstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this paper, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this paper is the proposal of a reproducible method for film thickness determination.

  18. Quantitative Energy-filtering Transmission Electron Microscopy in Materials Science.

    PubMed

    Grogger; Hofer; Warbichler; Kothleitner

    2000-03-01

    Energy-filtered transmission electron microscopy (EFTEM) can be used to acquire elemental distribution images at high lateral resolution within short acquisition times. In this article, we present an overview of typical problems from materials science which can be preferentially solved by means of EFTEM. In the first example, we show how secondary phases in a steel specimen can be easily detected by recording jump ratio images of the matrix element under rocking beam illumination. Secondly, we describe how elemental maps can be converted into concentration maps. A Ba-Nd-titanate ceramics serves as a typical materials science example exhibiting three different compounds with varying composition. In order to reduce diffraction and/or thickness variation effects which may be a problem for quantification of crystalline specimens, we calculated atomic ratio maps by dividing two elemental maps and subsequent normalizing by the partial ionization cross-sections (or k-factors). Additionally, the atomic ratio maps are correlated using the scatter diagram technique thus leading to quantitative chemical phase maps. Finally, we show how the near-edge structures (electron energy-loss near edge fine structures, or ELNES) can be used for mapping chemical bonding states thus differentiating between various modifications of an element. In order to distinguish between diamond and non-diamond carbon in diamond coated materials, we have investigated a diamond layer on a substrate with the help of ELNES mapping utilizing the pi*-peak of the C-K ionization edge. PMID:10742404

  19. High Resolution Transmission Electron Microscopy (HRTEM) of nanophase ferric oxides

    NASA Technical Reports Server (NTRS)

    Golden, D. C.; Morris, R. V.; Ming, D. W.; Lauer, H. V., Jr.

    1994-01-01

    Iron oxide minerals are the prime candidates for Fe(III) signatures in remotely sensed Martian surface spectra. Magnetic, Mossbauer, and reflectance spectroscopy have been carried out in the laboratory in order to understand the mineralogical nature of Martian analog ferric oxide minerals of submicron or nanometer size range. Out of the iron oxide minerals studied, nanometer sized ferric oxides are promising candidates for possible Martian spectral analogs. 'Nanophase ferric oxide (np-Ox)' is a generic term for ferric oxide/oxihydroxide particles having nanoscale (less than 10 nm) particle dimensions. Ferrihydrite, superparamagnetic particles of hematite, maghemite and goethite, and nanometer sized particles of inherently paramagnetic lepidocrocite are all examples of nanophase ferric oxides. np-Ox particles in general do not give X-ray diffraction (XRD) patterns with well defined peaks and would often be classified as X-ray amorphous. Therefore, different np-Oxs preparations should be characterized using a more sensitive technique e.g., high resolution transmission electron microscopy (HRTEM). The purpose of this study is to report the particle size, morphology and crystalline order, of five np-Ox samples by HRTEM imaging and electron diffraction (ED).

  20. Monte Carlo simulation of secondary electron images for real sample structures in scanning electron microscopy.

    PubMed

    Zhang, P; Wang, H Y; Li, Y G; Mao, S F; Ding, Z J

    2012-01-01

    Monte Carlo simulation methods for the study of electron beam interaction with solids have been mostly concerned with specimens of simple geometry. In this article, we propose a simulation algorithm for treating arbitrary complex structures in a real sample. The method is based on a finite element triangular mesh modeling of sample geometry and a space subdivision for accelerating simulation. Simulation of secondary electron image in scanning electron microscopy has been performed for gold particles on a carbon substrate. Comparison of the simulation result with an experiment image confirms that this method is effective to model complex morphology of a real sample.

  1. Scanning electron microscopy and electron probe X-ray microanalysis (SEM-EPMA) of pink teeth

    SciTech Connect

    Ikeda, N.; Watanabe, G.; Harada, A.; Suzuki, T.

    1988-11-01

    Samples of postmortem pink teeth were investigated by scanning electron microscopy and electron probe X-ray microanalysis. Fracture surfaces of the dentin in pink teeth were noticeably rough and revealed many more smaller dentinal tubules than those of the control white teeth. Electron probe X-ray microanalysis showed that the pink teeth contained iron which seemed to be derived from blood hemoglobin. The present study confirms that under the same circumstance red coloration of teeth may occur more easily in the teeth in which the dentin is less compact and contains more dentinal tubules.

  2. Interfacial ultramorphology evaluation of resin luting cements to dentin: a correlative scanning electron microscopy and transmission electron microscopy analysis.

    PubMed

    Aguiar, Thaiane Rodrigues; Vermelho, Paulo Moreira; André, Carolina Bosso; Giannini, Marcelo

    2013-12-01

    The objective of this study was to analyze the dentin-resin cements interfacial ultramorphologies using two different methods: scanning (SEM) and transmission electron microscopy (TEM). Four commercial products were evaluated: two conventional cementing system (RelyX ARC/Adper™ Scotchbond™ Multi-Purpose Plus, 3M ESPE and Clearfil Esthetic Cement/DC Bond, Kuraray) and two self-adhesive resin cements (RelyX Unicem, 3M ESPE and Clearfil SA Cement, Kuraray). Prepolymerized resin disks (Sinfony, 3M ESPE) were cemented on oclusal dentin surfaces of 24 third human molars, simulating the indirect restorations. After 24 h, teeth were sectioned into 0.9-mm thick slabs and processed for microscopy analyses (SEM or TEM/ n = 3). Qualitative characterization of dentin-resin cement interface was performed. Hybrid layer formation with long and dense resin tags was observed only for RelyX ARC cementing system. Clearfil Esthetic Cement/DC Bond system revealed few and short resin tags formation, whereas no hybridization and resin tags were detected for self-adhesive resin cements. Some interfacial regions exhibited that the self-adhesive resin cements were not bonded to dentin, presenting bubbles or voids at the interfaces. In conclusion, TEM and SEM bonding interface analyses showed ultramorphological variations among resin cements, which are directly related to dental bonding strategies used for each resin cement tested.

  3. Effects of ultramorphological changes on adhesion to lased dentin-Scanning electron microscopy and transmission electron microscopy analysis.

    PubMed

    Moretto, Simone G; Azambuja, Nilton; Arana-Chavez, Victor E; Reis, Andre F; Giannini, Marcelo; Eduardo, Carlos de P; De Freitas, Patricia M

    2011-08-01

    Dentin irradiation with erbium lasers has been reported to alter the composite resin bond to this treated surface. There is still a lack of studies reporting the effect of erbium lasers on dentin organic content and elucidating how laser treatment could interfere in the quality of the resin-dentin interface. This study aimed to evaluate the effect of erbium laser irradiation on dentin morphology and microtensile bond strength (μTBS) of an adhesive to dentin. Seventy-two dentin disks were divided into nine groups (n = 8): G1-Control (600-grit SiC paper); Er:YAG groups: G2- 250 mJ/4 Hz; G3- 200 mJ/4 Hz; G4- 180 mJ/10 Hz; G5- 160 mJ/10 Hz; Er,Cr:YSGG groups: G6- 2 W/20 Hz; G7- 2.5 W/20 Hz; G8- 3 W/20 Hz; G9- 4 W/20 Hz. Specimens were processed for cross-sectional analysis by scanning electron microscopy (SEM) (n = 3), transmission electron microscopy (TEM) (n = 2), and adhesive interface (n = 3). Forty-five dentin samples (n = 5) were restored and submitted to μTBS testing. ANOVA (α = 5%) revealed that G1 presented the highest μTBS values and irradiated groups did not differ from each other. TEM micrographs showed a superficial layer of denatured collagen fibrils. For SEM micrographs, it was possible to verify the laser effects extending to dentin subsurface presenting a rough aspect. Cross-sectional dentin micrographs of this hybridized surface revealed a pattern of modified tags with ringlike structures around it. This in vitro study showed that erbium laser irradiation interacts with the dental hard tissue resulting in a specific morphological pattern of dentin and collagen fibrils that negatively affected the bond strength to composite resin.

  4. Visualization of Clusters in Polymer Electrolyte Membranes by Electron Microscopy

    PubMed Central

    Yakovlev, Sergey

    2014-01-01

    The morphology of ionic clusters that form in polyelectrolyte membranes has a strong effect on transport and electrical properties. In spite of considerable research efforts the link between morphology and properties has not been clearly established, mainly due to difficulties in assessing nanoscale morphology. Electron microscopy (EM) has the potential to visualize morphology. However success in visualization has so far been moderate. In this review we focus on the potential of EM techniques to characterize the ionic domains. We use both experimental data and models to compare the capabilities of several EM techniques: BF TEM, HAADF, core-loss EELS, and low-loss EELS in projection imaging and STEM modes. The main problems common for all these EM modes are radiation damage and overlap of features in projection. Our models show that core loss EELS with exposures that are below the typical damage threshold is incapable of resolving 2 nm diameter sulfur-rich clusters in PEMs. While low loss EELS requires lower exposure the insight it can provide is quite limited. HAADF and BF TEM present the most effective modes for imaging the sulfur clusters in PEMs. While BF TEM uses scattered electrons more efficiently, HAADF using slightly higher doses can provide unique information due to in-focus imaging and transparent interpretation of the images. Fortunately, in at least some interesting cases the clusters themselves are much more radiation resistant than the polymer and can be studied at exposures high enough to obtain clear images. Our simulations also show that tomographic 3D reconstruction provides the best approach for solving the overlap problem. In spite of the abilities of electron tomography, data obtained from all EM techniques improves if thin sections are studied. We briefly discuss methods for obtaining such sections. PMID:23165242

  5. Electron microscopy analysis of skin biopsies in CADASIL disease.

    PubMed

    Cotrutz, Carmen Elena; Indrei, Anca; Bădescu, L; Dacălu, Cristina; Neamţu, Monica; Dumitrescu, Gabriela Florenţa; Stefanache, Felicia; Petreuş, T

    2010-01-01

    Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is an inherited vascular disorder, non-amyloid and non-atherosclerotic, affecting predominantly the central nervous system. We examined samples of skin biopsies from six patients (men, 43-52-year-old), admitted for treatment in the Neurology Clinic regarding the presence of partial motor impairment on upper and lower right limbs, facial asymmetry and phrasing impairment (three of the patients); These three patients had family history remarkable for early-onset strokes: mother and two brothers deceased by early strokes (40-50-year-old). Skin biopsy samples were fixed in glutaraldehyde and post-fixed in osmium tetroxyde. After dehydration, tissue samples were embedded in Epon. Ultrathin sections were mounted on copper grids and stained with uranyl acetate and lead citrate as usual and examined with a transmission electron microscope Phillips CM100. In all cases ultrastructural study showed granular osmiophilic material (GOM) in extracellular locations, between degenerating smooth muscle cells in dermal arteries or in their indentations. Deposits of GOM varied in size and electron density. Degeneration and loss of smooth muscle cells (SMCs) leads to abnormal enlargement of the space between these cells Ultrastructural analysis in three cases showed chromatin condensation and peripheral aggregation of nuclear material suggesting cells entry to apoptosis. These aspects and the marked destruction of the vascular wall were correlated with MRI findings and the severity of clinical manifestations at these patients. Our study showed that findings of GOM deposits, degeneration and loss of SMCs (probably by apoptosis), cell adhesion elements disturbance are characteristic for CADASIL disease and sufficient for diagnose of certainty. Moreover, electron microscopy analysis of skin biopsies is a useful tool for a differential diagnosis and can be considered as first choice method

  6. 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

  7. Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

    DOE PAGES

    Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R.; Chess, Jordan; McMorran, Benjamin J.; Czarnik, Cory; Rose, Harald H.; Ercius, Peter

    2016-02-29

    The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, makingmore » it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Ultimately, simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.« less

  8. Probing Individual Ice Nucleation Events with Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Bingbing; China, Swarup; Knopf, Daniel; Gilles, Mary; Laskin, Alexander

    2016-04-01

    Heterogeneous ice nucleation is one of the processes of critical relevance to a range of topics in the fundamental and the applied science and technologies. Heterogeneous ice nucleation initiated by particles proceeds where microscopic properties of particle surfaces essentially control nucleation mechanisms. Ice nucleation in the atmosphere on particles governs the formation of ice and mixed phase clouds, which in turn influence the Earth's radiative budget and climate. Heterogeneous ice nucleation is still insufficiently understood and poses significant challenges in predictive understanding of climate change. We present a novel microscopy platform allowing observation of individual ice nucleation events at temperature range of 193-273 K and relative humidity relevant for ice formation in the atmospheric clouds. The approach utilizes a home built novel ice nucleation cell interfaced with Environmental Scanning Electron Microscope (IN-ESEM system). The IN-ESEM system is applied for direct observation of individual ice formation events, determining ice nucleation mechanisms, freezing temperatures, and relative humidity onsets. Reported microanalysis of the ice nucleating particles (INP) include elemental composition detected by the energy dispersed analysis of X-rays (EDX), and advanced speciation of the organic content in particles using scanning transmission x-ray microscopy with near edge X-ray absorption fine structure spectroscopy (STXM/NEXAFS). The performance of the IN-ESEM system is validated through a set of experiments with kaolinite particles with known ice nucleation propensity. We demonstrate an application of the IN-ESEM system to identify and characterize individual INP within a complex mixture of ambient particles.

  9. Scanning transmission electron microscopy strain measurement from millisecond frames of a direct electron charge coupled device

    SciTech Connect

    Mueller, Knut; Rosenauer, Andreas; Ryll, Henning; Ordavo, Ivan; Ihle, Sebastian; Soltau, Heike; Strueder, Lothar; Volz, Kerstin; Zweck, Josef

    2012-11-19

    A high-speed direct electron detection system is introduced to the field of transmission electron microscopy and applied to strain measurements in semiconductor nanostructures. In particular, a focused electron probe with a diameter of 0.5 nm was scanned over a fourfold quantum layer stack with alternating compressive and tensile strain and diffracted discs have been recorded on a scintillator-free direct electron detector with a frame time of 1 ms. We show that the applied algorithms can accurately detect Bragg beam positions despite a significant point spread each 300 kV electron causes during detection on the scintillator-free camera. For millisecond exposures, we find that strain can be measured with a precision of 1.3 Multiplication-Sign 10{sup -3}, enabling, e.g., strain mapping in a 100 Multiplication-Sign 100 nm{sup 2} region with 0.5 nm resolution in 40 s.

  10. Scanning transmission electron microscopy strain measurement from millisecond frames of a direct electron charge coupled device

    NASA Astrophysics Data System (ADS)

    Müller, Knut; Ryll, Henning; Ordavo, Ivan; Ihle, Sebastian; Strüder, Lothar; Volz, Kerstin; Zweck, Josef; Soltau, Heike; Rosenauer, Andreas

    2012-11-01

    A high-speed direct electron detection system is introduced to the field of transmission electron microscopy and applied to strain measurements in semiconductor nanostructures. In particular, a focused electron probe with a diameter of 0.5 nm was scanned over a fourfold quantum layer stack with alternating compressive and tensile strain and diffracted discs have been recorded on a scintillator-free direct electron detector with a frame time of 1 ms. We show that the applied algorithms can accurately detect Bragg beam positions despite a significant point spread each 300 kV electron causes during detection on the scintillator-free camera. For millisecond exposures, we find that strain can be measured with a precision of 1.3 × 10-3, enabling, e.g., strain mapping in a 100×100 nm2 region with 0.5 nm resolution in 40 s.

  11. Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.

    PubMed

    Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir

    2013-01-01

    Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

  12. Influence of acceleration voltage on scanning electron microscopy of human blood platelets.

    PubMed

    Pretorius, E

    2010-03-01

    Scanning electron microscopy (SEM) is used to view a variety of surface structures, molecules, or nanoparticles of different materials, ranging from metals, dental and medical instruments, and chemistry (e.g. polymer analysis) to biological material. Traditionally, the operating conditions of the SEM are very important in the material sciences, particularly the acceleration voltage. However, in biological sciences, it is not typically seen as an important parameter. Acceleration voltage allows electrons to penetrate the sample; thus, the higher the acceleration voltage the more penetration into the sample will occur. As a result, ultrastructural information from deeper layers will interfere with the actual surface morphology that is seen. Therefore, ultimately, if acceleration voltage is lower, a better quality of the surface molecules and structures will be produced. However, in biological sciences, this is an area that is not well-documented. Typically, acceleration voltages of between 5 and 20 kV are used. This manuscript investigates the influence of acceleration voltages ranging from 5 kV to as low as 300 V, by studying surface ultrastructure of a human platelet aggregate. It is concluded that, especially at higher magnifications, much more surface detail is visible in biological samples when using an acceleration voltage between 2 kV and 300 V.

  13. Evaluation of virus inactivation by formaldehyde to enhance biosafety of diagnostic electron microscopy.

    PubMed

    Möller, Lars; Schünadel, Livia; Nitsche, Andreas; Schwebke, Ingeborg; Hanisch, Manuela; Laue, Michael

    2015-02-10

    Formaldehyde (FA) fixation of infectious samples is a well-established protocol in diagnostic electron microscopy of viruses. However, published experimental data that demonstrate virus inactivation by these fixation procedures are lacking. Usually, fixation is performed immediately before the sample preparation for microscopy. The fixation procedure should transform viruses in a non-infectious but nonetheless structurally intact form in order to allow a proper diagnosis based on morphology. FA provides an essential advantage in comparison to other disinfectants, because it preserves the ultrastructure of biological material without interfering significantly with the preparation (i.e., the negative staining) and the detection of viruses. To examine the efficiency of FA inactivation, we used Vaccinia virus, Human adenovirus and Murine norovirus as models and treated them with FA under various conditions. Critical parameters for the inactivation efficiency were the temperature, the duration of the FA treatment, and the resistance of the virus in question. Our results show that FA inactivation at low temperature (4 °C) bears a high risk of incomplete inactivation. Higher temperatures (25 °C) are more efficient, although they still require rather long incubation times to fully inactivate a complex and highly robust virus like Vaccinia. A protocol, which applied 2% buffered FA for 60 min and a temperature-shift from 25 to 37 °C after 30 min was efficient for the complete inactivation of all test viruses, and therefore has the potential to improve both biosafety and speed of diagnostic electron microscopy.

  14. Correlative in-resin super-resolution and electron microscopy using standard fluorescent proteins

    PubMed Central

    Johnson, Errin; Seiradake, Elena; Jones, E. Yvonne; Davis, Ilan; Grünewald, Kay; Kaufmann, Rainer

    2015-01-01

    We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge. We identified key aspects of the sample preparation procedure of high pressure freezing, freeze substitution and resin embedding that are critical for preserving fluorescence and photo-switching of standard fluorescent proteins, such as mGFP, mVenus and mRuby2. This enabled us to combine single molecule localization microscopy with transmission electron microscopy imaging of standard fluorescent proteins in cryo-fixed resin embedded cells. We achieved a structural resolution of 40–50 nm (~17 nm average single molecule localization accuracy) in the fluorescence images without the use of chemical fixation or special fluorophores. Using this approach enabled the correlation of fluorescently labeled structures to the ultrastructure in the same cell at the nanometer level and superior structural preservation. PMID:25823571

  15. Evaluation of Virus Inactivation by Formaldehyde to Enhance Biosafety of Diagnostic Electron Microscopy

    PubMed Central

    Möller, Lars; Schünadel, Livia; Nitsche, Andreas; Schwebke, Ingeborg; Hanisch, Manuela; Laue, Michael

    2015-01-01

    Formaldehyde (FA) fixation of infectious samples is a well-established protocol in diagnostic electron microscopy of viruses. However, published experimental data that demonstrate virus inactivation by these fixation procedures are lacking. Usually, fixation is performed immediately before the sample preparation for microscopy. The fixation procedure should transform viruses in a non–infectious but nonetheless structurally intact form in order to allow a proper diagnosis based on morphology. FA provides an essential advantage in comparison to other disinfectants, because it preserves the ultrastructure of biological material without interfering significantly with the preparation (i.e., the negative staining) and the detection of viruses. To examine the efficiency of FA inactivation, we used Vaccinia virus, Human adenovirus and Murine norovirus as models and treated them with FA under various conditions. Critical parameters for the inactivation efficiency were the temperature, the duration of the FA treatment, and the resistance of the virus in question. Our results show that FA inactivation at low temperature (4 °C) bears a high risk of incomplete inactivation. Higher temperatures (25 °C) are more efficient, although they still require rather long incubation times to fully inactivate a complex and highly robust virus like Vaccinia. A protocol, which applied 2% buffered FA for 60 min and a temperature–shift from 25 to 37 °C after 30 min was efficient for the complete inactivation of all test viruses, and therefore has the potential to improve both biosafety and speed of diagnostic electron microscopy. PMID:25674771

  16. Optical and scanning electron microscopy in the single osteoclast resorption assay.

    PubMed

    Boyde, A; Ali, N N; Jones, S J

    1985-01-01

    The present studies relate to the single or isolated osteoclastic resorption function assay which we introduced in 1983 to overcome objections to assays based upon measurements of calcium release from bones, in which it was never strictly controlled whether the mechanism involved the destruction of bone with the formation of classical Howship's lacunae. The method may prove to be quite popular in the near future and has already been adopted by other research groups. In previous work, we had utilised stereophotogrammetry of scanning electron micrographs to measure the depth, volume and other parameters of the individual lacunae. However, increasing experience with the method has suggested that we can await a wide range of biological variability in single cell function in any one experiment. We have therefore tested other methods from which data could be obtained more rapidly to permit a better statistical analysis, albeit with reduced accuracy, of each resorption complex. The main aim of the studies reported here was to evaluate various methods of optical and scanning electron microscopy that can be used for the visualization of osteoclasts and their associated resorption lacunae generated in vitro in slabs of dentine and bone. Optical microscopy was found to be complementary to SEM, enabling vital microscopy of unstained and stained cells. In particular, oblique illumination LM and tandem scanning reflected LM (TSRLM) proved to be of paramount value for this purpose. Fixed coated specimens could be most rapidly scanned for resorption lacunae using darkfield reflected LM or TSRLM.

  17. A scanning transmission electron microscopy approach to analyzing large volumes of tissue to detect nanoparticles.

    PubMed

    Kempen, Paul J; Thakor, Avnesh S; Zavaleta, Cristina; Gambhir, Sanjiv S; Sinclair, Robert

    2013-10-01

    The use of nanoparticles for the diagnosis and treatment of cancer requires the complete characterization of their toxicity, including accurately locating them within biological tissues. Owing to their size, traditional light microscopy techniques are unable to resolve them. Transmission electron microscopy provides the necessary spatial resolution to image individual nanoparticles in tissue, but is severely limited by the very small analysis volume, usually on the order of tens of cubic microns. In this work, we developed a scanning transmission electron microscopy (STEM) approach to analyze large volumes of tissue for the presence of polyethylene glycol-coated Raman-active-silica-gold-nanoparticles (PEG-R-Si-Au-NPs). This approach utilizes the simultaneous bright and dark field imaging capabilities of STEM along with careful control of the image contrast settings to readily identify PEG-R-Si-Au-NPs in mouse liver tissue without the need for additional time-consuming analytical characterization. We utilized this technique to analyze 243,000 mm³ of mouse liver tissue for the presence of PEG-R-Si-Au-NPs. Nanoparticles injected into the mice intravenously via the tail vein accumulated in the liver, whereas those injected intrarectally did not, indicating that they remain in the colon and do not pass through the colon wall into the systemic circulation.

  18. Biological x-ray microscopy: from biochemical mapping to lensless imaging

    NASA Astrophysics Data System (ADS)

    Jacobsen, Chris

    2005-03-01

    Cell structure has been very succesfully studied using light and electron microscopy. However, x rays ofer new insights, by imaging whole cells at 20-40 nm resolution using zone plate lenses, and in particular by combining this with spectroscopic sensitivity to organic functional groups. While spectra of single compounds can provide exquisite information on electronic states, a cell is much more complex. Pattern recognition algorithms provide a way to deal with this complexity and obtain insights into biochemical organization at a fine spatial scale, as illustrated in an ongoing study of the correlation of morphology with biochemical content in sperm. Another approach to biological imaging is to abandon the use of lenses and their resolution limits. The purest form of measurement is to collect x rays scattered by a cell with no optics-imposed losses. By using iterative phasing algorithms, this diffraction data can be phased to deliver a real-space image of a complex cell (at present, 30 nm resolution in studies of freeze-dried yeast) with a possible ultimate extension to atomic resolution imaging of proteins using x-ray free electron lasers.

  19. Maskiton: Interactive, Web-based Classification of Single-Particle Electron Microscopy Images

    PubMed Central

    Yoshioka, Craig; Lyumkis, Dmitry; Carragher, Bridget; Potter, Clinton S.

    2013-01-01

    Electron microscopy (EM) is an important tool for determining the composition, arrangement and structure of biological macromolecules. When studying structurally heterogeneous samples using EM, classification is a critical step toward achieving higher resolution and identifying biologically significant conformations. We have developed an interactive, web-based tool, called Maskiton, for creating custom masks and performing 2D classifications on aligned single-particle EM images. The Maskiton interface makes it considerably easier and faster to explore the significance of heterogeneity in single-particle datasets. Maskiton features include: resumable uploads to facilitate transfer of large datasets to the server, custom mask creation in the browser, continual progress updates, and interactive viewing of classification results. To demonstrate the value of this tool, we provide examples of its use on several experimental datasets and include analyses of the independent terminus mobility within the Ltn1 E3 ubiquitin ligase, the in-vitro assembly of 30S ribosomal subunits, and classification complexity reduction within Immunoglobulin M. This work also serves as a proof-of-concept for the development of future cross-platform, interactive user interfaces for electron microscopy data processing. PMID:23428431

  20. Droplets on superhydrophobic surfaces: visualization of the contact area by cryo-scanning electron microscopy.

    PubMed

    Ensikat, Hans J; Schulte, Anna J; Koch, Kerstin; Barthlott, Wilhelm

    2009-11-17

    The contact area between liquids and solid surfaces plays the crucial role in the wetting and self-cleaning properties of surfaces. In this study, we have developed a cryo-preparation method to visualize the contact area between liquids and superhydrophobic biological surfaces by scanning electron microscopy. Aqueous liquids that do not crystallize during freezing, such as glycerol and phosphoric acid, were used. First, the samples in contact with the liquid droplets were cooled with liquid nitrogen. After this, the droplets were separated and the contact areas on the frozen droplets were visualized by scanning electron microscopy. The contact areas of droplets on various biological and artificial surfaces with microstructure, nanostructure, and hierarchical structures are shown in detail. It could be shown that spaces between nanostructures were not penetrated by the droplet, which rested only on top of the structures. Measurements of the contact areas showed the largest reduction in the solid-liquid contact area on hierarchically structured leaf surfaces. On these surfaces, the droplets are in the "Cassie state" at both levels of surface structuring. On plant surfaces, the varying height of the epidermal cells and the surface relief caused considerable variations in the contact between droplet and surface. The examples demonstrate that this new approach provides detailed insights into the wetting behavior of surfaces in the Cassie state with partial contact with the liquid. PMID:19899819

  1. Rare-earth-doped nanophosphors for multicolor cathodoluminescence nanobioimaging using scanning transmission electron microscopy.

    PubMed

    Furukawa, Taichi; Fukushima, Shoichiro; Niioka, Hirohiko; Yamamoto, Naoki; Miyake, Jun; Araki, Tsutomu; Hashimoto, Mamoru

    2015-05-01

    We describe rare-earth-doped nanophosphors (RE-NPs) for biological imaging using cathodoluminescence(CL) microscopy based on scanning transmission electron microscopy (STEM). We report the first demonstration of multicolor CL nanobioimaging using STEM with nanophosphors. The CL spectra of the synthesized nanophosphors (Y2O3∶Eu, Y2O3∶Tb) were sufficiently narrow to be distinguished. From CL images of RE-NPs on an elastic carbon-coated copper grid, the spatial resolution was beyond the diffraction limit of light.Y2O3∶Tb and Y2O3∶Eu RE-NPs showed a remarkable resistance against electron beam exposure even at high acceleration voltage (80 kV) and retained a CL intensity of more than 97% compared with the initial intensity for 1 min. In biological CL imaging with STEM, heavy-metal-stained cell sections containing the RE-NPs were prepared,and both the CL images of RE-NPs and cellular structures, such as mitochondria, were clearly observed from STEM images with high contrast. The cellular CL imaging using RE-NPs also had high spatial resolution even though heavy-metal-stained cells are normally regarded as highly scattering media. Moreover, since theRE-NPs exhibit photoluminescence (PL) excited by UV light, they are useful for multimodal correlative imaging using CL and PL.

  2. Preparation of the planarian Schmidtea mediterranea for high-resolution histology and transmission electron microscopy

    PubMed Central

    Brubacher, John L.; Vieira, Ana P.; Newmark, Phillip A.

    2014-01-01

    The flatworm Schmidtea mediterranea is an emerging model species in such fields as stem-cell biology, regeneration, and evolutionary biology. Excellent molecular tools have been developed for S. mediterranea, but ultrastructural techniques have received far less attention. Processing specimens for histology and transmission electron microscopy is notoriously idiosyncratic for particular species or specimen types. Unfortunately however, most methods for S. mediterranea described in the literature lack numerous essential details, and those few that do provide them rely on specialized equipment that may not be readily available. Here we present an optimized protocol for ultrastructural preparation of S. mediterranea. The protocol can be completed in six days, much of which is “hands-off” time. To aid with troubleshooting, we also illustrate the significant effects of seemingly minor variations in fixative, buffer concentration, and dehydration steps. This procedure will be useful for all planarian researchers, particularly those with relatively little experience in tissue processing. PMID:24556788

  3. Compressive fluorescence microscopy for biological and hyperspectral imaging.

    PubMed

    Studer, Vincent; Bobin, Jérome; Chahid, Makhlad; Mousavi, Hamed Shams; Candes, Emmanuel; Dahan, Maxime

    2012-06-26

    The mathematical theory of compressed sensing (CS) asserts that one can acquire signals from measurements whose rate is much lower than the total bandwidth. Whereas the CS theory is now well developed, challenges concerning hardware implementations of CS-based acquisition devices--especially in optics--have only started being addressed. This paper presents an implementation of compressive sensing in fluorescence microscopy and its applications to biomedical imaging. Our CS microscope combines a dynamic structured wide-field illumination and a fast and sensitive single-point fluorescence detection to enable reconstructions of images of fluorescent beads, cells, and tissues with undersampling ratios (between the number of pixels and number of measurements) up to 32. We further demonstrate a hyperspectral mode and record images with 128 spectral channels and undersampling ratios up to 64, illustrating the potential benefits of CS acquisition for higher-dimensional signals, which typically exhibits extreme redundancy. Altogether, our results emphasize the interest of CS schemes for acquisition at a significantly reduced rate and point to some remaining challenges for CS fluorescence microscopy. PMID:22689950

  4. Study of titanate nanotubes by X-ray and electron diffraction and electron microscopy

    SciTech Connect

    Brunatova, Tereza; Popelkova, Daniela; Wan, Wei; Oleynikov, Peter; Danis, Stanislav; Zou, Xiaodong; Kuzel, Radomir

    2014-01-15

    The structure of titanate nanotubes (Ti-NTs) was studied by a combination of powder X-ray diffraction (PXRD), electron diffraction and high resolution transmission electron microscopy (HRTEM). Ti-NTs are prepared by hydrothermal treatment of TiO{sub 2} powder. The structure is identified by powder X-ray diffraction as the one based on the structure of H{sub 2}Ti{sub 2}O{sub 5}·H{sub 2}O phase. The same structure is obtained by projected potential from HRTEM through-focus image series. The structure is verified by simulated PXRD pattern with the aid of the Debye formula. The validity of the model is tested by computing Fourier transformation of a single nanotube which is proportional to measured electron diffraction intensities. A good agreement of this calculation with measured precession electron diffraction data is achieved. - Highlights: • Titanate nanotubes were prepared by hydrothermal method. • X-ray powder diffraction indicated their structure based on that of H{sub 2}Ti{sub 2}O{sub 5}·H{sub 2}O. • Structural model was created with the aid of high-resolution electron microscopy. • The model was verified with electron diffraction data. • X-ray powder diffraction pattern was calculated with the aid of the Debye formula.

  5. Using advanced electron microscopy for the characterization of catalytic materials

    NASA Astrophysics Data System (ADS)

    Pyrz, William D.

    Catalysis will continue to be vitally important to the advancement and sustainability of industrialized societies. Unfortunately, the petroleum-based resources that currently fuel the energy and consumer product needs of an advancing society are becoming increasingly difficult and expensive to extract as supplies diminish and the quality of sources degrade. Therefore, the development of sustainable energy sources and the improvement of the carbon efficiency of existing chemical processes are critical. Further challenges require that these initiatives are accomplished in an environmentally friendly fashion since the effects of carbon-based emissions are proving to be a serious threat to global climate stability. In this dissertation, materials being developed for sustainable energy and process improvement initiatives are studied. Our approach is to use materials characterization, namely advanced electron microscopy, to analyze the targeted systems at the nano- or Angstrom-scale with the goal of developing useful relationships between structure, composition, crystalline order, morphology, and catalytic performance. One area of interest is the complex Mo-V-M-O (M=Te, Sb, Ta, Nb) oxide system currently being developed for the selective oxidation/ammoxidation of propane to acrylic acid or acrylonitrile, respectively. Currently, the production of acrylic acid and acrylonitrile rely on propylene-based processes, yet significant cost savings could be realized if the olefin-based feeds could be replaced by paraffin-based ones. The major challenge preventing this feedstock replacement is the development of a suitable paraffin-activating catalyst. Currently, the best candidate is the Mo-V-Nb-Te-O complex oxide catalyst that is composed of two majority phases that are commonly referred to as M1 and M2. However, there is a limited understanding of the roles of each component with respect to how they contribute to catalyst stability and the reaction mechanism. Aberration

  6. Electron Microscopy and Image Analysis for Selected Materials

    NASA Technical Reports Server (NTRS)

    Williams, George

    1999-01-01

    This particular project was completed in collaboration with the metallurgical diagnostics facility. The objective of this research had four major components. First, we required training in the operation of the environmental scanning electron microscope (ESEM) for imaging of selected materials including biological specimens. The types of materials range from cyanobacteria and diatoms to cloth, metals, sand, composites and other materials. Second, to obtain training in surface elemental analysis technology using energy dispersive x-ray (EDX) analysis, and in the preparation of x-ray maps of these same materials. Third, to provide training for the staff of the metallurgical diagnostics and failure analysis team in the area of image processing and image analysis technology using NIH Image software. Finally, we were to assist in the sample preparation, observing, imaging, and elemental analysis for Mr. Richard Hoover, one of NASA MSFC's solar physicists and Marshall's principal scientist for the agency-wide virtual Astrobiology Institute. These materials have been collected from various places around the world including the Fox Tunnel in Alaska, Siberia, Antarctica, ice core samples from near Lake Vostoc, thermal vents in the ocean floor, hot springs and many others. We were successful in our efforts to obtain high quality, high resolution images of various materials including selected biological ones. Surface analyses (EDX) and x-ray maps were easily prepared with this technology. We also discovered and used some applications for NIH Image software in the metallurgical diagnostics facility.

  7. Capturing enveloped viruses on affinity grids for downstream cryo-electron microscopy applications

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Electron microscopy cryo-electron microscopy and cryo-electron tomography are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pa...

  8. Imaging doped silicon test structures using low energy electron microscopy.

    SciTech Connect

    Nakakura, Craig Yoshimi; Anderson, Meredith Lynn; Kellogg, Gary Lee

    2010-01-01

    This document is the final SAND Report for the LDRD Project 105877 - 'Novel Diagnostic for Advanced Measurements of Semiconductor Devices Exposed to Adverse Environments' - funded through the Nanoscience to Microsystems investment area. Along with the continuous decrease in the feature size of semiconductor device structures comes a growing need for inspection tools with high spatial resolution and high sample throughput. Ideally, such tools should be able to characterize both the surface morphology and local conductivity associated with the structures. The imaging capabilities and wide availability of scanning electron microscopes (SEMs) make them an obvious choice for imaging device structures. Dopant contrast from pn junctions using secondary electrons in the SEM was first reported in 1967 and more recently starting in the mid-1990s. However, the serial acquisition process associated with scanning techniques places limits on the sample throughput. Significantly improved throughput is possible with the use of a parallel imaging scheme such as that found in photoelectron emission microscopy (PEEM) and low energy electron microscopy (LEEM). The application of PEEM and LEEM to device structures relies on contrast mechanisms that distinguish differences in dopant type and concentration. Interestingly, one of the first applications of PEEM was a study of the doping of semiconductors, which showed that the PEEM contrast was very sensitive to the doping level and that dopant concentrations as low as 10{sup 16} cm{sup -3} could be detected. More recent PEEM investigations of Schottky contacts were reported in the late 1990s by Giesen et al., followed by a series of papers in the early 2000s addressing doping contrast in PEEM by Ballarotto and co-workers and Frank and co-workers. In contrast to PEEM, comparatively little has been done to identify contrast mechanisms and assess the capabilities of LEEM for imaging semiconductor device strictures. The one exception is the

  9. Automated Quantitative Rare Earth Elements Mineralogy by Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Sindern, Sven; Meyer, F. Michael

    2016-09-01

    Increasing industrial demand of rare earth elements (REEs) stems from the central role they play for advanced technologies and the accelerating move away from carbon-based fuels. However, REE production is often hampered by the chemical, mineralogical as well as textural complexity of the ores with a need for better understanding of their salient properties. This is not only essential for in-depth genetic interpretations but also for a robust assessment of ore quality and economic viability. The design of energy and cost-efficient processing of REE ores depends heavily on information about REE element deportment that can be made available employing automated quantitative process mineralogy. Quantitative mineralogy assigns numeric values to compositional and textural properties of mineral matter. Scanning electron microscopy (SEM) combined with a suitable software package for acquisition of backscatter electron and X-ray signals, phase assignment and image analysis is one of the most efficient tools for quantitative mineralogy. The four different SEM-based automated quantitative mineralogy systems, i.e. FEI QEMSCAN and MLA, Tescan TIMA and Zeiss Mineralogic Mining, which are commercially available, are briefly characterized. Using examples of quantitative REE mineralogy, this chapter illustrates capabilities and limitations of automated SEM-based systems. Chemical variability of REE minerals and analytical uncertainty can reduce performance of phase assignment. This is shown for the REE phases parisite and synchysite. In another example from a monazite REE deposit, the quantitative mineralogical parameters surface roughness and mineral association derived from image analysis are applied for automated discrimination of apatite formed in a breakdown reaction of monazite and apatite formed by metamorphism prior to monazite breakdown. SEM-based automated mineralogy fulfils all requirements for characterization of complex unconventional REE ores that will become

  10. Transmission electron microscopy analysis of corroded metal waste forms.

    SciTech Connect

    Dietz, N. L.

    2005-04-15

    This report documents the results of analyses with transmission electron microscopy (TEM) combined with energy dispersive X-ray spectroscopy (EDS) and selected area electron diffraction (ED) of samples of metallic waste form (MWF) materials that had been subjected to various corrosion tests. The objective of the TEM analyses was to characterize the composition and microstructure of surface alteration products which, when combined with other test results, can be used to determine the matrix corrosion mechanism. The examination of test samples generated over several years has resulted in refinements to the TEM sample preparation methods developed to preserve the orientation of surface alteration layers and the underlying base metal. The preservation of microstructural spatial relationships provides valuable insight for determining the matrix corrosion mechanism and for developing models to calculate radionuclide release in repository performance models. The TEM results presented in this report show that oxide layers are formed over the exposed steel and intermetallic phases of the MWF during corrosion in aqueous solutions and humid air at elevated temperatures. An amorphous non-stoichiometric ZrO{sub 2} layer forms at the exposed surfaces of the intermetallic phases, and several nonstoichiometric Fe-O layers form over the steel phases in the MWF. These oxide layers adhere strongly to the underlying metal, and may be overlain by one or more crystalline Fe-O phases that probably precipitated from solution. The layer compositions are consistent with a corrosion mechanism of oxidative dissolution of the steel and intermetallic phases. The layers formed on the steel and intermetallic phases form a continuous layer over the exposed waste form, although vertical splits in the layer and corrosion in pits and crevices were seen in some samples. Additional tests and analyses are needed to verify that these layers passivate the underlying metals and if passivation can break

  11. Environmental Scanning Electron Microscopy of Ice Crystal Nucleation and Growth

    NASA Astrophysics Data System (ADS)

    Amaral, M.; Miller, A. L.; Magee, N. B.

    2012-12-01

    Ice crystal nucleation and growth are dual processes that can be studied uniquely through Environmental Scanning Electron Microscopy (ESEM). By utilizing differential pumping systems and a Peltier element to vary the vapor pressure and to achieve temperatures below the freezing point, respectively, it is possible to obtain supersaturated conditions relative to ice in the sample chamber of an Environmental Scanning Electron Microscope. Ice crystals were nucleated on a variety of atmospherically relevant substrates and grown in a pure water vapor environment in the chamber of a FEI-Quanta 200 ESEM. To initiate ice crystal nucleation, the Peltier element was set at a temperature between -10°C and -25°C, while the chamber water vapor pressure was adjusted to just below the frost point. Ice crystal nucleation and growth was then controlled by careful adjustments of chamber pressure and temperature, where high-magnification images of hexagonal ice crystals were acquired at nanoscale resolution. These images display prominent mesoscopic surface topography including linear strands, crevasses, islands, and steps. The surface features are seen to be ubiquitously present at all observed temperatures, at many supersaturated and subsaturated conditions, and on all crystal facets. Additionally, a pre-growth "shadow" resembling a dark spot sometimes appeared on areas of the sample stage immediately preceding ice crystal nucleation and growth. The observations represent the most highly magnified images of ice surfaces yet reported and significantly expand the range of ambient conditions where the features are conspicuous. New knowledge of the presence and characteristics of these features could transform the fundamental understanding of ice crystal growth kinetics and its physical parameterization in the context of atmospheric and cryospheric science. To the extent these observations are applicable to atmospheric ice, the results suggest that the radiative representation of ice

  12. Total Sample Conditioning and Preparation of Nanoliter Volumes for Electron Microscopy.

    PubMed

    Arnold, Stefan A; Albiez, Stefan; Opara, Nadia; Chami, Mohamed; Schmidli, Claudio; Bieri, Andrej; Padeste, Celestino; Stahlberg, Henning; Braun, Thomas

    2016-05-24

    Electron microscopy (EM) entered a new era with the emergence of direct electron detectors and new nanocrystal electron diffraction methods. However, sample preparation techniques have not progressed and still suffer from extensive blotting steps leading to a massive loss of sample. Here, we present a simple but versatile method for the almost lossless sample conditioning and preparation of nanoliter volumes of biological samples for EM, keeping the sample under close to physiological condition. A microcapillary is used to aspirate 3-5 nL of sample. The microcapillary tip is immersed into a reservoir of negative stain or trehalose, where the sample becomes conditioned by diffusive exchange of salt and heavy metal ions or sugar molecules, respectively, before it is deposited as a small spot onto an EM grid. We demonstrate the use of the method to prepare protein particles for imaging by transmission EM and nanocrystals for analysis by electron diffraction. Furthermore, the minute sample volume required for this method enables alternative strategies for biological experiments, such as the analysis of the content of a single cell by visual proteomics, fully exploiting the single molecule detection limit of EM. PMID:27074622

  13. Non-thermal plasma mills bacteria: Scanning electron microscopy observations

    SciTech Connect

    Lunov, O. Churpita, O.; Zablotskii, V.; Jäger, A.; Dejneka, A.; Deyneka, I. G.; Meshkovskii, I. K.; Syková, E.; Kubinová, Š.

    2015-02-02

    Non-thermal plasmas hold great promise for a variety of biomedical applications. To ensure safe clinical application of plasma, a rigorous analysis of plasma-induced effects on cell functions is required. Yet mechanisms of bacteria deactivation by non-thermal plasma remain largely unknown. We therefore analyzed the influence of low-temperature atmospheric plasma on Gram-positive and Gram-negative bacteria. Using scanning electron microscopy, we demonstrate that both Gram-positive and Gram-negative bacteria strains in a minute were completely destroyed by helium plasma. In contrast, mesenchymal stem cells (MSCs) were not affected by the same treatment. Furthermore, histopathological analysis of hematoxylin and eosin–stained rat skin sections from plasma–treated animals did not reveal any abnormalities in comparison to control ones. We discuss possible physical mechanisms leading to the shred of bacteria under non-thermal plasma irradiation. Our findings disclose how helium plasma destroys bacteria and demonstrates the safe use of plasma treatment for MSCs and skin cells, highlighting the favorability of plasma applications for chronic wound therapy.

  14. Electron microscopy of iron chalcogenide FeTe(Se) films

    NASA Astrophysics Data System (ADS)

    Shchichko, I. O.; Presnyakov, M. Yu.; Stepantsov, E. A.; Kazakov, S. M.; Antipov, E. V.; Makarova, I. P.; Vasil'ev, A. L.

    2015-05-01

    The structure of Fe1 + δTe1 - x Se x films ( x = 0; 0.05) grown on single-crystal MgO and LaAlO3 substrates has been investigated by transmission and scanning transmission electron microscopy. The study of Fe1.11Te/MgO structures has revealed two crystallographic orientation relationships between the film and substrate. It is shown that the lattice mismatch between the film and substrate is compensated for by the formation of misfit dislocations. The Burgers vector projection is determined. The stresses in the film can partially be compensated for due to the formation of an intermediate disordered layer. It is shown that a FeTe0.5Se0.5 film grown on a LaAlO3 substrate is single-crystal and that the FeTe0.5Se0.5/LaAlO3 interface in a selected region is coherent. The orientation relationships between the film and substrate are also determined for this case.

  15. Measurement of Globular Protein Molecules by Electron Microscopy

    PubMed Central

    Hall, Cecil E.

    1960-01-01

    A series of molecular species with approximately spherical shape and with molecular weights between 35,000 and 250,000 were shadowed with platinum while resting on a cleaved mica surface. They were backed, stripped from the surface, and examined by electron microscopy. Materials examined were: pepsin, liver alcohol dehydrogenase, yeast alcohol dehydrogenase, glutamic dehydrogenase, polyhedral virus protein (insect), fibrinogen substructure, alkaline phosphatase, and microsomal particles from Escherichia coli. Measurements were made of widths perpendicular to the shadowing direction and heights were deduced from shadow lengths. For those molecular species with well established molecular weights the average heights correlate very well with the diameter of the theoretical sphere but the average widths are too great by 50 to 80 A due to the lateral growth of the deposited metal. Although the distortion in shape of shadowed particles is relatively large, with standardized conditions for shadowing, it is possible to make allowance for the distortion and to obtain reasonably reliable estimates of the dimensions of spherical organic particles down to a molecular weight of about 35,000. PMID:14399016

  16. Scanning electron microscopy and roughness study of dental composite degradation.

    PubMed

    Soares, Luís Eduardo Silva; Cortez, Louise Ribeiro; Zarur, Raquel de Oliveira; Martin, Airton Abrahão

    2012-04-01

    Our aim was to test the hypothesis that the use of mouthwashes, consumption of soft drinks, as well as the type of light curing unit (LCU), would change the surface roughness (Ra) and morphology of a nanofilled composite resin (Z350® 3M ESPE). Samples (80) were divided into eight groups: Halogen LCU, group 1, saliva (control); group 2, Pepsi Twist®; group 3, Listerine®; group 4, Colgate Plax®; LED LCU, group 5, saliva; group 6, Pepsi Twist®; group 7, Listerine®; group 8, Colgate Plax®. Ra values were measured at baseline, and after 7 and 14 days. One specimen of each group was prepared for scanning electron microscopy analysis after 14 days. The data were subjected to multifactor analysis of variance at a 95% confidence followed by Tukey's honestly significant difference post-hoc test. All the treatments resulted in morphological changes in composite resin surface, and the most significant change was in Pepsi Twist® groups. The samples of G6 had the greatest increase in Ra. The immersion of nanofilled resin in mouthwashes with alcohol and soft drink increases the surface roughness. Polymerization by halogen LCU (reduced light intensity) associated with alcohol contained mouthwash resulted in significant roughness on the composite.

  17. Scanning electron microscopy of xiphinema, longidorus, and californidorus stylet morphology.

    PubMed

    Cho, M R; Robbins, R T

    1990-04-01

    Stylet ultrastructure of five Xiphinema, four Longidorus, and three Californidorus species was compared by scanning electron microscopy. Morphological differences were seen in the odontophores and odontostyle bases between the genera and some of the species. All Xiphinema studied had well-developed odontophore flanges; the Longidorus species lacked flanges, except for weakly developed ones in L. diadecturus; and none of the Californidorus had flanges. Three sinuses were present in the odontophores of all species. The sinuses varied in length depending upon species. In Xiphinema and Californidorus the odontostyle bases had distinct overlapping collars, but in Longidorus the collars were absent except for L. diadecturus. The odontostyle-odontophore junction from a lateral view appeared as a slanted transverse line in all the species, but in a dorsal view of Xiphinema and Californidorus it was V-shaped. Dorsal longitudinal seams of the odontostyle and odontophore were observed in all the species. The dorsally located odontostyle aperture was ca. 1 mum from the anterior end in all species, except in one Longidorus sp. it was ca. 4 mum from the end.

  18. Preparing recombinant yeast septins and their analysis by electron microscopy.

    PubMed

    Bertin, A; Nogales, E

    2016-01-01

    Septins are highly conserved and essential eukaryotic cytoskeletal proteins that interact with the inner plasma membrane. They are involved in essential functions requiring cell membrane remodeling and compartmentalization, such as cell division and dendrite morphogenesis, and have been implicated in numerous diseases. Depending on the organisms and on the type of tissue, a specific set of septins genes are expressed, ranging from 2 to 13. Septins self-assemble into linear, symmetric rods that can further organize into linear filaments several microns in length. Only a subset of human septins has been described at high resolution by X-ray crystallography (Sirajuddin et al., 2007). Electron microscopy (EM) has proven to be a method of choice for analyzing the molecular organization of septins. It is possible to localize each septin subunit within the rod complex using genetic tags, such as maltose-binding protein or green fluorescent protein, to generate a visible label of a specific septin subunit in EM images that are processed using single-particle EM methodology. In this chapter we present, in detail, the methods that we have used to analyze the molecular organization of budding yeast septins (Bertin et al., 2008). These methods include purification of septin complexes, sample preparation for EM, and image processing procedures. Such methods can be generalized to analyze the organization of septins from any organism. PMID:27473901

  19. Scanning electron microscopy of eggs of Sabethes cyaneus.

    PubMed

    Santos-Mallet, Jacenir; Sarmento, Juliana Soares; Alencar, Jeronimo; Müller, Gerson Azulim; Oliveira, Eliana Medeiros; Foster, Woodbridge A; Marcondes, Carlos Brisola

    2013-03-01

    Mosquitoes of the Neotropical genus Sabethes, some species of which are yellow fever vectors, most often develop through the immature stages in tree holes. Sabethes eggs have not been previously characterized using scanning electron microscopy. Eggs of Sabethes cyaneus (length: 349.6 +/- 2.7 microm; width: 172.6 +/- 1.14 microm; n = 10) are almost biconical when examined from the top. From a lateral perspective 2 surfaces can be seen. One surface is smooth and more convex, whereas the other is less convex and partially covered by a network from which many fungiform tubercles arise. The micropyle is situated on the smooth surface of the pointed anterior tip and is surrounded by an irregular row of tubercles, some of which are leaf shaped. No structures possibly involved in adhesion to surfaces are visible. When hatching, the egg splits dorsoventrally approximately two-thirds of the length from the anterior end. The tubercles appear to be water repellent, and the more convex/smoother surface is downturned, and this position on water was confirmed by direct observation. The eggs float free on the water surface.

  20. Transmission electron microscopy (TEM) study of minerals in coal

    SciTech Connect

    Hsieh, Kuang-Chien

    1982-01-01

    Minerals in eight coals from different mines were characterized in the micron-size range by using analytical transmission electron microscopy. Specimens were thinned by ion-milling wafers cut from these coals; a cold stage cooled by liquid nitrogen was used to reduce thermal degradation of the minerals by the ion-beam. Different mineral compounds were observed in different coals. The major minerals are clays, sulfides, oxides, carbonates and some minor-element-bearing phosphates. Clays (kaolinite, illite and others) have been most commonly found as either flat sheets or round globules. Iron sulfide was mostly found in the No. 5 and No. 6 coals from Illinois, distributed as massive polycrystals, as clusters of single crystals (framboids) or as isolated single crystals with size range down to some 0.25 microns. Other sulfides and some oxides were found in other coals with particle size as small as some 200 angstroms. Quartz, titanium oxides and many other carbonates and phosphate compounds were also characterized. Brief TEM work in the organic mass of coal was also introduced to study the nature of the coal macerals.

  1. Analytical electron microscopy of biogenic and inorganic carbonates

    NASA Technical Reports Server (NTRS)

    Blake, David F.

    1989-01-01

    In the terrestrial sedimentary environment, the mineralogically predominant carbonates are calcite-type minerals (rhombohedral carbonates) and aragonite-type minerals (orthorhombic carbonates). Most common minerals precipitating either inorganically or biogenically are high magnesium calcite and aragonite. High magnesium calcite (with magnesium carbonate substituting for more than 7 mole percent of the calcium carbonate) is stable only at temperatures greater than 700 C or thereabouts, and aragonite is stable only at pressures exceeding several kilobars of confining pressure. Therefore, these carbonates are expected to undergo chemical stabilization in the diagenetic environment to ultimately form stable calcite and dolomite. Because of the strong organic control of carbonate deposition in organisms during biomineralization, the microchemistry and microstructure of invertebrate skeletal material is much different than that present in inorganic carbonate cements. The style of preservation of microstructural features in skeletal material is therefore often quite distinctive when compared to that of inorganic carbonate even though wholesale recrystallization of the sediment has taken place. Microstructural and microchemical comparisons are made between high magnesium calcite echinoderm skeletal material and modern inorganic high magnesium calcite inorganic cements, using analytical electron microscopy and related techniques. Similar comparisons are made between analogous materials which have undergone stabilization in the diagenetic environment. Similar analysis schemes may prove useful in distinguishing between biogenic and inorganic carbonates in returned Martian carbonate samples.

  2. Characterization of paired helical filaments by scanning transmission electron microscopy.

    PubMed

    Ksiezak-Reding, Hanna; Wall, Joseph S

    2005-07-01

    Paired helical filaments (PHFs) are abnormal twisted filaments composed of hyperphosphorylated tau protein. They are found in Alzheimer's disease and other neurodegenerative disorders designated as tauopathies. They are a major component of intracellular inclusions known as neurofibrillary tangles (NFTs). The objective of this review is to summarize various structural studies of PHFs in which using scanning transmission electron microscopy (STEM) has been particularly informative. STEM provides shape and mass per unit length measurements important for studying ultrastructural aspects of filaments. These include quantitative comparisons between dispersed and aggregated populations of PHFs as well as comparative studies of PHFs in Alzheimer's disease and other neurodegenerative disorders. Other approaches are also discussed if relevant or complementary to studies using STEM, e.g., application of a novel staining reagent, Nanovan. Our understanding of the PHF structure and the development of PHFs into NFTs is presented from a historical perspective. Others goals are to describe the biochemical and ultrastructural complexity of authentic PHFs, to assess similarities between authentic and synthetic PHFs, and to discuss recent advances in PHF modeling.

  3. Glycine receptor mechanism illuminated by electron cryo-microscopy

    PubMed Central

    Du, Juan; Lü, Wei; Wu, Shenping; Cheng, Yifan; Gouaux, Eric

    2015-01-01

    Summary The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of GlyRs has been hindered by a dearth of high-resolution structures. Here we report electron cryo-microscopy structures of the α1 GlyR with strychnine, glycine, or glycine/ivermectin. Strychnine arrests the receptor in an antagonist-bound, closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain ‘wrist’ interface, and leads to rotation of the transmembrane domain toward the pore axis, occluding the ion conduction pathway. These structures illuminate GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors. PMID:26344198

  4. Histological preparation of developing vestibular otoconia for scanning electron microscopy

    NASA Technical Reports Server (NTRS)

    Huss, D.; Dickman, J. D.

    2003-01-01

    The unique nature of vestibular otoconia as calcium carbonate biominerals makes them particularly susceptible to chemical deformation during histological processing. We fixed and stored otoconia from all three otolith endorgans of embryonic, hatchling and adult Japanese quail in glutaraldehyde containing either phosphate or non-phosphate buffers for varying lengths of time and processed them for scanning electron microscopy. Otoconia from all age groups and otolith endorgans processed in 0.1 M phosphate buffer (pH 7.4) showed abnormal surface morphology when compared to acetone fixed controls. Otoconia processed in 0.1 M sodium cacodylate or HEPES buffered artificial endolymph (pH 7.4) showed normal morphology that was similar to controls. The degree of otoconial deformation was directly related to the time exposed to phosphate buffer. Short duration exposure produced particulate deformations while longer exposures resulted in fused otoconia that formed solid sheets. Otoconial surface deformation and fusing was independent of the glutaraldehyde component of the histological processing. These findings should help vestibular researchers to develop appropriate histological processing protocols in future studies of otoconia.

  5. Cryogenic transmission electron microscopy nanostructural study of shed microparticles.

    PubMed

    Issman, Liron; Brenner, Benjamin; Talmon, Yeshayahu; Aharon, Anat

    2013-01-01

    Microparticles (MPs) are sub-micron membrane vesicles (100-1000 nm) shed from normal and pathologic cells due to stimulation or apoptosis. MPs can be found in the peripheral blood circulation of healthy individuals, whereas elevated concentrations are found in pregnancy and in a variety of diseases. Also, MPs participate in physiological processes, e.g., coagulation, inflammation, and angiogenesis. Since their clinical properties are important, we have developed a new methodology based on nano-imaging that provides significant new data on MPs nanostructure, their composition and function. We are among the first to characterize by direct-imaging cryogenic transmitting electron microscopy (cryo-TEM) the near-to-native nanostructure of MP systems isolated from different cell types and stimulation procedures. We found that there are no major differences between the MP systems we have studied, as most particles were spherical, with diameters from 200 to 400 nm. However, each MP population is very heterogeneous, showing diverse morphologies. We investigated by cryo-TEM the effects of standard techniques used to isolate and store MPs, and found that either high-g centrifugation of MPs for isolation purposes, or slow freezing to -80 °C for storage introduce morphological artifacts, which can influence MP nanostructure, and thus affect the efficiency of these particles as future diagnostic tools.

  6. Hybrid Electron Microscopy Normal Mode Analysis graphical interface and protocol.

    PubMed

    Sorzano, Carlos Oscar S; de la Rosa-Trevín, José Miguel; Tama, Florence; Jonić, Slavica

    2014-11-01

    This article presents an integral graphical interface to the Hybrid Electron Microscopy Normal Mode Analysis (HEMNMA) approach that was developed for capturing continuous motions of large macromolecular complexes from single-particle EM images. HEMNMA was shown to be a good approach to analyze multiple conformations of a macromolecular complex but it could not be widely used in the EM field due to a lack of an integral interface. In particular, its use required switching among different software sources as well as selecting modes for image analysis was difficult without the graphical interface. The graphical interface was thus developed to simplify the practical use of HEMNMA. It is implemented in the open-source software package Xmipp 3.1 (http://xmipp.cnb.csic.es) and only a small part of it relies on MATLAB that is accessible through the main interface. Such integration provides the user with an easy way to perform the analysis of macromolecular dynamics and forms a direct connection to the single-particle reconstruction process. A step-by-step HEMNMA protocol with the graphical interface is given in full details in Supplementary material. The graphical interface will be useful to experimentalists who are interested in studies of continuous conformational changes of macromolecular complexes beyond the modeling of continuous heterogeneity in single particle reconstruction.

  7. Analysis of virus textures in transmission electron microscopy images.

    PubMed

    Nanni, Loris; Paci, Michelangelo; Caetano Dos Santos, Florentino Luciano; Brahnam, Sheryl; Hyttinen, Jari

    2014-01-01

    In this paper we propose an ensemble of texture descriptors for analyzing virus textures in transmission electron microscopy images. Specifically, we present several novel multi-quinary (MQ) codings of local binary pattern (LBP) variants: the MQ version of the dense LBP, the MQ version of the rotation invariant co-occurrence among adjacent LBPs, and the MQ version of the LBP histogram Fourier. To reduce computation time as well as to improve performance, a feature selection approach is utilized to select the thresholds used in the MQ approaches. In addition, we propose new variants of descriptors where two histograms, instead of the standard one histogram, are produced for each descriptor. The two histograms (one for edge pixels and the other for non-edge pixels) are calculated for training two different SVMs, whose results are then combined by sum rule. We show that a bag of features approach works well with this problem. Our experiments, using a publicly available dataset of 1500 images with 15 classes and same protocol as in previous works, demonstrate the superiority of our new proposed ensemble of texture descriptors. The MATLAB code of our approach is available at https://www.dei.unipd.it/node/2357. PMID:25488214

  8. Scanning electron microscopy of the endometrium during the secretory phase.

    PubMed Central

    Motta, P M; Andrews, P M

    1976-01-01

    Scanning electron microscopy was used to study the surface morphology of the rabbit endometrium during the secretory phase of the oestrous cycle. The free surfaces of ciliated and of inactive active secretory cells are described. Changes in secretory cell surface morphology resulting from accumulation and secretion of material involve the apparent retraction of microvilli and the formation of one or more bulbous protrusions of the cell's apical surface. These protrusions may be relatively smooth surfaced or exhibit long slender micro-extensions. The protrusions grow in size and are eventually pinched off. Loss of the bulbous protrusions often leaves behind crater-like invaginations of the cell's surface. Secretory cells adjacent to the endometrial glands are the first to exhibit signs of mucin accumulation and secretion. The single cilium of a secretory cell is not apparently affected by the secretory process. Signs of ciliated and secretory cell degeneration, and possible sloughing, are also described. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:1033932

  9. Transmission electron microscopy of Terfenol-D crystals

    NASA Astrophysics Data System (ADS)

    Holden, A. P.; Lord, D. G.; Grundy, P. J.

    1996-04-01

    Magnetic domain and microstructure observations are presented from samples of pseudo-single-crystal Terfenol-D examined by transmission electron microscopy (TEM). This ternary alloy is of significant technological interest since it exhibits the highest known magnetostriction to anisotropy ratio near room temperature. Specimens for TEM studies in (110), (111), and (112) orientations have also shown regions of unusual diffraction contrast in bright field which appears to be very sensitive to specimen tilt. Lorentz mode TEM has subsequently shown such regions to correspond exactly with magnetic domains. This contrast is attributed to the high magnetostrictive strain causing a local distortion of the lattice and thus a local deviation from the Bragg condition. This conclusion has been investigated and supported by TEM observations with the samples cooled below the spin reorientation temperature. When this transition is reached the diffraction contrast in bright field is considerably decreased and cannot be made to vary by tilting the specimen. The latter experiments also indicate that the change from <111> to <100> easy axis is not a well-defined one but, rather, that the spin reorientation is a sluggish change. High-resolution lattice images show the coherency of the twin boundaries.

  10. Scanning electron microscopy of eggs of Sabethes cyaneus.

    PubMed

    Santos-Mallet, Jacenir; Sarmento, Juliana Soares; Alencar, Jeronimo; Müller, Gerson Azulim; Oliveira, Eliana Medeiros; Foster, Woodbridge A; Marcondes, Carlos Brisola

    2013-03-01

    Mosquitoes of the Neotropical genus Sabethes, some species of which are yellow fever vectors, most often develop through the immature stages in tree holes. Sabethes eggs have not been previously characterized using scanning electron microscopy. Eggs of Sabethes cyaneus (length: 349.6 +/- 2.7 microm; width: 172.6 +/- 1.14 microm; n = 10) are almost biconical when examined from the top. From a lateral perspective 2 surfaces can be seen. One surface is smooth and more convex, whereas the other is less convex and partially covered by a network from which many fungiform tubercles arise. The micropyle is situated on the smooth surface of the pointed anterior tip and is surrounded by an irregular row of tubercles, some of which are leaf shaped. No structures possibly involved in adhesion to surfaces are visible. When hatching, the egg splits dorsoventrally approximately two-thirds of the length from the anterior end. The tubercles appear to be water repellent, and the more convex/smoother surface is downturned, and this position on water was confirmed by direct observation. The eggs float free on the water surface. PMID:23687859

  11. Scanning electron microscopy and roughness study of dental composite degradation.

    PubMed

    Soares, Luís Eduardo Silva; Cortez, Louise Ribeiro; Zarur, Raquel de Oliveira; Martin, Airton Abrahão

    2012-04-01

    Our aim was to test the hypothesis that the use of mouthwashes, consumption of soft drinks, as well as the type of light curing unit (LCU), would change the surface roughness (Ra) and morphology of a nanofilled composite resin (Z350® 3M ESPE). Samples (80) were divided into eight groups: Halogen LCU, group 1, saliva (control); group 2, Pepsi Twist®; group 3, Listerine®; group 4, Colgate Plax®; LED LCU, group 5, saliva; group 6, Pepsi Twist®; group 7, Listerine®; group 8, Colgate Plax®. Ra values were measured at baseline, and after 7 and 14 days. One specimen of each group was prepared for scanning electron microscopy analysis after 14 days. The data were subjected to multifactor analysis of variance at a 95% confidence followed by Tukey's honestly significant difference post-hoc test. All the treatments resulted in morphological changes in composite resin surface, and the most significant change was in Pepsi Twist® groups. The samples of G6 had the greatest increase in Ra. The immersion of nanofilled resin in mouthwashes with alcohol and soft drink increases the surface roughness. Polymerization by halogen LCU (reduced light intensity) associated with alcohol contained mouthwash resulted in significant roughness on the composite. PMID:22325725

  12. Electron microscopy, tissue culture,and immunology of ovarian carcinoma.

    PubMed

    Ioachim, H L; Dorsett, B H; Sabbath, M; Barber, H R

    1975-10-01

    The ultrastructure of the major histologic types of ovarian carcinoma was investigated as part of a multilateral study of this tumor. The nuclear and nucleolar changes in size, shape, and structure correlated well with the degree of malignancy and tumor grading. Cytoplasmic organelles and intercellular junctions were abundant and fairly well differentiated even in ovarian carcinomas of higher grade and stage. Active processes of synthesis and secretion taking place in most of these tumors were suggested by the presence of a richly granulated endoplasmic reticulum, dilated cisternae, and numerous secretory granules. Seventy-eight different ovarian carcinomas of all histologic types were cultured in vitro for periods of up to 300 days, and their morphology in light and electron microscopy was compared to that of the original tumors. The cultures displayed a consistent pattern of growth which led to the conclusion that ovarian cancer cells in vitro preserve their salient features and are representative of the tumors of origin. Heterologous antisera raised with pooled extracts of various types of ovarian carcinomas reacted specifically in immunodiffusion and immunofluorescence tests only with ovarian carcinomas and not with normal ovaries, benigh ovarian tumors, and nonovarian malignant neoplasms, indicating the presence of a cross-reacting specific antigen for ovarian carcinomas. In other studies, autologous antibodies were isolated from antigen-antibody complexes recovered from peritoneal effusions of patients with ovarian carcinomas. These antibodies displayed a high degree of specificity against ovarian carcinoma cells when tested in immunofluorescence assays.

  13. Non-thermal plasma mills bacteria: Scanning electron microscopy observations

    NASA Astrophysics Data System (ADS)

    Lunov, O.; Churpita, O.; Zablotskii, V.; Deyneka, I. G.; Meshkovskii, I. K.; Jäger, A.; Syková, E.; Kubinová, Š.; Dejneka, A.

    2015-02-01

    Non-thermal plasmas hold great promise for a variety of biomedical applications. To ensure safe clinical application of plasma, a rigorous analysis of plasma-induced effects on cell functions is required. Yet mechanisms of bacteria deactivation by non-thermal plasma remain largely unknown. We therefore analyzed the influence of low-temperature atmospheric plasma on Gram-positive and Gram-negative bacteria. Using scanning electron microscopy, we demonstrate that both Gram-positive and Gram-negative bacteria strains in a minute were completely destroyed by helium plasma. In contrast, mesenchymal stem cells (MSCs) were not affected by the same treatment. Furthermore, histopathological analysis of hematoxylin and eosin-stained rat skin sections from plasma-treated animals did not reveal any abnormalities in comparison to control ones. We discuss possible physical mechanisms leading to the shred of bacteria under non-thermal plasma irradiation. Our findings disclose how helium plasma destroys bacteria and demonstrates the safe use of plasma treatment for MSCs and skin cells, highlighting the favorability of plasma applications for chronic wound therapy.

  14. Electron microscopy of iron chalcogenide FeTe(Se) films

    SciTech Connect

    Shchichko, I. O.; Presnyakov, M. Yu.; Stepantsov, E. A.; Kazakov, S. M.; Antipov, E. V.; Makarova, I. P.; Vasil’ev, A. L.

    2015-05-15

    The structure of Fe{sub 1+δ}Te{sub 1−x}Se{sub x} films (x = 0; 0.05) grown on single-crystal MgO and LaAlO{sub 3} substrates has been investigated by transmission and scanning transmission electron microscopy. The study of Fe{sub 1.11}Te/MgO structures has revealed two crystallographic orientation relationships between the film and substrate. It is shown that the lattice mismatch between the film and substrate is compensated for by the formation of misfit dislocations. The Burgers vector projection is determined. The stresses in the film can partially be compensated for due to the formation of an intermediate disordered layer. It is shown that a FeTe{sub 0.5}Se{sub 0.5} film grown on a LaAlO{sub 3} substrate is single-crystal and that the FeTe{sub 0.5}Se{sub 0.5}/LaAlO{sub 3} interface in a selected region is coherent. The orientation relationships between the film and substrate are also determined for this case.

  15. TRANSMISSION ELECTRON MICROSCOPY STUDY OF HELIUM BEARING FUSION WELDS

    SciTech Connect

    Tosten, M; Michael Morgan, M

    2008-12-12

    A transmission electron microscopy (TEM) study was conducted to characterize the helium bubble distributions in tritium-charged-and-aged 304L and 21Cr-6Ni-9Mn stainless steel fusion welds containing approximately 150 appm helium-3. TEM foils were prepared from C-shaped fracture toughness test specimens containing {delta} ferrite levels ranging from 4 to 33 volume percent. The weld microstructures in the low ferrite welds consisted mostly of austenite and discontinuous, skeletal {delta} ferrite. In welds with higher levels of {delta} ferrite, the ferrite was more continuous and, in some areas of the 33 volume percent sample, was the matrix/majority phase. The helium bubble microstructures observed were similar in all samples. Bubbles were found in the austenite but not in the {delta} ferrite. In the austenite, bubbles had nucleated homogeneously in the grain interiors and heterogeneously on dislocations. Bubbles were not found on any austenite/austenite grain boundaries or at the austenite/{delta} ferrite interphase interfaces. Bubbles were not observed in the {delta} ferrite because of the combined effects of the low solubility and rapid diffusion of tritium through the {delta} ferrite which limited the amount of helium present to form visible bubbles.

  16. High-performance probes for light and electron microscopy.

    PubMed

    Viswanathan, Sarada; Williams, Megan E; Bloss, Erik B; Stasevich, Timothy J; Speer, Colenso M; Nern, Aljoscha; Pfeiffer, Barret D; Hooks, Bryan M; Li, Wei-Ping; English, Brian P; Tian, Teresa; Henry, Gilbert L; Macklin, John J; Patel, Ronak; Gerfen, Charles R; Zhuang, Xiaowei; Wang, Yalin; Rubin, Gerald M; Looger, Loren L

    2015-06-01

    We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization. PMID:25915120

  17. Warthin's tumour in Jamaica. Incidence, electron microscopy and immunoenzyme studies.

    PubMed

    Shah, D; Williams, E; Brooks, S E

    1990-12-01

    Warthin's tumour has traditionally had a strong male association, and has been said to be rare in Blacks. Current studies describe a new trend; a rise in females, strongly linked to cigarette smoking. The tumour has eosinophilic epithelial cells packed with distinctive mitochondria, and a lymphoid stroma. Immunological investigations have demonstrated polyclonal B cells, T cells and macrophages. Views differ as to whether B or T cells predominate. Between 1958 and 1989, the Jamaica Cancer Registry recorded 491 benign and malignant salivary gland tumours. There were 18 cases of Warthin's tumour (3.7%), with a male: female ratio of 5:1. The low proportion of females is similar to the trend for female lung cancer in Kingston & St. Andrew. A case of Warthin's tumour was studied by light and electron microscopy and immunoenzyme methods. The epithelial cells contained numerous mitochondria with stacked cristae, as previously described. Similar morphology occurs in oncocytic tumours; riboflavin-deficient rats and mice; rats given non-lethal doses of hypoglycin; dogs treated with annatto extracts; and hibernating or starving frogs. The mitochondrial changes may be an adaptive response. The immunoenzyme studies utilized newly available monoclonal antibodies: UCHL1, L26, 4KB5, MT1 and LN2. The reaction patterns indicate a distribution of B and T cells in a manner expected in a lymph node. The interaction between mitochondrial changes, adaptive metabolic pathways, the immune cells and tobacco raises some interesting questions.

  18. An overview on bioaerosols viewed by scanning electron microscopy.

    PubMed

    Wittmaack, K; Wehnes, H; Heinzmann, U; Agerer, R

    2005-06-15

    Bioaerosols suspended in ambient air were collected with single-stage impactors at a semiurban site in southern Germany during late summer and early autumn. Sampling was mostly carried out at a nozzle velocity of 35 m/s, corresponding to a minimum aerodynamic diameter (cut-off diameter) of aerosol particles of 0.8 mum. The collected particles, sampled for short periods ( approximately 15 min) to avoid pile-up, were characterized by scanning electron microscopy (SEM). The observed bioaerosols include brochosomes, fungal spores, hyphae, insect scales, hairs of plants and, less commonly, bacteria and epicuticular wax. Brochosomes, which serve as a highly water repellent body coating of leafhoppers, are hollow spheroids with diameters around 400 nm, resembling C(60) or footballs (soccer balls). They are usually airborne not as individuals but in the form of large clusters containing up to 10,000 individual species or even more. Various types of spores and scales were observed, but assignment turned out be difficult due to the large number of fungi and insects from which they may have originated. Pollens were observed only once. The absence these presumably elastic particles suggests that they are frequently lost, at the comparatively high velocities, due to bounce-off from the nonadhesive impaction surfaces.

  19. Transcription mapping of the Escherichia coli chromosome by electron microscopy.

    PubMed

    French, S L; Miller, O L

    1989-08-01

    The distinctive double Christmas tree morphology of rRNA operons as visualized by electron microscopy makes them easy to recognize in chromatin spreads from Escherichia coli. On the basis of the pattern of nascent transcripts on nearby transcription units and the relative distances of the operons from one another and the replication origin, we are now able to specifically identify five of the seven rRNA operons in E. coli. The use of rRNA operons as markers of both position and distance has resulted in the morphological mapping of a significant portion of the E. coli chromosome; over 600 kilobase pairs in the 84- to 90-min and 72-min regions can now be recognized. Since individual rRNA operons could be identified, direct comparisons could be made of their transcriptional activities. As judged by the densities of RNA polymerases along the operons, rrnA, rrnB, rrnC, rrnD, and rrnE were all transcribed at similar levels, with one RNA polymerase every 85 base pairs. The ability to recognize individual operons and specific regions of the chromosome allows direct comparisons of various genetic parameters.

  20. Nanoparticle imaging. 3D structure of individual nanocrystals in solution by electron microscopy.

    PubMed

    Park, Jungwon; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A; Zettl, A; Alivisatos, A Paul

    2015-07-17

    Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale. PMID:26185247

  1. 3D structure of individual nanocrystals in solution by electron microscopy

    NASA Astrophysics Data System (ADS)

    Park, Jungwon; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T.; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A.; Zettl, A.; Alivisatos, A. Paul

    2015-07-01

    Knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale.

  2. Mass mapping of large globin complexes by scanning transmission electron microscopy.

    PubMed

    Wall, Joseph S; Simon, Martha N; Lin, Beth Y; Vinogradov, Serge N

    2008-01-01

    Scanning transmission electron microscopy (STEM) of unstained, freeze-dried biological macromolecules in the dark-field mode provides an image based on the number of electrons elastically scattered by the constituent atoms of the macromolecule. The image of each isolated particle provides information about the projected structure of the latter, and its integrated intensity is directly related to the mass of the selected particle. Particle images can be sorted by shape, providing independent histograms of mass to study assembly/disassembly intermediates. STEM is optimized for low-dose imaging and is suitable for accurate measurement of particle masses over the range from about 30 kDa to 1,000 MDa. This article describes the details of the method developed at the Brookhaven National Laboratory STEM facility and illustrates its application to the mass mapping of large globin complexes.

  3. Electron beam heating effects during environmental scanning electron microscopy imaging of water condensation on superhydrophobic surfaces

    NASA Astrophysics Data System (ADS)

    Rykaczewski, K.; Scott, J. H. J.; Fedorov, A. G.

    2011-02-01

    Superhydrophobic surfaces (SHSs) show promise as promoters of dropwise condensation. Droplets with diameters below ˜10 μm account for the majority of the heat transferred during dropwise condensation but their growth dynamics on SHS have not been systematically studied. Due to the complex topography of the surface environmental scanning electron microscopy is the preferred method for observing the growth dynamics of droplets in this size regime. By studying electron beam heating effects on condensed water droplets we establish a magnification limit below which the heating effects are negligible and use this insight to study the mechanism of individual drop growth.

  4. In situ transmission electron microscopy of electron-beam induced damage process in nuclear grade graphite

    SciTech Connect

    C. Karthik; J. Kane; D. P. Butt; W. E. Windes; R. Ubic

    2011-05-01

    Atomic level processes involved in the swelling and crack-closing in nuclear grade graphite under electron irradiation have been observed in real-time using transmission electron microscopy. Noise-filtered lattice images show the formation of vacancy loops, interstitial loops and resulting dislocations with unprecedented clarity. The dislocation dipoles formed via vacancy loops were found to undergo climb resulting in extra basal planes. Concurrent EELS studies showed a reduction in the atomic density because of the breakage of hexagonal carbon rings. The formation of new basal planes via dislocation climb in addition to the bending/breaking of basal planes leads to swelling and closing of micro-cracks.

  5. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells

    PubMed Central

    Bauluz, Blanca; Canudo, José Ignacio; Gasca, José Manuel; Torcida Fernández-Baldor, Fidel

    2016-01-01

    Abnormalities in the histo- and ultrastructure of the amniote eggshell are often related to diverse factors, such as ambient stress during egg formation, pathologies altering the physiology of the egg-laying females, or evolutionarily selected modifications of the eggshell structure that vary the physical properties of the egg, for example increasing its strength so as to avoid fracture during incubation. When dealing with fossil materials, all the above hypotheses are plausible, but a detailed taphonomical study has to be performed to rule out the possibility that secondary processes of recrystallization have occurred during fossilization. Traditional analyses, such as optical microscopy inspection and cathodoluminescence, have proven not to be enough to understand the taphonomic story of some eggshells. Recently, electron backscatter diffraction has been used, in combination with other techniques, to better understand the alteration of fossil eggshells. Here we present a combined study using scanning electron microscopy, optical microscopy, cathodoluminescence and electron backscatter diffraction of eggshell fragments assigned to Megaloolithus cf. siruguei from the Upper Cretaceous outcrops of the Cameros Basin. We focus our study on the presence of secondary shell units that mimic most aspects of the ultrastructure of the eggshell mammillae, but grow far from the inner surface of the eggshell. We call these structures extra-spherulites, describe their crystal structure and demonstrate their secondary origin. Our study has important implications for the interpretation of secondary shell units as biological or pathological structures. Thus, electron backscatter diffraction complements other microscope techniques as a useful tool for understanding taphonomical alterations in fossil eggshells. PMID:27144767

  6. New electron microscopy techniques of the study of meteoritic metal.

    SciTech Connect

    Michael, Joseph Richard; Goldstein, Joseph I.; Kotula, Paul Gabriel; Jones, R. H.

    2005-02-01

    Metallic Phases in extraterrestrial materials are composed of Fe-Ni with minor amounts of Co, P, Si, Cr, etc. Electron microscopy techniques (SEM, TEM, EPMA, AEM) have been used for almost 50 years to study micron and submicron microscopic features in the metal phases (Fig. 1) such as clear taenite, cloudy zone, plessite, etc [1,2]. However lack of instrumentation to prepare TEM thin foils in specific sample locations and to obtain micro-scale crystallographic data have limited these investigations. New techniques such as the focused ion beam (FIB) and the electron backscatter electron diffraction (EBSD) techniques have overcome these limitations. The application of the FIB instrument has allowed us to prepare {approx}10 um long by {approx} 5um deep TEM thin sections of metal phases from specific regions of metal particles, in chondrites, irons and stony iron meteorites, identified by optical and SEM observation. Using a FEI dual beam FIB we were able to study very small metal particles in samples of CH chondrites [3] and zoneless plessite (ZP) in ordinary chondrites. Fig. 2 shows a SEM photomicrograph of a {approx}40 um ZP particle in Kernouve, a H6 chondrite. Fig. 3a,b shows a TEM photograph of a section of the FIB prepared TEM foil of the ZP particle and a Ni trace through a tetrataenite/kamacite region of the particle. It has been proposed that the Widmanstatten pattern in low P iron meteorites forms by martensite decomposition, via the reaction {gamma} {yields} {alpha}{sub 2} + {gamma} {yields} {alpha} + {gamma} in which {alpha}{sub 2}, martensite, decomposes to the equilibrium {alpha} and {gamma} phases during the cooling process [4]. In order to show if this mechanism for Widmanstatten pattern formation is correct, crystallographic information is needed from the {gamma} or taenite phases throughout a given meteorite. The EBSD technique was employed in this study to obtain the orientation of the taenite surrounding the initial martensite phase and the

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

    SciTech Connect

    De Jonge, Niels; Peckys, Diana B; Veith, Gabriel M; Joy, David Charles

    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.

  8. EDITORIAL: Electron Microscopy and Analysis Group Conference 2011 (EMAG 2011)

    NASA Astrophysics Data System (ADS)

    Moebus, Guenter; Walther, Thomas; Brydson, Rik; Ozkaya, Dogan; MacLaren, Ian; Donnelly, Steve; Nellist, Pete; Li, Ziyou; Baker, Richard; Chiu, YuLung

    2012-07-01

    The biennial EMAG conference has established a strong reputation as a key event for the national and international electron microscopy community. In 2011 the meeting was held at The University of Birmingham, and I must first take this opportunity of thanking Birmingham for hosting the conference and for the excellent support we received from the local organisers. As a committee, we are delighted to see that enthusiasm for the EMAG conference series continues to be strong. We received more than 160 submitted abstracts, and 157 delegates attended the meeting. The scientific programme organiser, Ian MacLaren, put together an exciting programme. Plenary lectures were presented by Professor Knut Urban, Dr Frances Ross and Dr Richard Henderson. There were a further 10 invited speakers, from the UK, Continental Europe, Australia, the USA and Japan. The quality of the contributed oral and poster presentations was also very high. EMAG is keen to encourage student participation, and a winner and two runners-up were presented with prizes for the best oral and poster presentations from a student. I am always struck by the scientific quality of the oral and poster contributions and the vibrant discussions that occur both in the formal sessions and in the exhibition space at EMAG. I am convinced that a crucial part of maintaining that scientific quality is the opportunity that is offered of having a paper fully reviewed by two internationally selected referees and published in the Journal of Physics: Conference Series. For many students, this is the first fully reviewed paper they publish. I hope that you, like me, will be struck by the scientific quality of the 87 papers that follow, and that you will find them interesting and informative. Finally I must thank the platinum sponsors for their support of the meeting. These were Gatan, Zeiss, FEI, JEOL and Hitachi. I must also thank the European Microscopy Society for their generous sponsorship and support for the travel costs of

  9. Scanning transmission x-ray microscopy of unaltered biological specimens

    SciTech Connect

    Iskander, N.

    1987-05-01

    A scanning transmission x-ray microscope at the National Synchrotron Light Source was used to image fresh, wet biological specimens at 32 Angstroms, with resolution better than 750 Angstroms. A gold Fresnel zone plate (outer zone width 500 Angstroms) was used to focus the undulator radiation, and the sample was scanned through the spot. Absorption data was recorded digitally as a gridded array. The major accomplishment of the experiment was the demonstration of the ability to image biological samples in their natural state with high resolution and natural elemental contrast mechanisms. This was achieved through the design of a sample holder that maintains an aqueous environment for the sample, yet is transparent to x-rays at 32 Angstroms. The specimens used were isolated zymogen granules (approximately 1 micron diameter) from the pancreatic acinar cells of rats. The absorption data were correlated to protein concentration, and estimates of the protein concentrations within the granules were obtained. The data also yields some information about the spatial organization of the protein in the granules, and our data is compared to models for the internal structure. The success of this experiment points toward future opportunities for dynamical studies on living systems. 6 refs., 28 figs., 2 tabs.

  10. Functional Materials characterizations by Scanning/Transmission Electron Microscopy and Electron Energy Loss spectroscopy

    NASA Astrophysics Data System (ADS)

    Yang, Bo

    Along with the fast development of science and technology, the studied materials are becoming more complicated and smaller. All these achievements have advanced with the fast development of powerful tools currently, such as Scanning electron microscopy (SEM), Focused Ion Beam (FIB), Transmission electron microscopy (TEM), Energy dispersive X-ray spectroscopy (EDX), Electron energy loss spectroscopy (EELS) and so on. SiTiO3 thin film, which is grown on Si (100) single crystals, attracts a lot of interest in its structural and electronic properties close to its interface. Valence EELS is used to investigate the Plasmon excitations of the ultrathin SrTiO3 thin film which is sandwiched between amorphous Si and crystalline Si layers. On the other hand, theoretical simulations based on dielectric functions have been done to interpret the experimental results. Our findings demonstrate the value of valence electron energy-loss spectroscopy in detecting a local change in the effective electron mass. Recently it is reported that ZnO-LiYbO2 hybrid phosphor is an efficient UV-infrared convertor for silicon solar cell but the mechanism is still not very clear. The microstructure of Li and Yb co-doped ZnO has been studied by SEM and EDX, and our results suggest that a reaction (or diffusion) zone is very likely to exist between LiYbO2 and ZnO. Such diffusion regions may be responsible for the enhanced infrared emission in the Yb and Li co-doped ZnO. Furthermore, to help us study the diffusion zone under TEM in future, the radiation damage on synthesized LiYbO2 has been studied at first, and then the electronic structure of the synthesized LiYbO2 is compared with Yb2O 3 experimentally and theoretically, by EELS and FEFF8 respectively.

  11. PREFACE: Electron Microscopy and Analysis Group Conference 2009

    NASA Astrophysics Data System (ADS)

    Baker, Richard

    2010-04-01

    The latest biennial conference of the Electron Microscopy and Analysis Group (EMAG) of the Institute of Physics was held at the University of Sheffield on 9-11 September, 2009. In addition, the Advanced School associated with the conference was run at the University of Sheffield on 8 September. It was particularly pleasing to return to Sheffield after ten years, the successful and memorable EMAG 99 having been held here too. The subject areas covered at EMAG 2009 were advanced electron microscopy techniques; investigating structure-property relationships in advanced materials; nanophysics and nanotechnology. The EMAG 2009 conference attracted 172 delegates while the Advanced School had a full complement of eighteen attendees. Three plenary lectures were given to the whole conference and invited contributions were presented within the theme of each of nine parallel sessions. There were 54 contributed oral presentations within these parallel Sessions and a further 89 poster presentations. All authors were invited to contribute a paper to this Proceedings volume and 108 papers are presented here. I thank all who presented at EMAG 2009 and those who provided a paper for this Proceedings. Each paper was peer reviewed by two reviewers and I also want to thank those colleagues who helped with this essential task. In this volume, the plenary papers are presented first followed by all papers presented in each themed session. These sessions are ordered alphabetically. Within each Session, the invited presentations are presented first, followed by oral and poster contributions together. Another activity of EMAG which is directed primarily at less experienced scientists is the Advanced School. This year, this was on Nanofabrication and Nanomanipulation and I want to thank Guenter Moebus and his colleagues at th University of Sheffield for putting on such an excellent Advanced School. The EMAG series of conferences are well-known not only for the academic conference but also

  12. Transmission Electron Microscopy of Iron Metal in Almahata Sitta Ureilite

    NASA Technical Reports Server (NTRS)

    Mikouchi, T.; Yubuta, K.; Sugiyama, K.; Aoyagi, Y.; Yasuhara, A.; Mihira, T.; Zolensky, M. E.; Goodrich, C. A.

    2013-01-01

    Almahata Sitta (AS) is a polymict breccia mainly composed of variable ureilite lithologies with small amounts of chondritic lithologies [1]. Fe metal is a common accessory phase in ureilites, but our earlier study on Fe metals in one of AS fragments (#44) revealed a unique mineralogy never seen in other ureilites [2,3]. In this abstract we report detailed transmission electron microscopy (TEM) on these metal grains to better understand the thermal history of ureilites. We prepared FIB sections of AS#44 by JEOL JIB-4000 from the PTS that was well characterized by SEM-EBSD in our earlier study [2]. The sections were then observed by STEM (JEOL JEM- 2100F). One of the FIB sections shows a submicron-sized symplectic intergrown texture composed of Fe metal (kamacite), Fe carbide (cohenite), Fe phosphide (schreibersite), and Fe sulfide (troilite). Each phase has an identical SAED pattern in spite of its complex texture, suggesting co-crystallization of all phases. This is probably caused by shock re-melting of pre-existing metal + graphite to form a eutectic-looking texture. The other FIB section is mostly composed of homogeneous Fe metal (93 wt% Fe, 5 wt% Ni, and 2 wt% Si), but BF-STEM images exhibited the presence of elongated lathy grains (approx. 2 microns long) embedded in the interstitial matrix. The SAED patterns from these lath grains could be indexed by alpha-Fe (bcc) while interstitial areas are gamma-Fe (fcc). The elongated alpha-Fe grains show tweed-like structures suggesting martensite transformation. Such a texture can be formed by rapid cooling from high temperature where gamma-Fe was stable. Subsequently alpha-Fe crystallized, but gamma-Fe remained in the interstitial matrix due to quenching from high temperature. This scenario is consistent with very rapid cooling history of ureilites suggested by silicate mineralogy.

  13. Coliphage P1 morphogenesis: analysis of mutants by electron microscopy.

    PubMed Central

    Walker, J T; Walker, D H

    1983-01-01

    We used electron microscopy and serum blocking power tests to determine the phenotypes of 47 phage P1 amber mutants that have defects in particle morphogenesis. Eleven mutants showed head defects, 30 showed tail defects, and 6 had a defect in particle maturation (which could be either in the head or in the tail). Consideration of previous complementation test results, genetic and physical positions of the mutations, and phenotypes of the mutants allowed assignment of most of the 47 mutations to genes. Thus, a minimum of 12 tail genes, 4 head genes, and 1 particle maturation gene are now known for P1. Of the 12 tail genes, 1 (gene 19, located within the invertible C loop) codes for tail fibers, 6 (genes 3, 5, 16, 20, 21, and 26) code for baseplate components (although one of these genes could code for the tail tube), 1 (gene 22) codes for the sheath, 1 (gene 6) affects tail length, 2 (genes 7 and 25) are involved in tail stability, and 1 (gene 24) either codes for a baseplate component or is involved in tail stability. Of the four head genes, gene 9 codes for a protein required for DNA packaging. The function of head gene 4 is unclear. Head gene 8 probably codes for a minor head protein, whereas head gene 23 could code for either a minor head protein or the major head protein. Excluding the particle maturation gene (gene 1), the 12 tail genes are clustered in three regions of the P1 physical genome. The four head genes are at four separate locations. However, some P1 head genes have not yet been detected and could be located in two regions (for which there are no known genes) adjacent to genes 4 and 8. The P1 morphogenetic gene clusters are interrupted by many genes that are expressed in the prophage. Images PMID:6834479

  14. Transmission electron microscopy of subsolidus oxidation and weathering of olivine

    USGS Publications Warehouse

    Banfield, J.F.; Veblen, D.R.; Jones, B.F.

    1990-01-01

    Olivine crystals in basaltic andesites which crop out in the Abert Rim, south-central Oregon have been studied by high-resolution and analytical transmission electron microscopy. The observations reveal three distinct assemblages of alteration products that seem to correspond to three episodes of olivine oxidation. The olivine crystals contain rare, dense arrays of coherently intergrown Ti-free magnetite and inclusions of a phase inferred to be amorphous silica. We interpret this first assemblage to be the product of an early subsolidus oxidation event in the lava. The second olivine alteration assemblage contains complex ordered intergrowths on (001) of forsterite-rich olivine and laihunite (distorted olivine structure with Fe3+ charge balanced by vacancies). Based on experimental results for laihunite synthesis (Kondoh et al. 1985), these intergrowths probably formed by olivine oxidation between 400 and 800??C. The third episode of alteration involves the destruction of olivine by low-temperature hydrothermal alteration and weathering. Elongate etch-pits and channels in the margins of fresh olivine crystals contain semi-oriented bands of smectite. Olivine weathers to smectite and hematite, and subsequently to arrays of oriented hematite crystals. The textures resemble those reported by Eggleton (1984) and Smith et al. (1987). We find no evidence for a metastable phase intermediate between olivine and smectite ("M" - Eggleton 1984). The presence of laihunite exerts a strong control on the geometry of olivine weathering. Single laihunite layers and laihunite-forsteritic olivine intergrowths increase the resistance of crystals to weathering. Preferential development of channels between laihunite layers occurs where growth of laihunite produced compositional variations in olivine, rather than where coherency-strain is associated with laihunite-olivine interfaces. ?? 1990 Springer-Verlag.

  15. EDITORIAL: Electron Microscopy and Analysis Group Conference 2013 (EMAG2013)

    NASA Astrophysics Data System (ADS)

    Nellist, Pete

    2014-06-01

    It has once again been my pleasure to act as editor for these proceedings, and I must thank all those who have acted as reviewers. I am always struck by the scientific quality of the oral and poster contributions and the vibrant discussions that occur both in the formal sessions and in the exhibition space at EMAG. I am convinced that a crucial part of maintaining that scientific quality is the opportunity that is offered of having a paper fully reviewed by two internationally selected referees and published in the Journal of Physics: Conference Series. For many students, this is the first fully reviewed paper they publish. I hope that, like me, you will be struck by the scientific quality of the 80 papers that follow, and that you will find them interesting and informative. I must also personally thank all the organisers of EMAG2013 for arranging such an excellent meeting. Ian MacLaren, as Chair of the EMAG Group and of the meeting itself, has contributed a foreword to these proceedings describing the meeting in more detail. A particular highlight of the conference was the special symposium in honour of Professor Archie Howie. We all enjoyed a wonderful speech from Archie at the conference dinner, along with some of his electron microscopy-related poetry. I have great pleasure in publishing the conference dinner poems in this proceedings. I hope you will find these proceedings to be an interesting read and an invaluable resource. Pete Nellist Conference committee Conference chair: Dr I MacLaren Programme organiser: Dr C Ducati Proceedings editor: Prof P D Nellist Trade exhibition organiser: C Hockey (CEM Group) Local organisers: Professor E Boyes, Professor P Gai, Dr R Kröger, Dr V Lazarov, Dr P O'Toole, Dr S Tear and Professor J Yuan Advanced school organisers: Dr S Haigh, Dr A Brown Other committee members: Mr K Meade, Mr O Heyning, Dr M Crawford, Mr M Dixon and Dr Z Li

  16. Sample thickness determination by scanning transmission electron microscopy at low electron energies.

    PubMed

    Volkenandt, Tobias; Müller, Erich; Gerthsen, Dagmar

    2014-02-01

    Sample thickness is a decisive parameter for any quantification of image information and composition in transmission electron microscopy. In this context, we present a method to determine the local sample thickness by scanning transmission electron microscopy at primary energies below 30 keV. The image intensity is measured with respect to the intensity of the incident electron beam and can be directly compared with Monte Carlo simulations. Screened Rutherford and Mott scattering cross-sections are evaluated with respect to fitting experimental data with simulated image intensities as a function of the atomic number of the sample material and primary electron energy. The presented method is tested for sample materials covering a wide range of atomic numbers Z, that is, fluorenyl hexa-peri-hexabenzocoronene (Z = 3.5), carbon (Z = 6), silicon (Z = 14), gallium nitride (Z = 19), and tungsten (Z = 74). Investigations were conducted for two primary energies (15 and 30 keV) and a sample thickness range between 50 and 400 nm.

  17. High resolution analytical transmission electron microscopy of magnetic recording media

    NASA Astrophysics Data System (ADS)

    Risner, Juliet Danielle

    Since the invention of the hard disk drive in 1954, the density of bits per disk has increased exponentially. This trend is partly due to improvements to the magnetic recording media. In current hard disks, each bit is approximately 0.04 mum in its smallest dimension and comprises ˜100 hexagonal close packed Co-alloy magnetic grains. These grains have magnetic "easy" axes oriented longitudinally, or parallel to the film plane. Future recording media have easy axes oriented perpendicular to the film plane. Perpendicular media are expected to provide continued increases in storage density above the limit of longitudinal media. Quantum-mechanical exchange coupling between magnetic grains degrades the signal-to-noise ratio (SNR) and limits storage density in both media types. Controlling exchange coupling is possible by creating nonmagnetic grain boundaries which compositionally isolate the magnetic grains. High-resolution analytical transmission electron microscopy (TEM) is required to study these media because of their nano-scale grains and grain boundaries. Examining the microstructure and elemental distribution in these films at near atomic level is paramount to understanding their magnetic performance. The microstructure and elemental distribution in longitudinal and perpendicular media were examined using high resolution analytical TEM techniques, such as energy-filtered TEM (EFTEM), energy-dispersive x-ray spectroscopy (EDS) using a 1.5 nm electron probe, and spectrum imaging with a scanning TEM. These techniques successfully determined how grain boundary Cr segregation varies with grain orientation in longitudinal media. Boundaries misoriented by 0° and 90° commonly occur and were found to have minimal Cr segregation, which limits storage density improvement in these media. Analytical TEM techniques applied to oxygen-enriched perpendicular media, fabricated using different deposition methods, effectively related microstructure and composition to magnetic

  18. Transmission electron microscopy investigation of auto catalyst and cobalt germanide

    NASA Astrophysics Data System (ADS)

    Sun, Haiping

    The modern ceria-zirconia based catalysts are used in automobiles to reduce exhaust pollutants. Cobalt germanides have potential applications as electrical contacts in the future Ge-based semiconductor devices. In this thesis, transmission electron microscopy (TEM) techniques were used to study the atomic scale interactions between metallic nanostructures and crystalline substrates in the two material systems mentioned above. The model catalyst samples consisted of precious metal nano-particles (Pd, Rh) supported on the surface of (Ce,Zr)O2 thin films. The response of the microstructure of the metal-oxide interface to the reduction and oxidation treatments was investigated by cross-sectional high resolution TEM. Atomic detail of the metal-oxide interface was obtained. It was found that Pd and Rh showed different sintering and interaction behaviors on the oxide surface. The preferred orientation of Pd particles in this study was Pd(111)//CZO(111). Partial encapsulation of Pd particles by reduced (Ce,Zr)O 2 surface was observed and possible mechanisms of the encapsulation were discussed. The characteristics of the metal-oxide interaction depend on the properties of the oxide, as well as their relative orientation. The results provide experimental evidence for understanding the thermodynamics of the equilibrium morphology of a solid particle supported on a solid surface that is not considered as inert. The reaction of Co with Ge to form epitaxial Co5Ge7 was studied by in situ ultra-high vacuum (UHV) TEM using two methods. One was reactive deposition of Co on Ge, in which the Ge substrate was maintained at 350°C during deposition. The other method was solid state reaction, in which the deposition of Co on Ge was carried out at room temperature followed by annealing to higher temperatures. During reactive deposition, the deposited Co reacted with Ge to form nanosized 3D Co 5Ge7 islands. During solid state reaction, a continuous epitaxial Co5Ge7 film on the (001) Ge

  19. Practical performance evaluation of a 10k × 10k CCD for electron cryo-microscopy

    PubMed Central

    Bammes, Benjamin E.; Rochat, Ryan H.; Jakana, Joanita; Chiu, Wah

    2011-01-01

    Electron cryo-microscopy (cryo-EM) images are commonly collected using either charge-coupled devices (CCD) or photographic film. Both film and the current generation of 16 megapixel (4k × 4k) CCD cameras have yielded high-resolution structures. Yet, despite the many advantages of CCD cameras, more than two times as many structures of biological macromolecules have been published in recent years using photographic film. The continued preference to film, especially for subnanometer-resolution structures, may be partially influenced by the finer sampling and larger effective specimen imaging area offered by film. Large format digital cameras may finally allow them to overtake film as the preferred detector for cryo-EM. We have evaluated a 111-megapixel (10k × 10k) CCD camera with a 9 μm pixel size. The spectral signal-to-noise ratios of low dose images of carbon film indicate that this detector is capable of providing signal up to at least 2/5 Nyquist frequency potentially retrievable for 3-D reconstructions of biological specimens, resulting in more than double the effective specimen imaging area of existing 4k × 4k CCD cameras. We verified our estimates using frozen-hydrated ε15 bacteriophage as a biological test specimen with previously determined structure, yielding a ~7 Å resolution single particle reconstruction from only 80 CCD frames. Finally, we explored the limits of current CCD technology by comparing the performance of this detector to various CCD cameras used for recording data yielding subnanometer resolution cryo-EM structures submitted to the Electron Microscopy Data Bank (http://www.emdatabank.org/). PMID:21619932

  20. Focused ion beam (FIB)/scanning electron microscopy (SEM) in tissue structural research.

    PubMed

    Leser, Vladka; Milani, Marziale; Tatti, Francesco; Tkalec, Ziva Pipan; Strus, Jasna; Drobne, Damjana

    2010-10-01

    The focused ion beam (FIB) and scanning electron microscope (SEM) are commonly used in material sciences for imaging and analysis of materials. Over the last decade, the combined FIB/SEM system has proven to be also applicable in the life sciences. We have examined the potential of the focused ion beam/scanning electron microscope system for the investigation of biological tissues of the model organism Porcellio scaber (Crustacea: Isopoda). Tissue from digestive glands was prepared as for conventional SEM or as for transmission electron microscopy (TEM). The samples were transferred into FIB/SEM for FIB milling and an imaging operation. FIB-milled regions were secondary electron imaged, back-scattered electron imaged, or energy dispersive X-ray (EDX) analyzed. Our results demonstrated that FIB/SEM enables simultaneous investigation of sample gross morphology, cell surface characteristics, and subsurface structures. The same FIB-exposed regions were analyzed by EDX to provide basic compositional data. When samples were prepared as for TEM, the information obtained with FIB/SEM is comparable, though at limited magnification, to that obtained from TEM. A combination of imaging, micro-manipulation, and compositional analysis appears of particular interest in the investigation of epithelial tissues, which are subjected to various endogenous and exogenous conditions affecting their structure and function. The FIB/SEM is a promising tool for an overall examination of epithelial tissue under normal, stressed, or pathological conditions.