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Sample records for quantitative electron microscopy

  1. 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

  2. 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

  3. 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

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Factors influencing quantitative liquid (scanning) transmission electron microscopy.

    PubMed

    Abellan, P; Woehl, T J; Parent, L R; Browning, N D; Evans, J E; Arslan, I

    2014-05-18

    One of the experimental challenges in the study of nanomaterials in liquids in the (scanning) transmission electron microscope ((S)TEM) is gaining quantitative information. A successful experiment in the fluid stage will depend upon the ability to plan for sensitive factors such as the electron dose applied, imaging mode, acceleration voltage, beam-induced solution chemistry changes, and the specifics of solution reactivity. In this paper, we make use of a visual approach to show the extent of damage of different instrumental and experimental factors in liquid samples imaged in the (S)TEM. Previous results as well as new insights are presented to create an overview of beam-sample interactions identified for changing imaging and experimental conditions. This work establishes procedures to understand the effect of the electron beam on a solution, provides information to allow for a deliberate choice of the optimal experimental conditions to enable quantification, and identifies the experimental factors that require further analysis for achieving fully quantitative results in the liquid (S)TEM.

  9. 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)

  10. Some strategies for quantitative scanning Auger electron microscopy

    NASA Technical Reports Server (NTRS)

    Browning, R.; Peacock, D. C.; Prutton, M.

    1985-01-01

    The general applicability of power law forms of the background in electron spectra is pointed out and exploited for background removal from under Auger peaks. This form of B(E) is found to be extremely sensitive to instrumental alignment and to fault-free construction - an observation which can be used to set up analyser configurations in an accurate way. Also, differences between N(E) and B(E) can be used to derive a spectrometer transmission function T(E). The questions of information density in an energy-analysing spatially-resolving instrument are addressed after reliable instrumental characterization has been established. Strategies involving ratio histograms, showing the population distribution of the ratio of a pair of Auger peak heights, composition scatter diagrams and windowed imaging are discussed and illustrated.

  11. Quantitative fluctuation electron microscopy in the STEM: methods to identify, avoid, and correct for artifacts.

    PubMed

    Li, Tian T; Bogle, Stephanie N; Abelson, John R

    2014-10-01

    Fluctuation electron microscopy can reveal the nanoscale order in amorphous materials via the statistical variance in the scattering intensity as a function of position, scattering vector, and resolution. However, several sources of experimental artifacts can seriously affect the magnitude of the variance peaks. The use of a scanning transmission electron microscope for data collection affords a convenient means to check whether artifacts are present. As nanodiffraction patterns are collected in serial, any spatial or temporal dependence of the scattering intensity across the series can easily be detected. We present examples of the major types of artifact and methods to correct the data or to avoid the problem experimentally. We also re-cast the statistical formalism used to identify sources of noise in view of the present results. The present work provides a basis on which to perform fluctuation electron microscopy with a high level of reliability and confidence in the quantitative magnitude of the data.

  12. 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

  13. A methodology for the extraction of quantitative information from electron microscopy images at the atomic level

    NASA Astrophysics Data System (ADS)

    Galindo, P. L.; Pizarro, J.; Guerrero, E.; Guerrero-Lebrero, M. P.; Scavello, G.; Yáñez, A.; Núñez-Moraleda, B. M.; Maestre, J. M.; Sales, D. L.; Herrera, M.; Molina, S. I.

    2014-06-01

    In this paper we describe a methodology developed at the University of Cadiz (Spain) in the past few years for the extraction of quantitative information from electron microscopy images at the atomic level. This work is based on a coordinated and synergic activity of several research groups that have been working together over the last decade in two different and complementary fields: Materials Science and Computer Science. The aim of our joint research has been to develop innovative high-performance computing techniques and simulation methods in order to address computationally challenging problems in the analysis, modelling and simulation of materials at the atomic scale, providing significant advances with respect to existing techniques. The methodology involves several fundamental areas of research including the analysis of high resolution electron microscopy images, materials modelling, image simulation and 3D reconstruction using quantitative information from experimental images. These techniques for the analysis, modelling and simulation allow optimizing the control and functionality of devices developed using materials under study, and have been tested using data obtained from experimental samples.

  14. Second harmonic generation quantitative measurements on collagen fibrils through correlation to electron microscopy

    NASA Astrophysics Data System (ADS)

    Bancelin, S.; Aimé, C.; Gusachenko, I.; Kowalczuk, L.; Latour, G.; Coradin, T.; Schanne-Klein, M.-C.

    2015-03-01

    Type I collagen is a major structural protein in mammals that shows highly structured macromolecular organizations specific to each tissue. This biopolymer is synthesized as triple helices, which self-assemble into fibrils (Ø =10-300 nm) and further form various 3D organization. In recent years, Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique to probe in situ the fibrillar collagenous network within tissues. However, this optical technique cannot resolve most of the fibrils and is a coherent process, which has impeded quantitative measurements of the fibril diameter so far. In this study, we correlated SHG microscopy with Transmission Electron Microscopy to determine the sensitivity of SHG microscopy and to calibrate SHG signals as a function of the fibril diameter in reconstructed collagen gels. To that end, we synthetized isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter. We observed that SHG signals scaled as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. This calibration was then applied to diabetic rat cornea in which we successfully recovered the diameter of hyperglycemia-induced fibrils in the Descemet's membrane without having to resolve them. Finally we derived the first hyperpolarizability from a single collagen triple helix which validates the bottom-up approach used to calculate the non-linear response at the fibrillar scale and denotes a parallel alignment of triple helices within the fibrils. These results represent a major step towards quantitative SHG imaging of nm-sized collagen fibrils.

  15. Quantitative magnetic imaging at the nanometer scale by ballistic electron magnetic microscopy

    SciTech Connect

    Herve, M.; Tricot, S.; Guezo, S.; Delhaye, G.; Lepine, B.; Schieffer, P.; Turban, P.

    2013-06-21

    We demonstrate quantitative ballistic electron magnetic microscopy (BEMM) imaging of simple model Fe(001) nanostructures. We use in situ nanostencil shadow mask resistless patterning combined with molecular beam epitaxy deposition to prepare under ultra-high vacuum conditions nanostructured epitaxial Fe/Au/Fe/GaAs(001) spin-valves. In this epitaxial system, the magnetization of the bottom Fe/GaAs(001) electrode is parallel to the [110] direction, defining accurately the analysis direction for the BEMM experiments. The large hot-electron magnetoresistance of the Fe/Au/Fe/GaAs(001) epitaxial spin-valve allows us to image various stable magnetic configurations on the as-grown Fe(001) microstructures with a high sensitivity, even for small misalignments of both magnetic electrodes. The angular dependence of the hot-electron magnetocurrent is used to convert magnetization maps calculated by micromagnetic simulations into simulated BEMM images. The calculated BEMM images and magnetization rotation profiles show quantitative agreement with experiments and allow us to investigate the magnetic phase diagram of these model Fe(001) microstructures. Finally, magnetic domain reversals are observed under high current density pulses. This opens the way for further BEMM investigations of current-induced magnetization dynamics.

  16. Quantitative Description of Crystal Nucleation and Growth from in Situ Liquid Scanning Transmission Electron Microscopy.

    PubMed

    Ievlev, Anton V; Jesse, Stephen; Cochell, Thomas J; Unocic, Raymond R; Protopopescu, Vladimir A; Kalinin, Sergei V

    2015-12-22

    Recent advances in liquid cell (scanning) transmission electron microscopy (S)TEM has enabled in situ nanoscale investigations of controlled nanocrystal growth mechanisms. Here, we experimentally and quantitatively investigated the nucleation and growth mechanisms of Pt nanostructures from an aqueous solution of K2PtCl6. Averaged statistical, network, and local approaches have been used for the data analysis and the description of both collective particles dynamics and local growth features. In particular, interaction between neighboring particles has been revealed and attributed to reduction of the platinum concentration in the vicinity of the particle boundary. The local approach for solving the inverse problem showed that particles dynamics can be simulated by a stationary diffusional model. The obtained results are important for understanding nanocrystal formation and growth processes and for optimization of synthesis conditions.

  17. Quantitative optical phase microscopy.

    PubMed

    Barty, A; Nugent, K A; Paganin, D; Roberts, A

    1998-06-01

    We present a new method for the extraction of quantitative phase data from microscopic phase samples by use of partially coherent illumination and an ordinary transmission microscope. The technique produces quantitative images of the phase profile of the sample without phase unwrapping. The technique is able to recover phase even in the presence of amplitude modulation, making it significantly more powerful than existing methods of phase microscopy. We demonstrate the technique by providing quantitatively correct phase images of well-characterized test samples and show that the results obtained for more-complex samples correlate with structures observed with Nomarski differential interference contrast techniques.

  18. Quantitative transmission electron microscopy analysis of the nanocrystallization kinetics of soft magnetic alloys

    NASA Astrophysics Data System (ADS)

    Ramanujan, R. V.; Zhang, Y. R.

    2006-12-01

    Transmission electron microscopy was used for the first time to obtain quantitative values of the diffusional crystallization kinetics of initially amorphous Fe74.5Si13.5B9Nb3 , Fe76.5Si13.5B9Cu1 and Fe73.5Si13.5B9Nb3Cu1 and Fe77.5Si13.5Nb3Cu1 (Finemet) alloys. The role of Cu and Nb alloying additions was elucidated. Contrary to some models of the crystallization process, it is demonstrated that both Nb and Cu alloying additions can influence the nucleation and growth processes. Combined additions of both Cu and Nb induce drastic reduction in crystal size to about 10nm and 1000 times higher crystal number density. The high nucleation rate observed in the Fe-Si-B-Nb-Cu alloy was attributed to the formation of both Cu and Nb rich regions which provide a high number of heterogeneous nucleation sites, consistent with the Hampel and Pradell models, the low growth rate was consistent with the Hunziker model.

  19. Quantitative evaluation of monocyte differentiation by electron microscopy: impairment of differentiation ability in monocytes from children with acute lymphoblastic leukaemia.

    PubMed

    Tsukada, M; Hara, Y; Sugiyama, H; Yoda, S; Hara, T; Komiyama, A; Akabane, T

    1985-05-01

    The differentiation of monocytes was evaluated quantitatively by electron microscopy and was analyzed in relation to the clinical features of childhood acute lymphoblastic leukaemia (ALL). The monocyte cellular size increased and cell organellae became mature after a 72-h culture. Phagolysosome formation developed markedly- and the nucleus became circular--accompanied by chromatin deconcentration. The differentiation degree of the cell organellae was indexed and classified as mature by electron microscopy. The indexes of nuclear shape, chromatin deconcentration, cytoplasmonuclear ratio and vacuole formation increased with time. The total index increased linearly with time dependence. These results indicate that the ultrastructural parameters and indexes allowed a quantitative assessment of cell organellae and cellular differentiation of the monocyte. At the acute phase of ALL, the degrees of cell organnellae and cellular differentiation were significantly lower than the control. The above findings suggest that monocytes from children with ALL have impaired differentiation ability, which results in defective function of macrophages.

  20. Quantitative electron microscopy for the nanoscale analysis of membrane lipid distribution.

    PubMed

    Fujita, Akikazu; Cheng, Jinglei; Fujimoto, Toyoshi

    2010-04-01

    An important goal of membrane biology is to define the local heterogeneity of membrane lipid composition. Here we describe a quantitative electron microscopic method that enables the localization of specific membrane lipids at the nanometer scale. The method involves freezing cells rapidly to halt the molecular motion, physically stabilizing membrane molecules in the freeze-fracture replica by the deposition of evaporated platinum and carbon layers and labeling with specific probes for electron microscopic observation. Lipids in both the outer and inner membrane leaflets can thus be labeled, and their distributions can be analyzed quantitatively by statistical methods. A major advantage of this method is that it does not require the expression of artificial probes. Therefore, this method can be applied to any cell in vitro or in vivo, and the whole procedure can be completed in 1-2 d.

  1. Quantitative measurements in in situ straining experiments in transmission electron microscopy.

    PubMed

    Pettinari, F; Couret, A; Caillard, D; Molénat, G; Clément, N; Coujou, A

    2001-07-01

    Several examples of recent studies by in situ straining experiments in a transmission electron microscope performed in the Toulouse group (France) are presented. In particular, quantitative measurements of the features of the dislocation motion are described. These examples deal with individual or collective propagation of dislocations, which are submitted to various types of obstacle. Different metallic materials are investigated: magnesium, intermetallics, aluminium alloys and gamma phase of a superalloy. PMID:11454154

  2. Discrepancies in quantitative assessment of normal and regenerated peripheral nerve fibers between light and electron microscopy.

    PubMed

    Ronchi, Giulia; Jager, Sara Buskbjerg; Vaegter, Christian Bjerggaard; Raimondo, Stefania; Giacobini-Robecchi, Maria Giuseppina; Geuna, Stefano

    2014-09-01

    Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results.

  3. Dose limited reliability of quantitative annular dark field scanning transmission electron microscopy for nano-particle atom-counting.

    PubMed

    De Backer, A; Martinez, G T; MacArthur, K E; Jones, L; Béché, A; Nellist, P D; Van Aert, S

    2015-04-01

    Quantitative annular dark field scanning transmission electron microscopy (ADF STEM) has become a powerful technique to characterise nano-particles on an atomic scale. Because of their limited size and beam sensitivity, the atomic structure of such particles may become extremely challenging to determine. Therefore keeping the incoming electron dose to a minimum is important. However, this may reduce the reliability of quantitative ADF STEM which will here be demonstrated for nano-particle atom-counting. Based on experimental ADF STEM images of a real industrial catalyst, we discuss the limits for counting the number of atoms in a projected atomic column with single atom sensitivity. We diagnose these limits by combining a thorough statistical method and detailed image simulations.

  4. Quantitative high-angle annular dark field scanning transmission electron microscopy for materials science

    NASA Astrophysics Data System (ADS)

    Petrova, Rumyana V.

    Scanning transmission electron microscopy (STEM) has been widely used for characterization of materials; to identify micro- and nano-structures within a sample and to analyze crystal and defect structures. High-angle annular dark field (HAADF) STEM imaging using atomic number (Z) contrast has proven capable of resolving atomic structures with better than 2 A lateral resolution. In this work, the HAADF STEM imaging mode is used in combination with multislice simulations. This combination is applied to the investigation of the temperature dependence of the intensity collected by the HAADF detector in silicon, and to convergent beam electron diffraction (CBED) to measure the degree of chemical order in intermetallic nanoparticles. The experimental and simulation results on the high-angle scattering of 300 keV electrons in crystalline silicon provide a new contribution to the understanding of the temperature dependence of the HAADF intensity. In the case of 300 keV, the average high-angle scattered intensity slightly decreases as the temperature increases from 100 K to 300 K, and this is different from the temperature dependence at 100 keV and 200 keV where HAADF intensity increases with temperature, as had been previously reported by other workers. The L10 class of hard magnetic materials has attracted continuous attention as a candidate for high-density magnetic recording media, as this phase is known to have large magnetocrystalline anisotropy, with magnetocrystalline anisotropy constant, Ku, strongly dependent on the long-range chemical order parameter, S. A new method is developed to assess the degree of chemical order in small FePt L1 0 nanoparticles by implementing a CBED diffraction technique. Unexpectedly, the degree of order of individual particles is highly variable and not a simple function of particle size or sample composition. The particle-to-particle variability observed is an important new aspect to the understanding of phase transformations in

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

    PubMed

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

    2013-04-01

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

  6. Quantitative characterization of agglomerates and aggregates of pyrogenic and precipitated amorphous silica nanomaterials by transmission electron microscopy

    PubMed Central

    2012-01-01

    Background The interaction of a nanomaterial (NM) with a biological system depends not only on the size of its primary particles but also on the size, shape and surface topology of its aggregates and agglomerates. A method based on transmission electron microscopy (TEM), to visualize the NM and on image analysis, to measure detected features quantitatively, was assessed for its capacity to characterize the aggregates and agglomerates of precipitated and pyrogenic synthetic amorphous silicon dioxide (SAS), or silica, NM. Results Bright field (BF) TEM combined with systematic random imaging and semi-automatic image analysis allows measuring the properties of SAS NM quantitatively. Automation allows measuring multiple and arithmetically complex parameters simultaneously on high numbers of detected particles. This reduces operator-induced bias and assures a statistically relevant number of measurements, avoiding the tedious repetitive task of manual measurements. Access to multiple parameters further allows selecting the optimal parameter in function of a specific purpose. Using principle component analysis (PCA), twenty-three measured parameters were classified into three classes containing measures for size, shape and surface topology of the NM. Conclusion The presented method allows a detailed quantitative characterization of NM, like dispersions of precipitated and pyrogenic SAS based on the number-based distributions of their mean diameter, sphericity and shape factor. PMID:22709926

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

    PubMed

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

    2013-02-01

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

  8. Faults and foibles of quantitative scanning electron microscopy/energy dispersive x-ray spectrometry (SEM/EDS)

    NASA Astrophysics Data System (ADS)

    Newbury, Dale E.; Ritchie, Nicholas W. M.

    2012-06-01

    Scanning electron microscopy with energy dispersive x-ray spectrometry (SEM/EDS) is a powerful and flexible elemental analysis method that can identify and quantify elements with atomic numbers > 4 (Be) present as major constituents (where the concentration C > 0.1 mass fraction, or 10 weight percent), minor (0.01<= C <= 0.1) and trace (C < 0.01, with a minimum detectable limit of ~+/- 0.0005 - 0.001 under routine measurement conditions, a level which is analyte and matrix dependent ). SEM/EDS can select specimen volumes with linear dimensions from ~ 500 nm to 5 μm depending on composition (masses ranging from ~ 10 pg to 100 pg) and can provide compositional maps that depict lateral elemental distributions. Despite the maturity of SEM/EDS, which has a history of more than 40 years, and the sophistication of modern analytical software, the method is vulnerable to serious shortcomings that can lead to incorrect elemental identifications and quantification errors that significantly exceed reasonable expectations. This paper will describe shortcomings in peak identification procedures, limitations on the accuracy of quantitative analysis due to specimen topography or failures in physical models for matrix corrections, and quantitative artifacts encountered in xray elemental mapping. Effective solutions to these problems are based on understanding the causes and then establishing appropriate measurement science protocols. NIST DTSA II and Lispix are open source analytical software available free at www.nist.gov that can aid the analyst in overcoming significant limitations to SEM/EDS.

  9. Application of Quantitative Analytical Electron Microscopy to the Mineral Content of Insect Cuticle

    NASA Astrophysics Data System (ADS)

    Rasch, Ron; Cribb, Bronwen W.; Barry, John; Palmer, Christopher M.

    2003-04-01

    Quantification of calcium in the cuticle of the fly larva Exeretonevra angustifrons was undertaken at the micron scale using wavelength dispersive X-ray microanalysis, analytical standards, and a full matrix correction. Calcium and phosphorus were found to be present in the exoskeleton in a ratio that indicates amorphous calcium phosphate. This was confirmed through electron diffraction of the calcium-containing tissue. Due to the pragmatic difficulties of measuring light elements, it is not uncommon in the field of entomology to neglect the use of matrix corrections when performing microanalysis of bulk insect specimens. To determine, firstly, whether such a strategy affects the outcome and secondly, which matrix correction is preferable, phi-rho (z) and ZAF matrix corrections were contrasted with each other and without matrix correction. The best estimate of the mineral phase was found to be given by using the phi-rho (z) correction. When no correction was made, the ratio of Ca to P fell outside the range for amorphous calcium phosphate, possibly leading to flawed interpretation of the mineral form when used on its own.

  10. 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

  11. A combined quantitative mass spectrometry and electron microscopy analysis of ribosomal 30S subunit assembly in E. coli

    PubMed Central

    Sashital, Dipali G; Greeman, Candacia A; Lyumkis, Dmitry; Potter, Clinton S; Carragher, Bridget; Williamson, James R

    2014-01-01

    Ribosome assembly is a complex process involving the folding and processing of ribosomal RNAs (rRNAs), concomitant binding of ribosomal proteins (r-proteins), and participation of numerous accessory cofactors. Here, we use a quantitative mass spectrometry/electron microscopy hybrid approach to determine the r-protein composition and conformation of 30S ribosome assembly intermediates in Escherichia coli. The relative timing of assembly of the 3′ domain and the formation of the central pseudoknot (PK) structure depends on the presence of the assembly factor RimP. The central PK is unstable in the absence of RimP, resulting in the accumulation of intermediates in which the 3′-domain is unanchored and the 5′-domain is depleted for r-proteins S5 and S12 that contact the central PK. Our results reveal the importance of the cofactor RimP in central PK formation, and introduce a broadly applicable method for characterizing macromolecular assembly in cells. DOI: http://dx.doi.org/10.7554/eLife.04491.001 PMID:25313868

  12. 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

  13. 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.

  14. Quantitative Electron Nanodiffraction.

    SciTech Connect

    Spence, John

    2015-01-30

    This Final report summarizes progress under this award for the final reporting period 2002 - 2013 in our development of quantitive electron nanodiffraction to materials problems, especially devoted to atomistic processes in semiconductors and electronic oxides such as the new artificial oxide multilayers, where our microdiffraction is complemented with energy-loss spectroscopy (ELNES) and aberration-corrected STEM imaging (9). The method has also been used to map out the chemical bonds in the important GaN semiconductor (1) used for solid state lighting, and to understand the effects of stacking sequence variations and interfaces in digital oxide superlattices (8). Other projects include the development of a laser-beam Zernike phase plate for cryo-electron microscopy (5) (based on the Kapitza-Dirac effect), work on reconstruction of molecular images using the scattering from many identical molecules lying in random orientations (4), a review article on space-group determination for the International Tables on Crystallography (10), the observation of energy-loss spectra with millivolt energy resolution and sub-nanometer spatial resolution from individual point defects in an alkali halide, a review article for the Centenary of X-ray Diffration (17) and the development of a new method of electron-beam lithography (12). We briefly summarize here the work on GaN, on oxide superlattice ELNES, and on lithography by STEM.

  15. 3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy.

    PubMed

    Vahedi-Faridi, Ardeschir; Jastrzebska, Beata; Palczewski, Krzysztof; Engel, Andreas

    2013-02-01

    Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of >300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin.

  16. 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.

  17. A quantitative estimation of the exhaust, abrasion and resuspension components of particulate traffic emissions using electron microscopy

    NASA Astrophysics Data System (ADS)

    Weinbruch, Stephan; Worringen, Annette; Ebert, Martin; Scheuvens, Dirk; Kandler, Konrad; Pfeffer, Ulrich; Bruckmann, Peter

    2014-12-01

    The contribution of the three traffic-related components exhaust, abrasion, and resuspension to kerbside and urban background PM10 and PM1 levels was quantified based on the analysis of individual particles by scanning electron microscopy. A total of 160 samples was collected on 38 days between February and September 2009 at a kerbside and an urban background station in the urban/industrial Ruhr area (Germany). Based on size, morphology, chemical composition and stability under electron bombardment, the 111,003 particles studied in detail were classified into the following 14 particle classes: traffic/exhaust, traffic/abrasion, traffic/resuspension, carbonaceous/organic, industry/metallurgy, industry/power plants, secondary particles, (aged) sea salt, silicates, Ca sulfates, carbonates, Fe oxides/hydroxides, biological particles, and other particles. The traffic/exhaust component consists predominantly of externally mixed soot particles and soot internally mixed with secondary particles. The traffic/abrasion component contains all particles with characteristic tracer elements (Fe, Cu, Ba, Sb, Zn) for brake and tire abrasion. The traffic/resuspension component is defined by the mixing state and comprises all internally mixed particles with a high proportion of silicates or Fe oxides/hydroxides which contain soot or abrasion particles as minor constituent. In addition, silicates and Fe oxides/hydroxides internally mixed with chlorine and sulphur containing particles were also assigned to the traffic/resuspension component. The total contribution of traffic to PM10 was found to be 27% at the urban background station and 48% at the kerbside station, the corresponding values for PM1 are 15% and 39%. These values lie within the range reported in previous literature. The relative share of the different traffic components for PM10 at the kerbside station was 27% exhaust, 15% abrasion, and 58% resuspension (38%, 8%, 54% for PM1). For the urban background, the following

  18. 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).

  19. The effect of probe inaccuracies on the quantitative model-based analysis of high angle annular dark field scanning transmission electron microscopy images.

    PubMed

    Martinez, G T; De Backer, A; Rosenauer, A; Verbeeck, J; Van Aert, S

    2014-08-01

    Quantitative structural and chemical information can be obtained from high angle annular dark field scanning transmission electron microscopy (HAADF STEM) images when using statistical parameter estimation theory. In this approach, we assume an empirical parameterized imaging model for which the total scattered intensities of the atomic columns are estimated. These intensities can be related to the material structure or composition. Since the experimental probe profile is assumed to be known in the description of the imaging model, we will explore how the uncertainties in the probe profile affect the estimation of the total scattered intensities. Using multislice image simulations, we analyze this effect for Cs corrected and non-Cs corrected microscopes as a function of inaccuracies in cylindrically symmetric aberrations, such as defocus and spherical aberration of third and fifth order, and non-cylindrically symmetric aberrations, such as 2-fold and 3-fold astigmatism and coma.

  20. High speed quantitative digital microscopy

    NASA Technical Reports Server (NTRS)

    Castleman, K. R.; Price, K. H.; Eskenazi, R.; Ovadya, M. M.; Navon, M. A.

    1984-01-01

    Modern digital image processing hardware makes possible quantitative analysis of microscope images at high speed. This paper describes an application to automatic screening for cervical cancer. The system uses twelve MC6809 microprocessors arranged in a pipeline multiprocessor configuration. Each processor executes one part of the algorithm on each cell image as it passes through the pipeline. Each processor communicates with its upstream and downstream neighbors via shared two-port memory. Thus no time is devoted to input-output operations as such. This configuration is expected to be at least ten times faster than previous systems.

  1. 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

  2. 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.

  3. Resistivity of thin gold films on mica induced by electron-surface scattering: Application of quantitative scanning tunneling microscopy

    NASA Astrophysics Data System (ADS)

    Robles, Marcelo E.; Gonzalez-Fuentes, Claudio A.; Henriquez, Ricardo; Kremer, German; Moraga, Luis; Oyarzun, Simón; Suarez, Marco Antonio; Flores, Marcos; Munoz, Raul C.

    2012-02-01

    We report a comparison between the resistivity measured on thin gold films deposited on mica, with predictions based upon classical theories of size effects (Drude's, Sondheimer's and Calecki's), as well as predictions based upon quantum theories of electron-surface scattering (the modified theory of Sheng, Xing and Wang, the theory of Tesanovic, Jaric and Maekawa, and that of Trivedi and Aschroft). From topographic images of the surface recorded with a Scanning Tunneling Microscope, we determined the rms roughness amplitude, δ and the lateral correlation length, ξ corresponding to a Gaussian representation of the average height-height autocorrelation function, describing the roughness of each sample in the scale of length set by the Fermi wave length. Using (δ, ξ) as input data, we present a rigorous comparison between resistivity data and predictions based upon the theory of Calecki as well as quantum theoretical predictions without adjustable parameters. The resistivity was measured on gold films of different thickness evaporated onto mica substrates, between 4 K and 300 K. The resistivity data covers the range 0.1 < x(T) < 6.8, for 4 K < T < 300 K, where x(T) is the ratio between film thickness and electron mean free path in the bulk at temperature T. We experimentally identify electron-surface and electron-phonon scattering as the microscopic electron scattering mechanisms giving rise to the macroscopic resistivity. The different theories are all capable of estimating the thin film resistivity to an accuracy better than 10%; however the mean free path and the resistivity characterizing the bulk turn out to depend on film thickness. Surprisingly, only the Sondheimer theory and its quantum version, the modified theory of Sheng, Xing and Wang, predict and increase in resistivity induced by size effects that seems consistent with published galvanomagnetic phenomena also arising from electron-surface scattering measured at low temperatures.

  4. 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…

  5. FLIPPER, a combinatorial probe for correlated live imaging and electron microscopy, allows identification and quantitative analysis of various cells and organelles.

    PubMed

    Kuipers, Jeroen; van Ham, Tjakko J; Kalicharan, Ruby D; Veenstra-Algra, Anneke; Sjollema, Klaas A; Dijk, Freark; Schnell, Ulrike; Giepmans, Ben N G

    2015-04-01

    Ultrastructural examination of cells and tissues by electron microscopy (EM) yields detailed information on subcellular structures. However, EM is typically restricted to small fields of view at high magnification; this makes quantifying events in multiple large-area sample sections extremely difficult. Even when combining light microscopy (LM) with EM (correlated LM and EM: CLEM) to find areas of interest, the labeling of molecules is still a challenge. We present a new genetically encoded probe for CLEM, named "FLIPPER", which facilitates quantitative analysis of ultrastructural features in cells. FLIPPER consists of a fluorescent protein (cyan, green, orange, or red) for LM visualization, fused to a peroxidase allowing visualization of targets at the EM level. The use of FLIPPER is straightforward and because the module is completely genetically encoded, cells can be optimally prepared for EM examination. We use FLIPPER to quantify cellular morphology at the EM level in cells expressing a normal and disease-causing point-mutant cell-surface protein called EpCAM (epithelial cell adhesion molecule). The mutant protein is retained in the endoplasmic reticulum (ER) and could therefore alter ER function and morphology. To reveal possible ER alterations, cells were co-transfected with color-coded full-length or mutant EpCAM and a FLIPPER targeted to the ER. CLEM examination of the mixed cell population allowed color-based cell identification, followed by an unbiased quantitative analysis of the ER ultrastructure by EM. Thus, FLIPPER combines bright fluorescent proteins optimized for live imaging with high sensitivity for EM labeling, thereby representing a promising tool for CLEM.

  6. Bioaccumulation and localization of exogenous cadmium in a teleost by electron microscopy (TEM) and its specific quantitation by electron probe X-ray microanalysis (EPMA).

    PubMed

    Tayal, A K; Kaur, I; Mathur, R P

    2000-03-01

    A cadmium bioconcentration study was carried out in a fresh water teleost, Colisa fasciatus, to study the bioaccumulation kinetics and fate of exogenous cadmium (Cd) in biological tissues. Study shows that on exposure of the fish to a sublethal concentration of cadmium in test water, Cd uptake results in its bioconcentration in gills, liver and muscle tissues. To explore whether the accumulated Cd reaches the membranes or inside the cells, transmission electron microscopy (TEM) of the thin sections of tissues was done after histochemical localization of Cd in cells by modified SST method. TEM studies of sections of gills, liver and muscle tissues showed the deposits of exogenous Cd (visualized as dense clouds) in biological cells. This suggests the presence of free or loosely bound Cd on the membranes and inside the cells, which in the presence of Na2S is converted into insoluble metal sulfides. Electron probe X-ray microanalysis (EPMA) studies confirmed the presence of Cd on the membrane surface as well as inside the cells of bioindicator organs suggesting involvement of membrane transport of exogenous Cd inside the cells and its deposition as loosely bound insoluble metal complexes.

  7. Challenges in quantitative single molecule localization microscopy.

    PubMed

    Shivanandan, A; Deschout, H; Scarselli, M; Radenovic, A

    2014-10-01

    Single molecule localization microscopy (SMLM), which can provide up to an order of magnitude improvement in spatial resolution over conventional fluorescence microscopy, has the potential to be a highly useful tool for quantitative biological experiments. It has already been used for this purpose in varied fields in biology, ranging from molecular biology to neuroscience. In this review article, we briefly review the applications of SMLM in quantitative biology, and also the challenges involved and some of the solutions that have been proposed. Due to its advantages in labeling specificity and the relatively low overcounting caused by photoblinking when photo-activable fluorescent proteins (PA-FPs) are used as labels, we focus specifically on Photo-Activated Localization Microscopy (PALM), even though the ideas presented might be applicable to SMLM in general. Also, we focus on the following three quantitative measurements: single molecule counting, analysis of protein spatial distribution heterogeneity and co-localization analysis.

  8. 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

  9. 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.

  10. 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.

  11. Quantitative phase-amplitude microscopy I: optical microscopy.

    PubMed

    Barone-Nugent, E D; Barty, A; Nugent, K A

    2002-06-01

    In this paper, the application of a new optical microscopy method (quantitative phase-amplitude microscopy) to biological imaging is explored, and the issue of resolution and image quality is examined. The paper begins by presenting a theoretical analysis of the method using the optical transfer function formalism of Streibl (1985). The effect of coherence on the formation of the phase image is explored, and it is shown that the resolution of the method is not compromised over that of a conventional bright-field image. It is shown that the signal-to-noise ratio of the phase recovery, however, does depend on the degree of coherence in the illumination. Streibl (1985) notes that partially coherent image formation is a non-linear process because of the intermingling of amplitude and phase information. The work presented here shows that the quantitative phase-amplitude microscopy method acts to linearize the image formation process, and that the phase and amplitude information is properly described using a transfer function analysis. The theoretical conclusions are tested experimentally using an optical microscope and the theoretical deductions are confirmed. Samples for microscopy influence both the phase and amplitude of the light wave and it is demonstrated that the new phase recovery method can separate the amplitude and phase information, something not possible using traditional phase microscopy. In the case of a coherent wave, knowledge of the phase and amplitude constitutes complete information that can be used to emulate other forms of microscopy. This capacity is demonstrated by recovering the phase of a sample and using the data to emulate a differential interference contrast image.

  12. Quantitative study of particle size distribution in an in-situ grown Al–TiB{sub 2} composite by synchrotron X-ray diffraction and electron microscopy

    SciTech Connect

    Tang, Y.; Chen, Z.; Borbély, A.; Ji, G.; Zhong, S.Y.; Schryvers, D.; Ji, V.

    2015-04-15

    Synchrotron X-ray diffraction and transmission electron microscopy (TEM) were applied to quantitatively characterize the average particle size and size distribution of free-standing TiB{sub 2} particles and TiB{sub 2} particles in an in-situ grown Al–TiB{sub 2} composite. The detailed evaluations were carried out by X-ray line profile analysis using the restricted moment method and multiple whole profile fitting procedure (MWP). Both numerical methods indicate that the formed TiB{sub 2} particles are well crystallized and free of crystal defects. The average particle size determined from different Bragg reflections by the restricted moment method ranges between 25 and 55 nm, where the smallest particle size is determined using the 110 reflection suggesting the highest lateral-growth velocity of (110) facets. The MWP method has shown that the in-situ grown TiB{sub 2} particles have a very low dislocation density (~ 10{sup 11} m{sup −} {sup 2}) and their size distribution can be described by a log-normal distribution. Good agreement was found between the results obtained from the restricted moment and MWP methods, which was further confirmed by TEM. - Highlights: • Accurate quantitative characterization of in-situ grown T{sub i}B{sub 2} particles has been achieved. • Particle size anisotropy was revealed indicating 110 facets being largest during T{sub i}B{sub 2} growth. • A wide size distribution was observed for T{sub i}B{sub 2} particles with a dominant size smaller than 100 nm.

  13. 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

  14. 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

  15. 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.

  16. 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.

  17. 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

  18. 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.

  19. Quantitative confocal microscopy: beyond a pretty picture.

    PubMed

    Jonkman, James; Brown, Claire M; Cole, Richard W

    2014-01-01

    Quantitative optical microscopy has become the norm, with the confocal laser-scanning microscope being the workhorse of many imaging laboratories. Generating quantitative data requires a greater emphasis on the accurate operation of the microscope itself, along with proper experimental design and adequate controls. The microscope, which is more accurately an imaging system, cannot be treated as a "black box" with the collected data viewed as infallible. There needs to be regularly scheduled performance testing that will ensure that quality data are being generated. This regular testing also allows for the tracking of metrics that can point to issues before they result in instrument malfunction and downtime. In turn, images must be collected in a manner that is quantitative with maximal signal to noise (which can be difficult depending on the application) without data clipping. Images must then be processed to correct for background intensities, fluorophore cross talk, and uneven field illumination. With advanced techniques such as spectral imaging, Förster resonance energy transfer, and fluorescence-lifetime imaging microscopy, experimental design needs to be carefully planned out and include all appropriate controls. Quantitative confocal imaging in all of these contexts and more will be explored within the chapter. PMID:24974025

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. Prototype cantilevers for quantitative lateral force microscopy

    SciTech Connect

    Reitsma, Mark G.; Gates, Richard S.; Friedman, Lawrence H.; Cook, Robert F.

    2011-09-15

    Prototype cantilevers are presented that enable quantitative surface force measurements using contact-mode atomic force microscopy (AFM). The ''hammerhead'' cantilevers facilitate precise optical lever system calibrations for cantilever flexure and torsion, enabling quantifiable adhesion measurements and friction measurements by lateral force microscopy (LFM). Critically, a single hammerhead cantilever of known flexural stiffness and probe length dimension can be used to perform both a system calibration as well as surface force measurements in situ, which greatly increases force measurement precision and accuracy. During LFM calibration mode, a hammerhead cantilever allows an optical lever ''torque sensitivity'' to be generated for the quantification of LFM friction forces. Precise calibrations were performed on two different AFM instruments, in which torque sensitivity values were specified with sub-percent relative uncertainty. To examine the potential for accurate lateral force measurements using the prototype cantilevers, finite element analysis predicted measurement errors of a few percent or less, which could be reduced via refinement of calibration methodology or cantilever design. The cantilevers are compatible with commercial AFM instrumentation and can be used for other AFM techniques such as contact imaging and dynamic mode measurements.

  8. Nanoscale quantitative measurement of the potential of charged nanostructures by electrostatic and Kelvin probe force microscopy: unraveling electronic processes in complex materials.

    PubMed

    Liscio, Andrea; Palermo, Vincenzo; Samorì, Paolo

    2010-04-20

    In microelectronics and biology, many fundamental processes involve the exchange of charges between small objects, such as nanocrystals in photovoltaic blends or individual proteins in photosynthetic reactions. Because these nanoscale electronic processes strongly depend on the structure of the electroactive assemblies, a detailed understanding of these phenomena requires unraveling the relationship between the structure of the nano-object and its electronic function. Because of the fragility of the structures involved and the dynamic variance of the electric potential of each nanostructure during the charge generation and transport processes, understanding this structure-function relationship represents a great challenge. This Account discusses how our group and others have exploited scanning probe microscopy based approaches beyond imaging, particularly Kelvin probe force microscopy (KPFM), to map the potential of different nanostructures with a spatial and voltage resolution of a few nanometers and millivolts, respectively. We describe in detail how these techniques can provide researchers several types of chemical information. First, KPFM allows researchers to visualize the photogeneration and splitting of several unitary charges between well-defined nano-objects having complementary electron-acceptor and -donor properties. In addition, this method maps charge injection and transport in thin layers of polycrystalline materials. Finally, KPFM can monitor the activity of immobilized chemical components of natural photosynthetic systems. In particular, researchers can use KPFM to measure the electric potential without physical contact between the tip and the nanostructure studied. These measurements exploit long-range electrostatic interactions between the scanning probe and the sample, which scale with the square of the probe-sample distance, d. While allowing minimal perturbation, these long-range interactions limit the resolution attainable in the measurement

  9. Quantitative composition determination at the atomic level using model-based high-angle annular dark field scanning transmission electron microscopy.

    PubMed

    Martinez, G T; Rosenauer, A; De Backer, A; Verbeeck, J; Van Aert, S

    2014-02-01

    High angle annular dark field scanning transmission electron microscopy (HAADF STEM) images provide sample information which is sensitive to the chemical composition. The image intensities indeed scale with the mean atomic number Z. To some extent, chemically different atomic column types can therefore be visually distinguished. However, in order to quantify the atomic column composition with high accuracy and precision, model-based methods are necessary. Therefore, an empirical incoherent parametric imaging model can be used of which the unknown parameters are determined using statistical parameter estimation theory (Van Aert et al., 2009, [1]). In this paper, it will be shown how this method can be combined with frozen lattice multislice simulations in order to evolve from a relative toward an absolute quantification of the composition of single atomic columns with mixed atom types. Furthermore, the validity of the model assumptions are explored and discussed.

  10. 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.

  11. Development and Applications of Quantitative X - Microscopy with Chemical Sensitivity

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaodong

    1995-01-01

    X-ray microscopy has been developed to image biological samples with a resolution intermediate between visible light and electron microscopes. Interest in distinguishing different molecules using x-ray microscopy has motivated the effort to combine x-ray microscopy with x-ray absorption near-edge spectroscopy (XANES). This thesis includes three projects. The major part concerns the development and applications of XANES microscopy to quantitatively map DNA and proteins. The two other parts are image formation and image contrast enhancement through deconvolution and the use of carbon-XANES to study soft x-ray irradiation induced chemistry in PMMA (a widely used x-ray resist). For the major part of the thesis, XANES microscopy was applied to examine the content and distribution of DNA and protein in mature sperm cells. Sperm nuclei from five different species of mammals were examined, species chosen for analysis because their sperm contains markedly different proportion of protamine 1 and protamine 2 (which are the basic DNA packing proteins in sperm). This work provides new information about composition and structure of sperm chromatin by showing that the protein to DNA ratio in sperm nuclei is not strongly species dependent.

  12. Virus quantitation by transmission electron microscopy, TCID₅₀, and the role of timing virus harvesting: a case study of three animal viruses.

    PubMed

    Malenovska, Hana

    2013-08-01

    Quantitation of viruses is practised widely in both basic and applied virology. Infectious titration in cell cultures, the most common approach to it, is quite labour-intensive and alternative protocols are therefore sought. One of the alternatives is transmission electron microscope (TEM) quantitation using latex particles at a known concentration as a reference for counting virus particles. If virus TCID₅₀ is determined in parallel, the ratio of infectious to non-infectious virus particles may be established. This study employs such an approach to compute the number of virus particles and TCID₅₀, and establish their correlation for three viruses: Canine adenovirus 1 (CAdV-1), Feline calicivirus (FCV) and Bovine herpesvirus 1 (BoHV-1). Each of the viruses was grown in five replicates until complete cytopathology was recorded (time 0), then frozen. They were thawed, filter-sterilised and left for additional periods of 16, 32 and 48 h at 37°C. At each time point, the infectious ability of the virus was characterised by TCID50 and the number of virions quantified by TEM, in order to evaluate the influence of timing on virus harvest. The virus particle count determined by TEM did not change for any of the viruses throughout the experiment. The relationship between virus particle counts with TCID₅₀ at time 0 showed good linearity response; their ratio was almost constant. The virus particle-to-TCID₅₀ ratio varied between 146 and 426 (mean±SD: 282±103) for CAdV-1, between 36 and 79 (57±18) for FCV and between 110 and 249 (167±53) for BoHV-1. The proportion of non-infectious particles did not change throughout the experiment for either CAdV-1 or BoHV-1. However, a decrease in virus infectious ability disclosed by TCID₅₀ indicated that the fraction of non-infectious particles in FCV increased 300,000 times when time 0 and 48 h were compared. The quantitation of viruses with TEM is a simple and rapid protocol for virus quantitation but account must

  13. 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.

  14. 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.

  15. Quantitative transmission electron microscopy analysis of multi-variant grains in present L1{sub 0}-FePt based heat assisted magnetic recording media

    SciTech Connect

    Ho, Hoan; Zhu, Jingxi; Kulovits, Andreas; Laughlin, David E.; Zhu, Jian-Gang

    2014-11-21

    We present a study on atomic ordering within individual grains in granular L1{sub 0}-FePt thin films using transmission electron microscopy techniques. The film, used as a medium for heat assisted magnetic recording, consists of a single layer of FePt grains separated by non-magnetic grain boundaries and is grown on an MgO underlayer. Using convergent-beam techniques, diffraction patterns of individual grains are obtained for a large number of crystallites. The study found that although the majority of grains are ordered in the perpendicular direction, more than 15% of them are multi-variant, or of in-plane c-axis orientation, or disordered fcc. It was also found that these multi-variant and in-plane grains have always grown across MgO grain boundaries separating two or more MgO grains of the underlayer. The in-plane ordered portion within a multi-variant L1{sub 0}-FePt grain always lacks atomic coherence with the MgO directly underneath it, whereas, the perpendicularly ordered portion is always coherent with the underlying MgO grain. Since the existence of multi-variant and in-plane ordered grains are severely detrimental to high density data storage capability, the understanding of their formation mechanism obtained here should make a significant impact on the future development of hard disk drive technology.

  16. Lock-in thermography, penetrant inspection, and scanning electron microscopy for quantitative evaluation of open micro-cracks at the tooth-restoration interface

    NASA Astrophysics Data System (ADS)

    Streza, M.; Hodisan, I.; Prejmerean, C.; Boue, C.; Tessier, Gilles

    2015-03-01

    The evaluation of a dental restoration in a non-invasive way is of paramount importance in clinical practice. The aim of this study was to assess the minimum detectable open crack at the cavity-restorative material interface by the lock-in thermography technique, at laser intensities which are safe for living teeth. For the analysis of the interface, 18 box-type class V standardized cavities were prepared on the facial and oral surfaces of each tooth, with coronal margins in enamel and apical margins in dentine. The preparations were restored with the Giomer Beautifil (Shofu) in combination with three different adhesive systems. Three specimens were randomly selected from each experimental group and each slice has been analysed by visible, infrared (IR), and scanning electron microscopy (SEM). Lock-in thermography showed the most promising results in detecting both marginal and internal defects. The proposed procedure leads to a diagnosis of micro-leakages having openings of 1 µm, which is close to the diffraction limit of the IR camera. Clinical use of a thermographic camera in assessing the marginal integrity of a restoration becomes possible. The method overcomes some drawbacks of standard SEM or dye penetration testing. The results support the use of an IR camera in dentistry, for the diagnosis of micro-gaps at bio-interfaces.

  17. Quantitative phase-amplitude microscopy. III. The effects of noise.

    PubMed

    Paganin, D; Barty, A; McMahon, P J; Nugent, K A

    2004-04-01

    We explore the effect of noise on images obtained using quantitative phase-amplitude microscopy - a new microscopy technique based on the determination of phase from the intensity evolution of propagating radiation. We compare the predictions with experimental results and also propose an approach that allows good-quality quantitative phase retrieval to be obtained even for very noisy data.

  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. 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.

  20. The future of electron microscopy

    DOE PAGESBeta

    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

  1. Visualizing quantitative microscopy data: History and challenges

    PubMed Central

    Sailem, Heba Z.; Cooper, Sam; Bakal, Chris

    2016-01-01

    Abstract Data visualization is a fundamental aspect of science. In the context of microscopy-based studies, visualization typically involves presentation of the images themselves. However, data visualization is challenging when microscopy experiments entail imaging of millions of cells, and complex cellular phenotypes are quantified in a high-content manner. Most well-established visualization tools are inappropriate for displaying high-content data, which has driven the development of new visualization methodology. In this review, we discuss how data has been visualized in both classical and high-content microscopy studies; as well as the advantages, and disadvantages, of different visualization methods. PMID:26906253

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. Quantitative current measurements using scanning magnetoresistance microscopy.

    PubMed

    Takezaki, Taiichi; Sueoka, Kazuhisa

    2008-08-01

    We have demonstrated the capability of scanning magnetoresistance microscope (SMRM) to be used for quantitative current measurements. The SMRM is a magnetic microscope that is based on an atomic force microscope (AFM) and simultaneously measures the localized surface magnetic field distribution and surface topography. The proposed SMRM employs an in-house built AFM cantilever equipped with a miniaturized magnetoresistive (MR) sensor as a magnetic field sensor. In this study, a spin-valve type MR sensor with a width of 1 microm was used to measure the magnetic field distribution induced by a current carrying wire with a width of 5 microm and a spacing of 1.6 microm at room temperature and under ambient conditions. Simultaneous imaging of the magnetic field distribution and the topography was successfully performed in the DC current ranging from 500 microA to 8 mA. The characterized SV sensor, which has a linear response to magnetic fields, offers the quantitative analysis of a magnetic field and current. The measured magnetic field strength was in good agreement with the result simulated using Biot-Savart's law. PMID:18599218

  10. Imaging without fluorescence: nonlinear optical microscopy for quantitative cellular imaging.

    PubMed

    Streets, Aaron M; Li, Ang; Chen, Tao; Huang, Yanyi

    2014-09-01

    Quantitative single-cell analysis enables the characterization of cellular systems with a level of detail that cannot be achieved with ensemble measurement. In this Feature we explore quantitative cellular imaging applications with nonlinear microscopy techniques. We first offer an introductory tutorial on nonlinear optical processes and then survey a range of techniques that have proven to be useful for quantitative live cell imaging without fluorescent labels.

  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. 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.

  15. Quantitative Probes of Entanglement Using Collisional Microscopy

    NASA Astrophysics Data System (ADS)

    Price, Craig; Liu, Qi; Gemelke, Nathan

    2015-05-01

    Though entanglement is understood to play a critical role in determining the ground state structure and macroscopic properties of many known physical systems, its definitive quantification has until recently, through the creation of entanglement entropy (EE), spectrum and related measures, escaped a simple definition. Moreover, few if any of these measures have been directly extracted in experiments on strongly correlated matter. In this talk, we present a novel method to measure quantifiers of many-body entanglement by pair-wise entangling a small portion of an atomic gas with an optical-lattice-bound array of secondary atoms serving as quantum-non-destructive probes. For a sample with significant pre-existing long range entanglement, such as in a Bose-Hubbard system near its quantum critical point, the quantum back-action following probe detection affects the sample gas in regions spatially extended beyond where measured. This results in a non-local thermal effect; subsequent measurement of the thermal entropy through the local equation of state can reveal the EE. Quantitative analysis of thermodynamic back action and background effects, such as classical propagation of entropy after a measurement quench, will be discussed.

  16. 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

  17. 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.

  18. [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)

  19. New fluorescence microscopy methods for microbiology: sharper, faster, and quantitative.

    PubMed

    Gitai, Zemer

    2009-06-01

    In addition to the inherent interest stemming from their ecological and human health impacts, microbes have many advantages as model organisms, including ease of growth and manipulation and relatively simple genomes. However, the imaging of bacteria via light microscopy has been limited by their small sizes. Recent advances in fluorescence microscopy that allow imaging of structures at extremely high resolutions are thus of particular interest to the modern microbiologist. In addition, advances in high-throughput microscopy and quantitative image analysis are enabling cellular imaging to finally take advantage of the full power of bacterial numbers and ease of manipulation. These technical developments are ushering in a new era of using fluorescence microscopy to understand bacterial systems in a detailed, comprehensive, and quantitative manner.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: QA TESTS, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

    Confocal Microscopy System Performance: QA tests, Quantitation and Spectroscopy.

    Robert M. Zucker 1 and Jeremy M. Lerner 2,
    1Reproductive Toxicology Division, National Health and Environmental Effects Research Laboratory, Office of Research Development, U.S. Environmen...

  8. 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.

  9. Frontiers of in situ electron microscopy

    DOE PAGESBeta

    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

  10. A quantitative study of the microstructure and crystallographic fiber texture in nickel electrodeposits used in radio-frequency MEMS switches, including a new transmission electron microscopy (TEM) technique for polycrystalline films

    NASA Astrophysics Data System (ADS)

    Cantwell, Patrick R.

    The microstructure of electrodeposited nickel films in radio-frequency (RF) microelectromechanical systems (MEMS) switches has been quantitatively studied to inform and validate multi-scale, multi-physics computer simulations that aim to predict the lifetime and failure mechanisms of the RF MEMS switches. The RF MEMS switches are currently under study at the Purdue University center for the Prediction of Reliability, Integrity, and Survivability of Microsystems (PRISM). An array of microstructural characterization techniques including focused ion beam (FIB) microscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), and transmission electron microscopy have be used to study the nickel film and to quantify grain size and crystallographic texture and provide information about elemental impurities and surface roughness and impurity elements. Particular emphasis has been placed on quantifying the crystallographic fiber texture of the polycrystalline nickel film as a function of film height within a single specimen using a new transmission electron microscopy (TEM) microtexture method. The TEM method employs a special type of plan view TEM sample and uses hollow cone dark field (HCDF) TEM imaging to spatially map the orientation of individual crystallites at discrete film heights. A trend of increasing 001 fiber texture with film height was discovered, which has implications for the elastic behavior of the MEMS device. The method can be applied to study fiber texture evolution as a function of height in polycrystalline films to gather data that may elucidate fundamental film growth mechanisms. The method is explained in detail. It is well-known that the elastic properties of polycrystalline thin films used in MEMS devices can deviate from bulk isotropic values and become directionally-dependent if a crystallographic texture is present. Hence, the ability to predict the actual anisotropic elastic properties of textured films is important for MEMS design and

  11. 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

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. Quantitative low-cost webcam-based microscopy

    NASA Astrophysics Data System (ADS)

    Parikesit, Gea Oswah Fatah; Darmawan, Marten; Faisal, Amir

    2010-11-01

    Digital web cameras (popularly known as webcams) have recently gained a significant increase of relevance in the field of optical microscopy, in particular to allow for quick and do-it-yourself methods in developing low-cost and portable microscopes suitable for life sciences and engineering applications in low-resource areas. Unfortunately, these methods were published without any systematic explanation and quantitative assessment of the imaging performances. We reproduce these do-it-yourself methods, discuss the optical considerations that are relevant for them, and quantitatively compare their imaging performances to a commercial digital microscope in order to clarify both the advantages and disadvantages of the webcam-based microscopes.

  17. Quantitative sectioning and noise analysis for structured illumination microscopy

    PubMed Central

    Hagen, Nathan; Gao, Liang; Tkaczyk, Tomasz S.

    2011-01-01

    Structured illumination (SI) has long been regarded as a nonquantitative technique for obtaining sectioned microscopic images. Its lack of quantitative results has restricted the use of SI sectioning to qualitative imaging experiments, and has also limited researchers’ ability to compare SI against competing sectioning methods such as confocal microscopy. We show how to modify the standard SI sectioning algorithm to make the technique quantitative, and provide formulas for calculating the noise in the sectioned images. The results indicate that, for an illumination source providing the same spatially-integrated photon flux at the object plane, and for the same effective slice thicknesses, SI sectioning can provide higher SNR images than confocal microscopy for an equivalent setup when the modulation contrast exceeds about 0.09. PMID:22274364

  18. 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

  19. 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.

  20. 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

  1. Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

    PubMed Central

    Jensen, Thomas; Holten-Rossing, Henrik; Svendsen, Ida M H; Jacobsen, Christina; Vainer, Ben

    2016-01-01

    Background: The opportunity offered by whole slide scanners of automated histological analysis implies an ever increasing importance of digital pathology. To go beyond the importance of conventional pathology, however, digital pathology may need a basic histological starting point similar to that of hematoxylin and eosin staining in conventional pathology. This study presents an automated fluorescence-based microscopy approach providing highly detailed morphological data from unstained microsections. This data may provide a basic histological starting point from which further digital analysis including staining may benefit. Methods: This study explores the inherent tissue fluorescence, also known as autofluorescence, as a mean to quantitate cardiac tissue components in histological microsections. Data acquisition using a commercially available whole slide scanner and an image-based quantitation algorithm are presented. Results: It is shown that the autofluorescence intensity of unstained microsections at two different wavelengths is a suitable starting point for automated digital analysis of myocytes, fibrous tissue, lipofuscin, and the extracellular compartment. The output of the method is absolute quantitation along with accurate outlines of above-mentioned components. The digital quantitations are verified by comparison to point grid quantitations performed on the microsections after Van Gieson staining. Conclusion: The presented method is amply described as a prestain multicomponent quantitation and outlining tool for histological sections of cardiac tissue. The main perspective is the opportunity for combination with digital analysis of stained microsections, for which the method may provide an accurate digital framework. PMID:27141321

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Single beam Fourier transform digital holographic quantitative phase microscopy

    SciTech Connect

    Anand, A. Chhaniwal, V. K.; Mahajan, S.; Trivedi, V.; Faridian, A.; Pedrini, G.; Osten, W.; Dubey, S. K.; Javidi, B.

    2014-03-10

    Quantitative phase contrast microscopy reveals thickness or height information of a biological or technical micro-object under investigation. The information obtained from this process provides a means to study their dynamics. Digital holographic (DH) microscopy is one of the most used, state of the art single-shot quantitative techniques for three dimensional imaging of living cells. Conventional off axis DH microscopy directly provides phase contrast images of the objects. However, this process requires two separate beams and their ratio adjustment for high contrast interference fringes. Also the use of two separate beams may make the system more vulnerable to vibrations. Single beam techniques can overcome these hurdles while remaining compact as well. Here, we describe the development of a single beam DH microscope providing whole field imaging of micro-objects. A hologram of the magnified object projected on to a diffuser co-located with a pinhole is recorded with the use of a commercially available diode laser and an arrayed sensor. A Fourier transform of the recorded hologram directly yields the complex amplitude at the image plane. The method proposed was investigated using various phase objects. It was also used to image the dynamics of human red blood cells in which sub-micrometer level thickness variation were measurable.

  9. Surface plasmon resonance microscopy: Achieving a quantitative optical response

    NASA Astrophysics Data System (ADS)

    Peterson, Alexander W.; Halter, Michael; Plant, Anne L.; Elliott, John T.

    2016-09-01

    Surface plasmon resonance (SPR) imaging allows real-time label-free imaging based on index of refraction and changes in index of refraction at an interface. Optical parameter analysis is achieved by application of the Fresnel model to SPR data typically taken by an instrument in a prism based figuration. We carry out SPR imaging on a microscope by launching light into a sample and collecting reflected light through a high numerical aperture microscope objective. The SPR microscope enables spatial resolution that approaches the diffraction limit and has a dynamic range that allows detection of subnanometer to submicrometer changes in thickness of biological material at a surface. However, unambiguous quantitative interpretation of SPR changes using the microscope system could not be achieved using the Fresnel model because of polarization dependent attenuation and optical aberration that occurs in the high numerical aperture objective. To overcome this problem, we demonstrate a model to correct for polarization diattenuation and optical aberrations in the SPR data and develop a procedure to calibrate reflectivity to index of refraction values. The calibration and correction strategy for quantitative analysis was validated by comparing the known indices of refraction of bulk materials with corrected SPR data interpreted with the Fresnel model. Subsequently, we applied our SPR microscopy method to evaluate the index of refraction for a series of polymer microspheres in aqueous media and validated the quality of the measurement with quantitative phase microscopy.

  10. 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

  11. Quantitative phase microscopy and synthetic aperture tomography of live cells

    NASA Astrophysics Data System (ADS)

    Lue, Niyom

    For more than a decade MIT's George R. Harrison Spectroscopy Laboratory has been developing quantitative phase microscopy (QPM) for biological study. Measurements of a point field were made in the mid 90s, then extended to the full 2D field, and recently, to 3D by using tomography. In the first part of this thesis improvements in the techniques of Fourier Phase Microscopy (FPM) and Hilbert Phase Microscopy (HPM) and their applications to characterize cells and tissues are reported. Tomographic phase microscopy (TPM) provides quantitative information and highly detailed structural information about a live cell, but in its current form it can only examine one cell at a time. Many biological applications including statistical analysis of a large collection of cells such as flow cytometry need a tomography technique that can measure many cells at a time. For the second part of this thesis we have developed a new tomography technique that can measure many cells continuously. In this study we demonstrate the new technique by translating a live cell across a focused beam. This beam is composed of many angular plane waves, and by applying a so-called synthetic aperture algorithm we retrieve individual wave components of the focused beam. We demonstrate for the first time that we can retrieve the field of the focused beam and synthesize any arbitrary angular plane wave. We then construct a 3D map of the variations of the refractive index in a live cell from a series of these synthesized angular plane waves. This new technique is the first step needed to analyze cells flowing through a beam to provide a high-throughput 3D refractive index tomograms that can be used as a new kind of statistical optical assay of living cells.

  12. 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.

  13. Quantitative polarized light microscopy of human cochlear sections

    PubMed Central

    Low, Jacob C. M.; Ober, Thomas J.; McKinley, Gareth H.; Stankovic, Konstantina M.

    2015-01-01

    Dysfunction of the inner ear is the most common cause of sensorineural hearing loss, which is the most common sensory deficit worldwide. Conventional imaging modalities are unable to depict the microanatomy of the human inner ear, hence the need to explore novel imaging modalities. We provide the first characterization of the polarization dependent optical properties of human cochlear sections using quantitative polarized light microscopy (qPLM). Eight pediatric cadaveric cochlear sections, aged 0 (term) to 24 months, were selected from the US National Temporal Bone Registry, imaged with qPLM and analyzed using Image J. Retardance of the bony otic capsule and basilar membrane were substantially higher than that of the stria vascularis, spiral ganglion neurons, organ of Corti and spiral ligament across the half turns of the spiraling cochlea. qPLM provides quantitative information about the human inner ear, and awaits future exploration in vivo. PMID:25780749

  14. Quantitative microscopy characterization of hydrous niobium phosphate into bleached cellulose.

    PubMed

    Cruz, T G; Pereira, P H F; Silva, M L C P; Cioffi, M O H; Voorwald, H J C

    2010-07-01

    In this research the spatial distribution characterization of niobium phosphate into bleached cellulose was carried out combining processing and images analysis obtained by SEM and statistical methodologies. The objective is to investigate the deposit composition and phosphate morphology by using complementary analytical techniques. Based on the proposed methodology, parameters of niobium phosphate agglomerates (size and shape) and fiber morphology were evaluated depending on gray-levels (average luminance and fiber type): fiber characteristics (morphology) were measured. For the test method proposed, a specific region of cellulose/NbOPO(4) x nH(2)O composite was analyzed. This method involves area fraction measuring with a conditional probabilistic analysis. The analyzed fields were divided in different ways, called 'Scanning' and as a result, in quantitative terms, the phosphate deposition was described as spatial distribution homogeneous or inhomogeneous. The quantitative microscopy as a non-destructive testing provides relevant information when it is combined with statistic analysis.

  15. Rapid quantitative phase imaging for partially coherent light microscopy.

    PubMed

    Rodrigo, José A; Alieva, Tatiana

    2014-06-01

    Partially coherent light provides promising advantages for imaging applications. In contrast to its completely coherent counterpart, it prevents image degradation due to speckle noise and decreases cross-talk among the imaged objects. These facts make attractive the partially coherent illumination for accurate quantitative imaging in microscopy. In this work, we present a non-interferometric technique and system for quantitative phase imaging with simultaneous determination of the spatial coherence properties of the sample illumination. Its performance is experimentally demonstrated in several examples underlining the benefits of partial coherence for practical imagining applications. The programmable optical setup comprises an electrically tunable lens and sCMOS camera that allows for high-speed measurement in the millisecond range.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

    PubMed

    Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

    2014-02-01

    The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the

  5. Determination of elemental distribution in green micro-algae using synchrotron radiation nano X-ray fluorescence (SR-nXRF) and electron microscopy techniques--subcellular localization and quantitative imaging of silver and cobalt uptake by Coccomyxa actinabiotis.

    PubMed

    Leonardo, T; Farhi, E; Boisson, A-M; Vial, J; Cloetens, P; Bohic, S; Rivasseau, C

    2014-02-01

    The newly discovered unicellular micro-alga Coccomyxa actinabiotis proves to be highly radio-tolerant and strongly concentrates radionuclides, as well as large amounts of toxic metals. This study helps in the understanding of the mechanisms involved in the accumulation and detoxification of silver and cobalt. Elemental distribution inside Coccomyxa actinabiotis cells was determined using synchrotron nano X-ray fluorescence spectroscopy at the ID22 nano fluorescence imaging beamline of the European Synchrotron Radiation Facility. The high resolution and high sensitivity of this technique enabled the assessment of elemental associations and exclusions in subcellular micro-algae compartments. A quantitative treatment of the scans was implemented to yield absolute concentrations of each endogenous and exogenous element with a spatial resolution of 100 nm and compared to the macroscopic content in cobalt and silver determined using inductively coupled plasma-mass spectrometry. The nano X-ray fluorescence imaging was complemented by transmission electron microscopy coupled to X-ray microanalysis (TEM-EDS), yielding differential silver distribution in the cell wall, cytosol, nucleus, chloroplast and mitochondria with unique resolution. The analysis of endogenous elements in control cells revealed that iron had a unique distribution; zinc, potassium, manganese, molybdenum, and phosphate had their maxima co-localized in the same area; and sulfur, copper and chlorine were almost homogeneously distributed among the whole cell. The subcellular distribution and quantification of cobalt and silver in micro-alga, assessed after controlled exposure to various concentrations, revealed that exogenous metals were mainly sequestered inside the cell rather than on mucilage or the cell wall, with preferential compartmentalization. Cobalt was homogeneously distributed outside of the chloroplast. Silver was localized in the cytosol at low concentration and in the whole cell excluding the

  6. 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.

  7. Quantitative Phase Microscopy: how to make phase data meaningful

    PubMed Central

    Goldstein, Goldie; Creath, Katherine

    2014-01-01

    The continued development of hardware and associated image processing techniques for quantitative phase microscopy has allowed superior phase data to be acquired that readily shows dynamic optical volume changes and enables particle tracking. Recent efforts have focused on tying phase data and associated metrics to cell morphology. One challenge in measuring biological objects using interferometrically obtained phase information is achieving consistent phase unwrapping and -dimensions and correct for temporal discrepanices using a temporal unwrapping procedure. The residual background shape due to mean value fluctuations and residual tilts can be removed automatically using a simple object characterization algorithm. Once the phase data are processed consistently, it is then possible to characterize biological samples such as myocytes and myoblasts in terms of their size, texture and optical volume and track those features dynamically. By observing optical volume dynamically it is possible to determine the presence of objects such as vesicles within myoblasts even when they are co-located with other objects. Quantitative phase microscopy provides a label-free mechanism to characterize living cells and their morphology in dynamic environments, however it is critical to connect the measured phase to important biological function for this measurement modality to prove useful to a broader scientific community. In order to do so, results must be highly consistent and require little to no user manipulation to achieve high quality nynerical results that can be combined with other imaging modalities. PMID:25309099

  8. Quantitative electrostatic force microscopy with sharp silicon tips

    NASA Astrophysics Data System (ADS)

    Fumagalli, L.; Edwards, M. A.; Gomila, G.

    2014-12-01

    Electrostatic force microscopy (EFM) probes are typically coated in either metal (radius ˜ 30 nm) or highly-doped diamond (radius ˜ 100 nm). Highly-doped silicon probes, which offer a sharpened and stable tip apex (radius ˜ 1-10 nm) and are usually used only in standard atomic force microscopy, have been recently shown to allow enhanced lateral resolution in quantitative EFM and its application for dielectric constant measurement. Here we present the theoretical modelling required to quantitatively interpret the electrostatic force between these sharpened tips and samples. In contrast to a sphere-capped cone geometry used to describe metal/diamond-coated tips, modelling a sharpened silicon tip requires a geometry comprised of a cone with two different angles. Theoretical results are supported by experimental measurements of metallic substrates and ˜10 nm radius dielectric nanoparticles. This work is equally applicable to EFM and other electrical scanned probe techniques, where it allows quantifying electrical properties of nanomaterials and 3D nano-objects with higher resolution.

  9. [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.

  10. Morphological discretion of basidiospores of the puffball mushroom Calostoma by electron and atomic force microscopy.

    PubMed

    Kim, Misun; Kim, Ki Woo; Jung, Hack Sung

    2007-10-01

    Comparative morphology among species of the genus Calostoma, including C. cinnabarina, C. ravenelii, and C. japonicum, was investigated by scanning electron microscopy and atomic force microscopy. Spore morphology of cinnabarina and C. ravenelii showed no dramatic differences by light microcopy and scanning electron microscopy. To differentiate these species, atomic force microscopy was employed. Quantitative analysis of the surface roughness basidiospores revealed subtle differences in height fluctuation at the nanometer scale between the species of Calostoma. Basidiospores of C. cinnabarina had a relatively rougher surface than those of C. ravenelii at 2.0 x 2.0 micro m2 scan areas. PMID:18156793

  11. 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

  12. Quantitative thermal characterization of microelectronic devices by using CCD-based thermoreflectance microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Dong Uk; Ryu, Seon Young; Kim, Jun Ki; Chang, Ki Soo

    2014-03-01

    A thermoreflectance microscopy (TRM) system has emerged as a non-destructive and non-contact tool for a high resolution thermal imaging technique for micro-scale electronic and optoelectronic devices. Quantitative imaging of the temperature distribution is necessary for elaborate thermal characterization under operating conditions, such as thermal profiling and performance and reliability analysis. We introduce here a straightforward TRM system to perform quantitative thermal characterization of microelectronics devices. The quantitative imaging of the surface temperature distribution of a polysilicon micro-resistor is obtained by a lock-in measurement technique and calibration process in the conventional CCD-based widefield microscope. To confirm the quantitative thermal measurement, the measured thermal information is compared to that obtained with an infrared thermography (IRT) system. In addition to quantitative surface temperature distribution, the sub-micron defects on microelectronic devices can be clearly distinguished from the thermoreflectance images, which are hardly perceptible with a conventional widefield microscopy system. The thermal resolution of the proposed TRM system is experimentally determined by measuring standard deviation values of thermoreflectance data with respect to the iteration number. The spatial and thermal resolutions of our system are measured ~670 nm and ~13 mK, respectively. We believe that quantitative thermal imaging in the TRM system can be used for improvement of microelectronic devices and integrated circuit (IC) designs.

  13. HAADF-STEM atom counting in atom probe tomography specimens: Towards quantitative correlative microscopy.

    PubMed

    Lefebvre, W; Hernandez-Maldonado, D; Moyon, F; Cuvilly, F; Vaudolon, C; Shinde, D; Vurpillot, F

    2015-12-01

    The geometry of atom probe tomography tips strongly differs from standard scanning transmission electron microscopy foils. Whereas the later are rather flat and thin (<20 nm), tips display a curved surface and a significantly larger thickness. As far as a correlative approach aims at analysing the same specimen by both techniques, it is mandatory to explore the limits and advantages imposed by the particular geometry of atom probe tomography specimens. Based on simulations (electron probe propagation and image simulations), the possibility to apply quantitative high angle annular dark field scanning transmission electron microscopy to of atom probe tomography specimens has been tested. The influence of electron probe convergence and the benefice of deconvolution of electron probe point spread function electron have been established. Atom counting in atom probe tomography specimens is for the first time reported in this present work. It is demonstrated that, based on single projections of high angle annular dark field imaging, significant quantitative information can be used as additional input for refining the data obtained by correlative analysis of the specimen in APT, therefore opening new perspectives in the field of atomic scale tomography.

  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. 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

  16. 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.

  17. 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

  18. 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.

  19. High Resolution Quantitative Angle-Scanning Widefield Surface Plasmon Microscopy

    NASA Astrophysics Data System (ADS)

    Tan, Han-Min; Pechprasarn, Suejit; Zhang, Jing; Pitter, Mark C.; Somekh, Michael G.

    2016-02-01

    We describe the construction of a prismless widefield surface plasmon microscope; this has been applied to imaging of the interactions of protein and antibodies in aqueous media. The illumination angle of spatially incoherent diffuse laser illumination was controlled with an amplitude spatial light modulator placed in a conjugate back focal plane to allow dynamic control of the illumination angle. Quantitative surface plasmon microscopy images with high spatial resolution were acquired by post-processing a series of images obtained as a function of illumination angle. Experimental results are presented showing spatially and temporally resolved binding of a protein to a ligand. We also show theoretical results calculated by vector diffraction theory that accurately predict the response of the microscope on a spatially varying sample thus allowing proper quantification and interpretation of the experimental results.

  20. Fluorescent microscopy approaches of quantitative soil microbial analysis

    NASA Astrophysics Data System (ADS)

    Ivanov, Konstantin; Polyanskaya, Lubov

    2015-04-01

    Classical fluorescent microscopy method was used during the last decades in various microbiological studies of terrestrial ecosystems. The method provides representative results and simple application which is allow to use it both as routine part of amplitudinous research and in small-scaled laboratories. Furthermore, depending on research targets a lot of modifications of fluorescent microscopy method were established. Combination and comparison of several approaches is an opportunity of quantitative estimation of microbial community in soil. The first analytical part of the study was dedicated to soil bacterial density estimation by fluorescent microscopy in dynamic of several 30-days experiments. The purpose of research was estimation of changes in soil bacterial community on the different soil horizons under aerobic and anaerobic conditions with adding nutrients in two experimental sets: cellulose and chitin. Was modified the nalidixic acid method for inhibition of DNA division of gram-negative bacteria, and the method provides the quantification of this bacterial group by fluorescent microscopy. Established approach allowed to estimate 3-4 times more cells of gram-negative bacteria in soil. The functions of actinomyces in soil polymer destruction are traditionally considered as dominant in comparison to gram-negative bacterial group. However, quantification of gram-negative bacteria in chernozem and peatland provides underestimation of classical notion for this bacterial group. Chitin introduction had no positive effect to gram-negative bacterial population density changes in chernozem but concurrently this nutrient provided the fast growing dynamics at the first 3 days of experiment both under aerobic and anaerobic conditions. This is confirming chitinolytic activity of gram-negative bacteria in soil organic matter decomposition. At the next part of research modified method for soil gram-negative bacteria quantification was compared to fluorescent in situ

  1. 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

  2. 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

  3. Quantitative fluorescent speckle microscopy (QFSM) to measure actin dynamics.

    PubMed

    Mendoza, Michelle C; Besson, Sebastien; Danuser, Gaudenz

    2012-10-01

    Quantitative fluorescent speckle microscopy (QFSM) is a live-cell imaging method to analyze the dynamics of macromolecular assemblies with high spatial and temporal resolution. Its greatest successes were in the analysis of actin filament and adhesion dynamics in the context of cell migration and microtubule dynamics in interphase and the meiotic/mitotic spindle. Here, focus is on the former application to illustrate the procedures of FSM imaging and the computational image processing that extracts quantitative information from these experiments. QFSM is advantageous over other methods because it measures the movement and turnover kinetics of the actin filament (F-actin) network in living cells across the entire field of view. Experiments begin with the microinjection of fluorophore-labeled actin into cells, which generate a low ratio of fluorescently labeled to endogenously unlabeled actin monomers. Spinning disk confocal or wide-field imaging then visualizes fluorophore clusters (two to eight actin monomers) within the assembled F-actin network as speckles. QFSM software identifies and computationally tracks and utilizes the location, appearance, and disappearance of speckles to derive network flows and maps of the rate of filament assembly and disassembly. PMID:23042526

  4. Quantitative analysis of in vivo confocal microscopy images: a review.

    PubMed

    Patel, Dipika V; McGhee, Charles N

    2013-01-01

    In vivo confocal microscopy (IVCM) is a non-invasive method of examining the living human cornea. The recent trend towards quantitative studies using IVCM has led to the development of a variety of methods for quantifying image parameters. When selecting IVCM images for quantitative analysis, it is important to be consistent regarding the location, depth, and quality of images. All images should be de-identified, randomized, and calibrated prior to analysis. Numerous image analysis software are available, each with their own advantages and disadvantages. Criteria for analyzing corneal epithelium, sub-basal nerves, keratocytes, endothelium, and immune/inflammatory cells have been developed, although there is inconsistency among research groups regarding parameter definition. The quantification of stromal nerve parameters, however, remains a challenge. Most studies report lower inter-observer repeatability compared with intra-observer repeatability, and observer experience is known to be an important factor. Standardization of IVCM image analysis through the use of a reading center would be crucial for any future large, multi-centre clinical trials using IVCM.

  5. Extracting quantitative parameters from images in multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Zimmerley, Maxwell Stuart

    Coherent anti-Stokes Raman scattering (CARS) microscopy allows for fast, three-dimensionally resolved detection of molecules based on vibrational contrast. In CARS, the generated signal is nonlinearly dependent upon the concentration of the vibrational mode of interest. This makes it challenging to extract quantitative parameters (such as the concentration or orientation) from CARS images of biological and synthetic samples. Because of this, many investigations which employ CARS microscopy generally only report qualitative information extracted from these images. In this thesis, three methods have been developed to extract the quantitative concentration information from CARS images. In the first, the ratio of the forward-propagating and back-reflected CARS signal generated in tissue is used to monitor the percolation of DMSO into excised human cadaver skin. Through this, we find that the maximum clearing of skin with DMSO occurs at 40% v/v. We also combine CARS with second harmonic generation (SHG) to investigate the effects of DMSO on collagen. Up to a 20% v/v concentration of DMSO in the skin, the collagen becomes disrupted, resulting in a significant drop in the generated SHG. In the second method, the ratio between the CARS resonance peak and dip is correlated with the concentration to measure the concentration of water and deuterated glycine in hair. Both molecules are found to distribute throughout the hair fiber homogenously, water at a 34% v/v concentration, and d-glycine with a 0.22 M concentration. In the final method, CARS spectra over one vibrational mode are used to extract the imaginary part of the third-order nonlinear susceptibility. This quantity is linearly dependent upon the concentration of the vibrational mode of interest. This procedure is used to determine the degree of conversion of two-photon polymerized microstructures synthesized with varying writing powers. A sigmoidal relationship is observed between the applied intensity and the degree

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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

  12. 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.

  13. Quantitative thermal imaging of single-walled carbon nanotube devices by scanning Joule expansion microscopy.

    PubMed

    Xie, Xu; Grosse, Kyle L; Song, Jizhou; Lu, Chaofeng; Dunham, Simon; Du, Frank; Islam, Ahmad E; Li, Yuhang; Zhang, Yihui; Pop, Eric; Huang, Yonggang; King, William P; Rogers, John A

    2012-11-27

    Electrical generation of heat in single-walled carbon nanotubes (SWNTs) and subsequent thermal transport into the surroundings can critically affect the design, operation, and reliability of electronic and optoelectronic devices based on these materials. Here we investigate such heat generation and transport characteristics in perfectly aligned, horizontal arrays of SWNTs integrated into transistor structures. We present quantitative assessments of local thermometry at individual SWNTs in these arrays, evaluated using scanning Joule expansion microscopy. Measurements at different applied voltages reveal electronic behaviors, including metallic and semiconducting responses, spatial variations in diameter or chirality, and localized defect sites. Analytical models, validated by measurements performed on different device structures at various conditions, enable accurate, quantitative extraction of temperature distributions at the level of individual SWNTs. Using current equipment, the spatial resolution and temperature precision are as good as ∼100 nm and ∼0.7 K, respectively.

  14. 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

  15. Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

    PubMed Central

    Changou, Chun A.; Wolfson, Deanna L.; Ahluwalia, Balpreet Singh; Bold, Richard J.; Kung, Hsing-Jien; Chuang, Frank Y.S.

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine1. This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)1,10. Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)1,2,3. Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy11,12. Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early stages of

  16. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-01-01

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  17. Quantitative analysis of autophagy using advanced 3D fluorescence microscopy.

    PubMed

    Changou, Chun A; Wolfson, Deanna L; Ahluwalia, Balpreet Singh; Bold, Richard J; Kung, Hsing-Jien; Chuang, Frank Y S

    2013-05-03

    Prostate cancer is the leading form of malignancies among men in the U.S. While surgery carries a significant risk of impotence and incontinence, traditional chemotherapeutic approaches have been largely unsuccessful. Hormone therapy is effective at early stage, but often fails with the eventual development of hormone-refractory tumors. We have been interested in developing therapeutics targeting specific metabolic deficiency of tumor cells. We recently showed that prostate tumor cells specifically lack an enzyme (argininosuccinate synthase, or ASS) involved in the synthesis of the amino acid arginine(1). This condition causes the tumor cells to become dependent on exogenous arginine, and they undergo metabolic stress when free arginine is depleted by arginine deiminase (ADI)(1,10). Indeed, we have shown that human prostate cancer cells CWR22Rv1 are effectively killed by ADI with caspase-independent apoptosis and aggressive autophagy (or macroautophagy)(1,2,3). Autophagy is an evolutionarily-conserved process that allows cells to metabolize unwanted proteins by lysosomal breakdown during nutritional starvation(4,5). Although the essential components of this pathway are well-characterized(6,7,8,9), many aspects of the molecular mechanism are still unclear - in particular, what is the role of autophagy in the death-response of prostate cancer cells after ADI treatment? In order to address this question, we required an experimental method to measure the level and extent of autophagic response in cells - and since there are no known molecular markers that can accurately track this process, we chose to develop an imaging-based approach, using quantitative 3D fluorescence microscopy(11,12). Using CWR22Rv1 cells specifically-labeled with fluorescent probes for autophagosomes and lysosomes, we show that 3D image stacks acquired with either widefield deconvolution microscopy (and later, with super-resolution, structured-illumination microscopy) can clearly capture the early

  18. Uncertainty in Quantitative Electron Probe Microanalysis

    PubMed Central

    Heinrich, Kurt F. J.

    2002-01-01

    Quantitative electron probe analysis is based on models based on the physics or x-ray generation, empirically adjusted to the analyses of specimens of known composition. Their accuracy can be estimated by applying them to a set of specimens of presumably well-known composition. PMID:27446746

  19. Quantitative Connection between Ensemble Thermodynamics and Single-Molecule Kinetics: A Case Study Using Cryogenic Electron Microscopy and Single-Molecule Fluorescence Resonance Energy Transfer Investigations of the Ribosome.

    PubMed

    Kinz-Thompson, Colin D; Sharma, Ajeet K; Frank, Joachim; Gonzalez, Ruben L; Chowdhury, Debashish

    2015-08-27

    At equilibrium, thermodynamic and kinetic information can be extracted from biomolecular energy landscapes by many techniques. However, while static, ensemble techniques yield thermodynamic data, often only dynamic, single-molecule techniques can yield the kinetic data that describe transition-state energy barriers. Here we present a generalized framework based upon dwell-time distributions that can be used to connect such static, ensemble techniques with dynamic, single-molecule techniques, and thus characterize energy landscapes to greater resolutions. We demonstrate the utility of this framework by applying it to cryogenic electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET) studies of the bacterial ribosomal pre-translocation complex. Among other benefits, application of this framework to these data explains why two transient, intermediate conformations of the pre-translocation complex, which are observed in a cryo-EM study, may not be observed in several smFRET studies.

  20. 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.

  1. 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.

  2. 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

  3. Quantitative polarized light microscopy of unstained mammalian cochlear sections

    PubMed Central

    Kalwani, Neil M.; Ong, Cheng Ai; Lysaght, Andrew C.; Haward, Simon J.; McKinley, Gareth H.

    2013-01-01

    Abstract. Hearing loss is the most common sensory deficit in the world, and most frequently it originates in the inner ear. Yet, the inner ear has been difficult to access for diagnosis because of its small size, delicate nature, complex three-dimensional anatomy, and encasement in the densest bone in the body. Evolving optical methods are promising to afford cellular diagnosis of pathologic changes in the inner ear. To appropriately interpret results from these emerging technologies, it is important to characterize optical properties of cochlear tissues. Here, we focus on that characterization using quantitative polarized light microscopy (qPLM) applied to unstained cochlear sections of the mouse, a common animal model of human hearing loss. We find that the most birefringent cochlear materials are collagen fibrils and myelin. Retardance of the otic capsule, the spiral ligament, and the basilar membrane are substantially higher than that of other cochlear structures. Retardance of the spiral ligament and the basilar membrane decrease from the cochlear base to the apex, compared with the more uniform retardance of other structures. The intricate structural details revealed by qPLM of unstained cochlear sections ex vivo strongly motivate future application of polarization-sensitive optical coherence tomography to human cochlea in vivo. PMID:23407909

  4. Quantitative polarized light microscopy of unstained mammalian cochlear sections

    NASA Astrophysics Data System (ADS)

    Kalwani, Neil M.; Ong, Cheng Ai; Lysaght, Andrew C.; Haward, Simon J.; McKinley, Gareth H.; Stankovic, Konstantina M.

    2013-02-01

    Hearing loss is the most common sensory deficit in the world, and most frequently it originates in the inner ear. Yet, the inner ear has been difficult to access for diagnosis because of its small size, delicate nature, complex three-dimensional anatomy, and encasement in the densest bone in the body. Evolving optical methods are promising to afford cellular diagnosis of pathologic changes in the inner ear. To appropriately interpret results from these emerging technologies, it is important to characterize optical properties of cochlear tissues. Here, we focus on that characterization using quantitative polarized light microscopy (qPLM) applied to unstained cochlear sections of the mouse, a common animal model of human hearing loss. We find that the most birefringent cochlear materials are collagen fibrils and myelin. Retardance of the otic capsule, the spiral ligament, and the basilar membrane are substantially higher than that of other cochlear structures. Retardance of the spiral ligament and the basilar membrane decrease from the cochlear base to the apex, compared with the more uniform retardance of other structures. The intricate structural details revealed by qPLM of unstained cochlear sections ex vivo strongly motivate future application of polarization-sensitive optical coherence tomography to human cochlea in vivo.

  5. Silver nanoparticle-induced degranulation observed with quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Wenzhong; Lee, Seungrag; Lee, Jiyong; Bae, Yoonsung; Kim, Dugyoung

    2010-07-01

    Monitoring a degranulation process in a live mast cell is a quite important issue in immunology and pharmacology. Because the size of a granule is normally much smaller than the resolution limit of an optical microscope system, there is no direct real-time live cell imaging technique for observing degranulation processes except for fluorescence imaging techniques. In this research, we propose optical quantitative phase microscopy (QPM) as a new observation tool to study degranulation processes in a live mast cell without any fluorescence labeling. We measure the cell volumes and the cross sectional profiles (x-z plane) of an RBL-2H3 cell and a HeLa cell, before and after they are exposed to calcium ionophore A23187 and silver nanoparticles (AgNPs). We verify that the volume and the cross sectional line profile of the RBL-2H3 cell were changed significantly when it was exposed to A23187. When 50 μg/mL of AgNP is used instead of A23187, the measurements of cell volume and cross sectional profiles indicate that RBL-2H3 cells also follow degranulation processes. Degranulation processes for these cells are verified by monitoring the increase of intracellular calcium ([Ca2+]i) and histamine with fluorescent methods.

  6. Quantitative high dynamic range beam profiling for fluorescence microscopy

    SciTech Connect

    Mitchell, T. J. Saunter, C. D.; O’Nions, W.; Girkin, J. M.; Love, G. D.

    2014-10-15

    Modern developmental biology relies on optically sectioning fluorescence microscope techniques to produce non-destructive in vivo images of developing specimens at high resolution in three dimensions. As optimal performance of these techniques is reliant on the three-dimensional (3D) intensity profile of the illumination employed, the ability to directly record and analyze these profiles is of great use to the fluorescence microscopist or instrument builder. Though excitation beam profiles can be measured indirectly using a sample of fluorescent beads and recording the emission along the microscope detection path, we demonstrate an alternative approach where a miniature camera sensor is used directly within the illumination beam. Measurements taken using our approach are solely concerned with the illumination optics as the detection optics are not involved. We present a miniature beam profiling device and high dynamic range flux reconstruction algorithm that together are capable of accurately reproducing quantitative 3D flux maps over a large focal volume. Performance of this beam profiling system is verified within an optical test bench and demonstrated for fluorescence microscopy by profiling the low NA illumination beam of a single plane illumination microscope. The generality and success of this approach showcases a widely flexible beam amplitude diagnostic tool for use within the life sciences.

  7. Quantitative polarized light microscopy of unstained mammalian cochlear sections.

    PubMed

    Kalwani, Neil M; Ong, Cheng Ai; Lysaght, Andrew C; Haward, Simon J; McKinley, Gareth H; Stankovic, Konstantina M

    2013-02-01

    Hearing loss is the most common sensory deficit in the world, and most frequently it originates in the inner ear. Yet, the inner ear has been difficult to access for diagnosis because of its small size, delicate nature, complex three-dimensional anatomy, and encasement in the densest bone in the body. Evolving optical methods are promising to afford cellular diagnosis of pathologic changes in the inner ear. To appropriately interpret results from these emerging technologies, it is important to characterize optical properties of cochlear tissues. Here, we focus on that characterization using quantitative polarized light microscopy (qPLM) applied to unstained cochlear sections of the mouse, a common animal model of human hearing loss. We find that the most birefringent cochlear materials are collagen fibrils and myelin. Retardance of the otic capsule, the spiral ligament, and the basilar membrane are substantially higher than that of other cochlear structures. Retardance of the spiral ligament and the basilar membrane decrease from the cochlear base to the apex, compared with the more uniform retardance of other structures. The intricate structural details revealed by qPLM of unstained cochlear sections ex vivo strongly motivate future application of polarization-sensitive optical coherence tomography to human cochlea in vivo. PMID:23407909

  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. 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

  11. 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

  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. 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. 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.

  15. 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

  16. 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

  17. 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.

  18. Reliable strain measurement in transistor arrays by robust scanning transmission electron microscopy

    SciTech Connect

    Kim, Suhyun; Kim, Joong Jung; Jung, Younheum; Lee, Kyungwoo; Byun, Gwangsun; Hwang, KyoungHwan; Lee, Sunyoung; Lee, Kyupil

    2013-09-15

    Accurate measurement of the strain field in the channels of transistor arrays is critical for strain engineering in modern electronic devices. We applied atomic-resolution high-angle annular dark-field scanning transmission electron microscopy to quantitative measurement of the strain field in transistor arrays. The quantitative strain profile over 20 transistors was obtained with high reliability and a precision of 0.1%. The strain field was found to form homogeneously in the channels of the transistor arrays. Furthermore, strain relaxation due to the thin foil effect was quantitatively investigated for thicknesses of 35 to 275 nm.

  19. 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

  20. Silver nanoparticle-induced degranulation observed with quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Wenzhong; Lee, Seungrag; Lee, Jiyong; Bae, Yoonsung; Kim, Dugyoung

    2010-02-01

    The use of AgNP is becoming more and more widespread in biomedical field. But compared with the promising bactericidal function, other physiological effects of AgNP on cells are relatively scant. In this research, we propose quantitative phase microscopy (QPM) as a new method to study the degranulation, and AgNP-induced RBL-2H3 cell degranulation is studied as well. Firstly, HeLa cells as the cell control and PBS as the solvent control, we measured the cell volume and cross section profile (x-z plane) with QPM. The results showed that the volume and cross section profile changed only the RBL-2H3 cells exposed to calcium ionophore A23187, which demonstrates the validity of QPM in degranulation research. Secondly, 50μg/mL of AgNP was used instead of A23187, and the measurement of cell volume and cross section profile was carried out again. RBL-2H3 cell volume increased immediately after AgNP was added, and cross section profile showed that the cell surface became granulated, but HeLa cell was lack of that effect. Phase images obviously indicated the RBL-2H3 cell deformation. Thirdly, stained with Fluo-3/AM, intracellular calcium Ca2+]i of single RBL-2H3 cell treated with AgNP was observed with fluorescent microscopy; incubated with AgNP for 20min, the supernatant of RBL-2H3 cells was collected and reacted with o-phthalaldehyde (OPA), then the fluorescent intensity of histamine-OPA complex was assayed with spectrofluorometer. The results of Ca2+]i and histamine increase showed that degranulation of AgNP-induced RBL-2H3 cell occurred. So, the cell volume was used as a parameter of degranulation in our study and AgNP-induced RBL-2H3 cells degranulation was confirmed by the cell volume increment, cross section profile change, and [Ca2+]i and histamine in supernatant increase.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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)

  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 PAGESBeta

    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. Making microscopy count: quantitative light microscopy of dynamic processes in living plants.

    PubMed

    Fricker, Mark D; Moger, Julian; Littlejohn, George R; Deeks, Michael J

    2016-08-01

    Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking. PMID:27145353

  12. Making microscopy count: quantitative light microscopy of dynamic processes in living plants.

    PubMed

    Fricker, Mark D; Moger, Julian; Littlejohn, George R; Deeks, Michael J

    2016-08-01

    Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking.

  13. Quantitative X-ray projection microscopy: phase-contrast and multi-spectral imaging.

    PubMed

    Mayo, S C; Miller, P R; Wilkins, S W; Davis, T J; Gao, D; Gureyev, T E; Paganin, D; Parry, D J; Pogany, A; Stevenson, A W

    2002-08-01

    We outline a new approach to X-ray projection microscopy in a scanning electron microscope (SEM), which exploits phase contrast to boost the quality and information content of images. These developments have been made possible by the combination of a high-brightness field-emission gun (FEG)-based SEM, direct detection CCD technology and new phase retrieval algorithms. Using this approach we have been able to obtain spatial resolution of < 0.2 micro m and have demonstrated novel features such as: (i) phase-contrast enhanced visibility of high spatial frequency image features (e.g. edges and boundaries) over a wide energy range; (ii) energy-resolved imaging to simultaneously produce multiple quasi-monochromatic images using broad-band polychromatic illumination; (iii) easy implementation of microtomography; (iv) rapid and robust phase/amplitude-retrieval algorithms to enable new real-time and quantitative modes of microscopic imaging. These algorithms can also be applied successfully to recover object-plane information from intermediate-field images, unlocking the potentially greater contrast and resolution of the intermediate-field regime. Widespread applications are envisaged for fields such as materials science, biological and biomedical research and microelectronics device inspection. Some illustrative examples are presented. The quantitative methods described here are also very relevant to projection microscopy using other sources of radiation, such as visible light and electrons.

  14. 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.

  15. Relationship between the v2PO4/amide III ratio assessed by Raman spectroscopy and the calcium content measured by quantitative backscattered electron microscopy in healthy human osteonal bone

    NASA Astrophysics Data System (ADS)

    Roschger, Andreas; Gamsjaeger, Sonja; Hofstetter, Birgit; Masic, Admir; Blouin, Stéphane; Messmer, Phaedra; Berzlanovich, Andrea; Paschalis, Eleftherios P.; Roschger, Paul; Klaushofer, Klaus; Fratzl, Peter

    2014-06-01

    Raman microspectroscopy and quantitative backscattered electron imaging (qBEI) of bone are powerful tools to investigate bone material properties. Both methods provide information on the degree of bone matrix mineralization. However, a head-to-head comparison of these outcomes from identical bone areas has not been performed to date. In femoral midshaft cross sections of three women, 99 regions (20×20 μ) were selected inside osteons and interstitial bone covering a wide range of matrix mineralization. As the focus of this study was only on regions undergoing secondary mineralization, zones exhibiting a distinct gradient in mineral content close to the mineralization front were excluded. The same regions were measured by both methods. We found a linear correlation (R2=0.75) between mineral/matrix as measured by Raman spectroscopy and the wt. %Mineral/(100-wt. %Mineral) as obtained by qBEI, in good agreement with theoretical estimations. The observed deviations of single values from the linear regression line were determined to reflect biological heterogeneities. The data of this study demonstrate the good correspondence between Raman and qBEI outcomes in describing tissue mineralization. The obtained correlation is likely sensitive to changes in bone tissue composition, providing an approach to detect potential deviations from normal bone.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

    composite electrodes/electrolyte interfaces in conventional lithium ion batteries, depicted in Fig.1b, where quantitative interface characterization is extremely difficult if not impossible. A second strategy involves organic electrolyte, though this approach more closely resembles the actual operation conditions of a LIB, the extreme volatility In Situ Analytical Electron Microscopy for Probing Nanoscale Electrochemistry by Ying Shirley Meng, Thomas McGilvray, Ming-Che Yang, Danijel Gostovic, Feng Wang, Dongli Zeng, Yimei Zhu, and Jason Graetz of the organic electrolytes present significant challenges for designing an in situ cell that is suitable for the vacuum environment of the TEM. Significant progress has been made in the past few years on the development of in situ electron microscopy for probing nanoscale electrochemistry. In 2008, Brazier et al. reported the first cross-section observation of an all solid-state lithium ion nano-battery by TEM. In this study the FIB was used to make a 'nano-battery,' from an all solid-state battery prepared by pulsed laser deposition (PLD). In situ TEM observations were not possible at that time due to several key challenges such as the lack of a suitable biasing sample holder and vacuum transfer of sample. In 2010, Yamamoto et al. successfully observed changes of electric potential in an all-solid-state lithium ion battery in situ with electron holography (EH). The 2D potential distribution resulting from movement of lithium ions near the positive-electrode/electrolyte interface was quantified. More recently Huang et al. and Wang et al. reported the in situ observations of the electrochemical lithiation of a single SnO{sub 2} nanowire electrode in two different in situ setups. In their approach, a vacuum compatible ionic liquid is used as the electrolyte, eliminating the need for complicated membrane sealing to prevent the evaporation of carbonate based organic electrolyte into the TEM column. One main limitation of this approach

  3. 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.

  4. Quantitative WDS analysis using electron probe microanalyzer

    SciTech Connect

    Ul-Hamid, Anwar . E-mail: anwar@kfupm.edu.sa; Tawancy, Hani M.; Mohammed, Abdul-Rashid I.; Al-Jaroudi, Said S.; Abbas, Nureddin M.

    2006-04-15

    In this paper, the procedure for conducting quantitative elemental analysis by ZAF correction method using wavelength dispersive X-ray spectroscopy (WDS) in an electron probe microanalyzer (EPMA) is elaborated. Analysis of a thermal barrier coating (TBC) system formed on a Ni-based single crystal superalloy is presented as an example to illustrate the analysis of samples consisting of a large number of major and minor elements. The analysis was performed by known standards and measured peak-to-background intensity ratios. The procedure for using separate set of acquisition conditions for major and minor element analysis is explained and its importance is stressed.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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.

  11. 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

  12. Correlative Light and Electron Microscopy of Nucleolar Transcription in Saccharomyces cerevisiae.

    PubMed

    Normand, Christophe; Berthaud, Maxime; Gadal, Olivier; Léger-Silvestre, Isabelle

    2016-01-01

    Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller's spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated "pipeline" for imaging.

  13. Correlative Light and Electron Microscopy of Nucleolar Transcription in Saccharomyces cerevisiae.

    PubMed

    Normand, Christophe; Berthaud, Maxime; Gadal, Olivier; Léger-Silvestre, Isabelle

    2016-01-01

    Nucleoli form around RNA polymerase I transcribed ribosomal RNA (rRNA) genes. The direct electron microscopy observation of rRNA genes after nucleolar chromatin spreading (Miller's spreads) constitutes to date the only system to quantitatively assess transcription at a single molecule level. However, the spreading procedure is likely generating artifact and despite being informative, these spread rRNA genes are far from their in vivo situation. The integration of the structural characterization of spread rRNA genes in the three-dimensional (3D) organization of the nucleolus would represent an important scientific achievement. Here, we describe a correlative light and electron microscopy (CLEM) protocol allowing detection of tagged-Pol I by fluorescent microscopy and high-resolution imaging of the nucleolar ultrastructural context. This protocol can be implemented in laboratories equipped with conventional fluorescence and electron microscopes and does not require sophisticated "pipeline" for imaging. PMID:27576708

  14. The spatial coherence function in scanning transmission electron microscopy and spectroscopy.

    PubMed

    Nguyen, D T; Findlay, S D; Etheridge, J

    2014-11-01

    We investigate the implications of the form of the spatial coherence function, also referred to as the effective source distribution, for quantitative analysis in scanning transmission electron microscopy, and in particular for interpreting the spatial origin of imaging and spectroscopy signals. These questions are explored using three different source distribution models applied to a GaAs crystal case study. The shape of the effective source distribution was found to have a strong influence not only on the scanning transmission electron microscopy (STEM) image contrast, but also on the distribution of the scattered electron wavefield and hence on the spatial origin of the detected electron intensities. The implications this has for measuring structure, composition and bonding at atomic resolution via annular dark field, X-ray and electron energy loss STEM imaging are discussed.

  15. CONFOCAL MICROSCOPY SYSTEM PERFORMANCE: FOUNDATIONS FOR MEASUREMENTS, QUANTITATION AND SPECTROSCOPY

    EPA Science Inventory

    The confocal laser-scanning microscopy (CLSM) has enormous potential in many biological fields. The goal of a CLSM is to acquire and quantify fluorescence and in some instruments acquire spectral characterization of the emitted signal. The accuracy of these measurements demands t...

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. Using quantitative interference phase microscopy for sperm acrosome evaluation (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Balberg, Michal; Kalinowski, Ksawery; Levi, Mattan; Shaked, Natan T.

    2016-03-01

    We demonstrate quantitative assessment of sperm cell morphology, primarily acrosomal volume, using quantitative interference phase microscopy (IPM). Normally, the area of the acrosome is assessed using dyes that stain the acrosomal part of the cell. We have imaged fixed individual sperm cells using IPM. Following, the sample was stained and the same cells were imaged using bright field microscopy (BFM). We identified the acrosome using the stained BFM image, and used it to define a quantitative corresponding area in the IPM image and determine a quantitative threshold for evaluating the volume of the acrosome.

  4. 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

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Quantitative local photosynthetic flux measurements at isolated chloroplasts and thylakoid membranes using scanning electrochemical microscopy (SECM).

    PubMed

    McKelvey, Kim; Martin, Sophie; Robinson, Colin; Unwin, Patrick R

    2013-07-01

    Scanning electrochemical microscopy (SECM) offers a fast and quantitative method to measure local fluxes within photosynthesis. In particular, we have measured the flux of oxygen and ferrocyanide (Fe(CN)6(4-)), from the artificial electron acceptor ferricyanide (Fe(CN)6(3-)), using a stationary ultramicroelectrode at chloroplasts and thylakoid membranes (sourced from chloroplasts). Oxygen generation at films of chloroplasts and thylakoid membranes was detected directly during photosynthesis, but in the case of thylakoid membranes, this switched to sustained oxygen consumption at longer illumination times. An initial oxygen concentration spike was detected over both chloroplast and thylakoid membrane films, and the kinetics of the oxygen generation were extracted by fitting the experimental data to a finite element method (FEM) simulation. In contrast to previous work, the oxygen generation spike was attributed to the limited size of the plastoquinone pool, a key component in the linear electron transport pathway and a contributing factor in photoinhibition. Finally, the mobile nature of the SECM probe, and its high spatial resolution, also allowed us to detect ferrocyanide produced from a single thylakoid membrane. These results further demonstrate the power of SECM for localized flux measurements in biological processes, in this case photosynthesis, and that the high time resolution, combined with FEM simulations, allows the elucidation of quantitative kinetic information.

  12. 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.

  13. Quantitative thermal microscopy using thermoelectric probe in passive mode

    NASA Astrophysics Data System (ADS)

    Bontempi, A.; Thiery, L.; Teyssieux, D.; Briand, D.; Vairac, P.

    2013-10-01

    A scanning thermal microscope working in passive mode using a micronic thermocouple probe is presented as a quantitative technique. We show that actual surface temperature distributions of microsystems are measurable under conditions for which most of usual techniques cannot operate. The quantitative aspect relies on the necessity of an appropriate calibration procedure which takes into account of the probe-to-sample thermal interaction prior to any measurement. Besides this consideration that should be treated for any thermal contact probing system, the main advantages of our thermal microscope deal with the temperature available range, the insensitivity to the surface optical parameters, the possibility to image DC, and AC temperature components up to 1 kHz typically and a resolution limit related to near-field behavior.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. Segmentation and learning in the quantitative analysis of microscopy images

    NASA Astrophysics Data System (ADS)

    Ruggiero, Christy; Ross, Amy; Porter, Reid

    2015-02-01

    In material science and bio-medical domains the quantity and quality of microscopy images is rapidly increasing and there is a great need to automatically detect, delineate and quantify particles, grains, cells, neurons and other functional "objects" within these images. These are challenging problems for image processing because of the variability in object appearance that inevitably arises in real world image acquisition and analysis. One of the most promising (and practical) ways to address these challenges is interactive image segmentation. These algorithms are designed to incorporate input from a human operator to tailor the segmentation method to the image at hand. Interactive image segmentation is now a key tool in a wide range of applications in microscopy and elsewhere. Historically, interactive image segmentation algorithms have tailored segmentation on an image-by-image basis, and information derived from operator input is not transferred between images. But recently there has been increasing interest to use machine learning in segmentation to provide interactive tools that accumulate and learn from the operator input over longer periods of time. These new learning algorithms reduce the need for operator input over time, and can potentially provide a more dynamic balance between customization and automation for different applications. This paper reviews the state of the art in this area, provides a unified view of these algorithms, and compares the segmentation performance of various design choices.

  19. Quantitative imaging of intact cardiac tissue using remote focusing microscopy

    NASA Astrophysics Data System (ADS)

    Corbett, A. D.; Burton, R. A. B.; Bub, G.; Wilson, T.

    2015-03-01

    Remote focussing microscopy offers many advantages when acquiring volumetric data from living tissue. The all-optical means of refocussing does not agitate the specimen by moving either the stage or imaging objective. Aberrationcompensated imaging extends over volumes as large as 450 μm x 450 μm x 200 μm (X, Y and Z) allowing data to be collected from hundreds of cells. The speed with which refocussing can be achieved is limited only by the mechanical movement of a small (2 mm diameter) mirror. Using a pair of oblique imaging planes to rapidly acquire (<200ms) depth information temporally freezes residual tissue motion in the arrested heart. This paper discusses the progress of remote focussing microscopy from a novel imaging technique to a reliable tool in the life sciences. Specifically, we describe recent efforts to achieve the accurate calibration of both distance and orientation within the imaging volume. Using a laser machined fluorescent specimen it is possible to identify, with high sensitivity, small (<1%) depth-dependent magnification changes which are a linear function of axial misalignment of the imaging objective. The sensitivity of the calibration procedure limits distortion to <1 μm over the entire imaging volume. This work finds direct application in identifying the microscopic effects of chronic disease in the living heart.

  20. 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.

  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. 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…

  3. 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.

  4. 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.

  5. 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.

  6. 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

  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. 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.

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-01-01

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

  14. Correlative super-resolution fluorescence and electron microscopy of the nuclear pore complex with molecular resolution.

    PubMed

    Löschberger, Anna; Franke, Christian; Krohne, Georg; van de Linde, Sebastian; Sauer, Markus

    2014-10-15

    Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures.

  15. Quantitative flaw characterization with scanning laser acoustic microscopy

    NASA Technical Reports Server (NTRS)

    Generazio, E. R.; Roth, D. J.

    1986-01-01

    Surface roughness and diffraction are two factors that have been observed to affect the accuracy of flaw characterization with scanning laser acoustic microscopy. In accuracies can arise when the surface of the test sample is acoustically rough. It is shown that, in this case, Snell's law is no longer valid for determining the direction of sound propagation within the sample. The relationship between the direction of sound propagation within the sample, the apparent flaw depth, and the sample's surface roughness is investigated. Diffraction effects can mask the acoustic images of minute flaws and make it difficult to establish their size, depth, and other characteristics. It is shown that for Fraunhofer diffraction conditions the acoustic image of a subsurface defect corresponds to a two-dimensional Fourier transform. Transforms based on simulated flaws are used to infer the size and shape of the actual flaw.

  16. Quantitative flaw characterization with scanning laser acoustic microscopy

    NASA Technical Reports Server (NTRS)

    Generazio, E. R.; Roth, D. J.

    1986-01-01

    Surface roughness and diffraction are two factors that have been observed to affect the accuracy of flaw characterization with scanning laser acoustic microscopy. Inaccuracies can arise when the surface of the test sample is acoustically rough. It is shown that, in this case, Snell's law is no longer valid for determining the direction of sound propagation within the sample. The relationship between the direction of sound propagation within the sample, the apparent flaw depth, and the sample's surface roughness is investigated. Diffraction effects can mask the acoustic images of minute flaws and make it difficult to establish their size, depth, and other characteristics. It is shown that for Fraunhofer diffraction conditions the acoustic image of a subsurface defect corresponds to a two-dimensional Fourier transform. Transforms based on simulated flaws are used to infer the size and shape of the actual flaw.

  17. Insights into primary immune deficiency from quantitative microscopy.

    PubMed

    Mace, Emily M; Orange, Jordan S

    2015-11-01

    Recent advances in genomics-based technology have resulted in an increase in our understanding of the molecular basis of many primary immune deficiencies. Along with this increased knowledge comes an increased responsibility to understand the underlying mechanism of disease, and thus increasingly sophisticated technologies are being used to investigate the cell biology of human immune deficiencies. One such technology, which has itself undergone a recent explosion in innovation, is that of high-resolution microscopy and image analysis. These advances complement innovative studies that have previously shed light on critical cell biological processes that are perturbed by single-gene mutations in primary immune deficiency. Here we highlight advances made specifically in the following cell biological processes: (1) cytoskeletal-related processes; (2) cell signaling; (3) intercellular trafficking; and (4) cellular host defense. PMID:26078103

  18. 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

  19. Digital holographic microscopy for quantitative cell dynamic evaluation during laser microsurgery

    PubMed Central

    Yu, Lingfeng; Mohanty, Samarendra; Zhang, Jun; Genc, Suzanne; Kim, Myung K.; Berns, Michael W.; Chen, Zhongping

    2010-01-01

    Digital holographic microscopy allows determination of dynamic changes in the optical thickness profile of a transparent object with subwavelength accuracy. Here, we report a quantitative phase laser microsurgery system for evaluation of cellular/ sub-cellular dynamic changes during laser micro-dissection. The proposed method takes advantage of the precise optical manipulation by the laser microbeam and quantitative phase imaging by digital holographic microscopy with high spatial and temporal resolution. This system will permit quantitative evaluation of the damage and/or the repair of the cell or cell organelles in real time. PMID:19582118

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. Quantitative orientation-independent differential interference contrast (DIC) microscopy

    NASA Astrophysics Data System (ADS)

    Shribak, Michael; LaFountain, James; Biggs, David; Inoué, Shinya

    2007-02-01

    We describe a new DIC technique, which records phase gradients within microscopic specimens independently of their orientation. The proposed system allows the generation of images representing the distribution of dry mass (optical path difference) in the specimen. Unlike in other forms of interference microscopes, this approach does not require a narrow illuminating cone. The orientation-independent differential interference contrast (OI-DIC) system can also be combined with orientation-independent polarization (OI-Pol) measurements to yield two complementary images: one showing dry mass distribution (which is proportional to refractive index) and the other showing distribution of birefringence (due to structural or internal anisotropy). With a model specimen used for this work -- living spermatocytes from the crane fly, Nephrotoma suturalis --- the OI-DIC image clearly reveals the detailed shape of the chromosomes while the polarization image quantitatively depicts the distribution of the birefringent microtubules in the spindle, both without any need for staining or other modifications of the cell. We present examples of a pseudo-color combined image incorporating both orientation-independent DIC and polarization images of a spermatocyte at diakinesis and metaphase of meiosis I. Those images provide clear evidence that the proposed technique can reveal fine architecture and molecular organization in live cells without perturbation associated with staining or fluorescent labeling. The phase image was obtained using optics having a numerical aperture 1.4, thus achieving a level of resolution never before achieved with any interference microscope.

  9. Dynamic tunneling force microscopy for characterizing electronic trap states in non-conductive surfaces

    SciTech Connect

    Wang, R.; Williams, C. C.

    2015-09-15

    Dynamic tunneling force microscopy (DTFM) is a scanning probe technique for real space mapping and characterization of individual electronic trap states in non-conductive films with atomic scale spatial resolution. The method is based upon the quantum mechanical tunneling of a single electron back and forth between a metallic atomic force microscopy tip and individual trap states in completely non-conducting surface. This single electron shuttling is measured by detecting the electrostatic force induced on the probe tip at the shuttling frequency. In this paper, the physical basis for the DTFM method is unfolded through a physical model and a derivation of the dynamic tunneling signal as a function of several experimental parameters is shown. Experimental data are compared with the theoretical simulations, showing quantitative consistency and verifying the physical model used. The experimental system is described and representative imaging results are shown.

  10. Dynamic tunneling force microscopy for characterizing electronic trap states in non-conductive surfaces

    NASA Astrophysics Data System (ADS)

    Wang, R.; Williams, C. C.

    2015-09-01

    Dynamic tunneling force microscopy (DTFM) is a scanning probe technique for real space mapping and characterization of individual electronic trap states in non-conductive films with atomic scale spatial resolution. The method is based upon the quantum mechanical tunneling of a single electron back and forth between a metallic atomic force microscopy tip and individual trap states in completely non-conducting surface. This single electron shuttling is measured by detecting the electrostatic force induced on the probe tip at the shuttling frequency. In this paper, the physical basis for the DTFM method is unfolded through a physical model and a derivation of the dynamic tunneling signal as a function of several experimental parameters is shown. Experimental data are compared with the theoretical simulations, showing quantitative consistency and verifying the physical model used. The experimental system is described and representative imaging results are shown.

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Studying localized corrosion using liquid cell transmission electron microscopy

    DOE PAGESBeta

    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. 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

  4. 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.

  5. Quantitative phase-sensitive imaging in a transmission electron microscope

    PubMed

    Bajt; Barty; Nugent; McCartney; Wall; Paganin

    2000-05-01

    This paper presents a new technique for forming quantitative phase and amplitude electron images applicable to a conventional transmission electron microscope. With magnetised cobalt microstructures used as a test object, we use electron holography to obtain an independent measurement of the phase shift. After a suitable calibration of the microscope, we obtain quantitative agreement of the phase shift imposed on the 200 keV electrons passing through the sample.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. Quantitative temperature measurement of multi-layered semiconductor devices using spectroscopic thermoreflectance microscopy.

    PubMed

    Kim, Dong Uk; Park, Kwan Seob; Jeong, Chan Bae; Kim, Geon Hee; Chang, Ki Soo

    2016-06-27

    Thermoreflectance microscopy is essential in understanding the unpredictable local heating generation that occurs during microelectronic device operation. However, temperature measurements of multi-layered semiconductor devices represent a challenge because the thermoreflectance coefficient is quite small and is dramatically changed by the optical interference inside transparent layers of the device. Therefore, we propose a spectroscopic thermoreflectance microscopy system using a systematic approach for improving the quantitative temperature measurement of multi-layered semiconductor devices. We demonstrate the quantitative measurement of the temperature profile for physical defects on thin-film polycrystalline silicon resistors via thermoreflectance coefficient calibration and effective coefficient κ estimation.

  12. Quantitative temperature measurement of multi-layered semiconductor devices using spectroscopic thermoreflectance microscopy.

    PubMed

    Kim, Dong Uk; Park, Kwan Seob; Jeong, Chan Bae; Kim, Geon Hee; Chang, Ki Soo

    2016-06-27

    Thermoreflectance microscopy is essential in understanding the unpredictable local heating generation that occurs during microelectronic device operation. However, temperature measurements of multi-layered semiconductor devices represent a challenge because the thermoreflectance coefficient is quite small and is dramatically changed by the optical interference inside transparent layers of the device. Therefore, we propose a spectroscopic thermoreflectance microscopy system using a systematic approach for improving the quantitative temperature measurement of multi-layered semiconductor devices. We demonstrate the quantitative measurement of the temperature profile for physical defects on thin-film polycrystalline silicon resistors via thermoreflectance coefficient calibration and effective coefficient κ estimation. PMID:27410553

  13. 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

  14. 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

  15. 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

  16. 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.

  17. Nanoscale deformation analysis with high-resolution transmission electron microscopy and digital image correlation

    DOE PAGESBeta

    Wang, Xueju; Pan, Zhipeng; Fan, Feifei; Wang, Jiangwei; Liu, Yang; Mao, Scott X.; Zhu, Ting; Xia, Shuman

    2015-09-10

    We present an application of the digital image correlation (DIC) method to high-resolution transmission electron microscopy (HRTEM) images for nanoscale deformation analysis. The combination of DIC and HRTEM offers both the ultrahigh spatial resolution and high displacement detection sensitivity that are not possible with other microscope-based DIC techniques. We demonstrate the accuracy and utility of the HRTEM-DIC technique through displacement and strain analysis on amorphous silicon. Two types of error sources resulting from the transmission electron microscopy (TEM) image noise and electromagnetic-lens distortions are quantitatively investigated via rigid-body translation experiments. The local and global DIC approaches are applied for themore » analysis of diffusion- and reaction-induced deformation fields in electrochemically lithiated amorphous silicon. As a result, the DIC technique coupled with HRTEM provides a new avenue for the deformation analysis of materials at the nanometer length scales.« less

  18. Nanoscale deformation analysis with high-resolution transmission electron microscopy and digital image correlation

    SciTech Connect

    Wang, Xueju; Pan, Zhipeng; Fan, Feifei; Wang, Jiangwei; Liu, Yang; Mao, Scott X.; Zhu, Ting; Xia, Shuman

    2015-09-10

    We present an application of the digital image correlation (DIC) method to high-resolution transmission electron microscopy (HRTEM) images for nanoscale deformation analysis. The combination of DIC and HRTEM offers both the ultrahigh spatial resolution and high displacement detection sensitivity that are not possible with other microscope-based DIC techniques. We demonstrate the accuracy and utility of the HRTEM-DIC technique through displacement and strain analysis on amorphous silicon. Two types of error sources resulting from the transmission electron microscopy (TEM) image noise and electromagnetic-lens distortions are quantitatively investigated via rigid-body translation experiments. The local and global DIC approaches are applied for the analysis of diffusion- and reaction-induced deformation fields in electrochemically lithiated amorphous silicon. As a result, the DIC technique coupled with HRTEM provides a new avenue for the deformation analysis of materials at the nanometer length scales.

  19. 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.

  20. 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

  1. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    DOE PAGESBeta

    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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  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.

  11. 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

  12. 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

  13. 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

  14. 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.

  15. 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

  16. 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

  17. 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.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. Molecular and Cellular Quantitative Microscopy: theoretical investigations, technological developments and applications to neurobiology

    NASA Astrophysics Data System (ADS)

    Esposito, Alessandro

    2006-05-01

    This PhD project aims at the development and evaluation of microscopy techniques for the quantitative detection of molecular interactions and cellular features. The primarily investigated techniques are Fαrster Resonance Energy Transfer imaging and Fluorescence Lifetime Imaging Microscopy. These techniques have the capability to quantitatively probe the biochemical environment of fluorophores. An automated microscope capable of unsupervised operation has been developed that enables the investigation of molecular and cellular properties at high throughput levels and the analysis of cellular heterogeneity. State-of-the-art Förster Resonance Energy Transfer imaging, Fluorescence Lifetime Imaging Microscopy, Confocal Laser Scanning Microscopy and the newly developed tools have been combined with cellular and molecular biology techniques for the investigation of protein-protein interactions, oligomerization and post-translational modifications of α-Synuclein and Tau, two proteins involved in Parkinson’s and Alzheimer’s disease, respectively. The high inter-disciplinarity of this project required the merging of the expertise of both the Molecular Biophysics Group at the Debye Institute - Utrecht University and the Cell Biophysics Group at the European Neuroscience Institute - Gαttingen University. This project was conducted also with the support and the collaboration of the Center for the Molecular Physiology of the Brain (Göttingen), particularly with the groups associated with the Molecular Quantitative Microscopy and Parkinson’s Disease and Aggregopathies areas. This work demonstrates that molecular and cellular quantitative microscopy can be used in combination with high-throughput screening as a powerful tool for the investigation of the molecular mechanisms of complex biological phenomena like those occurring in neurodegenerative diseases.

  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. 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

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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

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

    PubMed

    Peckys, Diana B; de Jonge, Niels

    2011-04-13

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

  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. 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

  3. 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.

  4. 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

  5. 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.

  6. 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).

  7. 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).

  8. 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

  9. 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.

  10. Total internal reflection holographic microscopy (TIRHM) for quantitative phase characterization of cell-substrate adhesion

    NASA Astrophysics Data System (ADS)

    Ash, William Mason, III

    Total Internal Reflection Holographic Microscopy (TIRHM) combines near-field microscopy with digital holography to produce a new form of near-field phase microscopy. Using a prism in TIR as a near-field imager, the presence of microscopic organisms, cell-substrate interfaces, and adhesions, causes relative refractive index (RRI) and frustrated TIR (f-TIR) to modulate the object beam's evanescent wave phase front. Quantitative phase images of test specimens such as Amoeba proteus, Dictyostelium Discoideum and cells such as SKOV-3 ovarian cancer and 3T3 fibroblasts are produced without the need to introduce stains or fluorophores. The angular spectrum method of digital holography to compensate for tilt anamorphism due to the inclined TIR plane is also discussed. The results of this work conclusively demonstrate, for the first time, the integration of near-field microscopy with digital holography. The cellular images presented show a correlation between the physical extent of the Amoeba proteus plasma membrane and the adhesions that are quantitatively profiled by phase cross-sectioning of the holographic images obtained by digital holography. With its ability to quantitatively characterise cellular adhesion and motility, it is anticipated that TIRHM can be a tool for characterizing and combating cancer metastasis, as well as improving our understanding of morphogenesis and embryogenesis itself.

  11. Fast microstructure and phase analyses of nanopowders using combined analysis of transmission electron microscopy scattering patterns.

    PubMed

    Boullay, P; Lutterotti, L; Chateigner, D; Sicard, L

    2014-09-01

    The full quantitative characterization of nanopowders using transmission electron microscopy scattering patterns is shown. This study demonstrates the feasibility of the application of so-called combined analysis, a global approach for phase identification, structure refinement, characterization of anisotropic crystallite sizes and shapes, texture analysis and texture variations with the probed scale, using electron diffraction patterns of TiO2 and Mn3O4 nanocrystal aggregates and platinum films. Electron diffraction pattern misalignments, positioning, and slight changes from pattern to pattern are directly integrated and refined within this approach. The use of a newly developed full-pattern search-match methodology for phase identification of nanopowders and the incorporation of the two-wave dynamical correction for diffraction patterns are also reported and proved to be efficient. PMID:25176993

  12. Fast microstructure and phase analyses of nanopowders using combined analysis of transmission electron microscopy scattering patterns.

    PubMed

    Boullay, P; Lutterotti, L; Chateigner, D; Sicard, L

    2014-09-01

    The full quantitative characterization of nanopowders using transmission electron microscopy scattering patterns is shown. This study demonstrates the feasibility of the application of so-called combined analysis, a global approach for phase identification, structure refinement, characterization of anisotropic crystallite sizes and shapes, texture analysis and texture variations with the probed scale, using electron diffraction patterns of TiO2 and Mn3O4 nanocrystal aggregates and platinum films. Electron diffraction pattern misalignments, positioning, and slight changes from pattern to pattern are directly integrated and refined within this approach. The use of a newly developed full-pattern search-match methodology for phase identification of nanopowders and the incorporation of the two-wave dynamical correction for diffraction patterns are also reported and proved to be efficient.

  13. Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy.

    PubMed

    Ji, Minbiao; Lewis, Spencer; Camelo-Piragua, Sandra; Ramkissoon, Shakti H; Snuderl, Matija; Venneti, Sriram; Fisher-Hubbard, Amanda; Garrard, Mia; Fu, Dan; Wang, Anthony C; Heth, Jason A; Maher, Cormac O; Sanai, Nader; Johnson, Timothy D; Freudiger, Christian W; Sagher, Oren; Xie, Xiaoliang Sunney; Orringer, Daniel A

    2015-10-14

    Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a nondestructive, label-free optical method, to reveal glioma infiltration in animal models. We show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ = 0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density, and protein/lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density, and protein/lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery.

  14. Detection of human brain tumor infiltration with quantitative stimulated Raman scattering microscopy

    PubMed Central

    Ji, Minbiao; Lewis, Spencer; Camelo-Piragua, Sandra; Ramkissoon, Shakti H.; Snuderl, Matija; Venneti, Sriram; Fisher-Hubbard, Amanda; Garrard, Mia; Fu, Dan; Wang, Anthony C.; Heth, Jason A.; Maher, Cormac O.; Sanai, Nader; Johnson, Timothy D.; Freudiger, Christian W.; Sagher, Oren; Xie, Xiaoliang Sunney; Orringer, Daniel A.

    2016-01-01

    Differentiating tumor from normal brain is a major barrier to achieving optimal outcome in brain tumor surgery. New imaging techniques for visualizing tumor margins during surgery are needed to improve surgical results. We recently demonstrated the ability of stimulated Raman scattering (SRS) microscopy, a non-destructive, label-free optical method, to reveal glioma infiltration in animal models. Here we show that SRS reveals human brain tumor infiltration in fresh, unprocessed surgical specimens from 22 neurosurgical patients. SRS detects tumor infiltration in near-perfect agreement with standard hematoxylin and eosin light microscopy (κ=0.86). The unique chemical contrast specific to SRS microscopy enables tumor detection by revealing quantifiable alterations in tissue cellularity, axonal density and protein:lipid ratio in tumor-infiltrated tissues. To ensure that SRS microscopic data can be easily used in brain tumor surgery, without the need for expert interpretation, we created a classifier based on cellularity, axonal density and protein:lipid ratio in SRS images capable of detecting tumor infiltration with 97.5% sensitivity and 98.5% specificity. Importantly, quantitative SRS microscopy detects the spread of tumor cells, even in brain tissue surrounding a tumor that appears grossly normal. By accurately revealing tumor infiltration, quantitative SRS microscopy holds potential for improving the accuracy of brain tumor surgery. PMID:26468325

  15. 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).

  16. 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.

  17. Quantitative analysis of effective height of probes in microwave impedance microscopy

    NASA Astrophysics Data System (ADS)

    Wei, Zhun; Ma, Eric Yue; Cui, Yong-Tao; Johnston, Scott; Yang, Yongliang; Agarwal, Krishna; Kelly, Michael A.; Shen, Zhi-Xun; Chen, Xudong

    2016-09-01

    A quantitative approach is used to determine an effective height of probe beyond which the capacitance contribution is not significant in microwave impedance microscopy (MIM). We compare the effective height for three different modes of measurement, i.e., capacitance C(l) (l is the tip-sample distance), derivative of capacitance (C'(l)), and second derivative of capacitance (C″(l)). We discuss the effects of tip geometry and sample properties such as relative permittivity and sample height on the effective height with examples and analyze the implication on the spatial resolution of MIM. Finally, our results are verified by microwave impedance microscopy (MIM) measurement.

  18. Online quantitative phase imaging of vascular endothelial cells under fluid shear stress utilizing digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Odenthal-Schnittler, Maria; Schnittler, Hans Joachim; Kemper, Björn

    2016-03-01

    We have explored the utilization of quantitative phase imaging with digital holographic microscopy (DHM) as a novel tool for quantifying the dynamics of morphologic parameters (morphodynamics) of confluent endothelial cell layers under fluid shear stress conditions. Human umbilical vein endothelial cells (HUVECs) were exposed to fluid shear stress in a transparent cone/plate flow device (BioTech-Flow-System) and imaged with a modular setup for quantitative DHM phase imaging for up to 48 h. The resulting series of quantitative phase image sequences were analyzed for the average surface roughness of the cell layers and cell alignment. Our results demonstrate that quantitative phase imaging is a powerful and reliable tool to quantify the dynamics of morphological adaptation of endothelial cells to fluid shear stress.

  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. Exploring nanoscale electrical and electronic properties of organic and polymeric functional materials by atomic force microscopy based approaches.

    PubMed

    Palermo, Vincenzo; Liscio, Andrea; Palma, Matteo; Surin, Mathieu; Lazzaroni, Roberto; Samorì, Paolo

    2007-08-28

    Beyond imaging, atomic force microscopy (AFM) based methodologies enable the quantitative investigation of a variety of physico-chemical properties of (multicomponent) materials with a spatial resolution of a few nanometers. This Feature Article is focused on two AFM modes, i.e. conducting and Kelvin probe force microscopies, which allow the study of electrical and electronic properties of organic thin films, respectively. These nanotools provide a wealth of information on (dynamic) characteristics of tailor-made functional architectures, opening pathways towards their technological application in electronics, catalysis and medicine.

  1. Tunable PIE and synchronized gating detections by FastFLIM for quantitative microscopy measurements of fast dynamics of single molecules

    NASA Astrophysics Data System (ADS)

    Sun, Yuansheng; Coskun, Ulas; Ferreon, Allan Chris; Barbieri, Beniamino; Liao, Shih-Chu Jeff

    2016-03-01

    The crosstalk between two fluorescent species causes problems in fluorescence microscopy imaging, especially for quantitative measurements such as co-localization, Förster resonance energy transfer (FRET), fluorescence cross correlation spectroscopy (FCCS). In laser scanning confocal microscopy, the lasers can be switched on and off by acousto-optic tunable filters (AOTF) in the microsecond scale for alternative line scanning in order to avoid the crosstalk while minimizing the time delay between two lasers on the same pixel location. In contrast, the pulsed interleaved excitation (PIE) technique synchronizes two pulsed lasers of different wavelengths in the nanosecond scale to enable measuring superfast dynamics of two fluorescent species simultaneously and yet quantitatively without the crosstalk contamination. This feature is critical for many cell biology applications, e.g. accurate determination of stoichiometry in FRET measurements for studying protein-protein interactions or cell signal events, detection of weaker bindings in FCCS by eliminating the false cross correlation due to the crosstalk. The PIE has been used with the time correlated single photon counting (TCSPC) electronics. Here, we describe a novel PIE development using the digital frequency domain (DFD) technique -- FastFLIM, which provides tunable PIE setups and synchronized gating detections, tailored and optimized to specific applications. A few PIE setups by FastFLIM and measurement examples are described. Combined with the sensitivity of Alba and Q2 systems, the PIE allowed us to quantitatively measure the fast dynamics of single molecules.

  2. Combined scanning transmission X-ray and electron microscopy for the characterization of bacterial endospores.

    PubMed

    Jamroskovic, Jan; Shao, Paul P; Suvorova, Elena; Barak, Imrich; Bernier-Latmani, Rizlan

    2014-09-01

    Endospores (also referred to as bacterial spores) are bacterial structures formed by several bacterial species of the phylum Firmicutes. Spores form as a response to environmental stress. These structures exhibit remarkable resistance to harsh environmental conditions such as exposure to heat, desiccation, and chemical oxidants. The spores include several layers of protein and peptidoglycan that surround a core harboring DNA as well as high concentrations of calcium and dipicolinic acid (DPA). A combination of scanning transmission X-ray microscopy, scanning transmission electron microscopy, and energy dispersive spectroscopy was used for the direct quantitative characterization of bacterial spores. The concentration and localization of DPA, Ca(2+) , and other elements were determined and compared for the core and cortex of spores from two distinct genera: Bacillus subtilis and Desulfotomaculum reducens. This micro-spectroscopic approach is uniquely suited for the direct study of individual bacterial spores, while classical molecular and biochemical methods access only bulk characteristics.

  3. Microstructure-Sensitive Investigation of Fracture Using Acoustic Emission Coupled With Electron Microscopy

    NASA Technical Reports Server (NTRS)

    Wisner, Brian; Cabal, Mike; Vanniamparambiland, Prashanth A.; Leser, William; Hochhalter, Jacob; Kontsos, Antonios

    2015-01-01

    A novel technique using Scanning Electron Microscopy (SEM) in conjunction with Acoustic Emission (AE) monitoring is proposed to investigate microstructure-sensitive fatigue and fracture of metals. The coupling between quasi in situ microscopy with actual in situ nondestructive evaluation falls into the ICME framework and the idea of quantitative data-driven characterization of material behavior. To validate the use of AE monitoring inside the SEM chamber, Aluminum 2024-B sharp notch specimen were tested both inside and outside the microscope using a small scale mechanical testing device. Subsequently, the same type of specimen was tested inside the SEM chamber. Load data were correlated with both AE information and observations of microcracks around grain boundaries as well as secondary cracks, voids, and slip bands. The preliminary results are in excellent agreement with similar findings at the mesoscale. Extensions of the application of this novel technique are discussed.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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.

  10. 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

  11. Flipping interferometry and its application for quantitative phase microscopy in a micro-channel.

    PubMed

    Roitshtain, Darina; Turko, Nir A; Javidi, Bahram; Shaked, Natan T

    2016-05-15

    We present a portable, off-axis interferometric module for quantitative phase microscopy of live cells, positioned at the exit port of a coherently illuminated inverted microscope. The module creates on the digital camera an interference pattern between the image of the sample and its flipped version. The proposed simplified module is based on a retro-reflector modification in an external Michelson interferometer. The module does not contain any lenses, pinholes, or gratings and its alignment is straightforward. Still, it allows full control of the off-axis angle and does not suffer from ghost images. As experimentally demonstrated, the module is useful for quantitative phase microscopy of live cells rapidly flowing in a micro-channel. PMID:27177001

  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. 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.

  14. Simultaneous orientation and thickness mapping in transmission electron microscopy

    DOE PAGESBeta

    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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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.

  2. Advanced analytical electron microscopy for alkali-ion batteries

    DOE PAGESBeta

    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

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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

  9. 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

  10. Quantitative measurement of the nanoparticle size and number concentration from liquid suspensions by atomic force microscopy.

    PubMed

    Baalousha, M; Prasad, A; Lead, J R

    2014-05-01

    Microscopy techniques are indispensable to the nanoanalytical toolbox and can provide accurate information on the number size distribution and number concentration of nanoparticles (NPs) at low concentrations (ca. ppt to ppb range) and small sizes (ca. <20 nm). However, the high capabilities of microscopy techniques are limited by the traditional sample preparation based on drying a small volume of suspension of NPs on a microscopy substrate. This method is limited by low recovery of NPs (ca. <10%), formation of aggregates during the drying process, and thus, the complete misrepresentation of the NP suspensions under consideration. This paper presents a validated quantitative sampling technique for atomic force microscopy (AFM) that overcomes the above-mentioned shortcomings and allows full recovery and representativeness of the NPs under consideration by forcing the NPs into the substrate via ultracentrifugation and strongly attaches the NPs to the substrate by surface functionalization of the substrate or by adding cations to the NP suspension. The high efficiency of the analysis is demonstrated by the uniformity of the NP distribution on the substrate (that is low variability between the number of NPs counted on different images on different areas of the substrate), the high recovery of the NPs up to 71%) and the good correlation (R > 0.95) between the mass and number concentrations. Therefore, for the first time, we developed a validated quantitative sampling technique that enables the use of the full capabilities of microscopy tools to quantitatively and accurately determine the number size distribution and number concentration of NPs at environmentally relevant low concentrations (i.e. 0.34-100 ppb). This approach is of high environmental relevance and can be applied widely in environmental nanoscience and nanotoxicology for (i) measuring the number concentration dose in nanotoxicological studies and (ii) accurately measuring the number size distribution of

  11. Quantitative measurement of in-plane cantilever torsion for calibrating lateral piezoresponse force microscopy.

    SciTech Connect

    Choi, H.; Hong, S.; No, K.

    2011-01-01

    A simple quantitative measurement procedure of in-plane cantilever torsion for calibrating lateral piezoresponse force microscopy is presented. This technique enables one to determine the corresponding lateral inverse optical lever sensitivity (LIOLS) of the cantilever on the given sample. Piezoelectric coefficient, d{sub 31} of BaTiO{sub 3} single crystal (-81.62 {+-} 40.22 pm/V) which was calculated using the estimated LIOLS was in good agreement with the reported value in literature.

  12. Refractive-index profiling of optical fibers with axial symmetry by use of quantitative phase microscopy.

    PubMed

    Roberts, A; Ampem-Lassen, E; Barty, A; Nugent, K A; Baxter, G W; Dragomir, N M; Huntington, S T

    2002-12-01

    The application of quantitative phase microscopy to refractive-index profiling of optical fibers is demonstrated. Phase images of axially symmetric optical fibers immersed in index-matching fluid are obtained, and the inverse Abel transform is used to obtain the radial refractive-index profile. This technique is straightforward, nondestructive, repeatable, and accurate. Excellent agreement, to within approximately 0.0005, between this method and the index profile obtained with a commercial profiler is obtained.

  13. Quantitating therapeutic disruption of tumor blood flow with intravital video microscopy.

    PubMed

    Iga, Arthur M; Sarkar, Sandip; Sales, Kevin M; Winslet, Marc C; Seifalian, Alexander M

    2006-12-15

    Vascular-disrupting agents (VDA) kill tumor cells by selectively disrupting blood circulation in tumors. In vivo analysis of this intensely studied class of anticancer agents is invaluable for preclinical assessment of pharmacodynamic end points and effective therapeutic windows. In this review, we consider the role of intravital video microscopy in measuring tumor vascular response to VDAs, the potential of which lies in the opportunity to quantitate specific variables and to obtain real-time information on how VDAs affect tumor microcirculation.

  14. Analysis of mixed cell cultures with quantitative digital holographic phase microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Wibbeling, Jana; Ketelhut, Steffi

    2014-05-01

    In order to study, for example, the influence of pharmaceuticals or pathogens on different cell types under identical measurement conditions and to analyze interactions between different cellular specimens a minimally-invasive quantitative observation of mixed cell cultures is of particular interest. Quantitative phase microscopy (QPM) provides high resolution detection of optical path length changes that is suitable for stain-free minimally-invasive live cell analysis. Due to low light intensities for object illumination, QPM minimizes the interaction with the sample and is in particular suitable for long term time-lapse investigations, e.g., for the detection of cell morphology alterations due to drugs and toxins. Furthermore, QPM has been demonstrated to be a versatile tool for the quantification of cellular growth, the extraction morphological parameters and cell motility. We studied the feasibility of QPM for the analysis of mixed cell cultures. It was explored if quantitative phase images provide sufficient information to distinguish between different cell types and to extract cell specific parameters. For the experiments quantitative phase imaging with digital holographic microscopy (DHM) was utilized. Mixed cell cultures with different types of human pancreatic tumor cells were observed with quantitative DHM phase contrast up to 35 h. The obtained series of quantitative phase images were evaluated by adapted algorithms for image segmentation. From the segmented images the cellular dry mass and the mean cell thickness were calculated and used in the further analysis as parameters to quantify the reliability the measurement principle. The obtained results demonstrate that it is possible to characterize the growth of cell types with different morphologies in a mixed cell culture separately by consideration of specimen size and cell thickness in the evaluation of quantitative DHM phase images.

  15. Quantitative imaging of electrospun fibers by PeakForce Quantitative NanoMechanics atomic force microscopy using etched scanning probes.

    PubMed

    Chlanda, Adrian; Rebis, Janusz; Kijeńska, Ewa; Wozniak, Michal J; Rozniatowski, Krzysztof; Swieszkowski, Wojciech; Kurzydlowski, Krzysztof J

    2015-05-01

    Electrospun polymeric submicron and nanofibers can be used as tissue engineering scaffolds in regenerative medicine. In physiological conditions fibers are subjected to stresses and strains from the surrounding biological environment. Such stresses can cause permanent deformation or even failure to their structure. Therefore, there is a growing necessity to characterize their mechanical properties, especially at the nanoscale. Atomic force microscopy is a powerful tool for the visualization and probing of selected mechanical properties of materials in biomedical sciences. Image resolution of atomic force microscopy techniques depends on the equipment quality and shape of the scanning probe. The probe radius and aspect ratio has huge impact on the quality of measurement. In the presented work the nanomechanical properties of four different polymer based electrospun fibers were tested using PeakForce Quantitative NanoMechanics atomic force microscopy, with standard and modified scanning probes. Standard, commercially available probes have been modified by etching using focused ion beam (FIB). Results have shown that modified probes can be used for mechanical properties mapping of biomaterial in the nanoscale, and generate nanomechanical information where conventional tips fail.

  16. 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

  17. Quantitative aspects of digital microscopy applied to cellular localization of heparin in smooth muscle cells

    NASA Astrophysics Data System (ADS)

    Johnston, Richard F.; Hanzel, David K.; Stack, Bob; Brandley, Brian; Castellot, John

    1995-04-01

    High Resolution digital acquisition allows a great deal of flexibility in the types of questions that can be directed to microscopic samples. To eliminate subjective bias and provide quantitative results we have approached microscopy with an automated digital format. This mode can return quantitative data at high resolution over large fields. The digital format makes accessible data including [data segmentation]: multispectral colocalization, seeding and connectivity, particle size and shape distribution and population analysis. We have begun a program to investigate this approach using the confocal microscope. Scanning larger fields-of-view at lower spatial resolutions (e.g., low magnification objective) defines large maps that allow alignment of high spatial resolution (diffraction limited) sampling. The [objective] selection of the field-of-view with low spatial resolution reduces the subjective nature of the selection of a 'typical staining pattern'. High resolution digital scanning in three dimensions contribute both to the 'objective' nature of the analysis and allow for quantitation of characteristics not historically available/accessible. The complex carbohydrate heparin is implicated in tumor growth and wound healing by affecting angiogenesis, cell proliferation and motility. The internal localization of heparin within vascular cells appears to be a good predictor of the sensitivity of those cells to the action of heparin. Cells resistant to the antiproliferative action of heparin are able to sequester the heparin in large vacuoles whereas those cells sensitive to the carbohydrate do not exhibit these structures. We have applied our approach to QUANTITATIVE DIGITAL MICROSCOPY to the analysis of intracellular heparin distribution.

  18. 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

  19. 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.

  20. 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.

  1. 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.

  2. 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.

  3. [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

  4. 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.

  5. 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.

  6. 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.

  7. 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.

  8. 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).

  9. Advances in quantitative nanoscale subsurface imaging by mode-synthesizing atomic force microscopy

    SciTech Connect

    Vitry, P.; Bourillot, E.; Plassard, C.; Lacroute, Y.; Lesniewska, E.; Tetard, L.

    2014-08-04

    This paper reports on advances toward quantitative non-destructive nanoscale subsurface investigation of a nanofabricated sample based on mode synthesizing atomic force microscopy with heterodyne detection, addressing the need to correlate the role of actuation frequencies of the probe f{sub p} and the sample f{sub s} with depth resolution for 3D tomography reconstruction. Here, by developing a simple model and validating the approach experimentally through the study of the nanofabricated calibration depth samples consisting of buried metallic patterns, we demonstrate avenues for quantitative nanoscale subsurface imaging. Our findings enable the reconstruction of the sample depth profile and allow high fidelity resolution of the buried nanostructures. Non-destructive quantitative nanoscale subsurface imaging offers great promise in the study of the structures and properties of complex systems at the nanoscale.

  10. Quantitative vibrational imaging by hyperspectral stimulated Raman scattering microscopy and multivariate curve resolution analysis.

    PubMed

    Zhang, Delong; Wang, Ping; Slipchenko, Mikhail N; Ben-Amotz, Dor; Weiner, Andrew M; Cheng, Ji-Xin

    2013-01-01

    Spectroscopic imaging has been an increasingly critical approach for unveiling specific molecules in biological environments. Toward this goal, we demonstrate hyperspectral stimulated Raman loss (SRL) imaging by intrapulse spectral scanning through a femtosecond pulse shaper. The hyperspectral stack of SRL images is further analyzed by a multivariate curve resolution (MCR) method to reconstruct quantitative concentration images for each individual component and retrieve the corresponding vibrational Raman spectra. Using these methods, we demonstrate quantitative mapping of dimethyl sulfoxide concentration in aqueous solutions and in fat tissue. Moreover, MCR is performed on SRL images of breast cancer cells to generate maps of principal chemical components along with their respective vibrational spectra. These results show the great capability and potential of hyperspectral SRL microscopy for quantitative imaging of complicated biomolecule mixtures through resolving overlapped Raman bands.

  11. 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.

  12. 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.

  13. Segmentation of vascular structures and hematopoietic cells in 3D microscopy images and quantitative analysis

    NASA Astrophysics Data System (ADS)

    Mu, Jian; Yang, Lin; Kamocka, Malgorzata M.; Zollman, Amy L.; Carlesso, Nadia; Chen, Danny Z.

    2015-03-01

    In this paper, we present image processing methods for quantitative study of how the bone marrow microenvironment changes (characterized by altered vascular structure and hematopoietic cell distribution) caused by diseases or various factors. We develop algorithms that automatically segment vascular structures and hematopoietic cells in 3-D microscopy images, perform quantitative analysis of the properties of the segmented vascular structures and cells, and examine how such properties change. In processing images, we apply local thresholding to segment vessels, and add post-processing steps to deal with imaging artifacts. We propose an improved watershed algorithm that relies on both intensity and shape information and can separate multiple overlapping cells better than common watershed methods. We then quantitatively compute various features of the vascular structures and hematopoietic cells, such as the branches and sizes of vessels and the distribution of cells. In analyzing vascular properties, we provide algorithms for pruning fake vessel segments and branches based on vessel skeletons. Our algorithms can segment vascular structures and hematopoietic cells with good quality. We use our methods to quantitatively examine the changes in the bone marrow microenvironment caused by the deletion of Notch pathway. Our quantitative analysis reveals property changes in samples with deleted Notch pathway. Our tool is useful for biologists to quantitatively measure changes in the bone marrow microenvironment, for developing possible therapeutic strategies to help the bone marrow microenvironment recovery.

  14. 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.

  15. Detector non-uniformity in scanning transmission electron microscopy.

    PubMed

    Findlay, S D; LeBeau, J M

    2013-01-01

    A non-uniform response across scanning transmission electron microscope annular detectors has been found experimentally, but is seldom incorporated into simulations. Through case study simulations, we establish the nature and scale of the discrepancies which may arise from failing to account for detector non-uniformity. If standard detectors are used at long camera lengths such that the detector is within or near to the bright field region, we find errors in contrast of the order of 10%, sufficiently small for qualitative work but non-trivial as experiments become more quantitative. In cases where the detector has been characterized in advance, we discuss the detector response normalization and how it may be incorporated in simulations.

  16. Electron microscopy of an aluminum layer grown on the vicinal surface of a gallium arsenide substrate

    SciTech Connect

    Lovygin, M. V. Borgardt, N. I.; Kazakov, I. P.; Seibt, M.

    2015-03-15

    A thin Al layer grown by molecular-beam epitaxy on a misoriented GaAs (100) substrate is studied by transmission electron microscopy. Electron diffraction data and bright-field, dark-field, and high-resolution images show that, in the layer, there are Al grains of three types of crystallographic orientation: Al (100), Al (110), and Al (110)R. The specific structural features of the interfaces between the differently oriented grains and substrate are studied by digital processing of the high-resolution images. From quantitative analysis of the dark-field images, the relative content and sizes of the differently oriented grains are determined. It is found that atomic steps at the substrate surface cause an increase in the fraction and sizes of Al (110)R grains and a decrease in the fraction of Al (100) grains, compared to the corresponding fractions and sizes in the layer grown on a singular substrate surface.

  17. 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.

  18. 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

  19. 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

  20. 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.

  1. Imaging and quantitative data acquisition of biological cell walls with Atomic Force Microscopy and Scanning Acoustic Microscopy

    SciTech Connect

    Tittmann, B. R.; Xi, X.

    2014-09-01

    This chapter demonstrates the feasibility of Atomic Force Microscopy (AFM) and High Frequency Scanning Acoustic Microscopy (HF-SAM) as tools to characterize biological tissues. Both the AFM and the SAM have shown to provide imaging (with different resolution) and quantitative elasticity measuring abilities. Plant cell walls with minimal disturbance and under conditions of their native state have been examined with these two kinds of microscopy. After descriptions of both the SAM and AFM, their special features and the typical sample preparation is discussed. The sample preparation is focused here on epidermal peels of onion scales and celery epidermis cells which were sectioned for the AFM to visualize the inner surface (closest to the plasma membrane) of the outer epidermal wall. The nm-wide cellulose microfibrils orientation and multilayer structure were clearly observed. The microfibril orientation and alignment tend to be more organized in older scales compared with younger scales. The onion epidermis cell wall was also used as a test analog to study cell wall elasticity by the AFM nanoindentation and the SAM V(z) feature. The novelty in this work was to demonstrate the capability of these two techniques to analyze isolated, single layered plant cell walls in their natural state. AFM nanoindentation was also used to probe the effects of Ethylenediaminetetraacetic acid (EDTA), and calcium ion treatment to modify pectin networks in cell walls. The results suggest a significant modulus increase in the calcium ion treatment and a slight decrease in EDTA treatment. To complement the AFM measurements, the HF-SAM was used to obtain the V(z) signatures of the onion epidermis. These measurements were focused on documenting the effect of pectinase enzyme treatment. The results indicate a significant change in the V(z) signature curves with time into the enzyme treatment. Thus AFM and HF-SAM open the door to a systematic nondestructive structure and mechanical property

  2. 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.

  3. 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.

  4. 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.

  5. Low-energy electron scattering in carbon-based materials analyzed by scanning transmission electron microscopy and its application to sample thickness determination.

    PubMed

    Pfaff, M; Müller, E; Klein, M F G; Colsmann, A; Lemmer, U; Krzyzanek, V; Reichelt, R; Gerthsen, D

    2011-07-01

    High-angle annular dark-field scanning transmission electron microscopy (HAADF STEM) at low energies (≤30 keV) was used to study quantitatively electron scattering in amorphous carbon and carbon-based materials. Experimental HAADF STEM intensities from samples with well-known composition and thickness are compared with results of Monte Carlo simulations and semiempirical equations describing multiple electron scattering. A well-defined relationship is found between the maximum HAADF STEM intensity and sample thickness which is exploited (a) to derive a quantitative description for the mean quadratic scattering angle and (b) to calculate the transmitted HAADF STEM intensity as a function of the relevant materials parameters and electron energy. The formalism can be also applied to determine TEM sample thicknesses by minimizing the contrast of the sample as a function of the electron energy.

  6. Quantitative Thermal Microscopy Measurement with Thermal Probe Driven by dc+ac Current

    NASA Astrophysics Data System (ADS)

    Bodzenta, Jerzy; Juszczyk, Justyna; Kaźmierczak-Bałata, Anna; Firek, Piotr; Fleming, Austin; Chirtoc, Mihai

    2016-07-01

    Quantitative thermal measurements with spatial resolution allowing the examination of objects of submicron dimensions are still a challenging task. The quantity of methods providing spatial resolution better than 100 nm is very limited. One of them is scanning thermal microscopy (SThM). This method is a variant of atomic force microscopy which uses a probe equipped with a temperature sensor near the apex. Depending on the sensor current, either the temperature or the thermal conductivity distribution at the sample surface can be measured. However, like all microscopy methods, the SThM gives only qualitative information. Quantitative measuring methods using SThM equipment are still under development. In this paper, a method based on simultaneous registration of the static and the dynamic electrical resistances of the probe driven by the sum of dc and ac currents, and examples of its applications are described. Special attention is paid to the investigation of thin films deposited on thick substrates. The influence of substrate thermal properties on the measured signal and its dependence on thin film thermal conductivity and film thickness are analyzed. It is shown that in the case where layer thicknesses are comparable or smaller than the probe-sample contact diameter, a correction procedure is required to obtain actual thermal conductivity of the layer. Experimental results obtained for thin SiO2 and BaTiO_{3 }layers with thicknesses in the range from 11 nm to 100 nm are correctly confirmed with this approach.

  7. Quantitative Lifetime Unmixing of Multiexponentially Decaying Fluorophores Using Single-Frequency Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Kremers, Gert-Jan; van Munster, Erik B.; Goedhart, Joachim; Gadella, Theodorus W. J.

    2008-01-01

    Fluorescence lifetime imaging microscopy (FLIM) is a quantitative microscopy technique for imaging nanosecond decay times of fluorophores. In the case of frequency-domain FLIM, several methods have been described to resolve the relative abundance of two fluorescent species with different fluorescence decay times. Thus far, single-frequency FLIM methods generally have been limited to quantifying two species with monoexponential decay. However, multiexponential decays are the norm rather than the exception, especially for fluorescent proteins and biological samples. Here, we describe a novel method for determining the fractional contribution in each pixel of an image of a sample containing two (multiexponentially) decaying species using single-frequency FLIM. We demonstrate that this technique allows the unmixing of binary mixtures of two spectrally identical cyan or green fluorescent proteins, each with multiexponential decay. Furthermore, because of their spectral identity, quantitative images of the relative molecular abundance of these fluorescent proteins can be generated that are independent of the microscope light path. The method is rigorously tested using samples of known composition and applied to live cell microscopy using cells expressing multiple (multiexponentially decaying) fluorescent proteins. PMID:18359789

  8. Digital Holographic Microscopy: Quantitative Phase Imaging and Applications in Live Cell Analysis

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Langehanenberg, Patrik; Kosmeier, Sebastian; Schlichthaber, Frank; Remmersmann, Christian; von Bally, Gert; Rommel, Christina; Dierker, Christian; Schnekenburger, Jürgen

    The analysis of complex processes in living cells creates a high demand for fast and label-free methods for online monitoring. Widely used fluorescence methods require specific labeling and are often restricted to chemically fixated samples. Thus, methods that offer label-free and minimally invasive detection of live cell processes and cell state alterations are of particular interest. In combination with light microscopy, digital holography provides label-free, multi-focus quantitative phase imaging of living cells. In overview, several methods for digital holographic microscopy (DHM) are presented. First, different experimental setups for the recording of digital holograms and the modular integration of DHM into common microscopes are described. Then the numerical processing of digitally captured holograms is explained. This includes the description of spatial and temporal phase shifting techniques, spatial filtering based reconstruction, holographic autofocusing, and the evaluation of self-interference holograms. Furthermore, the usage of partial coherent light and multi-wavelength approaches is discussed. Finally, potentials of digital holographic microscopy for quantitative cell imaging are illustrated by results from selected applications. It is shown that DHM can be used for automated tracking of migrating cells and cell thickness monitoring as well as for refractive index determination of cells and particles. Moreover, the use of DHM for label-free analysis in fluidics and micro-injection monitoring is demonstrated. The results show that DHM is a highly relevant method that allows novel insights in dynamic cell biology, with applications in cancer research and for drugs and toxicity testing.

  9. 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.

  10. 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

  11. Scanning transmission and computer-aided volumic electron microscopy: 3-D modeling of entire cells by electronic imaging

    NASA Astrophysics Data System (ADS)

    Bron, Christophe; Gremillet, Philip; Launay, D.; Jourlin, Michel; Gautschi, H. P.; Baechi, Thomas; Schuepbach, Joerg

    1990-05-01

    The digital processing of electron microscopic images from serial sections containing laser-induced topographical references allows a 3-D reconstruction at a depth resolution of 30 to 40 nm of entire cells by the use of image analysis methods, as already demonstrated for Transmission Electron Microscopy (TEM) coupled with a video camera. We decided to use a Scanning Transmission Electron Microscope (STEM) to get higher contrast and better resolution at medium magnification. The scanning of our specimens at video frequencies is an attractive and easy way to link a STEM with an image processing system but the hysteresis of the electronic spools responsible for the magnetic deviation of the scanning electron beam induces deformations of images which have to be modelized and corrected before registration. Computer algorithms developed for image analysis and treatment correct the artifacts caused by the use of STEM and by serial sectioning to automatically reconstruct the third dimension of the cells. They permit the normalization of the images through logarithmic processing of the original grey level infonnation. The automatic extraction of cell limits allows to link the image analysis and treatments with image synthesis methods by minimal human intervention. The surface representation and the registered images provide an ultrastructural data base from which quantitative 3-D morphological parameters, as well as otherwise impossible visualizations, can be computed. This 3-D image processing named C.A.V.U.M. for Computer Aided Volumic Ultra-Microscopy offers a new tool for the documentation and analysis of cell ultrastructure and for 3-D morphometric studies at EM magnifications. Further, a virtual observer can be computed in such a way as to simulate a visit of the reconstructed object.

  12. 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...

  13. Confocal reflectance quantitative phase microscopy system for cell biology studies (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Singh, Vijay Raj; So, Peter T. C.

    2016-03-01

    Quantitative phase microscopy (QPM), used to measure the refractive index, provides the optical path delay measurement at each point of the specimen under study and becomes an active field in biological science. In this work we present development of confocal reflection phase microscopy system to provide depth resolved quantitative phase information for investigation of intracellular structures and other biological specimen. The system hardware development is mainly divided into two major parts. First, creates a pinhole array for parallel confocal imaging of specimen at multiple locations simultaneously. Here a digital micro mirror device (DMD) is used to generate pinhole array by turning on a subset micro-mirrors arranged on a grid. Second is the detection of phase information of confocal imaging foci by using a common path interferometer. With this novel approach, it is possible to measure the nuclei membrane fluctuations and distinguish them from the plasma membrane fluctuations. Further, depth resolved quantitative phase can be correlated to the intracellular contents and 3D map of refractive index measurements.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. 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.

  14. 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

  15. 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

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. 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.

  4. Quantitative tracking of tumor cells in phase-contrast microscopy exploiting halo artifact pattern

    NASA Astrophysics Data System (ADS)

    Kang, Mi-Sun; Song, Soo-Min; Lee, Hana; Kim, Myoung-Hee

    2012-03-01

    Tumor cell morphology is closely related to its invasiveness characteristics and migratory behaviors. An invasive tumor cell has a highly irregular shape, whereas a spherical cell is non-metastatic. Thus, quantitative analysis of cell features is crucial to determine tumor malignancy or to test the efficacy of anticancer treatment. We use phase-contrast microscopy to analyze single cell morphology and to monitor its change because it enables observation of long-term activity of living cells without photobleaching and phototoxicity, which is common in other fluorescence-labeled microscopy. Despite this advantage, there are image-level drawbacks to phase-contrast microscopy, such as local light effect and contrast interference ring, among others. Thus, we first applied a local filter to compensate for non-uniform illumination. Then, we used intensity distribution information to detect the cell boundary. In phase-contrast microscopy images, the cell normally appears as a dark region surrounded by a bright halo. As the halo artifact around the cell body is minimal and has an asymmetric diffusion pattern, we calculated the cross-sectional plane that intersected the center of each cell and was orthogonal to the first principal axis. Then, we extracted the dark cell region by level set. However, a dense population of cultured cells still rendered single-cell analysis difficult. Finally, we measured roundness and size to classify tumor cells into malignant and benign groups. We validated segmentation accuracy by comparing our findings with manually obtained results.

  5. Quantitative 3D molecular cutaneous absorption in human skin using label free nonlinear microscopy.

    PubMed

    Chen, Xueqin; Grégoire, Sébastien; Formanek, Florian; Galey, Jean-Baptiste; Rigneault, Hervé

    2015-02-28

    Understanding the penetration mechanisms of drugs into human skin is a key issue in pharmaceutical and cosmetics research. To date, the techniques available for percutaneous penetration of compounds fail to provide a quantitative 3D map of molecular concentration distribution in complex tissues as the detected microscopy images are an intricate combination of concentration distribution and laser beam attenuation upon deep penetration. Here we introduce and validate a novel framework for imaging and reconstructing molecular concentration within the depth of artificial and human skin samples. Our approach combines the use of deuterated molecular compounds together with coherent anti-Stokes Raman scattering spectroscopy and microscopy that permits targeted molecules to be unambiguously discriminated within skin layers. We demonstrate both intercellular and transcellular pathways for different active compounds, together with in-depth concentration profiles reflecting the detailed skin barrier architecture. This method provides an enabling platform for establishing functional activity of topically applied products. PMID:25550155

  6. Deformation of nanotubes in peeling contact with flat substrate: An in situ electron microscopy nanomechanical study

    NASA Astrophysics Data System (ADS)

    Chen, Xiaoming; Zheng, Meng; Wei, Qing; Signetti, Stefano; Pugno, Nicola M.; Ke, Changhong

    2016-04-01

    Peeling of one-dimensional (1D) nanostructures from flat substrates is an essential technique in studying their adhesion properties. The mechanical deformation of the nanostructure in the peeling experiment is critical to the understanding of the peeling process and the interpretation of the peeling measurements, but it is challenging to measure directly and quantitatively at the nanoscale. Here, we investigate the peeling deformation of a bundled carbon nanotube (CNT) fiber by using an in situ scanning electron microscopy nanomechanical peeling technique. A pre-calibrated atomic force microscopy cantilever is utilized as the peeling force sensor, and its back surface acts as the peeling contact substrate. The nanomechanical peeling scheme enables a quantitative characterization of the deformational behaviors of the CNT fiber in both positive and negative peeling configurations with sub-10 nm spatial and sub-nN force resolutions. Nonlinear continuum mechanics models and finite element simulations are employed to interpret the peeling measurements. The measurements and analysis reveal that the structural imperfections in the CNT fiber may have a substantial influence on its peeling deformations and the corresponding peeling forces. The research findings reported in this work are useful to the study of mechanical and adhesion properties of 1D nanostructures by using nanomechanical peeling techniques.

  7. 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.

  8. 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.

  9. 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

  10. Quantitative phase imaging by single-shot Hilbert-Huang phase microscopy.

    PubMed

    Trusiak, Maciej; Mico, Vicente; Garcia, Javier; Patorski, Krzysztof

    2016-09-15

    We propose a novel single-shot Hilbert-Huang transform-based algorithm applied to digital holographic microscopy (DHM) for robust, fast, and accurate single-shot quantitative phase imaging in on-axis and off-axis configurations. Fringe pattern with possible defects and closed fringes are adaptively filtered and accurately phase demodulated using local fringe direction estimation. Experimental validation of the proposed techniques is presented as the DHM study of microbeads and red blood cells phase samples. Obtained results compare very favorably with the Fourier approach (off-axis) and temporal phase shifting (on-axis). PMID:27628393

  11. Quantitative phase and refractive index measurements with point-source digital in-line holographic microscopy.

    PubMed

    Jericho, M H; Kreuzer, H J; Kanka, M; Riesenberg, R

    2012-04-01

    Point-source digital in-line holographic microscopy with numerical reconstruction is ideally suited for quantitative phase measurements to determine optical path lengths and to extract changes in refractive index within accuracy close to 0.001 on the submicrometer length scale. This is demonstrated with simulated holograms and with detailed measurements on a number of different micrometer-sized samples such as suspended drops, optical fibers, as well as organisms of biological interest such as E. coli bacteria, HeLa cells, and fibroblast cells.

  12. Quantitative detection of chemical compounds in human hair with coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Zimmerley, Maxwell; Lin, Chia-Yu; Oertel, David C.; Marsh, Jennifer M.; Ward, Jimmie L.; Potma, Eric Olaf

    2009-07-01

    Coherent anti-Stokes Raman scattering (CARS) microscopy is used to determine the distribution and concentration of selected compounds in intact human hair. By generating images based on ratiometric CARS contrast, quantitative concentration maps of both water and externally applied d-glycine are produced in the cortex of human hair fibers. Both water and d-glycine are found to homogeneously distribute throughout the cortical regions of the hair. The ability to selectively detect molecular agents in hair fibers is of direct relevance to understanding the chemical and physical mechanisms that underlie the performance of hair-care products.

  13. Spatially resolved quantitative mapping of thermomechanical properties and phase transition temperatures using scanning probe microscopy

    DOEpatents

    Jesse, Stephen; Kalinin, Sergei V; Nikiforov, Maxim P

    2013-07-09

    An approach for the thermomechanical characterization of phase transitions in polymeric materials (polyethyleneterephthalate) by band excitation acoustic force microscopy is developed. This methodology allows the independent measurement of resonance frequency, Q factor, and oscillation amplitude of a tip-surface contact area as a function of tip temperature, from which the thermal evolution of tip-surface spring constant and mechanical dissipation can be extracted. A heating protocol maintained a constant tip-surface contact area and constant contact force, thereby allowing for reproducible measurements and quantitative extraction of material properties including temperature dependence of indentation-based elastic and loss moduli.

  14. Quantitative analysis on collagen morphology in aging skin based on multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Shulian; Li, Hui; Yang, Hongqin; Zhang, Xiaoman; Li, Zhifang; Xu, Shufei

    2011-04-01

    Multiphoton microscopy was employed for monitoring the structure changes of mouse dermis collagen in the intrinsic- or the extrinsic-age-related processes in vivo. The characteristics of textures in different aging skins were uncovered by fast Fourier transform in which the orientation index and bundle packing of collagen were quantitatively analyzed. Some significant differences in collagen-related changes are found in different aging skins, which can be good indicators for the statuses of aging skins. The results are valuable to the study of aging skin and also of interest to biomedical photonics.

  15. 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

  16. Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy

    PubMed Central

    Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

    2016-01-01

    Local surface charge density of lipid membranes influences membrane–protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values. PMID:27561322

  17. New approaches for the analysis of confluent cell layers with quantitative phase digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Pohl, L.; Kaiser, M.; Ketelhut, S.; Pereira, S.; Goycoolea, F.; Kemper, Björn

    2016-03-01

    Digital holographic microscopy (DHM) enables high resolution non-destructive inspection of technical surfaces and minimally-invasive label-free live cell imaging. However, the analysis of confluent cell layers represents a challenge as quantitative DHM phase images in this case do not provide sufficient information for image segmentation, determination of the cellular dry mass or calculation of the cell thickness. We present novel strategies for the analysis of confluent cell layers with quantitative DHM phase contrast utilizing a histogram based-evaluation procedure. The applicability of our approach is illustrated by quantification of drug induced cell morphology changes and it is shown that the method is capable to quantify reliable global morphology changes of confluent cell layers.

  18. Mapping surface charge density of lipid bilayers by quantitative surface conductivity microscopy.

    PubMed

    Klausen, Lasse Hyldgaard; Fuhs, Thomas; Dong, Mingdong

    2016-01-01

    Local surface charge density of lipid membranes influences membrane-protein interactions leading to distinct functions in all living cells, and it is a vital parameter in understanding membrane-binding mechanisms, liposome design and drug delivery. Despite the significance, no method has so far been capable of mapping surface charge densities under physiologically relevant conditions. Here, we use a scanning nanopipette setup (scanning ion-conductance microscope) combined with a novel algorithm to investigate the surface conductivity near supported lipid bilayers, and we present a new approach, quantitative surface conductivity microscopy (QSCM), capable of mapping surface charge density with high-quantitative precision and nanoscale resolution. The method is validated through an extensive theoretical analysis of the ionic current at the nanopipette tip, and we demonstrate the capacity of QSCM by mapping the surface charge density of model cationic, anionic and zwitterionic lipids with results accurately matching theoretical values. PMID:27561322

  19. Quantitative determination of local potential values in inhomogeneously doped semiconductors by scanning tunneling microscopy

    NASA Astrophysics Data System (ADS)

    Weidlich, P. H.; Dunin-Borkowski, R. E.; Ebert, Ph.

    2011-08-01

    Local potential changes arising from nanoscale three-dimensional spatial fluctuations in the dopant distribution in Zn-doped GaAs were investigated quantitatively by scanning tunneling microscopy and spectroscopy at (110) cleavage surfaces. Tunneling spectra measured in areas with different local doping concentration show apparent shifts of the valence band edge and apparent changes of the band gap. A quantitative analysis, combined with band bending and tunnel current simulations, demonstrates that these effects arise from tip-induced band bending that modulates the real potential changes. It is illustrated how exact potential changes between locally high and low doped areas can be determined. It is found that the local potential fluctuations in three-dimensionally doped semiconductors are approximately one order of magnitude smaller that those observed in two-dimensionally doped semiconductors.

  20. Quantitative lateral and vertical piezoresponse force microscopy on a PbTiO3 single crystal

    NASA Astrophysics Data System (ADS)

    Lei, Shiming; Koo, Tae-Yeong; Cao, Wenwu; Eliseev, Eugene A.; Morozovska, Anna N.; Cheong, S.-W.; Gopalan, Venkatraman

    2016-09-01

    Piezoresponse force microscopy (PFM) has emerged as a powerful tool for research in ferroelectric and piezoelectric materials. While the vertical PFM (VPFM) mode is well understood and applied at a quantitative level, the lateral PFM (LPFM) mode is rarely quantified, mainly due to the lack of a practical calibration methodology. Here by PFM imaging on a LiNbO3 180° domain wall, we demonstrate a convenient way to achieve simultaneous VPFM and LPFM calibrations. Using these calibrations, we perform a full quantitative VPFM and LPFM measurement on a (001)-cut PbTiO3 single crystal. The measured effective piezoelectric coefficients d33 e f f and d35 e f f together naturally provide more information on a material's local tensorial electromechanical properties. The proposed approach can be applied to a wide variety of ferroelectric and piezoelectric systems.