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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. Determination of the mass of viruses by quantitative electron microscopy.

    PubMed

    Bahr, G F; Engler, W F; Mazzone, H M

    1976-11-01

    The photometric method of quantitative determination of dry mass by electron microscopy has been applied to the study of various types of viruses: animal, plant, insect, and bacterial. The method is applicable to all viruses having a mass of 1 x 10-18g or greater. The molecular weight of viruses can be calculated from the mass value by multiplying it by Avogadro's number. In comparison to other methods of determining the molecular weight of viruses, sedimentation and diffusion, sedimentation equilibrium, light scattering, and electron microscopy counting, the method of quantitative electron microscopy is competitive. In some ways quantitative electron microscopy is superior to other methods for the determination of molecular weight: There is no limitation to the size of the virus, no experimental time involved and no concentration and purity of virus preparations required, and finally it is independent of the geometry of the virion. In one important aspect it is unique when compared to other methods; namely, it affords one the capacity to analyse individual virus particles. PMID:189345

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

    PubMed

    Krause, Florian F; Schowalter, Marco; Grieb, Tim; Mller-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. PMID:26686661

  4. Quantitative analysis of mouse corpus callosum from electron microscopy images.

    PubMed

    West, Kathryn L; Kelm, Nathaniel D; Carson, Robert P; Does, Mark D

    2015-12-01

    This article provides morphometric analysis of 72 electron microscopy images from control (n=4) and hypomyelinated (n=2) mouse corpus callosum. Measures of axon diameter and g-ratio were tabulated across all brains from two regions of the corpus callosum and a non-linear relationship between axon diameter and g-ratio was observed. These data are related to the accompanying research article comparing multiple methods of measuring g-ratio entitled 'A revised model for estimating g-ratio from MRI' (West et al., NeuroImage, 2015). PMID:26504893

  5. 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. PMID:24643324

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

    SciTech Connect

    Abellan Baeza, Patricia; Woehl, Taylor J.; Parent, Lucas R.; Browning, Nigel D.; Evans, James E.; Arslan, Ilke

    2014-04-15

    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.

  7. Quantitative intercomparison of transmission electron microscopy, flow cytometry, and epifluorescence microscopy for nanometric particle analysis.

    PubMed

    Ferris, Matthew M; Stoffel, Carrie L; Maurer, Thain T; Rowlen, Kathy L

    2002-05-15

    Nanometric biological particles such as viruses have received increased attention in a wide range of scientific fields. Evaluation of viral contributions to environmental processes and the use of viruses in medical applications such as gene therapy require viruses to be routinely and accurately enumerated. There are a variety of existing techniques for counting viruses, namely, plaque assays, transmission electron microscopy (TEM), epifluorescence microscopy (EFM), and flow cytometry (FCM); each has advantages and disadvantages. While there have been attempts to intercompare some of these techniques to determine the most effective means to count viruses, no previous study used a technique-independent standard for quantitative comparison of collection efficiency, accuracy, and precision. In this work, polystyrene nanospheres were used as standards for the intercomparison of performance characteristics for TEM, EFM, FCM, as well as a custom-built flow cytometer (the Single Nanometric Particle Enumerator, SNaPE). EFM and SNaPE exhibited the highest degree of accuracy and precision, with particle concentrations deviating < or =5% from true and relative errors less than half that of TEM, EFM and SNaPE are also significantly more time and cost efficient than TEM. PMID:12009703

  8. Quantitative measurement of orbital angular momentum in electron microscopy

    NASA Astrophysics Data System (ADS)

    Clark, L.; Bch, A.; Guzzinati, G.; Verbeeck, J.

    2014-05-01

    Electron vortex beams have been predicted to enable atomic scale magnetic information measurement, via transfer of orbital angular momentum. Research so far has focused on developing production techniques and applications of these beams. However, methods to measure the outgoing orbital angular momentum distribution are also a crucial requirement towards this goal. Here, we use a method to obtain the orbital angular momentum decomposition of an electron beam, using a multipinhole interferometer. We demonstrate both its ability to accurately measure orbital angular momentum distribution, and its experimental limitations when used in a transmission electron microscope.

  9. Quantitative characterization of epitaxial superlattices by x-ray diffraction and high resolution electron microscopy

    SciTech Connect

    Fullerton, E.E. ); Cao, W.; Thomas, G. ); Schuller, I.K. ); Carey, M.J.; Berkowitz, A.E. )

    1993-07-26

    Quantitative x-ray diffraction (XRD) and high resolution electron microscopy (HREM) have been applied to the analysis of an epitaxial CoO/NiO superlattice. This example shows that the qualitative information determined directly from a XRD spectrum or HREM image is limited and can even be misleading. However, by a combination of quantitative intensity measurements and structural modeling, a detailed quantitative characterization of the superlattice structure is possible.

  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. 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)

  12. Compositional analysis of GaAs/AlGaAs heterostructures using quantitative scanning transmission electron microscopy

    SciTech Connect

    Kauko, H.; Helvoort, A. T. J. van; Zheng, C. L.; Glanvill, S.; Zhu, Y.; Etheridge, J.; Dwyer, C.; Ernst Ruska-Centre for Microscopy and Spectroscopy with Electrons, and Peter Grnberg Institute, Forschungszentrum Jlich, D-52425 Jlich ; Munshi, A. M.; Fimland, B. O.

    2013-12-02

    We demonstrate a method for compositional mapping of Al{sub x}Ga{sub 1x}As heterostructures with high accuracy and unit cell spatial resolution using quantitative high angle annular dark field scanning transmission electron microscopy. The method is low dose relative to spectroscopic methods and insensitive to the effective source size and higher order lens aberrations. We apply the method to study the spatial variation in Al concentration in cross-sectioned GaAs/AlGaAs core-shell nanowires and quantify the concentration in the Al-rich radial band and the AlGaAs shell segments.

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

    NASA Astrophysics Data System (ADS)

    Bancelin, S.; Aim, 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 ( =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.

  14. 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.

  15. Quantitative analysis of shadow x-ray magnetic circular dichroism photoemission electron microscopy

    NASA Astrophysics Data System (ADS)

    Jamet, S.; Da, S., Col; Rougemaille, N.; Wartelle, A.; Locatelli, A.; Mente?, T. O.; Santos Burgos, B.; Afid, R.; Cagnon, L.; Bochmann, S.; Bachmann, J.; Fruchart, O.; Toussaint, J. C.

    2015-10-01

    Shadow x-ray magnetic circular dichroism photoemission electron microscopy is a recent technique, in which the photon intensity in the shadow of an object lying on a surface may be used to gather information about the three-dimensional magnetization texture inside the object. Our purpose here is to lay the basis of a quantitative analysis of this technique. We first discuss the principle and implementation of a method to simulate the contrast expected from an arbitrary micromagnetic state. Textbook examples and successful comparison with experiments are then given. Instrumental settings are finally discussed, having an impact on the contrast and spatial resolution: photon energy, microscope extraction voltage and plane of focus, microscope background level, electric-field related distortion of three-dimensional objects, Fresnel diffraction, or photon scattering.

  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. PMID:26509714

  17. 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. PMID:25418762

  18. Clay microporosity in reservoir sandstones: An application of quantitative electron microscopy in petrophysical evaluation

    SciTech Connect

    Hurst, A.; Nadeau, P.H.

    1995-04-01

    Clay mineral microporosity in sandstones is measured using computer-assisted image analysis of back-scattered electron micrographs of petrographic sections. Diagenetic kaolinite has a variety of textures with microporosity values ranging from 15 to 61%. Diagenetic chlorite has a generally uniform grain-coating texture and microporosity of about 50%. Fibrous illitic clays are difficult to characterize by the same method (an average value of 63% microporosity was recorded), but analysis of stereo-pair micrographs from scanning-electron microscopy analyses reveals that illite commonly has microporosity of approximately 90%. Clay microporosity data are used to calculate effective pore volumes and volumes of clay-bound water for clay minerals in sandstones. Converting from weight percent clay to volume percent clay is important. Microporosity data are valuable input to V{sub shale} evaluation where water saturation is associated with clay mineral type, texture, and volume.

  19. Quantitative measurement of deformation field around low-angle grain boundaries by electron microscopy

    NASA Astrophysics Data System (ADS)

    Zhao, C. W.; Xing, Y. M.; Bai, P. C.; Hou, J. F.; Dai, X. J.

    2008-05-01

    The strain field of low-angle grain boundaries in gold was experimentally investigated. The grain boundaries consist of the arrangement of discrete dislocations. High-resolution transmission electron microscopy (HRTEM) and geometric phase analysis (GPA) were employed to map the strain field of grain boundaries. The numerical moir method was used to visualize the dislocations. The strain components ? xx, ? yy, ? xy and rigid rotation ? xy were mapped. The dislocation core regions are convergence regions of strain. The largest values of strain occur in the immediate dislocation core region. The strain field around an edge dislocation was compared with Peierls-Nabarro dislocation model. The comparison result has demonstrated that the Peierls-Nabarro model can describe the strain field around edge dislocation.

  20. Size-dependent second virial coefficients of quantum dots from quantitative cryogenic electron microscopy.

    PubMed

    van Rijssel, J; Peters, V F D; Meeldijk, J D; Kortschot, R J; van Dijk-Moes, R J A; Petukhov, A V; Ern, B H; Philipse, A P

    2014-09-18

    Cryogenic transmission electron microscopy (cryo-TEM) is utilized to determine the second virial coefficient of osmotic pressure of PbSe quantum dots (QDs) dispersed in apolar liquid. Cryo-TEM images from vitrified samples provide snapshots of the equilibrium distribution of the particles. These snapshots yield radial distribution functions from which second virial coefficients are calculated, which agree with second virial coefficients determined with analytical centrifugation and small-angle X-ray scattering. The size dependence of the second virial coefficient points to an interparticle interaction that is proportional to the QD surface area. A plausible cause for this attraction is the interaction between the surface ions on adjacent QDs. PMID:25153168

  1. Design and validation of a novel quantitative method for rapid bacterial enumeration using programmed stage movement scanning electron microscopy.

    PubMed

    Sanders, David L; Bond, Peter; Moate, Roy; Steer, Jane A

    2012-12-01

    The adhesion of bacteria to surgical implants is the first stage of implant infection. The method for detecting bound bacteria is an important consideration in the study of bacterial adherence and colonisation. Enumeration of bacteria by direct visualisation techniques is labour intensive and time consuming. We have developed and validated a method for enumerating bacteria on porous material surfaces using programmed stage movement scanning electron microscopy and compared cumulative counts after 1-10 stage movements with absolute bacterial counts. We describe this method with three commercially sourced meshes used for abdominal wall hernia repair and with three different inoculums of Staphylococcus epidermidis. The results demonstrate significant correlation to the absolute count after five cumulative counts for all meshes analysed. The mean time saved by the cumulative counting method was 1h and 9 min per mesh. We conclude that advances in scanning electron microscopy and the advent of precise automated stage control have facilitated rapid data acquisition for bacterial counting purposes and that five cumulative counts at 1000 or 2500 magnification are a valid quantitative method for enumerating S. epidermidis bacteria on porous surfaces (with a pore size of up to 1.3 mm). PMID:23041496

  2. 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

  3. Quantitative annular dark field scanning transmission electron microscopy for nanoparticle atom-counting: What are the limits?

    NASA Astrophysics Data System (ADS)

    De Backer, A.; De Wael, A.; Gonnissen, J.; Martinez, G. T.; Bch, A.; MacArthur, K. E.; Jones, L.; Nellist, P. D.; Van Aert, S.

    2015-10-01

    Quantitative atomic resolution annular dark field scanning transmission electron microscopy (ADF STEM) has become a powerful technique for nanoparticle atom-counting. However, a lot of nanoparticles provide a severe characterisation challenge because of their limited size and beam sensitivity. Therefore, quantitative ADF STEM may greatly benefit from statistical detection theory in order to optimise the instrumental microscope settings such that the incoming electron dose can be kept as low as possible whilst still retaining single-atom precision. The principles of detection theory are used to quantify the probability of error for atom-counting. This enables us to decide between different image performance measures and to optimise the experimental detector settings for atom-counting in ADF STEM in an objective manner. To demonstrate this, ADF STEM imaging of an industrial catalyst has been conducted using the near-optimal detector settings. For this experiment, we discussed the limits for atomcounting diagnosed by combining a thorough statistical method and detailed image simulations.

  4. 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.

  5. High Resolution Quantitative Lorentz Microscopy

    NASA Astrophysics Data System (ADS)

    McVitie, S.; McGrouther, D.; Krajnak, M.

    2015-10-01

    The advent of aberration corrected transmission electron microscopy has led to considerable improvements in the field of high resolution electron microscopy imaging. In this paper we show how these developments are applied to imaging of magnetic structure in field free or low field conditions. Whilst the capability of increased spatial resolution is demonstrated on magnetic layers with a width of < 20nm we also consider how a pixelated detector can be used to dramatically increase the efficiency of the detection of the magnetic signal variation in the presence of strong diffraction contrast.

  6. 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 200keV, 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

  7. Diagnostic electron microscopy

    SciTech Connect

    Dickersin, G.R.

    1988-01-01

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

  8. Conventional transmission electron microscopy.

    PubMed

    Winey, Mark; Meehl, Janet B; O'Toole, Eileen T; Giddings, Thomas H

    2014-02-01

    Researchers have used transmission electron microscopy (TEM) to make contributions to cell biology for well over 50 years, and TEM continues to be an important technology in our field. We briefly present for the neophyte the components of a TEM-based study, beginning with sample preparation through imaging of the samples. We point out the limitations of TEM and issues to be considered during experimental design. Advanced electron microscopy techniques are listed as well. Finally, we point potential new users of TEM to resources to help launch their project. PMID:24482357

  9. Analysis of Transient Polyhydroxybutyrate Production in Wautersia eutropha H16 by Quantitative Western Analysis and Transmission Electron Microscopy

    PubMed Central

    Tian, Jiamin; He, Aimin; Lawrence, Adam G.; Liu, Pinghua; Watson, Nicki; Sinskey, Anthony J.; Stubbe, JoAnne

    2005-01-01

    Polyhydroxybutyrates (PHBs) are polyoxoesters generated from (R)3-hydroxybutyryl coenzyme A by PHB synthase. During the polymerization reaction, the polymers undergo a phase transition and generate granules. Wautersia eutropha can transiently accumulate PHB when it is grown in a nutrient-rich medium (up to 23% of the cell dry weight in dextrose-free tryptic soy broth [TSB]). PHB homeostasis under these growth conditions was examined by quantitative Western analysis to monitor the proteins present, their levels, and changes in their levels over a 48-h growth period. The proteins examined include PhaC (the synthase), PhaP (a phasin), PhaR (a transcription factor), and PhaZ1a, PhaZ1b, and PhaZ1c (putative intracellular depolymerases), as well as PhaZ2 (a hydroxybutyrate oligomer hydrolase). The results show that PhaC and PhaZ1a were present simultaneously. No PhaZ1b or PhaZ1c was detected at any time throughout growth. PhaZ2 was observed and exhibited an expression pattern different from that of PhaZ1a. The levels of PhaP changed dramatically and corresponded kinetically to the levels of PHB. Transmission electron microscopy (TEM) provided the dimensions of the average cell and the average granule at 4 h and 24 h of growth (J. Tian, A. J. Sinskey, and J. Stubbe, J. Bacteriol. 187:3814-3824, 2005). This information allowed us to calculate the amount of each protein and number of granules per cell and the granule surface coverage by proteins. The molecular mass of PHB (106 Da) was determined by dynamic light scattering at 4 h, the time of maximum PHB accumulation. At this time, the surface area of the granules was maximally covered with PhaP (27 to 54%), and there were one or two PhaP molecules/PHB chain. The ratio of PHB chains to PhaC was ∼60, which required reinitiation of polymer formation on PhaC. The TEM studies of wild-type and ΔphaR strains in TSB provided further support for an alternative mechanism of granule formation (Tian et al., J. Bacteriol. 187:3814-3824, 2005). PMID:15901707

  10. 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

  11. 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.

  12. 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.

  13. Gallery | High Resolution Electron Microscopy

    Cancer.gov

    Skip to main content High Resolution Electron Microscopy High Resolution Electron Microscopy Center for Cancer Research at the National Institutes of Health Main menu Home Research 3D Correlative Imaging Methods Development Protein Complexes Viral Entry Publications Image

  14. Publications | High Resolution Electron Microscopy

    Cancer.gov

    Skip to main content High Resolution Electron Microscopy High Resolution Electron Microscopy Center for Cancer Research at the National Institutes of Health Main menu Home Research 3D Correlative Imaging Methods Development Protein Complexes Viral Entry Publications Image

  15. 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.

  16. 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 relative shares were obtained for PM10: 22% exhaust, 22% abrasion and 56% resuspension (40%, 27%, 33% for PM1). Compared to previous publications we have observed a significantly lower portion of exhaust particles and a significantly higher portion of resuspension particles. The high abundance of resuspension particles underlines their significance for the observed adverse health effects of traffic emissions and for mitigation measures.

  17. Publications | High Resolution Electron Microscopy

    Cancer.gov

    Imaging biological objects with electrons involves principles similar to those used in light microscopy, except that electrons are used for illumination instead of photons and the lenses are magnetic instead of being optical. In the last five decades, electron microscopy (EM) helped to reveal basic cell structures in great detail, allowing researchers to visualize internal structure at resolutions that were about 100 times better than that obtained by optical microscopy.

  18. Welcome | High Resolution Electron Microscopy

    Cancer.gov

    For many years, electron microscopy has been used to image cells and tissues at high resolution. This technology, invented in the early 20th century, provided breakthrough information in the virology and cell biology fields. Over the last 15 to 20 years, however, rapid advances in imaging and computation technologies have expanded the usefulness of electron microscopy into new realms. Electron microscopy is now poised to close a critical "gap" in the structural biology field.

  19. 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.

  20. Quantitative analysis on volcanic ash surfaces: application of extended depth-of-field (focus) algorithm for light and scanning electron microscopy and 3D reconstruction.

    PubMed

    Ersoy, Orkun; Aydar, Erkan; Gourgaud, Alain; Bayhan, Hasan

    2008-01-01

    The depth-of-field mainly affects the image quality either in scanning electron microscopy (SEM) or conventional light microscopy. The limited depth-of-field handicap of microscopy imaging can be used for obtaining "optically sectioned" specimens by moving the object along the optical axis. In this study, multiple images corresponding to different object planes were taken in order to overcome limited depth-of-field on conventional light microscope and SEM, estimation of an elevation surface and 3D reconstruction of different type volcanic ash surfaces. We used extended depth-of-field, a fusion algorithm that combines those images into one single sharp composite. Because of larger depth-of-field, we got higher-quality results even with image stacks taken by SEM with a fixed aperture in variable pressure mode. We calculated roughness descriptors, quadtree decomposition and greylevel standard deviation (sGL) and analyzed the shape of polar plots based on gradient analysis of constructed depth-maps. Furthermore, we calculated fractal dimensions of surfaces. Correlation analysis was performed to measure how these quantitative variables are related with different type ash surfaces. Roughness descriptors, quadtree decomposition, sGL and fractal dimension discriminate different types of volcanic ash surfaces. PMID:17208002

  1. Non-amplified Quantitative Detection of Nucleic Acid Sequences Using a Gold Nanoparticle Probe Set and Field-Emission Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Hyonchol; Kira, Atsushi; Yasuda, Kenji

    2010-06-01

    For the precise detection of the number of expressed biomarkers at the single-cell level, we have developed a method of quantifying and specifying target DNA fragments by using a set of gold nanoparticles as labels and field-emission scanning electron microscopy (FE-SEM) to measure the number and sizes of gold nanoparticles attached to target samples. One or more target DNAs on a substrate were labeled with a set of different-sized gold nanoparticle probes having complementary sequences to different target candidates. The type and number of the target DNAs having a specific sequence were identified by counting the attached nanoparticles of a specific size in FE-SEM images. The results evaluated using a DNA microarray showed high specificity and sensitivity, and a linear correlation between the number of attached particles and the target DNA concentration, indicating the feasibility of quantitative detection in the femtomolar to nanomolar concentration range.

  2. 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).

  3. 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. PMID:25786736

  4. 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 these particles. The 'more' hygroscopic particles appear to be liquid spheres and contained sulfur, oxygen, and sometimes cations such as sodium and potassium. In addition, carbon was sometimes found in the more hygroscopic particles.

  5. Quantitative analysis of the myelin g-ratio from electron microscopy images of the macaque corpus callosum

    PubMed Central

    Stikov, Nikola; Campbell, Jennifer S.W.; Stroh, Thomas; Lavele, Mariette; Frey, Stephen; Novek, Jennifer; Nuara, Stephen; Ho, Ming-Kai; Bedell, Barry J.; Dougherty, Robert F.; Leppert, Ilana R.; Boudreau, Mathieu; Narayanan, Sridar; Duval, Tanguy; Cohen-Adad, Julien; Picard, Paul-Alexandre; Gasecka, Alicja; Ct, Daniel; Pike, G. Bruce

    2015-01-01

    We provide a detailed morphometric analysis of eight transmission electron micrographs (TEMs) obtained from the corpus callosum of one cynomolgus macaque. The raw TEM images are included in the article, along with the distributions of the axon caliber and the myelin g-ratio in each image. The distributions are analyzed to determine the relationship between axon caliber and g-ratio, and compared against the aggregate metrics (myelin volume fraction, fiber volume fraction, and the aggregate g-ratio), as defined in the accompanying research article entitled In vivo histology of the myelin g-ratio with magnetic resonance imaging (Stikov et al., NeuroImage, 2015). PMID:26217818

  6. 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.

  7. Automated Quantitative Live Cell Fluorescence Microscopy

    PubMed Central

    Fero, Michael; Pogliano, Kit

    2010-01-01

    Advances in microscopy automation and image analysis have given biologists the tools to attempt large scale systems-level experiments on biological systems using microscope image readout. Fluorescence microscopy has become a standard tool for assaying gene function in RNAi knockdown screens and protein localization studies in eukaryotic systems. Similar high throughput studies can be attempted in prokaryotes, though the difficulties surrounding work at the diffraction limit pose challenges, and targeting essential genes in a high throughput way can be difficult. Here we will discuss efforts to make live-cell fluorescent microscopy based experiments using genetically encoded fluorescent reporters an automated, high throughput, and quantitative endeavor amenable to systems-level experiments in bacteria. We emphasize a quantitative data reduction approach, using simulation to help develop biologically relevant cell measurements that completely characterize the cell image. We give an example of how this type of data can be directly exploited by statistical learning algorithms to discover functional pathways. PMID:20591990

  8. 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…

  9. Research | High Resolution Electron Microscopy

    Cancer.gov

    Our research program primarily focuses on the development of technologies for 3D imaging using electron microscopy techniques, and on the use of these technologies to image cells, viruses and proteins at high resolution.

  10. Basic phage electron microscopy.

    PubMed

    Ackermann, Hans-W

    2009-01-01

    Negative staining of purified viruses is the most important electron microscopical technique in virology. The principal stains are phosphotungstate and uranyl acetate, both of which have problems and advantages. Particular problems are encountered in photography, calibration of magnification, measurements, and interpretation of artifacts. PMID:19066816

  11. Effects of non-rotationally symmetric aberrations on the quantitative measurement of lattice positions in a graphene monolayer using high-resolution transmission electron microscopy.

    PubMed

    Lin, Fang; Jian, Jiajun; Ye, Lvshan; Jin, Chuanhong

    2015-10-01

    It is crucial to determine the position of lattice atoms in a monolayer specimen with high precision using high-resolution transmission electron microscopy (HRTEM). Image simulations indicate that the intensity centers of periodic lattice atoms in graphene may deviate from their intrinsic positions if non-rotationally symmetric aberrations (except for three-fold and six-fold aberrations) exist in the HRTEM imaging system. In this letter, we quantitatively compared the deviations caused by non-rotationally symmetric aberrations, which are equivalent to individually produce a ?/4 phase shift in the wave aberration function at a given frequency of 7.2 nm(-1). A two-fold aberration caused a maximum shift of 0.3 , and in the images affected by the axial coma, graphene still maintained its hexagonal structure while all of the measured atomic positions deviated. Furthermore, we discovered that atoms on each sublattice tended to shift by similar distances in the image. Based on this rule, we retrieved the intrinsic bond length between neighboring carbon atoms by 'shifting' the measured atom positions in an experimental HRTEM image affected by residual aberrations. PMID:26070475

  12. Four-dimensional electron microscopy.

    PubMed

    Zewail, Ahmed H

    2010-04-01

    The discovery of the electron over a century ago and the realization of its dual character have given birth to one of the two most powerful imaging instruments: the electron microscope. The electron microscope's ability to resolve three-dimensional (3D) structures on the atomic scale is continuing to affect different fields, including materials science and biology. In this Review, we highlight recent developments and inventions made by introducing the fourth dimension of time in electron microscopy. Today, ultrafast electron microscopy (4D UEM) enables a resolution that is 10 orders of magnitude better than that of conventional microscopes, which are limited by the video-camera rate of recording. After presenting the central concept involved, that of single-electron stroboscopic imaging, we discuss prototypical applications, which include the visualization of complex structures when unfolding on different length and time scales. The developed UEM variant techniques are several, and here we illucidate convergent-beam and near-field imaging, as well as tomography and scanning-pulse microscopy. We conclude with current explorations in imaging of nanomaterials and biostructures and an outlook on possible future directions in space-time, 4D electron microscopy. PMID:20378810

  13. Four-Dimensional Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Zewail, Ahmed H.

    2010-04-01

    The discovery of the electron over a century ago and the realization of its dual character have given birth to one of the two most powerful imaging instruments: the electron microscope. The electron microscope’s ability to resolve three-dimensional (3D) structures on the atomic scale is continuing to affect different fields, including materials science and biology. In this Review, we highlight recent developments and inventions made by introducing the fourth dimension of time in electron microscopy. Today, ultrafast electron microscopy (4D UEM) enables a resolution that is 10 orders of magnitude better than that of conventional microscopes, which are limited by the video-camera rate of recording. After presenting the central concept involved, that of single-electron stroboscopic imaging, we discuss prototypical applications, which include the visualization of complex structures when unfolding on different length and time scales. The developed UEM variant techniques are several, and here we illucidate convergent-beam and near-field imaging, as well as tomography and scanning-pulse microscopy. We conclude with current explorations in imaging of nanomaterials and biostructures and an outlook on possible future directions in space-time, 4D electron microscopy.

  14. Dynamic imaging with electron microscopy

    ScienceCinema

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2014-05-30

    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.

  15. 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.

  16. 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

  17. From Quantitative Microscopy to Automated Image Understanding

    PubMed Central

    Huang, Kai; Murphy, Robert F.

    2005-01-01

    Quantitative microscopy has been extensively used in biomedical research and has provided significant insights into structure and dynamics at the cell and tissue level. The entire procedure of quantitative microscopy is comprised of specimen preparation, light absorption/reflection/emission from the specimen, microscope optical processing, optical/electrical conversion by a camera or detector, and computational processing of digitized images. Although many of the latest digital signal processing techniques have been successfully applied to compress, restore, and register digital microscope images, automated approaches for recognition and understanding of complex subcellular patterns in light microscope images have been far less widely used. In this review, we describe a systematic approach for interpreting protein subcellular distributions using various sets of Subcellular Location Features (SLF) in combination with supervised classification and unsupervised clustering methods. These methods can handle complex patterns in digital microscope images and the features can be applied for other purposes such as objectively choosing a representative image from a collection and performing statistical comparison of image sets. PMID:15447010

  18. Quantitative Aspects of Single Molecule Microscopy

    PubMed Central

    Ober, Raimund J.; Tahmasbi, Amir; Ram, Sripad; Lin, Zhiping; Ward, E. Sally

    2015-01-01

    Single molecule microscopy is a relatively new optical microscopy technique that allows the detection of individual molecules such as proteins in a cellular context. This technique has generated significant interest among biologists, biophysicists and biochemists, as it holds the promise to provide novel insights into subcellular processes and structures that otherwise cannot be gained through traditional experimental approaches. Single molecule experiments place stringent demands on experimental and algorithmic tools due to the low signal levels and the presence of significant extraneous noise sources. Consequently, this has necessitated the use of advanced statistical signal and image processing techniques for the design and analysis of single molecule experiments. In this tutorial paper, we provide an overview of single molecule microscopy from early works to current applications and challenges. Specific emphasis will be on the quantitative aspects of this imaging modality, in particular single molecule localization and resolvability, which will be discussed from an information theoretic perspective. We review the stochastic framework for image formation, different types of estimation techniques and expressions for the Fisher information matrix. We also discuss several open problems in the field that demand highly non-trivial signal processing algorithms. PMID:26167102

  19. ELECTRON MICROSCOPY OF RHODOTORULA GLUTINIS

    PubMed Central

    Thyagarajan, T. R.; Conti, S. F.; Naylor, H. B.

    1962-01-01

    Thyagarajan, T. R. (Dartmouth Medical School, Hanover, N. H.), S. F. Conti, and H. B. Naylor. Electron microscopy of Rhodotorula glutinis. J. Bacteriol. 83:381394. 1962.The structure and manner of nuclear division in Rhodotorula glutinis was studied by electron microscopy of ultrathin sections. Parallel studies with the light microscope, employing conventional staining techniques and phase-contrast microscope observations on nuclei in living cells, were carried out. The nucleus is spherical to oval and is bounded by a nuclear membrane. Intranuclear structures, identified as nucleoli, and electron-transparent areas were observed. The nuclear membrane persists throughout the various stages of cell division. Observations of the nucleus with the electron microscope revealed that nuclear division occurs by a process of elongation and constriction similar to that seen in both living and stained cells. The fine structure of mitochondria and other components of the yeast cell and their behavior during cell division are described. The absence of vacuoles in actively dividing cells of Rhodotorula glutinis lends further support to the view that the vacuole is not an integral part of the nucleus. The results with the electron microscope generally support and considerably extend those obtained with living and stained cells. Images PMID:13921132

  20. 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.

  1. Prototype cantilevers for quantitative lateral force microscopy

    NASA Astrophysics Data System (ADS)

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

    2011-09-01

    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

  2. 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.

  3. Correlative fluorescence and electron microscopy.

    PubMed

    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 associated with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology has led to rapid improvement in the protocols and has ushered in a new generation of instruments to reach the next level of correlation-integration. Curr. Protoc. Cytom. 70:12.36.1-12.36.10. 2014 by John Wiley & Sons, Inc. PMID:25271959

  4. Quantitative imaging of flux vortices in the type-II superconductor MgB2 using cryo-Lorentz transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Cottet, M. J. G.; Cantoni, M.; Mansart, B.; Alexander, D. T. L.; Hébert, C.; Zhigadlo, N. D.; Karpinski, J.; Carbone, F.

    2013-07-01

    Imaging of flux vortices in high quality MgB2 single crystals has been successfully performed in a commercial field-emission gun-based transmission electron microscope. In cryo-Lorentz microscopy, the sample quality and the vortex lattice can be monitored simultaneously, allowing one to relate microscopically the surface quality and the vortex dynamics. Such a vortex motion ultimately determines the flow resistivity ρf, the knowledge of which is indispensable for practical applications such as superconducting magnets or wires for magnetic resonance imaging. The observed patterns have been analyzed and compared with other studies by cryo-Lorentz microscopy or Bitter decoration. We find that the vortex lattice arrangement depends strongly on the surface quality obtained during the specimen preparation, and tends to form a hexagonal Abrikosov lattice at a relatively low magnetic field. Stripes or gossamerlike patterns, recently suggested as potential signatures of an unconventional behavior of MgB2, were not observed.

  5. Former Lab Members | High Resolution Electron Microscopy

    Cancer.gov

    Skip to main content High Resolution Electron Microscopy High Resolution Electron Microscopy Center for Cancer Research at the National Institutes of Health Main menu Home Research 3D Correlative Imaging Methods Development Protein Complexes Viral Entry Publications Image

  6. Journal Covers | High Resolution Electron Microscopy

    Cancer.gov

    Skip to main content High Resolution Electron Microscopy High Resolution Electron Microscopy Center for Cancer Research at the National Institutes of Health Main menu Home Research 3D Correlative Imaging Methods Development Protein Complexes Viral Entry Publications Image

  7. Correlative Stochastic Optical Reconstruction Microscopy and Electron Microscopy

    PubMed Central

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

    2015-01-01

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

  8. Low-magnification Quantitative X-ray Mapping of Grain-boundary Segregation in Aluminum-4 wt.% Copper by Analytical Electron Microscopy.

    PubMed

    Carpenter; Watanabe; Barmak; Williams

    1999-07-01

    : Quantitative X-ray mapping in the analytical electron microscope (AEM) could improve the statistics of grain-boundary segregation measurements if high spatial resolution can be maintained at lower magnifications (<500 kX). Typically, only about 10 boundaries are analyzed because of the difficulty of conventional AEM measurements; however, a low-magnification quantitative X-ray map could contain twice this number of boundaries in a single field of view. Microscope conditions and mapping parameters have been explored for operation at approximately 250 kX, under a variety of conditions to illustrate the trade-offs between various characteristics, such as analytical resolution, counting statistics, magnification, and acquisition time. From these data, it is possible to extrapolate to maps generated under different conditions and estimate their limitations with respect to these characteristics. A simple model has been developed to describe the behavior of inclined grain boundaries that can be used to estimate the detectability of segregant as a function of boundary tilt. Using quantitative X-ray maps, grain boundary Cu coverage has been measured from 55 boundaries in Al-4 wt.% Cu with minimal user effort. For fine-grained thin films, mapping is substantially more efficient than other methods of data acquisition and may be used to measure segregation at large numbers of boundaries. PMID:10421810

  9. Four-dimensional ultrafast electron microscopy

    PubMed Central

    Lobastov, Vladimir A.; Srinivasan, Ramesh; Zewail, Ahmed H.

    2005-01-01

    Electron microscopy is arguably the most powerful tool for spatial imaging of structures. As such, 2D and 3D microscopies provide static structures with subnanometer and increasingly with ngstrom-scale spatial resolution. Here we report the development of 4D ultrafast electron microscopy, whose capability imparts another dimension to imaging in general and to dynamics in particular. We demonstrate its versatility by recording images and diffraction patterns of crystalline and amorphous materials and images of biological cells. The electron packets, which were generated with femtosecond laser pulses, have a de Broglie wavelength of 0.0335 at 120 keV and have as low as one electron per pulse. With such few particles, doses of few electrons per square ngstrom, and ultrafast temporal duration, the long sought after but hitherto unrealized quest for ultrafast electron microscopy has been realized. Ultrafast electron microscopy should have an impact on all areas of microscopy, including biological imaging. PMID:15883380

  10. 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.

  11. 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.

  12. Quantitative transmission electron microscopy analysis of multi-variant grains in present L10-FePt based heat assisted magnetic recording media

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

    We present a study on atomic ordering within individual grains in granular L10-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 L10-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.

  13. 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.

  14. Atomic resolution 3D electron diffraction microscopy

    SciTech Connect

    Miao, Jianwei; Ohsuna, Tetsu; Terasaki, Osamu; O'Keefe, Michael A.

    2002-03-01

    Electron lens aberration is the major barrier limiting the resolution of electron microscopy. Here we describe a novel form of electron microscopy to overcome electron lens aberration. By combining coherent electron diffraction with the oversampling phasing method, we show that the 3D structure of a 2 x 2 x 2 unit cell nano-crystal (framework of LTA [Al12Si12O48]8) can be ab initio determined at the resolution of 1 Angstrom from a series of simulated noisy diffraction pattern projections with rotation angles ranging from -70 degrees to +70 degrees in 5 degrees increments along a single rotation axis. This form of microscopy (which we call 3D electron diffraction microscopy) does not require any reference waves, and can image the 3D structure of nanocrystals, as well as non-crystalline biological and materials science samples, with the resolution limited only by the quality of sample diffraction.

  15. 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

  16. 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.

  17. Contact | High Resolution Electron Microscopy

    Cancer.gov

    The long-term mission of our research program is to obtain an integrated, quantitative understanding of cells and viruses at molecular resolution. We take an interdisciplinary approach to this problem by combining novel technologies for 3D imaging with computational and cell biological tools.

  18. Fluorescence-integrated transmission electron microscopy images: integrating fluorescence microscopy with transmission electron microscopy.

    PubMed

    Sims, Paul A; Hardin, Jeff D

    2007-01-01

    This chapter describes high-pressure freezing (HPF) techniques for correlative light and electron microscopy on the same sample. Laser scanning confocal microscopy (LSCM) is exploited for its ability to collect fluorescent, as well as transmitted and back scattered light (BSL) images at the same time. Fluorescent information from a whole mount (preembedding) or from thin sections (post-embedding) can be displayed as a color overlay on transmission electron microscopy (TEM) images. Fluorescence-integrated TEM (F-TEM) images provide a fluorescent perspective to TEM images. The pre-embedding method uses a thin two-part agarose pad to immobilize live Caenorhabditis elegans embryos for LSCM, HPF, and TEM. Pre-embedding F-TEM images display fluorescent information collected from a whole mount of live embryos onto all thin sections collected from that sample. In contrast, the postembedding method uses HPF and freeze substitution with 1% paraformaldehyde in 95% ethanol followed by low-temperature embedding in methacrylate resin. This procedure preserves the structure and function of green fluorescent protein (GFP) as determined by immunogold labeling of GFP, when compared with GFP expression, both demonstrated in the same thin section. PMID:17656756

  19. 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.

  20. Publications | High Resolution Electron Microscopy

    Cancer.gov

    Zhang P, Land W, Lee S, Juliani J, Lefman J, Smith S, Germain D, Kessel M, Leapman R, Rouault TA and Subramaniam S. Electron tomography of degenerating neurons in mice with abnormal regulation of iron metabolism. J Struct Biol.

  1. Publications | High Resolution Electron Microscopy

    Cancer.gov

    Kuybeda O, Frank GA, Bartesaghi A, Borgnia M, Subramaniam S, and Sapiro G. A collaborative framework for 3D alignment and classification of heterogeneous subvolumes in cryo-electron tomography. J Struct Biol.

  2. Advance in orientation microscopy: quantitative analysis of nanocrystalline structures.

    PubMed

    Seyring, Martin; Song, Xiaoyan; Rettenmayr, Markus

    2011-04-26

    The special properties of nanocrystalline materials are generally accepted to be a consequence of the high density of planar defects (grain and twin boundaries) and their characteristics. However, until now, nanograin structures have not been characterized with similar detail and statistical relevance as coarse-grained materials, due to the lack of an appropriate method. In the present paper, a novel method based on quantitative nanobeam diffraction in transmission electron microscopy (TEM) is presented to determine the misorientation of adjacent nanograins and subgrains. Spatial resolution of <5 nm can be achieved. This method is applicable to characterize orientation relationships in wire, film, and bulk materials with nanocrystalline structures. As a model material, nanocrystalline Cu is used. Several important features of the nanograin structure are discovered utilizing quantitative analysis: the fraction of twin boundaries is substantially higher than that observed in bright-field images in the TEM; small angle grain boundaries are prominent; there is an obvious dependence of the grain boundary characteristics on grain size distribution and mean grain size. PMID:21375327

  3. Correlative light and electron microscopy of GFP.

    PubMed

    Grabenbauer, Markus

    2012-01-01

    The correlation of light and electron microscopy (EM) is a powerful tool as it combines the investigation of dynamic processes in vivo with the resolution power of the electron microscope. The green fluorescent proteins (GFPs) and its derivatives revolutionized live-cell light microscopy. Hence, this review outlines correlative microscopy of GFP through photo-oxidation, a method that allows for the direct ultrastructural visualization of fluorophores upon illumination. Oxygen radicals generated during the GFP bleaching process photo-oxidize diaminobenzidine (DAB) into an electron dense precipitate that can be visualized both by routine EM of thin sections and by electron tomography for 3D analysis. There are different levels of correlative microscopy, i.e. the correlation of certain areas, cells, or organelles from light to EM, where photo-oxidation of DAB through GFP allows the highest possible degree--the correlation of specific molecules. PMID:22857926

  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. PMID:25408379

  5. Scanning electron microscopy of bone.

    PubMed

    Boyde, Alan

    2012-01-01

    This chapter described methods for Scanning Electron Microscopical imaging of bone and bone cells. Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. This chapter considers preparing and imaging samples of unembedded bone having 3D detail in a 3D surface, topography-free, polished or micromilled, resin-embedded block surfaces, and resin casts of space in bone matrix. The chapter considers methods for fixation, drying, looking at undersides of bone cells, and coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralised matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples and recommendations for the types of resin embedding of bone for BSE imaging are given. Correlated confocal and SEM imaging of PMMA-embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualising fluorescent mineralising front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Environmental SEM (ESEM, controlled vacuum mode) is valuable in eliminating -"charging" problems which are common with complex, cancellous bone samples. PMID:22130941

  6. Quantitative interferometric microscopy cytometer based on regularized optical flow algorithm

    NASA Astrophysics Data System (ADS)

    Xue, Liang; Vargas, Javier; Wang, Shouyu; Li, Zhenhua; Liu, Fei

    2015-09-01

    Cell detections and analysis are important in various fields, such as medical observations and disease diagnoses. In order to analyze the cell parameters as well as observe the samples directly, in this paper, we present an improved quantitative interferometric microscopy cytometer, which can monitor the quantitative phase distributions of bio-samples and realize cellular parameter statistics. The proposed system is able to recover the phase imaging of biological samples in the expanded field of view via a regularized optical flow demodulation algorithm. This algorithm reconstructs the phase distribution with high accuracy with only two interferograms acquired at different time points simplifying the scanning system. Additionally, the method is totally automatic, and therefore it is convenient for establishing a quantitative phase cytometer. Moreover, the phase retrieval approach is robust against noise and background. Excitingly, red blood cells are readily investigated with the quantitative interferometric microscopy cytometer system.

  7. 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. PMID:20869522

  8. Transmission electron microscopy of composites

    NASA Technical Reports Server (NTRS)

    Pirouz, P.; Farmer, S. C.; Ernst, F.; Chung, J.

    1988-01-01

    Since interphase-interfaces are often both the structurally weakest and chemically least stable regions of a composite material, they are critical determinants of such macrostructural characteristics as tensile strength and fracture toughness. Attention is presently given to the use of TEM for the study of interfaces between dissimilar materials; electron-diffraction, analytical, and high-resolution forms of TEM are employed, for the cases of both structural and semiconductor composites. The materials studied are SiC/Si, GaP/Si, and SiC fiber- and whisker-reinforced Si3N4.

  9. Low voltage scanning electron microscopy.

    PubMed

    Pawley, J

    1984-10-01

    The scanning electron microscope (SEM) is usually operated with a beam voltage, V0, in the range of 10-30 kV, even though many early workers had suggested the use of lower voltages to increase topographic contrast and to reduce specimen charging and beam damage. The chief reason for this contradiction is poor instrumental performance when V0 = 1-3 kV, The problems include low source brightness, greater defocusing due to chromatic aberration greater sensitivity to stray fields, and difficulty in collecting the secondary electron signal. Responding to the needs of the semiconductor industry, which uses low V0 to reduce beam damage, considerable efforts have been made to overcome these problems. The resulting equipment has greatly improved performance at low kV and substantially removes the practical deterrents to operation in this mode. This paper reviews the advantages of low voltage operation, recent progress in instrumentation and describes a prototype instrument designed and built for optimum performance at 1 kV. Other limitations to high resolution topographic imaging such as surface contamination, the de-localized nature of the inelastic scattering event and radiation damage are also discussed. PMID:6512855

  10. Electron Microscopy of Natural and Epitaxial Diamond

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  11. Self-consistent absorption correction for quantitative energy- dispersive X-ray spectroscopy of InGaN layers in analytical transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Walther, T.; Wang, X.

    2015-10-01

    A new method of absorption correction for energy-dispersive X-ray spectroscopy in a transmission electron microscope is tested on InGaN samples. We simulate the effective k-factor for the In L line with respect to Ga L or Ga K and plot this as a function of the Ga K/L intensity ratio, which can be directly measured from experimental spectra. This basically performs an internal self-consistency check in the quantification using differently absorbed X-ray lines, which is in principle equivalent to an absorption correction as a function of specimen thickness but has the practical advantage that neither specimen thickness nor density or mass-thickness of the specimens need actually be measured.

  12. Experiences with remote electron microscopy

    SciTech Connect

    O'Keefe, Michael A.; Parvin, Bahram

    2002-02-22

    With the advent of a rapidly proliferating international computer network, it became feasible to consider remote operation of instrumentation normally operated locally. For modern electron microscopes, the growing automation and computer control of many instrumental operations facilitated the task of providing remote operation. In order to provide use of NCEM TEMs by distant users, a project was instituted in 1995 to place a unique instrument, a Kratos EM-1500 operating at 1.5MeV, on-line for remote use. In 1996, the Materials Microcharacterization Collaboratory (MMC) was created as a pilot project within the US Department of Energy's DOE2000 program to establish national collaboratories to provide access via the Internet to unique or expensive DOE research facilities as well as to expertise for remote collaboration, experimentation, production, software development, modeling, and measurement. A major LBNL contribution to the MMC was construction of DeepView, a microscope-independent computer-control system that could be ported to other MMC members to provide a common graphical user-interface (GUI) for control of any MMC instrument over the wide area network.

  13. 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

  14. 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.

  15. 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...

  16. 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.

  17. 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. PMID:18664385

  18. Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy.

    PubMed

    Liv, Nalan; van Oosten Slingeland, Daan S B; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W; Hoogenboom, Jacob P

    2016-01-26

    We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization. PMID:26580231

  19. 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.

  20. Low-pass secondary electron detector for outlens scanning electron microscopy

    NASA Astrophysics Data System (ADS)

    Sekiguchi, Takashi; Iwai, Hideo

    2015-08-01

    A low-pass secondary electron detector has been invented for outlens scanning electron microscopy. This detector is composed of a bias grid above and an electron detector below the specimen. The upward low-energy electrons emitted from the specimen are reflected downward by the bias grid and reach the secondary electron detector. The high-energy electrons penetrate the grid and are not detected. This detector has an advantage of quantitative analysis because the secondary electron trajectories are easily traced with simple parabolic motion. The energy-filtered images of the GaN/Si sample are obtained using this detector.

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

    PubMed

    Lalević-Vasić, B; Polić, D

    1988-01-01

    A case of ringed hair studied by light and electron microscopy is reported. The patient, a 20-year old girl, had been presenting with the hair abnormality since birth. At naked eye examination the hairs were dry, 6 to 7 cm long, and they showed dull and shining areas giving the scalp hair a scintillating appearance (fig. 1). Several samples of hair were taken and examined by light microscopy under white and polarized light. Hair shafts and cryo-fractured surfaces were examined by scanning electron microscopy. RESULTS. 1. Light microscopy. Lesions were found in every hair examined. There were abnormal, opaque and fusiform areas alternating with normal areas all along the hair shaft (fig. 2). The abnormal areas resulted from intracortical air-filled cavities. Fractures similar to those of trichorrhexis nodosa were found in the opaque areas of the distal parts of the hairs. 2. Scanning electron microscopy. A. Hair shaft surface. The abnormal areas showed a longitudinal, "curtain-like" folding of the cuticular cells which had punctiform depressions on their surface and worn free edges (fig. 4, 5, 6); trichorrhexis-type fractures were seen in the distal parts of the hair shafts (fig. 7, 8). Normal areas regularly presented with longitudinal, superficial, short and non-systematized depressions (fig. 9); the cuticular cells were worn, and there were places where the denuded cortex showed dissociated cortical fibres (fig. 10).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:3415147

  2. 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

  3. Spectral-domain interferometry for quantitative DIC microscopy

    NASA Astrophysics Data System (ADS)

    Li, Chengshuai; Zhu, Yizheng

    2014-03-01

    A spectral-domain differential interference contrast (SD-DIC) microscopy system is presented for quantitative imaging of both reflective and transparent samples. The spectral-domain interferometry, combined with the common-path DIC geometry, provides a shot noise-limited sensitivity of 14.3pm in optical pathlength gradient measurement. The optical resolution of the system was characterized using images of a USAF resolution target. Fused silica microspheres were imaged to demonstrate the reconstruction of two-dimensional optical pathlength topography from measured gradient fields. The exquisite sensitivity of the system showed potential in quantitative imaging of sub-diffraction limit objects such as gold nanoparticles.

  4. 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 analysis. An integrated technique combining X-ray diffraction (XRD) and density functional theory (DFT) simulation is presented here for the quantification and prediction of the elastic properties of crystallographically textured polycrystalline films used in MEMS devices. The technique is rapid, efficient, and capable of analyzing individual devices in an array, making it ideal for MEMS design, analysis, and quality control. Application of the technique to the electroplated nickel bridge of an RF MEMS switch, whose critical operating parameters depend on the in-plane Young's modulus, is demonstrated. It is shown that the in-plane Young's modulus of nickel films with a perfect, single fiber texture can vary over a large range from 172 GPa to 232 GPa. Experimental results significantly outside this range cannot be explained by crystallographic texture alone. The range of Young's modulus for real films is expected to be somewhat smaller because real films rarely have a near- perfect fiber texture and sometimes have a texture that cannot be described by a single fiber of orientation. The nickel bridge of the RF MEMS switch, which has a relatively strong 001 fiber texture component as well as a weak 111 fiber texture component, exemplifies such a case. The present technique takes these texture features into account to estimate the in-plane Young's modulus of the nickel bridge in several RF MEMS switches.

  5. Macrocyclization of polysaccharides visualized by electron microscopy.

    PubMed

    Stokke, B T; Elgsaeter, A; Kitamura, S

    1993-02-01

    Topological features of the polysaccharides schizophyllan, l-carrageenan and gellan gum were studied using electron microscopy. Electron micrographs of schizophyllan not subjected to any thermal or solvent composition history destabilizing the triple helix, show stiff, linear chains consistent with the structure being triple helical and with contour length proportional to the molecular weight in solution. A blend of linear, cyclic and hairpin topologies and higher molecular weight clusters were observed after renaturation, i.e. return to conditions favouring the triple helical structure, from solvent conditions dissociating the triple helix. Electron micrographs of l-carrageenan in salt-free solution reveal linear extended structures. Addition of 0.15 M LiI to the solution before preparation for electron microscopy, i.e. salt conditions that favour ordering but not gelation, yields a large fraction of cyclic structures with circumference of different lengths. Likewise, adding KCl to aqueous gellan gum changes their appearance from dispersed polymers to suprastrands with several associated chains. Macrocyclic species can also be observed in gellan gum after the addition of a gel-promoting salt. The tendency to form macrocyclic structures in competition with intermolecular aggregates is determined by the three factors: (1) chain stiffness relative to overall length; (2) parallel or antiparallel alignment of interacting chain segments; and (3) polymer concentration. The present study indicates that electron microscopy provides information about the topology adopted by polysaccharides. PMID:8443135

  6. Visualization of phytoplasmas using electron microscopy.

    PubMed

    Devonshire, B Jean

    2013-01-01

    The use of electron microscopy, both transmission and scanning, provides reliable and accurate methods for detecting phytoplasmas in plants. Our understanding of these pathogens, their morphology, development, and intracellular location in plants and insect vectors has been greatly increased through the use of these instruments. Development of techniques such as immunolabeling, cryofixation with freeze substitution or plunge freezing with direct transfer to the microscope stage, together with advances in instrumentation is enabling us to study these pathogens under conditions close to their native state. The visualization of fine detail and ultrastructure, using modern and established techniques, can only be appreciated by the magnification and spatial resolution offered in the electron microscopes. Now that the full sequencing of four phytoplasma genomes (to date) has been achieved, electron microscopy can play an important role in identifying and understanding specific gene functions. PMID:22987411

  7. 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

  8. Electronic imaging resolution criteria for light microscopy

    NASA Astrophysics Data System (ADS)

    Clarke, Theodore M.

    1995-08-01

    High resolution electronic imaging is certain to become the dominant method for recording microscope images (photomicrographs) in industrial laboratories. The rate of transition from film recording to electronic recording depends upon cost and quality. Cost, with little regard for image quality, has already caused a significant shift away from film recording. Recent technology developments appear to have eliminated the quality limitations of electronic imaging for industrial microscopy. These quality limitations are primarily related to the inability to match tradition 4 by 5 photomicrographs in field size and resolution. Analysis and limited experimental results indicate that 1024 by 1280 pixel imaging should be able to achieve the objective of matching 4 by 5 photomicrographs.

  9. Mudrocks examined by backscattered electron microscopy

    NASA Technical Reports Server (NTRS)

    Pye, K.; Krinsley, D.

    1983-01-01

    A method of studying mudrocks is developed using backscattered electrons (BSE) in scanning electron microscopy. Commercially available detectors are utilized to mix the BSE and secondary electron signals in order to obtain the optimum image for a particular material. Thin sections or polished rock chip surfaces are examined with BSE which provides both the atomic number contrast and topographic contrast. This technique provides very detailed information about the form and composition of individual grains in the mudrock thin sections and can be used in studies of the source, mode of deposition, diagenesis, and tectonic deformational history of mudrocks.

  10. Fast pixel shifting phase unwrapping algorithm in quantitative interferometric microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Mingfei; Shan, Yanke; Yan, Keding; Xue, Liang; Wang, Shouyu; Liu, Fei

    2014-11-01

    Quantitative interferometric microscopy is an important method for observing biological samples such as cells and tissues. In order to obtain continuous phase distribution of the sample from the interferogram, phase extracting and phase unwrapping are both needed in quantitative interferometric microscopy. Phase extracting includes fast Fourier transform method and Hilbert transform method, etc., almost all of them are rapid methods. However, traditional unwrapping methods such as least squares algorithm, minimum network flow method, etc. are time-consuming to locate the phase discontinuities which lead to low processing efficiency. Other proposed high-speed phase unwrapping methods always need at least two interferograms to recover final phase distributions which cannot realize real time processing. Therefore, high-speed phase unwrapping algorithm for single interferogram is required to improve the calculation efficiency. Here, we propose a fast phase unwrapping algorithm to realize high-speed quantitative interferometric microscopy, by shifting mod 2? wrapped phase map for one pixel, then multiplying the original phase map and the shifted one, then the phase discontinuities location can be easily determined. Both numerical simulation and experiments confirm that the algorithm features fast, precise and reliable.

  11. Quantitative phase imaging with scanning holographic microscopy: an experimental assesment

    PubMed Central

    Indebetouw, Guy; Tada, Yoshitaka; Leacock, John

    2006-01-01

    This paper demonstrates experimentally how quantitative phase information can be obtained in scanning holographic microscopy. Scanning holography can operate in both coherent and incoherent modes, simultaneously if desired, with different detector geometries. A spatially integrating detector provides an incoherent hologram of the object's intensity distribution (absorption and/or fluorescence, for example), while a point detector in a conjugate plane of the pupil provides a coherent hologram of the object's complex amplitude, from which a quantitative measure of its phase distribution can be extracted. The possibility of capturing simultaneously holograms of three-dimensional specimens, leading to three-dimensional reconstructions with absorption contrast, reflectance contrast, fluorescence contrast, as was previously demonstrated, and quantitative phase contrast, as shown here for the first time, opens up new avenues for multimodal imaging in biological studies. PMID:17132171

  12. 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

  13. Electron microscopy of desquamative interstitial pneumonia

    PubMed Central

    Shortland, J. R.; Darke, C. S.; Crane, W. A. J.

    1969-01-01

    The clinical, radiographical, and physiological picture of two patients suffering from desquamative interstitial pneumonia is described. The diagnosis was established by lung biopsy when the characteristic histological features were found on light microscopy. The dramatic response to adequate corticosteroid therapy is recorded, and attention is directed to the danger of serious relapse on early withdrawal of this treatment and the subsequent satisfactory response to a second course. Electron microscopical studies of the tissue from one patient add materially to the understanding of the clinical course and the nature of the tissue response. At the ultrastructural level the attenuated membranous (type 1) pneumonocytes which normally line the alveoli were replaced by granular (type 2) pneumonocytes. The desquamated intra-alveolar cells comprised two main groups. These were granular pneumonocytes, similar to those lining the alveoli, and smaller numbers of macrophages. The cytopathic effects of virus infection were not detected by light or electron microscopy. Images PMID:5821622

  14. Scanning electron microscopy of superficial white onychomycosis.

    PubMed

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

    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

  15. Scanning electron microscopy of superficial white onychomycosis*

    PubMed Central

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

    2015-01-01

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

  16. Correlative Photoactivated Localization and Scanning Electron Microscopy

    PubMed Central

    Kopek, Benjamin G.; Shtengel, Gleb; Grimm, Jonathan B.; Clayton, David A.; Hess, Harald F.

    2013-01-01

    The ability to localize proteins precisely within subcellular space is crucial to understanding the functioning of biological systems. Recently, we described a protocol that correlates a precise map of fluorescent fusion proteins localized using three-dimensional super-resolution optical microscopy with the fine ultrastructural context of three-dimensional electron micrographs. While it achieved the difficult simultaneous objectives of high photoactivated fluorophore preservation and ultrastructure preservation, it required a super-resolution optical and specialized electron microscope that is not available to many researchers. We present here a faster and more practical protocol with the advantage of a simpler two-dimensional optical (Photoactivated Localization Microscopy (PALM)) and scanning electron microscope (SEM) system that retains the often mutually exclusive attributes of fluorophore preservation and ultrastructure preservation. As before, cryosections were prepared using the Tokuyasu protocol, but the staining protocol was modified to be amenable for use in a standard SEM without the need for focused ion beam ablation. We show the versatility of this technique by labeling different cellular compartments and structures including mitochondrial nucleoids, peroxisomes, and the nuclear lamina. We also demonstrate simultaneous two-color PALM imaging with correlated electron micrographs. Lastly, this technique can be used with small-molecule dyes as demonstrated with actin labeling using phalloidin conjugated to a caged dye. By retaining the dense protein labeling expected for super-resolution microscopy combined with ultrastructural preservation, simplifying the tools required for correlative microscopy, and expanding the number of useful labels we expect this method to be accessible and valuable to a wide variety of researchers. PMID:24204771

  17. Connecting μ-fluidics to electron microscopy.

    PubMed

    Kemmerling, Simon; Ziegler, Jörg; Schweighauser, Gabriel; Arnold, Stefan A; Giss, Dominic; Müller, Shirley A; Ringler, Philippe; Goldie, Kenneth N; Goedecke, Nils; Hierlemann, Andreas; Stahlberg, Henning; Engel, Andreas; Braun, Thomas

    2012-01-01

    A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned. PMID:22094535

  18. 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 achievable with short intense pulses containing a large number of electrons, however, are limited to tens of nanometers and nanoseconds, respectively. This is because Coulomb repulsion is significant in such a pulse, and the electrons spread in space and time, thus limiting the beam coherence. It is therefore not possible to image the ultrafast elementary dynamics of complex transformations. The challenge was to retain the high spatial resolution of a conventional TEM while simultaneously enabling the temporal resolution required to visualize atomic-scale motions. In this Account, we discuss the development of four-dimensional ultrafast electron microscopy (4D UEM) and summarize techniques and applications that illustrate the power of the approach. In UEM, images are obtained either stroboscopically with coherent single-electron packets or with a single electron bunch. Coulomb repulsion is absent under the single-electron condition, thus permitting imaging, diffraction, and spectroscopy, all with high spatiotemporal resolution, the atomic scale (sub-nanometer and femtosecond). The time resolution is limited only by the laser pulse duration and energy carried by the electron packets; the CCD camera has no bearing on the temporal resolution. In the regime of single pulses of electrons, the temporal resolution of picoseconds can be attained when hundreds of electrons are in the bunch. The applications given here are selected to highlight phenomena of different length and time scales, from atomic motions during structural dynamics to phase transitions and nanomechanical oscillations. We conclude with a brief discussion of emerging methods, which include scanning ultrafast electron microscopy (S-UEM), scanning transmission ultrafast electron microscopy (ST-UEM) with convergent beams, and time-resolved imaging of biological structures at ambient conditions with environmental cells. PMID:22967215

  19. 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.

  20. Metallothioneins for correlative light and electron microscopy.

    PubMed

    Fernndez de Castro, Isabel; Sanz-Snchez, 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. PMID:25287836

  1. Quantitative analysis of transferrin cycling by automated fluorescence microscopy.

    PubMed

    Hirschmann, David T; Kasper, Christoph A; Spiess, Martin

    2015-01-01

    Surface receptors are transported between the plasma membrane and intracellular compartments by various endocytic mechanisms and by recycling via different pathways from sorting or recycling endosomes. The analysis of cellular components involved in mediating or regulating these transport steps is of high current interest and requires quantitative methods to determine rates of endocytosis and/or recycling. Various biochemical procedures to measure uptake of labeled ligand molecules or internalization and reappearance of surface-labeled receptors have been developed. Here, we describe a quantitative method based on fluorescence microscopy of adherent cells taking advantage of the transferrin (Tf) receptor as the prototype of cycling transport receptors. Tf is endocytosed with bound Fe(3+) and, upon release of the iron ion in endosomes, recycled as apo-Tf together with the receptor. To follow the ligand-receptor complex, fluorescently labeled Tf is used and detected microscopically with or without releasing Tf from cell surface receptors by acid stripping. To go beyond the observation of a few individual cells, automated fluorescence microscopy is employed to image thousands of cells at different time points and in parallel with different treatments (such as chemical inhibitors, siRNA silencing, or transfection of candidate genes) in a 96-well format. Computer-assisted image analysis allows unbiased quantitation of Tf content of each cell and to distinguish between different cell populations. PMID:25702129

  2. Quantitative X-ray Differential Interference Contrast Microscopy

    NASA Astrophysics Data System (ADS)

    Nakamura, Takashi

    Full-field soft x-ray microscopes are widely used in many fields of sciences. Advances in nanofabrication technology enabled short wavelength focusing elements with significantly improved spatial resolution. In the soft x-ray spectral region, samples as small as 12 nm can be resolved using micro zone-plates as the objective lens. In addition to conventional x-ray microscopy in which x-ray absorption difference provides the image contrast, phase contrast mechanisms such as differential phase contrast (DIC) and Zernike phase contrast have also been demonstrated These phase contrast imaging mechanisms are especially attractive at the x-ray wavelengths where phase contrast of most materials is typically 10 times stronger than the absorption contrast. With recent progresses in plasma-based x- ray sources and increasing accessibility to synchrotron user facilities, x-ray microscopes are quickly becoming standard measurement equipment in the laboratory. To further the usefulness of x-ray DIC microscopy this thesis explicitly addresses three known issues with this imaging modality by introducing new techniques and devices First, as opposed to its visible-light counterpart, no quantitative phase imaging technique exists for x-ray DIC microscopy. To address this issue, two nanoscale x-ray quantitative phase imaging techniques, using exclusive OR (XOR) patterns and zone-plate doublets, respectively, are proposed. Unlike existing x-ray quantitative phase imaging techniques such as Talbot interferometry and ptychography, no dedicated experimental setups or stringent illumination coherence are needed for quantitative phase retrieval. Second, to the best of our knowledge, no quantitative performance characterization of DIC microscopy exists to date. Therefore the imaging system's response to sample's spatial frequency is not known In order to gain in-depth understanding of this imaging modality, performance of x-ray DIC microscopy is quantified using modulation transfer function. A new illumination apparatus required for the transfer function analysis under partially coherent illumination is also proposed. Such a characterization is essential for a proper selection of DIC optics for various transparent samples under study. Finally, optical elements used for x-ray DIC microscopy are highly absorptive and high brilliance x-ray sources such as synchrotrons are generally needed for image contrast. To extend the use of x-ray DIC microscopy to a wider variety of applications, a high efficiency large numerical aperture optical element consisting of high reflective Bragg reflectors is proposed. Using Bragg reflectors, which have 70% 99% reflectivity at extreme ultraviolet and soft x-rays for all angles of glancing incidence, the first order focusing efficiency is expected to increase by 8 times compared to that of a typical Fresnel zone-plate. This thesis contributes to current nanoscale x-ray phase contrast imaging research and provides new insights for biological, material, and magnetic sciences

  3. Imaging intracellular quantum dots: fluorescence microscopy and transmission electron microscopy.

    PubMed

    Szymanski, Craig J; Yi, Hong; Liu, Joshua L; Wright, Elizabeth R; Payne, Christine K

    2013-01-01

    Quantum dots (QDs) and other nanoparticles require delivery and targeting for most intracellular applications. Despite many advances, intracellular delivery and targeting remains inefficient with many QDs remaining bound to the plasma membrane rather than internalized into the cell. The fluorescence resulting from these extracellular QDs results in a background signal that competes with intracellular QDs of interest. We present two methods for the reduction and discrimination of signal resulting from plasma membrane-bound QDs. The first method, a photophysical approach, uses an extracellular quencher to greatly reduce the fluorescence signal from extracellular QDs. This method is compatible with fast, widefield, fluorescence imaging in live cells. Results are presented for two extracellular quenchers, QSY-21 and trypan blue, used in combination with 655 nm emitting QDs. The use of an extracellular quencher can be extended to a wide variety of fluorophores. The second method uses transmission electron microscopy (TEM) to image thin (60-70 nm) slices of resin-embedded cells. The use of sectioned cells and high-resolution TEM makes it possible to discriminate between plasma membrane-bound and intracellular QDs. To overcome the difficulties associated with using TEM to image individual QDs in cells, we have utilized a silver enhancement method that significantly improves the contrast of QDs in TEM images. PMID:23749566

  4. Quantitative Fluorescence Microscopy Using Supported Lipid Bilayer Standards

    PubMed Central

    Galush, William J.; Nye, Jeffrey A.; Groves, Jay T.

    2008-01-01

    Routine quantitative analysis of biomolecule surface density by fluorescence microscopy has been limited by the difficulty of preparing appropriate calibration standards that relate measured fluorescence intensity to actual surface concentration. Supported lipid bilayers are planar fluid films of uniform density and composition which can incorporate a variety of lipidated fluorophores and work well as fluorescence standards. Here, we outline a straightforward strategy to calibrate digital micrographs of fluorescent surfaces such as planar cellular junctions for comparison to supported bilayer standards. It can be implemented with standard microscopy equipment. To illustrate the advantages of this approach, we quantify cell- and bilayer-side protein density patterns in a hybrid immunological synapse between a T-cell and a supported bilayer. PMID:18515392

  5. 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

  6. Quantitative single-molecule imaging by confocal laser scanning microscopy

    PubMed Central

    Vukojevi?, Vladana; Heidkamp, Marcus; Ming, Yu; Johansson, Bjrn; Terenius, Lars; Rigler, Rudolf

    2008-01-01

    A new approach to quantitative single-molecule imaging by confocal laser scanning microscopy (CLSM) is presented. It relies on fluorescence intensity distribution to analyze the molecular occurrence statistics captured by digital imaging and enables direct determination of the number of fluorescent molecules and their diffusion rates without resorting to temporal or spatial autocorrelation analyses. Digital images of fluorescent molecules were recorded by using fast scanning and avalanche photodiode detectors. In this way the signal-to-background ratio was significantly improved, enabling direct quantitative imaging by CLSM. The potential of the proposed approach is demonstrated by using standard solutions of fluorescent dyes, fluorescently labeled DNA molecules, quantum dots, and the Enhanced Green Fluorescent Protein in solution and in live cells. The method was verified by using fluorescence correlation spectroscopy. The relevance for biological applications, in particular, for live cell imaging, is discussed. PMID:19011092

  7. Quantitative polarized light microscopy of human cochlear sections.

    PubMed

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

    2015-02-01

    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

  8. Quantitative Phase Contrast Digital Holographic Microscopy in Biophotonics

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Langehanenberg, Patrik; von Bally, Gert

    2010-11-01

    Label-free, non-contact, non-destructive, on-line (video repetition rate), high resolution, full field (no scanning), quantitative analysis of morphology and dynamic processes in living cells are required features in life science research and medical diagnostics. Digital Holography combined with microscopic imaging provides these features simultaneously. The modular integration of digital holographic microscopy (DHM) into commercial microscopes yields an axial resolution with interferometric resolution while the lateral resolution is diffraction limited. As amplitude and phase are available by numerical reconstruction from a single digital hologram subsequent automated focus correction is enabled. The evaluation of quantitative digital holographic phase contrast images permits also an effective detection of lateral object movements. Thus, 3D tracking is achieved. The applicability of DHM techniques for dynamic live cell analysis is demonstrated by results from tumor cells and human erythrocytes.

  9. Scanning electron microscopy of lichen sclerosus*

    PubMed Central

    de Almeida, Hiram Larangeira; Bicca, Eduardo de Barros Coelho; Breunig, Juliano de Avelar; Rocha, Nara Moreira; Silva, Ricardo Marques e

    2013-01-01

    Lichen sclerosus is an acquired inflammatory condition characterized by whitish fibrotic plaques, with a predilection for the genital skin. We performed scanning electron microscopy of the dermis from a lesion of lichen sclerosus. Normal collagen fibers could be easily found in deeper layers of the specimen, as well as the transition to pathologic area, which seems homogenized. With higher magnifications in this transitional area collagen fibers are adherent to each other, and with very high magnifications a pearl chain aspect became evident along the collagen fibers. In the superficial dermis this homogenization is even more evident, collagen fibers are packed together and round structures are also observed. Rupture of collagen fibers and inflammatory cells were not found. These autoimmune changes of the extracellular matrix lead to the aggregation of immune complexes and/or changed matrix proteins along the collagen fibers, the reason why they seem hyalinized when examined by light microscopy. PMID:23739707

  10. Immunogold labelling for scanning electron microscopy.

    PubMed

    Goldberg, Martin W; Fiserova, Jindriska

    2010-01-01

    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 of anything from about x10 to x1,000,000. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualised. In order to identify that protein at high resolution, or to see and quantify its distribution at lower magnifications, samples can be labelled with antibodies. Any surface that can be exposed can potentially be studied in this way. Presented here is a generic method for immunogold labelling 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 labelling, 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:20602226

  11. Microfluidic system for transmission electron microscopy

    SciTech Connect

    Ring, Elisabeth A; De Jonge, Niels

    2010-01-01

    We present a microfluidic system that maintains liquid flow in a specimen chamber for (scanning) transmission electron microscope ((S)TEM) imaging. The specimen chamber consists of two ultra-thin silicon nitride windows supported by silicon microchips. They are placed in a specimen holder that seals the sample from the vacuum in the electron microscope, and incorporates tubing to and from the sample connected to a syringe pump outside the microscope. Using results obtained from fluorescence microscopy of microspheres flowing through the system, an equation to characterize the liquid flow through the system was calibrated. Gold nanoparticles of diameters of 30 and 100 nm moving in liquid were imaged with a 200 kV STEM. It was concluded that despite strong influences from Brownian motion, and sensitivity to small changes in the depth of the bypass channel, the electron microscopy flow data matched the calculated flow speed within an order of magnitude. The system allows for rapid (within a minute) liquid exchange, which can potentially be used, for example, to investigate the response of specimens, e.g., eukaryotic-, or bacterial cells, to certain stimuli.

  12. 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

  13. Quantitative electrostatic force microscopy with sharp silicon tips.

    PubMed

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

    2014-12-12

    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. PMID:25407683

  14. 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.

  15. Quantitative phase microscopy: how to make phase data meaningful

    NASA Astrophysics Data System (ADS)

    Goldstein, Goldie; Creath, Katherine

    2014-03-01

    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 background shape removal throughout a sequence of images. Work has been done to improve the phase unwrapping in two-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.

  16. HIV: The Initial Invasion | High Resolution Electron Microscopy

    Cancer.gov

    Skip to main content High Resolution Electron Microscopy High Resolution Electron Microscopy Center for Cancer Research at the National Institutes of Health Main menu Home Research 3D Correlative Imaging Methods Development Protein Complexes Viral Entry Publications Image

  17. Electron Microscopy of Botrytis cinerea Conidia

    PubMed Central

    Buckley, Patricia M.; Sjaholm, Virginia E.; Sommer, N. F.

    1966-01-01

    Buckley, Patricia M. (University of California, Davis), Virginia E. Sjaholm, and N. F. Sommer. Electron microscopy of Botrytis cinerea conidia. J. Bacteriol. 91:20372044. 1966.Sections of germinating and nongerminating Botrytis cinerea conidia were examined with an electron microscope. Uranyl acetate or lead citrate provided contrast between membranes and cytoplasm. Membrane-bounded, dense inclusions previously unreported in dormant spores were termed storage bodies. Whorled structures, spherules, granules, and membrane loops were seen within these inclusions. The various forms assumed by the enclosed materials closely resemble phospholipid inclusions described for other cells. It is suggested that the inclusions provide material for the assembly of membranous organelles during germination. Utilization of the stored material apparently results in extensive vacuolization in advanced germinants. Images PMID:5949251

  18. Scanning electron microscopy studies of bacterial cultures

    NASA Astrophysics Data System (ADS)

    Swinger, Tracy; Blust, Brittni; Calabrese, Joseph; Tzolov, Marian

    2012-02-01

    Scanning electron microscopy is a powerful tool to study the morphology of bacteria. We have used conventional scanning electron microscope to follow the modification of the bacterial morphology over the course of the bacterial growth cycle. The bacteria were fixed in vapors of Glutaraldehyde and ruthenium oxide applied in sequence. A gold film of about 5 nm was deposited on top of the samples to avoid charging and to enhance the contrast. We have selected two types of bacteria Alcaligenes faecalis and Kocuria rhizophila. Their development was carefully monitored and samples were taken for imaging in equal time intervals during their cultivation. These studies are supporting our efforts to develop an optical method for identification of the Gram-type of bacterial cultures.

  19. 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

  20. Direct imaging of crystal structure and defects in metastable Ge{sub 2}Sb{sub 2}Te{sub 5} by quantitative aberration-corrected scanning transmission electron microscopy

    SciTech Connect

    Ross, Ulrich; Lotnyk, Andriy Thelander, Erik; Rauschenbach, Bernd

    2014-03-24

    Knowledge about the atomic structure and vacancy distribution in phase change materials is of foremost importance in order to understand the underlying mechanism of fast reversible phase transformation. In this Letter, by combining state-of-the-art aberration-corrected scanning transmission electron microscopy with image simulations, we are able to map the local atomic structure and composition of a textured metastable Ge{sub 2}Sb{sub 2}Te{sub 5} thin film deposited by pulsed laser deposition with excellent spatial resolution. The atomic-resolution scanning transmission electron microscopy investigations display the heterogeneous defect structure of the Ge{sub 2}Sb{sub 2}Te{sub 5} phase. The obtained results are discussed. Highly oriented Ge{sub 2}Sb{sub 2}Te{sub 5} thin films appear to be a promising approach for further atomic-resolution investigations of the phase change behavior of this material class.

  1. 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.

  2. High-resolution transmission electron microscopy: the ultimate nanoanalytical technique.

    PubMed

    Thomas, John Meurig; Midgley, Paul A

    2004-06-01

    To be able to determine the elemental composition and morphology of individual nanoparticles consisting of no more than a dozen or so atoms that weigh a few zeptograms (10(-21) g) is but one of the attainments of modern electron microscopy. With slightly larger specimens (embracing a few unit cells of the structure) their symmetry, crystallographic phase, unit-cell dimension, chemical composition and often the valence state (from parallel electron spectroscopic measurements) of the constituent atoms may also be determined using a scanning beam of electrons of ca. 0.5 nm diameter. Nowadays electron crystallography, which treats the digital data of electron diffraction (ED) and high-resolution transmission electron microscope (HRTEM) images of minute (ca. 10(-18)g) specimens in a quantitatively rigorous manner, solves hitherto unknown structures just as X-ray diffraction does with bulk single crystals. In addition, electron tomography (see cover photograph and its animation) enables a three-dimensional picture of the internal structure of minute objects, such as nanocatalysts in a single pore, as well as structural faults such as micro-fissures, to be constructed with a resolution of 1 nm from an angular series of two-dimensional (projected) images. Very recently (since this article was first written) a new meaning has been given to electron crystallography as a result of the spatio-temporal resolution of surface phenomena achieved on a femtosecond timescale. PMID:15154029

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

    PubMed Central

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

    2016-01-01

    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. PMID:26830146

  4. 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.

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

    PubMed

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

    2016-01-01

    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. PMID:26830146

  6. 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 hybridization method (FISH). This approach was used for evaluation of contribution of each gram-negative bactera group. No significant difference between the main soil gram-negative bacterial groups (phylum Proteobacteria and Bacteroidetes) was found both under anaerobic and anaerobic conditions in chernozem in the topsoil. Thus soil gram-negative bacteria play an important ecological role in natural polymer degradation as common group of microorganisms. Another approach with using cascade filtration technique for bacterial population density estimation in chernozem was compared to classical method of fluorescent microscopy. Quantification of soil bacteria with cascade filtration provided by filters with different diameters and filtering of soil suspension in fixed amount. In comparison to the classical fluorescent microscopy method the modification with filtration of soil suspension provided to quantify more bacterial cells. Thus biomass calculation results of soil bacteria by using classical fluorescent microscopy could be underestimated and combination with cascade filtration technique allow to avoid potential experimental error. Thereby, combination and comparison of several fluorescent microscopy methods modifications established during the research provided miscellaneous approaches in soil bacteria quantification and analysis of ecological roles of soil microorganisms.

  7. Quantitative Imaging of Single Unstained Magnetotactic Bacteria by Coherent X-ray Diffraction Microscopy.

    PubMed

    Fan, Jiadong; Sun, Zhibin; Zhang, Jian; Huang, Qingjie; Yao, Shengkun; Zong, Yunbing; Kohmura, Yoshiki; Ishikawa, Tetsuya; Liu, Hong; Jiang, Huaidong

    2015-06-16

    Novel coherent diffraction microscopy provides a powerful lensless imaging method to obtain a better understanding of the microorganism at the nanoscale. Here we demonstrated quantitative imaging of intact unstained magnetotactic bacteria using coherent X-ray diffraction microscopy combined with an iterative phase retrieval algorithm. Although the signal-to-noise ratio of the X-ray diffraction pattern from single magnetotactic bacterium is weak due to low-scattering ability of biomaterials, an 18.6 nm half-period resolution of reconstructed image was achieved by using a hybrid input-output phase retrieval algorithm. On the basis of the quantitative reconstructed images, the morphology and some intracellular structures, such as nucleoid, poly?-hydroxybutyrate granules, and magnetosomes, were identified, which were also confirmed by scanning electron microscopy and energy dispersive spectroscopy. With the benefit from the quantifiability of coherent diffraction imaging, for the first time to our knowledge, an average density of magnetotactic bacteria was calculated to be ?1.19 g/cm(3). This technique has a wide range of applications, especially in quantitative imaging of low-scattering biomaterials and multicomponent materials at nanoscale resolution. Combined with the cryogenic technique or X-ray free electron lasers, the method could image cells in a hydrated condition, which helps to maintain their natural structure. PMID:26006162

  8. High voltage electron microscopy of lunar samples

    NASA Technical Reports Server (NTRS)

    Fernandez-Moran, H.

    1973-01-01

    Lunar pyroxenes from Apollo 11, 12, 14, and 15 were investigated. The iron-rich and magnesium-rich pyroxene specimens were crushed to a grain size of ca. 50 microns and studied by a combination of X-ray and electron diffraction, electron microscopy, 57 Fe Mossbauer spectroscopy and X-ray crystallography techniques. Highly ordered, uniform electron-dense bands, corresponding to exsolution lamellae, with average widths of ca. 230A to 1000A dependent on the source specimen were observed. These were?qr separated by wider, less-dense interband spacings with average widths of ca. 330A to 3100A. In heating experiments, splitting of the dense bands into finer structures, leading finally to obliteration of the exsolution lamellae was recorded. The extensive exsolution is evidence for significantly slower cooling rates, or possibly annealing, at temperatures in the subsolidus range, adding evidence that annealing of rock from the surface of the moon took place at ca. 600 C. Correlation of the band structure with magnetic ordering at low temperatures and iron clustering within the bands was studied.

  9. Hexamethyldisilazane for scanning electron microscopy of Gastrotricha.

    PubMed

    Hochberg, R; Litvaitis, M K

    2000-01-01

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

  10. Analyzing Tau Aggregation with Electron Microscopy.

    PubMed

    Huseby, Carol J; Kuret, Jeff

    2016-01-01

    Conversion of monomeric tau protein into filamentous aggregates is a defining event in the pathogenesis of Alzheimer's disease. To gain insight into disease pathogenesis, the mechanisms that trigger and mediate tau aggregation are under intense investigation. Characterization efforts have relied primarily on recombinant tau protein preparations and high-throughput solution-based detection methods such as thioflavin-dye fluorescence and laser-light-scattering spectroscopies. Transmission electron microscopy (TEM) is a static imaging tool that complements these approaches by detecting individual tau filaments at nanometer resolution. In doing so, it can provide unique insight into the quality, quantity, and composition of synthetic tau filament populations. Here we describe protocols for analysis of tau filament populations by TEM for purposes of dissecting aggregation mechanism. PMID:26453208

  11. 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.

  12. Toward site-specific dopant contrast in scanning electron microscopy.

    PubMed

    Druckmllerov, Zdena; Kolbal, Miroslav; Vystav?l, Tom; Sikola, Tom

    2014-08-01

    Since semiconductor devices are being scaled down to dimensions of several nanometers there is a growing need for techniques capable of quantitative analysis of dopant concentrations at the nanometer scale in all three dimensions. Imaging dopant contrast by scanning electron microscopy (SEM) is a very promising method, but many unresolved issues hinder its routine application for device analysis, especially in cases of buried layers where site-specific sample preparation is challenging. Here, we report on optimization of site-specific sample preparation by the focused Ga ion beam (FIB) technique that provides improved dopant contrast in SEM. Similar to FIB lamella preparation for transmission electron microscopy, a polishing sequence with decreasing ion energy is necessary to minimize the thickness of the electronically dead layer. We have achieved contrast values comparable to the cleaved sample, being able to detect dopant concentrations down to 110(16) cm-3. A theoretical model shows that the electronically dead layer corresponds to an amorphized Si layer formed during ion beam polishing. Our results also demonstrate that contamination issues are significantly suppressed for FIB-treated samples compared with cleaved ones. PMID:24844888

  13. Visualization of yeast cells by electron microscopy.

    PubMed

    Osumi, Masako

    2012-01-01

    In the 1970s, hydrocarbon or methanol utilizable yeasts were considered as a material for foods and ethanol production. During the course of studies into the physiology of yeasts, we found that these systems provide a suitable model for the biogenesis and ultrastructure research of microbodies (peroxisomes). Microbodies of hydrocarbon utilizing Candida tropicalis multiply profusely from the preexisting microbody. ? oxidation enzymes in the microbody were determined by means of immunoelectron microscopy. We examined the ultrastructure of Candida boidinii microbodies grown on methanol, and found a composite crystalloid of two enzymes, alcohol oxidase and catalase, by analyzing using the optical diffraction and filtering technique and computer simulation. We established methods for preparing the protoplasts of Schizosaccharomyces pombe and conditions for the complete regeneration of the cell wall. The dynamic process of cell wall formation was clarified through our study of the protoplasts, using an improved ultra high resolution (UHR) FESEM S-900 and an S-900LV. It was found that ?-1,3-glucan, ?-1,6-glucan and ?-1,3-glucan, as well as ?-galactomannan, are ingredients of the cell wall. The process of septum formation during cell division was examined after cryo-fixation by high pressure freezing (HPF). It was also found that ?-1,3- and ?-1,3-glucans were located in the invaginating nascent septum, and later, highly branched ?-1,6-glucan also appeared on the second septum. The micro-sampling method, using a focused ion beam (FIB), has been applied to our yeast cell wall research. A combination of FIB and scanning transmission electron microscopy is useful in constructing 3D images and analyzing the molecular architecture of cells, as well as for electron tomography of thick sections of biological specimens. PMID:23231852

  14. Comprehensive quantitative evaluation of FLIM-FRET microscopy

    NASA Astrophysics Data System (ADS)

    Wallrabe, Horst; Sun, Yuangsheng; Svindrych, Zdenek; Periasamy, Ammasi

    2015-03-01

    Average lifetime between the usually bi-exponential double-label specimen and a mono-exponential single donor sample serves as a basis for the calculation of the average energy transfer efficiency (E). This semi-quantitative approach however does not fully explore cellular functions, such as endosomal pH differences, specific morphological features, examining sub-populations and the like. We applied a different, quantitative Region-of-Interest (ROI)-based method in 2 live-cell assays by TCSPC FLIM-FRET microscopy: a 5 amino-acid linked FRET standard and mouse pituitary cells expressing a dimerized C/EBP?-bZip transcription factor in the nucleus, both tagged with Cerulean (C) and Venus (V). ROIs with different selection thresholds were generated and compared. Average lifetimes are similar, but ratios between them and other subtle differences are revealed by comprehensive distribution information. Following published references, we also explored 3 different methods to calculate FLIM-FRET energy transfer efficiencies for the Cerulean- Venus constructs, producing differences and supporting the long-held notion that E is called 'apparent' efficiency. FRET's greatest contribution continues to be exploring changes taking place at the cellular level and quantifying differences in relative terms between control and variables.

  15. 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 of conversion. The last chapter investigates another quantitative parameter, the orientation, of the CH2 vibrational mode in natural (cotton) and synthetic (rayon) cellulose fibers. Coupled with SHG, this method gives insight into the origins of observed optical nonlinearities in dry and hydrated cellulose fibers.

  16. Digital holographic microscopy: a quantitative label-free microscopy technique for phenotypic screening.

    PubMed

    Rappaz, Benjamin; Breton, Billy; Shaffer, Etienne; Turcatti, Gerardo

    2014-01-01

    Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays. PMID:24152227

  17. Digital Holographic Microscopy: A Quantitative Label-Free Microscopy Technique for Phenotypic Screening

    PubMed Central

    Rappaz, Benjamin; Breton, Billy; Shaffer, Etienne; Turcatti, Gerardo

    2014-01-01

    Digital Holographic Microscopy (DHM) is a label-free imaging technique allowing visualization of transparent cells with classical imaging cell culture plates. The quantitative DHM phase contrast image provided is related both to the intracellular refractive index and to cell thickness. DHM is able to distinguish cellular morphological changes on two representative cell lines (HeLa and H9c2) when treated with doxorubicin and chloroquine, two cytotoxic compounds yielding distinct phenotypes. We analyzed parameters linked to cell morphology and to the intracellular content in endpoint measurements and further investigated them with timelapse recording. The results obtained by DHM were compared with other optical label-free microscopy techniques, namely Phase Contrast, Differential Interference Contrast and Transport of Intensity Equation (reconstructed from three bright-field images). For comparative purposes, images were acquired in a common 96-well plate format on the different motorized microscopes. In contrast to the other microscopies assayed, images generated with DHM can be easily quantified using a simple automatized on-the-fly analysis method for discriminating the different phenotypes generated in each cell line. The DHM technology is suitable for the development of robust and unbiased image-based assays.

  18. Bright-field quantitative phase microscopy (BFQPM) for accurate phase imaging using conventional microscopy hardware

    NASA Astrophysics Data System (ADS)

    Jenkins, Micah; Gaylord, Thomas K.

    2015-03-01

    Most quantitative phase microscopy methods require the use of custom-built or modified microscopic configurations which are not typically available to most bio/pathologists. There are, however, phase retrieval algorithms which utilize defocused bright-field images as input data and are therefore implementable in existing laboratory environments. Among these, deterministic methods such as those based on inverting the transport-of-intensity equation (TIE) or a phase contrast transfer function (PCTF) are particularly attractive due to their compatibility with Khler illuminated systems and numerical simplicity. Recently, a new method has been proposed, called multi-filter phase imaging with partially coherent light (MFPI-PC), which alleviates the inherent noise/resolution trade-off in solving the TIE by utilizing a large number of defocused bright-field images spaced equally about the focal plane. Despite greatly improving the state-ofthe- art, the method has many shortcomings including the impracticality of high-speed acquisition, inefficient sampling, and attenuated response at high frequencies due to aperture effects. In this report, we present a new method, called bright-field quantitative phase microscopy (BFQPM), which efficiently utilizes a small number of defocused bright-field images and recovers frequencies out to the partially coherent diffraction limit. The method is based on a noiseminimized inversion of a PCTF derived for each finite defocus distance. We present simulation results which indicate nanoscale optical path length sensitivity and improved performance over MFPI-PC. We also provide experimental results imaging live bovine mesenchymal stem cells at sub-second temporal resolution. In all, BFQPM enables fast and accurate phase imaging with unprecedented spatial resolution using widely available bright-field microscopy hardware.

  19. Imaging Surface Topography using Lloyd's Mirror in Photoemission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Jesson, D. E.; Pavlov, K. M.; Morgan, M. J.; Usher, B. F.

    2007-07-01

    We use Lloyd’s mirror to modulate electron photoemission in photoemission electron microscopy. This results in the projection of Lloyd’s fringes on to three-dimensional (3D) surface objects. An iterative reconstruction method is used to correct for distortions in the fringe pattern due to the cathode immersion lens, thereby providing a quantitative interpretation of surface shape. It is therefore possible to extract 3D height information directly from a two-dimensional, plan-view image. The technique is of sufficient intensity and contrast to study real-time changes in surface topography and we apply the method to study unusual contact-line dynamics during the reactive wetting of metal droplets.

  20. Electron microscopy: cytology of cell fractions.

    PubMed

    NOVIKOFF, A B

    1956-11-16

    It should be evident from this brief account that electron microscopy of thin sections is an invaluable asset in the study of fractions isolated by differential centrifugation. I have tried to indicate how the integrity of particles, purity of fractions, and the existence of new particles can be established through its use. I have also suggested the desirability of a common terminology for all cytology -classic, electron-microscopic, and biochemical. Some have expressed the opinion that neutral terms such as alpha, beta, and gamma membranes (59) are more useful than ergastoplasm, Golgi apparatus, and so forth. As helpful as such neutral terms may be in describing intracellular structures, they do not appear to me to substitute for historically rooted cytological names. Note added in proof: Since this article went to press there has appeared an important article by G. E. Palade and P. Siekevitz (60). These authors consider that the vesicles without granules found in the microsome fraction were "probably derived from the smooth surfaced parts of the endoplasmic reticulum." The latter were found to be continuous with the granule-studded membranes; "the two varieties of profiles represent local differentiation within a common system." The authors confirm the finding of Rouiller (18) and Novikoff et al. (7) of the dense bodies adjacent to the bile canaliculi and describe their presence in the microsome fraction as a "minor component." PMID:13380407

  1. Low-energy electron point source microscopy and electron holography

    NASA Astrophysics Data System (ADS)

    Mutus, Josh

    Low energy electron point source (LEEPS) microscopy is the simplest embodiment of an electron microscope, consisting of only a source, a sample and a detector. In a specific regime, LEEPS may also be used to create in-line holograms; special interference patterns that contain the information about the entire electron wavefront, including the structure of the sample and electromagnetic field around it. This work describes the design, construction and characterization of a microscope designed to performs LEEPS microscopy and electron holography at the nanoscale. An overview of previous experimental apparatus are discussed. Also, the impact of spatial and energetic inhomogeneities of the electron source on the quality and resolution of the hologram, in terms of the numerical aperture of the microscope and the virtual source size of the electron emitter. The design of the microscope itself is presented including the system for isolating the microscope from contamination, mechanical vibration and electrical noise. Using scanning tunnelling microscopy (STM) the microscope is shown to be stable within 0.1 . Instructions for the maintenance of the system are presented for future users of the microscope and to illustrate many of the systems described in the design of the microscope. The source of electrons used in the LEEPS microscope is a tungsten tip sharpened so as to field-emit electrons from a single atom. The technique for crafting such tips by field-assisted etching with nitrogen is described along with a discussion of the parameters used to control the aspect-ratio of the tip. Several samples are investigated using LEEPS: a sharp silicon nitride edge, a carbon nanotube bundle and graphene. The sample preparation techniques are discussed for each sample. Also, simple models for describing the resulting fringe patterns are proposed. There are several benefits associated with using LEEPS, including the lack of beam induced morphological changes or contamination. The samples are used to elucidate many properties about the optical system of the microscope, most importantly the expected resolution of the system. The software designed for the microscope to acquire images with high fidelity and for post-processing and correcting data is demonstrated. The microscope is shown to have a virtual source size of 1.60.6 a figure that exceeds published results form similar instruments. Preliminary holographic reconstructions are shown. The work concludes with a discussion of the parameters to be optimized in order to reach atomic resolution.

  2. Electron Microscopy: Phase Transition Singled Out

    SciTech Connect

    Browning, Nigel D.

    2013-05-01

    One of the fundamental challenges within nanotechnology is to understand and control how nanoscale properties are initiated, evolve, and eventually terminate as a system moves from an individual nanostructure towards the meso- and macro-scale ensembles that are used in most applications. The ability to directly observe individual nanostructures and characterize their structure and composition has long been within the purview of transmission electron microscopy (TEM). The almost ubiquitous application of spherical aberration correction in TEM and in scanning-TEM (STEM) that has occurred over the last 10 years, now means it is possible to routinely characterize such nanostructures with both atomic resolution and sensitivity [1,2]. The development of temporal resolution in the TEM and the ability to study fast dynamics, on the other hand, has only recently come to the forefront of instrumentation development and is currently defined by two different approaches in the use of photoemission sources: the single shot s-ns dynamic TEM (DTEM) [3] and the stroboscopic ps-fs 4-D EM [4]. In the case of the DTEM, the goal is to observe the longer timescale irreversible structural changes that occur during nucleation and growth phenomena (here the single shot approach means there are enough electrons in a single pulsed beam to form a complete image). The 4-D EM focuses on a stroboscopic approach with the goal of studying very rapid reversible effects that occur during phase transitions (here an image is composed of thousands of pump-probe events each occurring with exactly the same time signature, with an individual pulse containing only a few electrons).

  3. 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 synthesis and growth mechanism of highly monodispersed Cu-Pt nanoclusters. The advance electron microscopy of microanalysis allowed us to study the distribution of Cu and Pt with atomistic resolution. The microanalysis revealed that Pt is embedded randomly in the Cu lattice. A novel grand canonical - Langevin dynamics simulation showed the formation of alloy structures in good agreement with the experimental evidence. Finally, we demonstrated the synthesis of AgPd-Pt trimetallic nanoparticles with two different morphologies: multiply twinned core-shell, and hollow particles. We also investigated the growth mechanism of the nanoparticles using grand canonical-Monte Carlo simulations. We found that the Pt regions grow at overpotentials on the AgPd nanoalloys, forming 3D islands at the early stages of the deposition process and presenting very good agreement between the simulated structures and those observed experimentally. Similarly, we also investigated AuCu/Pt core-shell trimetallic nanoparticles, presenting new way to control the nanoparticles morphologies due to the presence of third metal (Pt). Where, we observed the Pt layers are overgrowth on the as prepared AuCu core by Frank-van der Merwe (FM) and Stranski-Krastanov (SK) growth modes. In addition, these nanostructure presents high index facet surfaces with {211} and (321} families, that are highly open structure surfaces and interesting for the catalytic applications. The results of these studies will be useful for the future applications and the design of advanced functional nanomaterials.

  4. 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.

  5. 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.; Stankovic, Konstantina M.

    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

  6. 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.

  7. 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.

  8. Imaging Cytoskeleton Components by Electron Microscopy.

    PubMed

    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

  9. 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

  10. Quantitative Electron-Excited X-Ray Microanalysis of Borides, Carbides, Nitrides, Oxides, and Fluorides with Scanning Electron Microscopy/Silicon Drift Detector Energy-Dispersive Spectrometry (SEM/SDD-EDS) and NIST DTSA-II.

    PubMed

    Newbury, Dale E; Ritchie, Nicholas W M

    2015-10-01

    A scanning electron microscope with a silicon drift detector energy-dispersive X-ray spectrometer (SEM/SDD-EDS) was used to analyze materials containing the low atomic number elements B, C, N, O, and F achieving a high degree of accuracy. Nearly all results fell well within an uncertainty envelope of 5% relative (where relative uncertainty (%)=[(measured-ideal)/ideal]100%). Quantification was performed with the standards-based "k-ratio" method with matrix corrections calculated based on the Pouchou and Pichoir expression for the ionization depth distribution function, as implemented in the NIST DTSA-II EDS software platform. The analytical strategy that was followed involved collection of high count (>2.5 million counts from 100 eV to the incident beam energy) spectra measured with a conservative input count rate that restricted the deadtime to ~10% to minimize coincidence effects. Standards employed included pure elements and simple compounds. A 10 keV beam was employed to excite the K- and L-shell X-rays of intermediate and high atomic number elements with excitation energies above 3 keV, e.g., the Fe K-family, while a 5 keV beam was used for analyses of elements with excitation energies below 3 keV, e.g., the Mo L-family. PMID:26365439

  11. 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.

  12. 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. PMID:25785884

  13. Analytical electron microscopy in mineralogy; exsolved phases in pyroxenes

    USGS Publications Warehouse

    Nord, G.L., Jr.

    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.

  14. 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 application of the technique to solving seemingly intractable materials problems in addition to discovery-based research. Our goal with this Perspective is to bring the capabilities of TIEM to the-attention of materials scientists, chemists, physicists, and engineers in hopes that new,avenues of research emerge and to make clear the large parameter space that is opened by extending TEM, and the ability to readily manipulate electron trajectories and energies, into the ultrafast domain.

  15. 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. PMID:25885075

  16. 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 m-sized inclusions of FeS and have microstructurally complex rims. Par-ticle RA-QD02-0125 is rounded and has numerous sub-m 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 of [1]. The dis-ordered rim is nanocrystalline with minor amorphous material between crystalline domains. Quantitative element maps show the outermost 10 nm of the disordered rim is Si-rich. Discussion and Conclusions: Both particles record the ef-fects of space weathering processes on Itokawa. Noguchi et al. [2] proposed that the disordered rims they observed on Itokawa particles largely result from solar wind radiation damage and we arrive at a similar conclusion for the two particles we analyzed. The microstructure of the S-depleted layer on the pyrrhotite grain in RA-QD02-0125 is similar to that observed in troilite irradiated with 1018 4 kV He+ [3, 4]. Prolonged irradiation has also been shown to disorder pyrrhotite such that the superstructure reflec-tions are lost [5].

  17. 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 microscopy techniques in strongly correlated electron systems and nanostructured materials. As a first step, with the support of Materials Science Division, Office of Basic Energy Science, US Department of Energy, and the New York State Office of Science, Technology, and Academic Research, recently we acquired three aberration-corrected electron microscopes from the three major microscope manufacturers, i.e., JEOL, Hitachi, and FEI. The Hitachi HD2700C is equipped with a probe corrector, the FEI Titan 80-300 has an imaging corrector, while the JEOL2200MCO has both. All the correctors are of the dual-hexapole type, designed and manufactured by CEOS GmbH based on the design due to Rose and Haider [3, 18]. All these three are one-of-a-kind in the US, designed for specialized capabilities in characterizing nanoscale structure. In this chapter, we review the performance of these state-of-the art instruments and the new challenges associated with the improved spatial resolution, including the environment requirements of the laboratory that hosts these instruments. Although each instrument we describe here has its own strengths and drawbacks, it is not our intention to rank them in terms of their performance, especially their spatial resolution in imaging.

  18. Electron Microscopy for Rapid Diagnosis of Emerging Infectious Agents1

    PubMed Central

    Gelderblom, Hans R.

    2003-01-01

    Diagnostic electron microscopy has two advantages over enzyme-linked immunosorbent assay and nucleic acid amplification tests. After a simple and fast negative stain preparation, the undirected, open view of electron microscopy allows rapid morphologic identification and differential diagnosis of different agents contained in the specimen. Details for efficient sample collection, preparation, and particle enrichment are given. Applications of diagnostic electron microscopy in clinically or epidemiologically critical situations as well as in bioterrorist events are discussed. Electron microscopy can be applied to many body samples and can also hasten routine cell culture diagnosis. To exploit the potential of diagnostic electron microscopy fully, it should be quality controlled, applied as a frontline method, and be coordinated and run in parallel with other diagnostic techniques. PMID:12643823

  19. 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

  20. 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.

  1. Silicon nitride windows for electron microscopy of whole cells.

    PubMed

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

    2011-09-01

    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 there is 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 the microchips 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

  2. 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

  3. 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 theoretical predictions.

  4. Ion-induced electron emission microscopy

    DOEpatents

    Doyle, Barney L. (Albuquerque, NM); Vizkelethy, Gyorgy (Albuquerque, NM); Weller, Robert A. (Brentwood, TN)

    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.

  5. Soft X-ray contact microscopy and transmission electron microscopy: Comparative study of biological samples

    NASA Astrophysics Data System (ADS)

    Limongi, T.; Palladino, L.; Bernieri, E.; Tomassetti, G.; Reale, L.; Flora, F.; Cesare, P.; Ercole, C.; Aimola, P.; Ragnelli, A. M.

    2003-03-01

    Isolated cellular organelles (mitochondria, chloroplasts) and cultured bacteria were analysed both by soft X-ray contact microscopy (SXCM), and by transmission electron microscopy (TEM) after negative staining. For each sample, a comparison was performed between images obtained with either technique, with the aim of facilitating the interpretation of SXCM images. The validity and the limits of this comparative approach are discussed.

  6. 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.

  7. 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.

  8. Methods Development | High Resolution Electron Microscopy

    Cancer.gov

    The development of new technology and new methods has been central to our labs mission. One key development has been that of a complete framework for alignment, classification, and averaging of volumes derived by electron tomography that is computationally efficient and effectively accounts for the missing wedge that is inherent to limited angle electron tomography.

  9. Study of dynamic grain growth by electron microscopy and EBSD.

    PubMed

    Rofman, O V; Bate, P S; Brough, I; Humphreys, F J

    2009-03-01

    The effect of hot deformation on fully recrystallized aluminium-copper alloys (Al-4wt%Cu and Al-33wt%Cu) with different volume fractions of CuAl(2) has been studied. The alloys are Zener pinned systems with different superplastic properties. Strain-induced grain growth, observed in both alloys, was quantitatively estimated by means of electron microscopy and EBSD and compared with the rate of static grain growth. Surface marker observations and in situ hot-deformation experiments combined with EBSD were aimed at clarifying the mechanisms responsible for the changes in the deformed microstructures. A sequence of secondary and backscattered electron images and EBSD maps was obtained during in situ SEM deformation with different testing conditions. Overlaying EBSD maps for the Al-4wt%Cu with channelling contrast images showed that grain boundary motion occurred during deformation, creating a layered structure and leading to an increase in size of some grains and shrinkage of others. Of a particular interest are results related to behaviour of CuAl(2) in superplastic Al-33wt%Cu during deformation, including several problems with the use of EBSD in this alloy. PMID:19250464

  10. Carbon nanomaterial studied by atomic-force and electron microscopies

    SciTech Connect

    Bobrinetski, I. I.; Kukin, V. N.; Nevolin, V. K. Simunin, M. M.

    2008-12-15

    It is suggested to use the atomic-force microscopy (AFM) and transmission electron microscopy (TEM) to study carbon material synthesized by catalytic pyrolysis of ethanol. It is shown how AFM and TEM can be employed to determine the geometric parameters of carbon nanofibers and nanotubes, examine their mechanical and adhesion characteristics, and analyze their structure.

  11. 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)

  12. Outcome of the first electron microscopy validation task force meeting.

    PubMed

    Henderson, Richard; Sali, Andrej; Baker, Matthew L; Carragher, Bridget; Devkota, Batsal; Downing, Kenneth H; Egelman, Edward H; Feng, Zukang; Frank, Joachim; Grigorieff, Nikolaus; Jiang, Wen; Ludtke, Steven J; Medalia, Ohad; Penczek, Pawel A; Rosenthal, Peter B; Rossmann, Michael G; Schmid, Michael F; Schrder, Gunnar F; Steven, Alasdair C; Stokes, David L; Westbrook, John D; Wriggers, Willy; Yang, Huanwang; Young, Jasmine; Berman, Helen M; Chiu, Wah; Kleywegt, Gerard J; Lawson, Catherine L

    2012-02-01

    This Meeting Review describes the proceedings and conclusions from the inaugural meeting of the Electron Microscopy Validation Task Force organized by the Unified Data Resource for 3DEM (http://www.emdatabank.org) and held at Rutgers University in New Brunswick, NJ on September 28 and 29, 2010. At the workshop, a group of scientists involved in collecting electron microscopy data, using the data to determine three-dimensional electron microscopy (3DEM) density maps, and building molecular models into the maps explored how to assess maps, models, and other data that are deposited into the Electron Microscopy Data Bank and Protein Data Bank public data archives. The specific recommendations resulting from the workshop aim to increase the impact of 3DEM in biology and medicine. PMID:22325770

  13. 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.

  14. 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...

  15. DNA origami-based standards for quantitative fluorescence microscopy.

    PubMed

    Schmied, Jrgen J; Raab, Mario; Forthmann, Carsten; Pibiri, Enrico; Wnsch, Bettina; Dammeyer, Thorben; Tinnefeld, Philip

    2014-01-01

    Validating and testing a fluorescence microscope or a microscopy method requires defined samples that can be used as standards. DNA origami is a new tool that provides a framework to place defined numbers of small molecules such as fluorescent dyes or proteins in a programmed geometry with nanometer precision. The flexibility and versatility in the design of DNA origami microscopy standards makes them ideally suited for the broad variety of emerging super-resolution microscopy methods. As DNA origami structures are durable and portable, they can become a universally available specimen to check the everyday functionality of a microscope. The standards are immobilized on a glass slide, and they can be imaged without further preparation and can be stored for up to 6 months. We describe a detailed protocol for the design, production and use of DNA origami microscopy standards, and we introduce a DNA origami rectangle, bundles and a nanopillar as fluorescent nanoscopic rulers. The protocol provides procedures for the design and realization of fluorescent marks on DNA origami structures, their production and purification, quality control, handling, immobilization, measurement and data analysis. The procedure can be completed in 1-2 d. PMID:24833175

  16. Quantitative electron and gas cloud experiments

    NASA Astrophysics Data System (ADS)

    Molvik, A. W.; Kireeff Covo, M.; Cohen, R. H.; Friedman, A.; Sharp, W. M.; Baca, David; Bieniosek, F. M.; Leister, C.; Seidl, P. A.; Vay, J.-L.

    2007-07-01

    Electrons can accumulate in and degrade the quality of positively charged beams. This is a well-known problem in proton storage rings. Heavy-ion rings are more frequently limited by gas pressure-rise effects. Both effects may limit how closely the beam radius can approach the beam-tube radius in a heavy-ion linac. We study beams of 1 MeV K + with currents of up to 180 mA in the High-Current Experiment (HCX), and compare our work with simulations. The theory and simulation results are discussed in a companion papers. We have developed the first diagnostics that quantitatively measure the accumulation of electrons in a beam [M. Kireeff Covo, A. Molvik, A. Friedman, J.-L. Vay, P. Seidl, G. Logan, D. Baca, J.L. Vujic, Phys. Rev. Lett. 97 (2006) 054801; M. Kireeff Covo, et al., Nucl. Instr. and Meth. A, 2007, in press, doi:10.1016/j.nima.2007.02.045.]. This will enable the particle balance to be measured for each source of electrons in a linac: ionization of gas, emission from walls surrounding the beam, and emission from an end wall coupled with electron drifts upstream through quadrupole magnets, and electron-trapping efficiencies can be determined. Experiments where the heavy-ion beam is transported with solenoid magnetic fields, rather than with quadrupole magnetic or electrostatic fields, are being initiated. We discuss plans for experiments using electrode sets (in the middle and at the ends of magnets) to either expel or to trap electrons within the magnets. We observe oscillations of the electron density and position in the last quadrupole magnet when we flood the beam with electrons from an end wall. These oscillations, near 6 MHz, are observed to grow from the center of the magnet while drifting upstream against the beam, in good agreement with simulations.

  17. 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.

  18. 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. PMID:26206941

  19. Calibration procedures for quantitative multiple wavelengths reflectance microscopy

    NASA Astrophysics Data System (ADS)

    Fedala, Yasmina; Munteanu, Sorin; Kanoufi, Frédéric; Tessier, Gilles; Roger, Jean Paul; Wu, Chang; Amiot, Fabien

    2016-01-01

    In order to characterize surface chemo-mechanical phenomena driving micro-electro-mechanical systems (MEMSs) behavior, it has been previously proposed to use reflected intensity fields obtained from a standard microscope for different illumination wavelengths. Wavelength-dependent and -independent reflectivity fields are obtained from these images, provided the relative reflectance sensitivities ratio can be identified. This contribution focuses on the necessary calibration procedures and mathematical methods allowing for a quantitative conversion from a mechanically induced reflectivity field to a surface rotation field, therefore paving the way for a quantitative mechanical analysis of MEMS under chemical loading.

  20. Calibration procedures for quantitative multiple wavelengths reflectance microscopy.

    PubMed

    Fedala, Yasmina; Munteanu, Sorin; Kanoufi, Frdric; Tessier, Gilles; Roger, Jean Paul; Wu, Chang; Amiot, Fabien

    2016-01-01

    In order to characterize surface chemo-mechanical phenomena driving micro-electro-mechanical systems (MEMSs) behavior, it has been previously proposed to use reflected intensity fields obtained from a standard microscope for different illumination wavelengths. Wavelength-dependent and -independent reflectivity fields are obtained from these images, provided the relative reflectance sensitivities ratio can be identified. This contribution focuses on the necessary calibration procedures and mathematical methods allowing for a quantitative conversion from a mechanically induced reflectivity field to a surface rotation field, therefore paving the way for a quantitative mechanical analysis of MEMS under chemical loading. PMID:26827323

  1. Scanning Transmission Electron Microscopy at High Resolution

    PubMed Central

    Wall, J.; Langmore, J.; Isaacson, M.; Crewe, A. V.

    1974-01-01

    We have shown that a scanning transmission electron microscope with a high brightness field emission source is capable of obtaining better than 3 resolution using 30 to 40 keV electrons. Elastic dark field images of single atoms of uranium and mercury are shown which demonstrate this fact as determined by a modified Rayleigh criterion. Point-to-point micrograph resolution between 2.5 and 3.0 is found in dark field images of micro-crystallites of uranium and thorium compounds. Furthermore, adequate contrast is available to observe single atoms as light as silver. Images PMID:4521050

  2. 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.

  3. Photoacoustic microscopy for quantitative evaluation of angiogenesis inhibitor

    NASA Astrophysics Data System (ADS)

    Chen, Sung-Liang; Burnett, Joseph; Sun, Duxin; Xie, Zhixing; Wang, Xueding

    2014-03-01

    We present the photoacoustic microscopy (PAM) for evaluation of angiogenesis inhibitors on a chick embryo model. Microvasculature in the chorioallantoic membrane (CAM) of the chick embryos was imaged by PAM, and the optical microscopy (OM) images of the same set of CAMs were also acquired for comparisons, serving for validation of the results from PAM. The angiogenesis inhibitors, Sunitinib, with different concentrations applied to the CAM result in the change in microvascular density, which was quantified by both PAM and OM imaging. Similar change in microvascular density from PAM and OM imaging in response to angiogenesis inhibitor at different doses was observed, demonstrating that PAM has potential to provide objective evaluation of anti-angiogenesis medication. Besides, PAM is advantageous in three-dimensional and functional imaging compared with OM so that the emerging PAM technique may offer unique information on the efficacy of angiogenesis inhibitors and could benefit applications related to antiangiogenesis treatments.

  4. Electron Microscopy of Biological Materials at the Nanometer Scale

    NASA Astrophysics Data System (ADS)

    Kourkoutis, Lena Fitting; Plitzko, Jrgen 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.

  5. Preparation of Xenopus laevis retinal cryosections for electron microscopy.

    PubMed

    Tam, Beatrice M; Yang, Lee Ling; Bog?a, Tami H; Ross, Bradford; Martens, Garnet; Moritz, Orson L

    2015-07-01

    Transmission electron microscopy is the gold standard for examination of photoreceptor outer segment morphology and photoreceptor outer segment abnormalities in transgenic animal models of retinal disease. Small vertebrates such as zebrafish and Xenopus laevis tadpoles have been used to generate retinal disease models and to study outer segment processes such as protein trafficking, and their breeding capabilities facilitate experiments involving large numbers of animals and conditions. However, electron microscopy processing and analysis of these very small eyes can be challenging. Here we present a methodology that facilitates processing of X.laevis tadpole eyes for electron microscopy by introducing an intermediate cryosectioning step. This method reproducibly provides a well-oriented tissue block that can be sectioned with minimal effort by a non-expert, and also allows retroactive analysis of samples collected on slides for light microscopy. PMID:26008144

  6. Electron microscopy studies of CNT layers

    NASA Astrophysics Data System (ADS)

    Koz?owski, Miros?aw; Radomska, Joanna; Wronka, Halina; Czerwosz, El?bieta; Sobczak, Kamil

    2014-11-01

    SEM and TEM use in an investigation of CNT-Ni layers different properties is shown. We present the possibilities of using different SEM modes (SE - secondary electrons, LABE - low angle backscattered electrons) for studies of C-Ni and CNT-Ni layers topography, morphology and cross-sectional investigations (adhesion, pores size and shape, uniformity). Correlation between concentration of Ni in studied layers and technological parameters as well as in a case of CNT-Ni films correlations of Ni concentration and a diameter of carbon nanotubes are discussed. TEM studies concentrate on structure of Ni nanograins in C-Ni layers and CNT-Ni layers, CNT structure and defects, nanoonion structure. We present methods of determination of graphite plane number in MWCNTs, distance between these planes, role of catalyst position in CNT growth and interaction between catalyst and substrate. EDS method for qualitative analysis of Ni catalyst in these layers was also presented.

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

    NASA Astrophysics Data System (ADS)

    Bolze, Frdric; Ftouni, Hussein; Nicoud, Jean-Franois; 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.

  8. In Situ Analytical Electron Microscopy for Probing Nanoscale Electrochemistry

    SciTech Connect

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

    2011-10-31

    Oxides and their tailored structures are at the heart of electrochemical energy storage technologies and advances in understanding and controlling the dynamic behaviors in the complex oxides, particularly at the interfaces, during electrochemical processes will catalyze creative design concepts for new materials with enhanced and better-understood properties. Such knowledge is not accessible without new analytical tools. New innovative experimental techniques are needed for understanding the chemistry and structure of the bulk and interfaces, more importantly how they change with electrochemical processes in situ. Analytical Transmission Electron Microscopy (TEM) is used extensively to study electrode materials ex situ and is one of the most powerful tools to obtain structural, morphological, and compositional information at nanometer scale by combining imaging, diffraction and spectroscopy, e.g., EDS (energy dispersive X-ray spectrometry) and Electron Energy Loss Spectrometry (EELS). Determining the composition/structure evolution upon electrochemical cycling at the bulk and interfaces can be addressed by new electron microscopy technique with which one can observe, at the nanometer scale and in situ, the dynamic phenomena in the electrode materials. In electrochemical systems, for instance in a lithium ion battery (LIB), materials operate under conditions that are far from equilibrium, so that the materials studied ex situ may not capture the processes that occur in situ in a working battery. In situ electrochemical operation in the ultra-high vacuum column of a TEM has been pursued by two major strategies. In one strategy, a 'nano-battery' can be fabricated from an all-solid-state thin film battery using a focused ion beam (FIB). The electrolyte is either polymer based or ceramic based without any liquid component. As shown in Fig. 1a, the interfaces between the active electrode material/electrolyte can be clearly observed with TEM imaging, in contrast to the 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 is that EELS spectral imaging is not possible due to the high plasmon signal of the ionic liquid. To this end, we have developed a novel in situ instrumental system combining analytical electron microscopy with advanced spectroscopy to probe the dynamic phenomena in an all solid-state nano-battery. In situ electron microscopy is a versatile technique that yields insights into challenging questions that could not be obtained using other techniques. However, in order to fully exploit the capabilities, a very carefully thought-out plan of action is essential. It is important to recognize that this is not just a simple characterization tool, but a collection of tools that make up a complete experimental set-up: the choice of FIB operation conditions, specimen holder for biasing, grid materials and design as well as microscope environment must be thoroughly considered before performing an experiment.

  9. Quantitative thermal microscopy using thermoelectric probe in passive mode.

    PubMed

    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. PMID:24182115

  10. A Correlative Optical Microscopy and Scanning Electron Microscopy Approach to Locating Nanoparticles in Brain Tumors

    PubMed Central

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

    2014-01-01

    The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. PMID:25464144

  11. Photographic aspects of scanning electron microscopy.

    PubMed

    Malin, D F

    1975-01-01

    Factors governing photographic image quality in the scanning electron microscope are discussed with particular reference to the commonly used EXA camera on the Cambridge Stereoscan IIa. It is shown that the small image on the medium speed film suffers considerable loss of information due to the turbid nature of the photographic emulsion. Inexpensive modifications to the oscilloscope camera supplied with the Stereoscan are described which enable the superior quality of a larger format to be utilized. Appropriate settings of the brightness and contrast controls of the image tube with respect to the photographic system is discussed and the results illustrated in a series of micrographs. PMID:1173605

  12. Photon-induced near-field electron microscopy.

    PubMed

    Barwick, Brett; Flannigan, David J; Zewail, Ahmed H

    2009-12-17

    In materials science and biology, optical near-field microscopies enable spatial resolutions beyond the diffraction limit, but they cannot provide the atomic-scale imaging capabilities of electron microscopy. Given the nature of interactions between electrons and photons, and considering their connections through nanostructures, it should be possible to achieve imaging of evanescent electromagnetic fields with electron pulses when such fields are resolved in both space (nanometre and below) and time (femtosecond). Here we report the development of photon-induced near-field electron microscopy (PINEM), and the associated phenomena. We show that the precise spatiotemporal overlap of femtosecond single-electron packets with intense optical pulses at a nanostructure (individual carbon nanotube or silver nanowire in this instance) results in the direct absorption of integer multiples of photon quanta (nhomega) by the relativistic electrons accelerated to 200 keV. By energy-filtering only those electrons resulting from this absorption, it is possible to image directly in space the near-field electric field distribution, obtain the temporal behaviour of the field on the femtosecond timescale, and map its spatial polarization dependence. We believe that the observation of the photon-induced near-field effect in ultrafast electron microscopy demonstrates the potential for many applications, including those of direct space-time imaging of localized fields at interfaces and visualization of phenomena related to photonics, plasmonics and nanostructures. PMID:20016598

  13. 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.

  14. 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.

  15. 3D Correlative Imaging | High Resolution Electron Microscopy

    Cancer.gov

    One key area of interest for the lab has been to close the 3D imaging gap, finding ways to image whole cells and tissues at high resolution. Focused ion beam scanning electron microscopy (FIB-SEM, or otherwise known as ion abrasion scanning electron microscopy, IA-SEM) uses a scanning electron beam to image the face of a fixed, resin-embedded sample, and an ion beam to remove slices of the sample, resulting in a sequential stack of high resolution images.

  16. Novel Application of Fluorescence Lifetime and Fluorescence Microscopy Enables Quantitative Access to Subcellular Dynamics in Plant Cells

    PubMed Central

    Elgass, Kirstin; Caesar, Katharina; Schleifenbaum, Frank; Stierhof, York-Dieter; Meixner, Alfred J.; Harter, Klaus

    2009-01-01

    Background Optical and spectroscopic technologies working at subcellular resolution with quantitative output are required for a deeper understanding of molecular processes and mechanisms in living cells. Such technologies are prerequisite for the realisation of predictive biology at cellular and subcellular level. However, although established in the physical sciences, these techniques are rarely applied to cell biology in the plant sciences. Principal Findings Here, we present a combined application of one-chromophore fluorescence lifetime microscopy and wavelength-selective fluorescence microscopy to analyse the function of a GFP fusion of the Brassinosteroid Insensitive 1 Receptor (BRI1-GFP) with high spatial and temporal resolution in living Arabidopsis cells in their tissue environment. We show a rapid, brassinolide-induced cell wall expansion and a fast BR-regulated change in the BRI1-GFP fluorescence lifetime in the plasmamembrane in vivo. Both cell wall expansion and changes in fluorescence lifetime reflect early BR-induced and BRI1-dependent physiological or signalling processes. Our experiments also show the potential of one-chromophore fluorescence lifetime microscopy for the in vivo monitoring of the biochemical and biophysical subcellular environment using GFP fusion proteins as probes. Significance One-chromophore fluorescence lifetime microscopy, combined with wavelength-specific fluorescence microscopy, opens up new frontiers for in vivo dynamic and quantitative analysis of cellular processes at high resolution which are not addressable by pure imaging technologies or transmission electron microscopy. PMID:19492078

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

    NASA Astrophysics Data System (ADS)

    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.

  18. 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.

  19. 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. PMID:25638065

  20. Towards Automated Nanomanipulation under Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Ye, Xutao

    Robotic Nanomaterial Manipulation inside scanning electron microscopes (SEM) is useful for prototyping functional devices and characterizing one-dimensional nanomaterial's properties. Conventionally, manipulation of nanowires has been performed via teleoperation, which is time-consuming and highly skill-dependent. Manual manipulation also has the limitation of low success rates and poor reproducibility. This research focuses on a robotic system capable of automated pick-place of single nanowires. Through SEM visual detection and vision-based motion control, the system transferred individual silicon nanowires from their growth substrate to a microelectromechanical systems (MEMS) device that characterized the nanowires' electromechanical properties. The performances of the nanorobotic pick-up and placement procedures were quantified by experiments. The system demonstrated automated nanowire pick-up and placement with high reliability. A software system for a load-lock-compatible nanomanipulation system is also designed and developed in this research.

  1. Electron microscopy of biomaterials based on hydroxyapatite

    SciTech Connect

    Suvorova, E. I. Klechkovskaya, V. V.; Komarov, V. F.; Severin, A. V.; Melikhov, I. V.; Buffat, P. A.

    2006-10-15

    Three types of biomaterials based on hydroxyapatite are synthesized and investigated. Hydroxyapatite nanocrystals or microcrystals precipitated from low-temperature aqueous solutions serve as the initial material used for preparing spherical porous granules approximately 300-500 {mu}m in diameter. Sintering of hydroxyapatite crystals at a temperature of 870 deg. C for 2 h or at 1000 deg. C (for 3 h) + 1200 deg. C (for 2 h) brings about the formation of solid ceramics with different internal structures. According to the electron microscopic data, the ceramic material prepared at 870 deg. C is formed by agglomerated hydroxyapatite nanocrystals, whereas the ceramics sintered at 1200 deg. C (with a bending strength of the order of 100 MPa) are composed of crystal blocks as large as 2 {mu}m. It is established that all the biomaterials have a single-phase composition and consist of the hydroxyapatite with a structure retained up to a temperature of 1200 deg. C.

  2. Characterization of Metamict Structures by Electron Diffraction and Microscopy.

    NASA Astrophysics Data System (ADS)

    Qin, Lu-Chang

    Quantitative high-resolution electron microscopy and electron amorphography were used to investigate the metamictization of crystalline silica precursors and the structures of metamict silicas. Various polymorphs of crystalline silica, including quartz, cristobalite and tridymite, were examined, as well as some other forms of aperiodic silica such as vitreous silica, thermally-grown, and CVD-grown silica films on silicon substrates. Two major morphologies were observed in the crystalline -to-metamict transformation: in one, gradual transformation occurred without sharp boundaries visible between crystalline and metamict regions in images; in the other, sharp boundaries were visible. In quartz, both morphologies were observed, but in cristobalite and tridymite only the former was observed. Both forms of morphological evolution in quartz were attributed to nucleation-and-growth of amorphous inclusions via radiolysis. The appearance of a gradual transformation arises because the crystalline-metamict boundary is not parallel to the incident electron beam direction. Systematic image simulations were carried out to aid in the interpretation of experimental micrographs. It was also found that when the crystalline portion is greater than about one third of the specimen in thickness, the image may appear to be from wholly crystalline material, and the amorphous portion may not be detected in the images. Electron amorphography was developed and performed using energy-filtered electron diffraction data from the metamict states, collected in a scanning transmission electron microscope equipped with an electron energy-loss spectrometer. Exact formulae and numerical procedures were established to use the data to obtain radial distribution functions to characterize the metamict structures at the atomic level. By comparing the radial distribution functions for vitreous silica, electron-metamict quartz, and neutron-metamict quartz, it was found that, in the metamict states, a continuous -random-network structure was mostly preserved, but the coordination number for Si-O bonding was reduced in electron - and neutron-metamict quartz samples. In metamict cristobalite and metamict tridymite, the continuous random network structure was found to have been preserved also. Similar features were also recognizable in amorphous SiO_2 thin films grown on Si substrates. A new theory for the relationship of the first sharp diffraction peak to medium-range order in terms of ring contents is proposed. For electron metamict silicas, it is suggested that six-membered rings are indeed the dominant ones, as indicated by the position of the first sharp diffraction peaks in various samples. In Si ^+ ion-implanted quartz, however, the dominant rings are deduced to be of smaller size, which is consistent with the fact that the final product is Si rich. In comparison to X-ray and neutron amorphographies, electron amorphography offers certain unique features and advantages; for example, imaging can be performed simultaneously to provide complementary structural information and statistically valid data can be collected from microvolumes or thin films. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253 -1690.).

  3. 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

  4. Quantitative Localization Microscopy: Effects of Photophysics and Labeling Stoichiometry

    PubMed Central

    Szymborska, Anna; Lidke, Keith A.; Rieger, Bernd; Stallinga, Sjoerd

    2015-01-01

    Quantification in localization microscopy with reversibly switchable fluorophores is severely hampered by the unknown number of switching cycles a fluorophore undergoes and the unknown stoichiometry of fluorophores on a marker such as an antibody. We overcome this problem by measuring the average number of localizations per fluorophore, or generally per fluorescently labeled site from the build-up of spatial image correlation during acquisition. To this end we employ a model for the interplay between the statistics of activation, bleaching, and labeling stoichiometry. We validated our method using single fluorophore labeled DNA oligomers and multiple-labeled neutravidin tetramers where we find a counting error of less than 17% without any calibration of transition rates. Furthermore, we demonstrated our quantification method on nanobody- and antibody-labeled biological specimens. PMID:25992915

  5. Beam propagation analysis on thickness measurements in quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Bae, Yoon-Sung; Song, Jong-In; Har, Dongsoo; Kim, Dug Young

    2015-08-01

    The two-dimensional thickness profile of a phase object can be measured by phase microscopy by assuming that the light passes straight through the sample such that the measured phase profile is proportional to the thickness of the sample. However, any non-uniform index structure in a sample bends the straight light path by refraction and diffracts the non-uniform transverse phase structure of the wavefront along the propagation path within a sample. We investigated the consequence of these two effects within a phase object using a split-step beam propagation method that considers beam paths through a 3-μm-diameter bead sample. Our simulation results show that the phase profile of light just after passing through a sample differs significantly from an ideal phase profile. We verified these simulation results by comparing them with experimental data obtained with a Mach-Zehnder interferometer.

  6. 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

  7. Single-cell lysis for visual analysis by electron microscopy.

    PubMed

    Kemmerling, Simon; Arnold, Stefan A; Bircher, Benjamin A; Sauter, Nora; Escobedo, Carlos; Dernick, Gregor; Hierlemann, Andreas; Stahlberg, Henning; Braun, Thomas

    2013-09-01

    The stochastic nature of biological systems makes the study of individual cells a necessity in systems biology. Yet, handling and disruption of single cells and the analysis of the relatively low concentrations of their protein components still challenges available techniques. Transmission electron microscopy (TEM) allows for the analysis of proteins at the single-molecule level. Here, we present a system for single-cell lysis under light microscopy observation, followed by rapid uptake of the cell lysate. Eukaryotic cells were grown on conductively coated glass slides and observed by light microscopy. A custom-designed microcapillary electrode was used to target and lyse individual cells with electrical pulses. Nanoliter volumes were subsequently aspirated into the microcapillary and dispensed onto an electron microscopy grid for TEM inspection. We show, that the cell lysis and preparation method conserves protein structures well and is suitable for visual analysis by TEM. PMID:23816812

  8. Combined confocal Raman and quantitative phase microscopy system for biomedical diagnosis

    PubMed Central

    Kang, Jeon Woong; Lue, Niyom; Kong, Chae-Ryon; Barman, Ishan; Dingari, Narahara C.; Goldfless, Stephen J.; Niles, Jacquin C.; Dasari, Ramachandra R.; Feld, Michael S.

    2011-01-01

    We have developed a novel multimodal microscopy system that incorporates confocal Raman, confocal reflectance, and quantitative phase microscopy (QPM) into a single imaging entity. Confocal Raman microscopy provides detailed chemical information from the sample, while confocal reflectance and quantitative phase microscopy show detailed morphology. Combining these intrinsic contrast imaging modalities makes it possible to obtain quantitative morphological and chemical information without exogenous staining. For validation and characterization, we have used this multi-modal system to investigate healthy and diseased blood samples. We first show that the thickness of a healthy red blood cell (RBC) shows good correlation with its hemoglobin distribution. Further, in malaria infected RBCs, we successfully image the distribution of hemozoin (malaria pigment) inside the cell. Our observations lead us to propose morphological screening by QPM and subsequent chemical imaging by Raman for investigating blood disorders. This new approach allows monitoring cell development and cell-drug interactions with minimal perturbation of the biological system of interest. PMID:21991542

  9. Quantitative STEM normalisation: The importance of the electron flux.

    PubMed

    Martinez, G T; Jones, L; De Backer, A; Bch, A; Verbeeck, J; Van Aert, S; Nellist, P D

    2015-12-01

    Annular dark-field (ADF) scanning transmission electron microscopy (STEM) has become widely used in quantitative studies based on the opportunity to directly compare experimental and simulated images. This comparison merely requires the experimental data to be normalised and expressed in units of 'fractional beam-current'. However, inhomogeneities in the response of electron detectors can complicate this normalisation. The quantification procedure becomes both experiment and instrument specific, requiring new simulations for the particular response of each instrument's detector, and for every camera-length used. This not only impedes the comparison between different instruments and research groups, but can also be computationally very time consuming. Furthermore, not all image simulation methods allow for the inclusion of an inhomogeneous detector response. In this work, we propose an alternative method for normalising experimental data in order to compare these with simulations that consider a homogeneous detector response. To achieve this, we determine the electron flux distribution reaching the detector by means of a camera-length series or a so-called atomic column cross-section averaged convergent beam electron diffraction (XSACBED) pattern. The result is then used to determine the relative weighting of the detector response. Here we show that the results obtained by this new electron flux weighted (EFW) method are comparable to the currently used method, while considerably simplifying the needed simulation libraries. The proposed method also allows one to obtain a metric that describes the quality of the detector response in comparison with the 'ideal' detector response. PMID:26318098

  10. 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 photonelectron 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

  11. Photon gating in four-dimensional ultrafast electron microscopy.

    PubMed

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

    2015-10-20

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

  12. Transmission electron microscopy: A critical analytical tool for ULSI technology

    NASA Astrophysics Data System (ADS)

    Venables, David; Susnitzky, David W.; Mardinly, A. John

    1998-11-01

    An overview of the capabilities and limitations of transmission electron microscopy (TEM) based analysis techniques in the context of the electronics industry is presented. The electron-beam/specimen interactions that enable morphological, crystallographic and compositional characterization with modern TEMs are briefly reviewed. Diffraction contrast, lattice and energy filtered imaging; energy dispersive x-ray spectrometry (EDS), and electron energy loss spectrometry (EELS) are reviewed and discussed. These techniques are illustrated through specific applications and case studies in the electronics industry. Particular emphasis is placed on sample preparation concerns, which represent a practical limitation to an expanded role for TEM as a critical analytical tool for ULSI technology.

  13. Subsurface atomic force microscopy: towards a quantitative understanding

    NASA Astrophysics Data System (ADS)

    Verbiest, G. J.; Simon, J. N.; Oosterkamp, T. H.; Rost, M. J.

    2012-04-01

    Recent experiments in the field of subsurface atomic force microscopy have demonstrated that it is possible to nondestructively image micro- and even nanoparticles that are embedded significantly deep within the bulk of a sample. In order to get insights into the contrast formation mechanism, we performed a finite element analysis and an analytical study, in which we calculated the amplitude and phase variation on the surface of an ultrasound wave that has traveled through the sample. Our calculations were performed as closely as possible to the situation in the experiments to enable a (future) comparison based on our predictions. We show that Rayleigh scattering of acoustic waves accounts for the measured contrast and we verify the characteristic Rayleigh dependences. The numerical results show that the contrast is independent of the depth at which a particle is buried, whereas the analytical study reveals a 1/depth dependence. In addition, we find a large deviation in the width of the particle in the contrast at the surface when applying the numerical or the analytical calculation respectively. These results indicate the importance of both the reflections of sound waves at the sample interfaces and bulk damping, as both are treated differently in our two models.

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

    PubMed Central

    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

  15. 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

  16. 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.

  17. The CryoCapsule: Simplifying correlative light to electron microscopy

    PubMed Central

    Heiligenstein, Xavier; Heiligenstein, Jrme; Delevoye, Cdric; Hurbain, Ilse; Bardin, Sabine; Paul-Gilloteaux, Perrine; Sengmanivong, Lucie; Rgnier, 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

  18. 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.

  19. Microscopy with slow electrons: from LEEM to XPEEM

    ScienceCinema

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

    2010-01-08

    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.

  20. Evaluations of carbon nanotube field emitters for electron microscopy

    NASA Astrophysics Data System (ADS)

    Nakahara, Hitoshi; Kusano, Yoshikazu; Kono, Takumi; Saito, Yahachi

    2009-11-01

    Brightness of carbon nanotube (CNT) emitters was already reported elsewhere. However, brightness of electron emitter is affected by a virtual source size of the emitter, which strongly depends on electron optical configuration around the emitter. In this work, I- V characteristics and brightness of a CNT emitter are measured under a practical field emission electron gun (e-gun) configuration to investigate availability of CNT for electron microscopy. As a result, it is obtained that an emission area of MWNT is smaller than its tip surface area, and the emission area corresponds to a five-membered-ring with 2nd nearest six-membered-rings on the MWNT cap surface. Reduced brightness of MWNT is measured as at least 2.6109 A/m 2 sr V. It is concluded that even a thick MWNT has enough brightness under a practical e-gun electrode configuration and suitable for electron microscopy.

  1. Helium ion microscopy and energy selective scanning electron microscopy - two advanced microscopy techniques with complementary applications

    NASA Astrophysics Data System (ADS)

    Rodenburg, C.; Jepson, M. A. E.; Boden, Stuart A.; Bagnall, Darren M.

    2014-06-01

    Both scanning electron microscopes (SEM) and helium ion microscopes (HeIM) are based on the same principle of a charged particle beam scanning across the surface and generating secondary electrons (SEs) to form images. However, there is a pronounced difference in the energy spectra of the emitted secondary electrons emitted as result of electron or helium ion impact. We have previously presented evidence that this also translates to differences in the information depth through the analysis of dopant contrast in doped silicon structures in both SEM and HeIM. Here, it is now shown how secondary electron emission spectra (SES) and their relation to depth of origin of SE can be experimentally exploited through the use of energy filtering (EF) in low voltage SEM (LV-SEM) to access bulk information from surfaces covered by damage or contamination layers. From the current understanding of the SES in HeIM it is not expected that EF will be as effective in HeIM but an alternative that can be used for some materials to access bulk information is presented.

  2. 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.

  3. CI Slide: calibration slide for quantitative microscopy imaging in absorbance

    NASA Astrophysics Data System (ADS)

    Sheikhzadeh, Fahime; Ye, Qian; Zulkafly, Nasir; Carraro, Anita; Korbelic, Jagoda; Chen, Zhaoyang; Harrison, Alan; Follen, Michele; MacAulay, Calum; Ward, Rabab K.; Guillaud, Martial

    2014-03-01

    New imaging technologies are changing the field of digital pathology. This field faces numerous challenges and there is a pressing need for standardization, calibration protocols, quality control and quantitative assessment. We have designed a new calibration imaging slide (Cancer Imaging Slide), specifically to measure the characteristics of old or new imaging systems or scanners. The layout of the slide consists of 138 boxes with the side length of 1.6 mm, containing objects of known morphologic and photometric characteristics. Among them, 112 boxes contain different permutations of circles, ovals, and squares. The circles have different radii, radius/pitch ratios and step transmissions. The ovals have different sizes and orientations. The squares are consistent in size and orientation but have different step transmission values. Also, 16 boxes contain three resolution test targets: crosses, USAF target and Siemens star. The last 10 boxes are blank boxes with different transmission values. Four slides were scanned and imaged on one commercial whole-slide scanner and one high resolution imaging system. After segmenting the images, about 200 features (photometric, morphologic and architectural) were measured with our in-house image processing software. The objective of the project is to develop a statistical process control using this new slide. In this paper, we describe the characteristics of the slide and present our preliminary results.

  4. Directed evolution of APEX2 for electron microscopy and proteomics

    PubMed Central

    Lam, Stephanie S.; Martell, Jeffrey D.; Kamer, Kimberli J.; Deerinck, Thomas J.; Ellisman, Mark H.; Mootha, Vamsi K.; Ting, Alice Y.

    2014-01-01

    APEX is an engineered peroxidase that functions both as an electron microscopy tag, and as a promiscuous labeling enzyme for live-cell proteomics. Because the limited sensitivity of APEX precludes applications requiring low APEX expression, we used yeast display evolution to improve its catalytic efficiency. Our evolved APEX2 is far more active in cells, enabling the superior enrichment of endogenous mitochondrial and endoplasmic reticulum membrane proteins and the use of electron microscopy to resolve the sub-mitochondrial localization of calcium uptake regulatory protein MICU1. PMID:25419960

  5. 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

  6. Ultrastructural Analysis of Drosophila Ovaries by Electron Microscopy.

    PubMed

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

    2015-01-01

    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

  7. Dendritic Cell with HIV | High Resolution Electron Microscopy

    Cancer.gov

    In order to study the 3D structure of the site of HIV transfer from dendritic cells to T cells (the virological synapse), the Subramaniam group used a combination of scanning electron microscopy paired with an ion beam (FIB-SEM), for 3D imaging, and transmission electron microscopy (TEM), for visualizing virions. By reconstructing 3D FIB-SEM images of dendritic cells interacting with T cells, and pairing that with higher resolution TEM images of the virological synapse, the group pieced together the following story.

  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. Polyvinylidene fluoride molecules in nanofibers, imaged at atomic scale by aberration corrected electron microscopy

    NASA Astrophysics Data System (ADS)

    Lolla, Dinesh; Gorse, Joseph; Kisielowski, Christian; Miao, Jiayuan; Taylor, Philip L.; Chase, George G.; Reneker, Darrell H.

    2015-12-01

    Atomic scale features of polyvinylidene fluoride molecules (PVDF) were observed with aberration corrected transmission electron microscopy. Thin, self-supporting PVDF nanofibers were used to create images that show conformations and relative locations of atoms in segments of polymer molecules, particularly segments near the surface of the nanofiber. Rows of CF2 atomic groups, at 0.25 nm intervals, which marked the paths of segments of the PVDF molecules, were seen. The fact that an electron microscope image of a segment of a PVDF molecule depended upon the particular azimuthal direction, along which the segment was viewed, enabled observation of twist around the molecular axis. The 0.2 nm side-by-side distance between the two fluorine atoms attached to the same carbon atom was clearly resolved. Morphological and chemical changes produced by energetic electrons, ranging from no change to fiber scission, over many orders of magnitude of electrons per unit area, promise quantitative new insights into radiation chemistry. Relative movements of segments of molecules were observed. Promising synergism between high resolution electron microscopy and molecular dynamic modeling was demonstrated. This paper is at the threshold of growing usefulness of electron microscopy to the science and engineering of polymer and other molecules.Atomic scale features of polyvinylidene fluoride molecules (PVDF) were observed with aberration corrected transmission electron microscopy. Thin, self-supporting PVDF nanofibers were used to create images that show conformations and relative locations of atoms in segments of polymer molecules, particularly segments near the surface of the nanofiber. Rows of CF2 atomic groups, at 0.25 nm intervals, which marked the paths of segments of the PVDF molecules, were seen. The fact that an electron microscope image of a segment of a PVDF molecule depended upon the particular azimuthal direction, along which the segment was viewed, enabled observation of twist around the molecular axis. The 0.2 nm side-by-side distance between the two fluorine atoms attached to the same carbon atom was clearly resolved. Morphological and chemical changes produced by energetic electrons, ranging from no change to fiber scission, over many orders of magnitude of electrons per unit area, promise quantitative new insights into radiation chemistry. Relative movements of segments of molecules were observed. Promising synergism between high resolution electron microscopy and molecular dynamic modeling was demonstrated. This paper is at the threshold of growing usefulness of electron microscopy to the science and engineering of polymer and other molecules. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01619c

  10. Determination of mineral distributions in bituminous coals by electron microscopy

    SciTech Connect

    Harris, L.A.

    1982-01-01

    In recent transmission electron microscopical studies of coals, ultrafine minerals were observed (<1 ..mu..m). The observation and identity of these submicron minerals would have been difficult to achieve by use of the scanning electron microscope (SEM). However, the scanning transmission electron microscope (STEM) with energy dispersive x-ray analysis is an ideal analytical tool since it is capable of supplying elemental and diffraction data for particles as small as 30 nm in diameter. In this paper, we present observations and analyses of mineral matter in coals obtained through use of electron microscopes. These data significantly increase our knowledge of the mineral matter in coals as related to their affects on coal combustion: syngenetic and epigenetic minerals can be observed and identified by electron microscopy in conjunction with energy dispersive x-ray analysis; submicron micerals that are not readily identified or observed by scanning electron microscopy are easily viewed by use of transmission electron microscopy; calcite appears to be relatively scarce as a syngenetic mineral whereas calcite is an important epigenetic mineral usually occurring as cleat deposits; important minor syngenetic mineral assemblages appear to be associated with detritus. These minerals probably contain the major portion of minor and trace elements in coal; most of the epigenetic minerals should be readily removed from the coal resulting in a probable reduction in fouling and slagging.

  11. Charging processes in low vacuum scanning electron microscopy.

    PubMed

    Thiel, Bradley L; Toth, Milos; Craven, John P

    2004-12-01

    A framework is presented for understanding charging processes in low vacuum scanning electron microscopy. We consider the effects of electric fields generated above and below the specimen surface and their effects on various processes taking place in the system. These processes include the formation of an ionic space charge, field-enhanced electron emission, charge trapping and dissipation, and electron-ion recombination. The physical mechanisms behind each of these processes are discussed, as are the microscope operating conditions under which each process is most effective. Readily observable effects on gas gain curves, secondary electron images, and X-ray spectra are discussed. PMID:19780311

  12. Attosecond electron pulses for 4D diffraction and microscopy

    PubMed Central

    Baum, Peter; Zewail, Ahmed H.

    2007-01-01

    In this contribution, we consider the advancement of ultrafast electron diffraction and microscopy to cover the attosecond time domain. The concept is centered on the compression of femtosecond electron packets to trains of 15-attosecond pulses by the use of the ponderomotive force in synthesized gratings of optical fields. Such attosecond electron pulses are significantly shorter than those achievable with extreme UV light sources near 25 nm (?50 eV) and have the potential for applications in the visualization of ultrafast electron dynamics, especially of atomic structures, clusters of atoms, and some materials. PMID:18000040

  13. 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 work of Mankos et al., who evaluated the impact of high-throughput requirements on the LEEM designs and demonstrated new applications of imaging modes with a tilted electron beam. To assess its potential as a semiconductor device imaging tool and to identify contrast mechanisms, we used LEEM to investigate doped Si test structures. In section 2, Imaging Oxide-Covered Doped Si Structures Using LEEM, we show that the LEEM technique is able to provide reasonably high contrast images across lateral pn junctions. The observed contrast is attributed to a work function difference ({Delta}{phi}) between the p- and n-type regions. However, because the doped regions were buried under a thermal oxide ({approx}3.5 nm thick), e-beam charging during imaging prevented quantitative measurements of {Delta}{phi}. As part of this project, we also investigated a series of similar test structures in which the thermal oxide was removed by a chemical etch. With the oxide removed, we obtained intensity-versus-voltage (I-V) curves through the transition from mirror to LEEM mode and determined the relative positions of the vacuum cutoffs for the differently doped regions. Although the details are not discussed in this report, the relative position in voltage of the vacuum cutoffs are a direct measure of the work function difference ({Delta}{phi}) between the p- and n-doped regions.

  14. Direct investigation of subsurface interface electronic structure by ballistic-electron-emission microscopy

    NASA Technical Reports Server (NTRS)

    Kaiser, W. J.; Bell, L. D.

    1988-01-01

    A new technique for spectroscopic investigation of subsurface interface electronic structure has been developed. The method, ballistic-electron-emission microscopy (BEEM), is based on scanning tunneling microscopy. BEEM makes possible, for the first time, direct imaging of subsurface interface properties with nanometer spatial resolution. The first application of BEEM to subsurface Schottky-barrier interfaces is reported.

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

    PubMed

    Lschberger, 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. PMID:25146397

  16. 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 3256?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

  17. 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. PMID:24022088

  18. Breaking resolution limits in ultrafast electron diffraction and microscopy

    PubMed Central

    Baum, Peter; Zewail, Ahmed H.

    2006-01-01

    Ultrafast electron microscopy and diffraction are powerful techniques for the study of the time-resolved structures of molecules, materials, and biological systems. Central to these approaches is the use of ultrafast coherent electron packets. The electron pulses typically have an energy of 30 keV for diffraction and 100200 keV for microscopy, corresponding to speeds of 3370% of the speed of light. Although the spatial resolution can reach the atomic scale, the temporal resolution is limited by the pulse width and by the difference in group velocities of electrons and the light used to initiate the dynamical change. In this contribution, we introduce the concept of tilted optical pulses into diffraction and imaging techniques and demonstrate the methodology experimentally. These advances allow us to reach limits of time resolution down to regimes of a few femtoseconds and, possibly, attoseconds. With tilted pulses, every part of the sample is excited at precisely the same time as when the electrons arrive at the specimen. Here, this approach is demonstrated for the most unfavorable case of ultrafast crystallography. We also present a method for measuring the duration of electron packets by autocorrelating electron pulses in free space and without streaking, and we discuss the potential of tilting the electron pulses themselves for applications in domains involving nuclear and electron motions. PMID:17056711

  19. 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…

  20. 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

  1. 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

  2. 'GIARDIA MURIS': SCANNING ELECTRON MICROSCOPY OF IN VITRO EXCYSTATION

    EPA Science Inventory

    A recently developed in vitro excystation procedure results in almost total excystation of Giardia muris, an intestinal parasite of mice. The present experiment examines the G. muris cyst morphology by scanning electron microscopy and evaluates the efficacy of the excystation pro...

  3. 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

  4. 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.

  5. 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…

  6. Microstress contrast in scanning electron acoustic microscopy of ceramics

    NASA Technical Reports Server (NTRS)

    Cantrell, John H.; Qian, Menglu

    1991-01-01

    A mathematical model of image contrast in scanning electron acoustic microscopy (SEAM) due to the effect of residual stresses in materials is presented. It is found that in regions near the ends of the radial cracks induced by Vickers indentation the SEAM micrographs reveal a rather large variation of the acoustic output signal.

  7. Scanning electron microscopy analysis of corrosion degradation on tinplate substrates.

    PubMed

    Zumelzu, E; Cabezas, C; Vera, A

    2003-01-01

    The degradation of electrolytic tinplate used in food containers was analysed and evaluated, using scanning electron microscopy and electrochemical measurements of microcorrosion and ion dissolution by atomic absorption to prevent food contamination caused by metal traces and to increase the durability of such tinplates. PMID:12627896

  8. Cryo-electron microscopy of the giant Mimivirus.

    PubMed

    Xiao, Chuan; Chipman, Paul R; Battisti, Anthony J; Bowman, Valorie D; Renesto, Patricia; Raoult, Didier; Rossmann, Michael G

    2005-10-28

    Mimivirus is the largest known virus. Using cryo-electron microscopy, the virus was shown to be icosahedral, covered by long fibers, and appears to have at least two lipid membranes within its protein capsid. A unique vertex, presumably for attachment and infection of the host, can be seen for particles that have a suitable orientation on the micrographs. PMID:16185710

  9. 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.

  10. Quantitative imaging of cellular adhesion by total internal reflection holographic microscopy.

    PubMed

    Ash, William M; Krzewina, Leo; Kim, Myung K

    2009-12-01

    Total internal reflection (TIR) holographic microscopy uses a prism in TIR as a near-field imager to perform quantitative phase microscopy of cell-substrate interfaces. The presence of microscopic organisms, cell-substrate interfaces, adhesions, and tissue structures on the prism's TIR face causes relative index of refraction and frustrated TIR to modulate the object beam's evanescent wave phase front. We present quantitative phase images of test specimens such as Amoeba proteus and cells such as SKOV-3 and 3T3 fibroblasts. PMID:19956284

  11. Analytical quantitative theory of RF-SPM for nanocarbon electronics

    NASA Astrophysics Data System (ADS)

    Rotkin, Slava V.

    2015-03-01

    Among a variety of Scanning Probe Microscopy (SPM) tools RF- or microwave-SPM has recommended itself as a versatile characterization tool, recently demonstrated capability to map electronic properties of nanocarbon materials non-destructively and with nanometer resolution. The transparent theory of RF-SPM sensing mechanism is however lacking, mostly limited to numerical or empirical solutions, especially when studying low-dimensional quantum objects, such as nanotubes/nanowires (NT/NW), where the classical description is often invalid. One-dimensional electronic structure of the NT/NW, weak screening of Coulomb interaction and finite e-e compressibility were successfully taken into account to provide an analytic form of its quasi-stationary (due to low RF frequency of the excitation) selfconsistent response. SPM tip response function was, in turn, efficiently analyzed in multipole series, and non-perturbatively diagrammatically summed in the sense of the Random Phase Approximation. Resulting theory shows transparently the physics of RF-SPM sensing mechanism, simultaneously allowing a quantitative analysis of recent RF-SPM data on nanotube electronic devices [E. Seabron, S. MacLaren, X. Xie, SV. Rotkin, JA. Rogers, WL. Wilson, unpublished]. Support by AFOSR (# FA9550-11-1-0185) is acknowledged.

  12. Electron Microscopy of Probability Currents at Atomic Resolution

    NASA Astrophysics Data System (ADS)

    Lubk, A.; Bch, A.; Verbeeck, J.

    2015-10-01

    Atomic resolution transmission electron microscopy records the spatially resolved scattered electron density to infer positions, density, and species of atoms. These data are indispensable for studying the relation between structure and properties in solids. Here, we show how this signal can be augmented by the lateral probability current of the scattered electrons in the object plane at similar resolutions and fields of view. The currents are reconstructed from a series of three atomic resolution TEM images recorded under a slight difference of perpendicular line foci. The technique does not rely on the coherence of the electron beam and can be used to reveal electric, magnetic, and strain fields with incoherent electron beams as well as correlations in inelastic transitions, such as electron magnetic chiral dichroism.

  13. Electron Microscopy of Probability Currents at Atomic Resolution.

    PubMed

    Lubk, A; Bch, A; Verbeeck, J

    2015-10-23

    Atomic resolution transmission electron microscopy records the spatially resolved scattered electron density to infer positions, density, and species of atoms. These data are indispensable for studying the relation between structure and properties in solids. Here, we show how this signal can be augmented by the lateral probability current of the scattered electrons in the object plane at similar resolutions and fields of view. The currents are reconstructed from a series of three atomic resolution TEM images recorded under a slight difference of perpendicular line foci. The technique does not rely on the coherence of the electron beam and can be used to reveal electric, magnetic, and strain fields with incoherent electron beams as well as correlations in inelastic transitions, such as electron magnetic chiral dichroism. PMID:26551126

  14. Quantitative x-ray differential-interference-contrast microscopy with independently adjustable bias and shear

    SciTech Connect

    Nakamura, Takashi; Chang Chang

    2011-04-15

    We present a quantitative x-ray phase imaging method that can be readily implemented on existing x-ray microscopy facilities. This technique utilizes Fresnel zone plates both as imaging optical elements for magnification and as second-order grating structures for phase-shifting interferometry. By making high-resolution quantitative x-ray phase information widely available, we expect this work to have significant impact on nanoscale biological and material studies.

  15. Hybrid shear force feedback/scanning quantitative phase microscopy applied to subsurface imaging.

    PubMed

    Edward, Kert; Farahi, Faramarz; Hocken, Robert

    2009-10-12

    Quantitative phase microscopy allows for the study of the surface morphology and dynamics of transparent biological specimens. Although phase data often contains coupled subsurface information, decoupling the surface and subsurface components is often very difficult or impossible. We hereby present a simple procedure which exploits simultaneous obtained quantitative phase and shear-force feedback topography data to extract subsurface sample information. Our results reveal subsurface features in fabricated samples and fish erythrocytes. PMID:20372571

  16. 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. PMID:26680204

  17. Transmission Electron Microscopy of a Graphene-based Polymer Nanocomposite

    NASA Astrophysics Data System (ADS)

    Kohlhaas, Kevin; Dikin, Dmitriy; Stankovich, Sasha; Ruoff, Rodney; Stach, Eric

    2006-03-01

    A Polystyrene/CMG (chemically modified graphene) composite has been made by a solution-based processing technique followed by hot pressing or injection molding to form continuous specimens. Microtomed samples were prepared for study by transmission (TEM) and scanning (SEM) electron microscopy. The electron diffraction patterns and the resulting d-spacings, as well as high-resolution bright field TEM images, suggest that the platelets are individual graphene sheets randomly dispersed in the polymer matrix. Scanning electron microscopy observation indicates that the sheets are in a wrinkled conformation; this wrinkling has also been observed in TEM, in the form of 10 nm domains exhibiting lattice fringes of varying orientations. We gratefully acknowledge the NASA University Research, Engineering and Technology Institute on Bio Inspired Materials (BIMat; No. NCC-1-02037) and the National Science Foundation (No. DMR-0526959).

  18. 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.

  19. Biological imaging with 4D ultrafast electron microscopy

    PubMed Central

    Flannigan, David J.; Barwick, Brett; Zewail, Ahmed H.

    2010-01-01

    Advances in the imaging of biological structures with transmission electron microscopy continue to reveal information at the nanometer length scale and below. The images obtained are static, i.e., time-averaged over seconds, and the weak contrast is usually enhanced through sophisticated specimen preparation techniques and/or improvements in electron optics and methodologies. Here we report the application of the technique of photon-induced near-field electron microscopy (PINEM) to imaging of biological specimens with femtosecond (fs) temporal resolution. In PINEM, the biological structure is exposed to single-electron packets and simultaneously irradiated with fs laser pulses that are coincident with the electron pulses in space and time. By electron energy-filtering those electrons that gained photon energies, the contrast is enhanced only at the surface of the structures involved. This method is demonstrated here in imaging of protein vesicles and whole cells of Escherichia coli, both are not absorbing the photon energy, and both are of low-Z contrast. It is also shown that the spatial location of contrast enhancement can be controlled via laser polarization, time resolution, and tomographic tilting. The high-magnification PINEM imaging provides the nanometer scale and the fs temporal resolution. The potential of applications is discussed and includes the study of antibodies and immunolabeling within the cell. PMID:20479261

  20. Is transmission electron microscopy (TEM) a promising approach for qualitative and quantitative investigations of polymyxin B and miconazole interactions with cellular and subcellular structures of Staphylococcus pseudintermedius, Escherichia coli, Pseudomonas aeruginosa and Malassezia pachydermatis?

    PubMed

    Voget, Michael; Lorenz, Dorothea; Lieber-Tenorio, Elisabeth; Hauck, Ruediger; Meyer, Michael; Cieslicki, Michael

    2015-12-31

    Antimicrobial therapy using a combination of polymyxin B and miconazole is effective against the main bacterial pathogens associated with otitis externa in dogs, and a synergistic effect of both drugs has been shown previously. The objective of the present investigation was to visualize ultrastructural changes after exposure of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus pseudintermedius and Malassezia pachydermatis to polymyxin B and miconazole by transmission electron microscopic (TEM). For this, cultures of E. coli, P. aeruginosa, S. pseudintermedius and M. pachydermatis were exposed to polymyxin B and miconazole, alone or in combination for 24h. Ultrastructural changes were observed most frequently in the cell envelope of the four microorganisms. Exposure to polymyxin B seemed to cause more damage than miconazole within the range of concentrations applied. Treatment resulted in changes of the cell size: in E. coli, cell size increased significantly after treatment with either compound alone; in P. aeruginosa, cell size decreased significantly after treatment with polymyxin B and with miconazole; exposure of S. pseudintermedius to miconazole caused a decrease in cell size; in M. pachydermatis, cell size increased significantly after treatment with polymyxin B.; in E.coli, S. pseudintermedius and M. pachydermatis, cell size changed highly significant, in P. aeruginosa significantly after exposure to the combination of both compounds. In conclusion, by using a different approach than previous investigations, this study confirmed a clear combinatory effect of polymyxin B and miconazole against the tested microorganisms involved in canine otitis externa. It is the first time that visualization technologies were applied to compare the effect of single drugs to their combinatory effects on cellular and subcellular entities of selected bacterial and yeast species. PMID:26527257

  1. 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.

  2. Polyvinylidene fluoride molecules in nanofibers, imaged at atomic scale by aberration corrected electron microscopy.

    PubMed

    Lolla, Dinesh; Gorse, Joseph; Kisielowski, Christian; Miao, Jiayuan; Taylor, Philip L; Chase, George G; Reneker, Darrell H

    2015-12-17

    Atomic scale features of polyvinylidene fluoride molecules (PVDF) were observed with aberration corrected transmission electron microscopy. Thin, self-supporting PVDF nanofibers were used to create images that show conformations and relative locations of atoms in segments of polymer molecules, particularly segments near the surface of the nanofiber. Rows of CF2 atomic groups, at 0.25 nm intervals, which marked the paths of segments of the PVDF molecules, were seen. The fact that an electron microscope image of a segment of a PVDF molecule depended upon the particular azimuthal direction, along which the segment was viewed, enabled observation of twist around the molecular axis. The 0.2 nm side-by-side distance between the two fluorine atoms attached to the same carbon atom was clearly resolved. Morphological and chemical changes produced by energetic electrons, ranging from no change to fiber scission, over many orders of magnitude of electrons per unit area, promise quantitative new insights into radiation chemistry. Relative movements of segments of molecules were observed. Promising synergism between high resolution electron microscopy and molecular dynamic modeling was demonstrated. This paper is at the threshold of growing usefulness of electron microscopy to the science and engineering of polymer and other molecules. PMID:26369731

  3. 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.

  4. 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.

  5. Patterned Carbon Nanotube Applications for Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Zufelt, Kyle; Abbott, Jonathan; Davis, Robert; Vanfleet, Richard

    2010-10-01

    Transmission electron microscopy is a method for observing and characterizing thin films and other nanoscale samples. Carbon nanotubes were patterned and grown to function as disposable grids for transmission electron microscopy research. Patterned nanotube forests were infiltrated with carbon by chemical vapor deposition to provide greater strength. Carbon and polymer support films have been deposited in a batch process to provide a substrate for samples to be observed in the microscope. Grids are released from silicon wafer substrates by chemical etching. Carbon grids represent a significant improvement over traditional copper grids, which are not robust and must be individually coated in support films. These carbon support grids are also superior for use in spectroscopy applications (EELS, EDX) because of the low background signal.

  6. Macromolecular assembly structures by comparative modeling and electron microscopy.

    PubMed

    Lasker, Keren; Velázquez-Muriel, Javier A; Webb, Benjamin M; Yang, Zheng; Ferrin, Thomas E; Sali, Andrej

    2012-01-01

    Advances in electron microscopy allow for structure determination of large biological machines at increasingly higher resolutions. A key step in this process is fitting component structures into the electron microscopy-derived density map of their assembly. Comparative modeling can contribute by providing atomic models of the components, via fold assignment, sequence-structure alignment, model building, and model assessment. All four stages of comparative modeling can also benefit from consideration of the density map. In this chapter, we describe numerous types of modeling problems restrained by a density map and available protocols for finding solutions. In particular, we provide detailed instructions for density map-guided modeling using the Integrative Modeling Platform (IMP), MODELLER, and UCSF Chimera. PMID:22323229

  7. Scanning electron microscopy: preparation and imaging for SEM.

    PubMed

    Jones, Chris G

    2012-01-01

    Scanning electron microscopy (SEM) has been almost universally applied for the surface examination and characterization of both natural and man-made objects. Although an invasive technique, developments in electron microscopy over the years has given the microscopist a much clearer choice in how invasive the technique will be. With the advent of low vacuum SEM in the 1970s (The environmental cold stage, 1970) and environmental SEM in the late 1980s (J Microsc 160(pt. 1):9-19, 1989), it is now possible in some circumstances to examine samples without preparation. However, for the examination of biological tissue and cells it is still advisable to chemically fix, dehydrate, and coat samples for SEM imaging and analysis. This chapter aims to provide an overview of SEM as an imaging tool, and a general introduction to some of the methods applied for the preparation of samples. PMID:22907399

  8. Ultrafast electron microscopy in materials science, biology, and chemistry

    NASA Astrophysics Data System (ADS)

    King, Wayne E.; Campbell, Geoffrey H.; Frank, Alan; Reed, Bryan; Schmerge, John F.; Siwick, Bradley J.; Stuart, Brent C.; Weber, Peter M.

    2005-06-01

    The use of pump-probe experiments to study complex transient events has been an area of significant interest in materials science, biology, and chemistry. While the emphasis has been on laser pump with laser probe and laser pump with x-ray probe experiments, there is a significant and growing interest in using electrons as probes. Early experiments used electrons for gas-phase diffraction of photostimulated chemical reactions. More recently, scientists are beginning to explore phenomena in the solid state such as phase transformations, twinning, solid-state chemical reactions, radiation damage, and shock propagation. This review focuses on the emerging area of ultrafast electron microscopy (UEM), which comprises ultrafast electron diffraction (UED) and dynamic transmission electron microscopy (DTEM). The topics that are treated include the following: (1) The physics of electrons as an ultrafast probe. This encompasses the propagation dynamics of the electrons (space-charge effect, Child's law, Boersch effect) and extends to relativistic effects. (2) The anatomy of UED and DTEM instruments. This includes discussions of the photoactivated electron gun (also known as photogun or photoelectron gun) at conventional energies (60-200 keV) and extends to MeV beams generated by rf guns. Another critical aspect of the systems is the electron detector. Charge-coupled device cameras and microchannel-plate-based cameras are compared and contrasted. The effect of various physical phenomena on detective quantum efficiency is discussed. (3) Practical aspects of operation. This includes determination of time zero, measurement of pulse-length, and strategies for pulse compression. (4) Current and potential applications in materials science, biology, and chemistry. UEM has the potential to make a significant impact in future science and technology. Understanding of reaction pathways of complex transient phenomena in materials science, biology, and chemistry will provide fundamental knowledge for discovery-class science.

  9. Practical aspects of monochromators developed for transmission electron microscopy

    PubMed Central

    Kimoto, Koji

    2014-01-01

    A few practical aspects of monochromators recently developed for transmission electron microscopy are briefly reviewed. The basic structures and properties of four monochromators, a single Wien filter monochromator, a double Wien filter monochromator, an omega-shaped electrostatic monochromator and an alpha-shaped magnetic monochromator, are outlined. The advantages and side effects of these monochromators in spectroscopy and imaging are pointed out. A few properties of the monochromators in imaging, such as spatial or angular chromaticity, are also discussed. PMID:25125333

  10. 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.

  11. Practical aspects of monochromators developed for transmission electron microscopy.

    PubMed

    Kimoto, Koji

    2014-10-01

    A few practical aspects of monochromators recently developed for transmission electron microscopy are briefly reviewed. The basic structures and properties of four monochromators, a single Wien filter monochromator, a double Wien filter monochromator, an omega-shaped electrostatic monochromator and an alpha-shaped magnetic monochromator, are outlined. The advantages and side effects of these monochromators in spectroscopy and imaging are pointed out. A few properties of the monochromators in imaging, such as spatial or angular chromaticity, are also discussed. PMID:25125333

  12. 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.

  13. 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.

  14. Imaging and microanalysis of thin ionomer layers by scanning transmission electron microscopy

    SciTech Connect

    Cullen, David A; Koestner, Roland; Kukreja, Ratan; Minko, Sergiy; Trotsenko, Oleksandr; Tokarev, Alexander V; Guetaz, Laure; Meyer III, Harry M; Parish, Chad M; More, Karren Leslie

    2014-01-01

    Improved conditions for imaging and spectroscopic mapping of thin perfluorosulfonic acid (PFSA) ionomer layers in fuel cell electrodes by scanning transmission electron microscopy (STEM) have been investigated. These conditions are first identified on model systems of Nafion ionomer-coated nanostructured thin films and nanoporous Si. The optimized conditions are then applied in a quantitative study of the ionomer through-layer loading for two typical electrode catalyst coatings using electron energy loss and energy dispersive X-ray spectroscopy in the transmission electron microscope. The e-beam induced damage to the perfluorosulfonic acid (PFSA) ionomer is quantified by following the fluorine mass loss with electron exposure and is then mitigated by a few orders of magnitude using cryogenic specimen cooling and a higher incident electron voltage. Multivariate statistical analysis is also applied to the analysis of spectrum images for data denoising and unbiased separation of independent components related to the catalyst, ionomer, and support.

  15. Fixation methods for electron microscopy of human and other liver

    PubMed Central

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

    2010-01-01

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

  16. A scanning electron microscopy study of laser coating microstructures

    SciTech Connect

    Liu Jianglong; Ding Peidao; Shi Gongqi . Dept. of Metallurgy and Materials Engineering)

    1994-12-01

    Investigations were made, using scanning electron microscopy, to examine microstructural features of laser coatings. The scanning electron microscope make sit possible to observe clearly the detail of the structure in the laser-irradiated zone. It is shown that the microstructures in the laser-coated zone have various features, including a dendritic eutectic, dendritic solid solution, and residual graphite, and that the microstructures in the transitional zone are more complex, including cellular dendritic-cellular, dendritic eutectic, planar, and residual graphite. The laser coating process changes the nature of solidification of the coating melt, compared with common solidification.

  17. Transmission Electron Microscopy Study of InN Nanorods

    SciTech Connect

    Liliental-Weber, Z.; Li, X.; Kryliouk, Olga; Park, H.J.; Mangum,J.; Anderson, T.

    2006-07-13

    InN nanorods were grown on a, c-, and r-plane of sapphire and also on Si (111) and GaN (0001) by non-catalytic, template-free hydride metal-organic vapor phase epitaxy and studied by transmission electron microscopy, electron energy loss (EELS) and photoluminescence (PL) at room temperature. These nanocrystals have different shapes and different faceting depending on the substrate used and their crystallographic orientation. EELS measurements have confirmed the high purity of these crystals. The observed PL peak was in the range of 0.9-0.95 eV. The strongest PL intensity was observed for the nanocrystals with the larger diameters.

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

    NASA Astrophysics Data System (ADS)

    Cattle, J.; S'ari, M.; Hondow, N.; Abelln, 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.

  19. Three-dimensional volume imaging with electron microscopy toward connectome.

    PubMed

    Ohno, Nobuhiko; Katoh, Mitsuhiko; Saitoh, Yurika; Saitoh, Sei; Ohno, Shinichi

    2015-02-01

    Ultrastructural analyses with electron microscopy have provided indispensable information to understand physiology and pathology of the nervous system. Recent advancement in imaging methodology paved the way for complete reconstruction of the neuronal connection map in the central nervous system, which is termed 'connectome' and would provide key insights to understand the functions of the brain. The critical advancement includes serial ultrastructural observation with scanning electron microscopy (SEM) instead of conventional serial sectioning transmission electron microscopy along with specific tissue preparation methods to increase heavy metal deposition for efficient SEM imaging. The advanced imaging methods using SEM have distinct advantages and disadvantages in multiple aspects, such as resolution and imaging speed, and should be selected depending on the observation conditions, such as target tissue sizes, required spatial resolution and necessity for re-observation. Dealing with the huge dataset remained to be a major obstacle, and automation in segmentation and 3D reconstruction would be critical to understand neuronal circuits in a larger volume of the brain. Future improvement in acquisition and analyses of the morphological data obtained with the advanced SEM imaging is awaited to elucidate the significance of whole connectome as the structural basis of the consciousness, intelligence and memory of a subject. PMID:25550364

  20. 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.

  1. System and method for compressive scanning electron microscopy

    SciTech Connect

    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.

  2. 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.

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. Thickness determination of few-layer hexagonal boron nitride films by scanning electron microscopy and Auger electron spectroscopy

    SciTech Connect

    Sutter, P. Sutter, E.

    2014-09-01

    We assess scanning electron microscopy (SEM) and Auger electron spectroscopy (AES) for thickness measurements on few-layer hexagonal boron nitride (h-BN), the layered dielectric of choice for integration with graphene and other two-dimensional materials. Observations on h-BN islands with large, atomically flat terraces show that the secondary electron intensity in SEM reflects monolayer height changes in films up to least 10 atomic layers thickness. From a quantitative analysis of AES data, the energy-dependent electron escape depth in h-BN films is deduced. The results show that AES is suitable for absolute thickness measurements of few-layer h-BN of 1 to 6 layers.

  8. Direct Visualization of Dendrite Nucleation and Growth Kinetics during Lithium Deposition with in situ Electrochemical Transmission Electron Microscopy

    SciTech Connect

    Sacci, Robert L; Dudney, Nancy J; More, Karren Leslie; Browning, Nigel; Unocic, Raymond R

    2014-01-01

    Formation of Li dendrites is a major safety concern existing in Li-ion secondary batteries. A quantitative electrochemistry method to investigate the dendrite nucleation and growth mechanisms at high spatial is presented. Cyclic voltammetry, in combination with in situ electrochemical transmission electron microscopy (in situ ec-TEM), was used to quantitatively characterize dendrite nucleation and growth mechanisms from a Au working electrode and within a 1.2M LiPF6 EC:DMC electrolyte.

  9. 4D multiple-cathode ultrafast electron microscopy

    PubMed Central

    Baskin, John Spencer; Liu, Haihua; Zewail, Ahmed H.

    2014-01-01

    Four-dimensional multiple-cathode ultrafast electron microscopy is developed to enable the capture of multiple images at ultrashort time intervals for a single microscopic dynamic process. The dynamic process is initiated in the specimen by one femtosecond light pulse and probed by multiple packets of electrons generated by one UV laser pulse impinging on multiple, spatially distinct, cathode surfaces. Each packet is distinctly recorded, with timing and detector location controlled by the cathode configuration. In the first demonstration, two packets of electrons on each image frame (of the CCD) probe different times, separated by 19 picoseconds, in the evolution of the diffraction of a gold film following femtosecond heating. Future elaborations of this concept to extend its capabilities and expand the range of applications of 4D ultrafast electron microscopy are discussed. The proof-of-principle demonstration reported here provides a path toward the imaging of irreversible ultrafast phenomena of materials, and opens the door to studies involving the single-frame capture of ultrafast dynamics using single-pump/multiple-probe, embedded stroboscopic imaging. PMID:25006261

  10. Correlative cryo-electron tomography and optical microscopy of cells.

    PubMed

    Zhang, Peijun

    2013-10-01

    The biological processes occurring in a cell are complex and dynamic, and to achieve a comprehensive understanding of the molecular mechanisms underlying these processes, both temporal and spatial information is required. While cryo-electron tomography (cryoET) provides three-dimensional (3D) still pictures of near-native state cells and organelles at molecular resolution, fluorescence light microscopy (fLM) offers movies of dynamic cellular processes in living cells. Combining and integrating these two commonly used imaging modalities (termed correlative microscopy) provides a powerful means to not only expand the imaging scale and resolution but also to complement the dynamic information available from optical microscopy with the molecular-level, 3D ultrastructure detail provided by cryoET. As such, a correlative approach performed on a given specimen can provide high resolution snapshots of dynamic cellular events. In this article, I review recent advances in correlative light microscopy and cryoET and discuss major findings made available by applying this method. PMID:23962486

  11. Analysis of environmental particles by atomic force microscopy, scanning and transmission electron microscopy.

    PubMed

    Mavrocordatos, D; Pronk, W; Boiler, M

    2004-01-01

    Due to their large specific surface and their abundance, micro and nano particles play an important role in the transport of micropollutants in the environment. Natural particles are usually composed of a mixture of inorganic amorphous or crystalline material (mainly FeOOH, Fe(x)Oy, Mn(x)Oy and clays) and organic material (humics and polysaccharides). They all tend to occur as very small particles (1-1,000 nm in diameter). Most natural amorphous particles are unstable and tend to transform with time towards more crystalline forms, either by aging or possibly, by dissolution and re-crystallization. Such transformations affect the fate of sorbed micropollutants and the scavenging properties are therefore changed. As these entities are sensitive to dehydration (aggregation, changes in the morphology), it is highly important to observe their morphology in their natural environment and understand their composition at the scale of the individual particles. Also for the understanding and optimization of water treatment technologies, the knowledge of the occurrence and behavior of nano-particles is of high importance. Some of the possible particle analysis methods are presented: aggregation processes, biomineralization, bacterial adhesion, biofilms in freshwaters, ferrihydrite as heavy metals remover from storm water. These examples demonstrate the capabilities and focus of the microscopes. Atomic Force Microscopy (AFM) allows to analyze the particles in their own environment, meaning in air or in the water. Thus, native aspects of particles can be observed. As well, forces of interactions between particles or between particles and other surfaces such as membranes will be highly valuable data. Scanning Electron Microscopy (SEM) and for higher lateral resolution, Transmission Electron Microscopy (TEM) allow measurement of the morphology and composition. Especially, TEM coupled with Electron Energy Loss Spectroscopy (TEM-EELS) is a powerful technique for elemental analysis. Finally, general guidelines for the effective use of microscopic techniques are provided. PMID:15685998

  12. Opportunities for electron microscopy in space radiation biology

    SciTech Connect

    Lett, J.T.

    1986-01-01

    Densely ionizing, particulate radiations in outer space are likely to cause to mammalian tissues biological damage that is particularly amenable to examination by the techniques of electron microscopy. This situation arises primarily from the fact that once the density of ionization along the particle track exceeds a certain value, small discrete lesions involving many adjacent cells may be caused in organized tissues. Tissue damage produced by ionization densities below the critical value also afford opportunities for electron microscopic evaluation, as is shown by the damage produced in optic and proximate tissues of the New Zealand white rabbit in terrestrial experiments. Late radiation sequelae in nondividing, or terminally differentiating, tissues, and in stem cell populations, are of special importance in these regards. It is probable that evaluations of the hazards posed to astronauts by galactic particulate radiations during prolonged missions in outer space will not be complete without adequate electron microscopic evaluation of the damage those radiations cause to organized tissues.

  13. 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.

  14. Approach for investigating the astigmatism of a magnetic prism in low-energy electron microscopy

    NASA Astrophysics Data System (ADS)

    Kan, H.-C.; Auerbach, Daniel; Phaneuf, R. J.

    2003-02-01

    We report an approach for investigating the electron optical properties of a magnetic prism, which includes experimental measurement of the focal point positions and the focal lengths of a magnetic prism and a direct comparison between the measurement and the electron optical simulation. We applied this approach to the magnetic prism we constructed as the beam separator for our low-energy electron microscope (LEEM). The magnetic prism consists of two sets of concentric round pole pieces and a rectangular housing as the return circuit of the magnetic flux. The experimental measurements were obtained from images of a square array of submicron size silver dots patterned on a Si(100) substrate recorded with our LEEM operated in photoemission electron microscopy mode. The measurements were compared to results of numerical simulations done with two different approaches. Both calculations agree with the measurements quantitatively.

  15. Ultrahigh Voltage Electron Microscopy Links Neuroanatomy and Neuroscience/Neuroendocrinology

    PubMed Central

    Sakamoto, Hirotaka; Kawata, Mitsuhiro

    2012-01-01

    The three-dimensional (3D) analysis of anatomical ultrastructures is extremely important in most fields of biological research. Although it is very difficult to perform 3D image analysis on exact serial sets of ultrathin sections, 3D reconstruction from serial ultrathin sections can generally be used to obtain 3D information. However, this technique can only be applied to small areas of a specimen because of technical and physical difficulties. We used ultrahigh voltage electron microscopy (UHVEM) to overcome these difficulties and to study the chemical neuroanatomy of 3D ultrastructures. This methodology, which links UHVEM and light microscopy, is a useful and powerful tool for studying molecular and/or chemical neuroanatomy at the ultrastructural level. PMID:22567316

  16. Pars plana incisions of four patients: histopathology and electron microscopy.

    PubMed Central

    Koch, F H; Kreiger, A E; Spitznas, M; Glasgow, B; Foos, R Y; Yoshizumi, M O

    1995-01-01

    The pathology of pars plana incisions of four patients is described: three with light microscopy and one with light and electron microscopy. Two eyes were removed because of choroidal melanoma, immediately and 8 days after vitrectomy and transvitreous retinal biopsy. Considerable disruption of tissues surrounding the pars plana incisions was observed. Vitreous was incarcerated in the wounds, which healed with granulation tissue. One eye was examined 4 months after vitrectomy for diabetic retinopathy and a failed pars plana filtering operation. It contained fibrovascular ingrowth from all the incisions, infiltrating the vitreous base with granulation tissue and causing vitreous haemorrhage and retinal detachment. One eye was removed 1 year after vitrectomy for anterior hyaloidal fibrovascular proliferation and early phthisis. The wound had fibrous ingrowth histologically and evidence of active fibroplasia. Images PMID:7612564

  17. Phase measurements of erythrocytes affected by metal ions with quantitative interferometric microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Shouyu; Yan, Keding; Shan, Yanke; Xu, Mingfei; Liu, Fei; Xue, Liang

    2015-12-01

    Erythrocyte morphology is an important factor in disease diagnosis, however, traditional setups as microscopes and cytometers cannot provide enough quantitative information of cellular morphology for in-depth statistics and analysis. In order to capture variations of erythrocytes affected by metal ions, quantitative interferometric microscopy (QIM) is applied to monitor their morphology changes. Combined with phase retrieval and cell recognition, erythrocyte phase images, as well as phase area and volume, can be accurately and automatically obtained. The research proves that QIM is an effective tool in cellular observation and measurement.

  18. 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 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. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr08888g

  19. 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 GluR?2-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

  20. Quantitative phase and refractive index analysis of optical fibers using differential interference contrast microscopy.

    PubMed

    Kouskousis, Betty; Kitcher, Daniel J; Collins, Stephen; Roberts, Ann; Baxter, Greg W

    2008-10-01

    A systematic and straightforward image processing method to extract quantitative phase and refractive index data from weak phase objects is presented, obtained using differential interference contrast (DIC) microscopy. The method is demonstrated on DIC images of optical fibers where a directional integration routine is applied to the DIC images to extract phase and refractive index information using the data obtained across the whole DIC image. By applying the inverse Abel transform to the resultant phase images, an accurate refractive index profile is obtained. The method presented here is compared to the refracted near-field technique, typically used to obtain the refractive index profile of optical fibers, and shows excellent agreement. It is concluded that through careful image processing procedures, DIC microscopy can be successfully implemented to obtain quantitative phase and refractive index information of optical fibers. PMID:18830309

  1. An adjustable electron achromat for cathode lens microscopy.

    PubMed

    Tromp, R M

    2015-12-01

    Chromatic aberration correction in light optics began with the invention of a two-color-corrected achromatic crown/flint lens doublet by Chester Moore Hall in 1730. Such color correction is necessary because any single glass shows dispersion (i.e. its index of refraction changes with wavelength), which can be counteracted by combining different glasses with different dispersions. In cathode lens microscopes (such as Photo Electron Emission Microscopy - PEEM) we encounter a similar situation, where the chromatic aberration coefficient of the cathode lens shows strong dispersion, i.e. depends (non-linearly) on the energy with which the electrons leave the sample. Here I show how a cathode lens in combination with an electron mirror can be configured as an adjustable electron achromat. The lens/mirror combination can be corrected at two electron energies by balancing the settings of the electron mirror against the settings of the cathode lens. The achromat can be adjusted to deliver optimum performance, depending on the requirements of a specific experiment. Going beyond the achromat, an apochromat would improve resolution and transmission by a very significant margin. I discuss the requirements and outlook for such a system, which for now remains a wish waiting for fulfilment. PMID:25825026

  2. 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.

  3. Resinless section electron microscopy reveals the yeast cytoskeleton.

    PubMed

    Penman, J; Penman, S

    1997-04-15

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

  4. Scanning electron and tunneling microscopy of palladium barium emitters

    NASA Astrophysics Data System (ADS)

    Baiburin, V. B.; Volkov, U. P.; Semenov, S. V.; Semenov, A. S.

    2003-06-01

    The results of study of metal-alloyed palladium-barium emitters' of modern very high frequency high-powered electronic vacuum tubes by scanning electron microscopy (SEM) and scanning tunneling microscopy/spectroscopy (STM/STS) are presented. Since the Pd/Ba foil surface is fairly smooth and is not oxidized in air STM/STS investigations are carried out in air in normal laboratory environment. SEM and STM images show that the emitter surface has a complex porous structure. The cathode surface study by STS in tunneling gap modulation mode allowed to take a map of phase distribution with various work function values and high lateral resolution. Obtained images demonstrate the presence of three phases on the Pd/Ba emitter surface, viz. barium-oxygen compounds, intermetallic, and palladium. As it is seen from presented STS image the phase with a low work function value (barium oxides) is concentrated along boundaries of the substance inclusions with work function corresponding to the intemetallic compound Pd 5Ba. This supports the model of low work function areas obtained via Ba segregation from the intermetallic compound and oxidation. The presented methods may be used in the Pd/Ba cathode manufacturing process for increasing the yield of electronic devices in microwave tube production and optimize the emitters' characteristics.

  5. 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

  6. Quantitative self-organizing maps for clustering electron tomograms.

    PubMed

    Pascual-Montano, A; Taylor, K A; Winkler, H; Pascual-Marqui, R D; Carazo, J-M

    2002-01-01

    Tomography emerges as a powerful methodology for determining the complex architectures of biological specimens that are better regarded from the structural point of view as singular entities. However, once the structure of a sufficiently large number of singular specimens is solved, quite possibly structural patterns start to emerge. This latter situation is addressed here, where the clustering of a set of 3D reconstructions using a novel quantitative approach is presented. In general terms, we propose a new variant of a self-organizing neural network for the unsupervised classification of 3D reconstructions. The novelty of the algorithm lies in its rigorous mathematical formulation that, starting from a large set of noisy input data, finds a set of "representative" items, organized onto an ordered output map, such that the probability density of this set of representative items resembles at its possible best the probability density of the input data. In this study, we evaluate the feasibility of application of the proposed neural approach to the problem of identifying similar 3D motifs within tomograms of insect flight muscle. Our experimental results prove that this technique is suitable for this type of problem, providing the electron microscopy community with a new tool for exploring large sets of tomogram data to find complex patterns. PMID:12160707

  7. 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.

  8. Analysis of Electron Beam Damage of Crystalline Pharmaceutical Materials by Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    S'ari, M.; Cattle, J.; Hondow, N.; Blade, H.; Cosgrove, S.; Brydson, R. M.; Brown, A. P.

    2015-10-01

    We have studied the impact of transmission electron microscopy (TEM) and low dose electron diffraction on ten different crystalline pharmaceutical compounds, covering a diverse chemical space and with differing physical properties. The aim was to establish if particular chemical moieties were more susceptible to damage within the electron beam. We have measured crystalline diffraction patterns for each and indexed nine out of ten of them. Characteristic electron dosages are reported for each material, with no apparent correlation between chemical structure and stability within the electron beam. Such low dose electron diffraction protocols are suitable for the study of pharmaceutical compounds.

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

    NASA Astrophysics Data System (ADS)

    Kemper, Bjrn; 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.

  10. 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

  11. New immunolatex spheres: visual markers of antigens on lymphocytes for scanning electron microscopy

    PubMed Central

    1975-01-01

    New immunochemical reagents consisting of antibodies bound to small latex spheres were used as visual markers for the detection and localization of cell surface antigens by scanning electron microscopy. Cross-linked latex spheres of various sizes from 300 to 3,4000 A in diameter were synthesized by aqueous emulsion copolymerization of methacrylate derivatives containing hydroxyl and carboxyl functional groups. Proteins and other molecules containing primary amino groups were covalently bonded to the acrylic spheres under a variety of mild conditions by the aqueous carbodiimide, cyanogen bromide, and glutaraldehyde methods. For use in the indirect immunochemical-labeling technique, goat antibodies directed against rabbit immunoglobulins were bonded to the spheres. These immunolatex reagents were shown to bind only to cells (red blood and lymphocytes) which had previously been sensitized with rabbit antibodies against cell surface antigens. Mouse spleen lymphocytes with exposed immunoglobulins on their surface (B cells) were labeled with these spheres and distinguished from unlabeled or T lymphocytes by scanning electron microscopy. The distribution of Ig receptors on lymphocytes was also studied using the spheres as visual markers. When lymphocytes were fixed with glutaraldehyde and subsequently labeled with the immunolatex reagents, a random distribution was observed by scanning electron microscopy; a patchy distribution was observed when unfixed lymphocytes were used. These results are consistent with studies using ferritin-labeled antibodies (S. De Petris and M. Raff. 1973. Nature [Lond.]. 241:257.) and support the view that Ig receptors on lymphocytes undergo translational diffusion. In addition to serving as visual markers for scanning electron microscopy, these latex spheres tagged with fluorescent or radioactive molecules have applications as highly sensitive markers for fluorescent microscopy and as reagents for quantitative studies of cell surface antigens and other receptors. PMID:803228

  12. Investigation of the mechanism of transdermal penetration enhancer: a comparison of multiphoton microscopy and electron microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Sung-Jan; Lee, Jin-Ning; Lin, Chiao-Ying; Chan, Chih-Chieh; Lin, Ming-Gu; Wang, Chun-Chin; Tan, Hsin-Yuan; Tsai, Tsung-Hua; Jee, Shiou-Hwa; Dong, Chen-Yuan

    2008-02-01

    The aim of this study is to characterize the ability of multiphoton microscopy in monitoring the transdermal penetration enhancing effect of a depilatory agent and the associated structural alterations of stratum corneum. The result is compared with the electron microscopic findings. Our results show that the penetration of both hydrophilic and hydrophobic agents can be enhanced. The morphology of corneocytes becomes a homogenized pattern with focal detachment of surface corneocytes. In combination with Nile red staining, multiphoton imaging also shows that the regular motar-like distribution of lipid matrix was disrupted into a homogenized pattern of lipid distribution. These results are well correlated with the findings of ultrastructural analysis by electron micrographs showing disintegration of the protein envelope of coenocytes, disruption of intracellular keratin and loss of the regular lamellar packing of intercellular lipids. We conclude that, in addition to quantifying the permeation profiles of model drugs, multiphoton microscopy is able to detect the penetration enhancer-induced structural alterations of stratum corneum.

  13. Electron Microscopy Studies of Solid Surfaces and Interfaces.

    NASA Astrophysics Data System (ADS)

    Gajdardziska-Josifovska, Marija

    1991-02-01

    Electron microscopy techniques for study of surfaces and interfaces have been investigated and applied to (100) and (111) surfaces of MgO and to interfaces of Mo/Si multilayers and CoSi_2/Si epitaxial films. MgO surfaces subjected to different annealing and chemical treatments have been characterized by reflection electron microscopy imaging, reflection high-energy electron diffraction (RHEED), and reflection electron energy-loss spectroscopy (REELS). An oxygen rich (sqrt {3} times sqrt{3})R 30^circ reconstruction was found on the polar (111) surface upon annealing in oxygen at temperatures higher than 1500 ^circC. Transformation of the surface topography and segregation of calcium were observed on the cleaved (100) surface due to annealing. RHEED resonance conditions have been employed and studied with geometrical constructions, rocking curves and REELS. These conditions are associated with parabolas in the Kikuchi (K) patterns whose nature had been subject of much controversy. The parabolas have been explained as K lines of two-dimensional (2D) lattices in a general scheme which describes the K pattern geometry in terms of intersections of Brillouin zone boundaries with a sphere of reflections. Full treatment of the cases of 2D and 1D real lattices has revealed previously unknown boundaries in the form of parabolic surfaces (2D) and paraboloids of revolution (1D). These boundaries have been applied to lines which arise from electron channeling in 3D crystals and to RHEED parabolas from 2D surface reconstructions. Nanodiffraction, low angle dark-field imaging, electron holography, high spatial resolution EELS, and shadow imaging have been evaluated as means for measuring interface abruptness and change in mean-inner potential and compared to other microscopy techniques. Refraction effects at interfaces were observed as streaking of the nanodiffraction disks which was found to depend on the crystalline nature of the interface. For polycrystalline/amorphous interfaces asymmetric streaking towards the phase with larger mean-inner potentials was observed to depend on the interface abruptness and the thickness of the specimen. On crystal/crystal interfaces the streaking was found to be sensitive to interface reconstructions.

  14. 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.

  15. Investigation of human chromosome polymorphisms by scanning electron microscopy.

    PubMed

    Harrison, C J; Jack, E M; Allen, T D; Harris, R

    1985-02-01

    Human chromosome polymorphisms were investigated by scanning electron microscopy (SEM). Centromeric heterochromatin was of a constricted morphology. The extent of the C banded region was demarcated by a prominent circumferential groove in G banded chromosomes. Circumferential grooves were observed within the heterochromatin of chromosome 9, and the number of grooves present reflected the size of the region. Three dimensional viewing of satellites and short arms of acrocentric chromosomes, from different angles in the SEM, provided the opportunity for accurate assessment of the size of satellites to be made. Also, small morphological variations were defined in the SEM when definition was uncertain in the light microscope (LM). PMID:4039005

  16. Cryo-Electron Microscopy of Biological Macromolecular Structures

    NASA Astrophysics Data System (ADS)

    Yonekura, Koji

    There are many huge macromolecular complexes in living organisms. They are often hard to crystallize because of their size, complexity and heterogeneity. Cryo-electron microscopy (cryo-EM) is a suitable method to analyze the structures of such biological macromolecules, because it can be applied to various forms of samples, e.g. two-dimensional crystal, helical assembly, spherical virus, dispersed particle, cell organelle and cell, although attainable resolution depends on the system. In this review, I introduce these techniques and examples of the structure analysis, and briefly review the perspective of cryo-EM.

  17. Ballistic Electron Emission Microscopy of Metal/Group IV Interfaces

    NASA Technical Reports Server (NTRS)

    Hecht, M. H.; Kaiser, W. J.; Bell, L. D.; Fathauer, R.; Manion, S. J.

    1993-01-01

    Ballistic electron emission microscopy and spectroscopy, together with related techniques, have been applied with great success to the study of buried interfaces. These probes, known collectively as BEEM, have yielded important information on interface transport, interface band structure, and carrier scattering, with lateral spatial resolution on the nanometer scale. Recent applications of the technique to polycrystalline metal/semiconductor interfaces have demonstrated an ability to spatially map both conduction band and valence band semiconductor structure. BEEM studies of epitaxial silicide/silicon interfaces have been particularly fruitful, as the rich silicide band structure results in complex and often surprising transport behavior...

  18. Birefringence and transmission electron microscopy of monolayer and bilayer magnetoliposomes

    NASA Astrophysics Data System (ADS)

    Morais, P. C.; Skeff Neto, K.; Gravina, P. P.; Figueiredo, L. C.; Da Silva, M. F.; Lacava, Z. G. M.; Azevedo, R. B.; Silva, L. P.; De Cuyper, M.

    2002-11-01

    In this study, static magnetic birefringence (SMB) and transmission electron microscopy (TEM) were used to investigate magnetite-based monolayer and bilayer magnetoliposomes (MLs). The SMB data were analyzed using the recent model proposed by Skeff Neto et al. (J. Appl. Phys. 89 (2001) 3362). The SMB data indicate that monolayer-based MLs internalize magnetic nanoparticles as dimers while bilayer-based MLs internalize both isolated nanoparticles and dimers. The higher content of dimers inside monolayer MLs has been confirmed by TEM data.

  19. Confocal Microscopy for Modeling Electron Microbeam Irradiation of Skin

    SciTech Connect

    Miller, John H.; Chrisler, William B.; Wang, Xihai; Sowa, Marianne B.

    2011-08-01

    For radiation exposures employing targeted sources such as particle microbeams, the deposition of energy and dose will depend on the spatial heterogeneity of the spample. Although cell structural variations are relatively minor for two-dimensional cell cultures, they can vary significantly for fully differential tissues. Employing high-resolution confocal microscopy, we have determined the spatial distribution, size, and shape of epidermal kerantinocyte nuclei for the full-thickness EpiDerm skin model (MatTek, Ashland, VA). Application of these data to claculate the microdosimetry and microdistribution of energy deposition by an electron microbeam is discussed.

  20. Measurement of dihedral angles by scanning electron microscopy.

    NASA Technical Reports Server (NTRS)

    Achutaramayya, G.; Scott, W. D.

    1973-01-01

    The extension of Hoover's (1971) technique to the case of dihedral-angle measurement is described. Dihedral angles are often determined by interferometry on thermally grooved grain boundaries to obtain information on relative interfacial energies. In the technique considered the measured angles approach the true angles as the tilt angle approaches 90 deg. It is pointed out that the scanning electron microscopy method provides a means of seeing the real root of a groove at a lateral magnification which is higher than that obtainable with interferometry.

  1. 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.

  2. Cryogenic electron microscopy study of nanoemulsion formation from microemulsions.

    PubMed

    Lee, Han Seung; Morrison, Eric D; Frethem, Chris D; Zasadzinski, Joseph A; McCormick, Alon V

    2014-09-16

    We examine a process of preparing oil-in-water nanoemulsions by quenching (diluting and cooling) precursor microemulsions made with nonionic surfactants and a cosurfactant. The precursor microemulsion structure is varied by changing the concentration of the cosurfactant. Water-continuous microemulsions produce initial nanoemulsion structures that are small and simple, mostly unilamellar vesicles, but microemulsions that are not water-continuous produce initial nanoemulsion structures that are larger and multilamellar. Examination of these structures by cryo-electron microscopy supports the hypothesis that they are initially vesicular structures formed via lamellar intermediate structures, and that if the lamellar structures are too well ordered they fail to produce small simple structures. PMID:25141294

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

    PubMed

    Chang, Yi-Wei; Chen, Songye; Tocheva, Elitza I; Treuner-Lange, Anke; Lbach, Stephanie; Sgaard-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. PMID:24813625

  4. 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 gammasb2 phase which is the weakest component of amalgams. Ag-Hg-Sn (betasb1) and large grains of Cusb6Snsb5\\ (etasp') are found adjacent to the unreacted alloy particles. Tytin alloy particles contain Cusb3Sn\\ (epsilon) precipitates in a matrix of Agsb3Sn (gamma) and Agsb4Sn\\ (beta). SEM was used to correlate the TEM findings in the context of the general microstructure. The results are in good agreement with those published in the literature. The microstructural details reported here, many of which were not previously available, will help provide insight into the deformation mechanisms of dental amalgams.

  5. Immunochemistry and electron microscopy of head and neck rhabdomyoma.

    PubMed Central

    Helliwell, T R; Sissons, M C; Stoney, P J; Ashworth, M T

    1988-01-01

    Rhabdomyomas are rare benign tumours originating in skeletal or cardiac muscle. Extracardiac tumours are usually situated in the head and neck. Four cases are presented, three arising in the larynx and the other in the cervical region. All four cases were studied by light and electron microscopy, and in three immunohistochemical staining for myoglobin, desmin, and vimentin was carried out to study the diagnostic features of the lesions and their histogenesis. Images Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6 Fig 7 Fig 8 Fig 9 Fig 10 PMID:3056977

  6. Electron Spin Magnetic Resonance Force Microscopy of Nitroxide Spin Labels

    NASA Astrophysics Data System (ADS)

    Moore, Eric W.; Lee, Sanggap; Hickman, Steven A.; Wright, Sarah J.; Marohn, John A.

    2009-03-01

    Nitroxide spin labels are widely used in electron spin resonance studies of biological and polymeric systems. Magnetic resonance force microscopy (MRFM) is a magnetic resonance technique that couples the high spatial resolution of a scanning probe microscope with the species selectivity of magnetic resonance. We report on our investigations of 4-amino TEMPO, a nitroxide spin label, by force-gradient MRFM. Our microscope operates at high vacuum in liquid helium, using a custom fabricated ultra-soft silicon cantilever in the magnet-on-cantilever geometry. An 18 GHz gap coupled microstripline resonator supplies the transverse field.

  7. 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.

  8. Analytical electron microscopy of a hydrated interplanetary dust particle

    NASA Technical Reports Server (NTRS)

    Blake, David F.; Bunch, T. E.; Mardinly, A. J.; Echer, C. J.

    1988-01-01

    Properties of a hydrated interplanetary dust particle (IDP), Ames-Dec86-11, were investigated using TEM and analytical electron microscopy. The particle was found to have mineralogy and chondritic composition indicating an absence of direct kinship with known carbonaceous chondrites. The available data on the Ames-Dec86-11 suggest that at least one aqueous alteration event took place in this hydrated IDP, during which fine-grained material, possibly glass, was transformed to smectite. This event appears to be unique to hydrated IDPs.

  9. 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.

  10. Performance analysis of quantitative phase retrieval method in Zernike phase contrast X-ray microscopy

    NASA Astrophysics Data System (ADS)

    Heng, Chen; Kun, Gao; Da-Jiang, Wang; Li, Song; Zhi-Li, Wang

    2016-02-01

    Since the invention of Zernike phase contrast method in 1930, it has been widely used in optical microscopy and more recently in X-ray microscopy. Considering the image contrast is a mixture of absorption and phase information, we recently have proposed and demonstrated a method for quantitative phase retrieval in Zernike phase contrast X-ray microscopy. In this contribution, we analyze the performance of this method at different photon energies. Intensity images of PMMA samples are simulated at 2.5 keV and 6.2 keV, respectively, and phase retrieval is performed using the proposed method. The results demonstrate that the proposed phase retrieval method is applicable over a wide energy range. For weakly absorbing features, the optimal photon energy is 2.5 keV, from the point of view of image contrast and accuracy of phase retrieval. On the other hand, in the case of strong absorption objects, a higher photon energy is preferred to reduce the error of phase retrieval. These results can be used as guidelines to perform quantitative phase retrieval in Zernike phase contrast X-ray microscopy with the proposed method. Supported by the State Key Project for Fundamental Research (2012CB825801), National Natural Science Foundation of China (11475170, 11205157 and 11179004) and Anhui Provincial Natural Science Foundation (1508085MA20).

  11. Observations of Nanobubble Dynamics with Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Mohan, Meera Kanakamma; Arora, Manish; Mirsaidov, Utkur; Ohl, Claus-Dieter

    2013-11-01

    Recent developments in transmission electron microscopy (TEM) allow the imaging of liquids with high spatial resolution. Here we report on novel studies of water trapped between two monolayers of graphene sheets. The geometry prevents evaporation of the liquid into the low pressure environment of the TEM while providing excellent electron-optical properties for investigations. The graphene sheets are supported by a conventional TEM grid. We report on the nucleation of bubbles, the coalescence between neighbouring bubbles, rupture of thin liquid filaments, and their slow shrinkage. At a dose rate of 100-155 e--2s-1 these events are observed conveniently at video frame rate. The correlation with the local electron beam dose rate suggests that the radiolysis induced by the electron beam is the main driving force for most events. In general, we observed bubbles with lateral sizes between 20 nm and 100 nm and estimated heights between 6 nm and 30 nm. Likely, the bubbles connect both graphene sheets. In the absence of the electron beam the nanobubbles do not dissolve completely but surprisingly remain stable for even up to one hour. This resembles the stability of surface attached nanobubbles.

  12. 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-) 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 to near atomic (<4 ). 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. PMID:25747402

  13. Some applications of microanalytical electron microscopy in materials research

    SciTech Connect

    Thomas, G.

    1985-10-01

    Electron microscopy has made extraordinary progress over the past 30 years and has become an indispensible tool for research in materials science. In this paper a review is given of some applications of microdiffraction and microanalysis in our current materials science research projects at the University of California, Berkeley. The topics discussed include: (1) The problem of solute atom partitioning in steels; this includes the difficulties of measuring carbon contents and methods of utilizing diffraction, lattice imaging, energy dispersive x-ray (EDXS) and electron energy loss (EELS) spectroscopies and atom probe analysis will be illustrated. (2) Utilization of CBED and EDXS techniques in zirconia ceramics research. (3) Applications of CBED to the study of el-Fe2O3 particles used in magnetic recording systems. (4) Applications of CBED and EDXS to rare earth permanent magnets. (5) Channelling enhanced microanalysis. 50 refs., 21 figs.

  14. 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

  15. 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 Youngs 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

  16. Detection and identification of light impurities by electron microscopy

    SciTech Connect

    Idrobo Tapia, Juan C; Oxley, Mark P; Walkosz, Weronika; Klie, Robert F; Ogut, Serdar; Mikijelj, B; Pennycook, Stephen J; Pantelides, Sokrates T.

    2009-01-01

    For over 40 years impurities have been believed to stabilize the ceramic {alpha}-Si{sub 3}N{sub 4} but there is no direct evidence for their identity or lattice location. In bulk materials electron microscopy can generally image heavy impurities. Here we report direct imaging of N columns in {alpha}-Si{sub 3}N{sub 4} that suggests the presence of excess light elements in specific N columns. First-principles calculations rule out Si or N interstitials and suggest O impurities, which are then confirmed by atomically resolved electron-energy-loss spectroscopy. The result provides a possible explanation for the stability of {alpha}-Si{sub 3}N{sub 4} with implications for the design of next-generation structural ceramics.

  17. Electron microscopy and structural model of human fibronectin receptor.

    PubMed Central

    Nermut, M V; Green, N M; Eason, P; Yamada, S S; Yamada, K M

    1988-01-01

    Highly-purified human fibronectin receptor (a heterodimer of two distinct subunits, alpha and beta) was studied using electron microscopy and a variety of preparative procedures. It was found that the receptor consists of a globular head approximately 80 by 120 A and two tails about 20 A thick and 180-200 A long. The whole complex is approximately 280 A long. At low concentrations of detergent the receptor forms doublets, triplets or rosettes associated with the tails which possess the transmembrane portion of the molecule. Computer-assisted structure prediction using the published amino acid sequence of both subunits showed differences in the secondary structure of the tails, the alpha-tail being rich in beta-strands, the beta-tail having five cysteine-rich repeats analogous to the EGF-like repeats of laminin. Estimates of the length of the tails from the predicted structure conformed well with the dimensions obtained from electron micrographs. Images PMID:2977331

  18. 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.

  19. Electron microscopy of gold nanoparticles at atomic resolution

    PubMed Central

    Azubel, Maia; Koivisto, Jaakko; Malola, Sami; Bushnell, David; Hura, Greg L.; Koh, Ai Leen; Tsunoyama, Hironori; Tsukuda, Tatsuya; Pettersson, Mika; Hkkinen, Hannu; Kornberg, Roger D.

    2014-01-01

    Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, and only one AuNP larger than 1 nm in diameter, an Au102NP, has been solved to atomic resolution. Whereas the Au102NP structure was determined by X-ray crystallography, other large AuNPs have proved refractory to this approach. Here we report the structure determination of an Au68NP at atomic resolution by aberration-corrected transmission electron microscopy (AC-TEM), performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small angle X-ray scattering (SAXS) and by comparison of observed infrared (IR) absorption spectra with calculations by density functional theory (DFT). PMID:25146285

  20. Nanoparticle imaging. Electron microscopy of gold nanoparticles at atomic resolution.

    PubMed

    Azubel, Maia; Koivisto, Jaakko; Malola, Sami; Bushnell, David; Hura, Greg L; Koh, Ai Leen; Tsunoyama, Hironori; Tsukuda, Tatsuya; Pettersson, Mika; Hkkinen, Hannu; Kornberg, Roger D

    2014-08-22

    Structure determination of gold nanoparticles (AuNPs) is necessary for understanding their physical and chemical properties, but only one AuNP larger than 1 nanometer in diameter [a 102-gold atom NP (Au102NP)] has been solved to atomic resolution. Whereas the Au102NP structure was determined by x-ray crystallography, other large AuNPs have proved refractory to this approach. Here, we report the structure determination of a Au68NP at atomic resolution by aberration-corrected transmission electron microscopy, performed with the use of a minimal electron dose, an approach that should prove applicable to metal NPs in general. The structure of the Au68NP was supported by small-angle x-ray scattering and by comparison of observed infrared absorption spectra with calculations by density functional theory. PMID:25146285

  1. Four-dimensional ultrafast electron microscopy of phase transitions

    PubMed Central

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

    2006-01-01

    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 spacecharge 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 metalinsulator 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

  2. 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.

  3. 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. PMID:26638773

  4. 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.

  5. 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.

  6. Quantitative Vibrational Imaging by Hyperspectral Stimulated Raman Scattering Microscopy and Multivariate Curve Resolution Analysis

    PubMed Central

    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. Towards this goal, we demonstrate hyperspectral stimulated Raman loss (SRL) imaging by intra-pulse 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. PMID:23198914

  7. 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 various automotive catalysts. It was concluded that catalyst systems with wide size distribution should be characterized on the basis of surface area or area-weighted average size instead of simple average particle size. Area - and volume-weighted average particle sizes measured by TEM should be comparable to the average sizes measured by chemisorption and X-ray line broadening, respectively.

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

    PubMed

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

    2016-02-11

    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. PMID:26838122

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

    PubMed

    Saibil, Helen R; Grnewald, 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

  10. A national facility for biological cryo-electron microscopy

    PubMed Central

    Saibil, Helen R.; Grnewald, 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

  11. 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.

  12. 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).

  13. 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.

  14. Mixing state of soot particles analyzed using transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Adachi, K.; Zaizen, Y.; Igarashi, Y.

    2012-12-01

    Mixing state, shapes, and compositions of atmospheric aerosol particles influence their climate and health effects. Transmission electron microscopy (TEM) can reveal them at a single particle scale. This study shows these changes of aerosol particles collected from urban air (e.g., Los Angeles and Tokyo) and from a mountain site in Japan. We focused on soot particles since they absorb light and have great influence on the climate. To understand their optical properties accurately, their mixing states and shapes are important. Scanning transmission electron microscopy (STEM) with energy dispersive X-ray spectroscopy (EDS) analysis determines the compositions and mixing states of soot particles as well as elemental distribution within individual particles. Our STEM-EDS system revealed sizes, shape factors, and compositions of all aerosol particles within a field of view automatically (~300 particles). Using the STEM-EDS system, we investigate the soot mixing states, its sizes, and compositions of its coating. The preliminary results suggest that ~75% of soot particles were coated (internal mixture) at the mountain site (remote area) and the larger aerosol particles include the more soot particles. The soot particles are mostly coated by ammonium sulfate. In an urban area (Pasadena, CA) during the CalNex campaign, mixing states of soot particles varied largely within a day. Generally, during daytime, many soot particles were coated by mixtures of sulfate and organic matter. These data are useful to understand the optical properties, atmospheric lifetime, and climate effects of soot particles and to improve climate modeling.

  15. 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

  16. 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).

  17. Perspective: 4D ultrafast electron microscopy-Evolutions and revolutions.

    PubMed

    Shorokhov, Dmitry; Zewail, Ahmed H

    2016-02-28

    In this Perspective, the evolutionary and revolutionary developments of ultrafast electron imaging are overviewed with focus on the "single-electron concept" for probing methodology. From the first electron microscope of Knoll and Ruska [Z. Phys. 78, 318 (1932)], constructed in the 1930s, to aberration-corrected instruments and on, to four-dimensional ultrafast electron microscopy (4D UEM), the developments over eight decades have transformed humans' scope of visualization. The changes in the length and time scales involved are unimaginable, beginning with the micrometer and second domains, and now reaching the space and time dimensions of atoms in matter. With these advances, it has become possible to follow the elementary structural dynamics as it unfolds in real time and to provide the means for visualizing materials behavior and biological functions. The aim is to understand emergent phenomena in complex systems, and 4D UEM is now central for the visualization of elementary processes involved, as illustrated here with examples from past achievements and future outlook. PMID:26931672

  18. 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

  19. Engineering and Characterization of Collagen Networks Using Wet Atomic Force Microscopy and Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Osborn, Jenna; Coffey, Tonya; Conrad, Brad; Burris, Jennifer; Hester, Brooke

    2014-03-01

    Collagen is an abundant protein and its monomers covalently crosslink to form fibrils which form fibers which contribute to forming macrostructures like tendon or bone. While the contribution is well understood at the macroscopic level, it is not well known at the fibril level. We wish to study the mechanical properties of collagen for networks of collagen fibers that vary in size and density. We present here a method to synthesize collagen networks from monomers and that allows us to vary the density of the networks. By using biotynilated collagen and a surface that is functionalized with avidin, we generate two-dimensional collagen networks across the surface of a silicon wafer. During network synthesis, the incubation time is varied from 30 minutes to 3 hours or temperature is varied from 25C to 45C. The two-dimensional collagen network created in the process is characterized using environmental atomic force microscopy (AFM) and scanning electron microscopy (SEM). The network density is measured by the number of strands in one frame using SPIP software. We expect that at body temperature (37C) and with longer incubation times, the network density should increase.

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

    PubMed Central

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

    2011-01-01

    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. PMID:21750834

  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 study of complex biological cell walls. A unique feature of this approach is that both microscopes allow the biological samples to be examined in their natural fluid (water) environment.

  2. An investigation of nickel cobalt oxide nanorings using transmission electron, scanning electron and helium ion microscopy.

    PubMed

    Behan, G; Zhou, D; Boese, M; Wang, R M; Zhang, H Z

    2012-02-01

    Nickel cobalt oxide alloy nanorings have been synthesized using a wet chemistry method. Standard characterization techniques such as transmission electron microscopy (TEM) and scanning electron microscopy (SEM) have been used to characterize the nanorings. We, however, also examined the nanostructures in the helium ion microscope (HIM) and employed backscattered ion spectroscopy to determine thickness and composition of the nanostructure. The HIM provides complementary information of the nanostructures and the viability of using it as a tool for magnetic nanoparticle characterization was demonstrated by comparing the results from all three microscopes. PMID:22629901

  3. 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

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

    NASA Astrophysics Data System (ADS)

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

    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.

  5. Calibration of Wide-Field Deconvolution Microscopy for Quantitative Fluorescence Imaging

    PubMed Central

    Lee, Ji-Sook; Wee, Tse-Luen (Erika); Brown, Claire M.

    2014-01-01

    Deconvolution enhances contrast in fluorescence microscopy images, especially in low-contrast, high-background wide-field microscope images, improving characterization of features within the sample. Deconvolution can also be combined with other imaging modalities, such as confocal microscopy, and most software programs seek to improve resolution as well as contrast. Quantitative image analyses require instrument calibration and with deconvolution, necessitate that this process itself preserves the relative quantitative relationships between fluorescence intensities. To ensure that the quantitative nature of the data remains unaltered, deconvolution algorithms need to be tested thoroughly. This study investigated whether the deconvolution algorithms in AutoQuant X3 preserve relative quantitative intensity data. InSpeck Green calibration microspheres were prepared for imaging, z-stacks were collected using a wide-field microscope, and the images were deconvolved using the iterative deconvolution algorithms with default settings. Afterwards, the mean intensities and volumes of microspheres in the original and the deconvolved images were measured. Deconvolved data sets showed higher average microsphere intensities and smaller volumes than the original wide-field data sets. In original and deconvolved data sets, intensity means showed linear relationships with the relative microsphere intensities given by the manufacturer. Importantly, upon normalization, the trend lines were found to have similar slopes. In original and deconvolved images, the volumes of the microspheres were quite uniform for all relative microsphere intensities. We were able to show that AutoQuant X3 deconvolution software data are quantitative. In general, the protocol presented can be used to calibrate any fluorescence microscope or image processing and analysis procedure. PMID:24688321

  6. 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

  7. Quantitative sub-surface and non-contact imaging using scanning microwave microscopy

    NASA Astrophysics Data System (ADS)

    Gramse, Georg; Brinciotti, Enrico; Lucibello, Andrea; Patil, Samadhan B.; Kasper, Manuel; Rankl, Christian; Giridharagopal, Rajiv; Hinterdorfer, Peter; Marcelli, Romolo; Kienberger, Ferry

    2015-03-01

    The capability of scanning microwave microscopy for calibrated sub-surface and non-contact capacitance imaging of silicon (Si) samples is quantitatively studied at broadband frequencies ranging from 1 to 20 GHz. Calibrated capacitance images of flat Si test samples with varying dopant density (1015-1019 atoms cm-3) and covered with dielectric thin films of SiO2 (100-400 nm thickness) are measured to demonstrate the sensitivity of scanning microwave microscopy (SMM) for sub-surface imaging. Using standard SMM imaging conditions the dopant areas could still be sensed under a 400 nm thick oxide layer. Non-contact SMM imaging in lift-mode and constant height mode is quantitatively demonstrated on a 50 nm thick SiO2 test pad. The differences between non-contact and contact mode capacitances are studied with respect to the main parameters influencing the imaging contrast, namely the probe tip diameter and the tip-sample distance. Finite element modelling was used to further analyse the influence of the tip radius and the tip-sample distance on the SMM sensitivity. The understanding of how the two key parameters determine the SMM sensitivity and quantitative capacitances represents an important step towards its routine application for non-contact and sub-surface imaging.

  8. Spatial-domain low-coherence quantitative phase microscopy for cancer diagnosis

    NASA Astrophysics Data System (ADS)

    Wang, Pin; Bista, Rajan; Bhargava, Rohit; Brand, Randall E.; Liu, Yang

    2011-03-01

    A novel microscopy technique, spatial-domain low-coherence quantitative phase microscopy (SL-QPM), is proposed to obtain quantitative phase imaging of sub-cellular structures with sub-nanometer sensitivity. This technique utilizes a low spatial-coherence from a thermal light source and produces a speckle-free, nanoscale-sensitive quantitative phase map of scattering objects. With this technique, for the first time to our knowledge, we quantified the refractive index of the cell nuclei on the original unmodified histology specimens. The results show that the average refractive index of the cell nucleus is significantly increased in cells from cancer patients compared to that of the histologically normal cells from healthy patients. More importantly, we demonstrate the superior sensitivity of refractive index of cell nucleus in detecting cancer from histologically normal cells from cancer patients. Because this technique is simple, sensitive, does not require special tissue processing, and can be applied to archived specimens, it can be disseminated to all clinical settings.

  9. Charging in scanning electron microscopy "from inside and outside".

    PubMed

    Cazaux, Jacques

    2004-01-01

    This paper is an attempt to analyse most of the complicated mechanisms involved in charging and discharging of insulators investigated by scanning electron microscopy (SEM). Fundamental concepts on the secondary electron emission (SEE) yield from insulators combined with electrostatics arguments permit to reconsider, first, the widespread opinion following which charging is minimised when the incident beam energy E0 is chosen to be equal to the critical energy E(o)2, where the nominal total yield delta(o) + eta(o) = 1. For bare insulators submitted to a defocused irradiation, it is suggested here that the critical energy under permanent irradiation EC2 corresponds to a range of primary electrons, R, and nearly equals the maximum escape depth of the secondary electrons, r. This suggestion is supported by a comparison between published data of the SEE yield delta(o) of insulators (short pulse experiments) and experimental results obtained from a permanent irradiation for EC2. New SEE effects are also predicted at the early beginning of irradiation when finely focused probes are used. Practical considerations are also developed, with specific attention given to the role of a contamination layer where a negative charging may occur at any beam energy. The role of the various time constants involved in charging and discharging is also investigated, with special attention given to the dielectric time constant, which explains the dose rate-dependent effects on the effective landing energy in the steady state. Numerical applications permit to give orders of magnitude of various effects, and several other practical consequences are deduced and illustrated. Some new mechanisms for the contrast reversal during irradiation or with the change of the primary electron (PE) energy are also suggested. PMID:15473270

  10. Immune electron microscopy of avian infectious bronchitis virus serotypes.

    PubMed Central

    Odenwald, W F; Johnson, R B; Marquardt, W W; Hetrick, F M

    1978-01-01

    An immune electron microscopy agglutination technique in which emphasis is placed upon the importance of antigen-antibody equivalence has been developed as a possible method for the serotyping of avian infectious bronchitis viruses. The Connecticut and Massachusetts 41 serotypes were used as a model system. Stock virus concentrations were standardized by physical particle counts of virions sedimented directly onto electron microscope specimen grids. Suspensions containing approximately 150 virions per grid square were allowed to react with dilutions of homologous and heterologous antisera. Virions in these constant virus-variable serum mixtures were sedimented directly onto electron microscope specimen grids, and the relative degree of aggregation per grid was determined from the mean percent aggregation of five randomly selected grid squares. In homologous assays, regions of relative antibody excess, of equivalence, and of relative antigen excess were clearly evident. At equivalence, the mean percent aggregation was significantly higher than in the regions of relative antibody or antigen excess. In the heterologous systems, the degree of aggregation differed little from that of the virus controls containing no antiserum. Images PMID:209056

  11. Electron microscopy studies on DNA recognition by DNA-PK.

    PubMed

    Llorca, Oscar; Pearl, Laurence H

    2004-01-01

    Advances in transmission electron microscopy coupled to increasingly powerful biocomputing techniques are opening enormous possibilities to understand the structure and function of complex biological processes performed by large multi-protein assemblies. This is an exciting time for electron microscopists because we can combine our efforts with X-ray crystallographers and NMR spectroscopists to reach the prospect of studying the structure and dynamics of the so-called 'molecular machines'. One of these fascinating systems is the macromolecular complex formed around double-stranded DNA breaks (DSBs). Non-homologous end-joining (NHEJ) is the main DSBs repair pathway in mammalian cells, where a collection of proteins interact to rejoin two broken DNA ends. During NHEJ, DNA-dependent protein kinase (DNA-PK) binds damaged DNA with high affinity and acts as the main scaffold for other repair factors. Several studies have made use of the electron microscope to reveal the three-dimensional architecture of DNA-PK and the structural basis for the recognition of damaged DNA and the activation of DNA-PK's kinase activity. PMID:15288642

  12. Immuno-Electron Microscopy of the Morphogenesis of Mumps Virus

    PubMed Central

    Duc-Nguyen, Huu; Rosenblum, Edith N.

    1967-01-01

    The fine structure of mumps virus-infected chick embryo fibroblastic cells was examined sequentially after viral inoculation. Intracytoplasmic nucleoprotein strands, similar to those described for parainfluenza viruses, were detectable in small aggregates between 36 and 48 hr. The peripheral strands of this viral component lie beneath and along an antigenically altered bulging portion of the cell membrane. The outermost strands are consistently parallel to the differentiated segment of the plasma membrane, which is invariably associated with surface projections. As has been found with other myxoviruses, mumps virus replicates by budding from the cell surface. The virus particle, roughly spherical in shape, has a size ranging from 1,000 to 8,000 A. Filamentous forms are rarely observed in the present culture system. Ferritin-conjugated antibody specifically labels the cytoplasmic nucleoprotein, the modified cell membrane, and the virus particle. Intranuclear inclusions of low electron density and morphologically different from those described in measles virus-infected HeLa and amnion cells were observed in the nucleus of several infected cells. Immuno-electron microscopic observations suggest that the nucleoprotein synthesis rate exceeds that of cell membrane differentiation into viral envelope. This difference results in the accumulation of viral nucleoprotein in large intracytoplasmic masses which can be demonstrated by electron microscopy. Images PMID:5630382

  13. 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.

  14. Revealing the Formation of Copper Nanoparticles from a Homogeneous Solid Precursor by Electron Microscopy.

    PubMed

    van den Berg, Roy; Elkjaer, Christian F; Gommes, Cedric J; Chorkendorff, Ib; Sehested, Jens; de Jongh, Petra E; de Jong, Krijn P; Helveg, Stig

    2016-03-16

    The understanding of processes leading to the formation of nanometer-sized particles is important for tailoring of their size, shape and location. The growth mechanisms and kinetics of nanoparticles from solid precursors are, however, often poorly described. Here we employ transmission electron microscopy (TEM) to examine the formation of copper nanoparticles on a silica support during the reduction by H2 of homogeneous copper phyllosilicate platelets, as a prototype precursor for a coprecipitated catalyst. Specifically, time-lapsed TEM image series acquired of the material during the reduction process provide a direct visualization of the growth dynamics of an ensemble of individual nanoparticles and enable a quantitative evaluation of the nucleation and growth of the nanoparticles. This quantitative information is compared with kinetic models and found to be best described by a nucleation-and-growth scenario involving autocatalytic reduction of the copper phyllosilicate followed by diffusion-limited or reaction-limited growth of the copper nanoparticles. The plate-like structure of the precursor restricted the diffusion of copper and the autocatalytic reduction limited the probability for secondary nucleation. The combination of a uniform size of precursor particles and the autocatalytic reduction thus offers means to synthesize nanoparticles with well-defined sizes in large amounts. In this way, in situ observations made by electron microscopy provide mechanistic and kinetic insights into the formation of supported nanoparticles, essential for the rational design of nanomaterials. PMID:26891132

  15. 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.

  16. Quantitative imaging of collective cell migration during Drosophila gastrulation: multiphoton microscopy and computational analysis

    PubMed Central

    Supatto, Willy; McMahon, Amy; Fraser, Scott E.; Stathopoulos, Angelike

    2010-01-01

    This protocol describes imaging and computational tools to collect and analyze live imaging data of embryonic cell migration. Our five step protocol requires a few weeks to move through embryo preparation and four-dimensional (4D) live imaging using multiphoton microscopy, to 3D cell-tracking using image processing, registration of tracking data, and their quantitative analysis using computational tools. It uses commercially available equipment, and requires expertise in microscopy and programming that is appropriate for a biology laboratory. Custom-made scripts are provided, as well as sample datasets to permit readers without experimental data to perform the analysis. The protocol has offered new insights into the genetic control of cell migration during Drosophila gastrulation. With simple changes, this systematic analysis could be applied to any developing system to define cell positions in accordance with the body plan, to decompose complex 3D movements, and to quantify the collective nature of cell migration. PMID:19745822

  17. Quantitative phase imaging with molecular sensitivity using photoacoustic microscopy with a miniature ring transducer.

    PubMed

    Sheinfeld, Adi; Eldridge, Will J; Wax, Adam

    2015-08-01

    We present a dual-modality system for both structural and molecular cell imaging based on coregistered quantitative phase imaging (QPI) and photoacoustic microscopy (PAM). The QPI system was based on off-axis holography, whereas the PAM system comprised a sinusoidally modulated optical source for excitation and a narrow-band low profile and low-cost ring ultrasonic transducer for detection. This approach facilitated a simple confocal alignment of the excitation beams of both modalities and the ultrasonic detector. This system was demonstrated by imaging endogenous molecules in red blood cells (RBCs) as well as by imaging exogenous molecular labels on cancer cells using gold nanoparticles (GNPs) functionalized to target epidermal growth factor receptor. QPI provided high resolution imaging of the cellular structures while PAM provided molecular contrast. This dual-modality microscopy method can potentially be implemented as a compact and low cost cellular diagnostic assay. PMID:26263416

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

    PubMed

    Chen, Xueqin; Grgoire, Sbastien; Formanek, Florian; Galey, Jean-Baptiste; Rigneault, Herv

    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

  19. Quantitative segmentation of fluorescence microscopy images of heterogeneous tissue: Approach for tuning algorithm parameters

    NASA Astrophysics Data System (ADS)

    Mueller, Jenna L.; Harmany, Zachary T.; Mito, Jeffrey K.; Kennedy, Stephanie A.; Kim, Yongbaek; Dodd, Leslie; Geradts, Joseph; Kirsch, David G.; Willett, Rebecca M.; Brown, J. Quincy; Ramanujam, Nimmi

    2013-02-01

    The combination of fluorescent contrast agents with microscopy is a powerful technique to obtain real time images of tissue histology without the need for fixing, sectioning, and staining. The potential of this technology lies in the identification of robust methods for image segmentation and quantitation, particularly in heterogeneous tissues. Our solution is to apply sparse decomposition (SD) to monochrome images of fluorescently-stained microanatomy to segment and quantify distinct tissue types. The clinical utility of our approach is demonstrated by imaging excised margins in a cohort of mice after surgical resection of a sarcoma. Representative images of excised margins were used to optimize the formulation of SD and tune parameters associated with the algorithm. Our results demonstrate that SD is a robust solution that can advance vital fluorescence microscopy as a clinically significant technology.

  20. 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

  1. Electron microscopy of gallium nitride growth on polycrystalline diamond

    NASA Astrophysics Data System (ADS)

    Webster, R. F.; Cherns, D.; Kuball, M.; Jiang, Q.; Allsopp, D.

    2015-11-01

    Transmission and scanning electron microscopy were used to examine the growth of gallium nitride (GaN) on polycrystalline diamond substrates grown by metalorganic vapour phase epitaxy with a low-temperature aluminium nitride (AlN) nucleation layer. Growth on unmasked substrates was in the (0001) orientation with threading dislocation densities ?7 109 cm-2. An epitaxial layer overgrowth technique was used to reduce the dislocation densities further, by depositing silicon nitride stripes on the surface and etching the unmasked regions down to the diamond substrate. A re-growth was then performed on the exposed side walls of the original GaN growth, reducing the threading dislocation density in the overgrown regions by two orders of magnitude. The resulting microstructures and the mechanisms of dislocation reduction are discussed.

  2. Neuron Segmentation in Electron Microscopy Images Using Partial Differential Equations.

    PubMed

    Jones, Cory; Sayedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2013-01-01

    In connectomics, neuroscientists seek to identify the synaptic connections between neurons. Segmentation of cell membranes using supervised learning algorithms on electron microscopy images of brain tissue is often done to assist in this effort. Here we present a partial differential equation with a novel growth term to improve the results of a supervised learning algorithm. We also introduce a new method for representing the resulting image that allows for a more dynamic thresholding to further improve the result. Using these two processes we are able to close small to medium sized gaps in the cell membrane detection and improve the Rand error by as much as 9% over the initial supervised segmentation. PMID:25143802

  3. Transmission Electron Microscopy (TEM) investigations of ancient Egyptian cosmetic powders

    NASA Astrophysics Data System (ADS)

    Deeb, C.; Walter, P.; Castaing, J.; Penhoud, P.; Veyssire, P.

    The processing technologies available during the time of ancient Egypt are of present concern to the field of Archaeology and Egyptology. Materials characterization is the best tool for establishing the processing history of archaeological objects. In this study, transmission electron microscopy (TEM) is used, in addition to other techniques, for phase identification and study of the microstructure and characteristic defect structures in ancient Egyptian cosmetic powders. These powders generally consist of a mix of Pb-containing mineral phases: galena (PbS), cerussite (PbCO3), and phosgenite (Pb2Cl2CO3), among others. Modern materials are fabricated according to recipes found in ancient texts to mimic the processing of ancient times and to compare with the archaeological specimens. In particular, a comparison between the dislocation structures of PbS crystals deformed in the laboratory and PbS from archaeological specimens from the collections of the Louvre Museum is presented .

  4. Nanocrystal size distribution analysis from transmission electron microscopy images.

    PubMed

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

    2015-12-28

    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. PMID:26593390

  5. 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.

  6. 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

  7. Neuron Segmentation in Electron Microscopy Images Using Partial Differential Equations

    PubMed Central

    Jones, Cory; Sayedhosseini, Mojtaba; Ellisman, Mark; Tasdizen, Tolga

    2014-01-01

    In connectomics, neuroscientists seek to identify the synaptic connections between neurons. Segmentation of cell membranes using supervised learning algorithms on electron microscopy images of brain tissue is often done to assist in this effort. Here we present a partial differential equation with a novel growth term to improve the results of a supervised learning algorithm. We also introduce a new method for representing the resulting image that allows for a more dynamic thresholding to further improve the result. Using these two processes we are able to close small to medium sized gaps in the cell membrane detection and improve the Rand error by as much as 9% over the initial supervised segmentation. PMID:25143802

  8. 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. PMID:26611544

  9. Cryo-electron microscopy of GDP-tubulin rings.

    PubMed

    Nicholson, W V; Lee, M; Downing, K H; Nogales, E

    1999-01-01

    Rings of guanosine diphosphate (GDP)-tubulin formed in the presence of divalent cations have been studied using conventional negative stain and cryo-electron microscopy. The structure of such rings resembles that of depolymerizing microtubule ends and corresponds to an "unconstrained" conformation of tubulin in its GDP state. The use of cryo-techniques has allowed us to image the ring polymers free from dehydration and flattening artifacts. Preparations of frozen-hydrated GDP-tubulin rings are generally heterogeneous and contain a mixture of double, triple, and incomplete rings, as well as spirals and some rare single rings. Images of different polymer types can be identified and classified into groups that are then amenable for averaging and single particle reconstruction methods. Identifying the differences in tubulin structure, between straight and curve protofilaments, will be important to understand the molecular bases of dynamic instability in microtubules. PMID:10593258

  10. Collaborative Computational Project for Electron cryo-Microscopy

    PubMed Central

    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) 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. PMID:25615866

  11. Scanning electron microscopy of intact colonies of microorganisms.

    PubMed

    Whittaker, D K; Drucker, D B

    1970-11-01

    Colonies of S. mutans OMZ61, Streptococcus sp. D182, Staphylococcus aureus Oxford NCTC 6571, and Candida albicans type A, MRL 3153 were grown on various media. Cubes of agar bearing two to three colonies were excised and processed for scanning electron microscopy. The characteristic shape of the colonies was seen when examined at low magnifications. At a magnification of 2,000 diameters, the arrangement of individual organisms within the colonies was observed. Plano-convex colonies consisted of uniformly distributed organisms, whereas S. mutans colonies presented a more complex arrangement possibly associated with the production of extracellular polysaccharides. Certain colonies were totally or partially covered by an adherent film through which the outline of the organisms could be distinguished. PMID:5489440

  12. 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.

  13. Electron microscopy of Co/Fe/B/Si amorphous alloys

    SciTech Connect

    Rabenberg, L.; Mishra, R.K.; Thomas, G.; Kohmoto, O.; Ojima, T.

    1980-09-01

    Changes in magnetic structures with annealing are studied using Lorentz electron microscopy and are correlated with changes in magnetic properties for the Co/sub 71/ /sub 4/Fe/sub 4/ /sub 6/Si/sub 9/ /sub 6/B/sub 14/ /sub 4/ amorphous alloy. Domain wall stablization is shown to be the dominant factor resulting in decreasing ..mu.. and increasing H/sub c/ and K during low temperature annealing. Annealing near T/sub c/ results in an isotropic magnetic structure due to domain wall relaxation, and annealing above T/sub cry/ results in magnetically hard crystalline particles. It is concluded that treatments capable of producing a magnetically isotropic structure can produce the best soft magnetic materials.

  14. Electron microscopy of Co/Fe/B/Si amorphous alloys

    SciTech Connect

    Rabenberg, L.; Mishra, R.K.; Thomas, G.; Kohmoto, O.; Ojima, T.

    1980-09-01

    Changes in magnetic structures with annealing are studied using Lorentz electron microscopy and are correlated with changes in magnetic properties for the Co/sub 71/./sub 4/Fe/sub 4/./sub 6/Si/sub 9/./sub 6/B/sub 14/./sub 4/ amorphous alloy. Domain wall stabilization is shown to be the dominant factor resulting in decreasing ..mu.. and increasing H/sub c/ and K during low temperature annealing. Annealing near T/sub c/ results in an isotropic magnetic structure due to domain wall relaxation, and annealing above T/sub cry/ results in magnetically hard crystalline particles. It is concluded that treatments capable of producing a magnetically isotropic structure can produce the best soft magnetic materials.

  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. Electron affinity of tin measured by photodetachment microscopy

    NASA Astrophysics Data System (ADS)

    Vandevraye, M.; Drag, C.; Blondel, C.

    2013-06-01

    A beam of Sn- ions produced by a caesium sputtering ion source is photodetached in the presence of an electric field, with a single-mode ring Ti:Sa laser. The laser wavelength, about 806 nm, is set just above the excitation threshold of the 3P2, highest fine-structure sublevel of the 3P ground-term of Sn I. The photoelectron energy is measured by photodetachment microscopy. The measured photodetachment threshold is 1239?711.8?(11)?m-1, from which an improved value of the electron affinity of tin can be deduced: 896?944.7?(13)?m-1 or 1.112?070?(2)?eV.

  17. 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

  18. Electron Microscopy Analysis of the Nucleolus of Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

    Lpez-Velzquez, Gabriel; Hernndez, Roberto; Lpez-Villaseor, Imelda; Reyes-Vivas, Horacio; Segura-Valdez, Mara De L.; Jimnez-Garca, 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.

  19. Electron tomography and immunonanogold electron microscopy for investigating intracellular trafficking and secretion in human eosinophils.

    PubMed

    Melo, Rossana C N; Dvorak, Ann M; Weller, Peter F

    2008-08-01

    Electron tomography (ET) has increasingly been used to understand the complexity of membrane systems and protein-trafficking events. By ET and immunonanogold electron microscopy, we recently defined a route for vesicular transport and release of granule-stored products from within activated human eosinophils, cells specialized in the secretion of numerous cytokines and other proteins during inflammatory responses. Here, we highlight these techniques as important tools to unveil a distinct eosinophil vesicular system and secretory pathway. PMID:18410520

  20. 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.

  1. Structured illumination diffraction phase microscopy for broadband, sub-diffraction resolution, quantitative phase imaging

    PubMed Central

    Chowdhury, Shwetadwip; Izatt, Joseph A.

    2015-01-01

    Structured illumination microscopy (SIM) is an established technique that allows sub-diffraction resolution imaging by heterodyning high sample frequencies into the system’s passband via structured illumination. However, until now, SIM has been typically used to achieve sub-diffraction resolution for intensity-based imaging. Here, we present a novel optical setup that uses structured illumination with a broadband-light source to obtain noise-reduced, sub-diffraction resolution, quantitative-phase (QPM) imaging of cells. We compare this with a previous work for sub-diffraction QPM imaging via SIM that used a laser source, and was thus still corrupted by coherent noise. PMID:24562266

  2. Nanoscale nuclear architecture for cancer diagnosis by spatial-domain low-coherence quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Pin; Bista, Rajan K.; Khalbuss, Walid E.; Qiu, Wei; Staton, Kevin D.; Zhang, Lin; Brentnall, Teresa A.; Brand, Randall E.; Liu, Yang

    2011-03-01

    Alterations in nuclear architecture are the hallmark diagnostic characteristic of cancer cells. In this work, we show that the nuclear architectural characteristics quantified by spatial-domain low-coherence quantitative phase microscopy (SL-QPM), is more sensitive for the identification of cancer cells than conventional cytopathology. We demonstrated the importance of nuclear architectural characteristics in both an animal model of intestinal carcinogenesis - APC/Min mouse model and human cytology specimens with colorectal cancer by identifying cancer from cytologically noncancerous appearing cells. The determination of nanoscale nuclear architecture using this simple and practical optical instrument is a significant advance towards cancer diagnosis.

  3. Time-resolved imaging refractometry of microbicidal films using quantitative phase microscopy.

    PubMed

    Rinehart, Matthew T; Drake, Tyler K; Robles, Francisco E; Rohan, Lisa C; Katz, David; Wax, Adam

    2011-12-01

    Quantitative phase microscopy is applied to image temporal changes in the refractive index (RI) distributions of solutions created by microbicidal films undergoing hydration. We present a novel method of using an engineered polydimethylsiloxane structure as a static phase reference to facilitate calibration of the absolute RI across the entire field. We present a study of dynamic structural changes in microbicidal films during hydration and subsequent dissolution. With assumptions about the smoothness of the phase changes induced by these films, we calculate absolute changes in the percentage of film in regions across the field of view. PMID:22191912

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

    PubMed Central

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

    2010-01-01

    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. PMID:19725730

  5. Quantitative assessment of noise reduction with partial spatial coherence illumination in digital holographic microscopy.

    PubMed

    Dohet-Eraly, Jrme; Yourassowsky, Catherine; Mallahi, Ahmed El; Dubois, Frank

    2016-01-01

    Improving image quality in digital holographic microscopy is achievable by using partial spatial coherence (PSC) illumination instead of fully coherent illumination. This Letter presents simple theoretical models to quantitatively assess the reduction of noise as a function of both the spatial coherence of the illumination and the defocus distance of the noise source. The first developed model states that the effect of the PSC can be studied by discretizing the field of view in the plane of the noise source. The second model, following a continuous approach, corroborates the discrete model and extends it. Experimental results confirm theoretical expectations. PMID:26696171

  6. Quantitative analysis of platelets aggregates in 3D by digital holographic microscopy.

    PubMed

    Boudejltia, Karim Zouaoui; Ribeiro de Sousa, Daniel; Uzureau, Pierrick; Yourassowsky, Catherine; Perez-Morga, David; Courbebaisse, Guy; Chopard, Bastien; Dubois, Frank

    2015-09-01

    Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs. PMID:26417523

  7. Quantitative analysis of platelets aggregates in 3D by digital holographic microscopy

    PubMed Central

    Boudejltia, Karim Zouaoui; Ribeiro de Sousa, Daniel; Uzureau, Pierrick; Yourassowsky, Catherine; Perez-Morga, David; Courbebaisse, Guy; Chopard, Bastien; Dubois, Frank

    2015-01-01

    Platelet spreading and retraction play a pivotal role in the platelet plugging and the thrombus formation. In routine laboratory, platelet function tests include exhaustive information about the role of the different receptors present at the platelet surface without information on the 3D structure of platelet aggregates. In this work, we develop, a method in Digital Holographic Microscopy (DHM) to characterize the platelet and aggregate 3D shapes using the quantitative phase contrast imaging. This novel method is suited to the study of platelets physiology in clinical practice as well as the development of new drugs. PMID:26417523

  8. 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. PMID:22505068

  9. 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.

  10. Accurate single-shot quantitative phase imaging of biological specimens with telecentric digital holographic microscopy.

    PubMed

    Doblas, Ana; Snchez-Ortiga, Emilio; Martnez-Corral, Manuel; Saavedra, Genaro; Garcia-Sucerquia, Jorge

    2014-04-01

    The advantages of using a telecentric imaging system in digital holographic microscopy (DHM) to study biological specimens are highlighted. To this end, the performances of nontelecentric DHM and telecentric DHM are evaluated from the quantitative phase imaging (QPI) point of view. The evaluated stability of the microscope allows single-shot QPI in DHM by using telecentric imaging systems. Quantitative phase maps of a section of the head of the drosophila melanogaster fly and of red blood cells are obtained via single-shot DHM with no numerical postprocessing. With these maps we show that the use of telecentric DHM provides larger field of view for a given magnification and permits more accurate QPI measurements with less number of computational operations. PMID:24781590

  11. Quantitative Measurements of Grain Boundary Sliding in an Ultrafine-Grained Al Alloy by Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Han, Jung H.; Mohamed, Farghalli A.

    2011-12-01

    In the current study, quantitative measurements for grain boundary sliding (GBS) in ultrafine-grained (UFG) 5083 Al by atomic force microscopy (AFM) were performed. An ion beam polishing and etching technique was used to reveal grain boundaries in the alloy for AFM characterization. A comparison between the average grain sizes measured from AFM images and those estimated from transmission electron microscopy micrographs and electron backscatter diffraction (EBSD) maps showed excellent agreement. The vertical offset of GBS was measured by comparing predeformation and postdeformation AFM images. By analyzing these measurements, the contribution of GBS to the total tensile strain in 5083 Al was estimated as 25 pct at a strain rate of 10-4 seconds-1 and a temperature of 473 K (200 C). It was demonstrated that the relatively low value of the contribution of GBS to the total strain is most likely the result of testing UFG 5083 Al under experimental conditions that favor the dominance of region I (low-stress region) of the sigmoidal behavior characterizing high-strain-rate superplasticity, which was reported previously for the alloy.

  12. 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

  13. Dynamic phase imaging of host cells attacked by Vibrio vulnificus using quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Seungrag; Yang, Wenzhong; Lee, Ji Yong; Cha, Mi Hye; Kim, Young Ran; Kim, Dug Young

    2010-02-01

    We present the real time quantitative analysis of Vibrio vulnificus-infected host cells using high stability quantitative phase microscopy (HSQPM). It provides the ability to retrieve the phase or optical path length distribution over the cell from a single interferogram image, which has been measured with nanometer path length sensitivity for long periods of time. We have applied HSQPM to study dynamic cell morphologic changes and to quantify noninvasively cell volumes of rat basophilic leukemia RBL-2H3 cells infected with pathogenic bacteria V. vulnificus strains, wild type (MO6-24/O) and RTX toxin mutant (CMM770). During the process of V. vulnificus wild type infection to RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes and areas were observed in real time using HSQPM. In contrast, the dramatic changes were not detected in RBL-2H3 cells infected with RTX toxin mutant. The results showed the good correlation between HSQPM analysis and biochemical assays such as lactate dehydrogenase (LDH) assay and ?-hexosaminidase release assay. We suggest that HSQPM is useful real time quantitative method to study the dynamic process of host cells infected with pathogen in a noninvasive manner.

  14. Dynamic analysis of pathogen-infected host cells using quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Seungrag; Kim, Young Ran; Lee, Ji Yong; Rhee, Joon Haeng; Park, Chang-Soo; Kim, Dug Young

    2011-03-01

    We present the real-time quantitative analysis of Vibrio vulnificus-infected host cells using quantitative phase microscopy (QPM) based on interferometric techniques. This provides the ability to retrieve the phase or optical path-length distribution over the cell with nanometer path-length sensitivity from a single interferogram image. We have used QPM to study dynamic cell morphologic changes and to noninvasively quantify the cell volumes of rat basophilic leukemia RBL-2H3 cells infected with V. vulnificus strains: wild type (MO6-24/O) and RtxA1 toxin mutant (CMM770). During the process of V. vulnificus infection in RBL-2H3 cells, the dynamic changes of quantitative phase images, cell volumes, and areas were observed in real time using QPM. In contrast, dramatic changes were not detected in RBL-2H3 cells infected with the noncytotoxic RtxA1 toxin mutant. The results showed good correlation between QPM analysis and biochemical assays, such as lactate dehydrogenase assay or ?-hexosaminidase release assay. We suggest that QPM is a powerful quantitative method to study the dynamic process of host cells infected with pathogens in a noninvasive manner.

  15. Quantitative imaging of cell dynamics in mouse embryos using light-sheet microscopy

    PubMed Central

    Udan, Ryan S.; Piazza, Victor G.; Hsu, Chih-wei; Hadjantonakis, Anna-Katerina; Dickinson, Mary E.

    2014-01-01

    Single/selective-plane illumination, or light-sheet, systems offer several advantages over other fluorescence microscopy methods for live, 3D microscopy. These systems are valuable for studying embryonic development in several animal systems, such as Drosophila, C. elegans and zebrafish. The geometry of the light path in this form of microscopy requires the sample to be accessible from multiple sides and fixed in place so that it can be rotated around a single axis. Popular methods for mounting include hanging the specimen from a pin or embedding it in 1-2% agarose. These methods can be particularly problematic for certain samples, such as post-implantation mouse embryos, that expand significantly in size and are very delicate and sensitive to mounting. To overcome the current limitations and to establish a robust strategy for long-term (24 h) time-lapse imaging of E6.5-8.5 mouse embryos with light-sheet microscopy, we developed and tested a method using hollow agarose cylinders designed to accommodate for embryonic growth, yet provide boundaries to minimize tissue drift and enable imaging in multiple orientations. Here, we report the first 24-h time-lapse sequences of post-implantation mouse embryo development with light-sheet microscopy. We demonstrate that light-sheet imaging can provide both quantitative data for tracking changes in morphogenesis and reveal new insights into mouse embryogenesis. Although we have used this approach for imaging mouse embryos, it can be extended to imaging other types of embryos as well as tissue explants. PMID:25344073

  16. 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; Andr, 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. PMID:24030836

  17. Aberration Corrected Photoemission Electron Microscopy with Photonics Applications

    NASA Astrophysics Data System (ADS)

    Fitzgerald, Joseph P. S.

    Photoemission electron microscopy (PEEM) uses photoelectrons excited from material surfaces by incident photons to probe the interaction of light with surfaces with nanometer-scale resolution. The point resolution of PEEM images is strongly limited by spherical and chromatic aberration. Image aberrations primarily originate from the acceleration of photoelectrons and imaging with the objective lens and vary strongly in magnitude with specimen emission characteristics. Spherical and chromatic aberration can be corrected with an electrostatic mirror, and here I develop a triode mirror with hyperbolic geometry that has two adjacent, field-adjustable regions. I present analytic and numerical models of the mirror and show that the optical properties agree to within a few percent. When this mirror is coupled with an electron lens, it can provide a large dynamic range of correction and the coefficients of spherical and chromatic aberration can be varied independently. I report on efforts to realize a triode mirror corrector, including design, characterization, and alignment in our microscope at Portland State University (PSU). PEEM may be used to investigate optically active nanostructures, and we show that photoelectron emission yields can be identified with diffraction, surface plasmons, and dielectric waveguiding. Furthermore, we find that photoelectron micrographs of nanostructured metal and dielectric structures correlate with electromagnetic field calculations. We conclude that photoemission is highly spatially sensitive to the electromagnetic field intensity, allowing the direct visualization of the interaction of light with material surfaces at nanometer scales and over a wide range of incident light frequencies.

  18. Nanomusical systems visualized and controlled in 4D electron microscopy.

    PubMed

    Baskin, J Spencer; Park, Hyun Soon; Zewail, Ahmed H

    2011-05-11

    Nanomusical systems, nanoharp and nanopiano, fabricated as arrays of cantilevers by focused ion beam milling of a layered Ni/Ti/Si(3)N(4) thin film, have been investigated in 4D electron microscopy. With the imaging and selective femtosecond and nanosecond control combinations, full characterization of the amplitude and phase of the resonant response of a particular cantilever relative to the optical pulse train was possible. Using a high repetition rate, low energy optical pulse train for selective, resonant excitation, coupled with pulsed and steady-state electron imaging for visualization in space and time, both the amplitude on the nanoscale and resonance of motion on the megahertz scale were resolved for these systems. Tilting of the specimen allowed in-plane and out-of-plane cantilever bending and cantilever torsional motions to be identified in stroboscopic measurements of impulsively induced free vibration. Finally, the transient, as opposed to steady state, thermostat effect was observed for the layered nanocantilevers, with a sufficiently sensitive response to demonstrate suitability for in situ use in thin-film temperature measurements requiring resolutions of <10 K and 10 μm on time scales here mechanically limited to microseconds and potentially at shorter times. PMID:21513332

  19. Photooxidation technology for correlative light and electron microscopy.

    PubMed

    Meisslitzer-Ruppitsch, Claudia; Rhrl, Clemens; Ranftler, Carmen; Stangl, Herbert; Neumller, Josef; Pavelka, Margit; Ellinger, Adolf

    2013-01-01

    Correlative microscopic approaches combine the advantages of both light and electron microscopy. Here we show a correlative approach that uses the photooxidation capacity of fluorescent dyes. Through illumination with high energetic light, the chromogen diaminobenzidine is oxidized and stable deposits are formed at the sites of the former fluorescent signals, which after osmification are then visible in the electron microscope. The potential of the method is illustrated by tracing the endocytic pathway of three different ligands: the lipid ceramide, high density lipoproteins, and the lectin wheat germ agglutinin. The ligands were labeled either with BODIPY or Alexa dyes. Following cell surface binding, uptake, and time-dependent intracellular progression, the route taken by these molecules together with the organelles that have been visited is characterized. Correlative microscopic data are recorded at various levels. First, by fluorescence and phase contrast illumination with the light microscope, followed by the analysis of semithin sections after photooxidation, and finally of thin sections at the ultrastructural level. PMID:23027015

  20. Advanced scanning transmission electron microscopy characterization of UV LED nanowires

    NASA Astrophysics Data System (ADS)

    Phillips, Patrick; Kumar, Rajan; Carnevale, Santino; Myers, Roberto; Klie, Robert

    2013-03-01

    The role of aberration-corrected scanning transmission electron microscopy (STEM) in materials characterization is examined in regards to Al(x)Ga(1-x)N nanowires. Wires were graded from x =0 to x =1 and then from x =1 to x =0 with a small active quantum disk region located between the two gradations. This configuration is the basis for previously reported UV light emitting diodes. However, to assist subsequent growth processes while striving for optimum efficiency, both structural and chemical characterization methods are necessary, which can be provided at sufficiently high resolutions by advanced STEM instruments. Specifically, structural characterization will focus on determining layer thicknesses and wire polarity, as well as visualizing any short-range ordering and/or stacking faults that may be present. STEM multislice image simulations will also be discussed. Chemically, both energy dispersive X-ray (EDX) and electron energy loss (EEL) spectroscopies will be discussed in various capacities, ranging from quantum well composition (EDX) to N K-edge fine structure of both GaN and AlN (EELS).

  1. 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.

  2. ATOMIC RESOLUTION CRYO ELECTRON MICROSCOPY OF MACROMOLECULAR COMPLEXES

    PubMed Central

    ZHOU, Z. HONG

    2013-01-01

    Single-particle cryo electron microscopy (cryoEM) is a technique for determining three-dimensional (3D) structures from projection images of molecular complexes preserved in their native, noncrystalline state. Recently, atomic or near-atomic resolution structures of several viruses and protein assemblies have been determined by single-particle cryoEM, allowing ab initio atomic model building by following the amino acid side chains or nucleic acid bases identifiable in their cryoEM density maps. In particular, these cryoEM structures have revealed extended arms contributing to molecular interactions that are otherwise not resolved by the conventional structural method of X-ray crystallography at similar resolutions. High-resolution cryoEM requires careful consideration of a number of factors, including proper sample preparation to ensure structural homogeneity, optimal configuration of electron imaging conditions to record high-resolution cryoEM images, accurate determination of image parameters to correct image distortions, efficient refinement and computation to reconstruct a 3D density map, and finally appropriate choice of modeling tools to construct atomic models for functional interpretation. This progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines. PMID:21501817

  3. 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.

  4. Electron microscopy analysis of mineral fibers in human lung tissue

    SciTech Connect

    Friedrichs, K.H.; Brockmann, M.; Fischer, M.; Wick, G. )

    1992-01-01

    In the present study, lung samples from 126 autopsied cases were examined to determine the content of mineral fibers using analytical transmission electron microscopy (ATEM). The cases were divided into four groups (22 lungs of persons exposed to ambient environmental pollution, 32 cases of mesothelioma, 38 cases of primary lung cancer, and 34 asbestosis cases, 13 of these with additional pleural plaques). Fibers were counted, measured, and mineralogically identified using a combination of X-ray microanalysis and electron diffraction of the non-oriented fiber. Concentration of fibrous particles (defined as particles above 1 micron in length with roughly parallel long sides and an aspect ratio of 5:1 and greater) was calculated as fibers 10(6)/g dry lung weight. The concentration of chrysotile was found to be similar throughout the groups except for two cases in the asbestosis group with comparably high numbers of chrysotile. However, a remarkable difference for amphiboles could be observed between the groups. Asbestos bodies were mostly found in the asbestosis group. There was a rather good correlation between numbers of amphibole fibers and asbestos bodies, with an average ratio of 10:1. For comparison purposes between occupationally exposed/non-exposed individuals, a transition was found in the concentration range of 3-10(7) asbestos fibers/g dried lung weight.

  5. Combined scanning transmission electron microscopy tilt- and focal series.

    PubMed

    Dahmen, Tim; Baudoin, Jean-Pierre; Lupini, Andrew R; Kbel, 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. PMID:24548618

  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. 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).

  8. Label-free characterization of living human induced pluripotent stem cells by subcellular topographic imaging technique using full-field quantitative phase microscopy coupled with interference reflection microscopy.

    PubMed

    Sugiyama, Norikazu; Asai, Yasuyuki; Yamauchi, Toyohiko; Kataoka, Takuji; Ikeda, Takahiro; Iwai, Hidenao; Sakurai, Takashi; Mizuguchi, Yoshinori

    2012-09-01

    There is a need for a noninvasive technique to monitor living pluripotent stem cell condition without any labeling. We present an optical imaging technique that is able to capture information about optical path difference through the cell and cell adhesion properties simultaneously using a combination of quantitative phase microscopy (QPM) and interference reflection microscopy (IRM) techniques. As a novel application of QPM and IRM, this multimodal imaging technique demonstrated its ability to distinguish the undifferentiated status of human induced pluripotent stem (hiPS) cells quantitatively based on the variation of optical path difference between the nucleus and cytoplasm as well as hiPS cell-specific cell adhesion properties. PMID:23024911

  9. Oufti: an integrated software package for high-accuracy, high-throughput quantitative microscopy analysis.

    PubMed

    Paintdakhi, Ahmad; Parry, Bradley; Campos, Manuel; Irnov, Irnov; Elf, Johan; Surovtsev, Ivan; Jacobs-Wagner, Christine

    2016-02-01

    With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills. PMID:26538279

  10. Atomic-resolution electron energy loss spectroscopy imaging in aberration corrected scanning transmission electron microscopy.

    PubMed

    Allen, L J; Findlay, S D; Lupini, A R; Oxley, M P; Pennycook, S J

    2003-09-01

    The "delocalization" of inelastic scattering is an important issue for the ultimate spatial resolution of innershell spectroscopy in the electron microscope. It is demonstrated in a nonlocal model for electron energy loss spectroscopy (EELS) that delocalization of scanning transmission electron microscopy (STEM) images for single, isolated atoms is primarily determined by the width of the probe, even for light atoms. We present experimental data and theoretical simulations for Ti L-shell EELS in a [100] SrTiO3 crystal showing that, in this case, delocalization is not significantly increased by dynamical propagation. Issues relating to the use of aberration correctors in the STEM geometry are discussed. PMID:14525490

  11. 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.

  12. Electron microscopy analysis of skin biopsies in CADASIL disease.

    PubMed

    Cotrutz, Carmen Elena; Indrei, Anca; B?descu, L; Dac?lu, Cristina; Neam?u, Monica; Dumitrescu, Gabriela Floren?a; Stefanache, Felicia; Petreu?, 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. PMID:20809020

  13. Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

    PubMed Central

    Karreman, Matthia A.; Mercier, Luc; Schieber, Nicole L.; Shibue, Tsukasa; Schwab, Yannick; Goetz, Jacky G.

    2014-01-01

    Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. PMID:25479106

  14. Bone-titanium oxide interface in humans revealed by transmission electron microscopy and electron tomography.

    PubMed

    Palmquist, Anders; Grandfield, Kathryn; Norlindh, Birgitta; Mattsson, Torsten; Brnemark, Rickard; Thomsen, Peter

    2012-02-01

    Osseointegration, the direct contact between an implant surface and bone tissue, plays a critical role in interfacial stability and implant success. Analysis of interfacial zones at the micro- and nano-levels is essential to determine the extent of osseointegration. In this paper, a series of state-of-the-art microscopy techniques are used on laser-modified implants retrieved from humans. Partially laser-modified implants were retrieved after two and a half months' healing and processed for light and electron microscopy. Light microscopy showed osseointegration, with bone tissue growing both towards and away from the implant surface. Transmission electron microscopy revealed an intimate contact between mineralized bone and the laser-modified surface, including bone growth into the nano-structured oxide. This novel observation was verified by three-dimensional Z-contrast electron tomography, enabling visualization of an apatite layer, with different crystal direction compared with the apatite in the bone tissue, encompassing the nano-structured oxide. In conclusion, the present study demonstrates the nano-scale osseointegration and bonding between apatite and surface-textured titanium oxide. These observations provide novel data in human specimens on the ultrastructure of the titanium-bone interface. PMID:21849383

  15. Bonetitanium oxide interface in humans revealed by transmission electron microscopy and electron tomography

    PubMed Central

    Palmquist, Anders; Grandfield, Kathryn; Norlindh, Birgitta; Mattsson, Torsten; Brnemark, Rickard; Thomsen, Peter

    2012-01-01

    Osseointegration, the direct contact between an implant surface and bone tissue, plays a critical role in interfacial stability and implant success. Analysis of interfacial zones at the micro- and nano-levels is essential to determine the extent of osseointegration. In this paper, a series of state-of-the-art microscopy techniques are used on laser-modified implants retrieved from humans. Partially laser-modified implants were retrieved after two and a half months' healing and processed for light and electron microscopy. Light microscopy showed osseointegration, with bone tissue growing both towards and away from the implant surface. Transmission electron microscopy revealed an intimate contact between mineralized bone and the laser-modified surface, including bone growth into the nano-structured oxide. This novel observation was verified by three-dimensional Z-contrast electron tomography, enabling visualization of an apatite layer, with different crystal direction compared with the apatite in the bone tissue, encompassing the nano-structured oxide. In conclusion, the present study demonstrates the nano-scale osseointegration and bonding between apatite and surface-textured titanium oxide. These observations provide novel data in human specimens on the ultrastructure of the titaniumbone interface. PMID:21849383

  16. Activated sludge characterization through microscopy: a review on quantitative image analysis and chemometric techniques.

    PubMed

    Mesquita, Daniela P; Amaral, A Lus; Ferreira, Eugnio C

    2013-11-13

    In wastewater treatment processes, and particularly in activated sludge systems, efficiency is quite dependent on the operating conditions, and a number of problems may arise due to sludge structure and proliferation of specific microorganisms. In fact, bacterial communities and protozoa identification by microscopy inspection is already routinely employed in a considerable number of cases. Furthermore, quantitative image analysis techniques have been increasingly used throughout the years for the assessment of aggregates and filamentous bacteria properties. These procedures are able to provide an ever growing amount of data for wastewater treatment processes in which chemometric techniques can be a valuable tool. However, the determination of microbial communities' properties remains a current challenge in spite of the great diversity of microscopy techniques applied. In this review, activated sludge characterization is discussed highlighting the aggregates structure and filamentous bacteria determination by image analysis on bright-field, phase-contrast, and fluorescence microscopy. An in-depth analysis is performed to summarize the many new findings that have been obtained, and future developments for these biological processes are further discussed. PMID:24176501

  17. Coherence-controlled holographic microscopy for live-cell quantitative phase imaging

    NASA Astrophysics Data System (ADS)

    Slab, Tom.; K?ov, Aneta; Lot'k, Martin; ?ollkov, Jana; Jůzov, Veronika; Vesel, Pavel; Chmelk, Radim

    2015-03-01

    In this paper we present coherence-controlled holographic microscopy (CCHM) and various examples of observations of living cells including combination of CCHM with fluorescence microscopy. CCHM is a novel technique of quantitative phase imaging (QPI). It is based on grating off-axis interferometer, which is fully adapted for the use of incoherent illumination. This enables high-quality QPI free from speckles and parasitic interferences and lateral resolution of classical widefield microscopes. Label-free nature of QPI makes CCHM a useful tool for long-term observations of living cells. Moreover, coherence-gating effect induced by the use of incoherent illumination enables QPI of cells even in scattering media. Combination of CCHM with common imaging techniques brings the possibility to exploit advantages of QPI while simultaneously identifying the observed structures or processes by well-established imaging methods. We used CCHM for investigation of general parameters of cell life cycles and for research of cells reactions to different treatment. Cells were also visualized in 3D collagen gel with the use of CCHM. It was found that both the cell activity and movement of the collagen fibers can be registered. The method of CCHM in combination with fluorescence microscopy was used in order to obtain complementary information about cell morphology and identify typical morphological changes associated with different types of cell death. This combination of CCHM with common imaging technique has a potential to provide new knowledge about various processes and simultaneously their confirmation by comparison with known imaging method.

  18. Quantitative visualization of colloidal and intracellular gold nanoparticles by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Klein, Sabine; Petersen, Svea; Taylor, Ulrike; Rath, Detlef; Barcikowski, Stephan

    2010-05-01

    Gold nanoparticles (AuNPs) have the potential to become a versatile biomarker. For further use of AuNPs labeled with functionalized molecules, their visualization in biological systems by routine laboratory tools such as light microscopy is crucial. However, the size far below the diffraction limit affords specialized parameters for microscopical detection, which stimulated the current study, aimed to determine from which size onward AuNPs, either in dispersion or cell-associated, can be reliably detected by standard confocal microscopy. First, gold colloids of size-restricted fractions are examined in dispersion. At a minimum particle size of 60 nm, detection appears to be reliable. Particle counts in dilution series confirm these results by revealing single particle detection of 60-nm colloids. Second, AuNPs are visualized and quantified in cells, which interestingly cause a phase shift in the reflection of AuNPs. Gold mass spectroscopy confirms the number of AuNPs counted microscopically inside cells. Furthermore, it demonstrates for the first time a very high diffusion rate of 15-nm particles into the cells. In conclusion, the results back the suitability of confocal microscopy for the quantitative tracking of colloidal and intracellular gold nanoparticles sized 60 nm.

  19. Zebrafish Caudal Fin Angiogenesis Assay—Advanced Quantitative Assessment Including 3-Way Correlative Microscopy

    PubMed Central

    Correa Shokiche, Carlos; Schaad, Laura; Triet, Ramona; Jazwinska, Anna; Tschanz, Stefan A.; Djonov, Valentin

    2016-01-01

    Background Researchers evaluating angiomodulating compounds as a part of scientific projects or pre-clinical studies are often confronted with limitations of applied animal models. The rough and insufficient early-stage compound assessment without reliable quantification of the vascular response counts, at least partially, to the low transition rate to clinics. Objective To establish an advanced, rapid and cost-effective angiogenesis assay for the precise and sensitive assessment of angiomodulating compounds using zebrafish caudal fin regeneration. It should provide information regarding the angiogenic mechanisms involved and should include qualitative and quantitative data of drug effects in a non-biased and time-efficient way. Approach & Results Basic vascular parameters (total regenerated area, vascular projection area, contour length, vessel area density) were extracted from in vivo fluorescence microscopy images using a stereological approach. Skeletonization of the vasculature by our custom-made software Skelios provided additional parameters including “graph energy” and “distance to farthest node”. The latter gave important insights into the complexity, connectivity and maturation status of the regenerating vascular network. The employment of a reference point (vascular parameters prior amputation) is unique for the model and crucial for a proper assessment. Additionally, the assay provides exceptional possibilities for correlative microscopy by combining in vivo-imaging and morphological investigation of the area of interest. The 3-way correlative microscopy links the dynamic changes in vivo with their structural substrate at the subcellular level. Conclusions The improved zebrafish fin regeneration model with advanced quantitative analysis and optional 3-way correlative morphology is a promising in vivo angiogenesis assay, well-suitable for basic research and preclinical investigations. PMID:26950851

  20. Transmission electron microscopy of polymer blends and block copolymers

    NASA Astrophysics Data System (ADS)

    Gomez, Enrique Daniel

    Transmission electron microscopy (TEM) of soft matter is a field that warrants further investigation. Developments in sample preparation, imaging and spectroscopic techniques could lead to novel experiments that may further our understanding of the structure and the role structure plays in the functionality of various organic materials. Unlike most hard materials, TEM of organic molecules is limited by the amount of radiation damage the material can withstand without changing its structure. Despite this limitation, TEM has been and will be a powerful tool to study polymeric materials and other soft matter. In this dissertation, an introduction of TEM for polymer scientists is presented. The fundamentals of interactions of electrons with matter are described using the Schrodinger wave equation and scattering cross-sections to fully encompass coherent and incoherent scattering. The intensity, which is the product of the wave function and its complex conjugate, shows no perceptible change due to the sample. Instead, contrast is generated through the optical system of the microscope by removing scattered electrons or by generating interference due to material-induced phase changes. Perhaps the most challenging aspect of taking TEM images, however, is sample preparation, because TEM experiments require materials with approximately 50 nm thickness. Although ultramicrotomy is a well-established powerful tool for preparing biological and polymeric sections for TEM, the development of cryogenic Focused Ion Beam may enable unprecedented cross-sectional TEM studies of polymer thin films on arbitrary substrates with nanometer precision. Two examples of TEM experiments of polymeric materials are presented. The first involves quantifying the composition profile across a lamellar phase obtained in a multicomponent blend of saturated poly(butadiene) and poly(isobutylene), stabilized by a saturated poly(butadiene) copolymer serving as a surfactant, using TEM and self-consistent field theory (SCFT). The liquid-like nature of this system at room temperature makes traditional staining methods for the enhancement of contrast ineffective. As an alternative, we take advantage of the large inelastic scattering cross-section of soft materials to generate contrast in zero-loss TEM images. Independent spatially resolved thickness measurements enable quantification of electron scattering. This enabled a comparison between the TEM data and predictions based on SCFT without any adjustable parameters. The second example involves the utilization of energy-filtered transmission electron microscopy (EFTEM) to compute elemental maps by taking advantage of ionization events. Elemental mapping of lithium is used to determine the distribution of salt in nanostructured poly(styrene-block-ethylene oxide) (SEO) copolymer/lithium salt electrolytes. Surprisingly, the concentration of lithium within a poly(ethylene oxide) (PEO) domain is found to be inhomogeneous; the salt is localized to the middle of the channels. Self-consistent field theory simulations suggest that localization of lithium is due to chain stretching at the interface, which increases with molecular weight. EFTEM and SCFT results show that the segregation of lithium salt to the middle of the PEO lamellae is greater for higher molecular weight polymers. This is correlated with the ionic conductivity of the copolymer electrolyte, which is found to show a higher conductivity for thinner lithium lamellae.

  1. Identification and quantitive analysis of calcium phosphate microparticles in intestinal tissue by nuclear microscopy

    NASA Astrophysics Data System (ADS)

    Gomez-Morilla, Inmaculada; Thoree, Vinay; Powell, Jonathan J.; Kirkby, Karen J.; Grime, Geoffrey W.

    2006-08-01

    Microscopic particles (0.5-2 ?m diameter), rich in calcium and phosphorus, are found in the lumen of the mid-distal gut of all mammals investigated, including humans, and these may play a role in immuno-surveillance and immune regulation of antigens from food and symbiotic bacteria that are contained in the gut. Whether these particles can cross in to tissue of the intestinal mucosa is unclear. If so, characterising their morphology and chemical composition is an important task in elucidating their function. The analysis of calcium phosphate in biological tissues has been approached in several ways including optical microscopy, scanning electron microscopy and, most recently in this work, with nuclear microscopy. In this paper, we describe the use of microPIXE and microRBS to locate these particles and to determine, accurately, the ratio of phosphorus to calcium using the information on sample thickness obtained from RBS to allow the PIXE ratios to be corrected. A commercial sample of hydroxy apatite was used to demonstrate accuracy and precision of the technique. Then, in a pilot study on intestinal tissue of mice, we demonstrated the presence of calcium phosphate microparticles, consistent with confocal microscopy observations, and we identified the average molar P:Ca molar ratio as 1.0. Further work will confirm the exact chemical speciation of these particles and will examine the influence of differing calcium containing diets on the formation of these microparticles.

  2. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis

    PubMed Central

    Silvestri, Ludovico; Paciscopi, Marco; Soda, Paolo; Biamonte, Filippo; Iannello, Giulio; Frasconi, Paolo; Pavone, Francesco S.

    2015-01-01

    Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells (PCs) across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all PCs are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent PCs. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of PCs, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of PCs with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments. PMID:26074783

  3. Quantitative neuroanatomy of all Purkinje cells with light sheet microscopy and high-throughput image analysis.

    PubMed

    Silvestri, Ludovico; Paciscopi, Marco; Soda, Paolo; Biamonte, Filippo; Iannello, Giulio; Frasconi, Paolo; Pavone, Francesco S

    2015-01-01

    Characterizing the cytoarchitecture of mammalian central nervous system on a brain-wide scale is becoming a compelling need in neuroscience. For example, realistic modeling of brain activity requires the definition of quantitative features of large neuronal populations in the whole brain. Quantitative anatomical maps will also be crucial to classify the cytoarchtitectonic abnormalities associated with neuronal pathologies in a high reproducible and reliable manner. In this paper, we apply recent advances in optical microscopy and image analysis to characterize the spatial distribution of Purkinje cells (PCs) across the whole cerebellum. Light sheet microscopy was used to image with micron-scale resolution a fixed and cleared cerebellum of an L7-GFP transgenic mouse, in which all PCs are fluorescently labeled. A fast and scalable algorithm for fully automated cell identification was applied on the image to extract the position of all the fluorescent PCs. This vectorized representation of the cell population allows a thorough characterization of the complex three-dimensional distribution of the neurons, highlighting the presence of gaps inside the lamellar organization of PCs, whose density is believed to play a significant role in autism spectrum disorders. Furthermore, clustering analysis of the localized somata permits dividing the whole cerebellum in groups of PCs with high spatial correlation, suggesting new possibilities of anatomical partition. The quantitative approach presented here can be extended to study the distribution of different types of cell in many brain regions and across the whole encephalon, providing a robust base for building realistic computational models of the brain, and for unbiased morphological tissue screening in presence of pathologies and/or drug treatments. PMID:26074783

  4. Cryo-Electron Microscopy Structure of Lactococcal Siphophage 1358 Virion

    PubMed Central

    Spinelli, Silvia; Bebeacua, Cecilia; Orlov, Igor; Tremblay, Denise; Klaholz, Bruno P.

    2014-01-01

    ABSTRACT Lactococcus lactis, a Gram+ lactic acid-producing bacterium used for the manufacture of several fermented dairy products, is subject to infection by diverse virulent tailed phages, leading to industrial fermentation failures. This constant viral risk has led to a sustained interest in the study of their biology, diversity, and evolution. Lactococcal phages now constitute a wide ensemble of at least 10 distinct genotypes within the Caudovirales order, many of them belonging to the Siphoviridae family. Lactococcal siphophage 1358, currently the only member of its group, displays a noticeably high genomic similarity to some Listeria phages as well as a host range limited to a few L. lactis strains. These genomic and functional characteristics stimulated our interest in this phage. Here, we report the cryo-electron microscopy structure of the complete 1358 virion. Phage 1358 exhibits noteworthy features, such as a capsid with dextro handedness and protruding decorations on its capsid and tail. Observations of the baseplate of virion particles revealed at least two conformations, a closed and an open, activated form. Functional assays uncovered that the adsorption of phage 1358 to its host is Ca2+ independent, but this cation is necessary to complete its lytic cycle. Taken together, our results provide the complete structural picture of a unique lactococcal phage and expand our knowledge on the complex baseplate of phages of the Siphoviridae family. IMPORTANCE Phages of Lactococcus lactis are investigated mainly because they are sources of milk fermentation failures in the dairy industry. Despite the availability of several antiphage measures, new phages keep emerging in this ecosystem. In this study, we provide the cryo-electron microscopy reconstruction of a unique lactococcal phage that possesses genomic similarity to particular Listeria phages and has a host range restricted to only a minority of L. lactis strains. The capsid of phage 1358 displays the almost unique characteristic of being dextro handed. Its capsid and tail exhibit decorations that we assigned to nonspecific sugar binding modules. We observed the baseplate of 1358 in two conformations, a closed and an open form. We also found that the adsorption to its host, but not infection, is Ca2+ independent. Overall, this study advances our understanding of the adhesion mechanisms of siphophages. PMID:24872584

  5. Using advanced electron microscopy for the characterization of catalytic materials

    NASA Astrophysics Data System (ADS)

    Pyrz, William D.

    Catalysis will continue to be vitally important to the advancement and sustainability of industrialized societies. Unfortunately, the petroleum-based resources that currently fuel the energy and consumer product needs of an advancing society are becoming increasingly difficult and expensive to extract as supplies diminish and the quality of sources degrade. Therefore, the development of sustainable energy sources and the improvement of the carbon efficiency of existing chemical processes are critical. Further challenges require that these initiatives are accomplished in an environmentally friendly fashion since the effects of carbon-based emissions are proving to be a serious threat to global climate stability. In this dissertation, materials being developed for sustainable energy and process improvement initiatives are studied. Our approach is to use materials characterization, namely advanced electron microscopy, to analyze the targeted systems at the nano- or Angstrom-scale with the goal of developing useful relationships between structure, composition, crystalline order, morphology, and catalytic performance. One area of interest is the complex Mo-V-M-O (M=Te, Sb, Ta, Nb) oxide system currently being developed for the selective oxidation/ammoxidation of propane to acrylic acid or acrylonitrile, respectively. Currently, the production of acrylic acid and acrylonitrile rely on propylene-based processes, yet significant cost savings could be realized if the olefin-based feeds could be replaced by paraffin-based ones. The major challenge preventing this feedstock replacement is the development of a suitable paraffin-activating catalyst. Currently, the best candidate is the Mo-V-Nb-Te-O complex oxide catalyst that is composed of two majority phases that are commonly referred to as M1 and M2. However, there is a limited understanding of the roles of each component with respect to how they contribute to catalyst stability and the reaction mechanism. Aberration-corrected electron microscopy was used to systematically examine, atomic column by atomic column, the effect of elemental substitution on the long-range crystalline order, atomic coordinates, and site occupancies of the various formulations such that trends could be developed linking these properties to catalytic yields. To accomplish this task, an algorithm was developed that enabled the direct extraction of atomic coordinates and site occupancies from high-angle annular dark-field (HAADF) images to within 1% and 15% uncertainty, respectively. Furthermore, this general method could be applied to various crystalline systems and may dramatically improve the quality of initial structural models used in Rietveld refinements. Improvement in the quality of starting models may increase the structural and chemical complexity of inorganic structures that can be solved by using "powder methods" alone. In addition to the development of these trends, HAADF analyses also revealed the presence of coherent compositional miscibility gaps, rotational twin domains, and structural intergrowths in the complex Mo-V-M-O oxide system. Other catalytic systems that are addressed in this dissertation include Pd, Ag, and bimetallic Pd-Ag catalysts for the selective hydrogenation of acetylene in excess ethylene, alkali and alkaline earth promoted Ru catalysts for the production of clean hydrogen through the decomposition of ammonia, the production of Pt nanoparticles using dendrimer templates, and Pt-Re bimetallic catalysts for the conversion of glycerol to hydrocarbons and syn gas. In each of these studies, electron microscopy was used as a complimentary tool to synthetic and reaction studies to better understand interactions between the nanoparticles and the support/template, to determine the effect of adding various promoters, or to understand the nanoscale structural and chemical changes associated with the formation of bimetallic nanoparticles. A final area addressed in this dissertation is the interaction between the electron beam and the specimen. In one particular study directed toward the characterization of Ni-Bi nanomaterials, it was discovered that exposure to an intense electron beam initiated particle fragmentation that led to the formation of a field of nanoparticles. Subsequent microscopy studies of the resulting nanoparticle field revealed bimetallic nanoparticles, core-shell structures, Angstrom-scale atomic clusters, and individual atoms that decorated the surrounding carbon substrate. The identification of these structures along with the analysis of the parent material enabled the development of a fragmentation mechanism.

  6. Quantitative morphological evaluation of laser ablation on calculus using full-field optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Xiao, Q.; Lü, T.; Li, Z.; Fu, L.

    2011-10-01

    The quantitative morphological evaluation at high resolution is of significance for the study of laser-tissue interaction. In this paper, a full-field optical coherence microscopy (OCM) system with high resolution of ˜2 μm was developed to investigate the ablation on urinary calculus by a free-running Er:YAG laser. We studied the morphological variation quantitatively corresponding to change of energy setting of the Er:YAG laser. The experimental results show that the full-field OCM enables quantitative evaluation of the morphological shape of craters and material removal, and particularly the fine structure. We also built a heat conduction model to simulate the process of laser-calculus interaction by using finite element method. Through the simulation, the removal region of the calculus was calculated according to the temperature distribution. As a result, the depth, width, volume, and the cross-sectional profile of the crater in calculus measured by full-field OCM matched well with the theoretical results based on the heat conduction model. Both experimental and theoretical results confirm that the thermal interaction is the dominant effect in the ablation of calculus by Er:YAG laser, demonstrating the effectiveness of full-field OCM in studying laser-tissue interactions.

  7. A quantitative lateral force Microscopy study of the dolomite (104)-water interface.

    SciTech Connect

    Higgins, S. R.; Hu, X.; Fentert, P.; Wright State Univ.

    2007-08-14

    The friction and lateral stiffness of the contact between an atomic force microscopy (AFM) probe tip and an atomically flat dolomite (104) surface were investigated in contact with two aqueous solutions that were in equilibrium and supersaturated with respect to dolomite, respectively. The two aqueous solutions yielded negligible differences in friction at the native dolomite-water interface. However, the growth of a Ca-rich film from the supersaturated solution, revealed by X-ray reflectivity measurements, altered the probe-dolomite contact region sufficiently to observe distinct friction forces on the native dolomite and the film-covered surface regions. Quantitative friction-load relationships demonstrated three physically distinct load regimes for applied loads up to 200 nN. Similar friction forces were observed on both surfaces below 50 nN load and above 100 nN load. The friction forces on the two surfaces diverged at intermediate loads. Quantitative measurements of dynamic friction forces at low load were consistent with the estimated energy necessary to dehydrate the surface ions, whereas differences in mechanical properties of the Ca-rich film and dolomite surfaces were evidently important above 50 nN load. Attempts to fit the quantitative stiffness-load data using a Hertzian contact mechanical model based on bulk material properties yielded physically unrealistic fitting coefficients, suggesting that the interfacial contact region must be explicitly considered in describing the static and dynamic contact mechanics of this and similar systems.

  8. Quantitative confocal fluorescence microscopy of dynamic processes by multifocal fluorescence correlation spectroscopy

    NASA Astrophysics Data System (ADS)

    Krmpot, Aleksandar J.; Nikolić, Stanko N.; Vitali, Marco; Papadopoulos, Dimitrios K.; Oasa, Sho; Thyberg, Per; Tisa, Simone; Kinjo, Masataka; Nilsson, Lennart; Gehring, Walter J.; Terenius, Lars; Rigler, Rudolf; Vukojevic, Vladana

    2015-07-01

    Quantitative confocal fluorescence microscopy imaging without scanning is developed for the study of fast dynamical processes. The method relies on the use of massively parallel Fluorescence Correlation Spectroscopy (mpFCS). Simultaneous excitation of fluorescent molecules across the specimen is achieved by passing a single laser beam through a Diffractive Optical Element (DOE) to generate a quadratic illumination matrix of 32×32 light sources. Fluorescence from 1024 illuminated spots is detected in a confocal arrangement by a matching matrix detector consisting of the same number of single-photon avalanche photodiodes (SPADs). Software was developed for data acquisition and fast autoand cross-correlation analysis by parallel signal processing using a Graphic Processing Unit (GPU). Instrumental performance was assessed using a conventional single-beam FCS instrument as a reference. Versatility of the approach for application in biomedical research was evaluated using ex vivo salivary glands from Drosophila third instar larvae expressing a fluorescently-tagged transcription factor Sex Combs Reduced (Scr) and live PC12 cells stably expressing the fluorescently tagged mu-opioid receptor (MOPeGFP). We show that quantitative mapping of local concentration and mobility of transcription factor molecules across the specimen can be achieved using this approach, which paves the way for future quantitative characterization of dynamical reaction-diffusion landscapes across live cells/tissue with a submillisecond temporal resolution (presently 21 μs/frame) and single-molecule sensitivity.

  9. Three-dimensional quantitative phase imaging via tomographic deconvolution phase microscopy.

    PubMed

    Jenkins, Micah H; Gaylord, Thomas K

    2015-11-01

    The field of three-dimensional quantitative phase imaging (3D QPI) is expanding rapidly with applications in biological, medical, and industrial research, development, diagnostics, and metrology. Much of this research has centered on developing optical diffraction tomography (ODT) for biomedical applications. In addition to technical difficulties associated with coherent noise, ODT is not congruous with optical microscopy utilizing partially coherent light, which is used in most biomedical laboratories. Thus, ODT solutions have, for the most part, been limited to customized optomechanical systems which would be relatively expensive to implement on a wide scale. In the present work, a new phase reconstruction method, called tomographic deconvolution phase microscopy (TDPM), is described which makes use of commercial microscopy hardware in realizing 3D QPI. TDPM is analogous to methods used in deconvolution microscopy which improve spatial resolution and 3D-localization accuracy of fluorescence micrographs by combining multiple through-focal scans which are deconvolved by the system point spread function. TDPM is based on the 3D weak object transfer function theory which is shown here to be capable of imaging "nonweak" phase objects with large phase excursions. TDPM requires no phase unwrapping and recovers the entire object spectrum via object rotation, mitigating the need to fill in the "missing cone" of spatial frequencies algorithmically as in limited-angle ODT. In the present work, TDPM is demonstrated using optical fibers, including single-mode, polarization-maintaining, and photonic-crystal fibers as well as an azimuthally varying CO2-laser-induced long-period fiber grating period as test phase objects. PMID:26560576

  10. Visualization and Quantitative Analysis of Reconstituted Tight Junctions Using Localization Microscopy

    PubMed Central

    Kaufmann, Rainer; Piontek, Jörg; Grüll, Frederik; Kirchgessner, Manfred; Rossa, Jan; Wolburg, Hartwig; Blasig, Ingolf E.; Cremer, Christoph

    2012-01-01

    Tight Junctions (TJ) regulate paracellular permeability of tissue barriers. Claudins (Cld) form the backbone of TJ-strands. Pore-forming claudins determine the permeability for ions, whereas that for solutes and macromolecules is assumed to be crucially restricted by the strand morphology (i.e., density, branching and continuity). To investigate determinants of the morphology of TJ-strands we established a novel approach using localization microscopy. TJ-strands were reconstituted by stable transfection of HEK293 cells with the barrier-forming Cld3 or Cld5. Strands were investigated at cell-cell contacts by Spectral Position Determination Microscopy (SPDM), a method of localization microscopy using standard fluorophores. Extended TJ-networks of Cld3-YFP and Cld5-YFP were observed. For each network, 200,000 to 1,100,000 individual molecules were detected with a mean localization accuracy of ∼20 nm, yielding a mean structural resolution of ∼50 nm. Compared to conventional fluorescence microscopy, this strongly improved the visualization of strand networks and enabled quantitative morphometric analysis. Two populations of elliptic meshes (mean diameter <100 nm and 300–600 nm, respectively) were revealed. For Cld5 the two populations were more separated than for Cld3. Discrimination of non-polymeric molecules and molecules within polymeric strands was achieved. For both subtypes of claudins the mean density of detected molecules was similar and estimated to be ∼24 times higher within the strands than outside the strands. The morphometry and single molecule information provided advances the mechanistic analysis of paracellular barriers. Applying this novel method to different TJ-proteins is expected to significantly improve the understanding of TJ on the molecular level. PMID:22319608

  11. 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...

  12. 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 -10C and -25C, 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 and mixed-phase cloud properties in climate models could be markedly affected.

  13. 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 down as the MWF corrodes. The importance of localized corrosion should also be determined.

  14. 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.

  15. Scanning electron microscopy applied to seed-borne fungi examination.

    PubMed

    Alves, Marcelo de Carvalho; Pozza, Edson Ampélio

    2009-07-01

    The aim of this study was to test the standard scanning electron microscopy (SEM) as a potential alternative to study seed-borne fungi in seeds, by two different conditions of blotter test and water restriction treatment. In the blotter test, seeds were subjected to conditions that enabled pathogen growth and expression, whereas the water restriction method consisted in preventing seed germination during the incubation period, resulting in the artificial inoculation of fungi. In the first condition, seeds of common bean (Phaseolus vulgaris L.), maize (Zea mays L.), and cotton (Gossypium hirsutum L.) were submitted to the standard blotter test and then prepared and observed with SEM. In the second condition, seeds of cotton (G. hirsutum), soybean (Glycine max L.), and common bean (P. vulgaris L.) were, respectively, inoculated with Colletotrichum gossypii var. cephalosporioides, Colletotrichum truncatum, and Colletotrichum lindemuthianum by the water restriction technique, followed by preparation and observation with SEM. The standard SEM methodology was adopted to prepare the specimens. Considering the seeds submitted to the blotter test, it was possible to identify Fusarium sp. on maize, C. gossypii var. cephalosporioides, and Fusarium oxysporum on cotton, Aspergillus flavus, Penicillium sp., Rhizopus sp., and Mucor sp. on common bean. Structures of C. gossypii var. cephalosporioides, C. truncatum, and C. lindemuthianum were observed in the surface of inoculated seeds. PMID:19204924

  16. Cryogenic Transmission Electron Microscopy Nanostructural Study of Shed Microparticles

    PubMed Central

    Issman, Liron; Brenner, Benjamin; Talmon, Yeshayahu; Aharon, Anat

    2013-01-01

    Microparticles (MPs) are sub-micron membrane vesicles (1001000 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 80C for storage introduce morphological artifacts, which can influence MP nanostructure, and thus affect the efficiency of these particles as future diagnostic tools. PMID:24386253

  17. Scanning electron microscopy of lung following alpha irradiation

    SciTech Connect

    Sanders, C.L.; Lauhala, K.E.; McDonald, K.E. )

    1989-09-01

    Pulmonary aggregation of inhaled {sup 239}PuO{sub 2} particles leads to a cellular evolution of focal inflammation, fibrosis, epithelial dysplasia and lung tumor formation. Female Wistar rats were exposed to an aerosol of high-fired {sup 239}PuO{sub 2} (initial lung burden, 3.9 kBq) and the lungs examined at intervals from 1 day to 700 days after exposure by light and scanning electron microscopy and autoradiography. Peribronchiolar Pu particle aggregation increased with time, resulting in well-defined focal inflammatory lesions after 120 days and fibrotic lesions after 180 days. A generalized hypertrophy and hyperplasia of nonciliated bronchiolar cells was seen at 15 days and type II cell hyperplasia by 30 days after exposure. Focal dysplastic changes in type II alveolar epithelium and terminal nonciliated bronchiolar epithelium preceded carcinoma formation. Alveolar bronchiolarization was first noted at 120 days, squamous metaplasia at 210 days, squamous carcinoma at 270 days and adenocarcinoma at 600 days after exposure.

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Scanning electron microscopy of isolated dog renal tubules.

    PubMed

    Jones, D B

    1982-01-01

    Isolation of canine renal tubular segments, cell clusters and individual cells permits direct observation by scanning electron microscopy (SEM) of the three dimensional cell shapes and the complex lateral and basal labyrinth structures. These functionally vital areas of the various tubular segments have not been previously observed in such detail. The tubular segments were freed of the obscuring basement membranes by prolonged tryptic digestion of tissue perfusion-fixed with glutaraldehyde, followed by attachment to gelatine coated coverslips and preparation for SEM. The tubular basal labyrinth is found to consist of a mosaic of basal processes arising from the lateral processes and the base of the cell body. The lateral processes and basal processes interdigitate with adjacent cells. Each tubular segment exhibits its own characteristic patterns of basal processes. The basal processes of the proximal convoluted tubule (PCT) are shaped like an inverted anvil, often branching. The basal processes of the proximal straight tubule (PST) and thin limbs are short microvilli. The distal straight tubule (DST) have fishtail-like basal processes. The distal convoluted tubule (DCT) show a swirling mixture of anvil shaped and microvillous basal processes. The collecting ducts exhibit short basal microvilli, plicae and villi lined basal infoldings. The looser structure of the basal labyrinth of cell clusters and isolated cells produced by this isolation procedure facilitated recognition of these complex structures. PMID:7167772

  3. 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.

  4. 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.

  5. Investigating surface magnetism by means of photoexcitation electron emission microscopy

    NASA Astrophysics Data System (ADS)

    Schneider, Claus M.; Schnhense, Gerd

    2002-12-01

    The imaging of surfaces by means of photoexcitation electron emission microscopy (PEEM) has recently received considerable interest. This is mainly due to the extended use and availability of brilliant synchrotron radiation in the soft x-ray regime which generally facilitates studies with surface specificity and chemical selectivity. The most popular application of the x-ray PEEM (XPEEM) technique concerns studies of magnetic systems and phenomena. By exploiting the high degree of circular or linear polarization of the synchrotron light, the magnetic microstructure in both ferromagnets and antiferromagnets can be visualized. In this contribution we demonstrate the unique potential and the versatility of the PEEM approach, and review the current status with a certain emphasis on experiments with soft x-ray excitation. In some cases, the high-energy excitation studies can be complemented by laboratory experiments employing threshold photoemission with ultraviolet light (UV-PEEM). Current limitations and future developments and perspectives of the PEEM technique applied to magnetic systems are discussed.

  6. Surface treatment of feldspathic porcelain: scanning electron microscopy analysis

    PubMed Central

    Valian, Azam

    2014-01-01

    PURPOSE Topographic analysis of treated ceramics provides qualitative information regarding the surface texture affecting the micromechanical retention and locking of resin-ceramics. This study aims to compare the surface microstructure following different surface treatments of feldspathic porcelain. MATERIALS AND METHODS This in-vitro study was conducted on 72 porcelain discs randomly divided into 12 groups (n=6). In 9 groups, feldspathic surfaces were subjected to sandblasting at 2, 3 or 4 bar pressure for 5, 10 or 15 seconds with 50 µm alumina particles at a 5 mm distance. In group 10, 9.5% hydrofluoric acid (HF) gel was applied for 120 seconds. In group 11, specimens were sandblasted at 3 bar pressure for 10 seconds and then conditioned with HF. In group 12, specimens were first treated with HF and then sandblasted at 3 bar pressure for 10 seconds. All specimens were then evaluated under scanning electron microscopy (SEM) at different magnifications. RESULTS SEM images of HF treated specimens revealed deep porosities of variable sizes; whereas, the sandblasted surfaces were more homogenous and had sharper peaks. Increasing the pressure and duration of sandblasting increased the surface roughness. SEM images of the two combined techniques showed that in group 11 (sandblasted first), HF caused deeper porosities; whereas in group 12 (treated with HF first) sandblasting caused irregularities with less homogeneity. CONCLUSION All surface treatments increased the surface area and caused porous surfaces. In groups subjected to HF, the porosities were deeper than those in sandblasted only groups. PMID:25352961

  7. 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.

  8. Orthogonal Matrix Retrieval In Cryo-Electron Microscopy

    PubMed Central

    Bhamre, Tejal; Zhang, Teng; Singer, Amit

    2015-01-01

    In single particle reconstruction (SPR) from cryo-electron microscopy (EM), the 3D structure of a molecule needs to be determined from its 2D projection images taken at unknown viewing directions. Zvi Kam showed already in 1980 that the autocorrelation function of the 3D molecule over the rotation group SO(3) can be estimated from 2D projection images whose viewing directions are uniformly distributed over the sphere. The autocorrelation function determines the expansion coefficients of the 3D molecule in spherical harmonics up to an orthogonal matrix of size (2l + 1) (2l + 1) for each l = 0,1,2,. In this paper we show how techniques for solving the phase retrieval problem in X-ray crystallography can be modified for the cryo-EM setup for retrieving the missing orthogonal matrices. Specifically, we present two new approaches that we term Orthogonal Extension and Orthogonal Replacement, in which the main algorithmic components are the singular value decomposition and semidefinite programming. We demonstrate the utility of these approaches through numerical experiments on simulated data. PMID:26677402

  9. Candidate sampling for neuron reconstruction from anisotropic electron microscopy volumes.

    PubMed

    Funke, Jan; Martel, Julien N P; Gerhard, Stephan; Andres, Bjoern; Cire?an, Dan C; Giusti, Alessandro; Gambardella, Luca M; Schmidhuber, Jrgen; Pfister, Hanspeter; Cardona, Albert; Cook, Matthew

    2014-01-01

    The automatic reconstruction of neurons from stacks of electron microscopy sections is an important computer vision problem in neuroscience. Recent advances are based on a two step approach: First, a set of possible 2D neuron candidates is generated for each section independently based on membrane predictions of a local classifier. Second, the candidates of all sections of the stack are fed to a neuron tracker that selects and connects them in 3D to yield a reconstruction. The accuracy of the result is currently limited by the quality of the generated candidates. In this paper, we propose to replace the heuristic set of candidates used in previous methods with samples drawn from a conditional random field (CRF) that is trained to label sections of neural tissue. We show on a stack of Drosophila melanogaster neural tissue that neuron candidates generated with our method produce 30% less reconstruction errors than current candidate generation methods. Two properties of our CRF are crucial for the accuracy and applicability of our method: (1) The CRF models the orientation of membranes to produce more plausible neuron candidates. (2) The interactions in the CRF are restricted to form a bipartite graph, which allows a great sampling speed-up without loss of accuracy. PMID:25333096

  10. 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.

  11. 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. PMID:15993698

  12. High-performance probes for light and electron microscopy

    PubMed Central

    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.

    2015-01-01

    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) distribute well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localizes 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 allow robust, orthogonal multi-color visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers, greatly increase the number of simultaneous imaging channels, and perform well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improve single-molecule image tracking and increase yield for RNA-Seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization. PMID:25915120

  13. Customized patterned substrates for highly versatile correlative light-scanning electron microscopy

    PubMed Central

    Benedetti, Lorena; Sogne, Elisa; Rodighiero, Simona; Marchesi, Davide; Milani, Paolo; Francolini, Maura

    2014-01-01

    Correlative light electron microscopy (CLEM) combines the advantages of light and electron microscopy, thus making it possible to follow dynamic events in living cells at nanometre resolution. Various CLEM approaches and devices have been developed, each of which has its own advantages and technical challenges. We here describe our customized patterned glass substrates, which improve the feasibility of correlative fluorescence/confocal and scanning electron microscopy. PMID:25391455

  14. A technique to prepare asbestos air samples for light and electron microscopy.

    PubMed Central

    Pang, T W; Robinson, A E

    1981-01-01

    The direct transfer technique used in preparing waterborne asbestos for analysis by transmission electron microscopy is also suitable for preparing airborne asbestos collected on polycarbonate filters for light microscopy, scanning, and transmission electron microscopy. The same area and even the same fibre may be examined by the three microscopic methods and individual fibres identified by optical and electron-optical techniques and by x-ray microanalysis. Images PMID:7317304

  15. 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 kamacite which forms as {alpha}{sub 2} or as Widmanstatten plates in a series of IVB irons. Fig. 4a,b shows EBSD orientation maps of taenite and kamacite from the Tawallah Valley IVB iron. We observe that the orientation of the taenite in the IVB meteorites is the same throughout the sample consistent with the orientation of the high temperature single phase taenite before formation of the Widmanstatten pattern.

  16. Towards quantitative, atomic-resolution reconstruction of the electrostatic potential via differential phase contrast using electrons.

    PubMed

    Close, R; Chen, Z; Shibata, N; Findlay, S D

    2015-12-01

    Differential phase contrast images in scanning transmission electron microscopy can be directly and quantitatively related to the gradient of the projected specimen potential provided that (a) the specimen can be treated as a phase object and (b) full 2D diffraction patterns as a function of probe position can be obtained. Both are challenging to achieve in atomic resolution imaging. The former is fundamentally limited by probe spreading and dynamical electron scattering, and we explore its validity domain in the context of atomic resolution differential phase contrast imaging. The latter, for which proof-of-principle experimental data sets exist, is not yet routine. We explore the extent to which more established segmented detector geometries can instead be used to reconstruct a quantitatively good approximation to the projected specimen potential. PMID:26381331

  17. A general way for quantitative magnetic measurement by transmitted electrons

    NASA Astrophysics Data System (ADS)

    Song, Dongsheng; Li, Gen; Cai, Jianwang; Zhu, Jing

    2016-01-01

    EMCD (electron magnetic circular dichroism) technique opens a new door to explore magnetic properties by transmitted electrons. The recently developed site-specific EMCD technique makes it possible to obtain rich magnetic information from the Fe atoms sited at nonequivalent crystallographic planes in NiFe2O4, however it is based on a critical demand for the crystallographic structure of the testing sample. Here, we have further improved and tested the method for quantitative site-specific magnetic measurement applicable for more complex crystallographic structure by using the effective dynamical diffraction effects (general routine for selecting proper diffraction conditions, making use of the asymmetry of dynamical diffraction for design of experimental geometry and quantitative measurement, etc), and taken yttrium iron garnet (Y3Fe5O12, YIG) with more complex crystallographic structure as an example to demonstrate its applicability. As a result, the intrinsic magnetic circular dichroism signals, spin and orbital magnetic moment of iron with site-specific are quantitatively determined. The method will further promote the development of quantitative magnetic measurement with high spatial resolution by transmitted electrons.

  18. A general way for quantitative magnetic measurement by transmitted electrons.

    PubMed

    Song, Dongsheng; Li, Gen; Cai, Jianwang; Zhu, Jing

    2016-01-01

    EMCD (electron magnetic circular dichroism) technique opens a new door to explore magnetic properties by transmitted electrons. The recently developed site-specific EMCD technique makes it possible to obtain rich magnetic information from the Fe atoms sited at nonequivalent crystallographic planes in NiFe2O4, however it is based on a critical demand for the crystallographic structure of the testing sample. Here, we have further improved and tested the method for quantitative site-specific magnetic measurement applicable for more complex crystallographic structure by using the effective dynamical diffraction effects (general routine for selecting proper diffraction conditions, making use of the asymmetry of dynamical diffraction for design of experimental geometry and quantitative measurement, etc), and taken yttrium iron garnet (Y3Fe5O12, YIG) with more complex crystallographic structure as an example to demonstrate its applicability. As a result, the intrinsic magnetic circular dichroism signals, spin and orbital magnetic moment of iron with site-specific are quantitatively determined. The method will further promote the development of quantitative magnetic measurement with high spatial resolution by transmitted electrons. PMID:26726959

  19. A general way for quantitative magnetic measurement by transmitted electrons

    PubMed Central

    Song, Dongsheng; Li, Gen; Cai, Jianwang; Zhu, Jing

    2016-01-01

    EMCD (electron magnetic circular dichroism) technique opens a new door to explore magnetic properties by transmitted electrons. The recently developed site-specific EMCD technique makes it possible to obtain rich magnetic information from the Fe atoms sited at nonequivalent crystallographic planes in NiFe2O4, however it is based on a critical demand for the crystallographic structure of the testing sample. Here, we have further improved and tested the method for quantitative site-specific magnetic measurement applicable for more complex crystallographic structure by using the effective dynamical diffraction effects (general routine for selecting proper diffraction conditions, making use of the asymmetry of dynamical diffraction for design of experimental geometry and quantitative measurement, etc), and taken yttrium iron garnet (Y3Fe5O12, YIG) with more complex crystallographic structure as an example to demonstrate its applicability. As a result, the intrinsic magnetic circular dichroism signals, spin and orbital magnetic moment of iron with site-specific are quantitatively determined. The method will further promote the development of quantitative magnetic measurement with high spatial resolution by transmitted electrons. PMID:26726959

  20. 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 two invited speakers from Continental Europe. Finally, keep an eye on www.emag-iop.org for details on EMAG 2013, which is to be held at the University of York. P D Nellist University of Oxford