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Sample records for quantitative electron microscopy

  1. Quantitative Phase Retrieval in Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    McLeod, Robert Alexander

    Phase retrieval in the transmission electron microscope offers the unique potential to collect quantitative data regarding the electric and magnetic properties of materials at the nanoscale. Substantial progress in the field of quantitative phase imaging was made by improvements to the technique of off-axis electron holography. In this thesis, several breakthroughs have been achieved that improve the quantitative analysis of phase retrieval. An accurate means of measuring the electron wavefront coherence in two-dimensions was developed and pratical applications demonstrated. The detector modulation-transfer function (MTF) was assessed by slanted-edge, noise, and the novel holographic techniques. It was shown the traditional slanted-edge technique underestimates the MTF. In addition, progress was made in dark and gain reference normalization of images, and it was shown that incomplete read-out is a concern for slow-scan CCD detectors. Last, the phase error due to electron shot noise was reduced by the technique of summation of hologram series. The phase error, which limits the finest electric and magnetic phenomena which can be investigated, was reduced by over 900 % with no loss of spatial resolution. Quantitative agreement between the experimental root-mean-square phase error and the analytical prediction of phase error was achieved.

  2. Quantitative characterization of electron detectors for transmission electron microscopy

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-12-01

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

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

    NASA Astrophysics Data System (ADS)

    Yankovich, Andrew B.

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

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

    PubMed

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

    2016-02-01

    Several instrumental imperfections of transmission electron microscopes are characterized and their effects on the results of quantitative scanning electron microscopy (STEM) are investigated and quantified using simulations. Methods to either avoid influences of these imperfections during acquisition or to include them in reference calculations are proposed. Particularly, distortions inflicted on the diffraction pattern by an image-aberration corrector can cause severe errors of more than 20% if not accounted for. A procedure for their measurement is proposed here. Furthermore, afterglow phenomena and nonlinear behavior of the detector itself can lead to incorrect normalization of measured intensities. Single electrons accidentally impinging on the detector are another source of error but can also be exploited for threshold-less calibration of STEM images to absolute dose, incident beam current determination and measurement of the detector sensitivity. PMID:26686661

  6. Quantitative Energy-filtering Transmission Electron Microscopy in Materials Science.

    PubMed

    Grogger; Hofer; Warbichler; Kothleitner

    2000-03-01

    Energy-filtered transmission electron microscopy (EFTEM) can be used to acquire elemental distribution images at high lateral resolution within short acquisition times. In this article, we present an overview of typical problems from materials science which can be preferentially solved by means of EFTEM. In the first example, we show how secondary phases in a steel specimen can be easily detected by recording jump ratio images of the matrix element under rocking beam illumination. Secondly, we describe how elemental maps can be converted into concentration maps. A Ba-Nd-titanate ceramics serves as a typical materials science example exhibiting three different compounds with varying composition. In order to reduce diffraction and/or thickness variation effects which may be a problem for quantification of crystalline specimens, we calculated atomic ratio maps by dividing two elemental maps and subsequent normalizing by the partial ionization cross-sections (or k-factors). Additionally, the atomic ratio maps are correlated using the scatter diagram technique thus leading to quantitative chemical phase maps. Finally, we show how the near-edge structures (electron energy-loss near edge fine structures, or ELNES) can be used for mapping chemical bonding states thus differentiating between various modifications of an element. In order to distinguish between diamond and non-diamond carbon in diamond coated materials, we have investigated a diamond layer on a substrate with the help of ELNES mapping utilizing the pi*-peak of the C-K ionization edge. PMID:10742404

  7. 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)

  8. 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.

  9. Quantitative high resolution electron microscopy of III-V compounds: A fuzzy logic approach

    NASA Astrophysics Data System (ADS)

    Hillebrand, R.; Hofmeister, H.; Werner, P.; Gösele, U.

    1995-09-01

    In the study of interdiffusion phenomena in layered structures of III-V compounds by high resolution electron microscopy, contrast features in the micrographs can be correlated with the variation of the chemical composition of the crystals. For quantitative interpretation of the micrographs a fuzzy logic approach is adapted to extract chemical information. The linguistic variable ``similarity of images'' is derived from the standard deviation (SD) of their difference patterns, which proved to be an appropriate measure. The approach developed is used to analyze simulated contrast tableaus of GaAs/P (As/P variation) and experimental micrographs of Al/GaAs (Al/Ga variation).

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

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  12. 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.

  13. Quantitative analysis of sharp-force trauma: an application of scanning electron microscopy in forensic anthropology.

    PubMed

    Bartelink, E J; Wiersema, J M; Demaree, R S

    2001-11-01

    Scanning electron microscopy (SEM) has occasionally been used by anthropologists and forensic scientists to look at morphological characteristics that certain implements leave on bone. However, few studies have addressed techniques or protocols for assessing quantitative differences between tool marks on bone made by different bladed implements. In this study, the statistical variation in cut mark width was examined between control and test samples on bone using a scalpel blade, paring knife, and kitchen utility knife. Statistically significant differences (p < .0005) were found between cut marks made by the same knife under control and test conditions for all three knife types used in the study. When the control sample and test samples were examined individually for differences in mean variation between knife types, significant differences were also found (p < .0005). While significant differences in cut mark width were found, caution should be used in trying to classify individual cut marks as being inflicted by a particular implement, due to the overlap in cut mark width that exists between different knife types. When combined, both quantitative and qualitative analyses of cut marks should prove to be more useful in trying to identify a suspect weapon. Furthermore, the application of SEM can be particularly useful for assessing many of these features. PMID:11714137

  14. Barriers to Quantitative Electron Probe X-Ray Microanalysis for Low Voltage Scanning Electron Microscopy

    PubMed Central

    Newbury, Dale E.

    2002-01-01

    Low voltage x-ray microanalysis, defined as being performed with an incident beam energy ≤5 keV, can achieve spatial resolution, laterally and in depth, of 100 nm or less, depending on the exact selection of beam energy and the composition of the target. The shallow depth of beam penetration, with the consequent short path length for x-ray absorption, and the low overvoltage, the ratio of beam energy to the critical ionization energy, both contribute to minimizing the matrix effects in quantitative x-ray microanalysis when the unknown is compared to pure element standards. The low beam energy restricts the energy of the atomic shells that can be excited, forcing the analyst to choose unfamiliar shells/characteristic peaks. The low photon energy shells are subject to low fluorescence yield, so that the peak-to-continuum background is reduced, severely limiting detectability. The limited resolution of semiconductor energy dispersive spectrometry results in frequent peak interference situations and further exacerbates detection limits. Future improvements to the x-ray spectrometry limitations are possible with x-ray optics-augmented wavelength dispersive spectrometry and microcalorimeter energy dispersive spectrometry.

  15. Scanning electron microscopy of growing dental plaque: a quantitative study with different mouth rinses.

    PubMed

    Jentsch, Holger; Mozaffari, Eshan; Jonas, Ludwig

    2013-08-01

    The aim of this study was to quantify the influence of different mouth rinses on dental plaque. Wearing splints with enamel pieces 24 volunteers rinsed with essential oils, amine/stannous fluoride, or chlorhexidine digluconate (0.12%) mouth rinses. After 24, 48, 72, and 96 h the enamel pieces were analyzed by scanning electron microscopy. The counts of cocci and bacilli in different plaque layers and the plaque thickness were almost similar using essential oils and amine/stannous fluoride. These results differed significantly from those of chlorhexidine digluconate mouth rinses. The results for plaque thickness were without significant differences between the groups at any appointment. PMID:23758106

  16. 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.

  17. Phase-shifting by means of an electronically tunable lens: quantitative phase imaging of biological specimens with digital holographic microscopy.

    PubMed

    Trujillo, Carlos; Doblas, Ana; Saavedra, Genaro; Martínez-Corral, Manuel; García-Sucerquia, Jorge

    2016-04-01

    The use of an electronically tunable lens (ETL) to produce controlled phase shifts in interferometric arrangements is shown. The performance of the ETL as a phase-shifting device is experimentally validated in phase-shifting digital holographic microscopy. Quantitative phase maps of a section of the thorax of a Drosophila melanogaster fly and of human red blood cells have been obtained using our proposal. The experimental results validate the possibility of using the ETL as a reliable phase-shifter device. PMID:27192250

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

    NASA Astrophysics Data System (ADS)

    Petrova, Rumyana V.

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

  19. Accessibility of gonococcal and meningococcal surface antigens: immunogold labeling for quantitative electron microscopy.

    PubMed Central

    Pâques, M; Teppema, J S; Beuvery, E C; Abdillahi, H; Poolman, J T; Verkleij, A J

    1989-01-01

    The parallel application of two electron microscopic immunogold labeling procedures was used to assess the surface exposure and accessibility of gonococcal and meningococcal surface antigens. Monoclonal antibodies were used as markers for the surface antigens, i.e., outer membrane proteins and lipooligosaccharides. To evaluate the labeling densities obtained after incubation of whole bacteria in suspension or ultrathin cryosections of bacteria, a method of electron microscopic quantitation was developed. Incubation of whole bacterial suspensions with monoclonal antibodies and protein A-gold resulted in specific labeling of the bacterial surfaces. However, the labeling densities varied largely in each cell. By contrast, cryosections showed uniform heavy labeling densities at the surface of the outer membranes of all cells. Apparently, by sectioning the cells the antigen-masking barrier could be evaded, and steric hindrance was no longer restrictive. Thus, a better estimate of both the presence and the surface exposure, i.e., the accessibility of antigens, could be made. Such information is essential for us to better understand host-bacterial interactions and to develop new vaccines. Images PMID:2492264

  20. 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.; Béché, 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.

  1. 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.

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

    PubMed Central

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

    2014-01-01

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

  3. Mitochondrial morphology, topology, and membrane interactions in skeletal muscle: a quantitative three-dimensional electron microscopy study.

    PubMed

    Picard, Martin; White, Kathryn; Turnbull, Douglass M

    2013-01-15

    Dynamic remodeling of mitochondrial morphology through membrane dynamics are linked to changes in mitochondrial and cellular function. Although mitochondrial membrane fusion/fission events are frequent in cell culture models, whether mitochondrial membranes dynamically interact in postmitotic muscle fibers in vivo remains unclear. Furthermore, a quantitative assessment of mitochondrial morphology in intact muscle is lacking. Here, using electron microscopy (EM), we provide evidence of interacting membranes from adjacent mitochondria in intact mouse skeletal muscle. Electron-dense mitochondrial contact sites consistent with events of outer mitochondrial membrane tethering are also described. These data suggest that mitochondrial membranes interact in vivo among mitochondria, possibly to induce morphology transitions, for kiss-and-run behavior, or other processes involving contact between mitochondrial membranes. Furthermore, a combination of freeze-fracture scanning EM and transmission EM in orthogonal planes was used to characterize and quantify mitochondrial morphology. Two subpopulations of mitochondria were studied: subsarcolemmal (SS) and intermyofibrillar (IMF), which exhibited significant differences in morphological descriptors, including form factor (means ± SD for SS: 1.41 ± 0.45 vs. IMF: 2.89 ± 1.76, P < 0.01) and aspect ratio (1.97 ± 0.83 vs. 3.63 ± 2.13, P < 0.01) and circularity (0.75 ± 0.16 vs. 0.45 ± 0.22, P < 0.01) but not size (0.28 ± 0.31 vs. 0.27 ± 0.20 μm(2)). Frequency distributions for mitochondrial size and morphological parameters were highly skewed, suggesting the presence of mechanisms to influence mitochondrial size and shape. In addition, physical continuities between SS and IMF mitochondria indicated mixing of both subpopulations. These data provide evidence that mitochondrial membranes interact in vivo in mouse skeletal muscle and that factors may be involved in regulating skeletal muscle mitochondrial morphology. PMID

  4. QEMSCAN° (Quantitative Evaluation of Minerals by Scanning Electron Microscopy): capability and application to fracture characterization in geothermal systems

    NASA Astrophysics Data System (ADS)

    Ayling, B.; Rose, P. E.; Zemach, E.; Drakos, P. S.; Petty, S.

    2011-12-01

    Fractures are important conduits for fluids in geothermal systems, and the creation and maintenance of fracture permeability is a fundamental aspect of EGS (Engineered Geothermal System) development. Hydraulic or chemical stimulation techniques are often employed to achieve this. In the case of chemical stimulation, an understanding of the minerals present in the fractures themselves is desirable to better design a stimulation effort (i.e. which chemical to use and how much). Borehole televiewer surveys provide important information about regional and local stress regimes and fracture characteristics (e.g. fracture aperture), and XRD is useful for examining bulk rock mineralogy, but neither technique is able to quantify the distribution of these minerals in fractures. QEMSCAN° is a fully-automated micro-analysis system that enables quantitative chemical analysis of materials and generation of high-resolution mineral maps and images as well as porosity structure. It uses a scanning electron microscopy platform (SEM) with an electron beam source in combination with four energy-dispersive X-ray spectrometers (EDS). The measured backscattered electron and electron-induced secondary X-ray emission spectra are used to classify sample mineralogy. Initial applications of QEMSCAN° technology were predominantly in the minerals industry and application to geothermal problems has remained limited to date. In this pilot study, the potential application of QEMSCAN° technology to fracture characterization was evaluated using samples of representative mineralized fractures in two geothermal systems (Newberry Volcano, Oregon and Brady's geothermal field, Nevada). QEMSCAN° results were compared with XRD and petrographic techniques. Nine samples were analyzed from each field, collected from the drill core in the 1000-1500 m depth range in two shallow wells (GEO-N2 at Newberry Volcano and BCH-3 at Brady's). The samples were prepared as polished thin sections for QEMSCAN° analysis

  5. 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.

  6. 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.

  7. 3D imaging and quantitative analysis of small solubilized membrane proteins and their complexes by transmission electron microscopy

    PubMed Central

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

    2013-01-01

    Inherently unstable, detergent-solubilized membrane protein complexes can often not be crystallized. For complexes that have a mass of >300 kDa, cryo-electron microscopy (EM) allows their three-dimensional (3D) structure to be assessed to a resolution that makes secondary structure elements visible in the best case. However, many interesting complexes exist whose mass is below 300 kDa and thus need alternative approaches. Two methods are reviewed: (i) Mass measurement in a scanning transmission electron microscope, which has provided important information on the stoichiometry of membrane protein complexes. This technique is applicable to particulate, filamentous and sheet-like structures. (ii) 3D-EM of negatively stained samples, which determines the molecular envelope of small membrane protein complexes. Staining and dehydration artifacts may corrupt the quality of the 3D map. Staining conditions thus need to be optimized. 3D maps of plant aquaporin SoPIP2;1 tetramers solubilized in different detergents illustrate that the flattening artifact can be partially prevented and that the detergent itself contributes significantly. Another example discussed is the complex of G protein-coupled receptor rhodopsin with its cognate G protein transducin. PMID:23267047

  8. Characterization of individual submicrometer aerosol particles collected in Incheon, Korea, by quantitative transmission electron microscopy energy-dispersive X-ray spectrometry

    NASA Astrophysics Data System (ADS)

    Geng, Hong; Kang, Sujin; Jung, Hae-Jin; ChoëL, Marie; Kim, Hyekyeong; Ro, Chul-Un

    2010-08-01

    For the last decade the Monte Carlo calculation method has been proven to be an excellent tool for accurately simulating electron-solid interactions in atmospheric individual particles of micrometer size. Although it was designed for application to scanning electron microscopy, in the present study it is demonstrated that the Monte Carlo calculation can also be applied in a quantitative single particle analysis using transmission electron microscopy (TEM) with an ultrathin window energy-dispersive X-ray (EDX) spectrometer with a high accelerating voltage (200 kV). By utilizing an iterative reverse Monte Carlo simulation combined with successive approximation, atomic elemental concentrations (including low-Z elements) of submicrometer standard particles were determined with high accuracy for electron beam refractory particles such as NaCl, KCl, SiO2, Fe2O3, Na2SO4, K2SO4, CaCO3, and CaSO4. On the basis of quantitative X-ray analysis together with morphological information from TEM images, overall 1638 submicrometer individual particles from 10 sets of aerosol samples collected in Incheon, Korea, were identified. The most frequently encountered particle types are carbonaceous and (NH4)2SO4/NH4HSO4-containing particles, followed by mineral (e.g., aluminosilicate, SiO2, CaCO3), sea salt, K-rich (e.g., K2SO4 and KCl), Fe-rich, fly ash, and transition or heavy-metal-containing (e.g., ZnSO4, ZnCl2, PbSO4) particles. The relative abundances of the submicrometer particle types vary among samples collected in different seasons and also depend on different air mass transport routes. This study demonstrates that the quantitative TEM-EDX individual particle analysis is a useful and reliable technique in characterizing urban submicrometer aerosol particles.

  9. 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

  10. 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.

  11. Rigorous quantitative elemental microanalysis by scanning electron microscopy/energy dispersive x-ray spectrometry (SEM/EDS) with spectrum processing by NIST DTSA-II

    NASA Astrophysics Data System (ADS)

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

    2014-09-01

    Quantitative electron-excited x-ray microanalysis by scanning electron microscopy/silicon drift detector energy dispersive x-ray spectrometry (SEM/SDD-EDS) is capable of achieving high accuracy and high precision equivalent to that of the high spectral resolution wavelength dispersive x-ray spectrometer even when severe peak interference occurs. The throughput of the SDD-EDS enables high count spectra to be measured that are stable in calibration and resolution (peak shape) across the full deadtime range. With this high spectral stability, multiple linear least squares peak fitting is successful for separating overlapping peaks and spectral background. Careful specimen preparation is necessary to remove topography on unknowns and standards. The standards-based matrix correction procedure embedded in the NIST DTSA-II software engine returns quantitative results supported by a complete error budget, including estimates of the uncertainties from measurement statistics and from the physical basis of the matrix corrections. NIST DTSA-II is available free for Java-platforms at: http://www.cstl.nist.gov/div837/837.02/epq/dtsa2/index.html).

  12. 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).

  13. 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.

  14. 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

  15. Hormonal regulation of capillary fenestrae in the rat adrenal cortex: quantitative studies using objective lens staging scanning electron microscopy.

    PubMed

    Apkarian, R P; Curtis, J C

    1986-01-01

    High magnification studies of the fenestrated capillary endothelium in the zona fasciculata (ZF) of rat adrenal glands were performed using the objective lens stage of an analytical scanning electron microscope (SEM) equipped with a lanthanum hexaboride emitter (LaB6). Resolution of surface substructure of the luminal membrane obtained with specimens decorated with gold/palladium (Au/Pd) was compared with that observed in others sputter coated with tantalum (Ta). High magnification (50,000x) of the fenestrated endothelium demonstrates that tantalum coating of the cryofractured adrenals improves the substructural detail compared to that seen in Au/Pd decorated specimens. The procedures used in specimen preparation, metal deposition and secondary electron imaging (SEI) are described. Quality imaging achieved using the objective lens stage is a result of the elimination of the SE-III component derived from backscattered electrons. Rat adrenals exhibited uniformly patent capillaries. High magnification micrographs of capillary walls were randomly recorded in two morphometric studies of the fenestral content of capillaries in the rat adrenal cortex. Adrenocorticotropic hormone (ACTH), when administered to rats following dexamethasone (DEX) treatment, significantly reduced the fenestrae/micron 2 of endothelial surface and increased the mean size of fenestrae. After hypophysectomy, the number of fenestrae/micron 2 declined over 48 h; within 2 h after ACTH was given to rats hypophysectomized 48 hours earlier, the fenestrae/micron 2 had increased two-fold. These studies indicate that ACTH plays an important role in modulating fenestral content of the capillary endothelium in the adrenal cortex. PMID:3027881

  16. 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…

  17. Scanning tunneling microscopy on rough surfaces-quantitative image analysis

    NASA Astrophysics Data System (ADS)

    Reiss, G.; Brückl, H.; Vancea, J.; Lecheler, R.; Hastreiter, E.

    1991-07-01

    In this communication, the application of scanning tunneling microscopy (STM) for a quantitative evaluation of roughnesses and mean island sizes of polycrystalline thin films is discussed. Provided strong conditions concerning the resolution are satisfied, the results are in good agreement with standard techniques as, for example, transmission electron microscopy. Owing to its high resolution, STM can supply a better characterization of surfaces than established methods, especially concerning the roughness. Microscopic interpretations of surface dependent physical properties thus can be considerably improved by a quantitative analysis of STM images.

  18. 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

  19. 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.

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

    SciTech Connect

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

    2015-04-15

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

  1. Electron microscopy of electromagnetic waveforms.

    PubMed

    Ryabov, A; Baum, P

    2016-07-22

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

  2. Electron microscopy of electromagnetic waveforms

    NASA Astrophysics Data System (ADS)

    Ryabov, A.; Baum, P.

    2016-07-01

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

  3. 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.

  4. 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.

  5. Electron Diffraction Using Transmission Electron Microscopy

    PubMed Central

    Bendersky, Leonid A.; Gayle, Frank W.

    2001-01-01

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

  6. 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

  7. Localisation and semi-quantitative measurement of lipocortin 1 in rat anterior pituitary cells by fluorescence-activated cell analysis/sorting and electron microscopy.

    PubMed

    Christian, H C; Flower, R J; Morris, J F; Buckingham, J C

    1999-09-01

    Lipocortin 1 (LC1, also called annexin 1), a Ca2(+)- and phospholipid-binding protein, is an important mediator of glucocorticoid action in the anterior pituitary gland. Previous studies based on immunoprecipitation and Western blot analysis suggest that LC1 is found intracellularly both in the cytoplasm and in association with membranes and also on the cell surface where it attaches to the membrane by a Ca2(+)-dependent mechanism. However, as yet it is unclear which anterior pituitary cell types express the protein. Accordingly, we have developed a method based on a combination of fluorescence activated cell (FAC) analysis/sorting and electron microscopy to detect and quantify intracellular LC1 in rat anterior pituitary cells and to identify the cell types in which it is expressed. In addition, we have measured cell surface LC1 and examined the influence of glucocorticoids on the cellular disposition of the protein. Anterior pituitary cells were dispersed with collagenase. For experiments measuring intracellular LC1, three cell fixation/permeabilisation methods were examined initially, i.e. (1) Zamboni's fluid (30 min) and Triton-X-100 (0.12%, 1 or 12 h); (2) paraformaldehyde (2%, 1 h) and Triton-X-100 (0.2%, 10 min); and (3) paraformaldehyde (0.2%, 15 min) and saponin (0.1%, 5 min). The protocol using paraformaldehyde/Triton-X-100 provided optimal preservation of cell ultrastructure and of LC1 immunoreactivity (ir-LC1) while also effectively permeabilising the cells; it was therefore used in subsequent studies. Using an anti-LC1 monoclonal antibody as a probe, 82+/-5% of the secretory cells in the heterogeneous anterior pituitary cell preparation were shown by FAC analysis to display specific fluorescence for intracellular ir-LC1. Morphological analysis and immunogold-histochemistry of cells separated by FAC sorting identified corticotrophs, lactotrophs, somatotrophs and gonadotrophs in the population displaying LC1 immunofluorescence. LC1 was also detected on

  8. 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

  9. 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

  10. 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.

  11. Direct Detectors for Electron Microscopy

    NASA Astrophysics Data System (ADS)

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

    2014-06-01

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

  12. 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.

  13. Electron microscopy of pharmaceutical systems.

    PubMed

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

    2013-01-01

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

  14. Liquid Cell Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-01

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

  15. Liquid Cell Transmission Electron Microscopy.

    PubMed

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-27

    Liquid cell transmission electron microscopy (TEM) has attracted significant interest in recent years. With nanofabricated liquid cells, it has been possible to image through liquids using TEM with subnanometer resolution, and many previously unseen materials dynamics have been revealed. Liquid cell TEM has been applied to many areas of research, ranging from chemistry to physics, materials science, and biology. So far, topics of study include nanoparticle growth and assembly, electrochemical deposition and lithiation for batteries, tracking and manipulation of nanoparticles, catalysis, and imaging of biological materials. In this article, we first review the development of liquid cell TEM and then highlight progress in various areas of research. In the study of nanoparticle growth, the electron beam can serve both as the illumination source for imaging and as the input energy for reactions. However, many other research topics require the control of electron beam effects to minimize electron beam damage. We discuss efforts to understand electron beam-liquid matter interactions. Finally, we provide a perspective on future challenges and opportunities in liquid cell TEM. PMID:27215823

  16. 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

  17. 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 ångstrom-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 Å at 120 keV and have as low as one electron per pulse. With such few particles, doses of few electrons per square ångstrom, 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

  18. Automated quantitative cytological analysis using portable microfluidic microscopy.

    PubMed

    Jagannadh, Veerendra Kalyan; Murthy, Rashmi Sreeramachandra; Srinivasan, Rajesh; Gorthi, Sai Siva

    2016-06-01

    In this article, a portable microfluidic microscopy based approach for automated cytological investigations is presented. Inexpensive optical and electronic components have been used to construct a simple microfluidic microscopy system. In contrast to the conventional slide-based methods, the presented method employs microfluidics to enable automated sample handling and image acquisition. The approach involves the use of simple in-suspension staining and automated image acquisition to enable quantitative cytological analysis of samples. The applicability of the presented approach to research in cellular biology is shown by performing an automated cell viability assessment on a given population of yeast cells. Further, the relevance of the presented approach to clinical diagnosis and prognosis has been demonstrated by performing detection and differential assessment of malaria infection in a given sample. PMID:25990413

  19. PLS photoemission electron microscopy beamline

    NASA Astrophysics Data System (ADS)

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

    2001-07-01

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

  20. 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.

  1. 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.

  2. Visualizing quantitative microscopy data: History and challenges

    PubMed Central

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

    2016-01-01

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

  3. Visualizing quantitative microscopy data: History and challenges.

    PubMed

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

    2016-01-01

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

  4. Analytical transmission electron microscopy in materials science

    SciTech Connect

    Fraser, H.L.

    1980-01-01

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

  5. 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.

  6. 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.

  7. 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

  8. Cell cycle imaging with quantitative differential interference contrast microscopy

    NASA Astrophysics Data System (ADS)

    Kostyk, Piotr; Phelan, Shelley; Xu, Min

    2013-02-01

    We report a microscopic approach for determining cell cycle stages by measuring the nuclear optical path length (OPL) with quantitative differential interference contrast (DIC) microscopy. The approach is validated by the excellent agreement between the proportion of proliferating-to-quiescent cancerous breast epithelial cells obtained from DIC microscopy, and that from a standard immunofluorescence assay.

  9. Quantitative comparison of the void distribution in a. beta. '-phase Ni-Al-In alloy using x-ray small-angle scattering and transmission-electron microscopy. [Ni-51. 2 at. % Al-2. 6 at. % In

    SciTech Connect

    Epperson, J.E.; Loomis, B.A.; Lin, J.S.

    1981-11-01

    Small-angle scattering is a rather mature discipline which can yield valuable information on the size, amount, and distribution of inhomogeneities encountered in materials-science research. Methods have been publisheed which permit one to extend the standard analysis of data from a small-angle-scattering experiment to include determination of the distribution of particle sizes. This extended analysis has been carried out for voids in a ..beta..'-phase Ni-Al-In alloy, and, in order to assess the reliability of the procedure, the identical void distribution as been characterized by transmission-electron microscopy. A quantitative comparison is made of the results from thses two independent experiments, and the general performance of the Brill-Schmidt method for particle-size determinations is discussed. 6 figures, 1 table.

  10. Photoemission electron microscopy of graphene

    NASA Astrophysics Data System (ADS)

    Saliba, Sebastian; Wardini, Jenna; Fitzgerald, J. P. S.; Word, Robert C.; Kevek, Josh; Minot, Ethan; Koenenkamp, Rolf

    2012-10-01

    A study of chemical vapor deposited graphene on copper foil is conducted using an aberration-corrected photoemission electron microscope (PEEM). We demonstrate the efficacy such a PEEM has in identifying multi-layer graphene, defects and cracking. A model is developed to describe the observed reduction in photoemission rate where electrons originate from the copper foil and scatter through the graphene. A survey of several multi-layer feature line profiles demonstrates the reduced photoemission rate as the number of graphene layers increases. A mean-free-path length of l=3.8±0.8 nm is inferred assuming the layer spacing in graphene is δz=0.35 nm. The PEEM's high spatial resolution and surface sensitivity combined with no electron beam damage are promising for characterizing biosensors and other nanoscale graphene devices.

  11. 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.

  12. Resolution measures in molecular electron microscopy

    PubMed Central

    Penczek, Pawel A.

    2011-01-01

    Resolution measures in molecular electron microscopy provide means to evaluate quality of macromolecular structures computed from sets of their two-dimensional line projections. When the amount of detail in the computed density map is low there are no external standards by which the resolution of the result can be judged. Instead, resolution measures in molecular electron microscopy evaluate consistency of the results in reciprocal space and present it as a one-dimensional function of the modulus of spatial frequency. Here we provide description of standard resolution measures commonly used in electron microscopy. We point out that the organizing principle is the relationship between these measures and the Spectral Signal-to-Noise Ratio of the computed density map. Within this framework it becomes straightforward to describe the connection between the outcome of resolution evaluations and the quality of electron microscopy maps, in particular, the optimum filtration, in the Wiener sense, of the computed map. We also provide a discussion of practical difficulties of evaluation of resolution in electron microscopy, particularly in terms of its sensitivity to data processing operations used during structure determination process in single particle analysis and in electron tomography. PMID:20888958

  13. 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.

  14. Quantitative chemical imaging with multiplex stimulated Raman scattering microscopy.

    PubMed

    Fu, Dan; Lu, Fa-Ke; Zhang, Xu; Freudiger, Christian; Pernik, Douglas R; Holtom, Gary; Xie, Xiaoliang Sunney

    2012-02-29

    Stimulated Raman scattering (SRS) microscopy is a newly developed label-free chemical imaging technique that overcomes the speed limitation of confocal Raman microscopy while avoiding the nonresonant background problem of coherent anti-Stokes Raman scattering (CARS) microscopy. Previous demonstrations have been limited to single Raman band measurements. We present a novel modulation multiplexing approach that allows real-time detection of multiple species using the fast Fourier transform. We demonstrate the quantitative determination of chemical concentrations in a ternary mixture. Furthermore, two imaging applications are pursued: (1) quantitative determination of oil content as well as pigment and protein concentration in microalgae cultures; and (2) 3D high-resolution imaging of blood, lipids, and protein distribution in ex vivo mouse skin tissue. We believe that quantitative multiplex SRS uniquely combines the advantage of fast label-free imaging with the fingerprinting capability of Raman spectroscopy and enables numerous applications in lipid biology as well as biomedical imaging. PMID:22316340

  15. New Fluorescence Microscopy Methods for Microbiology: Sharper, Faster, and Quantitative

    PubMed Central

    Gitai, Zemer

    2009-01-01

    Summary In addition to the inherent interest stemming from their ecological and human health impacts, microbes have many advantages as model organisms, including ease of growth and manipulation and relatively simple genomes. However, the imaging of bacteria via light microscopy has been limited by their small sizes. Recent advances in fluorescence microscopy that allow imaging of structures at extremely high resolutions are thus of particular interest to the modern microbiologist. In addition, advances in high-throughput microscopy and quantitative image analysis are enabling cellular imaging to finally take advantage of the full power of bacterial numbers and ease of manipulation. These technical developments are ushering in a new era of using fluorescence microscopy to understand bacterial systems in a detailed, comprehensive, and quantitative manner. PMID:19356974

  16. Electron Microscopy Characterization of Hybrid Metallic Nanomaterials

    NASA Astrophysics Data System (ADS)

    Shindo, Daisuke; Akase, Zentaro

    In order to understand the excellent properties of nanoscale hybridized materials, it is very important to investigate the microstructures and interfaces of these materials at the nanometer scale. In this chapter, we present the basic principles of transmission electron microscopy and its applications to these materials. In addition to high-resolution transmission electron microscopy (HREM) and high-angle annular dark-field (HAADF) scanning transmission electron microscopy (STEM), analytical electron microscopy, including energy dispersive X-ray spectroscopy (EDS) and electron energyloss spectroscopy (EELS) as well as elemental mapping methods using these spectroscopy techniques will be presented. Also, the electron holographic technique for characterization of magnetic fields of nanohybridized materials will be explained. In addition to electron microscopic observation techniques, recently developed specimen preparation techniques, which are indispensable for obtaining homogeneous and thin films of nanohybridized materials, will be presented. In particular, a focused ion beam (FIB) method will be emphasized. The nanohybridized materials discussed in this chapter include carbon-based core-shell structure, nanocrystalline soft magnetic materials, nanocomposite magnets, and high-T c superconducting oxides. Application data will be provided in order to explain the usefulness of these analytical techniques for characterization of nanohybridized materials.

  17. 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

  18. 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.

  19. Optical microscopy versus scanning electron microscopy in urolithiasis.

    PubMed

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

    2009-10-01

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

  20. 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.

  1. 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...

  2. 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.

  3. [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

  4. Quantitative biological imaging by ptychographic x-ray diffraction microscopy

    PubMed Central

    Giewekemeyer, Klaus; Thibault, Pierre; Kalbfleisch, Sebastian; Beerlink, André; Kewish, Cameron M.; Dierolf, Martin; Pfeiffer, Franz; Salditt, Tim

    2010-01-01

    Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·105 Gy, and were shown to scale with photon fluence. PMID:20018650

  5. Quantitative coherent anti-Stokes Raman scattering (CARS) microscopy.

    PubMed

    Day, James P R; Domke, Katrin F; Rago, Gianluca; Kano, Hideaki; Hamaguchi, Hiro-o; Vartiainen, Erik M; Bonn, Mischa

    2011-06-23

    The ability to observe samples qualitatively at the microscopic scale has greatly enhanced our understanding of the physical and biological world throughout the 400 year history of microscopic imaging, but there are relatively few techniques that can truly claim the ability to quantify the local concentration and composition of a sample. We review coherent anti-Stokes Raman scattering (CARS) as a quantitative, chemically specific, and label-free microscopy. We discuss the complicating influence of the nonresonant response on the CARS signal and the various experimental and mathematical approaches that can be adopted to extract quantitative information from CARS. We also review the uses to which CARS has been employed as a quantitative microscopy to solve challenges in material and biological science. PMID:21526785

  6. Wet electron microscopy with quantum dots.

    PubMed

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

    2006-09-01

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

  7. 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

  8. The rapidly changing face of electron microscopy

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  9. Photon-induced near field electron microscopy

    NASA Astrophysics Data System (ADS)

    Park, Sang Tae; Zewail, Ahmed H.

    2013-09-01

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

  10. Chemistry of coal from electron microscopy measurements

    SciTech Connect

    Wert, C.A.; Hsieh, K.C.; Fraser, H.

    1986-04-01

    Well established techniques of analytical electron microscopy have applications to the chemistry of coal. The techniques use one or another of several interactions which occur when electrons are incident on a specimen. Two such interactions are discussed in this paper: 1: X-ray emission spectroscopy and 2: Electron energy loss spectroscopy. Both methods are used in the study of metallic and ceramic systems. The principles of the technique are illustrated by applications to metallic and ceramic systems; initial applications to coal are then described.

  11. 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

  12. Quantitative low-cost webcam-based microscopy

    NASA Astrophysics Data System (ADS)

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

    2010-11-01

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

  13. 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.

  14. 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

  15. 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 2π 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.

  16. 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

  17. 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

  18. Quantitative analysis of myocardial tissue with digital autofluorescence microscopy

    PubMed Central

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

    2016-01-01

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

  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. Immunogold Labeling for Scanning Electron Microscopy.

    PubMed

    Goldberg, Martin W; Fišerová, Jindřiška

    2016-01-01

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

  1. Electron Microscopy of Young Candida albicans Chlamydospores

    PubMed Central

    Miller, Sara E.; Spurlock, Ben O.; Michaels, G. E.

    1974-01-01

    One- to three-day-old cultures of Candida albicans bearing chlamydospores were grown and harvested by a special technique, free of agar, and prepared for ultramicrotomy and electron microscopy. These young chlamydospores exhibited a subcellular structure similar to that of the yeast phase, e.g., cytoplasmic membrane, ribosomes, and mitochondria. Other structural characteristics unique to chlamydospores were a very thick, layered cell wall, the outer layer of which was continuous with the outer layer of the suspensor cell wall and was covered by hair-like projections; membrane bound organelles; and large lipoid inclusions. Only young chlamydospores less than 3 to 4 days old exhibited these ultrastructural characteristics. Images PMID:4368664

  2. Electron microscopy methods for studying plasma membranes.

    PubMed

    Beckett, Alison J; Prior, Ian A

    2015-01-01

    Electron microscopy allows direct visualization of the underlying organization of cell surface components on a nano-scale. Immuno-gold labelling of isolated plasma membranes generates point patterns that enable mapping of protein and lipid distributions. 2D spatial statistics reveals the extent to which these distributions are clustered or dispersed and allows the extent of co-localization between different cell surface components to be precisely determined. This approach has been successfully applied to the study of signalling network organization and the consequences of physiological changes in modulating cell surface function. PMID:25331134

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

    NASA Astrophysics Data System (ADS)

    Lue, Niyom

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

  4. 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.

  5. Feature Adaptive Sampling for Scanning Electron Microscopy.

    PubMed

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

    2016-01-01

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

  6. Feature Adaptive Sampling for Scanning Electron Microscopy

    PubMed Central

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

    2016-01-01

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

  7. Feature Adaptive Sampling for Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  8. 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.

  9. 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. 91:2037–2044. 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

  10. Electron microscopy of compound oxide laser materials

    NASA Astrophysics Data System (ADS)

    Eakins, Daniel E.; LeBret, Joel B.; Norton, M. G.; Bahr, David F.; Dumm, John Q.

    2003-06-01

    Oxide single crystals, such as yttrium aluminum garnet (YAG) and yttrium orthovanadate (YVO4), are important host crystals for solid-state laser applications. These crystals are often grown by the Czochralski process and are doped with neodymium during growth. The microstructure of the resultant crystal affects the overall laser performance and it is necessary to be able to characterize grown-in defects in the material. Scanning electron microscopy has been used to examine the fracture surfaces of YAG and has shown the presence of microscopic voids, which act as stress concentrators and in some cases appear to be the cause of fracture. Transmission electron microscopy (TEM) has been used to characterize various defects in both YAG and YVO4 crystals. The defects found depend on the growth conditions, specifically the Nd concentration in the crystal and the position within the boule. One of the most common defects identified in both materials were microscopic spherical particles. In YAG these particles appeared to be located primarily in the core regions and analysis of high resolution images indicate that they are due to regions that are both compositionally and orientationally different from the matrix phase. Direct observation of dislocations in YVO4 was made using TEM. In YAG only indirect evidence for dislocations could be found from the observation of river marks on fracture surfaces.

  11. Electron Microscopy: an Analytical Tool for Solid State Physicists

    NASA Astrophysics Data System (ADS)

    van Tendeloo, Gustaaf

    2013-03-01

    For too long the electron microscope has been considered as ``a big magnifying glass.'' Modern electron microscopy however has evolved into an analytical technique, able to provide quantitative data on structure, composition, chemical bonding and magnetic properties. Using lens corrected instruments it is now possible to determine atom shifts at interfaces with a precision of a few picometer; chemical diffusion at these interfaces can be imaged down to atomic scale. The chemical nature of the surface atoms can be visualized and even the bonding state of the elements (e.g. Mn2+ versus Mn3+) can be detected on an atomic scale. Electron microscopy is by principle a projection technique, but the final dream is to obtain atomic info of materials in three dimensions. We will show that this is no longer a dream, but that it is possible using advanced microscopy. We will show evidence of determining the valence change Ce4+ versus Ce3+ at the surface of a CeO2 nanocrystal; the atomic shifts at the interface between LaAlO3 and SrTiO3 and the 3D relaxation of a Au nanocrystal.

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

    PubMed

    Goldstein, Goldie; Creath, Katherine

    2014-03-12

    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. Electron Microscopy of Chromatophores of Rhodopseudomonas spheroides

    PubMed Central

    Gibson, K. D.

    1965-01-01

    Gibson, K. D. (St. Mary's Hospital Medical School, London, England). Electron microscopy of Rhodopseudomonas spheroides. J. Bacteriol. 90:1059–1072. 1965.—Fixed and stained chromatophores and whole cells of anaerobically grown Rhodopseudomonas spheroides were examined in thin sections in the electron microscope. Both purified chromatophores and intracellular membrane-bound vesicles had exactly the same appearance, namely that of spheres or ellipsoids with a thin electron-dense shell surrounding an electron-lucent interior. The distribution of diameters in the two types of structure was also found to be the same, and was compatible with a normal distribution, with a mean of 570 A and a standard deviation 40 A. Negatively stained chromatophores appeared like discs or collapsed spheres. The presence of invaginations of the cytoplasmic membrane in this species was confirmed, and a new structure resembling a twin chromatophore was observed. The bearing of these results on theories of the origin of chromatophores is discussed, and it is concluded that they offer some support for each one of the three main theories about the origin of particulate organelles. Images PMID:5847796

  15. 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.

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

    PubMed

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

    2014-02-01

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

  17. IMAGING RED BLOOD CELL DYNAMICS BY QUANTITATIVE PHASE MICROSCOPY

    PubMed Central

    Popescu, Gabriel; Park, YoungKeun; Choi, Wonshik; Dasari, Ramachandra R.; Feld, Michael S.; Badizadegan, Kamran

    2008-01-01

    Red blood cells (RBCs) play a crucial role in health and disease, and structural and mechanical abnormalities of these cells have been associated with important disorders such as Sickle cell disease and hereditary cytoskeletal abnormalities. Although several experimental methods exist for analysis of RBC mechanical properties, optical methods stand out as they enable collecting mechanical and dynamic data from live cells without physical contact and without the need for exogenous contrast agents. In this report, we present quantitative phase microscopy techniques that enable imaging RBC membrane fluctuations with nanometer sensitivity at arbitrary time scales from milliseconds to hours. We further provide a theoretical framework for extraction of membrane mechanical and dynamical properties using time series of quantitative phase images. Finally, we present an experimental approach to extend quantitative phase imaging to 3-dimensional space using tomographic methods. By providing non-invasive methods for imaging mechanics of live cells, these novel techniques provide an opportunity for high-throughput analysis and study of RBC mechanical properties in health and disease. PMID:18387320

  18. Computer-aided application of quantitative microscopy in diagnostic pathology.

    PubMed

    Baak, J P; Kurver, P H; Boon, M E

    1982-01-01

    classified, whereas frozen-section diagnosis often gave erroneous or inconclusive results. The quantitative microscopic techniques that we have used are simple, inexpensive, and can be applied in most pathology laboratories. The classification rules can also be used in cases submitted for consultation. The pathologist must use his or her full diagnostic knowledge when applying these techniques. In doing so, he or she will learn that quantitative microscopy has an educative function, automatically results in an increase in the quality of histopathologic work, and supports and sometimes corrects the diagnosis in an objective, consistent way. PMID:7182754

  19. 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.

  20. Improved methods for high resolution electron microscopy

    NASA Astrophysics Data System (ADS)

    Taylor, J. R.

    1987-04-01

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

  1. Hexamethyldisilazane for scanning electron microscopy of Gastrotricha.

    PubMed

    Hochberg, R; Litvaitis, M K

    2000-01-01

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

  2. Electric fields in Scanning Electron Microscopy simulations

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  3. Scanning electron microscopy of tinea nigra*

    PubMed Central

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

    2014-01-01

    Tinea nigra is a rare superficial mycosis caused by Hortaea werneckii. This infection presents as asymptomatic brown to black maculae mostly in palmo-plantar regions. We performed scanning electron microscopy of a superficial shaving of a tinea nigra lesion. The examination of the outer surface of the sample showed the epidermis with corneocytes and hyphae and elimination of fungal filaments. The inner surface of the sample showed important aggregation of hyphae among keratinocytes, which formed small fungal colonies. The ultrastructural findings correlated with those of dermoscopic examination - the small fungal aggregations may be the dark spicules seen on dermoscopy - and also allowed to document the mode of dissemination of tinea nigra, showing how hyphae are eliminated on the surface of the lesion. PMID:24770516

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. 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

  9. Image restoration in cryo-electron microscopy.

    PubMed

    Penczek, Pawel A

    2010-01-01

    Image restoration techniques are used to obtain, given experimental measurements, the best possible approximation of the original object within the limits imposed by instrumental conditions and noise level in the data. In molecular electron microscopy (EM), we are mainly interested in linear methods that preserve the respective relationships between mass densities within the restored map. Here, we describe the methodology of image restoration in structural EM, and more specifically, we will focus on the problem of the optimum recovery of Fourier amplitudes given electron microscope data collected under various defocus settings. We discuss in detail two classes of commonly used linear methods, the first of which consists of methods based on pseudoinverse restoration, and which is further subdivided into mean-square error, chi-square error, and constrained based restorations, where the methods in the latter two subclasses explicitly incorporates non-white distribution of noise in the data. The second class of methods is based on the Wiener filtration approach. We show that the Wiener filter-based methodology can be used to obtain a solution to the problem of amplitude correction (or "sharpening") of the EM map that makes it visually comparable to maps determined by X-ray crystallography, and thus amenable to comparative interpretation. Finally, we present a semiheuristic Wiener filter-based solution to the problem of image restoration given sets of heterogeneous solutions. We conclude the chapter with a discussion of image restoration protocols implemented in commonly used single particle software packages. PMID:20888957

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

    PubMed

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

    2012-10-01

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

  11. Advanced electron microscopy characterization of multimetallic nanoparticles

    NASA Astrophysics Data System (ADS)

    Khanal, Subarna Raj

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

  12. 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.

  13. 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 Köhler 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.

  14. Electron microscopy of seed-storage globulins.

    PubMed

    Tulloch, P A; Blagrove, R J

    1985-09-01

    The quaternary structures of a range of seed globulins, including examples of both the so-called 7 S and 11 S types, have been examined by electron microscopy. The legume 7 S proteins, phaseolin (bean), beta-conglycinin (soybean), and vicilin (pea), appear as flat discs of diameter ca. 8.5 nm and thickness ca. 3.5 nm formed by association of three subunit domains. Phaseolin converts to an 18 S tetramer at acid pH, and images recorded under these conditions suggest that four of the 7 S protomer discs associate to form the faces of a regular tetrahedron. The classical 11 S seed globulins, cucurbitin (pumpkin) and legumin (pea), are approximately spherical molecules of diameter ca. 8.8 nm composed of six subunits. In contrast, the hexameric 10 S storage protein from lupin seed, conglutin gamma, appears toroidal in shape with outer diameter ca. 10.3 nm and thickness ca. 2.2 nm. These results indicate that constraints imposed on seed proteins by their role in sustaining the germinating plant may have allowed a variety of different globulin structures to accumulate in the protein-storage bodies of seeds. PMID:4037802

  15. Imaging Cytoskeleton Components by Electron Microscopy

    PubMed Central

    Svitkina, Tatyana

    2016-01-01

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

  16. Quantitative pathology in virtual microscopy: history, applications, perspectives.

    PubMed

    Kayser, Gian; Kayser, Klaus

    2013-07-01

    With the emerging success of commercially available personal computers and the rapid progress in the development of information technologies, morphometric analyses of static histological images have been introduced to improve our understanding of the biology of diseases such as cancer. First applications have been quantifications of immunohistochemical expression patterns. In addition to object counting and feature extraction, laws of thermodynamics have been applied in morphometric calculations termed syntactic structure analysis. Here, one has to consider that the information of an image can be calculated for separate hierarchical layers such as single pixels, cluster of pixels, segmented small objects, clusters of small objects, objects of higher order composed of several small objects. Using syntactic structure analysis in histological images, functional states can be extracted and efficiency of labor in tissues can be quantified. Image standardization procedures, such as shading correction and color normalization, can overcome artifacts blurring clear thresholds. Morphometric techniques are not only useful to learn more about biological features of growth patterns, they can also be helpful in routine diagnostic pathology. In such cases, entropy calculations are applied in analogy to theoretical considerations concerning information content. Thus, regions with high information content can automatically be highlighted. Analysis of the "regions of high diagnostic value" can deliver in the context of clinical information, site of involvement and patient data (e.g. age, sex), support in histopathological differential diagnoses. It can be expected that quantitative virtual microscopy will open new possibilities for automated histological support. Automated integrated quantification of histological slides also serves for quality assurance. The development and theoretical background of morphometric analyses in histopathology are reviewed, as well as their application

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

    PubMed

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

    2013-01-01

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

  18. 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

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

    PubMed

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

    2013-02-01

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

  20. 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.

  1. 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.

  2. Quantitative high dynamic range beam profiling for fluorescence microscopy.

    PubMed

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

    2014-10-01

    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. PMID:25362409

  3. 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.

  4. 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

  5. Structural examination of lithium niobate ferroelectric crystals by combining scanning electron microscopy and atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Efremova, P. V.; Ped'ko, B. B.; Kuznecova, Yu. V.

    2016-02-01

    The structure of lithium niobate single crystals is studied by a complex technique that combines scanning electron microscopy and atomic force microscopy. By implementing the piezoresponse force method on an atomic force microscope, the domain structure of lithium niobate crystals, which was not revealed without electron beam irradiation, is visualized

  6. 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.

  7. Ballistic-electron-emission Microscopy of Semiconductor Heterostructures

    NASA Technical Reports Server (NTRS)

    Bell, L. Douglas; Narayanamurti, Venkatesh

    1997-01-01

    Balistic-electron-emission microscopy has developed from its beginning as a probe of Schottky barriers into a powerful nanometer-scale method for characterizing semiconductor interfaces and hot-electron transport.

  8. Transmission Electron Microscopy of Itokawa Regolith Grains

    NASA Technical Reports Server (NTRS)

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

    2013-01-01

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

  9. Quantitative metallography by electron backscattered diffraction.

    PubMed

    Humphreys

    1999-09-01

    Although electron backscattered diffraction (EBSD) in the scanning electron microscope is used mainly to investigate the relationship between local textures and microstructures, the technique has now developed to the stage where it requires serious consideration as a tool for routine quantitative characterization of microstructures. This paper examines the application of EBSD to the characterization of phase distributions, grain and subgrain structures and also textures. Comparisons are made with the standard methods of quantitative metallography and it is shown that in many cases EBSD can produce more accurate and detailed measurements than the standard methods and that the data may sometimes be obtained more rapidly. The factors which currently limit the use of EBSD for quantitative microstructural characterization, including the speed of data acquisition and the angular and spatial resolutions, are discussed, and future developments are considered. PMID:10460682

  10. 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.

  11. Imaging Bioorthogonal Groups in Their Ultrastructural Context with Electron Microscopy.

    PubMed

    van Elsland, Daphne M; van Kasteren, Sander I

    2016-08-01

    Spitting image: Herein a recent paper on the imaging of bioorthogonal groups using three-dimensional electron microscopy is discussed. The work has demonstrated electron microscopy imaging as a technique suitable for gaining structural information on bioorthogonal groups in their cellular context. PMID:27346592

  12. 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

  13. 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

  14. 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.

  15. 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

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

    PubMed

    Begemann, Isabell; Galic, Milos

    2016-01-01

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

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

    PubMed Central

    Begemann, Isabell; Galic, Milos

    2016-01-01

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

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

    PubMed

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

    2016-08-01

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

  19. Fluctuation Electron Microscopy of Amorphous and Polycrystalline Materials

    NASA Astrophysics Data System (ADS)

    Rezikyan, Aram

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

  20. Ion-induced electron emission microscopy

    DOEpatents

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

    2001-01-01

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

  1. 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.

  2. Plasma Cleaning and Its Applications for Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Isabell, Thomas C.; Fischione, Paul E.; O'Keefe, Catherine; Guruz, Murat U.; Dravid, Vinayak P.

    1999-03-01

    The effectiveness of applying a high-frequency, low-energy, reactive gas plasma for the removal of hydrocarbon contamination from specimens and components for electron microscopy has been investigated with a variety of analytical techniques. Transmission electron microscopy (TEM) analysis of specimens that have been plasma cleaned shows an elimination of the carbonaceous contamination from the specimen. With extended cleaning times the removal of existing carbon contamination debris due to previously conducted microanalysis is shown. Following plasma cleaning, specimens may be examined in the electron microscope for several hours without exhibiting evidence of recontamination. The effectiveness of plasma cleaning is not limited to applications for TEM specimens. Scanning electron microscopy (SEM) specimens that have been plasma cleaned likewise show an elimination of carbonaceous contamination. Furthermore, other electron microscopy parts and accessories, such as aperture strips, specimen clamping rings, and Wehnelts, among others, can benefit from plasma cleaning.

  3. Electron microscopy study of zeolite ZK-14; a synthetic chabazite

    NASA Astrophysics Data System (ADS)

    Cartlidge, S.; Wessicken, R.; Nissen, H.-U.

    1983-03-01

    The defect structure of zeolite (K+, TMA+) — ZK-14, a synthetic chabazite, has been studied using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). SEM together with TEM bright field (BF) and dark field (DF) micrographs indicate that the hexagonal, platelet ZK-14 crystals are built up of crystalline blocks joined by twinning along (00.1). High resolution transmission electron microscopy (HRTEM) reveals faulting of the ideal AABBCC single 6-ring stacking sequence of ZK-14. This is consistent with an observed line broadening in its X-ray powder diffraction profile. Channel apertures are imaged, even for thick specimens.

  4. Phase contrast in high resolution electron microscopy

    DOEpatents

    Rose, H.H.

    1975-09-23

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

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

    PubMed

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

    2016-04-14

    We visualize atomic level dynamics during Si nanowire growth using aberration corrected environmental transmission electron microscopy, and compare with lower pressure results from ultra-high vacuum microscopy. We discuss the importance of higher pressure observations for understanding growth mechanisms and describe protocols to minimize effects of the higher pressure background gas. PMID:27041654

  6. [Electron microscopy study of artificial vitreous gel].

    PubMed

    Ehgartner, E M; Schmut, O; Hofmann, H

    1986-04-01

    Artificial gels prepared from Cu2+-ions and hyaluronic acid were studied in the electron microscope and compared with the native vitreous body. Additionally, the authors attempted to produce transparent gels from the native constituents of the vitreous body, namely collagen and hyaluronic acid. Mixing of solutions of these constituents formed no gels but white precipitates. The ultrastructure of these precipitates was also studied in the electron microscope. PMID:3723971

  7. 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.

  8. Transmission electron microscopy characterisation of 0-D nanomaterials

    NASA Astrophysics Data System (ADS)

    Turner, Stuart Matthew

    When materials are scaled down to the nanometre level, a change in physical behaviour is frequently observed. In so-called 0-D nanomaterials (nanoparticles), these unique nanoscale properties are most abundant and are usually linked to either a change in (electronic) structure of the material or to the dominating influence of the particle surface at the nanometre scale. In this doctoral work the nanoscale properties of several nanoparticle systems have been studied using advanced transmission electron microscopy (TEM). Every material that was studied required for its solution a unique approach and a host of transmission electron microscopy techniques. The title of this doctoral work can be freely translated as "retrieving quantitatively the maximal and most accurate chemical, structural and morphological information from nanoparticles by advanced transmission electron microscopy, to uncover and explain their unique properties". Chapter 1 gives a brief general introduction to the world of nanomaterials and nanotechnology in general and more specifically to 0-D nanomaterials (nanoparticles). The unique properties and potential applications of these materials are described. The production of 0-D nanomaterials is not covered in this chapter, as this is an extremely broad field to cover in only a few pages. Instead, the production method for each of the materials is left to the detailed chapters that follow. In Chapter 2 the main transmission electron microscopy techniques used to characterise the materials in the further chapters are described together with the microscopes used to perform these techniques and their parameters of operation. Again, the sample-specific setups are listed in the detailed chapters that follow. Chapter 3 covers all work carried out on luminescent detonation nanodiamond powder for drug delivery and bio-medical imaging applications. Specific attention is paid to the morphology, surface chemistry and nitrogen incorporation of detonation

  9. 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

  10. 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.

  11. Electron microscopy - A glimpse into the future.

    NASA Technical Reports Server (NTRS)

    Fernandez-Moran, H.

    1972-01-01

    A forecast attempt is presented on future advances in electron microscopic studies of membrane systems. A review of recent advances and present trends is followed by a discussion of prerequisites to further progress. Special attention is given to research areas of particular promise.

  12. Electron Microscopy of Biological Materials at the Nanometer Scale

    NASA Astrophysics Data System (ADS)

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

    2012-08-01

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

  13. 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.

  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. Correlated light and electron microscopy: ultrastructure lights up!

    PubMed

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

    2015-06-01

    Microscopy has gone hand in hand with the study of living systems since van Leeuwenhoek observed living microorganisms and cells in 1674 using his light microscope. A spectrum of dyes and probes now enable the localization of molecules of interest within living cells by fluorescence microscopy. With electron microscopy (EM), cellular ultrastructure has been revealed. Bridging these two modalities, correlated light microscopy and EM (CLEM) opens new avenues. Studies of protein dynamics with fluorescent proteins (FPs), which leave the investigator 'in the dark' concerning cellular context, can be followed by EM examination. Rare events can be preselected at the light microscopy level before EM analysis. Ongoing development-including of dedicated probes, integrated microscopes, large-scale and three-dimensional EM and super-resolution fluorescence microscopy-now paves the way for broad CLEM implementation in biology. PMID:26020503

  16. Transmission electron microscopy of mercury metal.

    PubMed

    Anjum, Dalaver H; Sougrat, Rachid

    2016-09-01

    Transmission electron microcopy (TEM) analysis of liquid metals, especially mercury (Hg), is difficult to carry out because their specimen preparation poses a daunting task due to the unique surface properties of these metals. This paper reports a cryoTEM study on Hg using a novel specimen preparation technique. Hg metal is mixed with water using sonication and quenched in liquid ethane cryogen. This technique permits research into the morphological, phase and structural properties of Hg at nanoscale dimensions. PMID:27018645

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

    NASA Astrophysics Data System (ADS)

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

    2013-03-01

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

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  20. 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.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. Thickness-dependent thin-film resistivity: Application of quantitative scanning-tunneling-microscopy imaging

    NASA Astrophysics Data System (ADS)

    Reiss, G.; Hastreiter, E.; Brückl, H.; Vancea, J.

    1991-02-01

    The dependence of thin-film resistivity on the thickness is known to be strongly influenced by the interaction of the conduction electrons with the surface. Great efforts have been made in recent years, mainly concerning the quantum-mechanical description of the surface scattering. Detailed discussions of this problem, however, suffer from the lack of information concerning the real topography of thin-film surfaces. The development of scanning tunneling microscopy (STM) now gives the chance of direct, quantitative imaging. In this paper, we use the topographic information of STM to improve the fitting of theoretical descriptions to the measured thickness-dependence of the resistivity. The transport parameters obtained from these calculations show a high degree of physical consistency.

  6. Persistent misconceptions about incoherence in electron microscopy.

    PubMed

    Van Dyck, D

    2011-06-01

    Incoherence in electron microscopic imaging occurs when during the observation the microscope and the object are subject to fluctuations. In order to speed up the computer simulation of the images, approximations are used that are considered as valid. In this paper we will question the validity of these approximations and show that in specific cases they can lead to erroneous results. It is shown in particular in the case of one single vibrating atom that the thermal diffuse scattering that causes the signal in HAADF STEM is not only dependent on Z but also on the mean square displacement of the atom so that it can even be large for light atoms in soft matter, provided the right HAADF aperture is used. In HREM imaging the diffuse scattering leaks out of the coherent (elastic) wave and is redistributed in the background. This might explain the mismatch in elastic contrast (Stobbs factor) especially for crystals with a thickness beyond the extinction distance, where also the HAADF signal saturates and the elastic (coherent) component vanishes. PMID:21664551

  7. Electron microscopy of low iodinated thyroglobulin molecules.

    PubMed

    Berg, G; Ekholm, R

    1975-04-29

    Thyroglobulin molecules were studied in the electron microscope with negative staining technique. In a first series of experiments samples of thyroglobulin varying in iodine content from 0.5 to 0.03% were prepared from the thyroids of mice and rats kept on iodine-poor diets. All samples contained thyroglobulin molecules of the normal ovoid shape, not deviating in size or shape from molecules obtained from normal thyroids. However, in addition, another type of molecule having a cylindrical shape was observed in all samples. The proportion of these cylindrical molecules increased from a few per cent in the moderately iodine-poor thyroglobulin samples to more than 80% in the highly iodine-deficient thyroglobulin (0.03%). In a second series of experiments extremely iodine-poor thyroglobulin (smaller than 0.005%) was obtained from propylthiouracil-treated rats. In these preparations practically all molecules had a cylindrical shape. These samples also contained smaller particles interpreted to be dissociation products. The cylindrical molecules were of two types, one appearing compact and measuring 250 times 135 A (length times diameter) and the other appearing porous and having a length of 145 and a diameter of 205 A. It is concluded that the cylindrical molecules represent non- or low-iodinated thyroglobulin and it is suggested that the porous cylindrical molecule is an unfolded form of the compact cylinder. PMID:1138879

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

    PubMed

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

    2016-01-01

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

  9. Photon gating in four-dimensional ultrafast electron microscopy

    PubMed Central

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

    2015-01-01

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

  10. 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.

  11. Label-free quantitative cell division monitoring of endothelial cells by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Bauwens, Andreas; Vollmer, Angelika; Ketelhut, Steffi; Langehanenberg, Patrik; Müthing, Johannes; Karch, Helge; von Bally, Gert

    2010-05-01

    Digital holographic microscopy (DHM) enables quantitative multifocus phase contrast imaging for nondestructive technical inspection and live cell analysis. Time-lapse investigations on human brain microvascular endothelial cells demonstrate the use of DHM for label-free dynamic quantitative monitoring of cell division of mother cells into daughter cells. Cytokinetic DHM analysis provides future applications in toxicology and cancer research.

  12. 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.

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

    SciTech Connect

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

    2011-02-01

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

  14. The CryoCapsule: Simplifying correlative light to electron microscopy

    PubMed Central

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

    2014-01-01

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

  15. Imaging hydrated microbial extracellular polymers: comparative analysis by electron microscopy.

    PubMed

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

    2011-02-01

    Microbe-mineral and -metal interactions represent a major intersection between the biosphere and geosphere but require high-resolution imaging and analytical tools for investigation of 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 by conventional electron microscopy approaches with 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 the nature of interactions between microbial extracellular polymers and their environment. PMID:21169451

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Quantitative flaw characterization with scanning laser acoustic microscopy

    NASA Technical Reports Server (NTRS)

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

    1986-01-01

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

  1. 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

  2. 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.

  3. 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

  4. Structured illumination quantitative phase microscopy for enhanced resolution amplitude and phase imaging

    PubMed Central

    Chowdhury, Shwetadwip; Izatt, Joseph

    2013-01-01

    Structured illumination microscopy (SIM) is an established microscopy technique typically used to image samples at resolutions beyond the diffraction limit. Until now, however, achieving sub-diffraction resolution has predominantly been limited to intensity-based imaging modalities. Here, we introduce an analogue to conventional SIM that allows sub-diffraction resolution, quantitative phase-contrast imaging of optically transparent objects. We demonstrate sub-diffraction resolution amplitude and quantitative-phase imaging of phantom targets and enhanced resolution quantitative-phase imaging of cells. We report a phase accuracy to within 5% and phase noise of 0.06 rad. PMID:24156044

  5. 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

  6. Surface morphology of Trichinella spiralis by scanning electron microscopy

    SciTech Connect

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

    1980-02-01

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

  7. 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

  8. Transmission Electron Microscopy Characterization of Helium Bubbles in Aged Plutonium

    SciTech Connect

    Schwartz, A J; Wall, M A; Zocco, T G; Blobaum, K M

    2004-11-02

    The self-irradiation damage generated by alpha decay of plutonium results in the formation of lattice defects, helium, and uranium atoms. Over time, microstructural evolution resulting from the self-irradiation may influence the physical and mechanical properties of the material. In order to assess microstructural changes, we have developed and applied procedures for the specimen preparation, handling, and transmission electron microscopy characterization of Pu alloys. These transmission electron microscopy investigations of Pu-Ga alloys ranging in age up to 42-years old reveal the presence of nanometer-sized helium bubbles. The number density of bubbles and the average size have been determined for eight different aged materials.

  9. 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

  10. 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

  11. Fibrin Architecture in Clots: A Quantitative Polarized Light Microscopy Analysis

    PubMed Central

    Whittaker, Peter; Przyklenk, Karin

    2009-01-01

    Fibrin plays a vital structural role in thrombus integrity. Thus, the ability to assess fibrin architecture has potential to provide insight into thrombosis and thrombolysis. Fibrin has an anisotropic molecular structure, which enables it to be seen with polarized light. Therefore, we aimed to determine if automated polarized light microscopy methods of quantifying two structural parameters; fibrin fiber bundle orientation and fibrin's optical retardation (OR: a measure of molecular anisotropy) could be used to assess thrombi. To compare fibrin fiber bundle orientation we analyzed picrosirius red-stained sections obtained from clots formed: (A) in vitro, (B) in injured and stenotic coronary arteries, and (C) in surgically created aortic aneurysms (n = 6 for each group). To assess potential changes in OR, we examined fibrin in picrosirius red-stained clots formed after ischemic preconditioning (10 minutes ischemia + 10 minutes reflow; a circumstance shown to enhance lysability) and in control clots (n = 8 each group). The degree of fibrin organization differed significantly according to the location of clot formation; fibrin was most aligned in the aneurysms and least aligned in vitro whereas fibrin in the coronary clots had an intermediate organization. The OR of fibrin in the clots formed after ischemic preconditioning was lower than that in controls (2.9 ± 0.5 nm versus 5.4 ± 1.0 nm, P < 0.05). The automated polarized light analysis methods not only enabled fibrin architecture to be assessed, but also revealed structural differences in clots formed under different circumstances. PMID:19054699

  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

  14. 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.

  15. 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.

  16. Nano-fEM: Protein Localization Using Photo-activated Localization Microscopy and Electron Microscopy

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2014-10-15

    Here, we combine super-resolution fluorescence localization microscopy with scanning electron microscopy to map the position of proteins of nuclear pore complexes in isolated Xenopus laevis oocyte nuclear envelopes with molecular resolution in both imaging modes. We use the periodic molecular structure of the nuclear pore complex to superimpose direct stochastic optical reconstruction microscopy images with a precision of <20 nm on electron micrographs. The correlative images demonstrate quantitative molecular labeling and localization of nuclear pore complex proteins by standard immunocytochemistry with primary and secondary antibodies and reveal that the nuclear pore complex is composed of eight gp210 (also known as NUP210) protein homodimers. In addition, we find subpopulations of nuclear pore complexes with ninefold symmetry, which are found occasionally among the more typical eightfold symmetrical structures. PMID:25146397

  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 100–200 keV for microscopy, corresponding to speeds of 33–70% 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. 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

  1. '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...

  2. 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…

  3. 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

  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. Electron microscopy of Mycoplasma pneumoniae microcolonies grown on solid surfaces.

    PubMed Central

    Kim, C K; Pfister, R M; Somerson, N L

    1977-01-01

    Mycoplasma pneumoniae sprain CL-8 was studied by using various surfaces for adherence and growth. Cells grown on Epon 812, Formvar, carbon, and glass were of similar morphology. Thin Epon pieces were good material for culturing the organisms and examining thin-sectioned microcolonies by transmission electron microscopy. Images PMID:931378

  6. Improved handling of embedding plastics for electron microscopy.

    PubMed

    Shannon, W A

    1982-08-01

    An improved, safer, rapid method for preparing embedding plastics for electron microscopy is described. The method consists of contained storage and dispensing of individual plastic components on an automatic tare balance. The proportions are based on weight measurements and may be calculated from volume or proportion recipes. The usual problems in and resulting from embedding plastic handling have been eliminated. PMID:6750130

  7. 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.

  8. DICHOTOMOUS SAMPLERS MODIFIED FOR USE WITH ELECTRON MICROSCOPY

    EPA Science Inventory

    Large sulfate artifacts up to 2 um in diameter were observed by scanning electron microscopy for the fine particle fraction collected in dichotomous samplers. he artifacts were attributed to small liquid particles that piled up on the filter, coalesced, and later dried as larger ...

  9. Detective quantum efficiency of electron area detectors in electron microscopy

    PubMed Central

    McMullan, G.; Chen, S.; Henderson, R.; Faruqi, A.R.

    2009-01-01

    Recent progress in detector design has created the need for a careful side-by-side comparison of the modulation transfer function (MTF) and resolution-dependent detective quantum efficiency (DQE) of existing electron detectors with those of detectors based on new technology. We present MTF and DQE measurements for four types of detector: Kodak SO-163 film, TVIPS 224 charge coupled device (CCD) detector, the Medipix2 hybrid pixel detector, and an experimental direct electron monolithic active pixel sensor (MAPS) detector. Film and CCD performance was measured at 120 and 300 keV, while results are presented for the Medipix2 at 120 keV and for the MAPS detector at 300 keV. In the case of film, the effects of electron backscattering from both the holder and the plastic support have been investigated. We also show that part of the response of the emulsion in film comes from light generated in the plastic support. Computer simulations of film and the MAPS detector have been carried out and show good agreement with experiment. The agreement enables us to conclude that the DQE of a backthinned direct electron MAPS detector is likely to be equal to, or better than, that of film at 300 keV. PMID:19497671

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

    PubMed

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

    2016-07-01

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

  11. Environmental scanning electron microscopy gold immunolabeling in cell biology.

    PubMed

    Rosso, Francesco; Papale, Ferdinando; Barbarisi, Alfonso

    2013-01-01

    Immunogold labeling (IGL) technique has been utilized by many authors in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to obtain the identification/localization of receptors and antigens, both in cells and tissues. Environmental scanning electron microscopy (ESEM) represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artifacts and interfere with the IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labeling detection is based on the atomic number difference between nanogold spheres and the biological material. Using the gaseous secondary electron detector, compositional contrast is easily revealed by the backscattered electron component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimized to improve the intensity and the specificity of the labeling signal, in order to obtain a semiquantitative evaluation of the labeling signal.In particular, we used a combination of IGL and ESEM to detect the presence of a protein on the cell surface. To achieve this purpose, we chose as an experimental system 3T3 Swiss albino mouse fibroblasts and galectin-3. PMID:23027021

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

    PubMed

    Killingsworth, Murray C; Bobryshev, Yuri V

    2016-01-01

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

  13. 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.

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

    SciTech Connect

    Wang, R.; Williams, C. C.

    2015-09-15

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

  15. Ballistic electron magnetic microscopy on epitaxial spin valves

    NASA Astrophysics Data System (ADS)

    Heindl, E.; Vancea, J.; Back, C. H.

    2007-02-01

    The tip of a scanning tunneling microscope has been used as an injector of hot electrons or hot holes into a spin valve epitaxially grown on n-GaAs67P33 . Spin-dependent transport of injected and hole excited electrons has been studied in an external magnetic field at room temperature. Significant variations in the collector current due to the spin-dependent inelastic decay of the hot charge carriers have been measured for parallel and antiparallel configurations of the magnetization of the individual layers. We found magnetocurrent effects on the order of 600% and relative large transmission values compared to other ballistic electron magnetic microscopy studies. In addition, we investigated the excitation of electron-hole pairs with its subsequent electron transport in the spin valve and found a magnetocurrent effect with positive sign.

  16. Scanning quantum dot microscopy: A quantitative method to measure local electrostatic potential near surfaces

    NASA Astrophysics Data System (ADS)

    Green, Matthew F. B.; Wagner, Christian; Leinen, Philipp; Deilmann, Thorsten; Krüger, Peter; Rohlfing, Michael; Tautz, F. Stefan; Temirov, Ruslan

    2016-08-01

    In this paper we review a recently introduced microscopy technique, scanning quantum dot microscopy (SQDM), which delivers quantitative maps of local electrostatic potential near surfaces in three dimensions. The key to achieving SQDM imaging is the functionalization of a scanning probe microscope tip with a π-conjugated molecule that acts as a gateable QD. Mapping of electrostatic potential with SQDM is performed by gating the QD by the bias voltage applied to the scanning probe microscope junction and registering changes of the QD charge state with frequency-modulated atomic force microscopy.

  17. Reactive gas plasma specimen processing for use in microanalysis and imaging in analytical electron microscopy

    SciTech Connect

    Zaluzec, N.J.; Kestel, B.J.; Henriks, D.

    1997-01-01

    It has long been the bane of analytical electron microscopy (AEM) that the use of focused probes during microanalysis of specimens increases the local rate of hydrocarbon contamination. This is most succinctly observed by the formation of contamination deposits during focused probe work typical of AEM studies. While serving to indicate the location of the electron probe, the contamination obliterates the area of the specimen being analyzed and adversely affects all quantitative microanalysis methodologies. A variety of methods including: UV, electron beam flooding, heating and/or cooling can decrease the rate of contamination, however, none of these methods directly attack the source of specimen borne contamination. Research has shown that reactive gas plasmas may be used to clean both the specimen and stage for AEM, in this study the authors report on quantitative measurements of the reduction in contamination rates in an AEM as a function of operating conditions and plasma gases.

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

    PubMed

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

    2014-04-01

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

  19. 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

    2016-01-01

    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. PMID:26369731

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

    PubMed

    Faulkner, Gary; Garduño, Rafael A

    2013-01-01

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

  1. Microfabricated high-bandpass foucault aperture for electron microscopy

    SciTech Connect

    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.

  2. 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

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

    PubMed Central

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

    2016-01-01

    Electron microscopy was used to test whether or not (a) in statu nascendi synthesized, and in situ measured, nanoparticle size does not differ significantly from the size of nanoparticles after their purification; and (b) the generation of selenium is detrimental to the bacterial strains that produce them. Elemental nano-sized selenium produced by probiotic latic acid bacteria was used as a lactomicroselenium (lactomicroSel) inhibitor of cell growth in the presence of lactomicroSel, and was followed by time-lapse microscopy. The size of lactomicroSel produced by probiotic bacteria was measured in situ and after isolation and purification. For these measurements the TESLA BS 540 transmission electron microscope was converted from analog (aTEM) to digital processing (dTEM), and further to remote-access internet electron microscopy (iTEM). Lactobacillus acidophilus produced fewer, but larger, lactomicroSel nanoparticles (200–350 nm) than Lactobacillus casei (L. casei), which generated many, smaller lactomicroSel particles (85–200 nm) and grains as a cloudy, less electrodense material. Streptococcus thermophilus cells generated selenoparticles (60–280 nm) in a suicidic manner. The size determined in situ in lactic acid bacteria was significantly lower than those measured by scanning electron microscopy after the isolation of lactomicroSel particles obtained from lactobacilli (100–500 nm), but higher relative to those isolated from Streptococcus thermopilus (50–100 nm). These differences indicate that smaller lactomicroSel particles could be more toxic to the producing bacteria themselves and discrepancies in size could have implications with respect to the applications of selenium nanoparticles as prebiotics. PMID:27376279

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

    PubMed

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

    2016-01-01

    Electron microscopy was used to test whether or not (a) in statu nascendi synthesized, and in situ measured, nanoparticle size does not differ significantly from the size of nanoparticles after their purification; and (b) the generation of selenium is detrimental to the bacterial strains that produce them. Elemental nano-sized selenium produced by probiotic latic acid bacteria was used as a lactomicroselenium (lactomicroSel) inhibitor of cell growth in the presence of lactomicroSel, and was followed by time-lapse microscopy. The size of lactomicroSel produced by probiotic bacteria was measured in situ and after isolation and purification. For these measurements the TESLA BS 540 transmission electron microscope was converted from analog (aTEM) to digital processing (dTEM), and further to remote-access internet electron microscopy (iTEM). Lactobacillus acidophilus produced fewer, but larger, lactomicroSel nanoparticles (200-350 nm) than Lactobacillus casei (L. casei), which generated many, smaller lactomicroSel particles (85-200 nm) and grains as a cloudy, less electrodense material. Streptococcus thermophilus cells generated selenoparticles (60-280 nm) in a suicidic manner. The size determined in situ in lactic acid bacteria was significantly lower than those measured by scanning electron microscopy after the isolation of lactomicroSel particles obtained from lactobacilli (100-500 nm), but higher relative to those isolated from Streptococcus thermopilus (50-100 nm). These differences indicate that smaller lactomicroSel particles could be more toxic to the producing bacteria themselves and discrepancies in size could have implications with respect to the applications of selenium nanoparticles as prebiotics. PMID:27376279

  5. 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

  6. 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.

  7. 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.

  8. Preparation of gold nanocluster bioconjugates for electron microscopy.

    PubMed

    Heinecke, Christine L; Ackerson, Christopher J

    2013-01-01

    In this chapter, we describe types of gold nanoparticle-biomolecule conjugates and their use in electron microscopy. Included are two detailed protocols for labeling an IgG antibody with gold monolayer protected clusters. The first approach is a direct bonding approach that utilizes the ligand place exchange reaction. The second approach describes NHS-EDC coupling of Au(144)(pMBA)(60) with IgG. Also included are various characterization techniques for determining labeling efficiency. PMID:23086882

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

    NASA Astrophysics Data System (ADS)

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

    2015-10-01

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

  10. Vertically integrated optics for ballistic electron emission luminescence microscopy

    NASA Astrophysics Data System (ADS)

    Appelbaum, Ian; Yi, Wei; Russell, K. J.; Narayanamurti, V.; Hanson, M. P.; Gossard, A. C.

    2005-02-01

    We have integrated a photon detector directly into a ballistic electron emission luminescence (BEEL) heterostructure, just below a luminescent quantum well. Results from solid-state metal-base hot-electron transistors fabricated with this collector design indicate that more than 10% of the photons emitted by the quantum well excite photoelectrons in the detector region. The improved photonic coupling and effective collection angle in this scheme improves the BEEL signal by many orders of magnitude as compared to far-field detection with the most sensitive single-photon counters, enabling BEEL microscopy in systems with no optical components.

  11. Imaging Nanobubbles in Water with Scanning Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    White, Edward R.; Mecklenburg, Matthew; Singer, Scott B.; Aloni, Shaul; Regan, Brian Christopher

    2011-05-01

    We present a technique based on scanning transmission electron microscopy (STEM) that is capable of probing nanobubble dynamics with nanometer spatial resolution. A vacuum-tight vessel holds a sub-micrometer layer of water between two electron-transparent dielectric membranes. Electrical current pulses passing through a platinum wire on one of the membranes inject sufficient heat locally to initiate single bubble formation. In the absence of power input, all bubbles are observed to be unstable against collapse, but the STEM beam alone can cause a shrinking bubble to grow.

  12. 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

  13. 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.

  14. Experiments in electron microscopy: from metals to nerves

    NASA Astrophysics Data System (ADS)

    Unwin, Nigel

    2015-04-01

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

  15. Reflection Electron Microscopy and Spectroscopy for Surface Analysis

    NASA Astrophysics Data System (ADS)

    Wang, Zhong Lin

    2005-08-01

    This book is a comprehensive review of the theories, techniques and applications of reflection electron microscopy (REM), reflection high-energy electron diffraction (RHEED) and reflection electron energy-loss spectroscopy (REELS). The book is divided into three parts: diffraction, imaging and spectroscopy. The text is written to combine basic techniques with special applications, theories with experiments, and the basic physics with materials science, so that a full picture of RHEED and REM emerges. An entirely self-contained study, the book contains much invaluable reference material, including FORTRAN source codes for calculating crystal structures data and electron energy-loss spectra in different scattering geometries. This and many other features makes the book an important and timely addition to the materials science literature for researchers and graduate students in physics and materials science.

  16. Reflection Electron Microscopy and Spectroscopy for Surface Analysis

    NASA Astrophysics Data System (ADS)

    Wang, Zhong Lin

    1996-05-01

    This book is a comprehensive review of the theories, techniques and applications of reflection electron microscopy (REM), reflection high-energy electron diffraction (RHEED) and reflection electron energy-loss spectroscopy (REELS). The book is divided into three parts: diffraction, imaging and spectroscopy. The text is written to combine basic techniques with special applications, theories with experiments, and the basic physics with materials science, so that a full picture of RHEED and REM emerges. An entirely self-contained study, the book contains much invaluable reference material, including FORTRAN source codes for calculating crystal structures data and electron energy-loss spectra in different scattering geometries. This and many other features makes the book an important and timely addition to the materials science literature for researchers and graduate students in physics and materials science.

  17. System and method for compressive scanning electron microscopy

    DOEpatents

    Reed, Bryan W

    2015-01-13

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

  18. Electron microscopy study of antioxidant interaction with bacterial cells

    NASA Astrophysics Data System (ADS)

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

    2000-10-01

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

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

    PubMed

    Shotton, D M

    1995-08-01

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

  20. 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

  1. 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.

  2. 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

  3. 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.

  4. The use of transmission electron microscopy in the quantification of nanoparticle dose

    NASA Astrophysics Data System (ADS)

    Hondow, N.; Brydson, R.; Brown, A.

    2014-06-01

    There are an increasing number of potential applications for nanoparticles in clinical medicine, including targeted drug delivery and contrast agents for biomedical imaging. Current in vitro studies are concerned with the biological impact of nanoparticles, with electron microscopy commonly employed to image their intracellular location. It is critical to quantify the absolute nanoparticle dose internalized by cells in a given exposure, and to understand the factors which affect this. In this work we are aiming to develop a full quantitative description of quantum dot uptake by an in vitro cell line. Transmission electron microscopy of thin cell sections provides the location and number of cellular vesicles per 2-D cell slice plus the number of quantum dots per vesicle. These results can then be correlated to other techniques to quantify the internalized nanoparticle dose distribution for whole cells.

  5. 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

  6. Color metallography and electron microscopy techniques applied to the characterization of 413.0 aluminum alloys.

    PubMed

    Vander Voort, George; Asensio-Lozano, Juan; Suárez-Peña, Beatriz

    2013-08-01

    The influence on alloy 413.0 of the refinement and modification of its microstructure was analyzed by means of several microscopy techniques, as well as the effect of the application of high pressure during solidification. For each treatment and solidification pressure condition employed, the most suitable microscopy techniques for identifying and characterizing the phases present were investigated. Color metallography and electron microscopy techniques were applied to the qualitative microstructural analysis. Volume fraction and grain size of the primary α-Al were characterized by quantitative metallographic techniques. The results show that the effect caused by applying high pressure during solidification of the alloy is more pronounced than that caused by modification and refinement of the microstructure when it solidifies at atmospheric pressure. Furthermore, it has been shown that, for Al-Si alloy characterization, when aiming to characterize the primary α-Al phase, optical color metallography observed under crossed polarized light plus a sensitive tint filter is the most suitable technique. When the goal is to characterize the eutectic Si, the use of optical color metallography or electron microscopy is equally valid. The characterization of iron-rich intermetallic compounds should preferably be performed by means of backscattered electron imaging. PMID:23701972

  7. Molybdenum work function determined by electron emission microscopy.

    NASA Technical Reports Server (NTRS)

    Jacobson, D. L.; Campbell, A. E.

    1971-01-01

    A polycrystalline molybdenum sample was recrystallized and thermally stabilized. Quantitative measurements of the emission from each individual grain were obtained with an electron emission microscope. The effective work function for each grain was then calculated. The crystallographic orientation of each grain was determined by Laue back-reflection techniques. A polar plot of effective work function vs crystallographic orientation for the sample was constructed to provide a correlation between effective work function and crystallographic orientation.

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

    PubMed

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

    2016-01-01

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

  9. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    PubMed Central

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

    2016-01-01

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

  10. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

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

    PubMed

    Kanagaraj, Palsamy; Riedel, Dietmar; Dosch, Roland

    2016-01-01

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

  12. Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

    DOE PAGESBeta

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

    2016-05-23

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

  13. Electron Microscopy Study of Exotic Nanostructures of Cadmium Sulfide

    NASA Astrophysics Data System (ADS)

    Dong, Lifeng; Jiao, Jun

    2005-04-01

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

  14. 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

  15. Minerals in coal: a transmission electron microscopy study

    SciTech Connect

    Wert, C.A.; Hsieh, K.C.

    1983-01-01

    Techniques of electron microscopy have been applied to identification of minerals in coal and coal conversion products. The principal problem is making satisfactory thin-samples. Ion-milling has been used, but grinding and microtoming also show promise. Principal attention has been given to characterization of sulfides and clays, but many other minerals have been identified. Application of the technique to identification of the minerals in oil shale has been demonstrated. The great value of this method is the extraordinary detail with which mineral inclusions can be characterized. General topography, crystal type (including space group of complex crystalline forms), planar spacing and chemical composition can be determined using the large array of techniques available - bright and dark field imaging, electron diffraction, including convergent beam electron diffraction, x-ray emission spectroscopy and energy loss spectroscopy. 63 refences, 10 figures.

  16. 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.

  17. 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.

  18. Scanning electron microscopy of Purkinje fibres of the pig heart.

    PubMed

    Bytzer, P

    1979-01-01

    Scanning electron microscopy (SEM) of Purkinje fibres (P-fibres) from the septal walls and the septomarginal trabecula was performed on deparaffinized sections, the identification in SEM made possible by comparative light microscopy. The myofibrils in P-fibres from the septal walls were arranged in a cart-wheel fashion, whereas P-fibres from the septomarginal trabecula showed a nearly parallel alignment of the contractile material. Z-line ridges resembling the T-tubules of the myocardial fibres were observed in both kinds of P-fibres. The myofibrillar arrangements are discussed in relation to the expected mechanical stress put upon P-fibres in the 2 locations during systolic-diastolic activity. An adaptive function of the contractile material to the mechanical stress is suggested and the possible need of a T-tubular system is discussed. PMID:507370

  19. 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

  20. 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.

  1. 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

  2. Installation of electric field electron beam blanker in high-resolution transmission electron microscopy

    SciTech Connect

    Hayashida, Misa; Kimura, Yoshihide; Taniguchi, Yoshifumi; Otsuka, Masayuki; Takai, Yoshizo

    2006-11-15

    We have newly installed an electric field electron beam blanker in a transmission electron microscopy, which chops an electron beam very quickly without the effect of hysteresis. The electric field, which is generated by the electron beam blanker, deflects the electron beam, and the electron beam is intercepted by an aperture. The response time of the beam blanker is 50 {mu}s. Therefore, a very short pulsed electron beam enables a charge-coupled device camera to directly expose an electron beam spot or diffraction pattern. Moreover, we measured the response of a deflector coil, which is usually used as an electron beam blanker, using our electron beam blanker. Our beam blanker will become a key component in a computer-assisted minimal dose system, which enables us to reduce the electron dose of the sample.

  3. 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.

  4. Measuring electron-phonon coupling with Scanning Tunneling Microscopy

    NASA Astrophysics Data System (ADS)

    Madhavan, Vidya

    Electron-boson interactions are ubiquitous in systems ranging from simple metals to novel materials such as graphene, high-temperature superconductors and topological insulators. Of particular interest is the coupling between electrons and phonons. In general, electron-phonon coupling gives rise to quasiparticles of decreased mobility and increased effective mass. Nearly all information about electron-phonon coupling is contained in the Eliashberg function (α2 F (ω k , E)) of the material. In this talk I discuss the various methods by which the effects of electron-phonon coupling can be measured by scanning tunneling microscopy. I will present STM data on a variety of systems ranging from metals to topological insulators and discuss the signatures of electron-phonon interactions in different types of STM data. In particular I discuss how high resolution measurements allow us to measure the dispersion and obtain the real part of the self-energy, which can in principle be inverted to obtain the Eliashberg function.

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

    PubMed

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

    2014-08-01

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

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

    PubMed Central

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

    2016-01-01

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

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

    PubMed

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

    2015-10-14

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

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

    PubMed

    Kubota, Yoshiyuki

    2015-02-01

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

  9. 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

  10. Electron microscopy study of direct laser deposited IN718

    SciTech Connect

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

    2015-08-15

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

  11. Analysis of electron emission from GaAs(Cs,O) by low energy electron microscopy

    NASA Astrophysics Data System (ADS)

    Jin, Xiuguang

    2015-10-01

    Low-energy electron microscopy was carried out to study the electron emission process from a GaAs photocathode with a negative electron affinity (NEA) surface. The relationship between emission current and electron affinity was investigated in detail to obtain information regarding the electron tunneling in the vacuum barrier and the electron distribution in the interior of GaAs, especially with respect to photoelectron capture in the band bending region. A comparison of the calculated quantized sub-band energies in the band bending region confirmed that the majority of photoelectrons fell within sub-bands, from where a large portion of the photoelectrons escape into the vacuum.

  12. 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.

  13. 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

  14. Copper Decoration of Carbon Nanotubes and High Resolution Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Probst, Camille

    A new process of decorating carbon nanotubes with copper was developed for the fabrication of nanocomposite aluminum-nanotubes. The process consists of three stages: oxidation, activation and electroless copper plating on the nanotubes. The oxidation step was required to create chemical function on the nanotubes, essential for the activation step. Then, catalytic nanoparticles of tin-palladium were deposited on the tubes. Finally, during the electroless copper plating, copper particles with a size between 20 and 60 nm were uniformly deposited on the nanotubes surface. The reproducibility of the process was shown by using another type of carbon nanotube. The fabrication of nanocomposites aluminum-nanotubes was tested by aluminum vacuum infiltration. Although the infiltration of carbon nanotubes did not produce the expected results, an interesting electron microscopy sample was discovered during the process development: the activated carbon nanotubes. Secondly, scanning transmitted electron microscopy (STEM) imaging in SEM was analysed. The images were obtained with a new detector on the field emission scanning electron microscope (Hitachi S-4700). Various parameters were analysed with the use of two different samples: the activated carbon nanotubes (previously obtained) and gold-palladium nanodeposits. Influences of working distance, accelerating voltage or sample used on the spatial resolution of images obtained with SMART (Scanning Microscope Assessment and Resolution Testing) were analysed. An optimum working distance for the best spatial resolution related to the sample analysed was found for the imaging in STEM mode. Finally, relation between probe size and spatial resolution of backscattered electrons (BSE) images was studied. An image synthesis method was developed to generate the BSE images from backscattered electrons coefficients obtained with CASINO software. Spatial resolution of images was determined using SMART. The analysis shown that using a probe

  15. Frontiers in Electron Microscopy: Probing the Nanoscale in Nanoseconds

    NASA Astrophysics Data System (ADS)

    Browning, Nigel

    2005-03-01

    Electron microscopy has traditionally been driven by the desire to investigate the result of a given materials process (e.g. nucleation and growth, fatigue etc) at the highest spatial resolution. However, this type of observation typically gives no indication as to how the material achieved its final state. With the nanotechnology revolution highlighting the novel properties that can be achieved by modifying the processing and ambient conditions a material is subjected to, the need to characterize the fundamentals behind the materials process itself has assumed critical importance. One of the developing methods to achieve this level of characterization is dynamic transmission electron microscopy (DTEM). Using a laser pulse to stimulate the electron emission, pulse durations of nanoseconds and shorter can be achieved with sufficient signal to obtain images and diffraction patterns from materials excited by a laser in a pump-probe configuration (with the probe being the electron beam). A novel nanosecond electron microscope incorporating this principle has been used initially to observe the hexagonal close packed (HCP) to body centered cubic (BCC) martensitic phase transformation in titanium. The general class of martensitic phase transformations occur by a rapid shear of the crystal lattice. No long range diffusion is required during these transformations, thus they propagate through a crystal with a speed that can approach the speed of sound. The images and diffraction patterns obtained can be interpreted in terms of the unusual vibrational stabilization of the high temperature BCC phase of Ti. An interesting observation is that the speed of the transition seems to be dependent on the history of the sample and appears to be linked to the presence of oxygen impurities. This work was performed in collaboration with A. Ziegler, G. H. Campbell, H. Kleinschmidt, and O. Bostonjoglo and supported by LLNL LDRD project 04-ERD-071. This work performed under the auspices of

  16. 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

  17. 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

  18. 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.

  19. Photonic near-field imaging in multiphoton photoemission electron microscopy

    NASA Astrophysics Data System (ADS)

    Fitzgerald, J. P. S.; Word, R. C.; Saliba, S. D.; Könenkamp, R.

    2013-05-01

    We report the observation of optical near fields in a photonic waveguide of conductive indium tin oxide (ITO) using multiphoton photoemission electron microscopy (PEEM). Nonlinear two-photon photoelectron emission is enhanced at field maxima created by interference between incident 410-nm and coherently excited guided photonic waves, providing strong phase contrast. Guided modes are observed under both transverse magnetic field (TM) and transverse electric field (TE) polarized illuminations and are consistent with classical electromagnetic theory. Implications on the role of multiphoton PEEM in optical near-field imaging are discussed.

  20. 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.

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

    NASA Astrophysics Data System (ADS)

    Hooghan, Tejpal Kaur

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

  2. 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.

  3. Fiber analysis vignettes: Electron microscopy to the rescue!

    PubMed

    Roggli, Victor L

    2016-01-01

    There has been considerable interest in the exposure doses that contribute to the various asbestos-associated diseases. Epidemiological studies have shown important differences in the contributions of the various fiber types to asbestos-related diseases, with the amphiboles showing a greater degree of potency as compared to chrysotile. However, epidemiological studies have occasionally provided misleading results. Over the past several decades, there have been several examples where fiber analysis using electron microscopy produced unexpected results which were important to our understanding of disease-exposure relationships. It is the purpose of this article to summarize these fiber analysis vignettes. PMID:26934117

  4. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  6. 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.

  7. Direct single electron detection with a CMOS detector for electron microscopy

    NASA Astrophysics Data System (ADS)

    Faruqi, A. R.; Henderson, R.; Pryddetch, M.; Allport, P.; Evans, A.

    2005-07-01

    We report the results of an investigation into the use of a monolithic active pixel sensor (MAPS) for electron microscopy. MAPS, designed originally for astronomers at the Rutherford Appleton Laboratories, was installed in a 120 kV electron microscope (Philips CM12) at the MRC Laboratory in Cambridge for tests which included recording single electrons at 40 and 120 keV, and measuring signal-to-noise ratio (SNR), spatial resolution and radiation sensitivity. Our results show that, due to the excellent SNR and resolution, it is possible to register single electrons. The radiation damage to the detector is apparent with low doses and gets progressively greater so that its lifetime is limited to 600,000-900,000 electrons/pixel (very approximately 10-15 krad). Provided this detector can be radiation hardened to reduce its radiation sensitivity several hundred fold and increased in size, it will provide excellent performance for all types of electron microscopy.

  8. Ballistic Electron Emission Microscopy Studies of Ferromagnet - Semiconductor Interfaces

    NASA Astrophysics Data System (ADS)

    Mather, P. G.; Perrella, A. C.; Yurtsever, A.; Buhrman, R. A.

    2004-03-01

    Devices that employ spin as well as charge effects have been the subjects of extensive study recently. The magnetic tunneling transistor (1) is one important device that demonstrates an electrical means of injecting spin-polarized electrons into a semiconductor. A Schottky barrier lies at the heart of the device, and a high quality spatially homogenous and uniform barrier formed on GaAs is highly desirable. We have used ballistic electron emission microscopy (BEEM) to study CoFe, Fe and permalloy deposited on a GaAs substrate to give nanometer resolved evaluation of hot electron transport through the films and across the Schottky barrier. All films give a homogenous, uniform barrier as compared with evaporated Au/GaAs and Ag/GaAs interfaces. We will report on BEEM measurements of the hot electron transfer ratio across the Schottky barrier for the different ferromagnetic materials, and on the energy and spin-dependent hot electron attenuation lengths of the CoFe, Fe, and permalloy films. (1) Sebastiaan van Dijken, Xin Jiang, Stuart S. P. Parkin, APL, 80, 3364.

  9. 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-Å-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.

  10. Correlative video-light-electron microscopy of mobile organelles.

    PubMed

    Beznoussenko, Galina V; Mironov, Alexander A

    2015-01-01

    Correlative microscopy is a method when for the analysis of the very same cell or tissue area, several different methods of light microscopy (LM) and then electron microscopy (EM) are used consecutively. The combination of LM and EM allows researchers to study phenomena at a global scale and then to look for unique or rare events for their subsequent EM examination. Unfortunately, the observation of living cells under EM is still impossible. LM provides the possibility to examine quickly many live cells, whereas EM provides the high level of resolution. On the other side, the final goal of any morphological analysis of a biological sample, whether it is an organism, organ, tissue, cell, organelle, or molecule, is to get an averaged three-dimensional model of the structure examined and to determine the chemical composition of it. This chapter describes the methodology of imaging with the help of CVLEM. The guidelines presented herein enable researchers to analyze structure of organelles and to obtain the three-dimensional model of the structure examined, and in particular rare events captured by low-resolution imaging of a population or transient events captured by live imaging can now also be studied at high resolution by EM. PMID:25702127

  11. Scanning gate microscopy of electronic inhomogeneities in single-walled carbon nanotube (SWCNT) devices

    NASA Astrophysics Data System (ADS)

    Hunt, Steven R.; Collins, Phillip G.

    2010-03-01

    The electronic properties of graphitic carbon devices are primarily determined by the contact metal and the carbon band structure. However, inhomogeneities such as substrate imperfections, surface defects, and mobile contaminants also contribute and can lead to transistor-like behaviors. We experimentally investigate this phenomena in the 1-D limit using metallic single-walled carbon nanotubes (SWCNTs) before and after the electrochemical creation of sidewall defects. While scanning gate microscopy readily identifies the defect sites, the energy-dependence of the technique allows quantitative analysis of the defects and discrimination of different defect types. This research is partly supported by the NSF (DMR 08-xxxx).

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

    PubMed

    Kohashi, Teruo; Motai, Kumi

    2013-08-01

    A sample-heating system for spin-polarized scanning electron microscopy (spin SEM) has been developed and used for microscopic magnetization analysis at temperatures up to 500°C. In this system, a compact ceramic heater and a preheating operation keep the ultra-high vacuum conditions while the sample is heated during spin SEM measurement. Moreover, the secondary-electron collector, which is arranged close to the sample, was modified so that it is not damaged at high temperatures. The system was used to heat a Co(1000) single-crystal sample from room temperature up to 500°C, and the magnetic-domain structures were observed. Changes of the domain structures were observed around 220 and 400°C, and these changes are considered to be due to phase transitions of this sample. PMID:23349241

  13. 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

  14. 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; Häkkinen, 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

  15. 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.

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

    PubMed

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

    2016-05-15

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

  17. High sensitivity piezomagnetic force microscopy for quantitative probing of magnetic materials at the nanoscale.

    PubMed

    Chen, Qian Nataly; Ma, Feiyue; Xie, Shuhong; Liu, Yuanming; Proksch, Roger; Li, Jiangyu

    2013-07-01

    Accurate scanning probing of magnetic materials at the nanoscale is essential for developing and characterizing magnetic nanostructures, yet quantitative analysis is difficult using the state of the art magnetic force microscopy, and has limited spatial resolution and sensitivity. In this communication, we develop a novel piezomagnetic force microscopy (PmFM) technique, with the imaging principle based on the detection of magnetostrictive response excited by an external magnetic field. In combination with the dual AC resonance tracking (DART) technique, the contact stiffness and energy dissipation of the samples can be simultaneously mapped along with the PmFM phase and amplitude, enabling quantitative probing of magnetic materials and structures at the nanoscale with high sensitivity and spatial resolution. PmFM has been applied to probe magnetic soft discs and cobalt ferrite thin films, demonstrating it as a powerful tool for a wide range of magnetic materials. PMID:23720016

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

    PubMed

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

    2015-02-15

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

  19. Electron Microscopy and Luminescence Study of Defects in Semiconductor Silicon

    NASA Astrophysics Data System (ADS)

    Johnson, Fiona Jane

    1990-03-01

    Available from UMI in association with The British Library. Dislocations and defects in semiconductor silicon have been the subject of much research in the last thirty years. However, to date some of the mechanisms involved in the nucleation and growth of oxidation induced defects remain uncertain, as does the origin of luminescence lines in heat treated and dislocated material. Defects introduced into silicon single crystals by three different mechanisms have been examined using Cathodoluminescence (CL), Photoluminescence (PL) and Transmission Electron Microscopy (TEM). The mechanisms employed were heat treatments, electron irradiation and plastic deformation. The CL system at Bristol had previously been developed for the study of II-VI and III-V semiconductors, the subsequent addition of the near infra-red detector enabled the luminescence from silicon to be studied. The first phase of this thesis explores the capability of the CL system in detecting and spatially resolving luminescence from silicon using the Germanium detector. The second phase involved the study of different defect systems with the aim of studying luminescence emitted from TEM thin samples. Transmission Electron Microscopy was used to examine large scale defects nucleated after single and multiple medium and high temperature anneals. Electron irradiation damage introduced in the 300kV TEM was studied in the CL system. The threshold energy for silicon displacement via 'knock-on' interactions was found to be 155keV. Dislocation related D line luminescence in plastically deformed silicon was examined using both PL and CL spectroscopy. D line emission from thermally annealed silicon, previously thought to be associated with energy states at the Si-SiO _{rm x} interface, was spatially resolved in the CL system. D line emission was detected close to the cleaved edges of samples studied.

  20. 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.

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

    NASA Astrophysics Data System (ADS)

    Kemper, Björn; Wibbeling, Jana; Ketelhut, Steffi

    2014-05-01

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

  2. A national facility for biological cryo-electron microscopy

    PubMed Central

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

    2015-01-01

    Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback. PMID:25615867

  3. 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).

  4. 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.

  5. Collaborative Computational Project for Electron cryo-Microscopy

    SciTech Connect

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

    2015-01-01

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

  6. Atomic scale characterization of materials using scanning transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Aguiar, Jeffery Andrew

    Coupling the development of emerging experimental techniques in STEM and EELS with a fundamental understanding of atomic electronic structure afforded by DFT represents the unique approach and intention of this thesis. Scanning transmission electron microscopes equipped with high-angle annular dark field (HAADF) detectors and Gatan image filters (GIF) provide images and spectra, where the image brightness is interpreted as a function of atomic mass and thickness, and elemental specific spectra provide a means for the exploration of electronic and chemical structure of materials at the angstrom size scale. Over the past 20 years, the application of EELS in STEM has enabled more accurate elemental identification and exploration of electronic and chemical structure on angstrom-length scales, and arguably has provided an unprecedented wealth of materials characterization compared to other available techniques. Many materials issues related to specific novel properties that cannot be analyzed using the traditional techniques of the past, however, still remain unanswered. These concepts require a married approach of experiment and theory to fully explain. The intent of this dissertation is the development of improved analysis techniques that derive quantitative atomic scale information in connection with unraveling the origins of materials properties linked to the electronic structure and chemistry of materials.

  7. 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).

  8. 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

  9. 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

  10. 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).

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

    NASA Astrophysics Data System (ADS)

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

    1995-04-01

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

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

    NASA Technical Reports Server (NTRS)

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

    2015-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    PubMed

    Prozorowska, Ewelina; Jackowiak, Hanna

    2015-03-01

    The present light microscopy (LM) and scanning electron microscopy (SEM) studies on porcine gallbladder mucosa provide a description of the microstructures of great functional importance such as mucosal folds, the epithelium, glands, and lymphatic nodules. The results showed the regional structural differences of the porcine gallbladder wall. Depending on the part of the gallbladder, three types of mucosal structures were described: simple and branched folds and mucosal crypts. An important structural feature found in the mucosa is connected with the structural variety of type of mucosal folds, which change from simple located in the neck, to most composed, i.e., branched or joined, in the polygonal crypts toward the fundus of the gallbladder. The morphometric analysis showed statistically significantly differences in the form and size of the folds and between the fundus, body, and neck of the gallbladder. Differences in the size of mucosal epithelium are discussed in terms of processes of synthesis and secretion of glycoproteins. Regional, species-specific differences in morphology of mucosal subepithelial glands, i.e., their secretory units and openings, and intensity of mucus secretion were described. Our results on the pig gallbladder show adaptation and/or specialization in particular areas of the mucosa for (1) secretion of mucus in the neck or body of gallbladder and (2) for cyclic volume changes, especially in the fundus of gallbladder. The description of the microstructures of mucosa in the porcine gallbladder could be useful as reference data for numerous experiments on the bile tract in the pig. PMID:25604381

  15. 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 25°C to 45°C. 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 (37°C) and with longer incubation times, the network density should increase.

  16. 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

  17. Cryo-electron microscopy of extracellular vesicles in fresh plasma

    PubMed Central

    Yuana, Yuana; Koning, Roman I.; Kuil, Maxim E.; Rensen, Patrick C. N.; Koster, Abraham J.; Bertina, Rogier M; Osanto, Susanne

    2013-01-01

    Introduction Extracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited. Objectives To image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM). Methods Fresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM. Results EV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25–260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules. Conclusions Cryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV. PMID:24455109

  18. Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

    PubMed Central

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

    2010-01-01

    A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset. PMID:20082729

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

    PubMed

    Harris, J Robin

    2015-09-01

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

  20. Electron microscopy investigations of nanoparticles for cancer diagnostic applications

    NASA Astrophysics Data System (ADS)

    Koh, Ai Leen

    This dissertation concerns electron microscopy characterization of magnetic (MNP) and surface enhanced Raman scattering (SERS) nanoparticles for in-vitro cancer diagnostic applications. Electron microscopy is an essential characterization tool owing to its (sub) nanometer spatial resolution. Structural information about the nanoparticles can be obtained using transmission electron microscopy (TEM), which can in turn be correlated to their physical characteristics. The scanning electron microscope (SEM) has excellent depth of field and can be effectively utilized to obtain high resolution information about nanoparticles binding onto cell surfaces. Part One of this thesis focuses on MNPs for bio-sensing and detection applications. As a preliminary study, chemically-synthesized, commercially-available iron oxide nanoparticles were compared against their laboratory-synthesized counterparts to assess their suitability for this application. The motivation for this initial study came about due to the lack of published data on commercially available iron oxide nanoparticles. TEM studies show that the latter are "beads" composed of multiple iron oxide cores encapsulated by a polymer shell, with large standard deviations in core diameter. Laboratory-synthesized iron oxide nanoparticles, on the other hand, are single core particles with small variations in diameter and therefore are expected to be better candidates for the required application. A key limitation in iron oxide nanoparticles is their relatively weak magnetic signals. The development of high moment Synthetic Anti-Ferromagnetic (SAF) nanoparticles aims to overcome this issue. SAFs are a novel class of MNPs fabricated using nanoimprint lithography, direct deposition of multilayer structure and final suspension into liquid medium (water). TEM analyses of cross-section specimens reveal that the SAFs possess characteristics similar to those of sputtered magnetic multilayer thin films. Their layered structure is

  1. Utility of transmission electron microscopy in small round cell tumors.

    PubMed

    Kim, Na Rae; Ha, Seung Yeon; Cho, Hyun Yee

    2015-03-01

    Small round cell tumors (SRCTs) are a heterogeneous group of neoplasms composed of small, primitive, and undifferentiated cells sharing similar histology under light microscopy. SRCTs include Ewing sarcoma/peripheral neuroectodermal tumor family tumors, neuroblastoma, desmoplastic SRCT, rhabdomyosarcoma, poorly differentiated round cell synovial sarcoma, mesenchymal chondrosarcoma, small cell osteosarcoma, small cell malignant peripheral nerve sheath tumor, and small cell schwannoma. Non-Hodgkin's malignant lymphoma, myeloid sarcoma, malignant melanoma, and gastrointestinal stromal tumor may also present as SRCT. The current shift towards immunohistochemistry and cytogenetic molecular techniques for SRCT may be inappropriate because of antigenic overlapping or inconclusive molecular results due to the lack of differentiation of primitive cells and unavailable genetic service or limited moleculocytogenetic experience. Although usage has declined, electron microscopy (EM) remains very useful and shows salient features for the diagnosis of SRCTs. Although EM is not always required, it provides reliability and validity in the diagnosis of SRCT. Here, the ultrastructural characteristics of SRCTs are reviewed and we suggest that EM would be utilized as one of the reliable modalities for the diagnosis of undifferentiated and poorly differentiated SRCTs. PMID:25812730

  2. Utility of Transmission Electron Microscopy in Small Round Cell Tumors

    PubMed Central

    Kim, Na Rae; Ha, Seung Yeon; Cho, Hyun Yee

    2015-01-01

    Small round cell tumors (SRCTs) are a heterogeneous group of neoplasms composed of small, primitive, and undifferentiated cells sharing similar histology under light microscopy. SRCTs include Ewing sarcoma/peripheral neuroectodermal tumor family tumors, neuroblastoma, desmoplastic SRCT, rhabdomyosarcoma, poorly differentiated round cell synovial sarcoma, mesenchymal chondrosarcoma, small cell osteosarcoma, small cell malignant peripheral nerve sheath tumor, and small cell schwannoma. Non-Hodgkin’s malignant lymphoma, myeloid sarcoma, malignant melanoma, and gastrointestinal stromal tumor may also present as SRCT. The current shift towards immunohistochemistry and cytogenetic molecular techniques for SRCT may be inappropriate because of antigenic overlapping or inconclusive molecular results due to the lack of differentiation of primitive cells and unavailable genetic service or limited moleculocytogenetic experience. Although usage has declined, electron microscopy (EM) remains very useful and shows salient features for the diagnosis of SRCTs. Although EM is not always required, it provides reliability and validity in the diagnosis of SRCT. Here, the ultrastructural characteristics of SRCTs are reviewed and we suggest that EM would be utilized as one of the reliable modalities for the diagnosis of undifferentiated and poorly differentiated SRCTs. PMID:25812730

  3. Procoagulant platelet balloons: evidence from cryopreparation and electron microscopy.

    PubMed

    Hess, M W; Siljander, P

    2001-05-01

    Visualisation of the procoagulant transformation of human platelets has recently become possible through use of an in vitro approach combined with fluorescence and phase contrast microscopy. Here, we extended these studies to the ultrastructural level by employing both rapid freezing/freeze-substitution and conventional ambient-temperature chemical fixation for transmission and scanning electron microscopy. Procoagulant transformation was only inducible by adhering platelets to collagen fibrils or to the collagen-related peptide and exposing them to physiological extracellular Ca2+ levels. Under these conditions prominent, 2- to 4-micron-wide balloon-like structures were regularly observed, regardless of the specimen fixation protocol. In strong contrast to normal platelets in their vicinity, the balloons' subcellular architecture proved remarkably poor: dilute cytoplasm, no cytoskeleton, only a few, randomly distributed organelles and/or their remnants. Cryofixed balloons displayed intact and smooth surfaces whereas conventional specimen processing caused plasma membrane perforations and shrinkage of the balloons. Our results clearly show that neither the balloons themselves, nor their simple ultrastructure reflect fixation artefacts caused by inadequate membrane stabilisation. The balloons are interpreted as to be transformed and/or fragmented procoagulant platelets. Thus, the generation of balloons represents a genuine, final stage of platelet ontogenesis, presumably occurring alternatively to aggregate formation. PMID:11449892

  4. 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.

  5. 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

  6. Advanced analytical electron microscopy for alkali-ion batteries

    SciTech Connect

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

    2015-01-01

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

  7. 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

  8. 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.

  9. 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

  10. Near field and exit wave computations for electron microscopy.

    PubMed

    Howie, A

    2013-11-01

    The partial wave phase shift formalism of atomic scattering is applied to compute exit wave functions for isolated Au and Si atoms under both plane wave and focused probe illumination. Connections between the far field and near field (exit) waves are clarified. This approach treats the Coulomb singularity properly though at 100 keV large numbers of phase shifts are required. In principle any form of incident wave can be handled so it may provide a means for testing traditional scattering theories used in electron microscopy. By applying the analysis to an atom embedded in a constant potential rather than free space, exit spheres of radius half the interatomic spacing can be used. PMID:23726769

  11. Scanning electron microscopy of a liver cavernous hemangioma.

    PubMed

    Yamamoto, K; Itoshima, T; Ito, T; Ukida, M; Ogawa, H; Kitadai, M; Hattori, S; Mizutani, S; Nagashima, H

    1983-02-01

    A 39-year-old female with a large cavernous hemangioma of the liver was successfully treated by ligation of the left hepatic artery. A wedge biopsy specimen of the hemangioma was obtained after the ligation and was examined by scanning electron microscopy. The hemangioma was demarcated from the surrounding normal liver parenchyma and had a labyrinth of caves 50-150 microns in diameter. The caves were separated by fibrous septa 20-40 microns in width. Endothelial cells of the caves were spindle-shaped and arranged in parallel. The surface property of the caves resembled that of the hepatic artery and differed from that of the portal vein or hepatic vein. These findings support that the cavernous hemangioma of the liver was supplied by the hepatic artery. The labyrinthine structure of the cavernous hemangioma may explain the long standing contrast enhancement of the hemangioma after hepatic arteriography. PMID:6832546

  12. A Primer to Single-Particle Cryo-Electron Microscopy

    PubMed Central

    Cheng, Yifan; Grigorieff, Nikolaus; Penczek, Pawel A.; Walz, Thomas

    2015-01-01

    Summary Cryo-electron microscopy (cryo-EM) of single-particle specimens is used to determine the structure of proteins and macromolecular complexes without the need for crystals. Recent advances in detector technology and software algorithms now allow images of unprecedented quality to be recorded and structures to be determined at near-atomic resolution. However, compared with X-ray crystallography, cryo-EM is a young technique with distinct challenges. This primer explains the different steps and considerations involved in structure determination by single-particle cryo-EM to provide an overview for scientists wishing to understand more about this technique and the interpretation of data obtained with it, as well as a starting guide for new practitioners. PMID:25910204

  13. High-resolution electron microscopy and its applications.

    PubMed

    Li, F H

    1987-12-01

    A review of research on high-resolution electron microscopy (HREM) carried out at the Institute of Physics, the Chinese Academy of Sciences, is presented. Apart from the direct observation of crystal and quasicrystal defects for some alloys, oxides, minerals, etc., and the structure determination for some minute crystals, an approximate image-contrast theory named pseudo-weak-phase object approximation (PWPOA), which shows the image contrast change with crystal thickness, is described. Within the framework of PWPOA, the image contrast of lithium ions in the crystal of R-Li2Ti3O7 has been observed. The usefulness of diffraction analysis techniques such as the direct method and Patterson method in HREM is discussed. Image deconvolution and resolution enhancement for weak-phase objects by use of the direct method are illustrated. In addition, preliminary results of image restoration for thick crystals are given. PMID:3505590

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

    NASA Astrophysics Data System (ADS)

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

  15. Electron microscopy analysis of microstructure of postannealed aluminum nitride template

    NASA Astrophysics Data System (ADS)

    Kaur, Jesbains; Kuwano, Noriyuki; Rijal Jamaludin, Khairur; Mitsuhara, Masatoshi; Saito, Hikaru; Hata, Satoshi; Suzuki, Shuhei; Miyake, Hideto; Hiramatsu, Kazumasa; Fukuyama, Hiroyuki

    2016-06-01

    The microstructure of an AlN template after high-temperature annealing was investigated by transmission electron microscopy (TEM). The AlN template was prepared by depositing an AlN layer of about 200 nm thickness on a sapphire (0001) substrate by metal–organic vapor phase epitaxy. The AlN template was annealed under (N2 + CO) atmosphere at 1500–1650 °C. TEM characterization was conducted to investigate the microstructural evolution, revealing that the postannealed AlN has a two-layer structure, the upper and lower layers of which exhibit Al and N polarities, respectively. It has been confirmed that postannealing is an effective treatment for controlling the microstructure.

  16. Electron Microscopy Analysis of the Nucleolus of Trypanosoma cruzi

    NASA Astrophysics Data System (ADS)

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

    2005-08-01

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

  17. Waveguide characterization with multi-photon photoemission electron microscopy

    NASA Astrophysics Data System (ADS)

    Fitzgerald, J. P. S.; Word, Robert C.; Saliba, Sebastian; Koenenkamp, Rolf

    2012-10-01

    Multi-photon photoemission electron microscopy (PEEM) images surface interactions of visible light with matter, showing electromagnetic (EM) waves that propagate at or near the surface. Images are interferometric, showing where incident and surface waves are in-phase (bright) and out-of-phase (dark), with strong contrast between regions of high and low rates of photoelectron emission. Interferogram analysis can determine the amplitude, wavelength, phase evolution, and propagation decay length of the surface waves. Most multi-photon PEEM studies focus on surface plasmon polaritons. We show that this technique can also be applied to conducting thin-film waveguides, measuring the properties of confined EM waves in a two-mode slab waveguide made of indium tin oxide on glass, which are consistent with waveguide theory. This research was funded by the US Department of Energy Basic Science Office under contract DE-FG02-10ER46406.

  18. 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.

  19. 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

  20. Advanced analytical electron microscopy for alkali-ion batteries

    DOE PAGESBeta

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

    2015-01-01

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

  1. 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

  2. Quantitative Electron Tomography of Rubber Composites

    NASA Astrophysics Data System (ADS)

    Staniewicz, Lech; Vaudey, Thomas; Degrandcourt, Christophe; Couty, Marc; Gaboriaud, Fabien; Midgley, Paul

    2014-06-01

    Rubber composite materials have many applications, one example being tyre manufacture. The presence of a filler material in the composite (such as carbon black or silica) causes its mechanical properties to differ in several ways when compared to pure rubber such as viscoelastic behaviour (the Payne effect), increased tensile strength and improved wear resistance. To fully understand these properties, it is necessary to characterise how the filler material is organised on the nanoscale. Using composite materials representative of those found in tyres, this work illustrates the use of electron tomography and machine learning methods as tools to describe the percolation behaviour of the filler; in this case, we focus on the largest proportion of particles absorbed into one single object as a function of particle spacing.

  3. Scanning electron microscopy of Strongylus spp. in zebra.

    PubMed

    Els, H J; Malan, F S; Scialdo-Krecek, R C

    1983-12-01

    The external ultrastructure of the anterior and posterior extremities of the nematodes, Strongylus asini , Strongylus vulgaris, Strongylus equinus and Strongylus edentatus, was studied with scanning electron microscopy (SEM). Fresh specimens of S. asini were collected from the caecum, ventral colon and vena portae of Equus burchelli and Equus zebra hartmannae ; S. vulgaris from the caecum, colon and arteria ileocolica of E. burchelli ; S. equinus from the ventral colon of E. z. hartmannae and S. edentatus from the caecum and ventral colon of both zebras , during surveys of parasites in zebras in the Etosha Game Reserve, South West Africa/Namibia, and the Kruger National Park, Republic of South Africa. The worms were cleaned, fixed and mounted by standard methods and photographed in a JEOL JSM - 35C scanning electron microscope (SEM) operating at 12kV . The SEM showed the following differences: the tips of the external leaf-crowns varied and were fine and delicate in S. asini , coarse and broad in S. vulgaris and, in S. equinus and S. edentatus, closely adherent, separating into single elements for half their length. The excretory pores showed only slight variation, and the morphology of the copulatory bursae did not differ from those seen with light microscopy. The genital cones differed markedly: S. asini had a ventral triangular projection and laterally 2 finger-like projections: in S. vulgaris there were numerous bosses on the lateral and ventral aspects of the cone; in S. equinus 2 finger-like processes projected laterocaudally ; and in S. edentatus 2 pairs of papilla-like processes projected laterally on the ventral aspects, and a pair of rounded projections and a pair of hair-like structures adorned the dorsal aspects.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:6676687

  4. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-07-01

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

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

    NASA Astrophysics Data System (ADS)

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

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

  7. 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

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

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  9. Intracellular subsurface imaging using a hybrid shear-force feedback/scanning quantitative phase microscopy technique

    NASA Astrophysics Data System (ADS)

    Edward, Kert

    Quantitative phase microscopy (QPM) allows for the imaging of translucent or transparent biological specimens without the need for exogenous contrast agents. This technique is usually applied towards the investigation of simple cells such as red blood cells which are typically enucleated and can be considered to be homogenous. However, most biological cells are nucleated and contain other interesting intracellular organelles. It has been established that the physical characteristics of certain subsurface structures such as the shape and roughness of the nucleus is well correlated with onset and progress of pathological conditions such as cancer. Although the acquired quantitative phase information of biological cells contains surface information as well as coupled subsurface information, the latter has been ignored up until now. A novel scanning quantitative phase imaging system unencumbered by 2pi ambiguities is hereby presented. This system is incorporated into a shear-force feedback scheme which allows for simultaneous phase and topography determination. It will be shown how subsequent image processing of these two data sets allows for the extraction of the subsurface component in the phase data and in vivo cell refractometry studies. Both fabricated samples and biological cells ranging from rat fibroblast cells to malaria infected human erythrocytes were investigated as part of this research. The results correlate quite well with that obtained via other microscopy techniques.

  10. A nondamaging electron microscopy approach to map In distribution in InGaN light-emitting diodes

    NASA Astrophysics Data System (ADS)

    Özdöl, V. B.; Koch, C. T.; van Aken, P. A.

    2010-09-01

    Dark-field inline electron holography and, for comparison, high-resolution transmission electron microscopy are used to investigate the distribution of indium in GaN-based commercial high-efficiency green light-emitting diodes consisting of InGaN multiquantum wells (QWs). Owing to the low electron doses used in inline holography measurements; this technique allows to map the indium distribution without introducing any noticeable electron beam-induced damage which is hardly avoidable in other quantitative transmission electron microscopy methods. Combining the large field of view with a spatial resolution better than 1 nm, we show that the InGaN QWs exhibit random alloy nature without any evidence of nanometer scale gross indium clustering in the whole active region.

  11. 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

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

    NASA Astrophysics Data System (ADS)

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

    2012-03-01

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

  13. Clinical applications of scanning electron microscopy and X-ray microanalysis in dermatology

    SciTech Connect

    Forslind, B.

    1984-01-01

    Scanning electron microscopy is frequently applied to dermatological problems, as is evident from a review of the recent literature. In this paper, preparation methods and new techniques allowing experimental studies on the integumentary system are emphasized. Quantitative analysis in the electron microscope by use of energy-dispersive X-ray microanalysis (EDX) has become an important accessory technique. EDX can, for instance, be used to study problems involving physiological changes induced in skin by agents causing contact reactions. Recently, it has been shown that treatment with DNCB, chromate and nickel causes changes in elemental distribution in guinea-pig skin. In addition, elemental uptake in the integumentary system and in pathological inclusions in skin can be analyzed.

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

    SciTech Connect

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

    2015-03-15

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

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

    PubMed

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

    2015-02-28

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

  16. 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).

  17. The characterization of nanoparticles using analytical electron microscopy

    NASA Astrophysics Data System (ADS)

    Hill, Whitney B.

    2011-06-01

    Nanoparticles are often overlooked during routine trace evidence analyses because of their small size and the degree of difficulty needed to efficiently characterize them. However, analytical electron microscopy (AEM) enables the characterization and/or identification of nanoparticles because of its high magnification capability, the ability to gather elemental data and also the ability to determine the internal structure of a single nanoparticles(1). There is a wide variety of natural and manufactured nanoparticles that are prominent within the environment and their presence becomes very valuable in the absence of larger particles. The combustion of materials produces by-products such as nano-sized carbon soot, fumes, fly ash and gun-shot residue (GSR). Using AEM, nano-sized carbon soot, fumes, fly ash and GSR can not only be distinguished from other nanoparticles within the environment but can also be distinguished from each other because of differences in morphology, elemental composition, and internal structure. The elemental information gathered from combustion by-products during AEM analysis can also give an indication of the original source material. Other nanoparticles such as paint pigments and fillers can also be characterized by AEM using morphology, electron diffraction and elemental composition.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  2. 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

  3. 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.

  4. 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.

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

    NASA Astrophysics Data System (ADS)

    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.

  6. High-sensitive and broad-dynamic-range quantitative phase imaging with spectral domain phase microscopy.

    PubMed

    Yan, Yangzhi; Ding, Zhihua; Shen, Yi; Chen, Zhiyan; Zhao, Chen; Ni, Yang

    2013-11-01

    Spectral domain phase microscopy for high-sensitive and broad-dynamic-range quantitative phase imaging is presented. The phase retrieval is realized in the depth domain to maintain a high sensitivity, while the phase information obtained in the spectral domain is exploited to extend the dynamic range of optical path difference. Sensitivity advantage of phase retrieved in the depth domain over that in the spectral domain is thoroughly investigated. The performance of the proposed depth domain phase based approach is illustrated by phase imaging of a resolution target and an onion skin. PMID:24216799

  7. 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

  8. 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

  9. Molecular tips for scanning tunneling microscopy: intermolecular electron tunneling for single-molecule recognition and electronics.

    PubMed

    Nishino, Tomoaki

    2014-01-01

    This paper reviews the development of molecular tips for scanning tunneling microscopy (STM). Molecular tips offer many advantages: first is their ability to perform chemically selective imaging because of chemical interactions between the sample and the molecular tip, thus improving a major drawback of conventional STM. Rational design of the molecular tip allows sophisticated chemical recognition; e.g., chiral recognition and selective visualization of atomic defects in carbon nanotubes. Another advantage is that they provide a unique method to quantify electron transfer between single molecules. Understanding such electron transfer is mandatory for the realization of molecular electronics. PMID:24420248

  10. 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.

  11. An open-source deconvolution software package for 3-D quantitative fluorescence microscopy imaging

    PubMed Central

    SUN, Y.; DAVIS, P.; KOSMACEK, E. A.; IANZINI, F.; MACKEY, M. A.

    2010-01-01

    Summary Deconvolution techniques have been widely used for restoring the 3-D quantitative information of an unknown specimen observed using a wide-field fluorescence microscope. Deconv, an open-source deconvolution software package, was developed for 3-D quantitative fluorescence microscopy imaging and was released under the GNU Public License. Deconv provides numerical routines for simulation of a 3-D point spread function and deconvolution routines implemented three constrained iterative deconvolution algorithms: one based on a Poisson noise model and two others based on a Gaussian noise model. These algorithms are presented and evaluated using synthetic images and experimentally obtained microscope images, and the use of the library is explained. Deconv allows users to assess the utility of these deconvolution algorithms and to determine which are suited for a particular imaging application. The design of Deconv makes it easy for deconvolution capabilities to be incorporated into existing imaging applications. PMID:19941558

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

    PubMed Central

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

    2016-01-01

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

  13. Quantitative surface topography determination by Nomarski reflection microscopy. 2: Microscope modification, calibration, and planar sample experiments.

    PubMed

    Hartman, J S; Gordon, R L; Lessor, D L

    1980-09-01

    The application of reflective Nomarski differential interference contrast microscopy for the determination of quantitative sample topography data is presented. The discussion includes a review of key theoretical results presented previously plus the experimental implementation of the concepts using a commercial Nomarski microscope. The experimental work included the modification and characterization of a commercial microscope to allow its use for obtaining quantitative sample topography data. System usage for the measurement of slopes on flat planar samples is also discussed. The discussion has been designed to provide the theoretical basis, a physical insight, and a cookbook procedure for implementation to allow these results to be of value to both those interested in the microscope theory and its practical usage in the metallography laboratory. PMID:20234540

  14. Isolated dystrophin molecules as seen by electron microscopy.

    PubMed

    Pons, F; Augier, N; Heilig, R; Léger, J; Mornet, D; Léger, J J

    1990-10-01

    Dystrophin, the protein product of the Duchenne muscular dystrophy locus [Hoffman, E. P., Brown, R. H., Jr., & Kunkel, L. M. (1987) Cell 51, 919-928], is expressed in striated and smooth muscles as well as in non-muscle tissues. Examination of its primary structure has revealed that the molecule is composed of four domains, three of which share many features with the membrane cytoskeletal proteins spectrin and actinin. Dystrophin has thus been predicted to adopt a rod shape [Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219-228]. In the present study, we describe its isolation from the chicken gizzard smooth muscle and present electron microscopic images of the molecule. Polyclonal antibodies were first prepared from a dystrophin fragment derived from the chicken skeletal muscle gene (residues 1173-1728). A dystrophin-enriched membrane preparation from chicken gizzard muscle was then purified by passing it through an affinity chromatography column made with the anti-dystrophin antibodies. Electron microscopy of isolated and rotatory-shadowed dystrophin molecules revealed that the lengths measured for the dystrophin monomers (175 +/- 15 nm) are compatible with a structural arrangement of the repeat sequence segments in triple-barrel alpha-helices connected by short-turn regions, as was earlier postulated for the repeat domains of spectrin and actinin. Electron microscopic images indicate that in addition the dystrophin molecules could present the same capacity of self-association in oligomeric structures as these cytoskeletal proteins and may thus be a part of a complex molecular meshwork essential to muscle cell function. PMID:2236001

  15. 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.

  16. Probing Individual Ice Nucleation Events with Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Bingbing; China, Swarup; Knopf, Daniel; Gilles, Mary; Laskin, Alexander

    2016-04-01

    Heterogeneous ice nucleation is one of the processes of critical relevance to a range of topics in the fundamental and the applied science and technologies. Heterogeneous ice nucleation initiated by particles proceeds where microscopic properties of particle surfaces essentially control nucleation mechanisms. Ice nucleation in the atmosphere on particles governs the formation of ice and mixed phase clouds, which in turn influence the Earth's radiative budget and climate. Heterogeneous ice nucleation is still insufficiently understood and poses significant challenges in predictive understanding of climate change. We present a novel microscopy platform allowing observation of individual ice nucleation events at temperature range of 193-273 K and relative humidity relevant for ice formation in the atmospheric clouds. The approach utilizes a home built novel ice nucleation cell interfaced with Environmental Scanning Electron Microscope (IN-ESEM system). The IN-ESEM system is applied for direct observation of individual ice formation events, determining ice nucleation mechanisms, freezing temperatures, and relative humidity onsets. Reported microanalysis of the ice nucleating particles (INP) include elemental composition detected by the energy dispersed analysis of X-rays (EDX), and advanced speciation of the organic content in particles using scanning transmission x-ray microscopy with near edge X-ray absorption fine structure spectroscopy (STXM/NEXAFS). The performance of the IN-ESEM system is validated through a set of experiments with kaolinite particles with known ice nucleation propensity. We demonstrate an application of the IN-ESEM system to identify and characterize individual INP within a complex mixture of ambient particles.

  17. Time-resolved quantitative-phase microscopy of laser-material interactions using a wavefront sensor.

    PubMed

    Gallais, Laurent; Monneret, Serge

    2016-07-15

    We report on a simple and efficient technique based on a wavefront sensor to obtain time-resolved amplitude and phase images of laser-material interactions. The main interest of the technique is to obtain quantitative self-calibrated phase measurements in one shot at the femtosecond time-scale, with high spatial resolution. The technique is used for direct observation and quantitative measurement of the Kerr effect in a fused silica substrate and free electron generation by photo-ionization processes in an optical coating. PMID:27420506

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

    NASA Astrophysics Data System (ADS)

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

    Atomic scale features of polyvinylidene fluoride molecules (PVDF) were observed. Electron micrographs of thin, self-supporting PVDF nanofibers showed conformations and relative locations of atoms in segments of polymer molecules. Rows of CF2 atomic groups, at 0.25 nm intervals, marked the paths of segments of the PVDF molecules. 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, provide quantitative new insights into radiation chemistry. Relative movements of segments of molecules were observed. Synergism between high resolution electron micrographs and images created by 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. Support from Coalescence Filtration Nanofiber Consortium and from the Office of Basic Energy Sciences Contract No. DE-AC02-05CH11231.

  19. 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

  20. 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

  1. Hot Electron Transport Properties of Thin Copper Films Using Ballistic Electron Emission Microscopy

    NASA Astrophysics Data System (ADS)

    Garramone, J. J.; Abel, J. R.; Sitnitsky, I. L.; Zhao, L.; Appelbaum, I.; Labella, V. P.

    2009-03-01

    Copper is widely used material for electrical interconnects within integrated circuits and recently as a base layer for hot electron spin injection and readout into silicon. Integral to both their applications is the knowledge of the electron scattering length. To the best of our knowledge, little work exists that directly measures the scattering length of electrons in copper. In this study we used ballistic electron emission microscopy (BEEM) to measure the hot electron attenuation length of copper thin films deposited on Si(001). BEEM is a three terminal scanning tunneling microcopy (STM) based technique that can measure transport and Schottky heights of metal/semiconductor systems. We find a Schottky height of 0.67 eV and an attenuation length approaching 40 nm just above the Schottky height at 77 K. We also measure a decrease in the attenuation length with increasing tip bias to determine the relative roles of inelastic and elastic scattering.

  2. Scanning transmission electron microscopy strain measurement from millisecond frames of a direct electron charge coupled device

    SciTech Connect

    Mueller, Knut; Rosenauer, Andreas; Ryll, Henning; Ordavo, Ivan; Ihle, Sebastian; Soltau, Heike; Strueder, Lothar; Volz, Kerstin; Zweck, Josef

    2012-11-19

    A high-speed direct electron detection system is introduced to the field of transmission electron microscopy and applied to strain measurements in semiconductor nanostructures. In particular, a focused electron probe with a diameter of 0.5 nm was scanned over a fourfold quantum layer stack with alternating compressive and tensile strain and diffracted discs have been recorded on a scintillator-free direct electron detector with a frame time of 1 ms. We show that the applied algorithms can accurately detect Bragg beam positions despite a significant point spread each 300 kV electron causes during detection on the scintillator-free camera. For millisecond exposures, we find that strain can be measured with a precision of 1.3 Multiplication-Sign 10{sup -3}, enabling, e.g., strain mapping in a 100 Multiplication-Sign 100 nm{sup 2} region with 0.5 nm resolution in 40 s.

  3. New innovations for contrast enhancement in electron microscopy

    NASA Astrophysics Data System (ADS)

    Mohan, A.

    In this study two techniques for producing and improving contrast in Electron Microscopy are discussed. The first technique deals with the production of secondary contrast in a Variable Pressure SEM under poor vacuum conditions using the specimen current signal. A review of the prior work in this field shows that the presence of the gas ions in the microscope column results in the amplification of the specimen current signal which is enriched in secondary content. The focus of this study is to establish practical conditions for imaging samples in the microscope using specimen current with gas amplification. This is done by understanding the different variables in the microscope which affect the image formation process and then finding out optimum conditions for obtaining the best possible image, i.e., the image most enhanced in secondary contrast. A few 'real life' samples analyzed using this technique show that the gas amplified specimen current images contain secondary information and, in some cases, provide clear advantages to imaging with conventional secondary and backscattered detectors. The second technique dealing with the production of phase contrast in the TEM for extremely thin, electron transparent samples, is analyzed. A review of the literature regarding prior work in the field shows that, while the theoretical aspects of production of phase contrast in the TEM using a phase plate are well understood, there have been problems in practically implementing this in the microscope. One major assumption with most of the studies is that a fiber, partially coated with gold, results in the formation of point charges which is an essential requirement for symmetrically shifting the phase of the electron beam. The focus of this portion of the dissertation is to image the type of fields associated with such a phase plate using the technique of electron holography. It is found that there are two types of fields associated with a phase plate of this sort. One is a

  4. Quantitative Analysis of Subcellular Distribution of the SUMO Conjugation System by Confocal Microscopy Imaging.

    PubMed

    Mas, Abraham; Amenós, Montse; Lois, L Maria

    2016-01-01

    Different studies point to an enrichment in SUMO conjugation in the cell nucleus, although non-nuclear SUMO targets also exist. In general, the study of subcellular localization of proteins is essential for understanding their function within a cell. Fluorescence microscopy is a powerful tool for studying subcellular protein partitioning in living cells, since fluorescent proteins can be fused to proteins of interest to determine their localization. Subcellular distribution of proteins can be influenced by binding to other biomolecules and by posttranslational modifications. Sometimes these changes affect only a portion of the protein pool or have a partial effect, and a quantitative evaluation of fluorescence images is required to identify protein redistribution among subcellular compartments. In order to obtain accurate data about the relative subcellular distribution of SUMO conjugation machinery members, and to identify the molecular determinants involved in their localization, we have applied quantitative confocal microscopy imaging. In this chapter, we will describe the fluorescent protein fusions used in these experiments, and how to measure, evaluate, and compare average fluorescence intensities in cellular compartments by image-based analysis. We show the distribution of some components of the Arabidopsis SUMOylation machinery in epidermal onion cells and how they change their distribution in the presence of interacting partners or even when its activity is affected. PMID:27424751

  5. 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

  6. 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...

  7. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2016-04-01

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

  9. Superhydrophobic and adhesive properties of surfaces: testing the quality by an elaborated scanning electron microscopy method.

    PubMed

    Ensikat, Hans J; Mayser, Matthias; Barthlott, Wilhelm

    2012-10-01

    In contrast to advancements in the fabrication of new superhydrophobic materials, the characterization of their water repellency and quality is often coarse and unsatisfactory. In view of the problems and inaccuracies, particularly in the measurement of very high contact angles, we developed alternative methods for the characterization of superhydrophobic surfaces. It was found that adhering water remnants after immersion are a useful criterion in determining the repellency quality. In this study, we introduce microscopy methods to detect traces of water-resembling test liquids on superhydrophobic surfaces by scanning electron microscopy (SEM) or fluorescence light microscopy (FLM). Diverse plant surfaces and some artificial superhydrophobic samples were examined. Instead of pure water, we used aqueous solutions containing a detectable stain and glycerol in order to prevent immediate evaporation of the microdroplets. For the SEM examinations, aqueous solutions of lead acetate were used, which could be detected in a frozen state at -90 °C with high sensitivity using a backscattered electron detector. For fluorescence microscopy, aqueous solutions of auramine were used. On different species of superhydrophobic plants, varying patterns of remaining microdroplets were found on their leaves. On some species, drop remnants occurred only on surface defects such as damaged epicuticular waxes. On others, microdroplets regularly decorated the locations of increased adhesion, particularly on hierarchically structured surfaces. Furthermore, it is demonstrated that the method is suitable for testing the limits of repellency under harsh conditions, such as drop impact or long-enduring contact. The supplementation of the visualization method by the measurement of the pull-off force between a water drop and the sample allowed us to determine the adhesive properties of superhydrophobic surfaces quantitatively. The results were in good agreement with former studies of the water

  10. Atomic-Resolution 3D Electron Microscopy with Dynamic Diffraction

    SciTech Connect

    O'Keefe, Michael A.; Downing, Kenneth H.; Wenk, Hans-Rudolf; Meisheng, Hu

    2005-02-15

    Achievement of atomic-resolution electron-beam tomography will allow determination of the three-dimensional structure of nanoparticles (and other suitable specimens) at atomic resolution. Three-dimensional reconstructions will yield ''section'' images that resolve atoms overlapped in normal electron microscope images (projections), resolving lighter atoms such as oxygen in the presence of heavier atoms, and atoms that lie on non-lattice sites such as those in non-periodic defect structures. Lower-resolution electron microscope tomography has been used to produce reconstructed 3D images of nanoparticles [1] but extension to atomic resolution is considered not to be straightforward. Accurate three-dimensional reconstruction from two-dimensional projections generally requires that intensity in the series of 2-D images be a monotonic function of the specimen structure (often specimen density, but in our case atomic potential). This condition is not satisfied in electron microscopy when specimens with strong periodicity are tilted close to zone-axis orientation and produce ''anomalous'' image contrast because of strong dynamic diffraction components. Atomic-resolution reconstructions from tilt series containing zone-axis images (with their contrast enhanced by strong dynamical scattering) can be distorted when the stronger zone-axis images overwhelm images obtained in other ''random'' orientations in which atoms do not line up in neat columns. The first demonstrations of 3-D reconstruction to atomic resolution used five zone-axis images from test specimens of staurolite consisting of a mix of light and heavy atoms [2,3]. Initial resolution was to the 1.6{angstrom} Scherzer limit of a JEOL-ARM1000. Later experiments used focal-series reconstruction from 5 to 10 images to produce staurolite images from the ARM1000 with resolution extended beyond the Scherzer limit to 1.38{angstrom} [4,5]. To obtain a representation of the three-dimensional structure, images were obtained

  11. 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

  12. In-vivo Candida biofilms in scanning electron microscopy.

    PubMed

    Paulitsch, Astrid Helga; Willinger, Birgit; Zsalatz, Benedikt; Stabentheiner, Edith; Marth, Egon; Buzina, Walter

    2009-11-01

    Candida biofilms on indwelling devices are an increasing problem in patients treated at intensive care units. The goal of this study was to examine the occurrence and frequency of these biofilms. A total of 172 catheters were collected from 105 male and 67 female patients (the age range of both patient groups was from 3 weeks to 98 years old). The catheters were incubated on blood agar plates and the resulting yeast colonies were subsequently identified. Furthermore, pieces of catheters were fixed, dried and sputter coated with gold for investigation with scanning electron microscopy (SEM). Yeasts were recovered from significantly more catheters obtained from men than from women (chi(2): n = 67; P < 0.01). In SEM, 56.4% catheters turned out to be positive for biofilm formation. Again catheters from male patients were statistically significant (chi(2): n = 40; P < 0.01) more often positive than those from women. Candida albicans (71.1%) was the most common species isolated from the catheters, followed by C. glabrata (10.3%), C. parapsilosis (8.2%) and C. tropicalis (5.2%). Based on the results of this investigation, the epidemiology of Candida biofilms on indwelling devices seems to be a promising target for future investigations. PMID:19888801

  13. Non-thermal plasma mills bacteria: Scanning electron microscopy observations

    NASA Astrophysics Data System (ADS)

    Lunov, O.; Churpita, O.; Zablotskii, V.; Deyneka, I. G.; Meshkovskii, I. K.; Jäger, A.; Syková, E.; Kubinová, Š.; Dejneka, A.

    2015-02-01

    Non-thermal plasmas hold great promise for a variety of biomedical applications. To ensure safe clinical application of plasma, a rigorous analysis of plasma-induced effects on cell functions is required. Yet mechanisms of bacteria deactivation by non-thermal plasma remain largely unknown. We therefore analyzed the influence of low-temperature atmospheric plasma on Gram-positive and Gram-negative bacteria. Using scanning electron microscopy, we demonstrate that both Gram-positive and Gram-negative bacteria strains in a minute were completely destroyed by helium plasma. In contrast, mesenchymal stem cells (MSCs) were not affected by the same treatment. Furthermore, histopathological analysis of hematoxylin and eosin-stained rat skin sections from plasma-treated animals did not reveal any abnormalities in comparison to control ones. We discuss possible physical mechanisms leading to the shred of bacteria under non-thermal plasma irradiation. Our findings disclose how helium plasma destroys bacteria and demonstrates the safe use of plasma treatment for MSCs and skin cells, highlighting the favorability of plasma applications for chronic wound therapy.

  14. Microstructural evaluation of ? bilayer film by transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Yuan; Liu, Wei; Wang, Ruilan; Xuan, Yi; Li, Lin; Li, Hongchen; Xi, Xiao Xing

    1998-07-01

    The microstructure of 0022-3727/31/14/005/img11Cu0022-3727/31/14/005/img12 bilayer film grown on 0022-3727/31/14/005/img13 substrate was studied by high-resolution transmission electron microscopy (HREM). The results showed that the 0022-3727/31/14/005/img14 film is epitaxially grown on the 0022-3727/31/14/005/img13 substrate with c axis orientation. Planar defects, grain boundaries, moiré patterns, a axis oriented 0022-3727/31/14/005/img14 and impurity particulates are found in the 0022-3727/31/14/005/img14 film. The 0022-3727/31/14/005/img18 film was grown on the 0022-3727/31/14/005/img14 film with a columnar structure. However, some region of the 0022-3727/31/14/005/img18 film is single crystalline, but with strain bands. The development of strain bands in the 0022-3727/31/14/005/img18 film could be a result of lattice mismatch between 0022-3727/31/14/005/img14 and 0022-3727/31/14/005/img18 films and the surface roughness of the 0022-3727/31/14/005/img14 film. In consequence, the dielectric properties of the strained STO film are greatly decreased compared to the bulk single crystalline STO.

  15. 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.

  16. 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.

  17. 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

  18. Glycine receptor mechanism elucidated by electron cryo-microscopy.

    PubMed

    Du, Juan; Lü, Wei; Wu, Shenping; Cheng, Yifan; Gouaux, Eric

    2015-10-01

    The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders, including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of glycine receptors has been hindered by a lack of high-resolution structures. Here we report electron cryo-microscopy structures of the zebrafish α1 GlyR with strychnine, glycine, or glycine and ivermectin (glycine/ivermectin). Strychnine arrests the receptor in an antagonist-bound closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain 'wrist' interface, and leads to rotation of the transmembrane domain towards the pore axis, occluding the ion conduction pathway. These structures illuminate the GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors. PMID:26344198

  19. Application of particle analysis to transmission electron microscopy (TEM)

    NASA Astrophysics Data System (ADS)

    DaPonte, J.; Sadowski, T.; Broadbridge, C. C.; Day, D.; Lehman, A. H.; Krishna, D.; Marinella, L.; Munhutu, P.; Sawicki, M.

    2007-04-01

    Nanoparticles, particles with a diameter of 1-100 nanometers (nm), are of interest in many applications including device fabrication, quantum computing, and sensing because their size may give them properties that are very different from bulk materials. Further advancement of nanotechnology cannot be obtained without an increased understanding of nanoparticle properties such as size (diameter) and size distribution frequently evaluated using transmission electron microscopy (TEM). In the past, these parameters have been obtained from digitized TEM images by manually measuring and counting many of these nanoparticles, a task that is highly subjective and labor intensive. More recently, computer imaging particle analysis has emerged as an objective alternative by counting and measuring objects in a binary image. This paper will describe the procedures used to preprocess a set of gray scale TEM images so that they could be correctly thresholded into binary images. This allows for a more accurate assessment of the size and frequency (size distribution) of nanoparticles. Several preprocessing methods including pseudo flat field correction and rolling ball background correction were investigated with the rolling ball algorithm yielding the best results. Examples of particle analysis will be presented for different types of materials and different magnifications. In addition, a method based on the results of particle analysis for identifying and removing small noise particles will be discussed. This filtering technique is based on identifying the location of small particles in the binary image and removing them without affecting the size of other larger particles.

  20. Analysis of virus textures in transmission electron microscopy images.

    PubMed

    Nanni, Loris; Paci, Michelangelo; Caetano Dos Santos, Florentino Luciano; Brahnam, Sheryl; Hyttinen, Jari

    2014-01-01

    In this paper we propose an ensemble of texture descriptors for analyzing virus textures in transmission electron microscopy images. Specifically, we present several novel multi-quinary (MQ) codings of local binary pattern (LBP) variants: the MQ version of the dense LBP, the MQ version of the rotation invariant co-occurrence among adjacent LBPs, and the MQ version of the LBP histogram Fourier. To reduce computation time as well as to improve performance, a feature selection approach is utilized to select the thresholds used in the MQ approaches. In addition, we propose new variants of descriptors where two histograms, instead of the standard one histogram, are produced for each descriptor. The two histograms (one for edge pixels and the other for non-edge pixels) are calculated for training two different SVMs, whose results are then combined by sum rule. We show that a bag of features approach works well with this problem. Our experiments, using a publicly available dataset of 1500 images with 15 classes and same protocol as in previous works, demonstrate the superiority of our new proposed ensemble of texture descriptors. The MATLAB code of our approach is available at https://www.dei.unipd.it/node/2357. PMID:25488214

  1. Life Cycle of Neurospora crassa Viewed by Scanning Electron Microscopy

    PubMed Central

    Seale, Thomas

    1973-01-01

    Scanning electron microscopy was used to examine the major stages of the life cycle of two wild-type strains of Neurospora crassa Shear and Dodge (St. Lawrence 3.1a and 74A): mycelia, protoperithecium formation, perithecia, ascospores, ascospore germination and outgrowth, macro and microconidia, and germination and outgrowth of macroconidia. Structures seen at the limit of resolution of bright-field and phase-contrast microscopes, e.g., the ribbed surface of ascospores, are well resolved. New details of conidial development and surface structure are revealed. There appears to be only one distinguishable morphological difference between the two strains. The pattern of germination and outgrowth which seems relatively constant for strain 74A or strain 3.1a, appears to be different for each. Conidia from strain 3.1a almost always germinate from a site between interconidial attachment points; whereas the germ tubes of strain 74A usually emerge from or very near the interconidial attachment site. These germination patterns usually do not segregate 2:2 in asci dissected in order. This observation suggests that conidial germination pattern is not under the control of a single gene. Images PMID:4266170

  2. Glycine receptor mechanism illuminated by electron cryo-microscopy

    PubMed Central

    Du, Juan; Lü, Wei; Wu, Shenping; Cheng, Yifan; Gouaux, Eric

    2015-01-01

    Summary The strychnine-sensitive glycine receptor (GlyR) mediates inhibitory synaptic transmission in the spinal cord and brainstem and is linked to neurological disorders including autism and hyperekplexia. Understanding of molecular mechanisms and pharmacology of GlyRs has been hindered by a dearth of high-resolution structures. Here we report electron cryo-microscopy structures of the α1 GlyR with strychnine, glycine, or glycine/ivermectin. Strychnine arrests the receptor in an antagonist-bound, closed ion channel state, glycine stabilizes the receptor in an agonist-bound open channel state, and the glycine/ivermectin complex adopts a potentially desensitized or partially open state. Relative to the glycine-bound state, strychnine expands the agonist-binding pocket via outward movement of the C loop, promotes rearrangement of the extracellular and transmembrane domain ‘wrist’ interface, and leads to rotation of the transmembrane domain toward the pore axis, occluding the ion conduction pathway. These structures illuminate GlyR mechanism and define a rubric to interpret structures of Cys-loop receptors. PMID:26344198

  3. 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.

  4. Electron microscopy of iron chalcogenide FeTe(Se) films

    SciTech Connect

    Shchichko, I. O.; Presnyakov, M. Yu.; Stepantsov, E. A.; Kazakov, S. M.; Antipov, E. V.; Makarova, I. P.; Vasil’ev, A. L.

    2015-05-15

    The structure of Fe{sub 1+δ}Te{sub 1−x}Se{sub x} films (x = 0; 0.05) grown on single-crystal MgO and LaAlO{sub 3} substrates has been investigated by transmission and scanning transmission electron microscopy. The study of Fe{sub 1.11}Te/MgO structures has revealed two crystallographic orientation relationships between the film and substrate. It is shown that the lattice mismatch between the film and substrate is compensated for by the formation of misfit dislocations. The Burgers vector projection is determined. The stresses in the film can partially be compensated for due to the formation of an intermediate disordered layer. It is shown that a FeTe{sub 0.5}Se{sub 0.5} film grown on a LaAlO{sub 3} substrate is single-crystal and that the FeTe{sub 0.5}Se{sub 0.5}/LaAlO{sub 3} interface in a selected region is coherent. The orientation relationships between the film and substrate are also determined for this case.

  5. 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

  6. 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.

  7. 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.

  8. Histological preparation of developing vestibular otoconia for scanning electron microscopy

    NASA Technical Reports Server (NTRS)

    Huss, D.; Dickman, J. D.

    2003-01-01

    The unique nature of vestibular otoconia as calcium carbonate biominerals makes them particularly susceptible to chemical deformation during histological processing. We fixed and stored otoconia from all three otolith endorgans of embryonic, hatchling and adult Japanese quail in glutaraldehyde containing either phosphate or non-phosphate buffers for varying lengths of time and processed them for scanning electron microscopy. Otoconia from all age groups and otolith endorgans processed in 0.1 M phosphate buffer (pH 7.4) showed abnormal surface morphology when compared to acetone fixed controls. Otoconia processed in 0.1 M sodium cacodylate or HEPES buffered artificial endolymph (pH 7.4) showed normal morphology that was similar to controls. The degree of otoconial deformation was directly related to the time exposed to phosphate buffer. Short duration exposure produced particulate deformations while longer exposures resulted in fused otoconia that formed solid sheets. Otoconial surface deformation and fusing was independent of the glutaraldehyde component of the histological processing. These findings should help vestibular researchers to develop appropriate histological processing protocols in future studies of otoconia.

  9. Applications of Direct Detection Device in Transmission Electron Microscopy

    PubMed Central

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

    2008-01-01

    A prototype Direct Detection Device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120–400 keV beam energies (Xuong, et al., 2007. Methods in Cell Biology, 79, 721–739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 μm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system. PMID:18054249

  10. 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

  11. 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.

  12. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy.

    PubMed

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Swann, Karl; Borri, Paola

    2016-06-15

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  13. Quantitative imaging of lipids in live mouse oocytes and early embryos using CARS microscopy

    PubMed Central

    Bradley, Josephine; Pope, Iestyn; Masia, Francesco; Sanusi, Randa; Langbein, Wolfgang; Borri, Paola

    2016-01-01

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. PMID:27151947

  14. 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

  15. 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 μm 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.

  16. 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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

    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.

  18. 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

  19. 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

  20. 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.

  1. 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.

  2. 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

  3. 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

  4. Large-scale automatic reconstruction of neuronal processes from electron microscopy images.

    PubMed

    Kaynig, Verena; Vazquez-Reina, Amelio; Knowles-Barley, Seymour; Roberts, Mike; Jones, Thouis R; Kasthuri, Narayanan; Miller, Eric; Lichtman, Jeff; Pfister, Hanspeter

    2015-05-01

    Automated sample preparation and electron microscopy enables acquisition of very large image data sets. These technical advances are of special importance to the field of neuroanatomy, as 3D reconstructions of neuronal processes at the nm scale can provide new insight into the fine grained structure of the brain. Segmentation of large-scale electron microscopy data is the main bottleneck in the analysis of these data sets. In this paper we present a pipeline that provides state-of-the art reconstruction performance while scaling to data sets in the GB-TB range. First, we train a random forest classifier on interactive sparse user annotations. The classifier output is combined with an anisotropic smoothing prior in a Conditional Random Field framework to generate multiple segmentation hypotheses per image. These segmentations are then combined into geometrically consistent 3D objects by segmentation fusion. We provide qualitative and quantitative evaluation of the automatic segmentation and demonstrate large-scale 3D reconstructions of neuronal processes from a 27,000 μm(3) volume of brain tissue over a cube of 30 μm in each dimension corresponding to 1000 consecutive image sections. We also introduce Mojo, a proofreading tool including semi-automated correction of merge errors based on sparse user scribbles. PMID:25791436

  5. An analytical electron microscopy study of paraequilibrium cementite precipitation in ultra-high-strength steel

    NASA Astrophysics Data System (ADS)

    Ghosh, G.; Olson, G. B.; Campbell, C. E.

    1999-03-01

    To support quantitative design of ultra-high-strength (UHS) secondary-hardening steels, the precipitation of cementite prior to the precipitation of the M2C phase is investigated using a model alloy. The microstructure of cementite is investigated by transmission electron microscopy (TEM) techniques. Consistent with earlier studies on tempering of Fe-C martensite, lattice imaging of cementite suggests microsyntactic intergrowth of M5C2 (Hägg carbide). The concentration of substitutional alloying elements in cementite are quantified by high-resolution analytical electron microscopy (AEM) using extraction replica specimens. Quantification of the substitutional elements in cementite confirms its paraequilibrium (PE) state with ferrite at the very early stage of tempering. The implications of these results are discussed in terms of the thermodynamic driving force for nucleation of the primary-strengthening, coherent M2C carbide phase. The ferrite-cementite PE condition reduces the carbon concentration in the ferrite matrix with a significant reduction of M2C driving force. The kinetics of dissolution of PE cementite and its transition to other intermediate states will also influence the kinetics of secondary hardening behavior in UHS steels.

  6. Characterizing nanoscale scanning probes using electron microscopy: A novel fixture and a practical guide

    NASA Astrophysics Data System (ADS)

    Jacobs, Tevis D. B.; Wabiszewski, Graham E.; Goodman, Alexander J.; Carpick, Robert W.

    2016-01-01

    The nanoscale geometry of probe tips used for atomic force microscopy (AFM) measurements determines the lateral resolution, contributes to the strength of the tip-surface interaction, and can be a significant source of uncertainty in the quantitative analysis of results. While inverse imaging of the probe tip has been used successfully to determine probe tip geometry, direct observation of the tip profile using electron microscopy (EM) confers several advantages: it provides direct (rather than indirect) imaging, requires fewer algorithmic parameters, and does not require bringing the tip into contact with a sample. In the past, EM-based observation of the probe tip has been achieved using ad hoc mounting methods that are constrained by low throughput, the risk of contamination, and repeatability issues. We report on a probe fixture designed for use in a commercial transmission electron microscope that enables repeatable mounting of multiple AFM probes as well as a reference grid for beam alignment. This communication describes the design, fabrication, and advantages of this probe fixture, including full technical drawings for machining. Further, best practices are discussed for repeatable, non-destructive probe imaging. Finally, examples of the fixture's use are described, including characterization of common commercial AFM probes in their out-of-the-box condition.

  7. Characterizing nanoscale scanning probes using electron microscopy: A novel fixture and a practical guide.

    PubMed

    Jacobs, Tevis D B; Wabiszewski, Graham E; Goodman, Alexander J; Carpick, Robert W

    2016-01-01

    The nanoscale geometry of probe tips used for atomic force microscopy (AFM) measurements determines the lateral resolution, contributes to the strength of the tip-surface interaction, and can be a significant source of uncertainty in the quantitative analysis of results. While inverse imaging of the probe tip has been used successfully to determine probe tip geometry, direct observation of the tip profile using electron microscopy (EM) confers several advantages: it provides direct (rather than indirect) imaging, requires fewer algorithmic parameters, and does not require bringing the tip into contact with a sample. In the past, EM-based observation of the probe tip has been achieved using ad hoc mounting methods that are constrained by low throughput, the risk of contamination, and repeatability issues. We report on a probe fixture designed for use in a commercial transmission electron microscope that enables repeatable mounting of multiple AFM probes as well as a reference grid for beam alignment. This communication describes the design, fabrication, and advantages of this probe fixture, including full technical drawings for machining. Further, best practices are discussed for repeatable, non-destructive probe imaging. Finally, examples of the fixture's use are described, including characterization of common commercial AFM probes in their out-of-the-box condition. PMID:26827324

  8. 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

  9. 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.

  10. 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

  11. EDITORIAL: Electron Microscopy and Analysis Group Conference 2013 (EMAG2013)

    NASA Astrophysics Data System (ADS)

    Nellist, Pete

    2014-06-01

    It has once again been my pleasure to act as editor for these proceedings, and I must thank all those who have acted as reviewers. I am always struck by the scientific quality of the oral and poster contributions and the vibrant discussions that occur both in the formal sessions and in the exhibition space at EMAG. I am convinced that a crucial part of maintaining that scientific quality is the opportunity that is offered of having a paper fully reviewed by two internationally selected referees and published in the Journal of Physics: Conference Series. For many students, this is the first fully reviewed paper they publish. I hope that, like me, you will be struck by the scientific quality of the 80 papers that follow, and that you will find them interesting and informative. I must also personally thank all the organisers of EMAG2013 for arranging such an excellent meeting. Ian MacLaren, as Chair of the EMAG Group and of the meeting itself, has contributed a foreword to these proceedings describing the meeting in more detail. A particular highlight of the conference was the special symposium in honour of Professor Archie Howie. We all enjoyed a wonderful speech from Archie at the conference dinner, along with some of his electron microscopy-related poetry. I have great pleasure in publishing the conference dinner poems in this proceedings. I hope you will find these proceedings to be an interesting read and an invaluable resource. Pete Nellist Conference committee Conference chair: Dr I MacLaren Programme organiser: Dr C Ducati Proceedings editor: Prof P D Nellist Trade exhibition organiser: C Hockey (CEM Group) Local organisers: Professor E Boyes, Professor P Gai, Dr R Kröger, Dr V Lazarov, Dr P O'Toole, Dr S Tear and Professor J Yuan Advanced school organisers: Dr S Haigh, Dr A Brown Other committee members: Mr K Meade, Mr O Heyning, Dr M Crawford, Mr M Dixon and Dr Z Li

  12. [High resolution scanning electron microscopy of isolated outer hair cells].

    PubMed

    Koitschev, A; Müller, H

    1996-11-01

    Isolated hair cell preparations have gained wide acceptance as a model for studying physiological and molecular properties of the sensory cells involved in the hearing process. Ultrastructural details, such as stereocilia links, lateral membrane substructure or synaptic links are of crucial importance for normal sensory transduction. For this reason, we developed a high-resolution scanning electron microscopy (SEM) procedure to study the surface of isolated hair cells. Cells were mechanically and/or enzymatically separated, isolated and immobilized on cover slips by alcian blue and fixed by 2% glutardialdehyde or 1% OsO4. After dehydration, preparations were critical point-dried and sputter-coated with gold-palladium (2-4 nm). Up to 5 nm resolution was achieved. Optimal fixation kept the cells in their typical cylindrical forms. Preservation of the stereocilia and the apical plates of the outer hair cells depended strongly on the fixation process. Tip- and side-links were observed only sporadically because of the aggressive preparation procedure. The lateral plasma membranes of the cell bodies showed regular granular structures of 5-7 nm diameter at maximal magnification. The granular structure of the cell membrane seemed to correspond to putative transmembrane proteins believed to generate membrane-based motility. The remnants of the nerve endings and/or supporting cells usually covered the cell base. The preservation of the cells was better when enzymatic isolation was omitted. The technique used allowed for high resolution ultrastructural examination of isolated hair cells and, when combined with immunological labeling, may permit the identification of proteins at a molecular level. PMID:9064297

  13. Transmission Electron Microscopy of Iron Metal in Almahata Sitta Ureilite

    NASA Technical Reports Server (NTRS)

    Mikouchi, T.; Yubuta, K.; Sugiyama, K.; Aoyagi, Y.; Yasuhara, A.; Mihira, T.; Zolensky, M. E.; Goodrich, C. A.

    2013-01-01

    Almahata Sitta (AS) is a polymict breccia mainly composed of variable ureilite lithologies with small amounts of chondritic lithologies [1]. Fe metal is a common accessory phase in ureilites, but our earlier study on Fe metals in one of AS fragments (#44) revealed a unique mineralogy never seen in other ureilites [2,3]. In this abstract we report detailed transmission electron microscopy (TEM) on these metal grains to better understand the thermal history of ureilites. We prepared FIB sections of AS#44 by JEOL JIB-4000 from the PTS that was well characterized by SEM-EBSD in our earlier study [2]. The sections were then observed by STEM (JEOL JEM- 2100F). One of the FIB sections shows a submicron-sized symplectic intergrown texture composed of Fe metal (kamacite), Fe carbide (cohenite), Fe phosphide (schreibersite), and Fe sulfide (troilite). Each phase has an identical SAED pattern in spite of its complex texture, suggesting co-crystallization of all phases. This is probably caused by shock re-melting of pre-existing metal + graphite to form a eutectic-looking texture. The other FIB section is mostly composed of homogeneous Fe metal (93 wt% Fe, 5 wt% Ni, and 2 wt% Si), but BF-STEM images exhibited the presence of elongated lathy grains (approx. 2 microns long) embedded in the interstitial matrix. The SAED patterns from these lath grains could be indexed by alpha-Fe (bcc) while interstitial areas are gamma-Fe (fcc). The elongated alpha-Fe grains show tweed-like structures suggesting martensite transformation. Such a texture can be formed by rapid cooling from high temperature where gamma-Fe was stable. Subsequently alpha-Fe crystallized, but gamma-Fe remained in the interstitial matrix due to quenching from high temperature. This scenario is consistent with very rapid cooling history of ureilites suggested by silicate mineralogy.

  14. Structure and assembly of haptoglobin polymers by electron microscopy.

    PubMed

    Wejman, J C; Hovsepian, D; Wall, J S; Hainfeld, J F; Greer, J

    1984-04-01

    Haptoglobin (Hp) consists of light (L) and heavy (H) chains, the latter of which combine with hemoglobin alpha beta dimers to form a highly stable complex. Human haptoglobin assembles as HL units that occur in two allelic forms; HL1 , which is monovalent, and HL2 , which is divalent. As a result, three phenotypic forms exist in the human population: Hp1-1, the homozygous form in which the monovalent HL1 unit occurs as a dimer; Hp2-2, the homozygous form of the divalent HL2 unit, which gives a series of polymers; and the heterozygous Hp2-1 form, which gives a different series of polymers. We have investigated the structures and assembly properties of these two haptoglobin polymeric series in their complexes with hemoglobin using high-resolution scanning transmission electron microscopy. Polymers of complex are composed of ellipsoidal or bilobal head groups, which are the H alpha beta subunits connected by thin filament-like structures, which are the L chains. Polymers of size up to pentamers can be identified easily by counting the number of head groups in the molecule. Complex 2-1 and complex 2-2 trimers were studied extensively. The differences in detailed morphology show that while the 2-1 trimer is a linear polymer, the 2-2 trimer is a closed circular molecule. The micrograph images suggest that complex 2-2 tetramers and pentamers, and perhaps higher forms may also be cyclic. The structure of the L2 subunit of haptoglobin is shown to be composed of two domains, which may be similar in structure to the single domain of the monovalent L1 chain. The two L2 domains are connected by a hinge that has quite limited flexibility. Using these structural models, assembly characteristics and structural properties of the trimers and tetramers of complex 2-1 and complex 2-2 are described. PMID:6716482

  15. Effects of Auger electron elastic scattering in quantitative AES

    NASA Astrophysics Data System (ADS)

    Jablonski, Aleksander

    1987-09-01

    The Monte Carlo algorithm was developed for simulating the trajectories of electrons elastically scattered in the solid. The distribution of scattering angles was determined using the partial wave expansion method. This algorithm was used to establish the influence of Auger electron elastic collisions on the results of quantitative AES analysis. The calculations were performed for the most pronounced KLL, L 3 MM and M 5NN Auger transitions. It turned out that due to the elastic collisions the Auger electron signal is decreased by up to 10%. The corresponding decreased of the escape depth of Auger electrons reaches 30% as compared with the value derived from the inelastic mean free path. The values of the inelastic mean free path resulting from the overalyer method may be strongly affected by elastic scattering of Auger electrons.

  16. Quantitative electron phase imaging with high sensitivity and an unlimited field of view

    PubMed Central

    Maiden, A. M.; Sarahan, M. C.; Stagg, M. D.; Schramm, S. M.; Humphry, M. J.

    2015-01-01

    As it passes through a sample, an electron beam scatters, producing an exit wavefront rich in information. A range of material properties, from electric and magnetic field strengths to specimen thickness, strain maps and mean inner potentials, can be extrapolated from its phase and mapped at the nanoscale. Unfortunately, the phase signal is not straightforward to obtain. It is most commonly measured using off-axis electron holography, but this is experimentally challenging, places constraints on the sample and has a limited field of view. Here we report an alternative method that avoids these limitations and is easily implemented on an unmodified transmission electron microscope (TEM) operating in the familiar selected area diffraction mode. We use ptychography, an imaging technique popular amongst the X-ray microscopy community; recent advances in reconstruction algorithms now reveal its potential as a tool for highly sensitive, quantitative electron phase imaging. PMID:26423558

  17. Transmission electron microscopy investigation of auto catalyst and cobalt germanide

    NASA Astrophysics Data System (ADS)

    Sun, Haiping

    The modern ceria-zirconia based catalysts are used in automobiles to reduce exhaust pollutants. Cobalt germanides have potential applications as electrical contacts in the future Ge-based semiconductor devices. In this thesis, transmission electron microscopy (TEM) techniques were used to study the atomic scale interactions between metallic nanostructures and crystalline substrates in the two material systems mentioned above. The model catalyst samples consisted of precious metal nano-particles (Pd, Rh) supported on the surface of (Ce,Zr)O2 thin films. The response of the microstructure of the metal-oxide interface to the reduction and oxidation treatments was investigated by cross-sectional high resolution TEM. Atomic detail of the metal-oxide interface was obtained. It was found that Pd and Rh showed different sintering and interaction behaviors on the oxide surface. The preferred orientation of Pd particles in this study was Pd(111)//CZO(111). Partial encapsulation of Pd particles by reduced (Ce,Zr)O 2 surface was observed and possible mechanisms of the encapsulation were discussed. The characteristics of the metal-oxide interaction depend on the properties of the oxide, as well as their relative orientation. The results provide experimental evidence for understanding the thermodynamics of the equilibrium morphology of a solid particle supported on a solid surface that is not considered as inert. The reaction of Co with Ge to form epitaxial Co5Ge7 was studied by in situ ultra-high vacuum (UHV) TEM using two methods. One was reactive deposition of Co on Ge, in which the Ge substrate was maintained at 350°C during deposition. The other method was solid state reaction, in which the deposition of Co on Ge was carried out at room temperature followed by annealing to higher temperatures. During reactive deposition, the deposited Co reacted with Ge to form nanosized 3D Co 5Ge7 islands. During solid state reaction, a continuous epitaxial Co5Ge7 film on the (001) Ge

  18. Unraveling irradiation induced grain growth with in situ transmission electron microscopy and coordinated modeling

    SciTech Connect

    Bufford, D. C.; Abdeljawad, F. F.; Foiles, S. M.; Hattar, K.

    2015-11-09

    Nanostructuring has been proposed as a method to enhance radiation tolerance, but many metallic systems are rejected due to significant concerns regarding long term grain boundary and interface stability. This work utilized recent advancements in transmission electron microscopy (TEM) to quantitatively characterize the grain size, texture, and individual grain boundary character in a nanocrystalline gold model system before and after in situ TEM ion irradiation with 10 MeV Si. The initial experimental measurements were fed into a mesoscale phase field model, which incorporates the role of irradiation-induced thermal events on boundary properties, to directly compare the observed and simulated grain growth with varied parameters. The observed microstructure evolution deviated subtly from previously reported normal grain growth in which some boundaries remained essentially static. In broader terms, the combined experimental and modeling techniques presented herein provide future avenues to enhance quantification and prediction of the thermal, mechanical, or radiation stability of grain boundaries in nanostructured crystalline systems.

  19. Automatic detection and morphological delineation of bacteriophages in electron microscopy images.

    PubMed

    Gelzinis, A; Verikas, A; Vaiciukynas, E; Bacauskiene, M; Sulcius, S; Simoliunas, E; Staniulis, J; Paskauskas, R

    2015-09-01

    Automatic detection, recognition and geometric characterization of bacteriophages in electron microscopy images was the main objective of this work. A novel technique, combining phase congruency-based image enhancement, Hough transform-, Radon transform- and open active contours with free boundary conditions-based object detection was developed to detect and recognize the bacteriophages associated with infection and lysis of cyanobacteria Aphanizomenon flos-aquae. A random forest classifier designed to recognize phage capsids provided higher than 99% accuracy, while measurable phage tails were detected and associated with a correct capsid with 81.35% accuracy. Automatically derived morphometric measurements of phage capsids and tails exhibited lower variability than the ones obtained manually. The technique allows performing precise and accurate quantitative (e.g. abundance estimation) and qualitative (e.g. diversity and capsid size) measurements for studying the interactions between host population and different phages that infect the same host. PMID:26164031

  20. Unraveling irradiation induced grain growth with in situ transmission electron microscopy and coordinated modeling

    NASA Astrophysics Data System (ADS)

    Bufford, D. C.; Abdeljawad, F. F.; Foiles, S. M.; Hattar, K.

    2015-11-01

    Nanostructuring has been proposed as a method to enhance radiation tolerance, but many metallic systems are rejected due to significant concerns regarding long term grain boundary and interface stability. This work utilized recent advancements in transmission electron microscopy (TEM) to quantitatively characterize the grain size, texture, and individual grain boundary character in a nanocrystalline gold model system before and after in situ TEM ion irradiation with 10 MeV Si. The initial experimental measurements were fed into a mesoscale phase field model, which incorporates the role of irradiation-induced thermal events on boundary properties, to directly compare the observed and simulated grain growth with varied parameters. The observed microstructure evolution deviated subtly from previously reported normal grain growth in which some boundaries remained essentially static. In broader terms, the combined experimental and modeling techniques presented herein provide future avenues to enhance quantification and prediction of the thermal, mechanical, or radiation stability of grain boundaries in nanostructured crystalline systems.

  1. Big Data and Deep data in scanning and electron microscopies: functionality from multidimensional data sets

    SciTech Connect

    Belianinov, Alex; Vasudevan, Rama K; Strelcov, Evgheni; Steed, Chad A; Yang, Sang Mo; Tselev, Alexander; Jesse, Stephen; Biegalski, Michael D; Shipman, Galen M; Symons, Christopher T; Borisevich, Albina Y; Archibald, Richard K; Kalinin, Sergei

    2015-01-01

    The development of electron, and scanning probe microscopies in the second half of the twentieth century have produced spectacular images of internal structure and composition of matter with, at nanometer, molecular, and atomic resolution. Largely, this progress was enabled by computer-assisted methods of microscope operation, data acquisition and analysis. The progress in imaging technologies in the beginning of the twenty first century has opened the proverbial floodgates of high-veracity information on structure and functionality. High resolution imaging now allows information on atomic positions with picometer precision, allowing for quantitative measurements of individual bond length and angles. Functional imaging often leads to multidimensional data sets containing partial or full information on properties of interest, acquired as a function of multiple parameters (time, temperature, or other external stimuli). Here, we review several recent applications of the big and deep data analysis methods to visualize, compress, and translate this data into physically and chemically relevant information from imaging data.

  2. Avoiding drying-artifacts in transmission electron microscopy: Characterizing the size and colloidal state of nanoparticles

    PubMed Central

    Michen, Benjamin; Geers, Christoph; Vanhecke, Dimitri; Endes, Carola; Rothen-Rutishauser, Barbara; Balog, Sandor; Petri-Fink, Alke

    2015-01-01

    Standard transmission electron microscopy nanoparticle sample preparation generally requires the complete removal of the suspending liquid. Drying often introduces artifacts, which can obscure the state of the dispersion prior to drying and preclude automated image analysis typically used to obtain number-weighted particle size distribution. Here we present a straightforward protocol for prevention of the onset of drying artifacts, thereby allowing the preservation of in-situ colloidal features of nanoparticles during TEM sample preparation. This is achieved by adding a suitable macromolecular agent to the suspension. Both research- and economically-relevant particles with high polydispersity and/or shape anisotropy are easily characterized following our approach (http://bsa.bionanomaterials.ch), which allows for rapid and quantitative classification in terms of dimensionality and size: features that are major targets of European Union recommendations and legislation. PMID:25965905

  3. Detection of Secondary and Supersecondary Structures of Proteins from Cryo-Electron Microscopy

    PubMed Central

    Bajaj, Chandrajit; Goswami, Samrat; Zhang, Qin

    2012-01-01

    Recent advances in three-dimensional electron microscopy (3D EM) have enabled the quantitative visualization of the structural building blocks of proteins at improved resolutions. We provide algorithms to detect the secondary structures (α-helices and β-sheets) from proteins for which the volumetric maps are reconstructed at 6–10Å resolution. Additionally, we show that when the resolution is coarser than 10Å, some of the super-secondary structures can be detected from 3D EM maps. For both these algorithms, we employ tools from computational geometry and differential topology, specifically the computation of stable/unstable manifolds of certain critical points of the distance function induced by the molecular surface. Our results connect mathematically well-defined constructions with bio-chemically induced structures observed in proteins. PMID:22186625

  4. Visualization of Microbial Biomarkers by Scanning Electron Microscopy

    NASA Technical Reports Server (NTRS)

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

    2001-01-01

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

  5. Ultra-high vacuum scanning thermal microscopy for nanometer resolution quantitative thermometry.

    PubMed

    Kim, Kyeongtae; Jeong, Wonho; Lee, Woochul; Reddy, Pramod

    2012-05-22

    Understanding energy dissipation at the nanoscale requires the ability to probe temperature fields with nanometer resolution. Here, we describe an ultra-high vacuum (UHV)-based scanning thermal microscope (SThM) technique that is capable of quantitatively mapping temperature fields with ∼15 mK temperature resolution and ∼10 nm spatial resolution. In this technique, a custom fabricated atomic force microscope (AFM) cantilever, with a nanoscale Au-Cr thermocouple integrated into the tip of the probe, is used to measure temperature fields of surfaces. Operation in an UHV environment eliminates parasitic heat transport between the tip and the sample enabling quantitative measurement of temperature fields on metal and dielectric surfaces with nanoscale resolution. We demonstrate the capabilities of this technique by directly imaging thermal fields in the vicinity of a 200 nm wide, self-heated, Pt line. Our measurements are in excellent agreement with computational results-unambiguously demonstrating the quantitative capabilities of the technique. UHV-SThM techniques will play an important role in the study of energy dissipation in nanometer-sized electronic and photonic devices and the study of phonon and electron transport at the nanoscale. PMID:22530657

  6. The probe profile and lateral resolution of scanning transmission electron microscopy of thick specimens.

    PubMed

    Demers, Hendrix; Ramachandra, Ranjan; Drouin, Dominique; de Jonge, Niels

    2012-06-01

    Lateral profiles of the electron probe of scanning transmission electron microscopy (STEM) were simulated at different vertical positions in a micrometers-thick carbon sample. The simulations were carried out using the Monte Carlo method in CASINO software. A model was developed to fit the probe profiles. The model consisted of the sum of a Gaussian function describing the central peak of the profile and two exponential decay functions describing the tail of the profile. Calculations were performed to investigate the fraction of unscattered electrons as a function of the vertical position of the probe in the sample. Line scans were also simulated over gold nanoparticles at the bottom of a carbon film to calculate the achievable resolution as a function of the sample thickness and the number of electrons. The resolution was shown to be noise limited for film thicknesses less than 1 μm. Probe broadening limited the resolution for thicker films. The validity of the simulation method was verified by comparing simulated data with experimental data. The simulation method can be used as quantitative method to predict STEM performance or to interpret STEM images of thick specimens. PMID:22564444

  7. Atomic force microscopy and scanning electron microscopy analysis of daily disposable limbal ring contact lenses

    PubMed Central

    Lorenz, Kathrine Osborn; Kakkassery, Joseph; Boree, Danielle; Pinto, David

    2014-01-01

    Background Limbal ring (also known as ‘circle’) contact lenses are becoming increasingly popular, especially in Asian markets because of their eye-enhancing effects. The pigment particles that give the eye-enhancing effects of these lenses can be found on the front or back surface of the contact lens or ‘enclosed’ within the lens matrix. The purpose of this research was to evaluate the pigment location and surface roughness of seven types of ‘circle’ contact lenses. Methods Scanning electron microscopic (SEM) analysis was performed using a variable pressure Hitachi S3400N instrument to discern the placement of lens pigments. Atomic force microscopy (Dimension Icon AFM from Bruker Nano) was used to determine the surface roughness of the pigmented regions of the contact lenses. Atomic force microscopic analysis was performed in fluid phase under contact mode using a Sharp Nitride Lever probe (SNL-10) with a spring constant of 0.06 N/m. Root mean square (RMS) roughness values were analysed using a generalised linear mixed model with a log-normal distribution. Least square means and their corresponding 95% confidence intervals were estimated for each brand, location and pigment combination. Results SEM cross-sectional images at 500× and 2,000× magnification showed pigment on the surface of six of the seven lens types tested. The mean depth of pigment for 1-DAY ACUVUE DEFINE (1DAD) lenses was 8.1 μm below the surface of the lens, while the remaining lens types tested had pigment particles on the front or back surface. Results of the atomic force microscopic analysis indicated that 1DAD lenses had significantly lower root mean square roughness values in the pigmented area of the lens than the other lens types tested. Conclusions SEM and AFM analysis revealed pigment on the surface of the lens for all types tested with the exception of 1DAD. Further research is required to determine if the difference in pigment location influences on-eye performance. PMID

  8. Quantitative X-Ray Magnetic Microscopy: from parallel stripe domains to buried topological defects

    NASA Astrophysics Data System (ADS)

    Velez, Maria; Blanco-Roldan, C.; Quiros, C.; Valdes-Bango, F.; Alvarez-Prado, L. M.; Martin, J. I.; Alameda, J. M.; Hierro-Rodriguez, A.; Duch, M.; Torras, N.; Esteve, J.; Sorrentino, A.; Valcarcel, R.; Pereiro, E.; Ferrer, S.

    Magnetic transmission X-ray microscopy (TXM) is a powerful imaging technique that can produce element specific images of magnetic domains with nanometric lateral resolution. Here we present a novel imaging method in which the angular dependence of the magnetic contrast in a series of high resolution TXM images is used to obtain quantitative descriptions of the magnetization (canting angles and sense). This has been applied first to analyze parallel stripe domains in weak perpendicular anisotropy ferromagnetic NdCo5 layers of different thickness, and in NdCo5/Permalloy bilayers. Then, our method has been used to identify complex topological defects (merons or 1/2 skyrmions) in a NdCo5 film that are only partially replicated by the Permalloy overlayer. Meron propagation in trilayers (across the thickness) and in hexagonal networks (across bifurcations) will be discussed in terms of their topological characteristics (chirality and polarity). Work supported by Spanish Grant FIS2013-45469.

  9. Robust high-resolution imaging and quantitative force measurement with tuned-oscillator atomic force microscopy.

    PubMed

    Dagdeviren, Omur E; Götzen, Jan; Hölscher, Hendrik; Altman, Eric I; Schwarz, Udo D

    2016-02-12

    Atomic force microscopy (AFM) and spectroscopy are based on locally detecting the interactions between a surface and a sharp probe tip. For highest resolution imaging, noncontact modes that avoid tip-sample contact are used; control of the tip's vertical position is accomplished by oscillating the tip and detecting perturbations induced by its interaction with the surface potential. Due to this potential's nonlinear nature, however, achieving reliable control of the tip-sample distance is challenging, so much so that despite its power vacuum-based noncontact AFM has remained a niche technique. Here we introduce a new pathway to distance control that prevents instabilities by externally tuning the oscillator's response characteristics. A major advantage of this operational scheme is that it delivers robust position control in both the attractive and repulsive regimes with only one feedback loop, thereby providing an easy-to-implement route to atomic resolution imaging and quantitative tip-sample interaction force measurement. PMID:26754332

  10. Quantitative phase microscopy using defocusing by means of a spatial light modulator.

    PubMed

    Camacho, Luis; Micó, Vicente; Zalevsky, Zeev; García, Javier

    2010-03-29

    A new method for recovery the quantitative phase information of microscopic samples is presented. It is based on a spatial light modulator (SLM) and digital image processing as key elements to extract the sample's phase distribution. By displaying a set of lenses with different focal power, the SLM produces a set of defocused images of the input sample at the CCD plane. Such recorded images are then numerically processed to retrieve phase information. This iterative process is based on the wave propagation equation and leads on a complex amplitude image containing information of both amplitude and phase distributions of the input sample diffracted wave front. The proposed configuration is a non-interferometric architecture (conventional transmission imaging mode) where no moving elements are included. Experimental results perfectly correlate with the results obtained by conventional digital holographic microscopy (DHM). PMID:20389696

  11. Quantitative microscopy and nanoscopy of sickle red blood cells performed by wide field digital interferometry

    NASA Astrophysics Data System (ADS)

    Shaked, Natan T.; Satterwhite, Lisa L.; Telen, Marilyn J.; Truskey, George A.; Wax, Adam

    2011-03-01

    We have applied wide-field digital interferometry (WFDI) to examine the morphology and dynamics of live red blood cells (RBCs) from individuals who suffer from sickle cell anemia (SCA), a genetic disorder that affects the structure and mechanical properties of RBCs. WFDI is a noncontact, label-free optical microscopy approach that can yield quantitative thickness profiles of RBCs and measurements of their membrane fluctuations at the nanometer scale reflecting their stiffness. We find that RBCs from individuals with SCA are significantly stiffer than those from a healthy control. Moreover, we show that the technique is sensitive enough to distinguish classes of RBCs in SCA, including sickle RBCs with apparently normal morphology, compared to the stiffer crescent-shaped sickle RBCs. We expect that this approach will be useful for diagnosis of SCA and for determining efficacy of therapeutic agents.

  12. Towards quantitative electrochemical measurements on the nanoscale by scanning probe microscopy: environmental and current spreading effects

    SciTech Connect

    Arruda, Thomas M; Kumar, Amit; Veith, Gabriel M; Jesse, Stephen; Tselev, Alexander; Baddorf, Arthur P; Balke, Nina; Kalinin, Sergei V

    2013-01-01

    The application of electric bias across tip-surface junctions in scanning probe microscopy can readily induce surface and bulk electrochemical processes that can be further detected though changes in surface topography, Faradaic or conductive currents, or electromechanical strain responses. However, the basic factors controlling tip-induced electrochemical processes, including the relationship between applied tip bias and the thermodynamics of local processes remains largely unexplored. Using the model Li-ion reduction reaction on the surface in Li-ion conducting glass ceramic, we explore the factors controlling Li-metal formation and find surprisingly strong effects of atmosphere and back electrode composition on the process. These studies suggest the feasibility of SPM-based quantitative electrochemical studies under proper environmental controls, extending the concepts of ultramicroelectrodes to the single-digit nanometer scale.

  13. Quantitative Characterization of Biological Liquids for Third-Harmonic Generation Microscopy

    PubMed Central

    Débarre, Delphine; Beaurepaire, Emmanuel

    2007-01-01

    Third-harmonic generation (THG) microscopy provides images of unstained biological samples based on spatial variations in third-order nonlinear susceptibility, refractive index, and dispersion. In this study, we establish quantitative values for the third-order nonlinear susceptibilities of several solvents (water, ethanol, glycerol), physiological aqueous (ions, amino acids, polypeptides, bovine serum albumin, glucose) and lipid (triglycerides, cholesterol) solutions as a function of solute concentration in the 1.05–1.25 μm excitation range. We use these data in conjunction with imaging experiments to show that THG imaging with ∼1.2 μm excitation lacks specificity and sensitivity to detect physiological ion concentration changes, and that nonaqueous structures such as lipid bodies provide a more robust source of signal. Finally, we illustrate the impact of index-matching liquids in THG images. These data provide a basis for interpreting biological THG images and for developing additional applications. PMID:17085492

  14. Introducing Simulated Cellular Architecture to the Quantitative Analysis of Fluorescent Microscopy

    PubMed Central

    DePristo, Mark A.; Chang, Lynne; Vale, Ronald D.; Khan, Shahid M.; Lipkow, Karen

    2009-01-01

    Biological cells are complex and highly dynamic: Many macromolecules are organized in loose assemblies, clusters or highly structured complexes, others exist most of the time as freely diffusing monomers. They move between regions and compartments through diffusion and enzyme-mediated transport, within a heavily crowded cytoplasm. To make sense of this complexity, computational models, and, in turn, quantitative in vivo data are needed. An array of fluorescent microscopy methods is available, but due to the inherent noise and complexity inside the cell, they are often hard to interpret. Using the example of fluorescence recovery after photobleaching (FRAP) and the bacterial chemotaxis system, we are here introducing detailed spatial simulations as a new approach in analysing such data. PMID:19628003

  15. Quantitative characterization of crosstalk effects for friction force microscopy with scan-by-probe SPMs.

    PubMed

    Prunici, Pavel; Hess, Peter

    2008-06-01

    If the photodetector and cantilever of an atomic force microscope (AFM) are not properly adjusted, crosstalk effects will appear. These effects disturb measurements of the absolute vertical and horizontal cantilever deflections, which are involved in friction force microscopy (FFM). A straightforward procedure is proposed to study quantitatively crosstalk effects observed in scan-by-probe SPMs. The advantage of this simple, fast, and accurate procedure is that no hardware change or upgrade is needed. The results indicate that crosstalk effects depend not only on the alignment of the detector but also on the cantilever properties, position, and detection conditions. The measurements may provide information on the origin of the crosstalk effect. After determination of its magnitude, simple correction formulas can be applied to correct the crosstalk effects and then the single-load wedge method, using a commercially available grating, can be employed for accurate calibration of the lateral force. PMID:18035500

  16. Quantitative analysis of Scanning Tunneling Microscopy images for surface structure determination: Sulfur on Re(0001)

    SciTech Connect

    Ogletree, D.F.; Dunphy, J.C.; Salmeron, M.B.; Sautet, P. |

    1993-02-01

    Scanning Tunneling Microscopy (STM) images of adsorbed atoms and molecules on single crystal substrates provide important information on surface structure and order. In many cases images are interpreted qualitatively based on other information on the system. To obtain quantitative information, a theoretical analysis of the STM image is required. A new method of calculating STM images is presented that includes a full description of the STM tip and surface structure. This method is applied to experimental STM images of sulfur adsorbed on Re(0001). Effects of adsorption site, adsorbate geometry, tip composition and tunnel gap resistance on STM image contrast are analyzed. The chemical identity of tip apex atom and substrate subsurface structure are both shown to significantly affect STM image contrast.

  17. Comparison of Scheimpflug-photography, specular microscopy and scanning electron microscopy to detect corneal changes in toxicity studies in rats

    SciTech Connect

    Boeker, T.W.; Wegener, A.; Koch, F.; Hockwin, O. )

    1990-01-01

    With an increasing number of in-vivo methods to examine the eyes of laboratory animals, the rat has become an important animal model in experimental eye research. Specular microscopy is a clinical tool to examine the corneal endothelium in-vivo. To evaluate the versatility of this method for small animal eyes, we studied both corneal endothelial cell-count and corneal thickness in normal rats as well as those with diabetic, naphthalene and UV-B cataract. As a reference scanning electron microscopy (SEM) of the corneal endothelium was performed. For cell-counts the correlation coefficient between both methods was found to be sufficient. The comparison of corneal thickness measurement (SEM-values) with specular microscopy and with Scheimpflugbiometry failed to show a satisfactory correlation. The study proves that specular microscopy is a useful tool to document changes also in the endothelium of the rat-cornea.

  18. Virtual unfolding of light sheet fluorescence microscopy dataset for quantitative analysis of the mouse intestine

    NASA Astrophysics Data System (ADS)

    Candeo, Alessia; Sana, Ilenia; Ferrari, Eleonora; Maiuri, Luigi; D'Andrea, Cosimo; Valentini, Gianluca; Bassi, Andrea

    2016-05-01

    Light sheet fluorescence microscopy has proven to be a powerful tool to image fixed and chemically cleared samples, providing in depth and high resolution reconstructions of intact mouse organs. We applied light sheet microscopy to image the mouse intestine. We found that large portions of the sample can be readily visualized, assessing the organ status and highlighting the presence of regions with impaired morphology. Yet, three-dimensional (3-D) sectioning of the intestine leads to a large dataset that produces unnecessary storage and processing overload. We developed a routine that extracts the relevant information from a large image stack and provides quantitative analysis of the intestine morphology. This result was achieved by a three step procedure consisting of: (1) virtually unfold the 3-D reconstruction of the intestine; (2) observe it layer-by-layer; and (3) identify distinct villi and statistically analyze multiple samples belonging to different intestinal regions. Even if the procedure has been developed for the murine intestine, most of the underlying concepts have a general applicability.

  19. Quantitative evaluation of software packages for single-molecule localization microscopy.

    PubMed

    Sage, Daniel; Kirshner, Hagai; Pengo, Thomas; Stuurman, Nico; Min, Junhong; Manley, Suliana; Unser, Michael

    2015-08-01

    The quality of super-resolution images obtained by single-molecule localization microscopy (SMLM) depends largely on the software used to detect and accurately localize point sources. In this work, we focus on the computational aspects of super-resolution microscopy and present a comprehensive evaluation of localization software packages. Our philosophy is to evaluate each package as a whole, thus maintaining the integrity of the software. We prepared synthetic data that represent three-dimensional structures modeled after biological components, taking excitation parameters, noise sources, point-spread functions and pixelation into account. We then asked developers to run their software on our data; most responded favorably, allowing us to present a broad picture of the methods available. We evaluated their results using quantitative and user-interpretable criteria: detection rate, accuracy, quality of image reconstruction, resolution, software usability and computational resources. These metrics reflect the various tradeoffs of SMLM software packages and help users to choose the software that fits their needs. PMID:26076424

  20. Nanoscale imaging of buried topological defects with quantitative X-ray magnetic microscopy

    PubMed Central

    Blanco-Roldán, C.; Quirós, C.; Sorrentino, A.; Hierro-Rodríguez, A.; Álvarez-Prado, L. M.; Valcárcel, R.; Duch, M.; Torras, N.; Esteve, J.; Martín, J. I.; Vélez, M.; Alameda, J. M.; Pereiro, E.; Ferrer, S.

    2015-01-01

    Advances in nanoscale magnetism increasingly require characterization tools providing detailed descriptions of magnetic configurations. Magnetic transmission X-ray microscopy produces element specific magnetic domain images with nanometric lateral resolution in films up to ∼100 nm thick. Here we present an imaging method using the angular dependence of magnetic contrast in a series of high resolution transmission X-ray microscopy images to obtain quantitative descriptions of the magnetization (canting angles relative to surface normal and sense). This method is applied to 55–120 nm thick ferromagnetic NdCo5 layers (canting angles between 65° and 22°), and to a NdCo5 film covered with permalloy. Interestingly, permalloy induces a 43° rotation of Co magnetization towards surface normal. Our method allows identifying complex topological defects (merons or ½ skyrmions) in a NdCo5 film that are only partially replicated by the permalloy overlayer. These results open possibilities for the characterization of deeply buried magnetic topological defects, nanostructures and devices. PMID:26337838

  1. Broadband quantitative phase microscopy with extended field of view using off-axis interferometric multiplexing.

    PubMed

    Girshovitz, Pinhas; Frenklach, Irena; Shaked, Natan T

    2015-11-01

    We propose a new portable imaging configuration that can double the field of view (FOV) of existing off-axis interferometric imaging setups, including broadband off-axis interferometers. This configuration is attached at the output port of the off-axis interferometer and optically creates a multiplexed interferogram on the digital camera, which is composed of two off-axis interferograms with straight fringes at orthogonal directions. Each of these interferograms contains a different FOV of the imaged sample. Due to the separation of these two FOVs in the spatial-frequency domain, they can be fully reconstructed separately, while obtaining two complex wavefronts from the sample at once. Since the optically multiplexed off-axis interferogram is recorded by the camera in a single exposure, fast dynamics can be recorded with a doubled imaging area. We used this technique for quantitative phase microscopy of biological samples with extended FOV. We demonstrate attaching the proposed module to a diffractive phase microscopy interferometer, illuminated by a broadband light source. The biological samples used for the experimental demonstrations include microscopic diatom shells, cancer cells, and flowing blood cells. PMID:26440914

  2. Quantitative Intracellular Localization of Cationic Lipid-Nucleic Acid Nanoparticles with Fluorescence Microscopy.

    PubMed

    Majzoub, Ramsey N; Ewert, Kai K; Safinya, Cyrus R

    2016-01-01

    Current activity in developing synthetic carriers of nucleic acids (NA) and small molecule drugs for therapeutic applications is unprecedented. One promising class of synthetic vectors for the delivery of therapeutic NA is PEGylated cationic liposome (CL)-NA nanoparticles (NPs). Chemically modified PEG-lipids can be used to surface-functionalize lipid-NA nanoparticles, allowing researchers to design active nanoparticles that can overcome the various intracellular and extracellular barriers to efficient delivery. Optimization of these functionalized vectors requires a comprehensive understanding of their intracellular pathways. In this chapter we present two distinct methods for investigating the intracellular activity of PEGylated CL-NA NPs using quantitative analysis with fluorescence microscopy.The first method, spatial localization, describes how to prepare fluorescently labeled CL-NA NPs, perform fluorescence microscopy and properly analyze the data to measure the intracellular distribution of nanoparticles and fluorescent signal. We provide software which allows data from multiple cells to be averaged together and yield statistically significant results. The second method, fluorescence colocalization, describes how to label endocytic organelles via Rab-GFPs and generate micrographs for software-assisted NP-endocytic marker colocalization measurements. These tools will allow researchers to study the endosomal trafficking of CL-NA NPs which can guide their design and improve their efficiency. PMID:27436314

  3. Backscattered Electron Microscopy as an Advanced Technique in Petrography.

    ERIC Educational Resources Information Center

    Krinsley, David Henry; Manley, Curtis Robert

    1989-01-01

    Three uses of this method with sandstone, desert varnish, and granite weathering are described. Background information on this technique is provided. Advantages of this type of microscopy are stressed. (CW)

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

    Cancer.gov

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

  5. Hyperspectral and differential CARS microscopy for quantitative chemical imaging in human adipocytes.

    PubMed

    Di Napoli, Claudia; Pope, Iestyn; Masia, Francesco; Watson, Peter; Langbein, Wolfgang; Borri, Paola

    2014-05-01

    In this work, we demonstrate the applicability of coherent anti-Stokes Raman scattering (CARS) micro-spectroscopy for quantitative chemical imaging of saturated and unsaturated lipids in human stem-cell derived adipocytes. We compare dual-frequency/differential CARS (D-CARS), which enables rapid imaging and simple data analysis, with broadband hyperspectral CARS microscopy analyzed using an unsupervised phase-retrieval and factorization method recently developed by us for quantitative chemical image analysis. Measurements were taken in the vibrational fingerprint region (1200-2000/cm) and in the CH stretch region (2600-3300/cm) using a home-built CARS set-up which enables hyperspectral imaging with 10/cm resolution via spectral focussing from a single broadband 5 fs Ti:Sa laser source. Through a ratiometric analysis, both D-CARS and phase-retrieved hyperspectral CARS determine the concentration of unsaturated lipids with comparable accuracy in the fingerprint region, while in the CH stretch region D-CARS provides only a qualitative contrast owing to its non-linear behavior. When analyzing hyperspectral CARS images using the blind factorization into susceptibilities and concentrations of chemical components recently demonstrated by us, we are able to determine vol:vol concentrations of different lipid components and spatially resolve inhomogeneities in lipid composition with superior accuracy compared to state-of-the art ratiometric methods. PMID:24877002

  6. Amplitude modulation atomic force microscopy, is acoustic driving in liquid quantitatively reliable?

    PubMed

    Liu, Fei; Zhao, Cunlu; Mugele, Frieder; van den Ende, Dirk

    2015-09-25

    Measuring quantitative tip-sample interaction forces in dynamic atomic force microscopy in fluids is challenging because of the strong damping of the ambient viscous medium and the fluid-mediated driving forces. This holds in particular for the commonly used acoustic excitation of the cantilever oscillation. Here we present measurements of tip-sample interactions due to conservative DLVO and hydration forces and viscous dissipation forces in aqueous electrolytes using tips with radii varying from typical 20 nm for the DLVO and hydration forces, to 1 μm for the viscous dissipation. The measurements are analyzed using a simple harmonic oscillator model, continuous beam theory with fluid-mediated excitation and thermal noise spectroscopy (TNS). In all cases consistent conservative forces, deviating less than 40% from each other, are obtained for all three approaches. The DLVO forces are even within 5% of the theoretical expectations for all approaches. Accurate measurements of dissipative forces within 15% of the predictions of macroscopic fluid dynamics require the use of TNS or continuous beam theory including fluid-mediated driving. Taking this into account, acoustic driving in liquid is quantitatively reliable. PMID:26335613

  7. Amplitude modulation atomic force microscopy, is acoustic driving in liquid quantitatively reliable?

    NASA Astrophysics Data System (ADS)

    Liu, Fei; Zhao, Cunlu; Mugele, Frieder; van den Ende, Dirk

    2015-09-01

    Measuring quantitative tip-sample interaction forces in dynamic atomic force microscopy in fluids is challenging because of the strong damping of the ambient viscous medium and the fluid-mediated driving forces. This holds in particular for the commonly used acoustic excitation of the cantilever oscillation. Here we present measurements of tip-sample interactions due to conservative DLVO and hydration forces and viscous dissipation forces in aqueous electrolytes using tips with radii varying from typical 20 nm for the DLVO and hydration forces, to 1 μm for the viscous dissipation. The measurements are analyzed using a simple harmonic oscillator model, continuous beam theory with fluid-mediated excitation and thermal noise spectroscopy (TNS). In all cases consistent conservative forces, deviating less than 40% from each other, are obtained for all three approaches. The DLVO forces are even within 5% of the theoretical expectations for all approaches. Accurate measurements of dissipative forces within 15% of the predictions of macroscopic fluid dynamics require the use of TNS or continuous beam theory including fluid-mediated driving. Taking this into account, acoustic driving in liquid is quantitatively reliable.

  8. Quantitative analysis of intrinsic skin aging in dermal papillae by in vivo harmonic generation microscopy

    PubMed Central

    Liao, Yi-Hua; Kuo, Wei-Cheng; Chou, Sin-Yo; Tsai, Cheng-Shiun; Lin, Guan-Liang; Tsai, Ming-Rung; Shih, Yuan-Ta; Lee, Gwo-Giun; Sun, Chi-Kuang

    2014-01-01

    Chronological skin aging is associated with flattening of the dermal-epidermal junction (DEJ), but to date no quantitative analysis focusing on the aging changes in the dermal papillae (DP) has been performed. The aim of the study is to determine the architectural changes and the collagen density related to chronological aging in the dermal papilla zone (DPZ) by in vivo harmonic generation microscopy (HGM) with a sub-femtoliter spatial resolution. We recruited 48 Asian subjects and obtained in vivo images on the sun-protected volar forearm. Six parameters were defined to quantify 3D morphological changes of the DPZ, which we analyzed both manually and computationally to study their correlation with age. The depth of DPZ, the average height of isolated DP, and the 3D interdigitation index decreased with age, while DP number density, DP volume, and the collagen density in DP remained constant over time. In vivo high-resolution HGM technology has uncovered chronological aging-related variations in DP, and sheds light on real-time quantitative skin fragility assessment and disease diagnostics based on collagen density and morphology. PMID:25401037

  9. Investigations of the ultrafast laser induced melt dynamics by means of transient quantitative phase microscopy (TQPm)

    NASA Astrophysics Data System (ADS)

    Mingareev, Ilya; Horn, Alexander

    2008-05-01

    Modifications of bulk aluminum irradiated well above ablation threshold (F < 300 J.cm-2) have been investigated in situ by means of shadowgraphy and transient quantitative phase microscopy (TQPm) using ultrafast laser radiation (tp=80 fs, λ=800 nm). This novel pump-probe technique enables quantitative time-resolved measurements of object's properties, e.g. dimensions of melt droplets and layer thickness or transient refractive index changes. A series of time-resolved phase images of vaporized material and/or melt, which are induced by n=1..8 pulses on an aluminum target, are obtained using TQPm. Dynamics and characteristics of melting, dependence of the ablated material volume on process parameters and thereby induced structural modifications have been studied. An increase of material ejection rate is observed at delay time of approximately τ=300 ns and τ>800 ns after the incident pulse. Transient refractive index modifications have been investigated in technical glass (Schott D263) by means of TQPm. By using high-repetition rate ultra-short pulsed laser radiation (tp=400 fs, λ=1045 nm, frep=1 MHz) focused by a microscope objective (w0 ~ 4 μm) heat accumulation and thereby glass melting as well as welding is enabled. Transient optical phase variation has been measured up to τ=2.1 μs after the incident pulse and can be attributed to the generation of free charge carriers and compression forces inside glass.

  10. Microscopy environment for quantitative spatial and temporal analysis of multicellular interactions

    NASA Astrophysics Data System (ADS)

    Sudar, Damir; Parvin, Bahram; Callahan, Daniel E.; Schwarz, Richard I.; Knowles, David W.; Ortiz de Solorzano, Carlos; Barcellos-Hoff, Mary H.

    2002-05-01

    Quantitative analysis of spatial and temporal concurrent responses of multiple markers in 3-dimensional cell cultures is hampered by the routine mode of sequential image acquisition, measurement and analysis of specific targets. A system was developed for detailed analysis of multi-dimensional, time-sequence responses and in order to relate features in novel and meaningful ways that will further our understanding of basic biology. Optical sectioning of the 3-dimensional structures is achieved with structured light illumination using the Wilson grating as described by Lanni. The automated microscopy system can image multicellular structures and track dynamic events, and is equipped for simultaneous/ sequential imaging of multiple fluorescent markers. Computer-controlled perfusion of external stimuli into the culture system allows (i) real-time observations of multiple cellular responses and (ii) automatic and intelligent adjustment of experimental parameters. This creates a feedback loop in real-time that directs desired responses in a given experiment. On-line image analysis routines provide cell-by-cell measurement results through segmentation and feature extraction (i.e. intensity, localization, etc.), and quantitation of meta-features such as dynamic responses of cells or correlations between different cells. Off-line image and data analysis is used to derive models of the processes involved, which will deepen the understanding of the basic biology.

  11. Quantitative Conductive Atomic Force Microscopy on Single-Walled Carbon Nanotube-Based Polymer Composites.

    PubMed

    Bârsan, Oana A; Hoffmann, Günter G; van der Ven, Leendert G J; de With, Gijsbertus

    2016-08-01

    Conductive atomic force microscopy (C-AFM) is a valuable technique for correlating the electrical properties of a material with its topographic features and for identifying and characterizing conductive pathways in polymer composites. However, aspects such as compatibility between tip material and sample, contact force and area between the tip and the sample, tip degradation and environmental conditions render quantifying the results quite challenging. This study aims at finding the suitable conditions for C-AFM to generate reliable, reproducible, and quantitative current maps that can be used to calculate the resistance in each point of a single-walled carbon nanotube (SWCNT) network, nonimpregnated as well as impregnated with a polymer. The results obtained emphasize the technique's limitation at the macroscale as the resistance of these highly conductive samples cannot be distinguished from the tip-sample contact resistance. Quantitative C-AFM measurements on thin composite sections of 150-350 nm enable the separation of sample and tip-sample contact resistance, but also indicate that these sections are not representative for the overall SWCNT network. Nevertheless, the technique was successfully used to characterize the local electrical properties of the composite material, such as sample homogeneity and resistance range of individual SWCNT clusters, at the nano- and microscale. PMID:27404764

  12. Lab on chip optical imaging of biological sample by quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Memmolo, P.; Miccio, L.; Merola, F.; Gennari, O.; Mugnano, M.; Netti, P. A.; Ferraro, P.

    2015-03-01

    Quantitative imaging and three dimensional (3D) morphometric analysis of flowing and not-adherent cells is an important aspect for diagnostic purposes at Lab on Chip scale. Diagnostics tools need to be quantitative, label-free and, as much as possible, accurate. In recent years digital holography (DH) has been improved to be considered as suitable diagnostic method in several research field. In this paper we demonstrate that DH can be used for retrieving 3D morphometric data for sorting and diagnosis aims. Several techniques exist for 3D morphological study as optical coherent tomography and confocal microscopy, but they are not the best choice in case of dynamic events as flowing samples. Recently, a DH approach, based on shape from silhouette algorithm (SFS), has been developed for 3D shape display and calculation of cells biovolume. Such approach, adopted in combination with holographic optical tweezers (HOT) was successfully applied to cells with convex shape. Unfortunately, it's limited to cells with convex surface as sperm cells or diatoms. Here, we demonstrate an improvement of such procedure. By decoupling thickness information from refractive index ones and combining this with SFS analysis, 3D shape of concave cells is obtained. Specifically, the topography contour map is computed and used to adjust the 3D shape retrieved by the SFS algorithm. We prove the new procedure for healthy red blood cells having a concave surface in their central region. Experimental results are compared with theoretical model.

  13. A CCD Camera with Electron Decelerator for Intermediate Voltage Electron Microscopy

    SciTech Connect

    Downing, Kenneth H; Downing, Kenneth H.; Mooney, Paul E.

    2008-03-17

    Electron microscopists are increasingly turning to Intermediate Voltage Electron Microscopes (IVEMs) operating at 300 - 400 kV for a wide range of studies. They are also increasingly taking advantage of slow-scan charge coupled device (CCD) cameras, which have become widely used on electron microscopes. Under some conditions CCDs provide an improvement in data quality over photographic film, as well as the many advantages of direct digital readout. However, CCD performance is seriously degraded on IVEMs compared to the more conventional 100 kV microscopes. In order to increase the efficiency and quality of data recording on IVEMs, we have developed a CCD camera system in which the electrons are decelerated to below 100 kV before impacting the camera, resulting in greatly improved performance in both signal quality and resolution compared to other CCDs used in electron microscopy. These improvements will allow high-quality image and diffraction data to be collected directly with the CCD, enabling improvements in data collection for applications including high-resolution electron crystallography, single-particle reconstruction of protein structures, tomographic studies of cell ultrastructure and remote microscope operation. This approach will enable us to use even larger format CCD chips that are being developed with smaller pixels.

  14. Dual-modality wide-field photothermal quantitative phase microscopy and depletion of cell populations

    NASA Astrophysics Data System (ADS)

    Turko, Nir A.; Barnea, Itay; Blum, Omry; Korenstein, Rafi; Shaked, Natan T.

    2015-03-01

    We review our dual-modality technique for quantitative imaging and selective depletion of populations of cells based on wide-field photothermal (PT) quantitative phase imaging and simultaneous PT cell extermination. The cells are first labeled by plasmonic gold nanoparticles, which evoke local plasmonic resonance when illuminated by light in a wavelength corresponding to their specific plasmonic resonance peak. This reaction creates changes of temperature, resulting in changes of phase. This phase changes are recorded by a quantitative phase microscope (QPM), producing specific imaging contrast, and enabling bio-labeling in phase microscopy. Using this technique, we have shown discrimination of EGFR over-expressing (EGFR+) cancer cells from EGFR under-expressing (EGFR-) cancer cells. Then, we have increased the excitation power in order to evoke greater temperatures, which caused specific cell death, all under real-time phase acquisition using QPM. Close to 100% of all EGFR+ cells were immediately exterminated when illuminated with the strong excitation beam, while all EGFR- cells survived. For the second experiment, in order to simulate a condition where circulating tumor cells (CTCs) are present in blood, we have mixed the EGFR+ cancer cells with white blood cells (WBCs) from a healthy donor. Here too, we have used QPM to observe and record the phase of the cells as they were excited for selective visualization and then exterminated. The WBCs survival rate was over 95%, while the EGFR+ survival rate was under 5%. The technique may be the basis for real-time detection and controlled treatment of CTCs.

  15. Nanoscale Electronic Inhomogeneity in In_2Se_3 Nanoribbons Revealed by Microwave Impedance Microscopy

    SciTech Connect

    Lai, K.J.

    2010-06-02

    Driven by interactions due to the charge, spin, orbital, and lattice degrees of freedom, nanoscale inhomogeneity has emerged as a new theme for materials with novel properties near multiphase boundaries. As vividly demonstrated in complex metal oxides and chalcogenides, these microscopic phases are of great scientific and technological importance for research in hightemperature superconductors, colossal magnetoresistance effect, phase-change memories, and domain switching operations. Direct imaging on dielectric properties of these local phases,however, presents a big challenge for existing scanning probe techniques. Here, we report the observation of electronic inhomogeneity in indium selenide (In{sub 2}Se{sub 3}) nanoribbons by near-field scanning microwave impedance microscopy. Multiple phases with local resistivity spanning six orders of magnitude are identified as the coexistence of superlattice, simple hexagonal lattice and amorphous structures with {approx}100nm inhomogeneous length scale, consistent with high-resolution transmission electron microscope studies. The atomic-force-microscope-compatible microwave probe is able to perform quantitative sub-surface electronic study in a noninvasive manner. Finally, the phase change memory function in In{sub 2}Se{sub 3} nanoribbon devices can be locally recorded with big signal of opposite signs.

  16. Atomic-Scale Imaging and Spectroscopy for In Situ Liquid Scanning Transmission Electron Microscopy

    SciTech Connect

    Jungjohann, K. L.; Evans, James E.; Aguiar, Jeff; Arslan, Ilke; Browning, Nigel D.

    2012-06-04

    Observation of growth, synthesis, dynamics and electrochemical reactions in the liquid state is an important yet largely unstudied aspect of nanotechnology. The only techniques that can potentially provide the insights necessary to advance our understanding of these mechanisms is simultaneous atomic-scale imaging and quantitative chemical analysis (through spectroscopy) under environmental conditions in the transmission electron microscope (TEM). In this study we describe the experimental and technical conditions necessary to obtain electron energy loss (EEL) spectra from a nanoparticle in colloidal suspension using aberration corrected scanning transmission electron microscopy (STEM) combined with the environmental liquid stage. At a fluid path length below 400 nm, atomic resolution images can be obtained and simultaneous compositional analysis can be achieved. We show that EEL spectroscopy can be used to quantify the total fluid path length around the nanoparticle, and demonstrate characteristic core-loss signals from the suspended nanoparticles can be resolved and analyzed to provide information on the local interfacial chemistry with the surrounding environment. The combined approach using aberration corrected STEM and EEL spectra with the in situ fluid stage demonstrates a plenary platform for detailed investigations of solution based catalysis and biological research.

  17. A toolkit for the characterization of CCD cameras for transmission electron microscopy.

    PubMed

    Vulovic, M; Rieger, B; van Vliet, L J; Koster, A J; Ravelli, R B G

    2010-01-01

    Charge-coupled devices (CCD) are nowadays commonly utilized in transmission electron microscopy (TEM) for applications in life sciences. Direct access to digitized images has revolutionized the use of electron microscopy, sparking developments such as automated collection of tomographic data, focal series, random conical tilt pairs and ultralarge single-particle data sets. Nevertheless, for ultrahigh-resolution work photographic plates are often still preferred. In the ideal case, the quality of the recorded image of a vitrified biological sample would solely be determined by the counting statistics of the limited electron dose the sample can withstand before beam-induced alterations dominate. Unfortunately, the image is degraded by the non-ideal point-spread function of the detector, as a result of a scintillator coupled by fibre optics to a CCD, and the addition of several inherent noise components. Different detector manufacturers provide different types of figures of merit when advertising the quality of their detector. It is hard for most laboratories to verify whether all of the anticipated specifications are met. In this report, a set of algorithms is presented to characterize on-axis slow-scan large-area CCD-based TEM detectors. These tools have been added to a publicly available image-processing toolbox for MATLAB. Three in-house CCD cameras were carefully characterized, yielding, among others, statistics for hot and bad pixels, the modulation transfer function, the conversion factor, the effective gain and the detective quantum efficiency. These statistics will aid data-collection strategy programs and provide prior information for quantitative imaging. The relative performance of the characterized detectors is discussed and a comparison is made with similar detectors that are used in the field of X-ray crystallography. PMID:20057054

  18. Characterization of gold nanoparticle films: Rutherford backscattering spectroscopy, scanning electron microscopy with image analysis, and atomic force microscopy

    SciTech Connect

    Lansåker, Pia C. Niklasson, Gunnar A.; Granqvist, Claes G.; Hallén, Anders

    2014-10-15

    Gold nanoparticle films are of interest in several branches of science and technology, and accurate sample characterization is needed but technically demanding. We prepared such films by DC magnetron sputtering and recorded their mass thickness by Rutherford backscattering spectroscopy. The geometric thickness d{sub g}—from the substrate to the tops of the nanoparticles—was obtained by scanning electron microscopy (SEM) combined with image analysis as well as by atomic force microscopy (AFM). The various techniques yielded an internally consistent characterization of the films. In particular, very similar results for d{sub g} were obtained by SEM with image analysis and by AFM.

  19. Fast Imaging with Inelastically Scattered Electrons by Off-Axis Chromatic Confocal Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne

    2014-04-01

    We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840 eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

  20. Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes

    PubMed Central

    Grassucci, Robert A; Taylor, Derek; Frank, Joachim

    2009-01-01

    This protocol details the steps used for visualizing the frozen-hydrated grids as prepared following the accompanying protocol entitled ‘Preparation of macromolecular complexes for visualization using cryo-electron microscopy.’ This protocol describes how to transfer the grid to the microscope using a standard cryo-transfer holder or, alternatively, using a cryo-cartridge loading system, and how to collect low-dose data using an FEI Tecnai transmission electron microscope. This protocol also summarizes and compares the various options that are available in data collection for three-dimensional (3D) single-particle reconstruction. These options include microscope settings, choice of detectors and data collection strategies both in situations where a 3D reference is available and in the absence of such a reference (random-conical and common lines). PMID:18274535