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Sample records for live microscopy film

  1. Scanning Ion Conductance Microscopy of Live Keratinocytes

    NASA Astrophysics Data System (ADS)

    Hegde, V.; Mason, A.; Saliev, T.; Smith, F. J. D.; McLean, W. H. I.; Campbell, P. A.

    2012-07-01

    Scanning ion conductance microscopy (SICM) is perhaps the least well known technique from the scanning probe microscopy (SPM) family of instruments. As with its more familiar counterpart, atomic force microscopy (AFM), the technique provides high-resolution topographic imaging, with the caveat that target structures must be immersed in a conducting solution so that a controllable ion current may be utilised as the basis for feedback. In operation, this non-contact characteristic of SICM makes it ideal for the study of delicate structures, such as live cells. Moreover, the intrinsic architecture of the instrument, incorporating as it does, a scanned micropipette, lends itself to combination approaches with complementary techniques such as patch-clamp electrophysiology: SICM therefore boasts the capability for both structural and functional imaging. For the present observations, an ICnano S system (Ionscope Ltd., Melbourn, UK) operating in 'hopping mode' was used, with the objective of assessing the instrument's utility for imaging live keratinocytes under physiological buffers. In scans employing cultured HaCaT cells (spontaneously immortalised, human keratinocytes), we compared the qualitative differences of live cells imaged with SICM and AFM, and also with their respective counterparts after chemical fixation in 4% paraformaldehyde. Characteristic surface microvilli were particularly prominent in live cell imaging by SICM. Moreover, time lapse SICM imaging on live cells revealed that changes in the pattern of microvilli could be tracked over time. By comparison, AFM imaging on live cells, even at very low contact forces (

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

    PubMed Central

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

    2016-01-01

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

  3. Live cell imaging by multifocal multiphoton microscopy.

    PubMed

    Straub, M; Lodemann, P; Holroyd, P; Jahn, R; Hell, S W

    2000-10-01

    Multifocal multiphoton microscopy (MMM) permits parallel multiphoton excitation by scanning an array of high numerical aperture foci across a plane in the sample. MMM is particularly suitable for live cell investigations since it combines advantages of standard multiphoton microscopy such as optical sectioning and suppression of out-of-focus phototoxicity with high recording speeds. Here we describe several applications of MMM to live cell imaging using the neuroendocrine cell line PC12 and bovine chromaffin cells. Stainings were performed with the acidophilic dye acridine orange and the lipophilic dyes FM1-43 and Fast DiA as well as by transfection of the cells with GFP. In both bovine chromaffin and PC12 cells structural elements of nuclear chromatin and the 3-D distribution of acidic organelles inside the cells were visualized. In PC12 cells differentiated by nerve growth factor examples of neurites were monitored. Stainings of membranes were used to reconstruct the morphology of cells and neurites in three dimensions by volume-rendering and by isosurface plots. 3-D reconstructions were composed from stacks of about 50 images each with a diameter of 30-100 microm that were acquired within a few seconds. We conclude that MMM proves to be a technically simple and very effective method for fast 3-D live cell imaging at high resolution.

  4. Circumventing photodamage in live-cell microscopy

    PubMed Central

    Magidson, Valentin; Khodjakov, Alexey

    2013-01-01

    Fluorescence microscopy has become an essential tool in cell biology. This technique allows researchers to visualize the dynamics of tissue, cells, individual organelles and macromolecular assemblies inside the cell. Unfortunately, fluorescence microscopy is not completely ‘non-invasive’ as the high-intensity excitation light required for excitation of fluorophores is inherently toxic for live cells. Physiological changes induced by excessive illumination can lead to artifacts and abnormal responses. In this chapter we review major factors that contribute to phototoxicity and discuss practical solutions for circumventing photodamage. These solutions include the proper choice of image acquisition parameters, optimization of filter sets, hardware synchronization, and the use of intelligent illumination to avoid unnecessary light exposure. PMID:23931522

  5. Multispectral imaging fluorescence microscopy for living cells.

    PubMed

    Hiraoka, Yasushi; Shimi, Takeshi; Haraguchi, Tokuko

    2002-10-01

    Multispectral imaging technologies have been widely used in fields of astronomy and remote sensing. Interdisciplinary approaches developed in, for example, the National Aeronautics and Space Administration (NASA, USA), the Jet Propulsion Laboratory (JPL, USA), or the Communications Research Laboratory (CRL, Japan) have extended the application areas of these technologies from planetary systems to cellular systems. Here we overview multispectral imaging systems that have been devised for microscope applications. We introduce these systems with particular interest in live cell imaging. Finally we demonstrate examples of spectral imaging of living cells using commercially available systems with no need for user engineering.

  6. Quantitative reflection contrast microscopy of living cells

    PubMed Central

    1979-01-01

    Mammalian cells in culture (BHK-21, PtK2, Friend, human flia, and glioma cells) have been observed by reflection contrast microscopy. Images of cells photographed at two different wavelengths (546 and 436 nm) or at two different angles of incidence allowed discrimination between reflected light and light that was both reflected and modulated by interference. Interference is involved when a change in reflected intensity (relative to glass/medium background reflected intensity) occurs on changing either the illumination wavelength or the reflection incidence angle. In cases where interference occurs, refractive indices can be determined at points where the optical path difference is known, by solving the given interference equation. Where cells are at least 50 nm distant from the glass substrate, intensities are also influenced by that distance as well as by the light's angle of incidence and wavelength. The reflected intensity at the glass/medium interface is used as a standard in calculating the refractive index of the cortical cytoplasm. Refractive indices were found to be higher (1.38--1.40) at points of focal contact, where stress fibers terminate, than in areas of close contact (1.354--1.368). In areas of the cortical cytoplasm, between focal contacts, not adherent to the glass substrate, refractive indices between 1.353 and 1.368 were found. This was thought to result from a microfilamentous network within the cortical cytoplasm. Intimate attachment of cells to their substrate is assumed to be characterized by a lack of an intermediate layer of culture medium. PMID:389938

  7. Live-Animal Imaging of Renal Function by Multiphoton Microscopy

    PubMed Central

    Dunn, Kenneth W.; Sutton, Timothy A.; Sandoval, Ruben M.

    2015-01-01

    Intravital microscopy, microscopy of living animals, is a powerful research technique that combines the resolution and sensitivity found in microscopic studies of cultured cells with the relevance and systemic influences of cells in the context of the intact animal. The power of intravital microscopy has recently been extended with the development of multiphoton fluorescence microscopy systems capable of collecting optical sections from deep within the kidney at subcellular resolution, supporting high-resolution characterizations of the structure and function of glomeruli, tubules, and vasculature in the living kidney. Fluorescent probes are administered to an anesthetized, surgically prepared animal, followed by image acquisition for up to 3 hr. Images are transferred via a high-speed network to specialized computer systems for digital image analysis. This general approach can be used with different combinations of fluorescent probes to evaluate processes such as glomerular permeability, proximal tubule endocytosis, microvascular flow, vascular permeability, mitochondrial function, and cellular apoptosis/necrosis. PMID:23042524

  8. Super-resolution Microscopy Approaches for Live Cell Imaging

    PubMed Central

    Godin, Antoine G.; Lounis, Brahim; Cognet, Laurent

    2014-01-01

    By delivering optical images with spatial resolutions below the diffraction limit, several super-resolution fluorescence microscopy techniques opened new opportunities to study biological structures with details approaching molecular structure sizes. They have now become methods of choice for imaging proteins and their nanoscale dynamic organizations in live cells. In this mini-review, we describe and compare the main far-field super-resolution approaches that allow studying endogenous or overexpressed proteins in live cells. PMID:25418158

  9. Scanned probe microscopy for thin film superconductor development

    SciTech Connect

    Moreland, J.

    1996-12-31

    Scanned probe microscopy is a general term encompassing the science of imaging based on piezoelectric driven probes for measuring local changes in nanoscale properties of materials and devices. Techniques like scanning tunneling microscopy, atomic force microscopy, and scanning potentiometry are becoming common tools in the production and development labs in the semiconductor industry. The author presents several examples of applications specific to the development of high temperature superconducting thin films and thin-film devices.

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

    NASA Astrophysics Data System (ADS)

    Raja Al-Ani, Ma'an Nassar

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

  11. Nanograting-based plasmon enhancement for total internal reflection fluorescence microscopy of live cells.

    PubMed

    Kim, Kyujung; Kim, Dong Jun; Cho, Eun-Jin; Suh, Jin-Suck; Huh, Yong-Min; Kim, Donghyun

    2009-01-07

    We investigated evanescent field enhancement based on subwavelength nanogratings for improved sensitivity in total internal reflection microscopy of live cells. The field enhancement is associated with subwavelength-grating-coupled plasmon excitation. An optimum sample employed a silver grating on a silver film and an SF10 glass substrate. Field intensity was enhanced by approximately 90% when measured by fluorescent excitation of microbeads relative to that on a bare prism as a control, which is in good agreement with numerical results. The subwavelength-grating-mediated field enhancement was also applied to live cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  12. Magnetic Resonance Force Microscopy Detected Long-Lived Spin Magnetization.

    PubMed

    Chen, Lei; Longenecker, Jonilyn G; Moore, Eric W; Marohn, John A

    2013-07-01

    Magnetic resonance force microscopy (MRFM), which combines magnetic resonance imaging with scanning probe microscopy together, is capable of performing ultra-sensitive detection of spin magnetization. In an attempt to observe dynamic nuclear polarization (DNP) in an MRFM experiment, which could possibly further improve its sensitivity towards a single proton spin, a film of perdeuterated polystyrene doped with a nitroxide electron-spin probe was prepared. A high-compliance cantilever with a 4 μm diameter magnetic tip was brought near the film at a temperature of 7.3 K and in a background magnetic field of ~0.6 T. The film was irradiated with 16.7 GHz microwaves while the resulting transient change in cantilever frequency was recorded in real time. In addition to observing the expected prompt change in cantilever frequency due to saturation of the nitroxide's electron-spin magnetization, we observed a persistent cantilever frequency change. Based on its magnitude, lifetime, and field dependence, we tentatively attribute the persistent signal to polarized deuteron magnetization created via transfer of magnetization from electron spins. Further measurements of the persistent signal's dependence on the cantilever amplitude and tip-sample separation are presented and explained by the cross-effect DNP mechanism in high magnetic field gradients.

  13. Scanning Tunneling Microscopy analysis of space-exposed polymer films

    NASA Technical Reports Server (NTRS)

    Kalil, Carol R.; Young, Philip R.

    1993-01-01

    The characterization of the surface of selected space-exposed polymer films by Scanning Tunneling Microscopy (STM) is reported. Principles of STM, an emerging new technique for materials analysis, are reviewed. The analysis of several films which received up to 5.8 years of low Earth orbital (LEO) exposure onboard the NASA Long Duration Exposure Facility (LDEF) is discussed. Specimens included FEP Teflon thermal blanket material, Kapton film, and several experimental polymer films. Ultraviolet and atomic oxygen-induced crazing and erosion are described. The intent of this paper is to demonstrate how STM is enhancing the understanding of LEO space environmental effects on polymer films.

  14. Generation of living cell arrays for atomic force microscopy studies.

    PubMed

    Formosa, Cécile; Pillet, Flavien; Schiavone, Marion; Duval, Raphaël E; Ressier, Laurence; Dague, Etienne

    2015-01-01

    Atomic force microscopy (AFM) is a useful tool for studying the morphology or the nanomechanical and adhesive properties of live microorganisms under physiological conditions. However, to perform AFM imaging, living cells must be immobilized firmly enough to withstand the lateral forces exerted by the scanning tip, but without denaturing them. This protocol describes how to immobilize living cells, ranging from spores of bacteria to yeast cells, into polydimethylsiloxane (PDMS) stamps, with no chemical or physical denaturation. This protocol generates arrays of living cells, allowing statistically relevant measurements to be obtained from AFM measurements, which can increase the relevance of results. The first step of the protocol is to generate a microstructured silicon master, from which many microstructured PDMS stamps can be replicated. Living cells are finally assembled into the microstructures of these PDMS stamps using a convective and capillary assembly. The complete procedure can be performed in 1 week, although the first step is done only once, and thus repeats can be completed within 1 d.

  15. Axially resolved polarisation microscopy of membrane dynamics in living cells

    NASA Astrophysics Data System (ADS)

    Wagner, Michael; Weber, Petra; Schneckenburger, Herbert

    2007-07-01

    Membrane dynamics has a large impact on cellular uptake and release of various metabolites or pharmaceutical agents. For a deeper understanding of the cellular processes involved, we used U373-MG human glioblastoma cells as a model system. As conventional microscopy does not permit to investigate individual layers in living cells, we used structured illumination techniques and total internal reflection fluorescence microscopy (TIRFM) to analyse the plasma membrane and intracellular membranes of living cells selectively. Optical image sections provide a high resolution and the possibility of 3D reconstruction. Membranes of living cells were characterized by the membrane marker 6-dodecanoyl-2-dimethylamino naphthalene (laurdan). Due to its spectral and kinetic properties this fluorescence marker appears appropriate for measuring membrane stiffness and fluidity. After excitation with linearly polarized laser pulses, membrane fluidity of human glioblastoma cells was determined by measurements of steady-state and time-resolved fluorescence anisotropy r(t), since with increasing viscosity of the environment, the rotation of an excited molecule is impeded. The corresponding time constant τ r of molecular relaxation decreased with temperature and increased with the amount of cholesterol. In addition, fluorescence anisotropy r(t) values of the plasma membrane were larger than the values of intracellular membranes for all temperatures in the range of 16°C<=T<=41°C.

  16. High-speed synthetic aperture microscopy for live cell imaging

    PubMed Central

    Kim, Moonseok; Choi, Youngwoon; Fang-Yen, Christopher; Sung, Yongjin; Dasari, Ramachandra R.; Feld, Michael S.; Choi, Wonshik

    2011-01-01

    We present a high-speed synthetic aperture microscopy for quantitative phase imaging of live biological cells. We measure 361 complex amplitude images of an object with various directions of illumination covering an NA of 0.8 in less than one-thirteenth of a second and then combine the images with a phase-referencing method to create a synthesized phase image. Because of the increased depth selectivity, artifacts from diffraction that are typically present in coherent imaging are significantly suppressed, and lateral resolution of phase imaging is improved. We use the instrument to demonstrate high-quality phase imaging of live cells, both static and dynamic, and thickness measurements of a nanoscale cholesterol helical ribbon. PMID:21263482

  17. Scanning Ion Conductance Microscopy for living cell membrane potential measurement

    NASA Astrophysics Data System (ADS)

    Panday, Namuna

    Recently, the existence of multiple micro-domains of extracellular potential around individual cells have been revealed by voltage reporter dye using fluorescence microscopy. One hypothesis is that these long lasting potential patterns play a vital role in regulating important cell activities such as embryonic patterning, regenerative repair and reduction of cancerous disorganization. We used multifunctional Scanning Ion Conductance Microscopy (SICM) to study these extracellular potential patterns of single cell with higher spatial resolution. To validate this novel technique, we compared the extracellular potential distribution on the fixed HeLa cell surface and Polydimethylsiloxane (PDMS) surface and found significant difference. We then measured the extracellular potential distributions of living melanocytes and melanoma cells and found both the mean magnitude and spatial variation of extracellular potential of the melanoma cells are bigger than those of melanocytes. As compared to the voltage reporter dye based fluorescence microscope method, SICM can achieve quantitative potential measurements of non-labeled living cell membranes with higher spatial resolution.

  18. STED microscopy of living cells--new frontiers in membrane and neurobiology.

    PubMed

    Eggeling, Christian; Willig, Katrin I; Barrantes, Francisco J

    2013-07-01

    Recent developments in fluorescence far-field microscopy such as STED microscopy have accomplished observation of the living cell with a spatial resolution far below the diffraction limit. Here, we briefly review the current approaches to super-resolution optical microscopy and present the implementation of STED microscopy for novel insights into live cell mechanisms, with a focus on neurobiology and plasma membrane dynamics.

  19. Dynamic Metabolism Studies of Live Bacterial Films

    SciTech Connect

    Majors, Paul D.; Mclean, Jeffrey S.

    2008-11-01

    Bacterial film (biofilm) microbes exist within spatial (nutrient, electron-acceptor, pH, etc.) gradients of their own making. Correspondingly, biofilm bacteria are physiologically and functionally distinct from free-floating bacteria and from their own species at differing biofilm depths. This article describes our efforts to develop noninvasive nuclear magnetic resonance (NMR) technologies for biofilm-metabolism studies. This involves integrating NMR with controlled-cultivation methods to interrogate microbial physiology live and under known growth conditions. NMR is uniquely capable of providing depth-resolved metabolic and transport information in a non-invasive, non-sample-consuming fashion, providing information required for experimental reactive transport studies. We have studied mono-species biofilms relevant to environment remediation and human health. We describe these technologies, discuss their advantages and limitations, and give examples of their application.

  20. Extrinsic Size Effect in Piezoresponse Force Microscopy of Thin Films

    SciTech Connect

    Morozovska, A. N.; Svechnikov, S. V.; Eliseev, E. A.; Kalinin, Sergei V

    2007-01-01

    The extrinsic size effect in piezoresponse force microscopy of ferroelectric and piezoelectric thin films on nonpolar dielectric substrate with matching elastic properties is analyzed. Analytical expressions for effective piezoresponse, object transfer function components, and Rayleigh two-point resolution are obtained. These results can be broadly applied for effective piezoelectric response calculations in thin piezoelectric and ferroelectric films as well as surface polar layers in, e.g., organic materials and biopolymers. In particular, the effective piezoresponse strongly decreases with film thickness, whereas the sharpness of domain stripe image increases due to the object transfer function spectrum broadening.

  1. Nonoptically probing near-field microscopy for the observation of biological living specimens

    NASA Astrophysics Data System (ADS)

    Kawata, Yoshimasa; Murakami, Manabu; Egami, Chikara; Sugihara, Okihiro; Okamoto, Naomichi; Tsuchimori, Masaaki; Watanabe, Osamu; Nakamura, Osamu

    2001-04-01

    We present the observation of living specimens with subwavelength resolution by using the nonoptically probing near-field microscopy we have developed recently. In the near-field microscope, the optical field distributions near the specimens are recorded as the surface topography of a photosensitive film, and the topographical distributions are readout with an atomic-force microscopy. Since the near-field microscope does not require the scanning of a probe tip for illumination or detection or scattering of light, it is possible to observe moving biological specimens and fast phenomena. We demonstrate the observation of a moving paramecium and euglena gracilis with subwavelength resolution. The observation of the nucleus inside a euglena cell was also demonstrated.

  2. Microscopy of Si films during laser melting

    SciTech Connect

    Lemons, R.A.; Boesch, M.A.

    1982-04-15

    By using an optical microscope to directly observe thin Si films as they are melted with a cw argon laser beam, the crystallization process can be better understood. In an environment containing oxygen, stable filaments of solid silicon precipitate from the molten pool at low laser power. The surrounding melt may contain dissolved oxygen which reduces the melting point, allowing the liquid and solid to coexist. As laser power is increased a uniform molten pool is achieved. In emitted light the pool is dark compared to the surrounding solid due to the melt's low emissivity. The spectrum of this emitted thermal radiation accurately fits the Planck law at 1740 /sup 0/K, confirming the temperature of the melt.

  3. Deconvolved spatial light interference microscopy for live cell imaging.

    PubMed

    Haldar, Justin P; Wang, Zhuo; Popescu, Gabriel; Liang, Zhi-Pei

    2011-09-01

    Spatial light interference microscopy (SLIM) is a recently developed method for the label-free imaging of live cells, using the quantitative optical path length through the sample as an endogenous source of contrast. In conventional SLIM, spatial resolution is limited by diffraction and aberrations. This paper describes a novel constrained deconvolution method for improving resolution in SLIM. Constrained deconvolution is enabled by experimental measurement of the system point-spread function and the modeling of coherent image formation in SLIM. Results using simulated and experimental data demonstrate that the proposed method leads to significant improvements in the resolution and contrast of SLIM images. The proposed method should prove useful for high-resolution label-free studies of biological cells and subcellular processes.

  4. Raman microscopy of individual living human embryonic stem cells

    NASA Astrophysics Data System (ADS)

    Novikov, S. M.; Beermann, J.; Bozhevolnyi, S. I.; Harkness, L. M.; Kassem, M.

    2010-04-01

    We demonstrate the possibility of mapping the distribution of different biomolecules in living human embryonic stem cells grown on glass substrates, without the need for fluorescent markers. In our work we improve the quality of measurements by finding a buffer that gives low fluorescence, growing cells on glass substrates (whose Raman signals are relatively weak compared to that of the cells) and having the backside covered with gold to improve the image contrast under direct white light illumination. The experimental setup used for Raman microscopy is the commercially available confocal scanning Raman microscope (Alpha300R) from Witec and sub-μm spatially resolved Raman images were obtained using a 532 nm excitation wavelength.

  5. Bioluminescence microscopy: application to ATP measurements in single living cells

    NASA Astrophysics Data System (ADS)

    Brau, Frederic; Helle, Pierre; Bernengo, Jean C.

    1997-12-01

    Bioluminescence microscopy can be used to measure intracellular cofactors and ionic concentrations (Ca2+, K+, ATP, NADH), as an alternative to micro- spectrophotometry and micro-fluorimetry, due to the development of sensitive detectors (cooled photomultipliers tubes and CCD). The main limitation comes from the very small and brief intensity of the emitted light. Our instrumentation based on an inverted microscope, equipped with high aperture immersion lenses is presented. Light intensity measurements are carried out through a photomultiplier sorted for low dark current and cooled at -5 degree(s)C to reduce thermal noise. Our first aim is to quantify ATP on single living cells using the firefly luciferin-luciferase couple. Experimental and kinetic aspects are presented to emphasize the potentialities of the technique.

  6. Quantitative analysis of live cells using digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Lewis, Tan Rongwei; Qu, Weijuan; Chee, Oi Choo; Singh, Vijay Raj; Asundi, Anand

    2010-03-01

    During the life time of a cell, it goes through changes to the plasma membrane as well as its internal structures especially distinctive during processes like cell division and death. Different types of microscope are used to fulfill the observation of the cell's variation. In our experiment, Vero cells have been investigated by using phase contrast microscopy and digital holographic microscopy (DHM). A comparison of the images obtained for cell division is presented here. The conventional phase contrast microscope provided a good imaging method in the real time analysis of cell division. The off-axis digital hologram recorded by the DHM system can be reconstructed to obtain both the intensity image and phase contrast image of the test object. These can be used for live cell imaging to provide multiple results from a single equipment setup. The DHM system, besides being a qualitative tool, is able to provide quantitative results and 3D images of the cell division process. The ability of DHM to provide quantitative analysis makes it an ideal tool for life science applications.

  7. Quantitative analysis of live cells using digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Lewis, Tan Rongwei; Qu, Weijuan; Chee, Oi Choo; Singh, Vijay Raj; Asundi, Anand

    2009-12-01

    During the life time of a cell, it goes through changes to the plasma membrane as well as its internal structures especially distinctive during processes like cell division and death. Different types of microscope are used to fulfill the observation of the cell's variation. In our experiment, Vero cells have been investigated by using phase contrast microscopy and digital holographic microscopy (DHM). A comparison of the images obtained for cell division is presented here. The conventional phase contrast microscope provided a good imaging method in the real time analysis of cell division. The off-axis digital hologram recorded by the DHM system can be reconstructed to obtain both the intensity image and phase contrast image of the test object. These can be used for live cell imaging to provide multiple results from a single equipment setup. The DHM system, besides being a qualitative tool, is able to provide quantitative results and 3D images of the cell division process. The ability of DHM to provide quantitative analysis makes it an ideal tool for life science applications.

  8. Enhanced live cell imaging via photonic crystal enhanced fluorescence microscopy.

    PubMed

    Chen, Weili; Long, Kenneth D; Yu, Hojeong; Tan, Yafang; Choi, Ji Sun; Harley, Brendan A; Cunningham, Brian T

    2014-11-21

    We demonstrate photonic crystal enhanced fluorescence (PCEF) microscopy as a surface-specific fluorescence imaging technique to study the adhesion of live cells by visualizing variations in cell-substrate gap distance. This approach utilizes a photonic crystal surface incorporated into a standard microscope slide as the substrate for cell adhesion, and a microscope integrated with a custom illumination source as the detection instrument. When illuminated with a monochromatic light source, angle-specific optical resonances supported by the photonic crystal enable efficient excitation of surface-confined and amplified electromagnetic fields when excited at an on-resonance condition, while no field enhancement occurs when the same photonic crystal is illuminated in an off-resonance state. By mapping the fluorescence enhancement factor for fluorophore-tagged cellular components between on- and off-resonance states and comparing the results to numerical calculations, the vertical distance of labelled cellular components from the photonic crystal substrate can be estimated, providing critical and quantitative information regarding the spatial distribution of the specific components of cells attaching to a surface. As an initial demonstration of the concept, 3T3 fibroblast cells were grown on fibronectin-coated photonic crystals with fluorophore-labelled plasma membrane or nucleus. We demonstrate that PCEF microscopy is capable of providing information about the spatial distribution of cell-surface interactions at the single-cell level that is not available from other existing forms of microscopy, and that the approach is amenable to large fields of view, without the need for coupling prisms, coupling fluids, or special microscope objectives.

  9. Scanning tunneling microscopy studies of diamond films and optoelectronic materials

    NASA Technical Reports Server (NTRS)

    Perez, Jose M.

    1993-01-01

    In this report, we report on progress achieved from 12/1/92 to 10/1/93 under the grant entitled 'Scanning Tunneling Microscopy Studies of Diamond Films and Optoelectronic Materials'. We have set-up a chemical vapor deposition (CVD) diamond film growth system and a Raman spectroscopy system to study the nucleation and growth of diamond films with atomic resolution using scanning tunneling microscopy (STM). A unique feature of the diamond film growth system is that diamond films can be transferred directly to the ultrahigh vacuum (UHV) chamber of a scanning tunneling microscope without contaminating the films by exposure to air. The University of North Texas (UNT) provided $20,000 this year as matching funds for the NASA grant to purchase the diamond growth system. In addition, UNT provided a Coherent Innova 90S Argon ion laser, a Spex 1404 double spectrometer, and a Newport optical table costing $90,000 to set-up the Raman spectroscopy system. The CVD diamond growth system and Raman spectroscopy system will be used to grow and characterize diamond films with atomic resolution using STM as described in our proposal. One full-time graduate student and one full-time undergraduate student are supported under this grant. In addition, several graduate and undergraduate students were supported during the summer to assist in setting-up the diamond growth and Raman spectroscopy systems. We have obtained research results concerning STM of the structural and electronic properties of CVD grown diamond films, and STM and scanning tunneling spectroscopy of carbon nanotubes. In collaboration with the transmission electron microscopy (TEM) group at UNT, we have also obtained results concerning the optoelectronic material siloxene. These results were published in refereed scientific journals, submitted for publication, and presented as invited and contributed talks at scientific conferences.

  10. Relative microelastic mapping of living cells by atomic force microscopy.

    PubMed Central

    A-Hassan, E; Heinz, W F; Antonik, M D; D'Costa, N P; Nageswaran, S; Schoenenberger, C A; Hoh, J H

    1998-01-01

    The spatial and temporal changes of the mechanical properties of living cells reflect complex underlying physiological processes. Following these changes should provide valuable insight into the biological importance of cellular mechanics and their regulation. The tip of an atomic force microscope (AFM) can be used to indent soft samples, and the force versus indentation measurement provides information about the local viscoelasticity. By collecting force-distance curves on a time scale where viscous contributions are small, the forces measured are dominated by the elastic properties of the sample. We have developed an experimental approach, using atomic force microscopy, called force integration to equal limits (FIEL) mapping, to produce robust, internally quantitative maps of relative elasticity. FIEL mapping has the advantage of essentially being independent of the tip-sample contact point and the cantilever spring constant. FIEL maps of living Madine-Darby canine kidney (MDCK) cells show that elasticity is uncoupled from topography and reveal a number of unexpected features. These results present a mode of high-resolution visualization in which the contrast is based on the mechanical properties of the sample. PMID:9512052

  11. Thin-film-based sensitivity enhancement for total internal reflection fluorescence live-cell imaging.

    PubMed

    Kim, Kyujung; Cho, Eun-Jin; Huh, Yong-Min; Kim, Donghyun

    2007-11-01

    We investigated experimentally the evanescent field enhancement based on dielectric thin films in total internal reflection microscopy. The sample employed two layers of Al2O3 and SiO2 deposited on an SF10 glass substrate. Field intensity enhancement measured by fluorescent excitation of microbeads relative to that of a control sample without dielectric films was polarization dependent, determined as 4.2 and 2.4 for TE and TM polarizations, respectively, and was in good agreement with numerical results. The thin-film-based field enhancement was also applied to live-cell imaging of quantum dots, which confirmed the sensitivity enhancement qualitatively.

  12. High-throughput characterization of film thickness in thin film materials libraries by digital holographic microscopy.

    PubMed

    Lai, Yiu Wai; Krause, Michael; Savan, Alan; Thienhaus, Sigurd; Koukourakis, Nektarios; Hofmann, Martin R; Ludwig, Alfred

    2011-10-01

    A high-throughput characterization technique based on digital holography for mapping film thickness in thin-film materials libraries was developed. Digital holographic microscopy is used for fully automatic measurements of the thickness of patterned films with nanometer resolution. The method has several significant advantages over conventional stylus profilometry: it is contactless and fast, substrate bending is compensated, and the experimental setup is simple. Patterned films prepared by different combinatorial thin-film approaches were characterized to investigate and demonstrate this method. The results show that this technique is valuable for the quick, reliable and high-throughput determination of the film thickness distribution in combinatorial materials research. Importantly, it can also be applied to thin films that have been structured by shadow masking.

  13. 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.

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

    SciTech Connect

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

    2015-05-15

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

  15. Organic Multilayer Films Studied by Scanning Tunneling Microscopy.

    PubMed

    He, Yang; Kröger, Jörg; Wang, Yongfeng

    2017-03-03

    This Minireview focuses exclusively on work with scanning tunneling microscopy to study the self-assembled multilayer films (SAMTs) of organic molecules. The π-conjugated organic molecules form different structures within different monolayers on various substrates. The interplay between molecule-substrate and intermolecular interactions plays a key role in determining the stacking mode of organic multilayer films. Different substrates strongly influence the organic-film growth and electronic properties of the organic molecules. Geometric and electronic structures of SAMTs are important factors that may determine device performance. In addition to the inorganic interface, this Minireview addresses the organic-organic interface. Homo- and hetero-SAMTs of organic molecules are also considered. The subtle interplay between structural and electronic characteristics, on one hand, and functionality and reactivity, on the other hand, are highlighted.

  16. NMR Spectroscopy for Thin Films by Magnetic Resonance Force Microscopy

    PubMed Central

    Won, Soonho; Saun, Seung-Bo; Lee, Soonchil; Lee, SangGap; Kim, Kiwoong; Han, Yunseok

    2013-01-01

    Nuclear magnetic resonance (NMR) is a fundamental research tool that is widely used in many fields. Despite its powerful applications, unfortunately the low sensitivity of conventional NMR makes it difficult to study thin film or nano-sized samples. In this work, we report the first NMR spectrum obtained from general thin films by using magnetic resonance force microscopy (MRFM). To minimize the amount of imaging information inevitably mixed into the signal when a gradient field is used, we adopted a large magnet with a flat end with a diameter of 336 μm that generates a homogeneous field on the sample plane and a field gradient in a direction perpendicular to the plane. Cyclic adiabatic inversion was used in conjunction with periodic phase inversion of the frequency shift to maximize the SNR. In this way, we obtained the 19F NMR spectrum for a 34 nm-thick CaF2 thin film. PMID:24217000

  17. Photoemission microscopy from magnetically coupled thin-film systems

    NASA Astrophysics Data System (ADS)

    Schneider, C. M.; de Haas, O.; Muschiol, U.; Cramer, N.; Oelsner, A.; Klais, M.; Schmidt, O.; Fecher, G. H.; Jark, W.; Schönhense, G.

    2001-07-01

    The magnetic microstructure and magnetic coupling phenomena in thin-film systems, relevant for applications in magneto-electronics, are investigated by means of photoemission electron microscopy. Element-selective magnetic information is obtained by exploiting magnetic circular dichroism in the soft X-ray regime. The domain shape and sizes found at the surface of antiferromagnetically coupled metallic multilayers indicate the presence of a ferromagnetic coupling contribution, presumably caused by a build-up of roughness during the growth process. The magnetic domain patterns in FeNi microstructures on sputtered NiO films reflect the presence of a local exchange anisotropy, causing the phenomenon of exchange biasing or pinning of the ferromagnetic layer.

  18. 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.

  19. Thin-film tunable filters for hyperspectral fluorescence microscopy.

    PubMed

    Favreau, Peter; Hernandez, Clarissa; Lindsey, Ashley Stringfellow; Alvarez, Diego F; Rich, Thomas; Prabhat, Prashant; Leavesley, Silas J

    2014-01-01

    Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging-the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes.

  20. Silicon Carbide Epitaxial Films Studied by Atomic Force Microscopy

    NASA Technical Reports Server (NTRS)

    1996-01-01

    Silicon carbide (SiC) holds great potential as an electronic material because of its wide band gap energy, high breakdown electric field, thermal stability, and resistance to radiation damage. Possible aerospace applications of high-temperature, high-power, or high-radiation SiC electronic devices include sensors, control electronics, and power electronics that can operate at temperatures up to 600 C and beyond. Commercially available SiC devices now include blue light-emitting diodes (LED's) and high-voltage diodes for operation up to 350 C, with other devices under development. At present, morphological defects in epitaxially grown SiC films limit their use in device applications. Research geared toward reducing the number of structural inhomogeneities can benefit from an understanding of the type and nature of problems that cause defects. The Atomic Force Microscope (AFM) has proven to be a useful tool in characterizing defects present on the surface of SiC epitaxial films. The in-house High-Temperature Integrated Electronics and Sensors (HTIES) Program at the NASA Lewis Research Center not only extended the dopant concentration range achievable in epitaxial SiC films, but it reduced the concentration of some types of defects. Advanced structural characterization using the AFM was warranted to identify the type and structure of the remaining film defects and morphological inhomogeneities. The AFM can give quantitative information on surface topography down to molecular scales. Acquired, in part, in support of the Advanced High Temperature Engine Materials Technology Program (HITEMP), the AFM had been used previously to detect partial fiber debonding in composite material cross sections. Atomic force microscopy examination of epitaxial SiC film surfaces revealed molecular-scale details of some unwanted surface features. Growth pits propagating from defects in the substrate, and hillocks due, presumably, to existing screw dislocations in the substrates, were

  1. Noncontact Viscoelastic Measurement of Polymer Thin Films in a Liquid Medium Using Long-Needle Atomic Force Microscopy.

    PubMed

    Guan, Dongshi; Barraud, Chloé; Charlaix, Elisabeth; Tong, Penger

    2017-02-14

    We report the noncontact measurement of the viscoelastic property of polymer thin films in a liquid medium using frequency-modulation atomic force microscopy with a newly developed long-needle probe. The probe contains a long vertical glass fiber with one end adhered to a cantilever beam and the other end with a sharp tip placed near the liquid-film interface. The nanoscale flow generated by the resonant oscillation of the needle tip provides a precise hydrodynamic force acting on the soft surface of the thin film. By accurately measuring the mechanical response of the thin film, we obtain the elastic and loss moduli of the thin film using the linear response theory of elastohydrodynamics. The experiment verifies the theory and demonstrates its applications. The technique can be used to accurately measure the viscoelastic property of soft surfaces, such as those made of polymers, nanobubbles, live cells, and tissues.

  2. 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.

  3. Immobilization Techniques of Bacteria for Live Super-resolution Imaging Using Structured Illumination Microscopy.

    PubMed

    Bottomley, Amy L; Turnbull, Lynne; Whitchurch, Cynthia B; Harry, Elizabeth J

    2017-01-01

    Advancements in optical microscopy technology have allowed huge progression in the ability to understand protein structure and dynamics in live bacterial cells using fluorescence microscopy. Paramount to high-quality microscopy is good sample preparation to avoid bacterial cell movement that can result in motion blur during image acquisition. Here, we describe two techniques of sample preparation that reduce unwanted cell movement and are suitable for application to a number of bacterial species and imaging methods.

  4. Scanning Tunneling Microscopy Studies of Diamond Films and Optoelectronic Materials

    NASA Technical Reports Server (NTRS)

    Perez, Jose M.

    1996-01-01

    We present a summary of the research, citations of publications resulting from the research and abstracts of such publications. We have made no inventions in the performance of the work in this project. The main goals of the project were to set up a Chemical Vapor Deposition (CVD) diamond growth system attached to an UltraHigh Vacuum (UHV) atomic resolution Scanning Tunneling Microscopy (STM) system and carry out experiments aimed at studying the properties and growth of diamond films using atomic resolution UHV STM. We successfully achieved these goals. We observed, for the first time, the atomic structure of the surface of CVD grown epitaxial diamond (100) films using UHV STM. We studied the effects of atomic hydrogen on the CVD diamond growth process. We studied the electronic properties of the diamond (100) (2x1) surface, and the effect of alkali metal adsorbates such as Cs on the work function of this surface using UHV STM spectroscopy techniques. We also studied, using STM, new electronic materials such as carbon nanotubes and gold nanostructures. This work resulted in four publications in refereed scientific journals and five publications in refereed conference proceedings.

  5. Characterization of konjac glucomannan-ethyl cellulose film formation via microscopy.

    PubMed

    Xiao, Man; Wan, Li; Corke, Harold; Yan, Wenli; Ni, Xuewen; Fang, Yapeng; Jiang, Fatang

    2016-04-01

    Konjac glucomannan-ethyl cellulose (KGM-EC, 7:3, w/w) blended film shows good mechanical and moisture resistance properties. To better understand the basis for the KGM-EC film formation, optical microscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) were used to observe the formation of the film from emulsion. Optical microscopy images showed that EC oil droplets were homogeneously dispersed in KGM water phase without obviously coalescence throughout the entire drying process. SEM images showed the surface and cross-sectional structures of samples maintained continuous and homogeneous appearance from the emulsion to dried film. AFM images indicated that KGM molecules entangled EC molecules in the emulsion. Interactions between KGM and EC improved the stability of KGM-EC emulsion, and contributed to uniformed structures of film formation. Based on these output information, a schematic model was built to elucidate KGM-EC film-forming process.

  6. High speed microscopy techniques for signaling detection in live cells

    NASA Astrophysics Data System (ADS)

    de Mauro, C.; Cecchetti, C. A.; Alfieri, D.; Borile, Giulia; Urbani, A.; Mongillo, M.; Pavone, F. S.

    2014-05-01

    Alterations in intracellular cardiomyocyte calcium handling have a key role in initiating and sustaining arrhythmias. Arrhythmogenic calcium leak from sarcoplasmic reticulum (SR) can be attributed to all means by which calcium exits the SR store in an abnormal fashion. Abnormal SR calcium exit maymanifest as intracellular Ca2+ sparks and/or Ca2+ waves. Ca2+ signaling in arrhythmogenesis has been mainly studied in isolated cardiomyocytes and given that the extracellular matrix influences both Ca2+ and membrane potential dynamics in the intact heart and underlies environmentally mediated changes, understanding how Ca2+ and voltage are regulated in the intact heart will represent a tremendous advancement in the understanding of arrhythmogenic mechanisms. Using novel high-speed multiphoton microscopy techinques, such as multispot and random access, we investigated animal models with inherited and acquired arrhythmias to assess the role of Ca2+ and voltage signals as arrhythmia triggers in cell and subcellular components of the intact heart and correlate these with electrophysiology.

  7. Wide-field optical sectioning for live-tissue imaging by plane-projection multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Yu, Jiun-Yann; Kuo, Chun-Hung; Holland, Daniel B.; Chen, Yenyu; Ouyang, Mingxing; Blake, Geoffrey A.; Zadoyan, Ruben; Guo, Chin-Lin

    2011-11-01

    Optical sectioning provides three-dimensional (3D) information in biological tissues. However, most imaging techniques implemented with optical sectioning are either slow or deleterious to live tissues. Here, we present a simple design for wide-field multiphoton microscopy, which provides optical sectioning at a reasonable frame rate and with a biocompatible laser dosage. The underlying mechanism of optical sectioning is diffuser-based temporal focusing. Axial resolution comparable to confocal microscopy is theoretically derived and experimentally demonstrated. To achieve a reasonable frame rate without increasing the laser power, a low-repetition-rate ultrafast laser amplifier was used in our setup. A frame rate comparable to that of epifluorescence microscopy was demonstrated in the 3D imaging of fluorescent protein expressed in live epithelial cell clusters. In this report, our design displays the potential to be widely used for video-rate live-tissue and embryo imaging with axial resolution comparable to laser scanning microscopy.

  8. Atomic force microscopy study of living diatoms in ambient conditions.

    PubMed

    Gebeshuber, I C; Kindt, J H; Thompson, J B; Del Amo, Y; Stachelberger, H; Brzezinski, M A; Stucky, G D; Morse, D E; Hansma, P K

    2003-12-01

    We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain-forming, benthic freshwater species -Eunotia sudetica, Navicula seminulum and a yet unidentified species - are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long-term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man-made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh- and in seawater environments. Our phase-imaging and force-pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man-made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge.

  9. Magnetic Force Microscopy Images of Magnetic Garnet With Thin-Film Magnetic Tip

    NASA Technical Reports Server (NTRS)

    Wadas, A.; Moreland, J.; Rice, P.; Katti, R.

    1993-01-01

    We present magnetic force microscopy images of YGdTmGa/YSmTmGa magnetic garnet, usinga thin Fe film deposited on Si_3N_5 tips. We have found correlations between the topography andthe magnetic domain structure. We have observed the domain wall contrast with a iron thin-film tip. We report on domain wall imaging of garnet with magnetic force microscopy.

  10. Labeling proteins inside living cells using external fluorophores for microscopy

    PubMed Central

    Teng, Kai Wen; Ishitsuka, Yuji; Ren, Pin; Youn, Yeoan; Deng, Xiang; Ge, Pinghua; Lee, Sang Hak; Belmont, Andrew S; Selvin, Paul R

    2016-01-01

    Site-specific fluorescent labeling of proteins inside live mammalian cells has been achieved by employing Streptolysin O, a bacterial toxin which forms temporary pores in the membrane and allows delivery of virtually any fluorescent probes, ranging from labeled IgG’s to small ligands, with high efficiency (>85% of cells). The whole process, including recovery, takes 30 min, and the cell is ready to be imaged immediately. A variety of cell viability tests were performed after treatment with SLO to ensure that the cells have intact membranes, are able to divide, respond normally to signaling molecules, and maintains healthy organelle morphology. When combined with Oxyrase, a cell-friendly photostabilizer, a ~20x improvement in fluorescence photostability is achieved. By adding in glutathione, fluorophores are made to blink, enabling super-resolution fluorescence with 20–30 nm resolution over a long time (~30 min) under continuous illumination. Example applications in conventional and super-resolution imaging of native and transfected cells include p65 signal transduction activation, single molecule tracking of kinesin, and specific labeling of a series of nuclear and cytoplasmic protein complexes. DOI: http://dx.doi.org/10.7554/eLife.20378.001 PMID:27935478

  11. Enlightening intracellular complexity of living cells with quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Martinez Torres, C.; Laperrousaz, B.; Berguiga, L.; Boyer Provera, E.; Elezgaray, J.; Nicolini, F. E.; Maguer-Satta, V.; Arneodo, A.; Argoul, F.

    2016-03-01

    The internal distribution of refractive indices (RIs) of a living cell is much more complex than usually admitted in multi-shell models. The reconstruction of RI maps from single phase images has rarely been achieved for several reasons: (i) we still have very little knowledge of the impact of internal macromolecular complexes on the local RI and (ii) phase changes produced by light propagation through the sample are mixed with diffraction effects by internal cell bodies. We propose the implementation a 2D wavelet-based contour chain detection method to distinguish internal boundaries thanks to their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are morphological indicators for distinguishing cells of different origins and to follow their transformation in pathologic situations. We use this method to compare non adherent blood cells from primary and laboratory culture origins, in healthy and pathological situations (chronic myelogenous leukaemia). In a second part of this presentation, we concentrate on the temporal dynamics of the phase contour chains and we discuss the spectral decomposition of their dynamics in both health and disease.

  12. Fluorescent proteins for FRET microscopy: monitoring protein interactions in living cells

    PubMed Central

    Day, Richard N.; Davidson, Michael W.

    2012-01-01

    Summary The discovery and engineering of novel fluorescent proteins (FPs) from diverse organisms is yielding fluorophores with exceptional characteristics for live-cell imaging. In particular, the development of FPs for fluorescence (or Förster) resonance energy transfer (FRET) microscopy is providing important tools for monitoring dynamic protein interactions inside living cells. The increased interest in FRET microscopy has driven the development of many different methods to measure FRET. However, the interpretation of FRET measurements is complicated by several factors including the high fluorescence background, the potential for photoconversion artifacts, and the relatively low dynamic range afforded by this technique. Here, we describe the advantages and disadvantages of four methods commonly used in FRET microscopy. We then discuss the selection of FPs for the different FRET methods, identifying the most useful FP candidates for FRET microscopy. The recent success in expanding the FP color palette offers the opportunity to explore new FRET pairs. PMID:22396229

  13. Two-Color STED Microscopy of Living Synapses Using A Single Laser-Beam Pair

    PubMed Central

    Tønnesen, Jan; Nadrigny, Fabien; Willig, Katrin I.; Wedlich-Söldner, Roland; Nägerl, U. Valentin

    2011-01-01

    The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited for imaging living synapses in brain slices using yellow fluorescent protein as a single label. However, it would be highly desirable to be able to image presynaptic boutons and postsynaptic spines, which together form synapses, using two different fluorophores. As STED microscopy uses separate laser beams for fluorescence excitation and quenching, incorporation of multicolor imaging for STED is more difficult than for conventional light microscopy. Although two-color schemes exist for STED microscopy, these approaches have several drawbacks due to their complexity, cost, and incompatibility with common labeling strategies and fluorophores. Therefore, we set out to develop a straightforward method for two-color STED microscopy that permits the use of popular green-yellow fluorescent labels such as green fluorescent protein, yellow fluorescent protein, Alexa Fluor 488, and calcein green. Our new (to our knowledge) method is based on a single-excitation/STED laser-beam pair to simultaneously excite and quench pairs of these fluorophores, whose signals can be separated by spectral detection and linear unmixing. We illustrate the potential of this approach by two-color superresolution time-lapse imaging of axonal boutons and dendritic spines in living organotypic brain slices. PMID:22098754

  14. Super-resolution video microscopy of live cells by structured illumination.

    PubMed

    Kner, Peter; Chhun, Bryant B; Griffis, Eric R; Winoto, Lukman; Gustafsson, Mats G L

    2009-05-01

    Structured-illumination microscopy can double the resolution of the widefield fluorescence microscope but has previously been too slow for dynamic live imaging. Here we demonstrate a high-speed structured-illumination microscope that is capable of 100-nm resolution at frame rates up to 11 Hz for several hundred time points. We demonstrate the microscope by video imaging of tubulin and kinesin dynamics in living Drosophila melanogaster S2 cells in the total internal reflection mode.

  15. Characterization of MSB synapses in dissociated hippocampal culture with simultaneous pre- and postsynaptic live microscopy.

    PubMed

    Reilly, James E; Hanson, Hugo H; Fernández-Monreal, Mónica; Wearne, Susan L; Hof, Patrick R; Phillips, Greg R

    2011-01-01

    Multisynaptic boutons (MSBs) are presynaptic boutons in contact with multiple postsynaptic partners. Although MSB synapses have been studied with static imaging techniques such as electron microscopy (EM), the dynamics of individual MSB synapses have not been directly evaluated. It is known that the number of MSB synapses increases with synaptogenesis and plasticity but the formation, behavior, and fate of individual MSB synapses remains largely unknown. To address this, we developed a means of live imaging MSB synapses to observe them directly over time. With time lapse confocal microscopy of GFP-filled dendrites in contact with VAMP2-DsRed-labeled boutons, we recorded both MSBs and their contacting spines hourly over 15 or more hours. Our live microscopy showed that, compared to spines contacting single synaptic boutons (SSBs), MSB-contacting spines exhibit elevated dynamic behavior. These results are consistent with the idea that MSBs serve as intermediates in synaptic development and plasticity.

  16. Live imaging of Tribolium castaneum embryonic development using light-sheet-based fluorescence microscopy.

    PubMed

    Strobl, Frederic; Schmitz, Alexander; Stelzer, Ernst H K

    2015-10-01

    Tribolium castaneum has become an important insect model organism for evolutionary developmental biology, genetics and biotechnology. However, few protocols for live fluorescence imaging of Tribolium have been reported, and little image data is available. Here we provide a protocol for recording the development of Tribolium embryos with light-sheet-based fluorescence microscopy. The protocol can be completed in 4-7 d and provides procedural details for: embryo collection, microscope configuration, embryo preparation and mounting, noninvasive live imaging for up to 120 h along multiple directions, retrieval of the live embryo once imaging is completed, and image data processing, for which exemplary data is provided. Stringent quality control criteria for developmental biology studies are also discussed. Light-sheet-based fluorescence microscopy complements existing toolkits used to study Tribolium development, can be adapted to other insect species, and requires no advanced imaging or sample preparation skills.

  17. Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy.

    PubMed

    Vélez-Ortega, A Catalina; Frolenkov, Gregory I

    2016-01-01

    The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3-4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette-which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier-is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface.Here, we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations.

  18. Visualization of Live Cochlear Stereocilia at a Nanoscale Resolution Using Hopping Probe Ion Conductance Microscopy

    PubMed Central

    Vélez-Ortega, A. Catalina; Frolenkov, Gregory I.

    2016-01-01

    The mechanosensory apparatus that detects sound-induced vibrations in the cochlea is located on the apex of the auditory sensory hair cells and it is made up of actin-filled projections, called stereocilia. In young rodents, stereocilia bundles of auditory hair cells consist of 3 to 4 rows of stereocilia of decreasing height and varying thickness. Morphological studies of the auditory stereocilia bundles in live hair cells have been challenging because the diameter of each stereocilium is near or below the resolution limit of optical microscopy. In theory, scanning probe microscopy techniques, such as atomic force microscopy, could visualize the surface of a living cell at a nanoscale resolution. However, their implementations for hair cell imaging have been largely unsuccessful because the probe usually damages the bundle and disrupts the bundle cohesiveness during imaging. We overcome these limitations by using hopping probe ion conductance microscopy (HPICM), a non-contact scanning probe technique that is ideally suited for the imaging of live cells with a complex topography. Organ of Corti explants are placed in a physiological solution and then a glass nanopipette –which is connected to a 3D-positioning piezoelectric system and to a patch clamp amplifier– is used to scan the surface of the live hair cells at nanometer resolution without ever touching the cell surface. Here we provide a detailed protocol for the imaging of mouse or rat stereocilia bundles in live auditory hair cells using HPICM. We provide information about the fabrication of the nanopipettes, the calibration of the HPICM setup, the parameters we have optimized for the imaging of live stereocilia bundles and, lastly, a few basic image post-processing manipulations. PMID:27259929

  19. Long-term time-lapse multimodal microscopy for tracking cell dynamics in live tissue

    NASA Astrophysics Data System (ADS)

    Graf, Benedikt W.; Valero, Maria C.; Chaney, Eric J.; Marjanovic, Marina; Boppart, Marni D.; Boppart, Stephen A.

    2011-02-01

    High speed intravital microscopy has emerged as an essential tool for studying cellular dynamics in live tissue. A limitation of this technique, however, is that the timescale that a sample can be continuously imaged is limited by practical considerations to several hours. Long term observation of live tissue is of great interest for a variety of research areas. We present methods for observing long term cellular dynamics in live tissue based on three-dimensional registration of time-lapse intravital microscopy images. For these experiments we utilized a custom multimodal microscope that allows simultaneous and co-registered acquisition of optical coherence (OCM) and multiphoton (MPM) microscopy images. OCM allows the structure of a sample to be visualized based on backscattered light while MPM excited fluorescence allows individual cells and cell function to be visualized. The OCM images of tissue structure are used to register data sets taken at different time points. The transformations of the OCM images are applied to MPM images to determine the migration of cell populations. This method of image registration is applied to in vivo tracking of bone-marrow derived GFP-labeled stem cells in mouse skin following bone marrow transplants from GFP donors into species-matched wildtype hosts. The use of three-dimensional image registration of time-lapse microscopy images enables tracking these cells after local cutaneous injury, and for investigating the role of skin stem cells in wound healing.

  20. Visualizing live dynamics and ultrastructure of intracellular organelles with preembedding correlative light-electron microscopy.

    PubMed

    Polishchuk, Roman S; Polishchuk, Elena V; Luini, Alberto

    2012-01-01

    One of the very effective methods to perform correlative light-electron microscopy (CLEM) is to combine video imaging of live cells with immuno-electron microscopy. This technique can thus provide detailed, high-resolution characterization of dynamic intracellular organelles. The use of green fluorescent protein (GFP)-tagged chimeras allows the movements and/or behavior of intracellular structures in a live cell to be followed, which can then be fixed at the moment of interest. The subsequent immuno-electron microscopy analysis reveals the three-dimensional (3D) architecture of the same structure, together with the precise identification of the GFP-labeled protein pattern. The process resembles taking a high-resolution snapshot of an interesting and/or rare live event. Conceptually, it consists of a switch of wavelengths, from that of photons to that of electrons, with the associated huge gain in resolution. In this respect, CLEM can be considered as the first, and probably one of the most powerful, super-resolution microscopy techniques. This switch, however, requires complex manipulations of the sample. Considering that CLEM is a very valuable but technically challenging and time-consuming method, accurate protocols are needed to simplify the efforts of researchers who are willing to apply this method for their own purposes. Here, we present a detailed description of the preembedding CLEM procedures that explains the know-how and the "tricks of the trade" that are involved in carrying out the crucial steps of CLEM.

  1. A Microfluidic Platform for Correlative Live-Cell and Super-Resolution Microscopy

    PubMed Central

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images. PMID:25545548

  2. 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

  3. A microfluidic platform for correlative live-cell and super-resolution microscopy.

    PubMed

    Tam, Johnny; Cordier, Guillaume Alan; Bálint, Štefan; Sandoval Álvarez, Ángel; Borbely, Joseph Steven; Lakadamyali, Melike

    2014-01-01

    Recently, super-resolution microscopy methods such as stochastic optical reconstruction microscopy (STORM) have enabled visualization of subcellular structures below the optical resolution limit. Due to the poor temporal resolution, however, these methods have mostly been used to image fixed cells or dynamic processes that evolve on slow time-scales. In particular, fast dynamic processes and their relationship to the underlying ultrastructure or nanoscale protein organization cannot be discerned. To overcome this limitation, we have recently developed a correlative and sequential imaging method that combines live-cell and super-resolution microscopy. This approach adds dynamic background to ultrastructural images providing a new dimension to the interpretation of super-resolution data. However, currently, it suffers from the need to carry out tedious steps of sample preparation manually. To alleviate this problem, we implemented a simple and versatile microfluidic platform that streamlines the sample preparation steps in between live-cell and super-resolution imaging. The platform is based on a microfluidic chip with parallel, miniaturized imaging chambers and an automated fluid-injection device, which delivers a precise amount of a specified reagent to the selected imaging chamber at a specific time within the experiment. We demonstrate that this system can be used for live-cell imaging, automated fixation, and immunostaining of adherent mammalian cells in situ followed by STORM imaging. We further demonstrate an application by correlating mitochondrial dynamics, morphology, and nanoscale mitochondrial protein distribution in live and super-resolution images.

  4. Live cell imaging based on surface plasmon-enhanced fluorescence microscopy using random nanostructures

    NASA Astrophysics Data System (ADS)

    Oh, Youngjin; Lee, Wonju; Son, Taehwang; Kim, Sook Young; Shin, Jeon-Soo; Kim, Donghyun

    2014-02-01

    Localized surface plasmon enhanced microscopy based on nanoislands of random spatial distribution was demonstrated for imaging live cells and molecular interactions. Nanoislands were produced without lithography by high temperature annealing under various processing conditions. The localization of near-field distribution that is associated with localized surface plasmon on metallic random nanoislands was analyzed theoretically and experimentally in comparison with periodic nanostructures. For experimental validation in live cell imaging, mouse macrophage-like cell line stained with Alexa Fluor 488 was prepared on nanoislands. The results suggest the possibility of attaining the imaging resolution on the order of 80 nm.

  5. Minimal tags for rapid dual-color live-cell labeling and super-resolution microscopy.

    PubMed

    Nikić, Ivana; Plass, Tilman; Schraidt, Oliver; Szymański, Jędrzej; Briggs, John A G; Schultz, Carsten; Lemke, Edward A

    2014-02-17

    The growing demands of advanced fluorescence and super-resolution microscopy benefit from the development of small and highly photostable fluorescent probes. Techniques developed to expand the genetic code permit the residue-specific encoding of unnatural amino acids (UAAs) armed with novel clickable chemical handles into proteins in living cells. Here we present the design of new UAAs bearing strained alkene side chains that have improved biocompatibility and stability for the attachment of tetrazine-functionalized organic dyes by the inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC). Furthermore, we fine-tuned the SPIEDAC click reaction to obtain an orthogonal variant for rapid protein labeling which we termed selectivity enhanced (se) SPIEDAC. seSPIEDAC and SPIEDAC were combined for the rapid labeling of live mammalian cells with two different fluorescent probes. We demonstrate the strength of our method by visualizing insulin receptors (IRs) and virus-like particles (VLPs) with dual-color super-resolution microscopy.

  6. Optical imaging of non-fluorescent nanodiamonds in live cells using transient absorption microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Tao; Lu, Feng; Streets, Aaron M.; Fei, Peng; Quan, Junmin; Huang, Yanyi

    2013-05-01

    We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism.We directly observe non-fluorescent nanodiamonds in living cells using transient absorption microscopy. This label-free technology provides a novel modality to study the dynamic behavior of nanodiamonds inside the cells with intrinsic three-dimensional imaging capability. We apply this method to capture the cellular uptake of nanodiamonds under various conditions, confirming the endocytosis mechanism. Electronic supplementary information (ESI) available. See DOI: 10.1039/c3nr00308f

  7. Wavelength-Dependent Differential Interference Contrast Microscopy: Selectively Imaging Nanoparticle Probes in Live Cells

    SciTech Connect

    Sun, Wei; Wang, Gufeng; Fang, Ning; and Yeung, Edward S.

    2009-11-15

    Gold and silver nanoparticles display extraordinarily large apparent refractive indices near their plasmon resonance (PR) wavelengths. These nanoparticles show good contrast in a narrow spectral band but are poorly resolved at other wavelengths in differential interference contrast (DIC) microscopy. The wavelength dependence of DIC contrast of gold/silver nanoparticles is interpreted in terms of Mie's theory and DIC working principles. We further exploit this wavelength dependence by modifying a DIC microscope to enable simultaneous imaging at two wavelengths. We demonstrate that gold/silver nanoparticles immobilized on the same glass slides through hybridization can be differentiated and imaged separately. High-contrast, video-rate images of living cells can be recorded both with and without illuminating the gold nanoparticle probes, providing definitive probe identification. Dual-wavelength DIC microscopy thus presents a new approach to the simultaneous detection of multiple probes of interest for high-speed live-cell imaging.

  8. Comparative three-dimensional imaging of living neurons with confocal and atomic force microscopy.

    PubMed

    McNally, Helen A; Rajwa, Bartek; Sturgis, Jennie; Robinson, J Paul

    2005-03-30

    Atomic force microscopy applications extend across a number of fields; however, limitations have reduced its effectiveness in live cell analysis. This report discusses the use of AFM to evaluate the three-dimensional (3-D) architecture of living chick dorsal root ganglia and sympathetic ganglia. These data sets were compared to similar images acquired with confocal laser scanning microscopy of identical cells. For this comparison we made use of visualization techniques which were applicable to both sets of data and identified several issues when coupling these technologies. These direct comparisons offer quantitative validation and confirmation of the character of novel images acquired by AFM. This paper is one in a series emphasizing various new applications of AFM in neurobiology.

  9. Comparative Analyses of Live-Action and Animated Film Remake Scenes: Finding Alternative Film-Based Teaching Resources

    ERIC Educational Resources Information Center

    Champoux, Joseph E.

    2005-01-01

    Live-action and animated film remake scenes can show many topics typically taught in organizational behaviour and management courses. This article discusses, analyses and compares such scenes to identify parallel film scenes useful for teaching. The analysis assesses the scenes to decide which scene type, animated or live-action, more effectively…

  10. Atomic force microscopy as a tool for the investigation of living cells.

    PubMed

    Morkvėnaitė-Vilkončienė, Inga; Ramanavičienė, Almira; Ramanavičius, Arūnas

    2013-01-01

    Atomic force microscopy is a valuable and useful tool for the imaging and investigation of living cells in their natural environment at high resolution. Procedures applied to living cell preparation before measurements should be adapted individually for different kinds of cells and for the desired measurement technique. Different ways of cell immobilization, such as chemical fixation on the surface, entrapment in the pores of a membrane, or growing them directly on glass cover slips or on plastic substrates, result in the distortion or appearance of artifacts in atomic force microscopy images. Cell fixation allows the multiple use of samples and storage for a prolonged period; it also increases the resolution of imaging. Different atomic force microscopy modes are used for the imaging and analysis of living cells. The contact mode is the best for cell imaging because of high resolution, but it is usually based on the following: (i) image formation at low interaction force, (ii) low scanning speed, and (iii) usage of "soft," low resolution cantilevers. The tapping mode allows a cell to behave like a very solid material, and destructive shear forces are minimized, but imaging in liquid is difficult. The force spectroscopy mode is used for measuring the mechanical properties of cells; however, obtained results strongly depend on the cell fixation method. In this paper, the application of 3 atomic force microscopy modes including (i) contact, (ii) tapping, and (iii) force spectroscopy for the investigation of cells is described. The possibilities of cell preparation for the measurements, imaging, and determination of mechanical properties of cells are provided. The applicability of atomic force microscopy to diagnostics and other biomedical purposes is discussed.

  11. Polarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in live cells

    PubMed Central

    McQuilken, Molly; Mehta, Shalin B.; Verma, Amitabh; Harris, Grant; Oldenbourg, Rudolf; Gladfelter, Amy S.

    2015-01-01

    The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with fluorescence microscopy and GFP-tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using polarized fluorescence microscopy and OpenPolScope open-access software (www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, polarized fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user-friendly image acquisition to measure and analyze polarized fluorescence. PMID:26061244

  12. N-way FRET microscopy of multiple protein-protein interactions in live cells.

    PubMed

    Hoppe, Adam D; Scott, Brandon L; Welliver, Timothy P; Straight, Samuel W; Swanson, Joel A

    2013-01-01

    Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

  13. Mapping nanomechanical properties of live cells using multi-harmonic atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Raman, A.; Trigueros, S.; Cartagena, A.; Stevenson, A. P. Z.; Susilo, M.; Nauman, E.; Contera, S. Antoranz

    2011-12-01

    The nanomechanical properties of living cells, such as their surface elastic response and adhesion, have important roles in cellular processes such as morphogenesis, mechano-transduction, focal adhesion, motility, metastasis and drug delivery. Techniques based on quasi-static atomic force microscopy techniques can map these properties, but they lack the spatial and temporal resolution that is needed to observe many of the relevant details. Here, we present a dynamic atomic force microscopy method to map quantitatively the nanomechanical properties of live cells with a throughput (measured in pixels/minute) that is ~10-1,000 times higher than that achieved with quasi-static atomic force microscopy techniques. The local properties of a cell are derived from the 0th, 1st and 2nd harmonic components of the Fourier spectrum of the AFM cantilevers interacting with the cell surface. Local stiffness, stiffness gradient and the viscoelastic dissipation of live Escherichia coli bacteria, rat fibroblasts and human red blood cells were all mapped in buffer solutions. Our method is compatible with commercial atomic force microscopes and could be used to analyse mechanical changes in tumours, cells and biofilm formation with sub-10 nm detail.

  14. Light sheet microscopy for tracking single molecules on the apical surface of living cells.

    PubMed

    Li, Yu; Hu, Ying; Cang, Hu

    2013-12-12

    Single particle tracking is a powerful tool to study single molecule dynamics in living biological samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single molecules on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF molcules on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temperature, the average diffusion coefficient of EGF on A549 cells was measured to be 0.13 μm(2)/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coefficients and an increase of the average diffusion coefficient at room temperature. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole "lifetime" of a particle from binding till photobleaching or internalization.

  15. Live imaging of nervous system development and function using light-sheet microscopy.

    PubMed

    Lemon, William C; Keller, Philipp J

    2015-01-01

    In vivo imaging applications typically require carefully balancing conflicting parameters. Often it is necessary to achieve high imaging speed, low photo-bleaching, and photo-toxicity, good three-dimensional resolution, high signal-to-noise ratio, and excellent physical coverage at the same time. Light-sheet microscopy provides good performance in all of these categories, and is thus emerging as a particularly powerful live imaging method for the life sciences. We see an outstanding potential for applying light-sheet microscopy to the study of development and function of the early nervous system in vertebrates and higher invertebrates. Here, we review state-of-the-art approaches to live imaging of early development, and show how the unique capabilities of light-sheet microscopy can further advance our understanding of the development and function of the nervous system. We discuss key considerations in the design of light-sheet microscopy experiments, including sample preparation and fluorescent marker strategies, and provide an outlook for future directions in the field.

  16. Intravital microscopy: a novel tool to study cell biology in living animals

    PubMed Central

    Weigert, Roberto; Sramkova, Monika; Parente, Laura; Masedunskas, Andrius

    2011-01-01

    Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criteria. Indeed, first we will focus on those studies in which organs where imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures. PMID:20372919

  17. Intravital microscopy: a novel tool to study cell biology in living animals.

    PubMed

    Weigert, Roberto; Sramkova, Monika; Parente, Laura; Amornphimoltham, Panomwat; Masedunskas, Andrius

    2010-05-01

    Intravital microscopy encompasses various optical microscopy techniques aimed at visualizing biological processes in live animals. In the last decade, the development of non-linear optical microscopy resulted in an enormous increase of in vivo studies, which have addressed key biological questions in fields such as neurobiology, immunology and tumor biology. Recently, few studies have shown that subcellular processes can be imaged dynamically in the live animal at a resolution comparable to that achieved in cell cultures, providing new opportunities to study cell biology under physiological conditions. The overall aim of this review is to give the reader a general idea of the potential applications of intravital microscopy with a particular emphasis on subcellular imaging. An overview of some of the most exciting studies in this field will be presented using resolution as a main organizing criterion. Indeed, first we will focus on those studies in which organs were imaged at the tissue level, then on those focusing on single cells imaging, and finally on those imaging subcellular organelles and structures.

  18. A bright and long-pulse illumination for ultrahigh-speed microscopy of living specimens.

    PubMed

    Nakano, Hitoshi; Yokoi, Sayoko; Yoshida, Shigeru; Yamada, Makoto; Takeuchi, Takeshi; Takehara, Kosei; Etoh, T Goji

    2010-01-01

    Ultrahigh-speed microscopy of living specimens requires ultrabright illumination. Moreover, the duration of illumination should be sufficiently long, on the order of at least several tens of milliseconds, in order to investigate the dynamic state of living specimens. However, specimens are exposed to a high risk of damage by the intense illumination. The brightness and pulse duration of illumination have to be continuously controlled for use in the ultrahigh-speed microscopy of living specimens. Commercial or laboratory-made illumination systems do not satisfy the abovementioned requirements. In this paper, the development of a bright and long-pulse illumination system for ultrahigh-speed microscopy of living specimens is presented. A xenon flashlamp with an arc length of 1.5 mm has been used as the light source. The electrical power supply consists of a voltage-regulated circuit, a capacitor bank, and a control circuit including an insulated-gate bipolar transistor as a gating device, which provides a large rectangular current pulse with the duration in the range to the order of several tens of milliseconds. The brightness, pulse duration, and repetition rate can be easily and continuously controlled. The illumination developed in the present study is installed in an inverted fluorescence microscope equipped with a high-speed camera in order to evaluate the performance as an illumination source. A fluorescent image of the living spermatozoa of a mouse obtained at a frame rate of 8 kHz shows good contrast. Such an image cannot be obtained using a commercial illumination system.

  19. A bright and long-pulse illumination for ultrahigh-speed microscopy of living specimens

    NASA Astrophysics Data System (ADS)

    Nakano, Hitoshi; Yokoi, Sayoko; Yoshida, Shigeru; Yamada, Makoto; Takeuchi, Takeshi; Takehara, Kosei; Etoh, T. Goji

    2010-01-01

    Ultrahigh-speed microscopy of living specimens requires ultrabright illumination. Moreover, the duration of illumination should be sufficiently long, on the order of at least several tens of milliseconds, in order to investigate the dynamic state of living specimens. However, specimens are exposed to a high risk of damage by the intense illumination. The brightness and pulse duration of illumination have to be continuously controlled for use in the ultrahigh-speed microscopy of living specimens. Commercial or laboratory-made illumination systems do not satisfy the abovementioned requirements. In this paper, the development of a bright and long-pulse illumination system for ultrahigh-speed microscopy of living specimens is presented. A xenon flashlamp with an arc length of 1.5 mm has been used as the light source. The electrical power supply consists of a voltage-regulated circuit, a capacitor bank, and a control circuit including an insulated-gate bipolar transistor as a gating device, which provides a large rectangular current pulse with the duration in the range to the order of several tens of milliseconds. The brightness, pulse duration, and repetition rate can be easily and continuously controlled. The illumination developed in the present study is installed in an inverted fluorescence microscope equipped with a high-speed camera in order to evaluate the performance as an illumination source. A fluorescent image of the living spermatozoa of a mouse obtained at a frame rate of 8 kHz shows good contrast. Such an image cannot be obtained using a commercial illumination system.

  20. Transparent thin-film characterization by using differential optical sectioning interference microscopy.

    PubMed

    Wang, Chun-Chieh; Lin, Jiunn-Yuan; Jian, Hung-Jhang; Lee, Chau-Hwang

    2007-10-20

    We propose an optical thin-film characterization technique, differential optical sectioning interference microscopy (DOSIM), for simultaneously measuring the refractive indices and thicknesses of transparent thin films with submicrometer lateral resolution. DOSIM obtains the depth and optical phase information of a thin film by using a dual-scan concept in differential optical sectioning microscopy combined with the Fabry-Perot interferometric effect and allows the solution of refractive index and thickness without the 2pi phase-wrapping ambiguity. Because DOSIM uses a microscope objective as the probe, its lateral resolution achieves the diffraction limit. As a demonstration, we measure the refractive indices and thicknesses of SiO2 thin films grown on Si substrate and indium-tin-oxide thin films grown on a glass substrate. We also compare the measurement results of DOSIM with those of a conventional ellipsometer and an atomic force microscope.

  1. Characterization of Homopolymer and Polymer Blend Films by Phase Sensitive Acoustic Microscopy

    NASA Astrophysics Data System (ADS)

    Ngwa, Wilfred; Wannemacher, Reinhold; Grill, Wolfgang

    2003-03-01

    CHARACTERIZATION OF HOMOPOLYMER AND POLYMER BLEND FILMS BY PHASE SENSITIVE ACOUSTIC MICROSCOPY W Ngwa, R Wannemacher, W Grill Institute of Experimental Physics II, University of Leipzig, 04103 Leipzig, Germany Abstract We have used phase sensitive acoustic microscopy (PSAM) to study homopolymer thin films of polystyrene (PS) and poly (methyl methacrylate) (PMMA), as well as PS/PMMA blend films. We show from our results that PSAM can be used as a complementary and highly valuable technique for elucidating the three-dimensional (3D) morphology and micromechanical properties of thin films. Three-dimensional image acquisition with vector contrast provides the basis for: complex V(z) analysis (per image pixel), 3D image processing, height profiling, and subsurface image analysis of the polymer films. Results show good agreement with previous studies. In addition, important new information on the three dimensional structure and properties of polymer films is obtained. Homopolymer film structure analysis reveals (pseudo-) dewetting by retraction of droplets, resulting in a morphology that can serve as a starting point for the analysis of polymer blend thin films. The outcome of confocal laser scanning microscopy studies, performed on the same samples are correlated with the obtained results. Advantages and limitations of PSAM are discussed.

  2. Characterization of organic film with scanning acoustic microscopy

    NASA Astrophysics Data System (ADS)

    Miyasaka, Chiaki; Du, Jikai; Tittmann, Bernhard R.

    2003-08-01

    The present article reports a technique to measure velocity of an organic film deposited on a homogeneous substrate, wherein the thickness of the film and the diameter of the measured area of the specimen are in the order of a few microns. A thinly sliced human kidney was selected as an example of an organic film. The thickness of the specimen was substantially 3 μm. For the substrate, fused quartz was used because its elastic properties are known and stable. The spherical acoustic lens was used to determine the position for measurement. The frequencies at 400 MHz and 600 MHz were used for the measurement and the visualization, respectively. The generation of the Rayleigh waves under the above conditions was simulated by numerical calculations based onthe wave propagation theory for layered media.

  3. Live cell response to mechanical stimulation studied by integrated optical and atomic force microscopy.

    PubMed

    Trache, Andreea; Lim, Soon-Mi

    2010-10-04

    To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a new technology able to investigate cells behavior at sub-cellular level with high spatial and temporal resolution was developed. Thus, an atomic force microscope (AFM) was integrated with total internal reflection fluorescence (TIRF) microscopy and fast-spinning disk (FSD) confocal microscopy. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real-time. Significant rearrangement of the actin filaments and focal adhesions was shown due to local mechanical stimulation at the apical cell surface that induced changes into the cellular structure throughout the cell body. These innovative techniques will provide new information for understanding live cell restructuring and dynamics in response to mechanical force. A detailed protocol and a representative data set that show live cell response to mechanical stimulation are presented.

  4. Study of environmental biodegradation of LDPE films in soil using optical and scanning electron microscopy.

    PubMed

    Mumtaz, Tabassum; Khan, M R; Hassan, Mohd Ali

    2010-07-01

    An outdoor soil burial test was carried out to evaluate the degradation of commercially available LDPE carrier bags in natural soil for up to 2 years. Biodegradability of low density polyethylene films in soil was monitored using both optical and scanning electron microscopy (SEM). After 7-9 months of soil exposure, microbial colonization was evident on the film surface. Exposed LDPE samples exhibit progressive changes towards degradation after 17-22 months. SEM images reveal signs of degradation such as exfoliation and formation of cracks on film leading to disintegration. The possible degradation mode and consequences on the use and disposal of LDPE films is discussed.

  5. Highly photostable, reversibly photoswitchable fluorescent protein with high contrast ratio for live-cell superresolution microscopy

    PubMed Central

    Zhang, Xi; Zhang, Mingshu; Li, Dong; He, Wenting; Peng, Jianxin; Betzig, Eric; Xu, Pingyong

    2016-01-01

    Two long-standing problems for superresolution (SR) fluorescence microscopy are high illumination intensity and long acquisition time, which significantly hamper its application for live-cell imaging. Reversibly photoswitchable fluorescent proteins (RSFPs) have made it possible to dramatically lower the illumination intensities in saturated depletion-based SR techniques, such as saturated depletion nonlinear structured illumination microscopy (NL-SIM) and reversible saturable optical fluorescence transition microscopy. The characteristics of RSFPs most critical for SR live-cell imaging include, first, the integrated fluorescence signal across each switching cycle, which depends upon the absorption cross-section, effective quantum yield, and characteristic switching time from the fluorescent “on” to “off” state; second, the fluorescence contrast ratio of on/off states; and third, the photostability under excitation and depletion. Up to now, the RSFPs of the Dronpa and rsEGFP (reversibly switchable EGFP) families have been exploited for SR imaging. However, their limited number of switching cycles, relatively low fluorescence signal, and poor contrast ratio under physiological conditions ultimately restrict their utility in time-lapse live-cell imaging and their ability to reach the desired resolution at a reasonable signal-to-noise ratio. Here, we present a truly monomeric RSFP, Skylan-NS, whose properties are optimized for the recently developed patterned activation NL-SIM, which enables low-intensity (∼100 W/cm2) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view. PMID:27562163

  6. Enhanced piezoelectric performance of composite sol-gel thick films evaluated using piezoresponse force microscopy.

    PubMed

    Liu, Yuanming; Lam, Kwok Ho; Kirk Shung, K; Li, Jiangyu; Zhou, Qifa

    2013-05-14

    Conventional composite sol-gel method has been modified to enhance the piezoelectric performance of ceramic thick films. Lead zirconate titanate (PZT) and lead magnesium niobate-lead titanate (PMN-PT) thick films were fabricated using the modified sol-gel method for ultrasonic transducer applications. In this work, piezoresponse force microscopy was employed to evaluate the piezoelectric characteristics of PZT and PMN-PT composite sol-gel thick films. The images of the piezoelectric response and the strain-electric field hysteresis loop behavior were measured. The effective piezoelectric coefficient (d33,eff) of the films was determined from the measured loop data. It was found that the effective local piezoelectric coefficient of both PZT and PMN-PT composite films is comparable to that of their bulk ceramics. The promising results suggest that the modified composite sol-gel method is a promising way to prepare the high-quality, crack-free ceramic thick films.

  7. Determination of the actuator sensitivity of electromechanical polypropylene films by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Peltonen, Jouko; Paajanen, Mika; Lekkala, Jukka

    2000-10-01

    The actuator functionality of electromechanical polypropylene films was studied using atomic force microscopy. The film carries a permanent electric charge and includes microbubbles as a result of two-dimensional stretching of the film. The thickness change of various film structures covered with electrodes was measured as a function of external voltage. The dependence was found to be nonlinear, the thickness change in the range 0.001%-0.1% of the total film thickness and affected by the internal charge density of the film. Applying a capacitor model including an air gap within the polymer layer enabled the determination of the Young's modulus, the interfacial charge density and the actuator sensitivity of the studied structures.

  8. Scanning Tunneling Microscopy of Multilayer Thin Film Solar Cell Materials^*

    NASA Astrophysics Data System (ADS)

    Mantovani, J. G.; Friedfeld, R.; Raffaelle, R. P.

    1996-03-01

    We have been investigating electrochemically deposited multilayer structures based on the Cu_xIn_2-xSe2 system for use in thin film solar cells. The interest in multilayer structures is due to their proposed use in increasing thin film solar cell efficiency. We have imaged the artificially imposed superstructure of our nanoscale multilayers using a scanning tunneling microscope. A comparison is made between the theoretically calculated modulation wavelengths and those generated by Fourier analysis of the scanning tunneling microscope images. A discussion of the use of photo-assisted tunneling spectroscopy in a modified STM is presented. * This work was supported by the Southeastern University Research Association in collaboration with Oak Ridge National Laboratory and the Florida Solar Energy Center.

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

    NASA Astrophysics Data System (ADS)

    Slabý, TomáÅ.¡; Křížová, Aneta; Lošt'ák, Martin; Čolláková, Jana; Jůzová, Veronika; Veselý, Pavel; Chmelík, Radim

    2015-03-01

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

  10. A simple, straightforward correlative live-cell-imaging-structured-illumination-microscopy approach for studying organelle dynamics.

    PubMed

    Sherman, Shachar; Nachmias, Dikla; Elia, Natalie

    2015-09-01

    Most cellular organelles are highly dynamic and continuously undergo membrane fission and fusion to mediate their function. Documenting organelle dynamics under physiological conditions, therefore, requires high temporal resolution of the recording system. Concurrently, these structures are relatively small and determining their substructural organization is often impossible using conventional microscopy. Structured Illumination Microscopy (SIM) is a super resolution technique providing a two-fold increase in resolution. Importantly, SIM is versatile because it allows the use of any fluorescent dye or protein and, hence, is highly applicable for cell biology. However, similar to other SR techniques, the applicability of SIM to high-speed live cell imaging is limited. Here we present an easy, straightforward methodology for coupling of high-speed live cell recordings, using spinning disk (SD) microscopy, with SIM. Using this simple methodology, we are able to track individual mitochondrial membrane fission and fusion events in real time and to determine the network connectivity and substructural organization of the membrane at high resolution. Applying this methodology to other cellular organelles such as, ER, golgi, and cilia will no doubt contribute to our understanding of membrane dynamics in cells.

  11. IQGAP1 Interactome Analysis by In Vitro Reconstitution and Live Cell 3-Color FRET Microscopy

    PubMed Central

    Wallrabe, Horst; Cai, Ying; Sun, Yuansheng; Periasamy, A.; Luzes, R.; Fang, Xiaolan; Kan, Ho-Man; Cameron, L. C.; Schafer, Dorothy A.; Bloom, George S.

    2014-01-01

    IQGAP1 stimulates branched actin filament nucleation by activating N-WASP, which then activates the Arp2/3 complex. N-WASP can be activated by other factors, including GTP-bound Cdc42 or Rac1, which also bind IQGAP1. Here we report the use of purified proteins for in vitro binding and actin polymerization assays, and Förster (or fluorescence) resonance energy transfer (FRET) microscopy of cultured cells to illuminate functional interactions among IQGAP1, N-WASP, actin, and either Cdc42 or Rac1. In pyrene-actin assembly assays containing N-WASP and Arp2/3 complex, IQGAP1 plus either small G protein cooperatively stimulated actin filament nucleation by reducing the lag time before 50% maximum actin polymerization was reached. Similarly, Cdc42 and Rac1 modulated the binding of IQGAP1 to N-WASP in a dose-dependent manner, with Cdc42 enhancing the interaction and Rac1 reducing the interaction. These in vitro reconstitution results suggested that IQGAP1 interacts by similar, yet distinct mechanisms with Cdc42 versus Rac1 to regulate actin filament assembly through N-WASP in vivo. The physiological relevance of these multi-protein interactions was substantiated by 3-color FRET microscopy of live MDCK cells expressing various combinations of fluorescent N-WASP, IQGAP1, Cdc42, Rac1 and actin. This study also establishes 3-color FRET microscopy as a powerful tool for studying dynamic intermolecular interactions in live cells. PMID:24124181

  12. Integrated microscopy for real-time imaging of mechanotransduction studies in live cells

    NASA Astrophysics Data System (ADS)

    Trache, Andreea; Lim, Soon-Mi

    2009-05-01

    Mechanical force is an important stimulus and determinant of many vascular smooth muscle cell functions including contraction, proliferation, migration, and cell attachment. Transmission of force from outside the cell through focal adhesions controls the dynamics of these adhesion sites and initiates intracellular signaling cascades that alter cellular behavior. To understand the mechanism by which living cells sense mechanical forces, and how they respond and adapt to their environment, a critical first step is to develop a new technology to investigate cellular behavior at subcellular level that integrates an atomic force microscope (AFM) with total internal reflection fluorescence (TIRF) and fast-spinning disk (FSD) confocal microscopy, providing high spatial and temporal resolution. AFM uses a nanosensor to measure the cell surface topography and can apply and measure mechanical force with high precision. TIRF microscopy is an optical imaging technique that provides high-contrast images with high z-resolution of fluorescently labeled molecules in the immediate vicinity of the cell-coverslip interface. FSD confocal microscopy allows rapid 3-D imaging throughout the cell in real time. The integrated system is broadly applicable across a wide range of molecular dynamic studies in any adherent live cells, allowing direct optical imaging of cell responses to mechanical stimulation in real time.

  13. Correlated Atomic Force Microscopy and Flourescence Lifetime Imaging of Live Bacterial Cells

    SciTech Connect

    Micic, Miodrag; Hu, Dehong; Suh, Yung D.; Newton, Greg J.; Romine, Margaret F.; Lu, H PETER.

    2004-04-01

    We report on the imaging of living bacterial cells by using a new correlated tapping-mode atomic force microscopy (AFM) and confocal al fluorescence lifetime imaging microscopy (FLIM). Different methods of preparing the bacterial sample were explored for optimal imaging of Gram-negative Shewanella oneidensis MR-1 cells on poly-1-lysine coated surfaces and agarose gel coated surfaces. We have found that the agarose gel containing 99% buffer can provide a local aqueous environment for single bacterial cells. Furthermore, the cell surface topography can be characterized by tapping-mode in-air AFM imaging for the single bacterial cells that are partially embedded. Using in-air rather than under-water AFM imaging of the living cells significantly enhanced the contrast and single-to-noise ration of the AFM images. Near-field AFM-tip enhanced fluorescence lifetime imaging (AFM-FLIM) holds great promise for obtaining fluorescence images beyond the optical diffraction limited spatial resolution. We have previously demonstrated near-field AFM-FLIM imaging of polymer beads beyond the diffraction limited spatial resolution. Here, as the first step of applying AFM-FLIM on imaging living bacterial cells, we demonstrate a correlated and consecutive AFM topographic imaging, fluorescence intensity imaging, and FLIM imaging to characterize cell polarity.

  14. Imaging and control of domain structures in ferroelectric thin films via scanning force microscopy.

    SciTech Connect

    Gruverman, A.; Auciello, O.; Tokumoto, H.; Materials Science Division; Joint Research Center for Atom Tech.; National Inst. for Advanced Interdisciplinary Research

    1998-01-01

    Scanning force microscopy (SFM) is becoming a powerful technique with great potential both for imaging and for control of domain structures in ferroelectric materials at the nanometer scale. Application of SFM to visualization of domain structures in ferroelectric thin films is described. Imaging methods of ferroelectric domains are based on the detection of surface charges in the noncontact mode of SFM and on the measurement of the piezoelectric response of a ferroelectric film to an external field applied by the tip in the SFM contact mode. This latter mode can be used for nondestructive evaluation of local ferroelectric and piezoelectric properties and for manipulation of domains of less than 50 nm in diameter. The effect of the film thickness and crystallinity on the imaging resolution is discussed. Scanning force microscopy is shown to be a technique well suited for nanoscale investigation of switching processes and electrical degradation effects in ferroelectric thin films.

  15. Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy.

    PubMed

    Almassalha, Luay M; Bauer, Greta M; Chandler, John E; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K; Subramanian, Hariharan; Chandel, Navdeep S; Szleifer, Igal; Backman, Vadim

    2016-10-18

    The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure-function relationship in live cells.

  16. Label-free imaging of the native, living cellular nanoarchitecture using partial-wave spectroscopic microscopy

    PubMed Central

    Almassalha, Luay M.; Bauer, Greta M.; Chandler, John E.; Gladstein, Scott; Cherkezyan, Lusik; Stypula-Cyrus, Yolanda; Weinberg, Samuel; Zhang, Di; Thusgaard Ruhoff, Peder; Roy, Hemant K.; Subramanian, Hariharan; Chandel, Navdeep S.; Szleifer, Igal; Backman, Vadim

    2016-01-01

    The organization of chromatin is a regulator of molecular processes including transcription, replication, and DNA repair. The structures within chromatin that regulate these processes span from the nucleosomal (10-nm) to the chromosomal (>200-nm) levels, with little known about the dynamics of chromatin structure between these scales due to a lack of quantitative imaging technique in live cells. Previous work using partial-wave spectroscopic (PWS) microscopy, a quantitative imaging technique with sensitivity to macromolecular organization between 20 and 200 nm, has shown that transformation of chromatin at these length scales is a fundamental event during carcinogenesis. As the dynamics of chromatin likely play a critical regulatory role in cellular function, it is critical to develop live-cell imaging techniques that can probe the real-time temporal behavior of the chromatin nanoarchitecture. Therefore, we developed a live-cell PWS technique that allows high-throughput, label-free study of the causal relationship between nanoscale organization and molecular function in real time. In this work, we use live-cell PWS to study the change in chromatin structure due to DNA damage and expand on the link between metabolic function and the structure of higher-order chromatin. In particular, we studied the temporal changes to chromatin during UV light exposure, show that live-cell DNA-binding dyes induce damage to chromatin within seconds, and demonstrate a direct link between higher-order chromatin structure and mitochondrial membrane potential. Because biological function is tightly paired with structure, live-cell PWS is a powerful tool to study the nanoscale structure–function relationship in live cells. PMID:27702891

  17. Influence of Cu-Ti thin film surface properties on antimicrobial activity and viability of living cells.

    PubMed

    Wojcieszak, Damian; Kaczmarek, Danuta; Antosiak, Aleksandra; Mazur, Michal; Rybak, Zbigniew; Rusak, Agnieszka; Osekowska, Malgorzata; Poniedzialek, Agata; Gamian, Andrzej; Szponar, Bogumila

    2015-11-01

    The paper describes properties of thin-film coatings based on copper and titanium. Thin films were prepared by co-sputtering of Cu and Ti targets in argon plasma. Deposited coatings consist of 90at.% of Cu and 10at.% of Ti. Characterization of the film was made on the basis of investigations of microstructure and physicochemical properties of the surface. Methods such as scanning electron microscopy, x-ray microanalysis, x-ray diffraction, x-ray photoelectron spectroscopy, atomic force microscopy, optical profilometry and wettability measurements were used to assess the properties of deposited thin films. An impact of Cu-Ti coating on the growth of selected bacteria and viability of the living cells (line L929, NCTC clone 929) was described in relation to the structure, surface state and wettability of the film. It was found that as-deposited films were amorphous. However, in such surroundings the nanocrystalline grains of 10-15nm and 25-35nm size were present. High surface active area with a roughness of 8.9nm, had an effect on receiving relatively high water contact angle value (74.1°). Such wettability may promote cell adhesion and result in an increase of the probability of copper ion transfer from the film surface into the cell. Thin films revealed bactericidal and fungicidal effects even in short term-contact. High activity of prepared films was directly related to high amount (ca. 51 %) of copper ions at 1+ state as x-ray photoelectron spectroscopy results have shown.

  18. Magnetic force microscopy of nano-size magnetic domain ordering in heavy ion irradiated fullerene films.

    PubMed

    Kumar, Amit; Avasthi, D K; Pivin, J C; Papaléo, R M; Tripathi, A; Singh, F; Sulania, I

    2007-06-01

    In the present work, magnetic force microscopy is employed to investigate the magnetic ordering in ion irradiated fullerene films. It is observed that magnetic domain size is approximately 100-200 nm and magnetic signal is stronger at the domain boundaries. Magnetic signal arise in irradiated films is confirmed by magnetic measurements using a superconducting quantum interference device which increases with the ion fluence. The induced magnetism is possibly due to structural defects in the amorphous carbon phase formed by ion irradiation.

  19. Integral refractive index determination of living suspension cells by multifocus digital holographic phase contrast microscopy.

    PubMed

    Kemper, Björn; Kosmeier, Sebastian; Langehanenberg, Patrik; von Bally, Gert; Bredebusch, Ilona; Domschke, Wolfram; Schnekenburger, Jürgen

    2007-01-01

    A method for the determination of the integral refractive index of living cells in suspension by digital holographic microscopy is described. Digital holographic phase contrast images of spherical cells in suspension are recorded, and the radius as well as the integral refractive index are determined by fitting the relation between cell thickness and phase distribution to the measured phase data. The algorithm only requires information about the refractive index of the suspension medium and the image scale of the microscope system. The specific digital holographic microscopy advantage of subsequent focus correction allows a simultaneous investigation of cells in different focus planes. Results obtained from human pancreas and liver tumor cells show that the integral cellular refractive index decreases with increasing cell radius.

  20. Multiparametric atomic force microscopy imaging of single bacteriophages extruding from living bacteria

    NASA Astrophysics Data System (ADS)

    Alsteens, David; Trabelsi, Heykel; Soumillion, Patrice; Dufrêne, Yves F.

    2013-12-01

    Force-distance (FD) curve-based atomic force microscopy is a valuable tool to simultaneously image the structure and map the biophysical properties of biological samples at the nanoscale. Traditionally, FD-based atomic force microscopy has been severely limited by its poor temporal and lateral resolutions. Here we report the use of advanced FD-based technology combined with biochemically sensitive tips to image filamentous bacteriophages extruding from living bacteria at unprecedented speed and resolution. Directly correlated multiparametric images of the structure, adhesion and elasticity of infected bacteria demonstrate that the sites of assembly and extrusion localize at the bacterial septum in the form of soft nanodomains surrounded by stiff cell wall material. The quantitative nano-bio-imaging method presented here offers a wealth of opportunities for mapping the physical properties and molecular interactions of complex biosystems, from viruses to tissues.

  1. Probing local water contents of in vitro protein films by ultrasonic force microscopy

    NASA Astrophysics Data System (ADS)

    Szoszkiewicz, Robert; Kulik, Andrzej J.; Gremaud, Gerard; Lekka, Malgorzata

    2005-03-01

    By means of ultrasonic force microscopy and lateral force microscopy we measure adhesion hysteresis and friction on protein films of bovine serum albumin and concanavalin A at local scales. Our investigations at different relative humidities (less than 5% and at 50% relative humidity) correspond to dehydrated and hydrated states of proteins. We demonstrate that a substantial increase of adhesion hysteresis with relative humidity is sensitive measure of protein-water binding capacity at local scales.

  2. Monitoring mitochondrial membranes permeability in live neurons and mitochondrial swelling through electron microscopy analysis.

    PubMed

    Arrázola, Macarena S; Inestrosa, Nibaldo C

    2015-01-01

    Maintenance of mitochondrial membrane integrity is essential for mitochondrial function and neuronal viability. Apoptotic stimulus or calcium overload leads to mitochondrial permeability transition pore (mPTP ) opening and induces mitochondrial swelling, a common feature of mitochondrial membrane permeabilization. The first phenomenon can be evaluated in cells loaded with the dye calcein -AM quenched by cobalt, and mitochondrial swelling can be detected by electron microscopy through the analysis of mitochondrial membrane integrity. Here, we describe a live cell imaging assay to detect mitochondrial permeability transition and the development of a detailed analysis of morphological and ultrastructural changes that mitochondria undergo during this process.

  3. Fluorescent Rhodamines and Fluorogenic Carbopyronines for Super-Resolution STED Microscopy in Living Cells.

    PubMed

    Butkevich, Alexey N; Mitronova, Gyuzel Yu; Sidenstein, Sven C; Klocke, Jessica L; Kamin, Dirk; Meineke, Dirk N H; D'Este, Elisa; Kraemer, Philip-Tobias; Danzl, Johann G; Belov, Vladimir N; Hell, Stefan W

    2016-03-01

    A range of bright and photostable rhodamines and carbopyronines with absorption maxima in the range of λ=500-630 nm were prepared, and enabled the specific labeling of cytoskeletal filaments using HaloTag technology followed by staining with 1 μm solutions of the dye-ligand conjugates. The synthesis, photophysical parameters, fluorogenic behavior, and structure-property relationships of the new dyes are discussed. Light microscopy with stimulated emission depletion (STED) provided one- and two-color images of living cells with an optical resolution of 40-60 nm.

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

    PubMed

    Peckys, Diana B; de Jonge, Niels

    2011-04-13

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

  5. Measuring the elastic properties of living cells with atomic force microscopy indentation.

    PubMed

    Mackay, Joanna L; Kumar, Sanjay

    2013-01-01

    Atomic force microscopy (AFM) is a powerful and versatile tool for probing the mechanical properties of biological samples. This chapter describes the procedures for using AFM indentation to measure the elastic moduli of living cells. We include step-by-step instructions for cantilever calibration and data acquisition using a combined AFM/optical microscope system, as well as a detailed protocol for data analysis. Our protocol is written specifically for the BioScope™ Catalyst™ AFM system (Bruker AXS Inc.); however, most of the general concepts can be readily translated to other commercial systems.

  6. Monitoring of relative mitochondrial membrane potential in living cells by fluorescence microscopy

    PubMed Central

    1981-01-01

    Permeant cationic fluorescent probes are shown to be selectively accumulated by the mitochondria of living cells. Mitochondria-specific interaction of such molecules is apparently dependent on the high trans- membrane potential (inside negative) maintained by functional mitochondria. Dissipation of the mitochondrial trans-membrane and potential by ionophores or inhibitors of electron transport eliminates the selective mitochondrial association of these compounds. The application of such potential-dependent probes in conjunction with fluorescence microscopy allows the monitoring of mitochondrial membrane potential in individual living cells. Marked elevations in mitochondria- associated probe fluorescence have been observed in cells engaged in active movement. This approach to the analysis of mitochondrial membrane potential should be of value in future investigations of the control of energy metabolism and energy requirements of specific biological functions at the cellular level. PMID:6783667

  7. Imaging Complex Protein Metabolism in Live Organisms by Stimulated Raman Scattering Microscopy with Isotope Labeling

    PubMed Central

    2016-01-01

    Protein metabolism, consisting of both synthesis and degradation, is highly complex, playing an indispensable regulatory role throughout physiological and pathological processes. Over recent decades, extensive efforts, using approaches such as autoradiography, mass spectrometry, and fluorescence microscopy, have been devoted to the study of protein metabolism. However, noninvasive and global visualization of protein metabolism has proven to be highly challenging, especially in live systems. Recently, stimulated Raman scattering (SRS) microscopy coupled with metabolic labeling of deuterated amino acids (D-AAs) was demonstrated for use in imaging newly synthesized proteins in cultured cell lines. Herein, we significantly generalize this notion to develop a comprehensive labeling and imaging platform for live visualization of complex protein metabolism, including synthesis, degradation, and pulse–chase analysis of two temporally defined populations. First, the deuterium labeling efficiency was optimized, allowing time-lapse imaging of protein synthesis dynamics within individual live cells with high spatial–temporal resolution. Second, by tracking the methyl group (CH3) distribution attributed to pre-existing proteins, this platform also enables us to map protein degradation inside live cells. Third, using two subsets of structurally and spectroscopically distinct D-AAs, we achieved two-color pulse–chase imaging, as demonstrated by observing aggregate formation of mutant hungtingtin proteins. Finally, going beyond simple cell lines, we demonstrated the imaging ability of protein synthesis in brain tissues, zebrafish, and mice in vivo. Hence, the presented labeling and imaging platform would be a valuable tool to study complex protein metabolism with high sensitivity, resolution, and biocompatibility for a broad spectrum of systems ranging from cells to model animals and possibly to humans. PMID:25560305

  8. Scanning Probe Microscopy on heterogeneous CaCu3Ti4O12 thin films

    PubMed Central

    2011-01-01

    The conductive atomic force microscopy provided a local characterization of the dielectric heterogeneities in CaCu3Ti4O12 (CCTO) thin films deposited by MOCVD on IrO2 bottom electrode. In particular, both techniques have been employed to clarify the role of the inter- and sub-granular features in terms of conductive and insulating regions. The microstructure and the dielectric properties of CCTO thin films have been studied and the evidence of internal barriers in CCTO thin films has been provided. The role of internal barriers and the possible explanation for the extrinsic origin of the giant dielectric response in CCTO has been evaluated. PMID:21711646

  9. Noncontact atomic force microscopy studies of ultrathin films of amorphous solid water deposited on Au(111).

    PubMed

    Donev, J M K; Yu, Q; Long, B R; Bollinger, R K; Fain, S C

    2005-07-22

    Noncontact atomic force microscopy was used to study the morphological changes of an ultrathin amorphous solid water (ASW) film as a function of deposition temperature, annealing temperature, and annealing time. ASW deposited at 80 or 108 K on Au(111) formed truncated hemispherical clusters of increasing size during annealing at 134 K; these clusters were inferred to be crystalline. The number of nuclei present at the outer surface of the film after deposition was greater for higher deposition temperature. For lower cluster densities, depletion of the ASW film around the clusters was observed when the clusters became larger and dendritic growth was observed when the apparent cluster footprint radius exceeded 100 nm.

  10. Nanocharacterization and nanofabrication of a Nafion thin film in liquids by atomic force microscopy.

    PubMed

    Umemura, Kazuo; Wang, Tong; Hara, Masahiko; Kuroda, Reiko; Uchida, On; Nagai, Masayuki

    2006-03-28

    We demonstrated the nanocharacterization and nanofabrication of a Nafion thin film using atomic force microscopy (AFM). AFM images showed that the Nafion molecules form nanoclusters in water, in 5% methanol, and in acetic acid. Young's modulus E of a Nafion film was estimated by sequential force curve measurements in water and in 5% methanol on one sample surface. Ewater/E5% methanol was 1.75 +/- 0.40, so the film was much softer in 5% methanol than in water. Even when solvent was replaced from 5% methanol to water, Young's modulus was not recovered soon. We showed the first example of the mechanical properties of a Nafion film on the nanoscale. Furthermore, we succeeded in fabricating 3D nanostructures on a Nafion surface by AFM nanolithography in liquids. Our results showed the new potential of the AFM nanolithography of a polymer film by softening the molecules in liquids.

  11. Monitoring Biosensor Activity in Living Cells with Fluorescence Lifetime Imaging Microscopy

    PubMed Central

    Hum, Julia M.; Siegel, Amanda P.; Pavalko, Fredrick M.; Day, Richard N.

    2012-01-01

    Live-cell microscopy is now routinely used to monitor the activities of the genetically encoded biosensor proteins that are designed to directly measure specific cell signaling events inside cells, tissues, or organisms. Most fluorescent biosensor proteins rely on Förster resonance energy transfer (FRET) to report conformational changes in the protein that occur in response to signaling events, and this is commonly measured with intensity-based ratiometric imaging methods. An alternative method for monitoring the activities of the FRET-based biosensor proteins is fluorescence lifetime imaging microscopy (FLIM). FLIM measurements are made in the time domain, and are not affected by factors that commonly limit intensity measurements. In this review, we describe the use of the digital frequency domain (FD) FLIM method for the analysis of FRET signals. We illustrate the methods necessary for the calibration of the FD FLIM system, and demonstrate the analysis of data obtained from cells expressing “FRET standard” fusion proteins. We then use the FLIM-FRET approach to monitor the changes in activities of two different biosensor proteins in specific regions of single living cells. Importantly, the factors required for the accurate determination and reproducibility of lifetime measurements are described in detail. PMID:23203070

  12. Live-cell superresolution microscopy reveals the organization of RNA polymerase in the bacterial nucleoid

    PubMed Central

    Stracy, Mathew; Lesterlin, Christian; Garza de Leon, Federico; Uphoff, Stephan; Zawadzki, Pawel; Kapanidis, Achillefs N.

    2015-01-01

    Despite the fundamental importance of transcription, a comprehensive analysis of RNA polymerase (RNAP) behavior and its role in the nucleoid organization in vivo is lacking. Here, we used superresolution microscopy to study the localization and dynamics of the transcription machinery and DNA in live bacterial cells, at both the single-molecule and the population level. We used photoactivated single-molecule tracking to discriminate between mobile RNAPs and RNAPs specifically bound to DNA, either on promoters or transcribed genes. Mobile RNAPs can explore the whole nucleoid while searching for promoters, and spend 85% of their search time in nonspecific interactions with DNA. On the other hand, the distribution of specifically bound RNAPs shows that low levels of transcription can occur throughout the nucleoid. Further, clustering analysis and 3D structured illumination microscopy (SIM) show that dense clusters of transcribing RNAPs form almost exclusively at the nucleoid periphery. Treatment with rifampicin shows that active transcription is necessary for maintaining this spatial organization. In faster growth conditions, the fraction of transcribing RNAPs increases, as well as their clustering. Under these conditions, we observed dramatic phase separation between the densest clusters of RNAPs and the densest regions of the nucleoid. These findings show that transcription can cause spatial reorganization of the nucleoid, with movement of gene loci out of the bulk of DNA as levels of transcription increase. This work provides a global view of the organization of RNA polymerase and transcription in living cells. PMID:26224838

  13. Accurate cell counts in live mouse embryos using optical quadrature and differential interference contrast microscopy

    NASA Astrophysics Data System (ADS)

    Warger, William C., II; Newmark, Judith A.; Zhao, Bing; Warner, Carol M.; DiMarzio, Charles A.

    2006-02-01

    Present imaging techniques used in in vitro fertilization (IVF) clinics are unable to produce accurate cell counts in developing embryos past the eight-cell stage. We have developed a method that has produced accurate cell counts in live mouse embryos ranging from 13-25 cells by combining Differential Interference Contrast (DIC) and Optical Quadrature Microscopy. Optical Quadrature Microscopy is an interferometric imaging modality that measures the amplitude and phase of the signal beam that travels through the embryo. The phase is transformed into an image of optical path length difference, which is used to determine the maximum optical path length deviation of a single cell. DIC microscopy gives distinct cell boundaries for cells within the focal plane when other cells do not lie in the path to the objective. Fitting an ellipse to the boundary of a single cell in the DIC image and combining it with the maximum optical path length deviation of a single cell creates an ellipsoidal model cell of optical path length deviation. Subtracting the model cell from the Optical Quadrature image will either show the optical path length deviation of the culture medium or reveal another cell underneath. Once all the boundaries are used in the DIC image, the subtracted Optical Quadrature image is analyzed to determine the cell boundaries of the remaining cells. The final cell count is produced when no more cells can be subtracted. We have produced exact cell counts on 5 samples, which have been validated by Epi-Fluorescence images of Hoechst stained nuclei.

  14. Nanomechanical and topographical imaging of living cells by atomic force microscopy with colloidal probes

    SciTech Connect

    Puricelli, Luca; Galluzzi, Massimiliano; Schulte, Carsten; Podestà, Alessandro Milani, Paolo

    2015-03-15

    Atomic Force Microscopy (AFM) has a great potential as a tool to characterize mechanical and morphological properties of living cells; these properties have been shown to correlate with cells’ fate and patho-physiological state in view of the development of novel early-diagnostic strategies. Although several reports have described experimental and technical approaches for the characterization of cellular elasticity by means of AFM, a robust and commonly accepted methodology is still lacking. Here, we show that micrometric spherical probes (also known as colloidal probes) are well suited for performing a combined topographic and mechanical analysis of living cells, with spatial resolution suitable for a complete and accurate mapping of cell morphological and elastic properties, and superior reliability and accuracy in the mechanical measurements with respect to conventional and widely used sharp AFM tips. We address a number of issues concerning the nanomechanical analysis, including the applicability of contact mechanical models and the impact of a constrained contact geometry on the measured Young’s modulus (the finite-thickness effect). We have tested our protocol by imaging living PC12 and MDA-MB-231 cells, in order to demonstrate the importance of the correction of the finite-thickness effect and the change in Young’s modulus induced by the action of a cytoskeleton-targeting drug.

  15. Characterization of thin film semiconductors by scanning probe microscopy and tunneling spectroscopy

    NASA Astrophysics Data System (ADS)

    Gichuhi, Anthony

    We have used scanning tunneling microscopy, atomic force microscopy, tunneling spectroscopy, resonance Raman spectroscopy and electrochemistry to study the electrosynthesis of II-VI compound semiconductors with special emphasis on ZnS, CdS, and HgS. This dissertation will focus mainly on the electrochemical and scanning probe (STM and AFM) applications to these compounds, in addition to novel materials such as CoSb. We hope to understand the structural, as well optical properties of these materials. Finally, we hope to develop a recipe for the electrosynthesis of high quality semiconductor films. In Chapter 2, we report an electrochemical, scanning probe microscopic and Raman spectroscopic investigation of thin US films grown by electrochemical atomic layer epitaxy (EC-ALE) aimed at understanding the role played by the order of deposition on film quality. In Chapter 3, we report a study of electrosynthesized CdS-HgS heterojunctions using scanning tunneling microscopy (STM), photoluminescence spectroscopy (PL), and electrochemistry. US thin films were grown by electrochemical atomic layer epitaxy onto Au(111) substrates and were terminated with a single HgS monolayer. In Chapter 4, the structure and chemical composition of electrosynthesized ZnS thin films on Au(111) substrates grown by alternating underpotential deposition and oxidative adsorption cycles of S and Zn from solution precursors was studied by scanning tunneling microscopy (STM) and X-ray photoelectron spectroscopy (XPS). In Chapter 5, conditions for the growth of. stable mercury sulfide (HgS) monolayers on Au(111) surfaces using electrochemical atomic layer epitaxy have been investigated. HgS thin films were characterized by X-ray photoelectron spectroscopy (XPS) and scanning tunneling microscopy (STM). Chapter 6: This chapter describes the use of resonance Raman spectroscopy to characterize thin films of the II-VI compound semiconductors electrosynthesized on metal surfaces. We describe how resonance

  16. Use of virtual microscopy for didactic live-audience presentation in anatomic pathology.

    PubMed

    Romer, David J; Suster, Saul

    2003-02-01

    Didactic presentations on the topic of anatomic pathology in front of a live audience have been largely dependent on the use of standard 2 x 2 inch projection slides (Kodachromes) of selected still images from the topic at hand. Because of the highly visual nature of the specialty of anatomic pathology, this method has had some serious limitations. With the advent of digital imaging techniques and the availability of new electronic software for the projection of images, new possibilities have become available for didactic presentations in anatomic pathology in front of a large, live audience. We describe a method whereby large digital images or "virtual slides" were produced from digitally scanned whole-mount sections of histologic glass slides and projected using a combination of PowerPoint (Microsoft Corp, Redmond, WA) and virtual microscopy in front of a live audience. To provide a seamless transition between the two presentation formats, the personal computer-based PowerPoint slides were hyperlinked to a browser-based virtual microscope viewer. The presenter, with the use of a mouse, was able to "move" the image of the scanned slide on the screen, to transition seamlessly among various magnifications, and to rapidly select from the whole-mount scanned slide among any areas of interest pertinent to the topic. Thus, the visual experience obtained by the audience simulated that of viewing a glass slide at a multi-headed microscope during a glass slide tutorial. Because this most closely approximates the experience of reviewing glass slides under the microscope for practicing pathologists, the educational experience of the presentation is greatly enhanced by the use of this technique. Also, this method permits making this type of presentation available to a much larger group of individuals in a live audience.

  17. Mapping chemical concentration in binary thin organic films via multi-wavelength scanning absorption microscopy (MWSAM)

    NASA Astrophysics Data System (ADS)

    Berriman, Garth; Routley, Ben; Holdsworth, John; Zhou, Xiaojing; Belcher, Warwick; Dastoor, Paul

    2014-09-01

    The composition and thickness of binary thin organic films is determined by measuring the optical absorption at multiple wavelengths across the film surface and performing a component analysis fit to absorption standards for the materials. The multiple laser wavelengths are focused onto the surface using microscope objectives and raster scanned across the film surface using a piezo-electric actuator X-Y stage. All of the wavelengths are scanned simultaneously with a frequency division multiplexing system used to separate the individual wavelength response. The composition values are in good quantitative agreement with measurements obtained by scanning transmission x-ray microscopy (STXM). This new characterization technique extends quantitative compositional mapping of thin films to thickness regimes beyond that accessible by STXM.

  18. Characterization of polysilicon films by Raman spectroscopy and transmission electron microscopy: A comparative study

    SciTech Connect

    Tallant, D.R.; Headley, T.J.; Medernach, J.W.; Geyling, F.

    1993-11-12

    Samples of chemically-vapor-deposited micrometer and sub-micrometer-thick films of polysilicon were analyzed by transmission electron microscopy (TEM) in cross-section and by Raman spectroscopy with illumination at their surface. TEM and Raman spectroscopy both find varying amounts of polycrystalline and amorphous silicon in the wafers. Raman spectra obtained using blue, green and red excitation wavelengths to vary the Raman sampling depth are compared with TEM cross-sections of these films. Films showing crystalline columnar structures in their TEM micrographs have Raman spectra with a band near 497 cm{sup {minus}1} in addition to the dominant polycrystalline silicon band (521 cm{sup {minus}1}). The TEM micrographs of these films have numerous faulted regions and fringes indicative of nanometer-scale silicon structures, which are believed to correspond to the 497cm{sup {minus}1} Raman band.

  19. Observation of Individual Fluorine Atom from Highly Oriented Poly (tetrafluoroethylene) Films by Atomic Force Microscopy

    NASA Technical Reports Server (NTRS)

    Lee, Jonathan A.,; Paley, Mark S.

    1999-01-01

    Direct observation of the film thickness, molecular structure and individual fluorine atoms from highly oriented poly(tetrafluoroethylene) (PTFE) films were achieved using atomic force microscopy (AFM). A thin PTFE film is mechanically deposited onto a smooth glass substrate at specific temperatures by a friction transfer technique. Atomic resolution images of these films show that the chain-like helical structures of the PTFE macromolecules are aligned parallel to each other with an intermolecular spacing of 5.72 A, and individual fluorine atoms are clearly observed along these twisted molecular chains with an interatomic spacing of 2.75 A. Furthermore, the first direct AFM measurements for the radius of the fluorine-helix, and of the carbon-helix in sub-angstrom scale are reported as 1.70 A and 0.54 A respectively.

  20. Observation of Individual Fluorine Atoms from Highly Oriented Poly(Tetrafluoroethylene) Films by Atomic Force Microscopy

    NASA Technical Reports Server (NTRS)

    Lee, J. A.

    2000-01-01

    Direct observation of the film thickness, molecular structure, and individual fluorine atoms from highly oriented poly(tetrafluoroethylene) (PTFE) films were achieved using atomic force microscopy (AFM). A thin PTFE film is mechanically deposited onto a smooth glass substrate at specific temperatures by a friction-transfer technique. Atomic resolution images of these films show that the chain-like helical structures of the PTFE macromolecules are aligned parallel to each other with an intermolecular spacing of 5.72 A, and individual fluorine atoms are clearly observed along these twisted molecular chains with an interatomic spacing of 2.75 A. Furthermore, the first direct AFM measurements for the radius of the fluorine-helix, and of the carbon-helix in sub-angstrom scale are reported as 1.7 and 0.54 A respectively.

  1. Mechanical characterization of porous nano-thin films by use of atomic force acoustic microscopy.

    PubMed

    Kopycinska-Müller, M; Clausner, A; Yeap, K-B; Köhler, B; Kuzeyeva, N; Mahajan, S; Savage, T; Zschech, E; Wolter, K-J

    2016-03-01

    The indentation modulus of thin films of porous organosilicate glass with a nominal porosity content of 30% and thicknesses of 350nm, 200nm, and 46nm is determined with help of atomic force acoustic microscopy (AFAM). This scanning probe microscopy based technique provides the highest possible depth resolution. The values of the indentation modulus obtained for the 350nm and 200nm thin films were respectively 6.3GPa±0.2GPa and 7.2GPa±0.2GPa and free of the substrate influence. The sample with the thickness of 46nm was tested in four independent measurement sets. Cantilevers with two different tip radii of about 150nm and less than 50nm were applied in different force ranges to obtain a result for the indentation modulus that was free of the substrate influence. A detailed data analysis yielded value of 8.3GPa±0.4GPa for the thinnest film. The values of the indentation modulus obtained for the thin films of porous organosilicate glasses increased with the decreasing film thickness. The stiffening observed for the porous films could be explained by evolution of the pore topology as a function of the film thickness. To ensure that our results were free of the substrate influence, we analyzed the ratio of the sample deformation as well as the tip radius to the film thickness. The results obtained for the substrate parameter were compared for all the measurement series and showed, which ones could be declared as free of the substrate influence.

  2. Scanning probe microscopy: instrumentation and applications on thin films and magnetic multilayers.

    PubMed

    Karoutsos, Vagelis

    2009-12-01

    In this article we present a review on instrumentation and the modes of operation of a scanning probe microscope. In detail, we review the main techniques of Scanning Probe Microscopy (SPM), which are Scanning Tunneling Microscopy (STM) and Atomic Force Microscopy (AFM), focusing our attention on the latter one. The AFM instrument provides information on the roughness and grain size of thin films. As an example we review recent results on two metallic thin film systems: thin Ag films deposited on glass, and Ni/Pt compositionally modulated multilayers deposited on glass, Si, and polyimide substrates. To show the validity of the grain size measurements, we compare the data with the ones resulting from X-ray diffraction (XRD) measurements. We show that the AFM results are reliable for grain diameters as small as 14 nm, which is approximately comparable to the tip radius. Finally, we deal with Magnetic Force Microscopy (MFM) results on Co/Pt and Co/Au multilayers. We observe perpendicularly magnetized domains. The domain configurations are correlated to the magnetization hysteresis curves.

  3. A simple and stable auto focusing protocol for long multidimensional live cell microscopy.

    PubMed

    Wolf, F; Geley, S

    2006-01-01

    Focus maintenance is a challenging problem in multidimensional wide-field microscopy. Most automated microscopes use software algorithms, which are applied to z-sections of the object, to select for the plane with the best signal to noise ratio. When applied automatically in multidimensional imaging applications, auto focus routines significantly increase light exposure and can become cytotoxic if applied too frequently. In addition, automated focusing procedures can readily focus on unwanted high contrast objects. By labelling a defined position with a fluorescent marker, we were able to separate the focusing procedure from the actual image acquisition positions and therefore overcome some of the major drawbacks of routine auto focus procedures. To implement this method in a multidimensional acquisition experiment, we created a visual basic-based program, which is run prior to each image acquisition. This technique allows tight control of focus whilst keeping light toxicity in live cell imaging experiments to a minimum.

  4. Hydroxylated fluorescent dyes for live cell labeling: synthesis, spectra and superresolution STED** microscopy.

    PubMed

    Belov, Vladimir N; Butkevich, Alexey N; Kolmakov, Kirill; Sokolov, Viktor V; Shojaei, Heydar; Sidenstein, Sven C; Kamin, Dirk; Matthias, Jessica; Vlijm, Rifka; Engelhardt, Johann; Hell, Stefan W

    2017-03-29

    Hydroxylated rhodamines, carbopyronines, silico- and germanorhodamines with absorption maxima in the range of 530-640 nm were prepared and applied in specific labeling of living cells. The direct and high-yielding entry to germa- and silaxanthones tolerates the presence of protected heteroatoms and may be considered for the syntheses of various sila- and germafluoresceins, as well as -rhodols. Application in stimulated emission depletion (STED) fluorescence microscopy revealed a resolution of 50-75 nm in one- and two-color imaging of vimentin-HaloTag fused protein and native tubulin. The established structure-property relationships allow prediction of the spectral properties and the positions of spirolactone/zwitterion equilibria for the new analogs of rhodamines, carbo-, silico- and germanorhodamines using simple additive schemes.

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

    PubMed Central

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

    2016-01-01

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

  6. A general method to improve fluorophores for live-cell and single-molecule microscopy

    PubMed Central

    Grimm, Jonathan B.; English, Brian P.; Chen, Jiji; Slaughter, Joel P.; Zhang, Zhengjian; Revyakin, Andrey; Patel, Ronak; Macklin, John J.; Normanno, Davide; Singer, Robert H.; Lionnet, Timothée; Lavis, Luke D.

    2014-01-01

    Specific labeling of biomolecules with bright fluorophores is the keystone of fluorescence microscopy. Genetically encoded self-labeling tag proteins can be coupled to synthetic dyes inside living cells, resulting in brighter reporters than fluorescent proteins. Intracellular labeling using these techniques requires cell-permeable fluorescent ligands, however, limiting utility to a small number of classic fluorophores. Here, we describe a simple structural modification that improves the brightness and photostability of dyes while preserving spectral properties and cell permeability. Inspired by molecular modeling, we replaced the N,N-dimethylamino substituents in tetramethylrhodamine with four-membered azetidine rings. This addition of two carbon atoms doubles the quantum efficiency and improves the photon yield of the dye in applications ranging from in vitro single-molecule measurements to super-resolution imaging. The novel substitution is generalizable, yielding a palette of chemical dyes with improved quantum efficiencies that spans the UV and visible range. PMID:25599551

  7. Parallel excitation-emission multiplexed fluorescence lifetime confocal microscopy for live cell imaging

    PubMed Central

    Zhao, Ming; Li, Yu; Peng, Leilei

    2014-01-01

    We present a novel excitation-emission multiplexed fluorescence lifetime microscopy (FLIM) method that surpasses current FLIM techniques in multiplexing capability. The method employs Fourier multiplexing to simultaneously acquire confocal fluorescence lifetime images of multiple excitation wavelength and emission color combinations at 44,000 pixels/sec. The system is built with low-cost CW laser sources and standard PMTs with versatile spectral configuration, which can be implemented as an add-on to commercial confocal microscopes. The Fourier lifetime confocal method allows fast multiplexed FLIM imaging, which makes it possible to monitor multiple biological processes in live cells. The low cost and compatibility with commercial systems could also make multiplexed FLIM more accessible to biological research community. PMID:24921725

  8. Directing the assembly of nanostructured films with living cells

    NASA Astrophysics Data System (ADS)

    Brinker, C. Jeffrey

    2007-03-01

    This talk describes our recent discovery of the ability of living cells to organize extended nanostructures and nano-objects in a manner that creates a unique, highly biocompatible nano//bio interface (Science 313, 337-340, 2006). We find that, using short chain phospholipids to direct the formation of thin film silica mesophases during evaporation-induced self-assembly, the introduction of cells (so far yeast and bacteria) alters profoundly the inorganic self-assembly pathway. Cells actively organize around themselves an ordered, multilayered lipid-membrane that interfaces coherently with a lipid-templated silica mesophase. This bio/nano interface is unique in that it withstands drying (even evacuation) without cracking or the development of tensile stresses -- yet it maintains accessibility to molecules, proteins/antibodies, plasmids, etc - introduced into the 3D silica host. Additionally cell viability is preserved for weeks to months in the absence of buffer, making these constructs useful as standalone cell-based sensors. The bio/nano interfaces we describe do not form `passively' -- rather they are a consequence of the cell's ability to sense and actively respond to external stimuli. During EISA, solvent evaporation concentrates the extracellular environment in osmolytes. In response to this hyperosmotic stress, the cells release water, creating a gradient in pH, which is maintained within the adjoining nanostructured host and serves to localize lipids, proteins, plasmids, lipidized nanocrystals, and a variety of other components at the cellular surface. This active organization of the bio/nano interface can be accomplished during ink-jet printing or selective wetting -- processes allowing patterning of cellular arrays - and even spatially-defined genetic modification.

  9. In-situ investigation of thermal instabilities and solid state dewetting in polycrystalline platinum thin films via confocal laser microscopy

    SciTech Connect

    Jahangir, S.; Cheng, Xuan; Huang, H. H.; Nagarajan, V.; Ihlefeld, J.

    2014-10-28

    Solid state dewetting and the subsequent morphological changes for platinum thin films grown on zinc oxide (ZnO) buffered (001) silicon substrates (Pt/ZnO/SiO{sub 2}/(001)Si system) is investigated under vacuum conditions via a custom-designed confocal laser microscope coupled with a laser heating system. Live imaging of thin film dewetting under a range of heating and quenching vacuum ambients reveals events including hillock formation, hole formation, and hole growth that lead to formation of a network of Pt ligaments, break up of Pt ligaments to individual islands and subsequent Pt islands shape reformation, in chronological fashion. These findings are corroborated by ex-situ materials characterization and quantitative electron microscopy analysis. A secondary hole formation via blistering before film rupture is revealed to be the critical stage, after which a rapid dewetting catastrophe occurs. This process is instantaneous and cannot be captured by ex-situ methods. Finally, an intermetallic phase forms at 900 °C and alters the morphology of Pt islands, suggesting a practical limit to the thermal environments that may be used for these platinized silicon wafers in vacuum conditions.

  10. Back-etch method for plan view transmission electron microscopy sample preparation of optically opaque films.

    PubMed

    Yao, Bo; Coffey, Kevin R

    2008-04-01

    Back-etch methods have been widely used to prepare plan view transmission electron microscopy (TEM) samples of thin films on membranes by removal of the Si substrate below the membrane by backside etching. The conventional means to determine when to stop the etch process is to observe the color of the light transmitted through the sample, which is sensitive to the remaining Si thickness. However, most metallic films thicker than 75 nm are opaque, and there is no detectable color change prior to film perforation. In this paper, a back-etch method based on the observation of an abrupt change of optical reflection contrast is introduced as a means to determine the etch endpoint to prepare TEM samples for these films. As the acid etchant removes the Si substrate material a rough interface is generated. This interface becomes a relatively smooth and featureless region when the etchant reaches the membrane (film/SiO2). This featureless region is caused by the mirror reflection of the film plane (film/SiO2 interface) through the optically transparent SiO2 layer. The lower etch rate of SiO2 (compared with Si) gives the operator enough time to stop the etching without perforating the film. A clear view of the morphology and control of Si roughness during etching are critical to this method, which are discussed in detail. The procedures of mounting wax removal and sample rinsing are also described in detail, as during these steps damage to the membrane may easily occur without appropriate consideration. As examples, the preparation of 100-nm-thick Fe-based amorphous alloy thin film and 160-nm-thick Cu-thin film samples for TEM imaging is described.

  11. Measuring Exciton Diffusion in Conjugated Polymer Films with Super-resolution Microscopy

    NASA Astrophysics Data System (ADS)

    Penwell, Samuel; Ginsberg, Lucas; Noriega Manez, Rodrigo; Ginsberg, Naomi

    2015-03-01

    Conjugated polymers are highly tunable organic semiconductors, which can be solution processed to form thin films, making them prime candidates for organic photovoltaic devices. One of the most important parameters in a conjugated polymer solar cell is the exciton diffusion length, which depends on intermolecular couplings, and is typically on the order of 10 nm. This mean exciton migration can vary dramatically between films and within a single film due to heterogeneities in morphology on length scales of 10's to 100's nm. To study the variability of exciton diffusion and morphology within individual conjugated polymer films, we are adapting stimulated emission depletion microscopy. STED is typically used in biology with well-engineered fluorescent labels or on NV-centers in diamond. I will, however, describe how we have demonstrated STED in conjugated polymer films of MEH-PPV and CN-PPV by taking care to first understand the film's photophysical properties. This new approach provides a way to study exciton diffusion by utilizing subdiffraction optical excitation volumes. In this way, we will obtain a spatiotemporal map of exciton distributions that will help to correlate the energetic landscape to film morphology at the nanoscale. This research is supported in part by the Department of Energy Office of Science Graduate Fellowship Program (DOE SCGF), made possible in part by the American Recovery and Reinvestment Act of 2009, administered by ORISE-ORAU under Contract No. DE-AC05-06.

  12. In situ atomic force microscopy observation of hydrogen absorption/desorption by Palladium thin film

    NASA Astrophysics Data System (ADS)

    Matsumoto, Itoko; Sakaki, Kouji; Nakamura, Yumiko; Akiba, Etsuo

    2011-12-01

    Grain structure changes in Pd thin film during hydrogen absorption and desorption were observed by in situ atomic force microscopy. The as-sputtered film had a smooth flat surface with 20-30 nm grains. Film that absorbed hydrogen showed buckling, caused by the compressive stress due to lattice expansion as Pd metal reacted with hydrogen to form the hydride. Grains on the buckles were agglomerated and deformed unlike those on flat areas beside the buckles. Film that absorbed and then desorbed hydrogen still showed some buckling; however, many buckles shrank and flattened when the compressive stress of lattice expansion was released during desorption. On both the remaining and the shrunken buckles, grain agglomeration was retained; whereas, the deformed grains reverted back to their original form. X-ray diffraction indicated compressive residual stress in the as-sputtered film and tensile residual stress in the film after hydrogen absorption/desorption. These results indicate that irreversible grain agglomeration is related to residual tensile stress in the film although agglomeration occurs only on the buckled areas.

  13. Hygroscopic Swelling Determination of Cellulose Nanocrystal (CNC) Films by Polarized Light Microscopy Digital Image Correlation.

    PubMed

    Shrestha, Shikha; Diaz, Jairo A; Ghanbari, Siavash; Youngblood, Jeffrey P

    2017-04-11

    The coefficient of hygroscopic swelling (CHS) of self-organized and shear-oriented cellulose nanocrystal (CNC) films was determined by capturing hygroscopic strains produced as result of isothermal water vapor intake in equilibrium. Contrast enhanced microscopy digital image correlation enabled the characterization of dimensional changes induced by the hygroscopic swelling of the films. The distinct microstructure and birefringence of CNC films served in exploring the in-plane hygroscopic swelling at relative humidity values ranging from 0% to 97%. Water vapor intake in CNC films was measured using dynamic vapor sorption (DVS) at constant temperature. The obtained experimental moisture sorption and kinetic profiles were analyzed by fitting with Guggenheim, Anderson, and deBoer (GAB) and Parallel Exponential Kinetics (PEK) models, respectively. Self-organized CNC films showed isotropic swelling, CHS ∼0.040 %strain/%C. By contrast, shear-oriented CNC films exhibited an anisotropic swelling, resulting in CHS ∼0.02 and ∼0.30 %strain/%C, parallel and perpendicular to CNC alignment, respectively. Finite element analysis (FEA) further predicted moisture diffusion as the predominant mechanism for swelling of CNC films.

  14. Refractive index sensing of green fluorescent proteins in living cells using fluorescence lifetime imaging microscopy.

    PubMed

    van Manen, Henk-Jan; Verkuijlen, Paul; Wittendorp, Paul; Subramaniam, Vinod; van den Berg, Timo K; Roos, Dirk; Otto, Cees

    2008-04-15

    We show that fluorescence lifetime imaging microscopy (FLIM) of green fluorescent protein (GFP) molecules in cells can be used to report on the local refractive index of intracellular GFP. We expressed GFP fusion constructs of Rac2 and gp91(phox), which are both subunits of the phagocyte NADPH oxidase enzyme, in human myeloid PLB-985 cells and showed by high-resolution confocal fluorescence microscopy that GFP-Rac2 and GFP-gp91(phox) are targeted to the cytosol and to membranes, respectively. Frequency-domain FLIM experiments on these PLB-985 cells resulted in average fluorescence lifetimes of 2.70 ns for cytosolic GFP-Rac2 and 2.31 ns for membrane-bound GFP-gp91(phox). By comparing these lifetimes with a calibration curve obtained by measuring GFP lifetimes in PBS/glycerol mixtures of known refractive index, we found that the local refractive indices of cytosolic GFP-Rac2 and membrane-targeted GFP-gp91(phox) are approximately 1.38 and approximately 1.46, respectively, which is in good correspondence with reported values for the cytosol and plasma membrane measured by other techniques. The ability to measure the local refractive index of proteins in living cells by FLIM may be important in revealing intracellular spatial heterogeneities within organelles such as the plasma and phagosomal membrane.

  15. Free-living spirochetes from Cape Cod microbial mats detected by electron microscopy

    NASA Technical Reports Server (NTRS)

    Teal, T. H.; Chapman, M.; Guillemette, T.; Margulis, L.

    1996-01-01

    Spirochetes from microbial mats and anaerobic mud samples collected in salt marshes were studied by light microscopy, whole mount and thin section transmission electron microscopy. Enriched in cellobiose-rifampin medium, selective for Spirochaeta bajacaliforniensis, seven distinguishable spirochete morphotypes were observed. Their diameters ranged from 0.17 micron to > 0.45 micron. Six of these morphotypes came from southwest Cape Cod, Massachusetts: five from Microcoleus-dominated mat samples collected at Sippewissett salt marsh and one from anoxic mud collected at School Street salt marsh (on the east side of Eel Pond). The seventh morphotype was enriched from anoxic mud sampled from the north central Cape Cod, at the Sandy Neck salt marsh. Five of these morphotypes are similar or identical to previously described spirochetes (Leptospira, Spirochaeta halophila, Spirochaeta bajacaliforniensis, Spirosymplokos deltaeiberi and Treponema), whereas the other two have unique features that suggest they have not been previously described. One of the morphotypes resembles Spirosymplokos deltaeiberi (the largest free-living spirochete described), in its large variable diameter (0.4-3.0 microns), cytoplasmic granules, and spherical (round) bodies with composite structure. This resemblance permits its tentative identification as a Sippewissett strain of Spirosymplokos deltaeiberi. Microbial mats samples collected in sterile Petri dishes and stored dry for more than four years yielded many organisms upon rewetting, including small unidentified spirochetes in at least 4 out of 100 enrichments.

  16. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart

    PubMed Central

    Huang, Chao; Kaza, Aditya K.; Hitchcock, Robert W.; Sachse, Frank B.

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5–9 lines, which is comparable to 4–8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery. PMID:25309455

  17. Local delivery of fluorescent dye for fiber-optics confocal microscopy of the living heart.

    PubMed

    Huang, Chao; Kaza, Aditya K; Hitchcock, Robert W; Sachse, Frank B

    2014-01-01

    Fiber-optics confocal microscopy (FCM) is an emerging imaging technology with various applications in basic research and clinical diagnosis. FCM allows for real-time in situ microscopy of tissue at sub-cellular scale. Recently FCM has been investigated for cardiac imaging, in particular, for discrimination of cardiac tissue during pediatric open-heart surgery. FCM relies on fluorescent dyes. The current clinical approach of dye delivery is based on systemic injection, which is associated with high dye consumption, and adverse clinical events. In this study, we investigated approaches for local dye delivery during FCM imaging based on dye carriers attached to the imaging probe. Using three-dimensional confocal microscopy, automated bench tests, and FCM imaging we quantitatively characterized dye release of carriers composed of open-pore foam only and foam loaded with agarose hydrogel. In addition, we compared local dye delivery with a model of systemic dye delivery in the isolated perfused rodent heart. We measured the signal-to-noise ratio (SNR) of images acquired in various regions of the heart. Our evaluations showed that foam-agarose dye carriers exhibited a prolonged dye release vs. foam-only carriers. Foam-agarose dye carriers allowed reliable imaging of 5-9 lines, which is comparable to 4-8 min of continuous dye release. Our study in the living heart revealed that the SNR of FCM images using local and systemic dye delivery is not different. However, we observed differences in the imaged tissue microstructure with the two approaches. Structural features characteristic of microvasculature were solely observed for systemic dye delivery. Our findings suggest that local dye delivery approach for FCM imaging constitutes an important alternative to systemic dye delivery. We suggest that the approach for local dye delivery will facilitate clinical translation of FCM, for instance, for FCM imaging during pediatric heart surgery.

  18. Localization of bleomycin in a single living cell using three-photon excitation microscopy

    NASA Astrophysics Data System (ADS)

    Abraham, Anil T.; Brautigan, David L.; Hecht, Sidney M.; Periasamy, Ammasi

    2001-04-01

    Bleomycin has been used in the clinic as a chemotherapeutic agent for the treatment of several neoplasms, including non-Hodgkins lymphomas, squamous cell carcinomas, and testicular tumors. The effectiveness of bleomycin is believed to be derived from its ability to bind and oxidatively cleave DNA in the presence of a iron cofactor in vivo. A substantial amount of data on BLM has been collected, there is little information concerning the effects of bleomycin in living cells. In order to obtain data pertinent to the effects of BLM in intact cells, we have exploited the intrinsic fluorescence property of bleomycin to monitor the uptake of the drug in mammalian cells. We employed two light microscopy techniques, a wide-field and three-photon excitation (760 nm) fluorescence microscopy. Treatment of HeLa cells with bleomycin resulted in rapid to localization within the cells. In addition data collected from the wide field experiments, three-photon excitation of BLM which considerably reduced the phototoxic effect compared with UV light excitation in the wide-field microscopy indicated co-localization of the drug to regions of the cytoplasm occupied by the endoplasmic reticulum probe, DiOC5. The data clearly indicates that the cellular uptake of bleomycin after one minute includes the nucleus as well as in cytoplasm. Contrary to previous studies, which indicate chromosomal DNA as the target of bleomycin, the current findings suggest that the drug is distributed to many areas within the cell, including the endoplasmic reticulum, an organelle that is known to contain ribonucleic acids.

  19. Scanning thermoelectric microscopy of local thermoelectric behaviors in (Bi,Sb)2Te3 films

    NASA Astrophysics Data System (ADS)

    Zhao, Kunyu; Zeng, Huarong; Xu, Kunqi; Yu, Huizhu; Li, Guorong; Song, Junqiang; Shi, Xun; Chen, Lidong

    2015-01-01

    In this paper we develop scanning thermoelectric microscopy (STeM) on the basis of commercial atomic force microscope. The nanoscale thermoelectric behaviors of (Bi,Sb)2Te3 (BST) thin films were studied. 3ω-technique was used for thermal conductivity imaging and quantitative thermal characterization. By acquiring the unique Seebeck information from 2ω frequency component, nanoscale thermoelectric images were firstly obtained, exhibiting remarkably inhomogeneous distribution of local Seebeck coefficient in the thin films. Positive thermoelectric response is revealed by the modulation of temperature difference between thermal tip and sample, corresponding to p-type conduction within BST sample.

  20. Preparation and atomic force microscopy of CTAB stabilized polythiophene nanoparticles thin film

    NASA Astrophysics Data System (ADS)

    Graak, Pinki; Devi, Ranjna; Kumar, Dinesh; Singh, Vishal; Kumar, Sacheen

    2016-05-01

    Polythiophene nanoparticles were synthesized by iron catalyzed oxidative polymerization method. Polythiophene formation was detected by UV-Visible spectroscopy with λmax 375nm. Thin films of CTAB stabilized polythiophene nanoparticles was deposited on n-type silicon wafer by spin coating technique at 3000rpm in three cycles. Thickness of the thin films was computed as 300-350nm by ellipsometry. Atomic force micrscopyrevealws the particle size of polymeric nanoparticles in the range of 30nm to 100nm. Roughness of thinfilm was also analyzed from the atomic force microscopy data by Picoimage software. The observed RMS value lies in the range of 6 nm to 12 nm.

  1. A promising new wavelength region for three-photon fluorescence microscopy of live cells.

    PubMed

    Norris, Greg; Amor, Rumelo; Dempster, John; Amos, William B; McConnell, Gail

    2012-06-01

    We report three-photon laser scanning microscopy (3PLSM) using a bi-directional pumped optical parametric oscillator (OPO) with signal wavelength output at λ= 1500 nm. This novel laser was used to overcome the high optical loss in the infrared spectral region observed in laser scanning microscopes and objective lenses that renders them otherwise difficult to use for imaging. To test our system, we performed 3PLSM auto-fluorescence imaging of live plant cells at λ= 1500 nm, specifically Spirogyra, and compared performance with two-photon excitation (2PLSM) imaging using a femtosecond pulsed Ti:Sapphire laser at λ= 780 nm. Analysis of cell viability based on cytoplasmic organelle streaming and structural changes of cells revealed that at similar peak powers, 2PLSM caused gross cell damage after 5 min but 3PLSM showed little or no interference with cell function after 15 min. The λ= 1500 nm OPO is thus shown to be a practical laser source for live cell imaging.

  2. Adaptive light-sheet microscopy for long-term, high-resolution imaging in living organisms.

    PubMed

    Royer, Loïc A; Lemon, William C; Chhetri, Raghav K; Wan, Yinan; Coleman, Michael; Myers, Eugene W; Keller, Philipp J

    2016-12-01

    Optimal image quality in light-sheet microscopy requires a perfect overlap between the illuminating light sheet and the focal plane of the detection objective. However, mismatches between the light-sheet and detection planes are common owing to the spatiotemporally varying optical properties of living specimens. Here we present the AutoPilot framework, an automated method for spatiotemporally adaptive imaging that integrates (i) a multi-view light-sheet microscope capable of digitally translating and rotating light-sheet and detection planes in three dimensions and (ii) a computational method that continuously optimizes spatial resolution across the specimen volume in real time. We demonstrate long-term adaptive imaging of entire developing zebrafish (Danio rerio) and Drosophila melanogaster embryos and perform adaptive whole-brain functional imaging in larval zebrafish. Our method improves spatial resolution and signal strength two to five-fold, recovers cellular and sub-cellular structures in many regions that are not resolved by non-adaptive imaging, adapts to spatiotemporal dynamics of genetically encoded fluorescent markers and robustly optimizes imaging performance during large-scale morphogenetic changes in living organisms.

  3. Live cell microscopy analysis of radiation-induced DNA double-strand break motion

    PubMed Central

    Jakob, B.; Splinter, J.; Durante, M.; Taucher-Scholz, G.

    2009-01-01

    We studied the spatiotemporal organization of DNA damage processing by live cell microscopy analysis in human cells. In unirradiated U2OS osteosarcoma and HeLa cancer cells, a fast confined and Brownian-like motion of DNA repair protein foci was observed, which was not altered by radiation. By analyzing the motional activity of GFP-53BP1 foci in live cells up to 12-h after irradiation, we detected an additional slower mobility of damaged chromatin sites showing a mean square displacement of ≈0.6 μm2/h after exposure to densely- or sparsely-ionizing radiation, most likely driven by normal diffusion of chromatin. Only occasionally, larger translational motion connected to morphological changes of the whole nucleus could be observed. In addition, there was no general tendency to form repair clusters in the irradiated cells. We conclude that long-range displacements of damaged chromatin domains do not generally occur during DNA double-strand break repair after introduction of multiple damaged sites by charged particles. The occasional and in part transient appearance of cluster formation of radiation-induced foci may represent a higher mobility of chromatin along the ion trajectory. These observations support the hypothesis that spatial proximity of DNA breaks is required for the formation of radiation-induced chromosomal exchanges. PMID:19221031

  4. Live endothelial cells imaged by Scanning Near-field Optical Microscopy (SNOM): capabilities and challenges.

    PubMed

    Bulat, Katarzyna; Rygula, Anna; Szafraniec, Ewelina; Ozaki, Yukihiro; Baranska, Malgorzata

    2016-08-22

    The scanning near-field optical microscopy (SNOM) shows a potential to study details of biological samples, since it provides the optical images of objects with nanometric spatial resolution (50-200 nm) and the topographic information at the same time. The goal of this work is to demonstrate the capabilities of SNOM in transmission configuration to study human endothelial cells and their morphological changes, sometimes very subtle, upon inflammation. Various sample preparations were tested for SNOM measurements and promising results are collected to show: 1) the influence of α tumor necrosis factor (TNF-α) on EA.hy 926 cells (measurements of the fixed cells); 2) high resolution images of various endothelial cell lines, i.e. EA.hy 926 and HLMVEC (investigations of the fixed cells in buffer environment); 3) imaging of live endothelial cells in physiological buffers. The study demonstrate complementarity of the SNOM measurements performed in air and in liquid environments, on fixed as well as on living cells. Furthermore, it is proved that the SNOM is a very useful method for analysis of cellular morphology and topography. Changes in the cell shape and nucleus size, which are the symptoms of inflammatory reaction, were noticed in TNF-α activated EA.hy 926 cells. The cellular structures of submicron size were observed in high resolution optical images of cells from EA.hy 926 and HLMVEC lines.

  5. Differentiation of live-viable versus dead bacterial endospores by calibrated hyperspectral reflectance microscopy.

    PubMed

    Anderson, J; Reynolds, C; Ringelberg, D; Edwards, J; Foley, K

    2008-10-01

    This paper describes the use of hyperspectral imaging microscopy (HIM) for the characterization and differentiation of live viable versus dead/non-viable bacterial endospores for two species of Bacillus. To accomplish this, endospore-forming Bacillus were cultured and differentiated into endospores. Non-viable endospores were produced using sporicidal methods representing standard decontamination procedures incorporating chlorine and peroxide. Finally, endospore samples were lyophilized to prepare them for spectral analysis. Prior to HIM, baseline spectral reflectance characterizing the endospores was measured using an ASD (400-900 nm) reflectance spectrometer. These data were used to calibrate the resulting spectral image data. HIM data comprising 32 images ranging from 400 to 720 nm (visible to near infrared) were recorded using a C-mounted VariSpec hyperspectral camera attached to an epifluorescent microscope. The images produced by the system record the reflectance and absorption features of endospores based on the structure of the outer coat. Analysis of the HIM data was performed using accepted image and spectral processing routines. Where peroxide was the sporicide, changes in the outer endospore coat contributed to structurally significant visible and near infrared signature differences between live-viable versus dead, non-viable endospores. A statistical test for divergence, a method for scoring spectral structural diversity, also showed the difference between viable and non-viable peroxide killed endospores to be statistically significant. These findings may lead to an improved optical procedure to rapidly identify viable and non-viable endospores in situations of decontamination.

  6. Subsurface defect of amorphous carbon film imaged by near field acoustic microscopy

    NASA Astrophysics Data System (ADS)

    Zeng, J. T.; Zhao, K. Y.; Zeng, H. R.; Song, H. Z.; Zheng, L. Y.; Li, G. R.; Yin, Q. R.

    2008-05-01

    Amorphous carbon films were examined by low frequency scanning-probe acoustic microscopy (LF-SPAM). Local elastic properties as well as topography were imaged in the acoustic mode. Two kinds of subsurface defects were revealed by the LF-SPAM method. The influence of the subsurface defects on the elastic properties was also discussed. The ability to image subsurface defects was dependent on the scan area and the scan speed. Our results showed that the low frequency scanning-probe acoustic microscopy is a useful method for imaging subsurface defects with high resolution.

  7. Scanning probe microscopy of atoms and molecules on insulating films: from imaging to molecular manipulation.

    PubMed

    Meyer, Gerhard; Gross, Leo; Mohn, Fabian; Repp, Jascha

    2012-01-01

    Scanning tunneling microscopy (STM) and atomic force microscopy (AFM) of single atoms and molecules on ultrathin insulating films have led to a wealth of novel observations and insights. Based on the reduced electronic coupling to the metallic substrate, these techniques allow the charge state of individual atoms to be controlled, orbitals of individual molecules to be imaged and metal-molecule complexes to be built up. Near-contact AFM adds the unique capabilities of imaging and probing the chemical structure of single molecules with atomic resolution. With the help of atomic/molecular manipulation techniques, chemical binding processes and molecular switches can be studied in detail.

  8. Comparative morphology analysis of live blood platelets using scanning ion conductance and robotic dark-field microscopy.

    PubMed

    Kraus, Max-Joseph; Seifert, Jan; Strasser, Erwin F; Gawaz, Meinrad; Schäffer, Tilman E; Rheinlaender, Johannes

    2016-09-01

    Many conventional microscopy techniques for investigating platelet morphology such as electron or fluorescence microscopy require highly invasive treatment of the platelets such as fixation, drying and metal coating or staining. Here, we present two unique but entirely different microscopy techniques for direct morphology analysis of live, unstained platelets: scanning ion conductance microscopy (SICM) and robotic dark-field microscopy (RDM). We demonstrate that both techniques allow for a quantitative evaluation of the morphological features of live adherent platelets. We show that their morphology can be quantified by both techniques using the same geometric parameters and therefore can be directly compared. By imaging the same identical platelets subsequently with SICM and RDM, we found that area, perimeter and circularity of the platelets are directly correlated between SICM and dark-field microscopy (DM), while the fractal dimension (FD) differed between the two microscopy techniques. We show that SICM and RDM are both valuable tools for the ex vivo investigation of the morphology of live platelets, which might contribute to new insights into the physiological and pathophysiological role of platelet spreading.

  9. The Growth and Mechanical Properties of Living Neurons Measured via Atomic Force and Fluorescence Microscopy

    NASA Astrophysics Data System (ADS)

    Spedden, Elise

    In this thesis we explore specific properties of the cytoskeleton and growth of living neurons via atomic force and fluorescence microscopies. We make the first comparative elastic modulus measurements on three types of neuronal cells plated on three types of substrate adhesion factors. We discover that during phases of active neurite extension the soma of cortical neurons stiffens reversibly due to changes in microtubule aggregation. Additionally, we demonstrate that mechanical properties of cortical neurons measured near physiological temperatures are primarily dependent on the microtubule component of the cytoskeleton. We further explore the response of the neuronal cytoskeleton to changes in ambient temperature. The elastic modulus of cortical neuron somas is discovered to increase dramatically upon a drop in ambient temperature. We determine through fluorescent staining and chemical modification of the cytoskeleton that this stiffening is due primarily to a change in the mechanically dominant component of the cytoskeleton from microtubules at 37ºC to actin at 25ºC precipitated by changes in myosin II dynamics within the cell. We make the first direct mechanical measurements of the pericellular brush layer on living neurons, demonstrating that the traditionally observed viscoelastic behavior of the neuronal soma is due to the properties of this brush layer. When the brush layer is excluded, the underlying soma is discovered to be both stiffer than previously observed, and elastic, with no loading-speed dependence to the elastic modulus under the test conditions. We additionally demonstrate that the soma elastic modulus, brush length, and brush density are all dependent on the ambient temperature. Finally, through fluorescent and bright field microscopies we track the outgrowth of living neurons on patterned directional surfaces, demonstrating that asymmetrical ratchet topographies unidirectionally bias axonal outgrowth. We model the outgrowth of the neurons

  10. Development of novel two-photon microscopy for living brain and neuron.

    PubMed

    Nemoto, Tomomi

    2014-11-01

    "In vivo" two-photon microscopy (TPLSM) has revealed vital information on neural activity for brain function, even in light of its limitation in imaging events at depths greater than a several hundred micrometers from the brain surface. To break the limit of this penetration depth, we introduced a novel light source based on a semiconductor laser [1]. The light source successfully visualized not only cortex layer V pyramidal neurons spreading to all cortex layers at a superior S/N ratio, but visualize hippocampal CA1 neurons in young adult mice [2]. These results indicate that the penetration depth of this laser was ∼1.4 mm. In vivo TPLSM with a laser emitting a longer wavelength might give us insights on activities of neurons in the cortex or the hippocampus. This deep imaging method could be applicable to other living organs including tumor tissues. In addition, we developed liquid crystal devices to convert linearly polarized beams (LP) to vector beams [3]. A liquid device generated a vector beam called higher-order radially polarized (HRP) beam, which enabled that each of the aggregated 0.17 m beads was distinguished individually, whereas in conventional confocal microscopy or TPLSM they could not. We also visualized the finer structures of networks of filamentous cytoskeleton microtubule fluorescently-labeled in the COS-7, and primary culture of mouse neurons. Moreover, by taking an advantage of the LCDs that can utilize various wavelengths including near-infrared, we could employ an HRP beam for improving TPLSM. An HRP beam visualized fine intracellular structures not only in fixed cells stained with various dyes, but also in living cells expressing a fluorescent protein [4]. HRP beam also visualized finer structures of microtubules in fixed cells. Here, we will discuss these improvements and future application on the basis of our recent data.jmicro;63/suppl_1/i7/DFU087F1F1DFU087F1Fig. 1."in vivo" imaging of living mouse brain (H-line).

  11. Transverse Shear Microscopy: A Novel Microstructural Probe for Organic Semiconductor Thin Films

    NASA Astrophysics Data System (ADS)

    Kalihari, Vivek

    The microstructure of ultrathin organic semiconductor films (1-2nm) on gate dielectrics plays a pivotal role in the electrical transport performance of these films in organic field effect transistors. Similarly, organic/organic interfaces play a crucial role in organic solar cells and organic light emitting diodes. Therefore, it is important to study these critical organic interfaces in order to correlate thin film microstructure and electrical performance. Conventional characterization techniques such as SEM and TEM cannot be used to probe these interfaces because of the requirement of conducting substrates and the issue of beam damage. Here, we introduce a novel contact mode variant of atomic force microscopy, termed transverse shear microscopy (TSM), which can be used to probe organic interfaces. TSM produces striking, high contrast images of grain size, shape, and orientation in ultrathin films of polycrystalline organic materials, which are hard to visualize by any other method. It can probe epitaxial relationships between organic semiconductor thin film layers, and can be used in conjunction with other techniques to investigate the dependence of thin film properties on film microstructure. In order to explain the TSM signal, we used the theory of linear elasticity and developed a model that agrees well with the experimental findings and can predict the signal based on the components of the in-plane elastic tensor of the sample. TSM, with its ability to image elastic anisotropy at high resolution, can be very useful for microstructural characterization of soft materials, and for understanding bonding anisotropy that impacts a variety of physical properties in molecular systems.

  12. Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells

    PubMed Central

    Shibata, Mikihiro; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

    2015-01-01

    Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons. PMID:25735540

  13. Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells

    NASA Astrophysics Data System (ADS)

    Shibata, Mikihiro; Uchihashi, Takayuki; Ando, Toshio; Yasuda, Ryohei

    2015-03-01

    Visualization of morphological dynamics of live cells with nanometer resolution under physiological conditions is highly desired, but challenging. It has been demonstrated that high-speed atomic force microscopy is a powerful technique for visualizing dynamics of biomolecules under physiological conditions. However, application of high-speed atomic force microscopy for imaging larger objects such as live mammalian cells has been complicated because of the collision between the cantilever and samples. Here, we demonstrate that attaching an extremely long (~3 μm) and thin (~5 nm) tip by amorphous carbon to the cantilever allows us to image the surface structure of live cells with the spatiotemporal resolution of nanometers and seconds. We demonstrate that long-tip high-speed atomic force microscopy is capable of imaging morphogenesis of filopodia, membrane ruffles, pit formation, and endocytosis in COS-7, HeLa cells and hippocampal neurons.

  14. Direct Measurements of the Penetration Depth in a Superconducting Film using Magnetic Force Microscopy

    SciTech Connect

    E Nazaretski; J Thibodaux; I Vekhter; L Civale; J Thompson; R Movshovich

    2011-12-31

    We report the local measurements of the magnetic penetration depth in a superconducting Nb film using magnetic force microscopy (MFM). We developed a method for quantitative extraction of the penetration depth from single-parameter simultaneous fits to the lateral and height profiles of the MFM signal, and demonstrate that the obtained value is in excellent agreement with that obtained from the bulk magnetization measurements.

  15. Thin-Film Phase Plates for Transmission Electron Microscopy Fabricated from Metallic Glasses.

    PubMed

    Dries, Manuel; Hettler, Simon; Schulze, Tina; Send, Winfried; Müller, Erich; Schneider, Reinhard; Gerthsen, Dagmar; Luo, Yuansu; Samwer, Konrad

    2016-10-01

    Thin-film phase plates (PPs) have become an interesting tool to enhance the contrast of weak-phase objects in transmission electron microscopy (TEM). The thin film usually consists of amorphous carbon, which suffers from quick degeneration under the intense electron-beam illumination. Recent investigations have focused on the search for alternative materials with an improved material stability. This work presents thin-film PPs fabricated from metallic glass alloys, which are characterized by a high electrical conductivity and an amorphous structure. Thin films of the zirconium-based alloy Zr65.0Al7.5Cu27.5 (ZAC) were fabricated and their phase-shifting properties were evaluated. The ZAC film was investigated by different TEM techniques, which reveal beneficial properties compared with amorphous carbon PPs. Particularly favorable is the small probability for inelastic plasmon scattering, which results from the combined effect of a moderate inelastic mean free path and a reduced film thickness due to a high mean inner potential. Small probability plasmon scattering improves contrast transfer at high spatial frequencies, which makes the ZAC alloy a promising material for PP fabrication.

  16. Characterization of ferroelectric lead zirconate titanate films by scanning force microscopy

    SciTech Connect

    Zavala, G.; Fendler, J.H.; Trolier-McKinstry, S.

    1997-06-01

    Scanning force microscopy (SFM) has been used for the determination of friction, phase transformation, piezoelectric behavior (in the contact mode), polarization state, and dielectric constant (in the noncontact mode) of nanometer regions of lead zirconate titanate (PZT) films. The use of the SFM tip in the contact mode, to polarize different nanoregions of the PZT film and to apply an oscillating field thereon, led to effective piezoelectric coefficients and piezoelectric loops. The measured effective piezoelectric coefficient was shown to depend appreciably on both the tip contact force and the quality of the tip-to-film electrical contact. In the noncontact mode, application of an ac signal (with a frequency {omega}) across the tip{emdash}PZT film{emdash}electrode system produced an oscillation of the tip at frequencies {omega} (fundamental or first harmonic) and 2{omega} (second harmonic). The signals at {omega} and 2{omega} were related to the state of polarization and the dielectric constant of the PZT film, respectively. Analysis of the combined contact, noncontact and friction force microscopic data provided insight into the structure and into the dielectric, ferroelectric, and piezoelectric properties of distinct nanoregions of the PZT film. {copyright} {ital 1997 American Institute of Physics.}

  17. Scanning tunneling microscopy study of the superconducting properties of three-atomic-layer Pb films

    SciTech Connect

    Wang, Yilin; Li, Zhi; Wang, Lili; He, Ke; Ma, Xucun; Chen, Mu; Xue, Qi-Kun

    2013-12-09

    Ultrathin Pb films with a thickness of three monolayers (ML) were prepared on α-√(3)×√(3)Pb/Si(111) (Pb-SIC) substrate by molecular beam epitaxy. Despite significant defect scattering, low temperature scanning tunneling microscopy reveals a high superconducting transition temperature T{sub c} of 6.9 K, compared with the bulk T{sub c} (7.2 K). By applying external magnetic field, magnetic vortices were directly imaged, which demonstrates the robustness of superconductivity. By comparing to nearly free-standing Pb films on graphitized SiC (0001) substrate, we suggest that the higher T{sub c} of 3 ML Pb films on Pb-SIC originates from the combined effects of quantum confinement and substrate-enhanced electron-phonon coupling.

  18. Molecular beam epitaxy growth and scanning tunneling microscopy study of TiSe2 ultrathin films

    NASA Astrophysics Data System (ADS)

    Peng, Jun-Ping; Guan, Jia-Qi; Zhang, Hui-Min; Song, Can-Li; Wang, Lili; He, Ke; Xue, Qi-Kun; Ma, Xu-Cun

    2015-03-01

    Molecular beam epitaxy is used to grow TiSe2 ultrathin films on a graphitized SiC(0001) substrate. TiSe2 films proceed via a nearly layer-by-layer growth mode and exhibit two dominant types of defects, identified as Se vacancy and interstitial, respectively. By means of scanning tunneling microscopy, we demonstrate that the well-established charge density waves can survive in a single unit-cell (one triple-layer) regime, and find a gradual reduction in their correlation length as the density of surface defects in TiSe2 ultrathin films increases. Our findings offer important insights into the nature of charge density waves in TiSe2, and also pave a material foundation for potential applications based on the collective electronic states.

  19. Three-dimensional readout of flash x-ray images of living sperm in water by atomic-force microscopy.

    PubMed

    Tomie, T; Shimizu, H; Majima, T; Yamada, M; Kanayama, T; Kondo, H; Yano, M; Ono, M

    1991-05-03

    The imaging of living specimens in water by x-ray microscopy can be greatly enhanced with the use of an intense flash x-ray source and sophisticated technologies for reading x-ray images. A subnanosecond [corrected] x-ray pulse from a laser-produced plasma was used to record the x-ray image of living sea urchin sperm in an x-ray resist. The resist relief was visualized at high resolution by atomic-force microscopy. Internal structure of the sperm head was evident, and the carbon density in a flagellum was estimated from the relief height.

  20. Bright Lu2O3:Eu thin-film scintillators for high-resolution radioluminescence microscopy

    PubMed Central

    Sengupta, Debanti; Miller, Stuart; Marton, Zsolt; Chin, Frederick; Nagarkar, Vivek

    2015-01-01

    We investigate the performance of a new thin-film Lu2O3:Eu scintillator for single-cell radionuclide imaging. Imaging the metabolic properties of heterogeneous cell populations in real time is an important challenge with clinical implications. We have developed an innovative technique called radioluminescence microscopy, to quantitatively and sensitively measure radionuclide uptake in single cells. The most important component of this technique is the scintillator, which converts the energy released during radioactive decay into luminescent signals. The sensitivity and spatial resolution of the imaging system depend critically on the characteristics of the scintillator, i.e. the material used and its geometrical configuration. Scintillators fabricated using conventional methods are relatively thick, and therefore do not provide optimal spatial resolution. We compare a thin-film Lu2O3:Eu scintillator to a conventional 500 μm thick CdWO4 scintillator for radioluminescence imaging. Despite its thinness, the unique scintillation properties of the Lu2O3:Eu scintillator allow us to capture single positron decays with over fourfold higher sensitivity, a significant achievement. The thin-film Lu2O3:Eu scintillators also yield radioluminescence images where individual cells appear smaller and better resolved on average than with the CdWO4 scintillators. Coupled with the thin-film scintillator technology, radioluminescence microscopy can yield valuable and clinically relevant data on the metabolism of single cells. PMID:26183115

  1. Domain imaging in ferroelectric thin films via channeling-contrast backscattered electron microscopy

    DOE PAGES

    Ihlefeld, Jon F.; Michael, Joseph R.; McKenzie, Bonnie B.; ...

    2016-09-16

    We report that ferroelastic domain walls provide opportunities for deterministically controlling mechanical, optical, electrical, and thermal energy. Domain wall characterization in micro- and nanoscale systems, where their spacing may be of the order of 100 nm or less is presently limited to only a few techniques, such as piezoresponse force microscopy and transmission electron microscopy. These respective techniques cannot, however, independently characterize domain polarization orientation and domain wall motion in technologically relevant capacitor structures or in a non-destructive manner, thus presenting a limitation of their utility. In this work, we show how backscatter scanning electron microscopy utilizing channeling contrast yieldmore » can image the ferroelastic domain structure of ferroelectric films with domain wall spacing as narrow as 10 nm.« less

  2. Domain imaging in ferroelectric thin films via channeling-contrast backscattered electron microscopy

    SciTech Connect

    Ihlefeld, Jon F.; Michael, Joseph R.; McKenzie, Bonnie B.; Scrymgeour, David A.; Maria, Jon-Paul; Paisley, Elizabeth A.; Kitahara, Andrew R.

    2016-09-16

    We report that ferroelastic domain walls provide opportunities for deterministically controlling mechanical, optical, electrical, and thermal energy. Domain wall characterization in micro- and nanoscale systems, where their spacing may be of the order of 100 nm or less is presently limited to only a few techniques, such as piezoresponse force microscopy and transmission electron microscopy. These respective techniques cannot, however, independently characterize domain polarization orientation and domain wall motion in technologically relevant capacitor structures or in a non-destructive manner, thus presenting a limitation of their utility. In this work, we show how backscatter scanning electron microscopy utilizing channeling contrast yield can image the ferroelastic domain structure of ferroelectric films with domain wall spacing as narrow as 10 nm.

  3. Fast intracellular motion in the living cell by video rate reflection confocal laser scanning microscopy

    PubMed Central

    VESELY, PAVEL; BOYDE, ALAN

    2001-01-01

    Fast intracellular motion (FIM) was first revealed by back scattered light (BSL) imaging in video rate confocal scanning laser microscopy (VRCSLM), beyond the limits of spatial and temporal resolution obtainable with conventional optical microscopy. BSL imaging enabled visualisation of intra and extracellular motion with resolution in space down to 0.2 μm and in time to 1/25th of a second. Mapping the cell space at 0.2 μm×0.2 μm (XY = in instantaneous best focal plane)×0.5 μm (Z = height/depth, optic axis direction) volume steps revealed a communication layer above the known contact layer and an integrated dynamic spatial network (IDSN) towards the cell centre. FIM was originally observed as localised quasichaotic dancing (dithering) or reflecting patches/spots in the cell centre, faster in the darker nuclear space. Later, a second type of FIM was recognised which differed by the presence of a varied proportion of centrifugal and centripetal directional movements and/or jumping of patches/spots in the cell centre and outside the nuclear space. The first type is characteristic for cells in slightly adverse conditions while the second type has so far only been found in eutrophic cells. Temporal speeding up and coarsening of FIM, followed by slowing and eventually cessation at cell death, was found on exposure to strong stressors. It was concluded that the state of FIM provides instantaneous information about individual cell reactions to actual treatment and about cell survival. A putative switch between the first and second type FIM could be considered as an indicator of timing of cellular processes. The significance of FIM for the biology of the cell is seen in the rapid assessment of the condition of an individual live cell investigated by combination of various methods. Requirements for further development of this approach are outlined. PMID:11465857

  4. Evaluating the Performance of Time-Gated Live-Cell Microscopy with Lanthanide Probes

    PubMed Central

    Rajendran, Megha; Miller, Lawrence W.

    2015-01-01

    Probes and biosensors that incorporate luminescent Tb(III) or Eu(III) complexes are promising for cellular imaging because time-gated microscopes can detect their long-lifetime (approximately milliseconds) emission without interference from short-lifetime (approximately nanoseconds) fluorescence background. Moreover, the discrete, narrow emission bands of Tb(III) complexes make them uniquely suited for multiplexed imaging applications because they can serve as Förster resonance energy transfer (FRET) donors to two or more differently colored acceptors. However, lanthanide complexes have low photon emission rates that can limit the image signal/noise ratio, which has a square-root dependence on photon counts. This work describes the performance of a wide-field, time-gated microscope with respect to its ability to image Tb(III) luminescence and Tb(III)-mediated FRET in cultured mammalian cells. The system employed a UV-emitting LED for low-power, pulsed excitation and an intensified CCD camera for gated detection. Exposure times of ∼1 s were needed to collect 5–25 photons per pixel from cells that contained micromolar concentrations of a Tb(III) complex. The observed photon counts matched those predicted by a theoretical model that incorporated the photophysical properties of the Tb(III) probe and the instrument’s light-collection characteristics. Despite low photon counts, images of Tb(III)/green fluorescent protein FRET with a signal/noise ratio ≥ 7 were acquired, and a 90% change in the ratiometric FRET signal was measured. This study shows that the sensitivity and precision of lanthanide-based cellular microscopy can approach that of conventional FRET microscopy with fluorescent proteins. The results should encourage further development of lanthanide biosensors that can measure analyte concentration, enzyme activation, and protein-protein interactions in live cells. PMID:26200860

  5. Piezoelectricity and ferroelectricity of cellular polypropylene electrets films characterized by piezoresponse force microscopy

    SciTech Connect

    Miao, Hongchen; Sun, Yao; Zhou, Xilong; Li, Yingwei; Li, Faxin

    2014-08-14

    Cellular electrets polymer is a new ferroelectret material exhibiting large piezoelectricity and has attracted considerable attentions in researches and industries. Property characterization is very important for this material and current investigations are mostly on macroscopic properties. In this work, we conduct nanoscale piezoelectric and ferroelectric characterizations of cellular polypropylene (PP) films using piezoresponse force microscopy (PFM). First, both the single-frequency PFM and dual-frequency resonance-tracking PFM testings were conducted on the cellular PP film. The localized piezoelectric constant d{sub 33} is estimated to be 7–11pC/N by correcting the resonance magnification with quality factor and it is about one order lower than the macroscopic value. Next, using the switching spectroscopy PFM (SS-PFM), we studied polarization switching behavior of the cellular PP films. Results show that it exhibits the typical ferroelectric-like phase hysteresis loops and butterfly-shaped amplitude loops, which is similar to that of a poly(vinylidene fluoride) (PVDF) ferroelectric polymer film. However, both the phase and amplitude loops of the PP film are intensively asymmetric, which is thought to be caused by the nonzero remnant polarization after poling. Then, the D-E hysteresis loops of both the cellular PP film and PVDF film were measured by using the same wave form as that used in the SS-PFM, and the results show significant differences. Finally, we suggest that the ferroelectric-like behavior of cellular electrets films should be distinguished from that of typical ferroelectrics, both macroscopically and microscopically.

  6. Piezoelectricity and ferroelectricity of cellular polypropylene electrets films characterized by piezoresponse force microscopy

    NASA Astrophysics Data System (ADS)

    Miao, Hongchen; Sun, Yao; Zhou, Xilong; Li, Yingwei; Li, Faxin

    2014-08-01

    Cellular electrets polymer is a new ferroelectret material exhibiting large piezoelectricity and has attracted considerable attentions in researches and industries. Property characterization is very important for this material and current investigations are mostly on macroscopic properties. In this work, we conduct nanoscale piezoelectric and ferroelectric characterizations of cellular polypropylene (PP) films using piezoresponse force microscopy (PFM). First, both the single-frequency PFM and dual-frequency resonance-tracking PFM testings were conducted on the cellular PP film. The localized piezoelectric constant d33 is estimated to be 7-11pC/N by correcting the resonance magnification with quality factor and it is about one order lower than the macroscopic value. Next, using the switching spectroscopy PFM (SS-PFM), we studied polarization switching behavior of the cellular PP films. Results show that it exhibits the typical ferroelectric-like phase hysteresis loops and butterfly-shaped amplitude loops, which is similar to that of a poly(vinylidene fluoride) (PVDF) ferroelectric polymer film. However, both the phase and amplitude loops of the PP film are intensively asymmetric, which is thought to be caused by the nonzero remnant polarization after poling. Then, the D-E hysteresis loops of both the cellular PP film and PVDF film were measured by using the same wave form as that used in the SS-PFM, and the results show significant differences. Finally, we suggest that the ferroelectric-like behavior of cellular electrets films should be distinguished from that of typical ferroelectrics, both macroscopically and microscopically.

  7. Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy.

    PubMed

    Lahne, Manuela; Gorsuch, Ryne A; Nelson, Craig M; Hyde, David R

    2017-02-24

    An endogenous regeneration program is initiated by Müller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The Müller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both Müller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled Müller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP](mi2004) zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

  8. Real-time phosphate sensing in living cells using fluorescence lifetime imaging microscopy (FLIM).

    PubMed

    Paredes, Jose M; Giron, Maria D; Ruedas-Rama, Maria J; Orte, Angel; Crovetto, Luis; Talavera, Eva M; Salto, Rafael; Alvarez-Pez, Jose M

    2013-07-11

    Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dye's nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process.

  9. Single-Molecule Imaging in Living Drosophila Embryos with Reflected Light-Sheet Microscopy

    PubMed Central

    Greiss, Ferdinand; Deligiannaki, Myrto; Jung, Christophe; Gaul, Ulrike; Braun, Dieter

    2016-01-01

    In multicellular organisms, single-fluorophore imaging is obstructed by high background. To achieve a signal/noise ratio conducive to single-molecule imaging, we adapted reflected light-sheet microscopy (RLSM) to image highly opaque late-stage Drosophila embryos. Alignment steps were modified by means of commercially available microprisms attached to standard coverslips. We imaged a member of the septate-junction complex that was used to outline the three-dimensional epidermal structures of Drosophila embryos. Furthermore, we show freely diffusing single 10 kDa Dextran molecules conjugated to one to two Alexa647 dyes inside living embryos. We demonstrate that Dextran diffuses quickly (∼6.4 μm2/s) in free space and obeys directional movement within the epidermal tissue (∼0.1 μm2/s). Our single-particle-tracking results are supplemented by imaging the endosomal marker Rab5-GFP and by earlier reports on the spreading of morphogens and vesicles in multicellular organisms. The single-molecule results suggest that RLSM will be helpful in studying single molecules or complexes in multicellular organisms. PMID:26910430

  10. Imaging Mitochondrial Organization in Living Primate Oocytes and Embryos using Multiphoton Microscopy

    NASA Astrophysics Data System (ADS)

    Squirrell, J. M.; Schramm, R. D.; Paprocki, A. M.; Wokosin, D. L.; Bavister, B. D.

    2003-06-01

    We employed multiphoton laser scanning microscopy (MPLSM) to image changes in mitochondrial distribution in living rhesus monkey embryos. This method of imaging does not impair development; thus, the same specimen can be visualized multiple times at various developmental stages. Not only does this increase the amount of information that can be gathered on a single specimen but it permits the correlation of early events with subsequent development in the same specimen. Here we demonstrate the utility of MPLSM for determining changes in mitochondrial organization at various developmental stages and show that rhesus zygotes possess a distinct accumulation of mitochondria between the pronuclei prior to syngamy. We present evidence that suggests that this pronuclear accumulation may be positively correlated with development to the blastocyst stage—in the same embryo—thereby illustrating how MPLSM can be used to correlate cellular dynamics of primate oocytes and early embryos with their developmental potential. Understanding the relationship between mitochondrial distribution and the subsequent development of mammalian embryos, particularly primates, will increase our ability to improve embryo culture technologies, including those used for human assisted reproduction.

  11. Chronic imaging of amyloid plaques in the live mouse brain using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Bacskai, Brian J.; Kajdasz, Stephen T.; Christie, R. H.; Zipfel, Warren R.; Williams, Rebecca M.; Kasischke, Karl A.; Webb, Watt W.; Hyman, B. T.

    2001-04-01

    Transgenic mice expressing the human Amyloid Precursor Protein (APP) develop amyloid plaques as they age. These plaques resemble those found in the human disease. Multiphoton laser scanning microscopy combined with a novel surgical approach was used to measure amyloid plaque dynamics chronically in the cortex of living transgenic mice. Thioflavine S (thioS) was used as a fluorescent marker of amyloid deposits. Multiphoton excitation allowed visualization of amyloid plaques up to 200 micrometers deep into the brain. The surgical site could be imaged repeatedly without overt damage to the tissue, and individual plaques within this volume could be reliably identified over periods of several days to several months. On average, plaque sizes remained constant over time, supporting a model of rapid deposition, followed by relative stability. Alternative reporters for in vivo histology include thiazine red, and FITC-labeled amyloid-(Beta) peptide. We also present examples of multi-color imaging using Hoechst dyes and FITC-labeled tomato lectin. These approaches allow us to observe cell nuclei or microglia simultaneously with amyloid-(Beta) deposits in vivo. Chronic imaging of a variety of reporters in these transgenic mice should provide insight into the dynamics of amyloid-(Beta) activity in the brain.

  12. Functional imaging of living Paramecium by means of confocal and two-photon excitation fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto; Fronte, Paola; Raimondo, Marco; Fato, Marco; DeLeo, Gianluca; Beltrame, Francesco; Cannone, Fabio; Chirico, Giberto; Ramoino, Paola

    2002-05-01

    Confocal and Two-photon excitation laser scanning microscopy allow gathering three-dimensional and temporal information from biological systems exploiting fluorescence labeling and autofluorescence properties. In this work we study biological events linked to functionality in Paramecium primaurelia. The internalization of material in ciliated one-celled organisms (protozoa) occurs via different mechanisms, even if most of nutrients, particulate or not, is taken up by food vacuoles formed at the bottom of the oral cavity. The endocytosis of small-sized molecules occurs at the parasomal sacs, located next the ciliar basal bodies. Vital fluorescent dyes (BSA-FITC, WGA-FITC, dextran-Texas Red, cholesteryl-Bodipy) and autofluorescence were used to study formation, movement, and fusion of vesicles during endocytosis and phagocytosis of Paramecium primaurelia. By immobilizing living cells pulsed with food vacuole and endosome markers at successive times after chasing in unlabeled medium, the intracellular movement and fusion of food vacuoles and of endosomes were visualized. A temporal analysis of fluorescence images and the false-color technique were used. Starting from time series or 3D data sets composite images were generated by associating with each originally acquired image a different color corresponding to each sampling point in time and along the z-axis. Second Harmonic Generation Imaging attempts are also outlined.

  13. Live Bacterial Physiology Visualized with 5 nm Resolution Using Scanning Transmission Electron Microscopy.

    PubMed

    Kennedy, Eamonn; Nelson, Edward M; Tanaka, Tetsuya; Damiano, John; Timp, Gregory

    2016-02-23

    It is now possible to visualize at nanometer resolution the infection of a living biological cell with virus without compromising cell viability using scanning transmission electron microscopy (STEM). To provide contrast while preserving viability, Escherichia coli and P1 bacteriophages were first positively stained with a very low concentration of uranyl acetate in minimal phosphate medium and then imaged with low-dose STEM in a microfluidic liquid flow cell. Under these conditions, it was established that the median lethal dose of electrons required to kill half the tested population was LD50 = 30 e(-)/nm(2), which coincides with the disruption of a wet biological membrane, according to prior reports. Consistent with the lateral resolution and high-contrast signal-to-noise ratio (SNR) inferred from Monte Carlo simulations, images of the E. coli membrane, flagella, and the bacteriophages were acquired with 5 nm resolution, but the cumulative dose exceeded LD50. On the other hand, with a cumulative dose below LD50 (and lower SNR), it was still possible to visualize the infection of E. coli by P1, showing the insertion of viral DNA within 3 s, with 5 nm resolution.

  14. The four dimensions of clathrin coats in living cells measured by advanced fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Davoust, Jean; Cosson, Pierre

    1991-05-01

    After microinjection into cultured Vero cells, rhodamine-labeled clathrin triskelions gave rise to a punctuate fluorescence pattern typical for clathrin, with two major localizations: the plasma membrane and the perinuclear region of the cells. We analyzed clathrin motion by Fluorescence Recovery After Photobleaching and its 3 dimensional distribution by Confocal Microscopy. Altogether, 55% of total clathrin is polymerized into coats which are turning over with a half time of 11 seconds and 45% of total diffuses freely in the cytoplasm. Various conditions known to affect membrane traffic were investigated. Cytosolic acidification or ATP depletion stabilized the polymerized clathrin coats without modifying the ratio of free versus polymerized clathrin. Low temperature (6 °C) or hypertonic media dramatically increased both the stability and the amount of the polymerized clathrin. We conclude that ATP and pH homeostasis are needed to support a very high turnover of the clathrin coats in living cells whereas low temperature and high osmotic strength promote an extensive polymerization of clathrin.

  15. Pulse splitter-based nonlinear microscopy for live-cardiomyocyte imaging

    NASA Astrophysics Data System (ADS)

    Wang, Zhonghai; Qin, Wan; Shao, Yonghong; Ma, Siyu; Borg, Thomas K.; Gao, Bruce Z.

    2014-02-01

    Second harmonic generation (SHG) microscopy is a new imaging technique used in sarcomeric-addition studies. However, during the early stage of cell culture in which sarcomeric additions occur, the neonatal cardiomyocytes that we have been working with are very sensitive to photodamage, the resulting high rate of cell death prevents systematic study of sarcomeric addition using a conventional SHG system. To address this challenge, we introduced use of the pulse-splitter system developed by Na Ji et al. in our two photon excitation fluorescence (TPEF) and SHG hybrid microscope. The system dramatically reduced photodamage to neonatal cardiomyocytes in early stages of culture, greatly increasing cell viability. Thus continuous imaging of live cardiomyocytes was achieved with a stronger laser and for a longer period than has been reported in the literature. The pulse splitter-based TPEF-SHG microscope constructed in this study was demonstrated to be an ideal imaging system for sarcomeric addition-related investigations of neonatal cardiomyocytes in early stages of culture.

  16. High-resolution, label-free imaging of living cells with direct electron-beam-excitation-assisted optical microscopy.

    PubMed

    Nawa, Yasunori; Inami, Wataru; Lin, Sheng; Kawata, Yoshimasa; Terakawa, Susumu

    2015-06-01

    High spatial resolution microscope is desired for deep understanding of cellular functions, in order to develop medical technologies. We demonstrate high-resolution imaging of un-labelled organelles in living cells, in which live cells on a 50 nm thick silicon nitride membrane are imaged by autofluorescence excited with a focused electron beam through the membrane. Electron beam excitation enables ultrahigh spatial resolution imaging of organelles, such as mitochondria, nuclei, and various granules. Since the autofluorescence spectra represent molecular species, this microscopy allows fast and detailed investigations of cellular status in living cells.

  17. Instabilities and Waves in Thin Films of Living Fluids

    NASA Astrophysics Data System (ADS)

    Sankararaman, Sumithra; Ramaswamy, Sriram

    2009-03-01

    We formulate the thin-film hydrodynamics of a suspension of polar self-driven particles and show that it is prone to several instabilities through the interplay of activity, polarity, and the existence of a free surface. Our approach extends, to self-propelling systems, the work of Ben Amar and Cummings [Phys. FluidsPHFLE61070-6631 13 1160 (2001)10.1063/1.1359748] on thin-film nematics. Based on our estimates the instabilities should be seen in bacterial suspensions and the lamellipodium, and are potentially relevant to the morphology of biofilms. We suggest several experimental tests of our theory.

  18. Instabilities and waves in thin films of living fluids

    NASA Astrophysics Data System (ADS)

    Sankararaman, Sumithra; Ramaswamy, Sriram

    2009-03-01

    We formulate the thin-film hydrodynamics of a suspension of polar self-driven particles and show that it is prone to several instabilities through the interplay of activity, polarity and the existence of a free surface. Our approach extends, to self-propelling systems, the work of Ben Amar and Cummings [Phys Fluids 13 (2001) 1160] on thin-film nematics. Based on our estimates the instabilities should be seen in bacterial suspensions and the lamellipodium, and are potentially relevant to the morphology of biofilms. We suggest several experimental tests of our theory.

  19. Analysis of Comparison of Human Meibomian Lipid Films and Mixtures with Cholesteryl Esters In Vitro Films using High Resolution Color Microscopy

    PubMed Central

    Millar, Thomas J.; King-Smith, P. Ewen

    2012-01-01

    Purpose. The lipid layer of the tears has been studied in vivo using high resolution color microscopy (HRCM). The purpose of these experiments was to gain further insight into the structure of the lipid layer by applying HRCM to in vitro meibomian lipid films. Methods. Films of human meibomian lipids, cholesteryl nervonate, cholesteryl palmitate, or their mixtures, were spread on a Langmuir trough. Changes to the films were monitored using HRCM as the films were compressed to different surface pressures. The penetration of albumin into a meibomian lipid film also was studied. Results. Small amounts of meibomian lipids at low pressures formed very thin films estimated to be 5.2 nm thick. Compression caused spots to appear in the films. At higher concentrations, micro lenses were a feature of the film. Cholesteryl nervonate formed a multilayered oil slick that did not change with surface pressure. Cholesteryl palmitate formed a stiff film that collapsed at high compression. Mixtures of cholesteryl nervonate and meibomian lipids showed that they mixed to increase surface pressures above that of the individual components. HRCM also allowed albumin to be seen penetrating the meibomian lipid film. Conclusions. HRCM combined with in vitro surface pressure measurements using a Langmuir trough is useful for modeling meibomian lipid films. The films often resemble the appearance of the lipid layer of in vivo films. The data indicate that the lipid layer might be modeled best as a duplex film containing an array of liquid crystals. PMID:22695957

  20. Aggregation quenching in thin films of meh-ppv studied by near-field scanning optical microscopy and spectroscopy

    SciTech Connect

    Huser, T; Yan, M

    2000-04-11

    Aggregates in thin films of conjugated polymers form excimer states and significantly reduce the photo- and electroluminescence efficiency in devices produced from these materials. We have studied the aggregate formation in thin films of MEH-PPV by near-field scanning optical microscopy and spectroscopy. Local photoluminescence spectroscopy and photo-bleaching experiments have been used to show that thin films of MEH-PPV are homogeneously aggregated and do not form aggregated domains.

  1. Two-photon excitation microscopy for the study of living cells and tissues.

    PubMed

    Benninger, Richard K P; Piston, David W

    2013-06-01

    Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application.

  2. Temperature dependence dynamical permeability characterization of magnetic thin film using near-field microwave microscopy.

    PubMed

    Hung, Le Thanh; Phuoc, Nguyen N; Wang, Xuan-Cong; Ong, C K

    2011-08-01

    A temperature dependence characterization system of microwave permeability of magnetic thin film up to 5 GHz in the temperature range from room temperature up to 423 K is designed and fabricated as a prototype measurement fixture. It is based on the near field microwave microscopy technique (NFMM). The scaling coefficient of the fixture can be determined by (i) calibrating the NFMM with a standard sample whose permeability is known; (ii) by calibrating the NFMM with an established dynamic permeability measurement technique such as shorted microstrip transmission line perturbation method; (iii) adjusting the real part of the complex permeability at low frequency to fit the value of initial permeability. The algorithms for calculating the complex permeability of magnetic thin films are analyzed. A 100 nm thick FeTaN thin film deposited on Si substrate by sputtering method is characterized using the fixture. The room temperature permeability results of the FeTaN film agree well with results obtained from the established short-circuited microstrip perturbation method. Temperature dependence permeability results fit well with the Landau-Lifshitz-Gilbert equation. The temperature dependence of the static magnetic anisotropy H(K)(sta), the dynamic magnetic anisotropy H(K)(dyn), the rotational anisotropy H(rot), together with the effective damping coefficient α(eff), ferromagnetic resonance f(FMR), and frequency linewidth Δf of the thin film are investigated. These temperature dependent magnetic properties of the magnetic thin film are important to the high frequency applications of magnetic devices at high temperatures.

  3. Measuring Exciton Migration in Conjugated Polymer Films with Ultrafast Time Resolved Stimulated Emission Depletion Microscopy

    NASA Astrophysics Data System (ADS)

    Penwell, Samuel

    Conjugated polymers are highly tunable organic semiconductors, which can be solution processed to form thin films, making them prime candidates for organic photovoltaic devices. One of the most important parameters in a conjugated polymer solar cell is the exciton diffusion length, which depends on intermolecular couplings, and is typically on the order of 10 nm. This mean exciton migration can vary dramatically between films and within a single film due to heterogeneities in morphology on length scales of 10's to 100's nm. To study the variability of exciton diffusion and morphology within individual conjugated polymer films, we are adapting stimulated emission depletion (STED) microscopy. STED is typically used in biology with sparse well-engineered fluorescent labels or on NV-centers in diamond. I will, however, describe how we have demonstrated the extension of STED to conjugated polymer films and nanoparticles of MEH-PPV and CN-PPV, despite the presence of two photon absorption, by taking care to first understand the material's photophysical properties. We then further adapt this approach, by introducing a second ultrafast STED pulse at a variable delay. Excitons that migrate away from the initial subdiffraction excitation volume during the ps-ns time delay, are preferentially quenched by the second STED pulse, while those that remain in the initial volume survive. The resulting effect of the second STED pulse is modulated by the degree of migration over the ultrafast time delay, thus providing a new method to study exciton migration. Since this technique utilizes subdiffraction optical excitation and detection volumes with ultrafast time resolution, it provides a means of spatially and temporally resolving measurements of exciton migration on the native length and time scales. In this way, we will obtain a spatiotemporal map of exciton distributions and migration that will help to correlate the energetic landscape to film morphology at the nanoscale.

  4. Confocal Raman microscopy for investigation of the level of differentiation in living neuroblastoma tumor cells

    NASA Astrophysics Data System (ADS)

    Scalfi-Happ, Claudia; Jauss, Andrea; Hollricher, Olaf; Fulda, Simone; Hauser, Carmen; Steiner, Rudolf; Rück, Angelika

    2007-07-01

    The investigation of living cells at physiological conditions requires very sensitive, sophisticated, non invasive methods. In this study, Raman spectral imaging is used to identify different biomolecules inside of cells. Raman spectroscopy, a chemically and structurally sensitive measuring technique, is combined with high resolution confocal microscopy. In Raman spectral imaging mode, a complete Raman spectrum is recorded at every confocal image point, giving insight into the chemical composition of each sample compartment. Neuroblastoma is the most common solid extra-cranial tumor in children. One of the unique features of neuroblastoma cells is their ability to differentiate spontaneously, eventually leading to complete remission. Since differentiation agents are currently used in the clinic for neuroblastoma therapy, there is a special need to develop non-invasive and sensitive new methods to monitor neuroblastoma cell differentiation. Neuroblastoma cells at different degrees of differentiation were analysed with the confocal Raman microscope alpha300 R (WITec GmbH, Germany), using a frequency doubled Nd:YAG laser at 532 nm and 10 mW for excitation. Integration time per spectrum was 80-100 ms. A lateral resolution in submicrometer range was achieved by using a 60x water immersion lens with a numerical aperture of 1,0. Raman images of cells were generated from these sets of data by either integrating over specific Raman bands, by basis analysis using reference spectra or by cluster analysis. The automated evaluation of all spectra results in spectral unmixed images providing insight into the chemical composition of the sample. With these procedures, different cell organelles, cytosol, membranes could be distinguished. Since neuroblastoma cells at high degree of differentiation overproduce noradrenaline, an attempt was made to trace the presence of this neurotransmitter as a marker for differentiation. The results of this work may have applications in the

  5. Lensfree super-resolution holographic microscopy using wetting films on a chip

    NASA Astrophysics Data System (ADS)

    Mudanyali, Onur; Bishara, Waheb; Ozcan, Aydogan

    2011-08-01

    We investigate the use of wetting films to significantly improve the imaging performance of lensfree pixel super-resolution on-chip microscopy, achieving < 1 μm spatial resolution over a large imaging area of ~24 mm2. Formation of an ultra-thin wetting film over the specimen effectively creates a micro-lens effect over each object, which significantly improves the signal-to-noise-ratio and therefore the resolution of our lensfree images. We validate the performance of this approach through lensfree on-chip imaging of various objects having fine morphological features (with dimensions of e.g., ≤0.5 μm) such as Escherichia coli (E. coli), human sperm, Giardia lamblia trophozoites, polystyrene micro beads as well as red blood cells. These results are especially important for the development of highly sensitive field-portable microscopic analysis tools for resource limited settings.

  6. Atomic force microscopy of AgBr crystals and adsorbed gelatin films

    SciTech Connect

    Haugstad, G.; Gladfelter, W.L.; Keyes, M.P.; Weberg, E.B.

    1993-06-01

    Atomic force microscopy of the (111) surface of macroscopic AgBr crystals revealed steps ranging in height from two atomic layers up to 10 nm, lying predominantly along the (110) and (112) families of crystal directions. Rods of elemental Ag, formed via photoreduction, were observed along the (110) family of directions. Images of adsorbed gelatin films revealed circular pores with diameters of order 10-100 nm, extending to the AgBr surface. The length of deposition time, the pH and concentration of the gelatin solution, and the presence of steps on the AgBr surface were observed to affect the size, number, and location of pores in the gelatin films. 12 refs., 7 figs.

  7. Formation and disruption of current paths of anodic porous alumina films by conducting atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Oyoshi, K.; Nigo, S.; Inoue, J.; Sakai, O.; Kitazawa, H.; Kido, G.

    2010-11-01

    Anodic porous alumina (APA) films have a honeycomb cell structure of pores and a voltage-induced bi-stable switching effect. We have applied conducting atomic force microscopy (CAFM) as a method to form and to disrupt current paths in the APA films. A bi-polar switching operation was confirmed. We have firstly observed terminals of current paths as spots or areas typically on the center of the triangle formed by three pores. In addition, though a part of the current path showed repetitive switching, most of them were not observed again at the same position after one cycle of switching operations in the present experiments. This suggests that a part of alumina structure and/or composition along the current paths is modified during the switching operations.

  8. Atomic-force-microscopy nanowriting on ultrathin tetrahedral amorphous carbon films

    NASA Astrophysics Data System (ADS)

    Pivovarov, Pavel A.; Zavedeev, Evgeny V.; Frolov, Vadim D.; Roch, Teja; Scheibe, Hans-Joachim; Pimenov, Sergei M.

    2016-11-01

    We report the atomic-force-microscopy (AFM)-based nanolithography writing of surface patterns on ultrathin (<100 nm thick) ta-C films in ambient air and following contact-mode AFM reading of nanoscale topography, nanofriction properties, and local electrical conductivity of the produced nanopatterns. AFM writing of various patterns such as single nanospots, linear nanostructures, and raster images (`nanoletters') is demonstrated, depending on the magnitude and duration of the voltage pulses applied between the ta-C film and conductive probe, and the relative humidity of ambient air. It is found that the AFM tip-assisted nanowriting process occurring under (1) the presence of adsorbed water layers on the ta-C surface, (2) the applied voltage of >4 V, and (3) the contact pressures in the GPa range results in the formation of a novel carbon phase in a nm-thick surface layer characterized by the lower density, lower mechanical strength, lower electrical conductivity, and increased nanofriction as compared to the original film. The structure of the tip-modified nm-thick layer on the ta-C film is assumed to be a structure of graphite oxide which can be further modified in the presence of water under high contact pressures.

  9. Quantifying charge carrier concentration in ZnO thin films by Scanning Kelvin Probe Microscopy

    PubMed Central

    Maragliano, C.; Lilliu, S.; Dahlem, M. S.; Chiesa, M.; Souier, T.; Stefancich, M.

    2014-01-01

    In the last years there has been a renewed interest for zinc oxide semiconductor, mainly triggered by its prospects in optoelectronic applications. In particular, zinc oxide thin films are being widely used for photovoltaic applications, in which the determination of the electrical conductivity is of great importance. Being an intrinsically doped material, the quantification of its doping concentration has always been challenging. Here we show how to probe the charge carrier density of zinc oxide thin films by Scanning Kelvin Probe Microscopy, a technique that allows measuring the contact potential difference between the tip and the sample surface with high spatial resolution. A simple electronic energy model is used for correlating the contact potential difference with the doping concentration in the material. Limitations of this technique are discussed in details and some experimental solutions are proposed. Two-dimensional doping concentration images acquired on radio frequency-sputtered intrinsic zinc oxide thin films with different thickness and deposited under different conditions are reported. We show that results inferred with this technique are in accordance with carrier concentration expected for zinc oxide thin films deposited under different conditions and obtained from resistivity and mobility measurements. PMID:24569599

  10. Local Electronic Characterization of Conjugated Polymer Films using Conducting-Probe Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    O'Brien, G.; Quinn, A. J.; Redmond, G.

    2004-03-01

    Correlation of local electronic properties with film morphology is a key challenge to be addressed in order to understand (and therefore control) charge injection, transport and recombination in organic electronic devices. We present a flexible method, Conducting-Probe Atomic Force Microscopy (CP-AFM), which can be used as a local probe of both film morphology and spectroscopy. MEH-PPV layers with thickness values comparable to films used in organic electronic devices (60 nm) are spun onto gold substrates under inert conditions. Tip-height vs bias voltage (z-V) sweeps taken at constant tunnel current (50 pA) show clear charge injection thresholds at both positive and negative bias (E_+,E_-). Statistical analysis of measured single-particle gap energies, E_gsp=E_+-E_-, reveals a distribution across the surface with peaks corresponding to (extracted) exciton binding energies of 100 meV and 400 meV respectively. Analysis of measured E_gsp values for films prepared under ambient conditions show a large density of mid-gap states confirming that the preparation route is critical for organic electronic devices.

  11. Growth of nanometer thin ice films from water vapor studied using scanning polarization force microscopy

    SciTech Connect

    Bluhm, H.; Salmeron, M.

    1999-10-01

    Atomic force microscopy (AFM) was used to study the growth and morphology of ice films on the cleavage surface of mica. Measurements performed in contact, as well as in noncontact operation modes of the microscope, allowed us to distinguish the solid and liquid parts of the film. At temperatures below {minus}30&hthinsp;{degree}C, supercooled water droplets formed on top of a thin (nanometer range) ice layer in contact with the substrate. After annealing, a contiguous flat film was formed. Between {minus}20 and {minus}10&hthinsp;{degree}C and at a relative humidity of {approximately}83{percent}, the film consisted of a solid ice layer {approximately}7 {Angstrom} thick, covered by a liquid-like layer 50{plus_minus}5&hthinsp;{Angstrom} thick. When the temperature was raised above 0&hthinsp;{degree}C, droplets formed, which subsequently evaporated. Comparison of results obtained in the various AFM operation modes allowed us to conclude the existence of a liquid-like layer on the ice surface. {copyright} {ital 1999 American Institute of Physics.}

  12. Nanoscale characterization of oxidized ultrathin Co-films by ballistic electron emission microscopy

    NASA Astrophysics Data System (ADS)

    Eng Johnson Goh, Kuan; Wang, Simin; Tan, Siew Ting Melissa; Zhang, Zheng; Kawai, Hiroyo; Troadec, Cedric; Ng, Vivian

    2016-01-01

    In anticipation of devices scaling down further to the few nanometer regime, the ability to characterize material localized within the few nm of a critical device region poses a current challenge, particularly when the material is already buried under other material layers such as under a metal contact. Conventional techniques typically provide indirect information of the nanoscale material quality through a surface or volume averaging perspective. Here we present a study of local (nm range) oxidation in few nanometer thick Co-films using Ballistic Electron Emission Microscopy/Spectroscopy (BEEM/BEES). Co films were grown on n-Si(111) substrates, oxidized in ambient atmosphere before capping with a thin Au film to prevent further oxidation and enable BEEM measurements. In addition to BEES, the temporal progression of Co oxidation was also tracked by X-ray Photoelectron Spectroscopy. At room temperature, we report that the electron injection thresholds are sufficiently different for local regions with Co and oxidized-Co enabling their distinction in BEEM measurements. Our results demonstrate the possibility of using BEEM for nanoscale spatial mapping of the oxidized regions in Co-films, and this can provide critical information toward the successful fabrication of next generation Co-based nano-devices.

  13. Critical invisible defect detection system of thin film transistor panels using Kelvin probe force microscopy

    NASA Astrophysics Data System (ADS)

    Park, Yonmook; Heo, Keun

    2016-07-01

    In this paper, a novel method that can perform measurements of the contact potential difference (CPD) between a tip and a thin film transistor (TFT) panel using the Kelvin probe force microscopy (KPFM) is proposed for inspection of critical invisible defects on TFT panels. In this application, the surface potential of a TFT panel is inferred from the electrostatic interaction force between a tip and a TFT panel induced by the electric field. The experimental results are given to illustrate that the KPFM provides a novel and feasible way to detect the most critical invisible defects on TFT panels.

  14. Work function of few layer graphene covered nickel thin films measured with Kelvin probe force microscopy

    SciTech Connect

    Eren, B.; Gysin, U.; Marot, L. Glatzel, Th.; Steiner, R.; Meyer, E.

    2016-01-25

    Few layer graphene and graphite are simultaneously grown on a ∼100 nm thick polycrystalline nickel film. The work function of few layer graphene/Ni is found to be 4.15 eV with a variation of 50 meV by local measurements with Kelvin probe force microscopy. This value is lower than the work function of free standing graphene due to peculiar electronic structure resulting from metal 3d-carbon 2p(π) hybridization.

  15. Defects in paramagnetic Co-doped ZnO films studied by transmission electron microscopy

    SciTech Connect

    Kovacs, Andras; Ney, A.; Duchamp, Martial; Ney, V.; Boothroyd, Chris; Galindo, Pedro L.; Kaspar, Tiffany C.; Chambers, Scott A.; Dunin-Borkowski, Rafal

    2013-12-23

    We have studied planar defects in epitaxial Co:ZnO dilute magnetic semiconductor thin films deposited on c-plane sapphire (Al2O3) and the Co:ZnO/Al2O3 interface structure at atomic resolution using aberration-corrected transmission electron microscopy (TEM) and electron energy-loss spectroscopy (EELS). Comparing Co:ZnO samples deposited by pulsed laser deposition and reactive magnetron sputtering, both exhibit extrinsic stacking faults, incoherent interface structures, and compositional variations within the first 3-4 Co:ZnO layers at the interface.. In addition, we have measured the local strain which reveals the lattice distortion around the stacking faults.

  16. CONDENSED MATTER: STRUCTURE, MECHANICAL AND THERMAL PROPERTIES: A Thin Liquid Film and Its Effects in an Atomic Force Microscopy Measurement

    NASA Astrophysics Data System (ADS)

    Lin, Jing; Zheng, Zhi-Jun; Yu, Ji-Lin; Bai, Yi-Long

    2009-08-01

    Recently, it has been observed that a liquid film spreading on a sample surface will significantly distort atomic force microscopy (AFM) measurements. In order to elaborate on the effect, we establish an equation governing the deformation of liquid film under its interaction with the AFM tip and substrate. A key issue is the critical liquid bump height y0c, at which the liquid film jumps to contact the AFM tip. It is found that there are three distinct regimes in the variation of y0c with film thickness H, depending on Hamaker constants of tip, sample and liquid. Noticeably, there is a characteristic thickness H* physically defining what a thin film is; namely, once the film thickness H is the same order as H*, the effect of film thickness should be taken into account. The value of H* is dependent on Hamaker constants and liquid surface tension as well as tip radius.

  17. Electrical Study of Trapped Charges in Copper-Doped Zinc Oxide Films by Scanning Probe Microscopy for Nonvolatile Memory Applications

    PubMed Central

    Su, Ting; Zhang, Haifeng

    2017-01-01

    Charge trapping properties of electrons and holes in copper-doped zinc oxide (ZnO:Cu) films have been studied by scanning probe microscopy. We investigated the surface potential dependence on the voltage and duration applied to the copper-doped ZnO films by Kelvin probe force microscopy. It is found that the Fermi Level of the 8 at.% Cu-doped ZnO films shifted by 0.53 eV comparing to undoped ZnO films. This shift indicates significant change in the electronic structure and energy balance in Cu-doped ZnO films. The Fermi Level (work function) of zinc oxide films can be tuned by Cu doping, which are important for developing this functional material. In addition, Kelvin probe force microscopy measurements demonstrate that the nature of contact at Pt-coated tip/ZnO:Cu interface is changed from Schottky contact to Ohmic contact by increasing sufficient amount of Cu ions. The charge trapping property of the ZnO films enhance greatly by Cu doping (~10 at.%). The improved stable bipolar charge trapping properties indicate that copper-doped ZnO films are promising for nonvolatile memory applications. PMID:28135335

  18. In vivo nanomechanical imaging of blood-vessel tissues directly in living mammals using atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Mao, Youdong; Sun, Quanmei; Wang, Xiufeng; Ouyang, Qi; Han, Li; Jiang, Lei; Han, Dong

    2009-07-01

    Atomic force microscopy (AFM) is difficult to achieve in living mammals but is necessary for understanding mechanical properties of tissues in their native form in organisms. Here we report in vivo nanomechanical imaging of blood-vessel tissues directly in living mammalians by AFM combined with surgical operations. Nanomechanical heterogeneity of blood vessels is observed across the diverse microenvironments of the same tissues in vivo. This method is further used to measure the counteractive nanomechanical changes in real time during drug-induced vasodilation and vasoconstriction in vivo, demonstrating appealing potential in characterization of in vivo nanomechanical dynamics of native tissues.

  19. Dynamic structure and protein expression of the live embryonic heart captured by 2-photon light sheet microscopy and retrospective registration

    PubMed Central

    Trivedi, Vikas; Truong, Thai V.; Trinh, Le A.; Holland, Daniel B.; Liebling, Michael; Fraser, Scott E.

    2015-01-01

    We present an imaging and image reconstruction pipeline that captures the dynamic three-dimensional beating motion of the live embryonic zebrafish heart at subcellular resolution. Live, intact zebrafish embryos were imaged using 2-photon light sheet microscopy, which offers deep and fast imaging at 70 frames per second, and the individual optical sections were assembled into a full 4D reconstruction of the beating heart using an optimized retrospective image registration algorithm. This imaging and reconstruction platform permitted us to visualize protein expression patterns at endogenous concentrations in zebrafish gene trap lines. PMID:26114028

  20. Simple and fast spectral domain algorithm for quantitative phase imaging of living cells with digital holographic microscopy.

    PubMed

    Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Engwer, Christian; Greve, Burkhard; Kemper, Björn

    2017-01-15

    We present a simple and fast phase aberration compensation method in digital holographic microscopy (DHM) for quantitative phase imaging of living cells. By analyzing the frequency spectrum of an off-axis hologram, phase aberrations can be compensated for automatically without fitting or pre-knowledge of the setup and/or the object. Simple and effective computation makes the method suitable for quantitative online monitoring with highly variable DHM systems. Results from automated quantitative phase imaging of living NIH-3T3 mouse fibroblasts demonstrate the effectiveness and the feasibility of the method.

  1. Correlation of Gear Surface Fatigue Lives to Lambda Ratio (Specific Film Thickness)

    NASA Technical Reports Server (NTRS)

    Krantz, Timothy Lewis

    2013-01-01

    The effect of the lubrication regime on gear performance has been recognized, qualitatively, for decades. Often the lubrication regime is characterized by the specific film thickness being the ratio of lubricant film thickness to the composite surface roughness. Three studies done at NASA to investigate gearing pitting life are revisited in this work. All tests were done at a common load. In one study, ground gears were tested using a variety of lubricants that included a range of viscosities, and therefore the gears operated with differing film thicknesses. In a second and third study, the performance of gears with ground teeth and superfinished teeth were assessed. Thicker oil films provided longer lives as did improved surface finish. These datasets were combined into a common dataset using the concept of specific film thickness. This unique dataset of more 258 tests provides gear designers with some qualitative information to make gear design decisions.

  2. Application of confocal Raman microscopy to investigate casein micro-particles in blend casein/pectin films.

    PubMed

    Zhuang, Yu; Sterr, Julia; Kulozik, Ulrich; Gebhardt, Ronald

    2015-03-01

    Pectin triggers formation of casein micro-particles during solution casting. Confocal Raman microscopy revealed their composition and spatial dimension in resulting films. Peaks in the Raman spectra corresponded to those found in films prepared by either casein or pectin. This suggested that no conformational changes occurred after mixing. Raman images revealed incompatibility of both polymers because particles consisted of casein only and the surrounding matrix of pectin. Deformation of micro-particles into an oblate shape took place during film formation. In dried films, an empty space between casein and pectin was found in lateral dimension. In contrast, casein micro-particles overlapped with the pectin matrix in the vertical dimension.

  3. 3D-localization microscopy and tracking of FoF1-ATP synthases in living bacteria

    NASA Astrophysics Data System (ADS)

    Renz, Anja; Renz, Marc; Klütsch, Diana; Deckers-Hebestreit, Gabriele; Börsch, Michael

    2015-03-01

    FoF1-ATP synthases are membrane-embedded protein machines that catalyze the synthesis of adenosine triphosphate. Using photoactivation-based localization microscopy (PALM) in TIR-illumination as well as structured illumination microscopy (SIM), we explore the spatial distribution and track single FoF1-ATP synthases in living E. coli cells under physiological conditions at different temperatures. For quantitative diffusion analysis by mean-squared-displacement measurements, the limited size of the observation area in the membrane with its significant membrane curvature has to be considered. Therefore, we applied a 'sliding observation window' approach (M. Renz et al., Proc. SPIE 8225, 2012) and obtained the one-dimensional diffusion coefficient of FoF1-ATP synthase diffusing on the long axis in living E. coli cells.

  4. Quantitative Visualization of Molecular Delivery and Uptake at Living Cells with Self-Referencing Scanning Ion Conductance Microscopy-Scanning Electrochemical Microscopy.

    PubMed

    Page, Ashley; Kang, Minkyung; Armitstead, Alexander; Perry, David; Unwin, Patrick R

    2017-03-07

    A multifunctional dual-channel scanning probe nanopipet that enables simultaneous scanning ion conductance microscopy (SICM) and scanning electrochemical microscopy (SECM) measurements is demonstrated to have powerful new capabilities for spatially mapping the uptake of molecules of interest at living cells. One barrel of the probe is filled with electrolyte and the molecules of interest and is open to the bulk solution for both topographical feedback and local delivery to a target interface, while a solid carbon electrode in the other barrel measures the local concentration and flux of the delivered molecules. This setup allows differentiation in molecular uptake rate across several regions of single cells with individual measurements at nanoscale resolution. Further, operating in a "hopping mode", where the probe is translated toward the interface (cell) at each point allows self-referencing to be employed, in which the carbon electrode response is calibrated at each and every pixel in bulk for comparison to the measurement near the surface. This is particularly important for measurements in living systems where an electrode response may change over time. Finite element method (FEM) modeling places the technique on a quantitative footing to allow the response of the carbon electrode and local delivery rates to be quantified. The technique is extremely versatile, with the local delivery of molecules highly tunable via control of the SICM bias to promote or restrict migration from the pipet orifice. It is expected to have a myriad of applications from drug delivery to screening catalysts.

  5. Atomic force imaging microscopy investigation of the interaction of ultraviolet radiation with collagen thin films

    NASA Astrophysics Data System (ADS)

    Stylianou, A.; Yova, D.; Alexandratou, E.; Petri, A.

    2013-02-01

    Collagen is the major fibrous protein in the extracellular matrix and consists a significant component of skin, bone, cartilage and tendon. Due to its unique properties, it has been widely used as scaffold or culture substrate for tissue regeneration or/and cell-substrate interaction studies. The ultraviolet light-collagen interaction investigations are crucial for the improvement of many applications such as that of the UV irradiation in the field of biomaterials, as sterilizing and photo-cross-linking method. The aim of this paper was to investigate the mechanisms of UV-collagen interactions by developing a collagen-based, well characterized, surface with controlled topography of collagen thin films in the nanoscale range. The methodology was to quantify the collagen surface modification induced on ultraviolet radiation and correlate it with changes induced in cells. Surface nanoscale characterization was performed by Atomic Force Microscopy (AFM) which is a powerful tool and offers quantitative and qualitative information with a non-destructive manner. In order to investigate cells behavior, the irradiated films were used for in vitro cultivation of human skin fibroblasts and the cells morphology, migration and alignment were assessed with fluorescence microscopy imaging and image processing methods. The clarification of the effects of UV light on collagen thin films and the way of cells behavior to the different modifications that UV induced to the collagen-based surfaces will contribute to the better understanding of cell-matrix interactions in the nanoscale and will assist the appropriate use of UV light for developing biomaterials.

  6. In situ fluorescent protein imaging with metal film-enhanced total internal reflection microscopy.

    PubMed

    Burghardt, Thomas P; Charlesworth, Jon E; Halstead, Miriam F; Tarara, James E; Ajtai, Katalin

    2006-06-15

    Fluorescence detection of single molecules provides a means to investigate protein dynamics minus ambiguities introduced by ensemble averages of unsynchronized protein movement or of protein movement mimicking a local symmetry. For proteins in a biological assembly, taking advantage of the single molecule approach could require single protein isolation from within a high protein concentration milieu. Myosin cross-bridges in a muscle fiber are proteins attaining concentrations of approximately 120 muM, implying single myosin detection volume for this biological assembly is approximately 1 attoL (10(-18) L) provided that just 2% of the cross-bridges are fluorescently labeled. With total internal reflection microscopy (TIRM) an exponentially decaying electromagnetic field established on the surface of a glass-substrate/aqueous-sample interface defines a subdiffraction limit penetration depth into the sample that, when combined with confocal microscopy, permits image formation from approximately 3 attoL volumes. Demonstrated here is a variation of TIRM incorporating a nanometer scale metal film into the substrate/glass interface. Comparison of TIRM images from rhodamine-labeled cross-bridges in muscle fibers contacting simultaneously the bare glass and metal-coated interface show the metal film noticeably reduces both background fluorescence and the depth into the sample from which fluorescence is detected. High contrast metal film-enhanced TIRM images allow secondary label visualization in the muscle fibers, facilitating elucidation of Z-disk structure. Reduction of both background fluorescence and detection depth will enhance TIRM's usefulness for single molecule isolation within biological assemblies.

  7. Photoemission Electron Microscopy of TiO2 Anatase Films Embedded with Rutile Nanocrystals

    SciTech Connect

    Xiong, Gang; Shao, Rui; Droubay, Timothy C.; Joly, Alan G.; Beck, Kenneth M.; Chambers, Scott A.; Hess, Wayne P.

    2007-09-03

    Photoemission electron microscopy (PEEM) excited by x-ray and UV sources is used to investigate epitaxial anatase thin films embedded with rutile nanocrystals, a model system for the study of heterocatalysis on mixed-phase TiO2. Both excitation sources show distinct contrast between the two TiO2 phases, however, the contrast is reversed. Rutile nanocrystals appear darker than the anatase film in X-ray PEEM images but brighter in UV-PEEM images. Topography-induced contrast is dominant X-ray PEEM imaging, whereas work function contrast, dominates for UV-PEEM. Work function contrast results from the differences in work function and surface defect state densities between the two phases near the Fermi level. This assertion is confirmed by UPS data that shows the rutile work function to be 0.2 eV lower and a greater occupied valence band density-of-states in rutile (100) than in anatase (001). Since the boundaries between rutile nanocrystals and the anatase film are clearly resolved, these results indicate that PEEM studies of excited state dynamics and heterocatalysis are possible at chemically intriguing mixed-phase TiO2 interfaces and grain boundaries.

  8. High resolution transmission electron microscopy study of diamond films grown from fullerene precursors

    SciTech Connect

    Luo, J.S.; Gruen, D.M.; Krauss, A.R.

    1995-07-01

    High-resolution transmission electron microscopy (HRTEM) has been used to investigate the microstructure of diamond films grown by plasma-assisted chemical vapor deposition using fullerene precursors. HRTEM observations of as-grown films revealed an array of larger crystals (>200 nm) within a polycrystalline matrix of much smaller crystallites (<20 nm). The randomly oriented small crystallites were nearly free of structural imperfections such as stacking faults or twins, while the larger ones had preferred <110> orientations with respect to the Si (100) substrate and showed evidence of structural defects on the periphery of the crystals. The most common defects were V-shaped {Sigma}9 twin boundaries, which are generally believed to serve as re-entrant sites for diamond nucleation and growth. The observation of growth steps on both (111) and (110) surfaces seems to support a reaction model in which fragments of C{sub 60}, including C{sub 2}, are considered the growth species. In particular, the nanocrystallinity of the films is most likely due to a high carbon cluster density from C{sub 60} fragmentation at or near the diamond surface, which can serve as nucleation sites for the growth of new crystallites.

  9. Förster resonance energy transfer microscopy and spectroscopy for localizing protein-protein interactions in living cells

    PubMed Central

    Sun, Yuansheng; Rombola, Christina; Jyothikumar, Vinod; Periasamy, Ammasi

    2014-01-01

    The fundamental theory of Förster resonance energy transfer (FRET) was established in the 1940's. Its great power was only realized in the past 20 years after different techniques were developed and applied to biological experiments. This success was made possible by the availability of suitable fluorescent probes, advanced optics, detectors, microscopy instrumentation and analytical tools. Combined with state-of-the-art microscopy and spectroscopy, FRET imaging allows scientists to study a variety of phenomena that produce changes in molecular proximity, thereby leading to many significant findings in the life sciences. In this review, we outline various FRET imaging techniques and their strengths and limitations; we also provide a biological model to demonstrate how to investigate protein-protein interactions in living cells using both intensity- and fluorescence lifetime-based FRET microscopy methods. PMID:23813736

  10. Live-cell analysis of plant reproduction: live-cell imaging, optical manipulation, and advanced microscopy technologies.

    PubMed

    Kurihara, Daisuke; Hamamura, Yuki; Higashiyama, Tetsuya

    2013-05-01

    Sexual reproduction ensures propagation of species and enhances genetic diversity within populations. In flowering plants, sexual reproduction requires complicated and multi-step cell-to-cell communications among male and female cells. However, the confined nature of plant reproduction processes, which occur in the female reproductive organs and several cell layers of the pistil, limits our ability to observe these events in vivo. In this review, we discuss recent live-cell imaging in in vitro systems and the optical manipulation techniques that are used to capture the dynamic mechanisms representing molecular and cellular communications in sexual plant reproduction.

  11. A practical method for monitoring FRET-based biosensors in living animals using two-photon microscopy

    PubMed Central

    Tao, Wen; Rubart, Michael; Ryan, Jennifer; Xiao, Xiao; Qiao, Chunping; Hato, Takashi; Davidson, Michael W.; Dunn, Kenneth W.

    2015-01-01

    The commercial availability of multiphoton microscope systems has nurtured the growth of intravital microscopy as a powerful technique for evaluating cell biology in the relevant context of living animals. In parallel, new fluorescent protein (FP) biosensors have become available that enable studies of the function of a wide range of proteins in living cells. Biosensor probes that exploit Förster resonance energy transfer (FRET) are among the most sensitive indicators of an array of cellular processes. However, differences between one-photon and two-photon excitation (2PE) microscopy are such that measuring FRET by 2PE in the intravital setting remains challenging. Here, we describe an approach that simplifies the use of FRET-based biosensors in intravital 2PE microscopy. Based on a systematic comparison of many different FPs, we identified the monomeric (m) FPs mTurquoise and mVenus as particularly well suited for intravital 2PE FRET studies, enabling the ratiometric measurements from linked FRET probes using a pair of experimental images collected simultaneously. The behavior of the FPs is validated by fluorescence lifetime and sensitized emission measurements of a set of FRET standards. The approach is demonstrated using a modified version of the AKAR protein kinase A biosensor, first in cells in culture, and then in hepatocytes in the liver of living mice. The approach is compatible with the most common 2PE microscope configurations and should be applicable to a variety of different FRET probes. PMID:26333599

  12. Nonperturbative Chemical Imaging of Organelle Transport in Living Cells with Coherent Anti-Stokes Raman Scattering Microscopy

    PubMed Central

    Nan, Xiaolin; Potma, Eric O.; Xie, X. Sunney

    2006-01-01

    Nonperturbative monitoring of intracellular organelle transport in unstained living cells was achieved with coherent anti-Stokes Raman scattering (CARS) microscopy. To avoid possible interference with the organelle transport introduced by laser radiation, we first examined different illumination conditions. Using a new photodamage criterion based on morphological changes of the cells, we determined the threshold values of both pulse energy and average power at relevant wavelengths. Under excitation conditions much milder than the threshold levels, we were able to monitor the motions of lipid droplet (LD) organelles in steroidogenic mouse adrenal cortical (Y-1) cells with CARS microscopy in real time without perturbations to the cells. Particle tracking analyses revealed subdiffusion as well as active transport of LDs along microtubules. Interestingly, LD active transport is only present in Y-1 cells that rounded up in culture, a morphological change associated with steroidogenesis, suggesting possible involvements of LD active transport in the latter. Simultaneous imaging of LDs and mitochondria with CARS and two-photon fluorescence microscopy clearly showed that interactions between the two organelles could be facilitated by high LD motility. These observations demonstrate CARS microscopy as a powerful noninvasive imaging tool for studying dynamic processes in living cells. PMID:16632501

  13. Prior Exposure to Creatures from a Horror Film: Live versus Photographic Representations.

    ERIC Educational Resources Information Center

    Weiss, Audrey J.; And Others

    1993-01-01

    Finds that exposure to graphic photographs of worms taken from a horror film increased children's enjoyment of the horror movie segment and reduced fear reactions to the scene. Shows that exposure to a live earthworm was effective in reducing fear reactions to the movie only among boys but did alter children's affective reactions to and judgments…

  14. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    PubMed Central

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, YongKeun

    2016-01-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated. PMID:27605489

  15. A practical approach for the detection of DNA nanostructures in single live human cells by fluorescence microscopy.

    PubMed

    Bergamini, C; Angelini, P; Rhoden, K J; Porcelli, A M; Fato, R; Zuccheri, G

    2014-05-15

    In the last decade, in vivo studies have revealed that even subtle differences in size, concentration of components, cell cycle stage, make the cells in a population respond differently to the same stimulus. In order to characterize such complexity of behavior and shed more light on the functioning and communication amongst cells, researchers are developing strategies to study single live cells in a population. In this paper, we describe the methods to design and prepare DNA-based fluorescent tetrahedral nanostructures, to deliver them to live cells and characterize such cells with epifluorescence microscopy. We report that HeLa cells internalize these nanostructures spontaneously with a higher efficiency with respect to single-stranded or double-stranded oligonucleotides. Our findings suggest that DNA tetrahedra could serve as a platform for the realization of a series of multifunctional intracellular biosensors for the analysis of single live cells.

  16. Holographic intravital microscopy for 2-D and 3-D imaging intact circulating blood cells in microcapillaries of live mice

    NASA Astrophysics Data System (ADS)

    Kim, Kyoohyun; Choe, Kibaek; Park, Inwon; Kim, Pilhan; Park, Yongkeun

    2016-09-01

    Intravital microscopy is an essential tool that reveals behaviours of live cells under conditions close to natural physiological states. So far, although various approaches for imaging cells in vivo have been proposed, most require the use of labelling and also provide only qualitative imaging information. Holographic imaging approach based on measuring the refractive index distributions of cells, however, circumvent these problems and offer quantitative and label-free imaging capability. Here, we demonstrate in vivo two- and three-dimensional holographic imaging of circulating blood cells in intact microcapillaries of live mice. The measured refractive index distributions of blood cells provide morphological and biochemical properties including three-dimensional cell shape, haemoglobin concentration, and haemoglobin contents at the individual cell level. With the present method, alterations in blood flow dynamics in live healthy and sepsis-model mice were also investigated.

  17. Thermodynamics and spreading behavior of thin perfluoropolyether films investigated with atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Bowles, Adam P.

    Computer hard drives utilize read/write heads mounted on long arms that are suspended only a few nanometers above the data storage disk. This configuration results in occasional contact between the two surfaces. These collisions are mediated by protective layers: carbon overcoats and a perfluoropolyether (PFPE) lubricant nano-film spread on the hard disk. The mechanism of lubrication at these length scales is not fully understood by the hard drive industry and to facilitate the design of better lubricants a substantial amount of research is being performed on this vital portion of the head-disk interface. A method of measuring disjoining pressure is proposed that could be applied to hard drive lubricant films. Disjoining pressure is a measure of the change in free energy of interaction between two half-spaces as the thickness of intervening layers is increased. Disjoining pressure plays a role or dictates many of the properties of films that are critical for lubrication including: wetting, adhesion, flow dynamics and volatility. Therefore, disjoining pressure provides a short cut to many properties of interest packaged in the form of an intrinsic property of the lubricant film. The technique for measuring disjoining pressure uses atomic force microscopy (AFM). During this procedure a meniscus of liquid is stretched between an AFM probe and a film. If the liquid bridge is stretched slowly enough, equilibrium between the film and meniscus is approximated. At equilibrium, the disjoining pressure of the film is balanced by the Laplace pressure of the meniscus. The Laplace pressure is described by the shape of the meniscus but for nanometer films it is difficult to actually view the tip-film contact. To remedy this, a meniscus reconstruction model is developed for spherical AFM probes that describes the force expected for constant Laplace pressure menisci as they are stretched. Fitting AFM force curve data with this model allows identification of Laplace pressure and

  18. Use of non-contact hopping probe ion conductance microscopy to investigate dynamic morphology of live platelets.

    PubMed

    Liu, Xiao; Li, Ying; Zhu, Hui; Zhao, Zilong; Zhou, Yuan; Zaske, Ana-Maria; Liu, Li; Li, Min; Lu, Hujie; Liu, Wei; Dong, Jing-Fei; Zhang, Jianning; Zhang, Yanjun

    2015-01-01

    Circulating platelets are anucleated and multi-functional cells that participate in hemostasis and arterial thrombosis. Multiple ligands and mechanical forces activate platelets, leading to cytoskeletal rearrangement and dramatic shape-changes. Such dramatic changes in platelets membrane structures are commonly detected by optical and electron microscopy after platelets are fixed. We have recently developed a method to study the membrane morphology of live platelets using Hopping Probe Ion Conductance Microscopy (HPICM). We have successfully used this technology to study the process of platelet microvesiculation upon exposure to selective agonists. Here, we further discussed technical details of using HPICM to study platelet biology and compared results from HPICM to those from conventional atomic force microscopy and scanning electron microscopy. This method offers several advantages over current technologies. First, it monitors morphological changes of platelets in response to agonists in real time. Second, platelets can be repeatedly scanned over time without damages brought by heat and prolong light exposure. Third, there is no direct contact with platelet surface so that there will no or minimal mechanical damages brought by a cantilever of a conventional atomic force microscopy. Finally, it offers the potential to study platelet membrane ion channels, which have been technically challenging up-to-date. Our data show that HPICM has high-resolution in delineating changes of platelet morphology in response to stimulations and could help to unravel the complex role of platelet in thrombus formation.

  19. Real time imaging of live cell ATP leaking or release events by chemiluminescence microscopy

    SciTech Connect

    Zhang, Yun

    2008-12-18

    The purpose of this research was to expand the chemiluminescence microscopy applications in live bacterial/mammalian cell imaging and to improve the detection sensitivity for ATP leaking or release events. We first demonstrated that chemiluminescence (CL) imaging can be used to interrogate single bacterial cells. While using a luminometer allows detecting ATP from cell lysate extracted from at least 10 bacterial cells, all previous cell CL detection never reached this sensitivity of single bacteria level. We approached this goal with a different strategy from before: instead of breaking bacterial cell membrane and trying to capture the transiently diluted ATP with the firefly luciferase CL assay, we introduced the firefly luciferase enzyme into bacteria using the modern genetic techniques and placed the CL reaction substrate D-luciferin outside the cells. By damaging the cell membrane with various antibacterial drugs including antibiotics such as Penicillins and bacteriophages, the D-luciferin molecules diffused inside the cell and initiated the reaction that produces CL light. As firefly luciferases are large protein molecules which are retained within the cells before the total rupture and intracellular ATP concentration is high at the millmolar level, the CL reaction of firefly luciferase, ATP and D-luciferin can be kept for a relatively long time within the cells acting as a reaction container to generate enough photons for detection by the extremely sensitive intensified charge coupled device (ICCD) camera. The result was inspiring as various single bacterium lysis and leakage events were monitored with 10-s temporal resolution movies. We also found a new way of enhancing diffusion D-luciferin into cells by dehydrating the bacteria. Then we started with this novel single bacterial CL imaging technique, and applied it for quantifying gene expression levels from individual bacterial cells. Previous published result in single cell gene expression quantification

  20. Focused ion beam patterned Fe thin films A study by selective area Stokes polarimetry and soft x-Ray microscopy

    SciTech Connect

    Cook, P. J.; Shen, T. H.; Grundy, P. J.; Im, M.-Y.; Fischer, P.; Morton, S. A.; Kilcoyne, A. L. D.

    2010-11-14

    We demonstrate the potential to modify the magnetic behavior and structural properties of ferromagnetic thin films using focused ion beam 'direct-write' lithography. Patterns inspired by the split-ring resonators often used as components in meta-materials were defined upon 15 nm Fe films using a 30 keV Ga{sup +} focused ion beam at a dose of 2 x 10{sup 16} ions cm{sup -2}. Structural, chemical and magnetic changes to the Fe were studied using transmission soft X-ray microscopy at the ALS, Berkeley CA. X-ray absorption spectra showed a 23% reduction in the thickness of the film in the Ga irradiated areas, but no chemical change to the Fe was evident. X-ray images of the magnetic reversal process show domain wall pinning around the implanted areas, resulting in an overall increase in the coercivity of the film. Transmission electron microscopy showed significant grain growth in the implanted regions.

  1. Imaging electronic trap states in perovskite thin films with combined fluorescence and femtosecond transient absorption microscopy

    DOE PAGES

    Xiao, Kai; Ma, Ying -Zhong; Simpson, Mary Jane; ...

    2016-04-22

    Charge carrier trapping degrades the performance of organometallic halide perovskite solar cells. To characterize the locations of electronic trap states in a heterogeneous photoactive layer, a spatially resolved approach is essential. Here, we report a comparative study on methylammonium lead tri-iodide perovskite thin films subject to different thermal annealing times using a combined photoluminescence (PL) and femtosecond transient absorption microscopy (TAM) approach to spatially map trap states. This approach coregisters the initially populated electronic excited states with the regions that recombine radiatively. Although the TAM images are relatively homogeneous for both samples, the corresponding PL images are highly structured. Themore » remarkable variation in the PL intensities as compared to transient absorption signal amplitude suggests spatially dependent PL quantum efficiency, indicative of trapping events. Furthermore, detailed analysis enables identification of two trapping regimes: a densely packed trapping region and a sparse trapping area that appear as unique spatial features in scaled PL maps.« less

  2. Imaging electronic trap states in perovskite thin films with combined fluorescence and femtosecond transient absorption microscopy

    SciTech Connect

    Xiao, Kai; Ma, Ying -Zhong; Simpson, Mary Jane; Doughty, Benjamin; Yang, Bin

    2016-04-22

    Charge carrier trapping degrades the performance of organometallic halide perovskite solar cells. To characterize the locations of electronic trap states in a heterogeneous photoactive layer, a spatially resolved approach is essential. Here, we report a comparative study on methylammonium lead tri-iodide perovskite thin films subject to different thermal annealing times using a combined photoluminescence (PL) and femtosecond transient absorption microscopy (TAM) approach to spatially map trap states. This approach coregisters the initially populated electronic excited states with the regions that recombine radiatively. Although the TAM images are relatively homogeneous for both samples, the corresponding PL images are highly structured. The remarkable variation in the PL intensities as compared to transient absorption signal amplitude suggests spatially dependent PL quantum efficiency, indicative of trapping events. Furthermore, detailed analysis enables identification of two trapping regimes: a densely packed trapping region and a sparse trapping area that appear as unique spatial features in scaled PL maps.

  3. Characterization of defect growth structure in ion plated films by scanning electron microscopy

    NASA Technical Reports Server (NTRS)

    Spalvins, T.

    1979-01-01

    Copper and gold films (0.2 to 2 microns) were ion plated onto polished 304-stainless-steel surfaces. These coatings were examined by scanning electron microscopy for coating growth defects. Three types of defects were distinguished: nodular growth, abnormal or runaway growth, and spits. The cause and origin for each type of defect was traced. Nodular growth is primarily due to inherent substrate microdefects, abnormal or runaway growth is due to external surface inclusions, and spits are due to nonuniform evaporation. All these defects have adverse effects on the coatings. They induce stresses and produce porosity in the coatings and thus weaken their mechanical properties. Friction and wear characteristics are affected by coating defects, since the large nodules are pulled out and additional wear debris is generated.

  4. Defects in paramagnetic Co-doped ZnO films studied by transmission electron microscopy

    SciTech Connect

    Kovács, A.; Duchamp, M.; Boothroyd, C. B.; Dunin-Borkowski, R. E.; Ney, A.; Ney, V.; Galindo, P. L.; Kaspar, T. C.; Chambers, S. A.

    2013-12-28

    We study planar defects in epitaxial Co:ZnO dilute magnetic semiconductor thin films deposited on c-plane sapphire (Al{sub 2}O{sub 3}), as well as the Co:ZnO/Al{sub 2}O{sub 3} interface, using aberration-corrected transmission electron microscopy and electron energy-loss spectroscopy. Co:ZnO samples that were deposited using pulsed laser deposition and reactive magnetron sputtering are both found to contain extrinsic stacking faults, incoherent interface structures, and compositional variations within the first 3–4 Co:ZnO layers next to the Al{sub 2}O{sub 3} substrate. The stacking fault density is in the range of 10{sup 17} cm{sup −3}. We also measure the local lattice distortions around the stacking faults. It is shown that despite the relatively high density of planar defects, lattice distortions, and small compositional variation, the Co:ZnO films retain paramagnetic properties.

  5. Scanning tunneling microscopy/spectroscopy of picene thin films formed on Ag(111)

    SciTech Connect

    Yoshida, Yasuo Yokosuka, Takuya; Hasegawa, Yukio; Yang, Hung-Hsiang; Huang, Hsu-Sheng; Guan, Shu-You; Su, Wei-Bin; Chang, Chia-Seng; Yanagisawa, Susumu; Lin, Minn-Tsong; Hoffmann, Germar

    2014-09-21

    Using ultrahigh-vacuum low-temperature scanning tunneling microscopy and spectroscopy combined with first principles density functional theory calculations, we have investigated structural and electronic properties of pristine and potassium (K)-deposited picene thin films formed in situ on a Ag(111) substrate. At low coverages, the molecules are uniformly distributed with the long axis aligned along the [112{sup ¯}] direction of the substrate. At higher coverages, ordered structures composed of monolayer molecules are observed, one of which is a monolayer with tilted and flat-lying molecules resembling a (11{sup ¯}0) plane of the bulk crystalline picene. Between the molecules and the substrate, the van der Waals interaction is dominant with negligible hybridization between their electronic states; a conclusion that contrasts with the chemisorption exhibited by pentacene molecules on the same substrate. We also observed a monolayer picene thin film in which all molecules were standing to form an intermolecular π stacking. Two-dimensional delocalized electronic states are found on the K-deposited π stacking structure.

  6. Measuring thermal conductivity of thin films by Scanning Thermal Microscopy combined with thermal spreading resistance analysis.

    PubMed

    Juszczyk, J; Kaźmierczak-Bałata, A; Firek, P; Bodzenta, J

    2017-01-27

    While measuring the thermal properties of a thin film, one of the most often encountered problems is the influence of the substrate thermal properties on measured signal and the need for its separation. In this work an approach for determining the thermal conductivity κ of a thin layer is presented. It bases on Scanning Thermal Microscopy (SThM) measurement combined with thermal spreading resistance analysis for a system consisting of a single layer on a substrate. Presented approach allows to take into account the influence of the substrate thermal properties on SThM signal and to estimate the true value of a thin film κ. It is based on analytical solution of the problem being a function of dimensionless parameters and requires numerical solution of relatively simple integral equation. As the analysis utilizes a solution in dimensionless parameters it can be used for any substrate-layer system. As an example, the method was applied for determination of the thermal conductivities of 4 different thin layers of thicknesses from 12 to 100nm. The impact of model parameters on the uncertainty of the estimated final κ value was analyzed.

  7. Elucidation of perovskite film micro-orientations using two-photon total internal reflectance fluorescence microscopy

    SciTech Connect

    Watson, Brianna R.; Yang, Bin; Xiao, Kai; Ma, Ying -Zhong; Doughty, Benjamin L.; Calhoun, Tessa R.

    2015-07-29

    The emergence of efficient hybrid organic-inorganic perovskite photovoltaic materials has caused the rapid development of a variety of preparation and processing techniques designed to maximize their performance. As processing methods continue to emerge, it is important to understand how the optical properties of these materials are affected on a microscopic scale. Here polarization resolved two-photon total internal reflectance microscopy (TIRFM) was used to probe changes in transition dipole moment orientation as a function of thermal annealing time in hybrid organic-inorganic lead iodide based perovskite (CH3NH3PbI3) thin films on glass. These results show that as thermal annealing time is increased the distribution of transition moments pointing out-of-plane decreases in favor of forming areas with increased in-plane orientations. As a result, it was also shown through the axial sensitivity of TIRFM that the surface topography is manifested in the signal intensity and can be used to survey aspects of morphology in coincidence with the optical properties of these films.

  8. Elucidation of perovskite film micro-orientations using two-photon total internal reflectance fluorescence microscopy

    DOE PAGES

    Watson, Brianna R.; Yang, Bin; Xiao, Kai; ...

    2015-07-29

    The emergence of efficient hybrid organic-inorganic perovskite photovoltaic materials has caused the rapid development of a variety of preparation and processing techniques designed to maximize their performance. As processing methods continue to emerge, it is important to understand how the optical properties of these materials are affected on a microscopic scale. Here polarization resolved two-photon total internal reflectance microscopy (TIRFM) was used to probe changes in transition dipole moment orientation as a function of thermal annealing time in hybrid organic-inorganic lead iodide based perovskite (CH3NH3PbI3) thin films on glass. These results show that as thermal annealing time is increased themore » distribution of transition moments pointing out-of-plane decreases in favor of forming areas with increased in-plane orientations. As a result, it was also shown through the axial sensitivity of TIRFM that the surface topography is manifested in the signal intensity and can be used to survey aspects of morphology in coincidence with the optical properties of these films.« less

  9. INFLUENCE OF FILM STRUCTURE AND LIGHT ON CHARGE TRAPPING AND DISSIPATION DYNAMICS IN SPUN-CAST ORGANIC THIN-FILM TRANSISTORS MEASURED BY SCANNING KELVIN PROBE MICROSCOPY

    SciTech Connect

    Teague, L.; Moth, M.; Anthony, J.

    2012-05-03

    Herein, time-dependent scanning Kelvin probe microscopy of solution processed organic thin film transistors (OTFTs) reveals a correlation between film microstructure and OTFT device performance with the location of trapped charge within the device channel. The accumulation of the observed trapped charge is concurrent with the decrease in I{sub SD} during operation (V{sub G}=-40 V, V{sub SD}= -10 V). We discuss the charge trapping and dissipation dynamics as they relate to the film structure and show that application of light quickly dissipates the observed trapped charge.

  10. Layer-Resolved Evolution of Organic Thin Films Monitored by Photoelectron Emission Microscopy and Optical Reflectance Spectroscopy

    PubMed Central

    2015-01-01

    Photoelectron emission microscopy (PEEM) and differential (optical) reflectance spectroscopy (DRS) have proven independently to be versatile analytical tools for monitoring the evolution of organic thin films during growth. In this paper, we present the first experiment in which both techniques have been applied simultaneously and synchronously. We illustrate how the combined PEEM and DRS results can be correlated to obtain an extended perspective on the electronic and optical properties of a molecular film dependent on the film thickness and morphology. As an example, we studied the deposition of the organic molecule α-sexithiophene on Ag(111) in the thickness range from submonolayers up to several monolayers. PMID:26523159

  11. A laminated polymer film formulation for enteric delivery of live vaccine and probiotic bacteria.

    PubMed

    de Barros, João M S; Scherer, Timothy; Charalampopoulos, Dimitrios; Khutoryanskiy, Vitaliy V; Edwards, Alexander D

    2014-07-01

    Live bacterial cells (LBCs) are administered orally as attenuated vaccines to deliver biopharmaceutical agents and as probiotics to improve gastrointestinal (GI) health. However, LBCs present unique formulation challenges and must survive GI antimicrobial defenses including gastric acid after administration. We present a simple new formulation concept, termed polymer film laminate (PFL). LBCs are ambient dried onto cast acid-resistant enteric polymer films that are then laminated together to produce a solid oral dosage form. LBC of a model live bacterial vaccine and a probiotic were dried directly onto a cast film of enteric polymer. The effectiveness at protecting dried cells in a simulated gastric fluid (SGF, pH 2.0) depended on the composition of enteric polymer film used, with a blend of ethylcellulose plus Eudragit L100 55 providing greater protection from acid than Eudragit alone. However, although PFL made from blended polymer films completely released low-molecular-weight dye into intestinal conditions (pH 7.0), they failed to release LBCs. In contrast, PFL made from Eudragit alone successfully protected dried probiotic or vaccine LBC from SGF for 2 h, and subsequently released all viable cells within 60 min of transfer into simulated intestinal fluid. Release kinetics could be controlled by modifying the lamination method.

  12. Nanomechanical properties of SiC films grown from C{sub 60} precursors using atomic force microscopy

    SciTech Connect

    Morse, K.; Balooch, M.; Hamza, A.V.; Belak, J.

    1994-12-01

    The mechanical properties of SiC films grown via C{sub 60} precursors were determined using atomic force microscopy (AFM). Conventional silicon nitride and modified diamond cantilever AFM tips were employed to determine the film hardness, friction coefficient, and elastic modulus. The hardness is found to be between 26 and 40 GPa by nanoindentation of the film with the diamond tip. The friction coefficient for the silicon nitride tip on the SiC film is about one third that for silicon nitride sliding on a silicon substrate. By combining nanoindentation and AFM measurements an elastic modulus of {approximately}300 GPa is estimated for these SiC films. In order to better understand the atomic scale mechanisms that determine the hardness and friction of SiC, we simulated the molecular dynamics of a diamond indenting a crystalline SiC substrate.

  13. Stain-Free Quantification of Chromosomes in Live Cells Using Regularized Tomographic Phase Microscopy

    PubMed Central

    Sung, Yongjin; Choi, Wonshik; Lue, Niyom; Dasari, Ramachandra R.; Yaqoob, Zahid

    2012-01-01

    Refractive index imaging is a label-free technique that enables long-term monitoring of the internal structures and molecular composition in living cells with minimal perturbation. Existing tomographic methods for the refractive index imaging lack 3-D resolution and result in artifacts that prevent accurate refractive index quantification. To overcome these limitations without compromising the capability to observe a sample in its most native condition, we have developed a regularized tomographic phase microscope (RTPM) enabling accurate refractive index imaging of organelles inside intact cells. With the enhanced accuracy, we quantify the mass of chromosomes in intact living cells, and differentiate two human colon cancer lines, HT-29 and T84 cells, solely based on the non-aqueous (dry) mass of chromosomes. In addition, we demonstrate chromosomal imaging using a dual-wavelength RTPM, which shows its potential to determine the molecular composition of cellular organelles in live cells. PMID:23166689

  14. High resolution imaging of the ultrastructure of living algal cells using soft x-ray contact microscopy

    SciTech Connect

    Ford, T.W.; Cotton, R.A.; Page, A.M.; Tomie, T.; Majima, T.; Stead, A.D.

    1995-12-31

    Soft x-ray contact microscopy provides the biologist with a technique for examining the ultrastructure of living cells at a much higher resolution than that possible by various forms of light microscopy. Readout of the developed photoresist using atomic force microscopy (AFM) produces a detailed map of the carbon densities generated in the resist following exposure of the specimen to water-window soft x-rays (2--4nm) produced by impact of a high energy laser onto a suitable target. The established high resolution imaging method of transmission electron microscopy (TEM) has inherent problems in the chemical pre-treatment required for producing the ultrathin sections necessary for this technique. Using the unicellular green alga Chlamydomonas the ultrastructural appearance of the cells following SXCM and TEM has been compared. While SXCM confirms the basic structural organization of the cell as seen by TEM (e.g., the organization of the thylakoid membranes within the chloroplast; flagellar insertion into the cytoplasm), there are important differences. These are in the appearance of the cell covering and the presence of carbon-dense spherical cellular inclusions.

  15. Ultrastructural imaging and molecular modeling of live bacteria using soft x-ray contact microscopy with nanoseconds laser plasma radiation

    SciTech Connect

    Kado, M.; Richardson, M.C.; Gabel, K.; Torres, D.; Rajyaguru, J.; Muszynski, M.J.

    1995-12-31

    Detection for clinical diagnosis and study of microbial cell is performed by a combination of low magnification optical microscopy and direct and indirect labeling techniques. Visual ultrastructural studies on subcellular organelles are possible with variations of electron microscopy (thin section, scanning and freeze fracture), although specimen preparation steps such as fixation, dehydration, resin embedding, ultra-thin sectioning, coating and staining are very specialized, extensive and may introduce artifacts in the original sample. The development of high resolution x-ray microscopy is a new technique well suited to observe the intact structure of a biological specimen at high resolution without any artifacts. Here, x ray images of the various live bacteria, such as Staphylococcus and Streptococcus, and micromolecule such as chromosomal DNA from Escherichia coli, and Lipopolysaccharide from Burkholderia cepacia, are obtained with soft x-ray contact microscopy. A compact tabletop type glass laser system is used to produce x rays from Al, Si, and Au targets. The PMMA photoresists are used to record x-ray images. An AFM (atomic force microscope) is used to reproduce the x-ray images from the developed photoresists. The performance of the 50 nm spatial resolutions are achieved and images are able to be discussed on the biological view.

  16. Single-Molecule Tracking Photoactivated Localization Microscopy to Map Nano-Scale Structure and Dynamics in Living Spines

    PubMed Central

    MacGillavry, Harold D.; Blanpied, Thomas A.

    2013-01-01

    Super-resolution microscopy has rapidly become an indispensable tool in cell biology and neuroscience by enabling measurement in live cells of structures smaller than the classical limit imposed by diffraction. The most widely applied super-resolution method currently is localization microscopy, which takes advantage of the ability to determine the position of individual fluorescent molecules with nanometer accuracy even in cells. By iteratively measuring sparse subsets of photoactivatable fluorescent proteins, protein distribution in macromolecular structures can be accurately reconstructed. Moreover, the motion trajectories of individual molecules within cells can be measured, providing unique ability to measure transport kinetics, exchange rates, and binding affinities of even small subsets of molecules with high temporal resolution and great spatial specificity. This unit describes protocols to measure and quantify the distribution of scaffold proteins within single synapses of cultured hippocampal neurons, and to track and measure the diffusion of intracellular constituents of the neuronal plasma membrane. PMID:25429311

  17. Enhanced multi-spectral imaging of live breast cancer cells using immunotargeted gold nanoshells and two-photon excitation microscopy

    NASA Astrophysics Data System (ADS)

    Bickford, Lissett; Sun, Jiantang; Fu, Kun; Lewinski, Nastassja; Nammalvar, Vengadesan; Chang, Joseph; Drezek, Rebekah

    2008-08-01

    We demonstrate the capability of using immunotargeted gold nanoshells as contrast agents for in vitro two-photon microscopy. The two-photon luminescence properties of different-sized gold nanoshells are first validated using near-infrared excitation at 780 nm. The utility of two-photon microscopy as a tool for imaging live HER2-overexpressing breast cancer cells labeled with anti-HER2-conjugated nanoshells is then explored and imaging results are compared to normal breast cells. Five different imaging channels are simultaneously examined within the emission wavelength range of 451-644 nm. Our results indicate that under near-infrared excitation, superior contrast of SK-BR-3 cancer cells labeled with immunotargeted nanoshells occurs at an emission wavelength ranging from 590 to 644 nm. Luminescence from labeled normal breast cells and autofluorescence from unlabeled cancer and normal cells remain imperceptible under the same conditions.

  18. Observation of Live Ticks (Haemaphysalis flava) by Scanning Electron Microscopy under High Vacuum Pressure

    PubMed Central

    Ishigaki, Yasuhito; Nakamura, Yuka; Oikawa, Yosaburo; Yano, Yasuhiro; Kuwabata, Susumu; Nakagawa, Hideaki; Tomosugi, Naohisa; Takegami, Tsutomu

    2012-01-01

    Scanning electron microscopes (SEM), which image sample surfaces by scanning with an electron beam, are widely used for steric observations of resting samples in basic and applied biology. Various conventional methods exist for SEM sample preparation. However, conventional SEM is not a good tool to observe living organisms because of the associated exposure to high vacuum pressure and electron beam radiation. Here we attempted SEM observations of live ticks. During 1.5×10−3 Pa vacuum pressure and electron beam irradiation with accelerated voltages (2–5 kV), many ticks remained alive and moved their legs. After 30-min observation, we removed the ticks from the SEM stage; they could walk actively under atmospheric pressure. When we tested 20 ticks (8 female adults and 12 nymphs), they survived for two days after SEM observation. These results indicate the resistance of ticks against SEM observation. Our second survival test showed that the electron beam, not vacuum conditions, results in tick death. Moreover, we describe the reaction of their legs to electron beam exposure. These findings open the new possibility of SEM observation of living organisms and showed the resistance of living ticks to vacuum condition in SEM. These data also indicate, for the first time, the usefulness of tick as a model system for biology under extreme condition. PMID:22431980

  19. Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

    PubMed Central

    de Jong, Imke G.; Beilharz, Katrin; Kuipers, Oscar P.; Veening, Jan- Willem

    2011-01-01

    During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy). Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ. PMID:21841760

  20. Scanning electrochemical microscopy studies of micropatterned copper sulfide (Cu(x)S) thin films fabricated by a wet chemistry method.

    PubMed

    Chen, Miao; Zhao, Jing; Zhao, Xiaocui

    2011-05-30

    Patterned copper sulfide (Cu(x)S) microstructures on Si (1 1 1) wafers were successfully fabricated by a relatively simple solution growth method using copper sulfate, ethylenediaminetetraacetate and sodium thiosulfate aqueous solutions as precursors. The Cu(x)S particles were selectively deposited on a patterned self-assembled monolayer of 3-aminopropyltriethoxysilane regions created by photolithography. To obtain high quality Cu(x)S films, preparative conditions such as concentration, proportion, pH and temperature of the precursor solutions were optimized. Various techniques such as optical microscopy, atomic force microscopy (AFM), X-ray diffraction, optical absorption and scanning electrochemical microscopy (SECM) were employed to examine the topography and properties of the micro-patterned Cu(x)S films. Optical microscopy and AFM results indicated that the Cu(x)S micro-pattern possessed high selectivity and clear edge resolution. From combined X-ray diffraction analysis and optical band gap calculations we conclude that Cu(9)S(5) (digenite) was the main phase within the resultant Cu(x)S film. Both SECM image and cyclic voltammograms confirmed that the Cu(x)S film had good electrical conductivity. Moreover, from SECM approach curve analysis, the apparent electron-transfer rate constant (k) in the micro-pattern of Cu(x)S dominated surface was estimated as 0.04 cm/s. The SECM current map showed high edge acuity of the micro-patterned Cu(x)S.

  1. Scanning electrochemical microscopy studies of micropatterned copper sulfide (CuxS) thin films fabricated by a wet chemistry method

    PubMed Central

    Chen, Miao; Zhao, Jing; Zhao, Xiaocui

    2011-01-01

    Patterned copper sulfide (CuxS) microstructures on Si (1 1 1) wafers were successfully fabricated by a relatively simple solution growth method using copper sulfate, ethylenediaminetetraacetate and sodium thiosulfate aqueous solutions as precursors. The CuxS particles were selectively deposited on a patterned self-assembled monolayer of 3-aminopropyltriethoxysilane regions created by photolithography. To obtain high quality CuxS films, preparative conditions such as concentration, proportion, pH and temperature of the precursor solutions were optimized. Various techniques such as optical microscopy, atomic force microscopy (AFM), X-ray diffraction, optical absorption and scanning electrochemical microscopy (SECM) were employed to examine the topography and properties of the micro-patterned CuxS films. Optical microscopy and AFM results indicated that the CuxS micro-pattern possessed high selectivity and clear edge resolution. From combined X-ray diffraction analysis and optical band gap calculations we conclude that Cu9S5 (digenite) was the main phase within the resultant CuxS film. Both SECM image and cyclic voltammograms confirmed that the CuxS film had good electrical conductivity. Moreover, from SECM approach curve analysis, the apparent electron-transfer rate constant (k) in the micro-pattern of CuxS dominated surface was estimated as 0.04 cm/s. The SECM current map showed high edge acuity of the micro-patterned CuxS. PMID:21785491

  2. Label-free imaging of gold nanoparticles in single live cells by photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Tian, Chao; Qian, Wei; Shao, Xia; Xie, Zhixing; Cheng, Xu; Liu, Shengchun; Cheng, Qian; Liu, Bing; Wang, Xueding

    2016-03-01

    Gold nanoparticles (AuNPs) have been extensively explored as a model nanostructure in nanomedicine and have been widely used to provide advanced biomedical research tools in diagnostic imaging and therapy. Due to the necessity of targeting AuNPs to individual cells, evaluation and visualization of AuNPs in the cellular level is critical to fully understand their interaction with cellular environment. Currently imaging technologies, such as fluorescence microscopy and transmission electron microscopy all have advantages and disadvantages. In this paper, we synthesized AuNPs by femtosecond pulsed laser ablation, modified their surface chemistry through sequential bioconjugation, and targeted the functionalized AuNPs with individual cancer cells. Based on their high optical absorption contrast, we developed a novel, label-free imaging method to evaluate and visualize intracellular AuNPs using photoacoustic microscopy (PAM). Preliminary study shows that the PAM imaging technique is capable of imaging cellular uptake of AuNPs in vivo at single-cell resolution, which provide an important tool for the study of AuNPs in nanomedicine.

  3. A coral-on-a-chip microfluidic platform enabling live-imaging microscopy of reef-building corals.

    PubMed

    Shapiro, Orr H; Kramarsky-Winter, Esti; Gavish, Assaf R; Stocker, Roman; Vardi, Assaf

    2016-03-04

    Coral reefs, and the unique ecosystems they support, are facing severe threats by human activities and climate change. Our understanding of these threats is hampered by the lack of robust approaches for studying the micro-scale interactions between corals and their environment. Here we present an experimental platform, coral-on-a-chip, combining micropropagation and microfluidics to allow direct microscopic study of live coral polyps. The small and transparent coral micropropagates are ideally suited for live-imaging microscopy, while the microfluidic platform facilitates long-term visualization under controlled environmental conditions. We demonstrate the usefulness of this approach by imaging coral micropropagates at previously unattainable spatio-temporal resolutions, providing new insights into several micro-scale processes including coral calcification, coral-pathogen interaction and the loss of algal symbionts (coral bleaching). Coral-on-a-chip thus provides a powerful method for studying coral physiology in vivo at the micro-scale, opening new vistas in coral biology.

  4. Label-free chemical imaging of live Euglena gracilis by high-speed SRS spectral microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Wakisaka, Yoshifumi; Suzuki, Yuta; Tokunaga, Kyoya; Hirose, Misa; Domon, Ryota; Akaho, Rina; Kuroshima, Mai; Tsumura, Norimichi; Shimobaba, Tomoyoshi; Iwata, Osamu; Suzuki, Kengo; Nakashima, Ayaka; Goda, Keisuke; Ozeki, Yasuyuki

    2016-03-01

    Microbes, especially microalgae, have recently been of great interest for developing novel biofuels, drugs, and biomaterials. Imaging-based screening of live cells can provide high selectivity and is attractive for efficient bio-production from microalgae. Although conventional cellular screening techniques use cell labeling, labeling of microbes is still under development and can interfere with their cellular functions. Furthermore, since live microbes move and change their shapes rapidly, a high-speed imaging technique is required to suppress motion artifacts. Stimulated Raman scattering (SRS) microscopy allows for label-free and high-speed spectral imaging, which helps us visualize chemical components inside biological cells and tissues. Here we demonstrate high-speed SRS imaging, with temporal resolution of 0.14 seconds, of intracellular distributions of lipid, polysaccharide, and chlorophyll concentrations in rapidly moving Euglena gracilis, a unicellular phytoflagellate. Furthermore, we show that our method allows us to analyze the amount of chemical components inside each living cell. Our results indicate that SRS imaging may be applied to label-free screening of living microbes based on chemical information.

  5. Video rate confocal laser scanning reflection microscopy in the investigation of normal and neoplastic living cell dynamics.

    PubMed

    Vesely, P; Boyde, A

    1996-01-01

    The introduction of video rate confocal laser scanning microscopes (VRCLSM) used in reflection mode with high magnification, high aperture objective lenses and with further magnification by a zoom facility allowed the first detailed observations of the activity of living cytoplasm and offered a new tool for investigation of the structural transition from the living state to the specimen fixed for electron microscopy (EM). We used a Noran Odyssey VRCLSM in reflection (backscattered) mode. A greater degree of oversampling and more comfortable viewing of the liver or taped video image was achieved at zoom factor 5, giving a display monitor field width of 10 microns. A series of mesenchyme derived cell lines--from normal cells to sarcoma cells of different malignancy--was used to compare behaviour of the observed intracellular structures and results of fixation. We contrasted the dynamic behaviour of fine features in the cytoplasm of normal and neoplastic living cells and changes induced by various treatments. The tubulomembraneous 3D structure of cytoplasm in living cells is dynamic with motion observable at the new limits of resolution provided by VRCLSM. All organelles appear integrated into one functional compartment supporting the continuous 3D trafficking of small particles (vesicles). This integrated dynamic spatial network (IDSN) was found to be largest in neoplastic cells.

  6. Magnetism of epitaxial Tb films on W(110) studied by spin-polarized low-energy electron microscopy

    NASA Astrophysics Data System (ADS)

    Prieto, J. E.; Chen, Gong; Schmid, A. K.; de la Figuera, J.

    2016-11-01

    Thin epitaxial films of Tb metal were grown on a clean W(110) substrate in ultrahigh vacuum and studied in situ by low-energy electron microscopy. Annealed films present magnetic contrast in spin-polarized low-energy electron microscopy. The energy dependence of the electron reflectivity was determined and a maximum value of its spin asymmetry of about 1% was measured. The magnetization direction of the Tb films is in-plane. Upon raising the temperature, no change in the domain distribution is observed, while the asymmetry in the electron reflectivity decreases when approaching the critical temperature, following a power law ˜(1-T /TC) β with a critical exponent β of 0.39.

  7. Mechanical sensing of the penetration of various nanoneedles into a living cell using atomic force microscopy.

    PubMed

    Obataya, Ikuo; Nakamura, Chikashi; Han, SungWoong; Nakamura, Noriyuki; Miyake, Jun

    2005-02-15

    Mechanical responses during insertion of a silicon nanoneedle into a living melanocyte were observed by using an atomic force microscope (AFM). In order to study the dependence of the mechanical response on the shape of the nanoneedle, we prepared various shapes of silicon AFM tips by focused-ion beam (FIB) etching. The force curves showed increases up to 0.65-1.9 nN after contact on the cell surface, and then the force dropped corresponding with the penetration of the needle through the cell membrane. The force required for penetration was significantly smaller than that using a normal pyramidal tip. The force curves with a cylindrical tip showed a shorter indenting distance before penetration than that with the cone-shaped tip. It is considered that the information about the geometry of penetrating material leads to the development of more suitable micro- and nano-materials to insert into a living cell for cell surgery.

  8. Live from the Mars Hotel - Space Locations and the Film Industry

    NASA Astrophysics Data System (ADS)

    Sivier, D.

    Space exploration is the subject of intense media interest in a way unparalleled in any other branch of science. It is the subject of countless films and television programmes, both fact and fiction, many using original footage from space. Astronauts have broadcast live from the Moon, and TV journalists have travelled to Mir, similar to the use of exotic terrestrial locations for filming by professional film crews. Although prohibitively expensive at the moment, the next generation of spacecraft may lower launch costs to an affordable level, so that space locations become competitive against computer graphics and model work. The construction of orbital hotels will create the demand for human interest stories similar to those set in holiday locations like the south of France and Italy made just after the Second World War, at a time when mass tourism on foreign holidays was just beginning, aided by the development of large transport aircraft able to cater to the demand for mass flight.

  9. Rapid telomere motions in live human cells analyzed by highly time-resolved microscopy

    PubMed Central

    Wang, Xueying; Kam, Zvi; Carlton, Peter M; Xu, Lifeng; Sedat, John W; Blackburn, Elizabeth H

    2008-01-01

    Background Telomeres cap chromosome ends and protect the genome. We studied individual telomeres in live human cancer cells. In capturing telomere motions using quantitative imaging to acquire complete high-resolution three-dimensional datasets every second for 200 seconds, telomere dynamics were systematically analyzed. Results The motility of individual telomeres within the same cancer cell nucleus was widely heterogeneous. One class of internal heterochromatic regions of chromosomes analyzed moved more uniformly and showed less motion and heterogeneity than telomeres. The single telomere analyses in cancer cells revealed that shorter telomeres showed more motion, and the more rapid telomere motions were energy dependent. Experimentally increasing bulk telomere length dampened telomere motion. In contrast, telomere uncapping, but not a DNA damaging agent, methyl methanesulfonate, significantly increased telomere motion. Conclusion New methods for seconds-scale, four-dimensional, live cell microscopic imaging and data analysis, allowing systematic tracking of individual telomeres in live cells, have defined a previously undescribed form of telomere behavior in human cells, in which the degree of telomere motion was dependent upon telomere length and functionality. PMID:19014413

  10. CRISPR/Cas9-mediated endogenous protein tagging for RESOLFT super-resolution microscopy of living human cells.

    PubMed

    Ratz, Michael; Testa, Ilaria; Hell, Stefan W; Jakobs, Stefan

    2015-04-20

    Overexpression is a notorious concern in conventional and especially in super-resolution fluorescence light microscopy studies because it may cause numerous artifacts including ectopic sub-cellular localizations, erroneous formation of protein complexes, and others. Nonetheless, current live cell super-resolution microscopy studies generally rely on the overexpression of a host protein fused to a fluorescent protein. Here, we establish CRISPR/Cas9-mediated generation of heterozygous and homozygous human knockin cell lines expressing fluorescently tagged proteins from their respective native genomic loci at close to endogenous levels. We tagged three different proteins, exhibiting various localizations and expression levels, with the reversibly switchable fluorescent protein rsEGFP2. We demonstrate the benefit of endogenous expression levels compared to overexpression and show that typical overexpression-induced artefacts were avoided in genome-edited cells. Fluorescence activated cell sorting analysis revealed a narrow distribution of fusion protein expression levels in genome-edited cells, compared to a pronounced variability in transiently transfected cells. Using low light intensity RESOLFT (reversible saturable optical fluorescence transitions) nanoscopy we show sub-diffraction resolution imaging of living human knockin cells. Our strategy to generate human cell lines expressing fluorescent fusion proteins at endogenous levels for RESOLFT nanoscopy can be extended to other fluorescent tags and super-resolution approaches.

  11. Low-cost multimodal light sheet microscopy for optically cleared tissues and living specimens

    NASA Astrophysics Data System (ADS)

    Rouger, Vincent; Alchini, Ricardo; Kazarine, Alexei; Gopal, Angelica A.; Girouard, Marie-Pier; Fournier, Alyson E.; Wiseman, Paul W.

    2016-12-01

    Light sheet microscopy techniques have expanded with designs to address many new applications. Due to rapid advancements in computing power, camera/detector technologies, and tissue clearing techniques, light sheet methods are becoming increasingly popular for biomedical imaging applications at the cellular and tissue levels. Light sheet imaging modalities couple rapid imaging rates, low-levels of phototoxicity, and excellent signal to noise ratios, contributing to their popularity for experimental biology. However, the current major limitation of light sheet microscopy arises from optical aberrations, with the main drawback being the defocusing introduced by refractive index variations that accompany clearing techniques. Here, we propose an inexpensive and easy to build light sheet based instrumentation to overcome this limitation by optomechanically decoupling the sample scanning movement from the detection step. Our solution is relatively simple to implement and also provides increased modularity by using a swappable excitation arm. This expands the range of samples we can image on a single system, from high resolution for single cells at μm spatial resolution, to tissues with mm spatial resolution. We demonstrate our approach, using the system to image iDISCO cleared embryos and sciatic nerves, and provide the full three-dimensional reconstruction of these objects in minutes.

  12. Measurement of intracellular calcium gradients in single living cells using optical sectioning microscopy

    NASA Astrophysics Data System (ADS)

    Yelamarty, Rao V.; Cheung, Joseph Y.

    1992-06-01

    Intracellular free calcium has been recognized as a regulator of many cellular processes and plays a key role in mediating actions of many drugs. To elucidate subcellular spatial calcium changes throughout the cell in three dimensions (3-D), optical sectioning microscopy was applied using digital imaging coupled fluorescence microscopy. The cell was loaded with a fluorescent indicator, fura-2, and a stack of sectional fluorescent images were acquired, digitized and finally stored on-line for post image analysis. Each sectional image was then deconvolved, to remove contaminating light signals from adjacent planes, using the Nearest Neighboring Deconvolution Algorithm (NNDA) and the overall imaging system's empirical Point Spread Function (PSF) that is measured with a 0.25 micrometers fluorescent bead. Using this technique, we measured that the addition of growth factors caused a 2 - 3 fold increase (1) in nuclear calcium compared to cytosolic calcium in blood cells and (2) in both nuclear and cytosolic calcium in liver cells. Such spatial information, which is important in understanding subcellular processes, would not be possible to measure with other methods.

  13. Silica colloidal crystals as porous substrates for total internal reflection fluorescence microscopy of live cells.

    PubMed

    Velarde, Tomika R C; Wirth, Mary J

    2008-06-01

    Total internal reflection fluorescence (TIRF) microscopy is a powerful means of probing biological cells because it reduces autofluorescence, but the need for direct contact between the cell surface and the microscope slide hinders chemical access to the cell surface. In this work, a submicrometer crystalline layer of colloidal silica on the microscope coverslip is shown to allow TIRF microscopy while also allowing chemical access to the cell surface. A 750 nm layer of 165 nm silica colloidal crystals was sintered onto a fused silica coverslip, and Chinese hamster ovary cells were successfully grown on this surface. This cell line over-expresses the human delta-opioid receptor, which enabled probing of the binding of a labeled ligand to the receptors on the cell surface. Total internal reflection and chemical access to the cell surface are demonstrated. The range of angles for total internal reflection is reduced only by 1/3 due to the lower index of refraction of the colloidal multilayer relative to fused silica.

  14. Measuring the elastic properties of living cells through the analysis of current-displacement curves in scanning ion conductance microscopy.

    PubMed

    Pellegrino, Mario; Pellegrini, Monica; Orsini, Paolo; Tognoni, Elisabetta; Ascoli, Cesare; Baschieri, Paolo; Dinelli, Franco

    2012-09-01

    Knowledge of mechanical properties of living cells is essential to understand their physiological and pathological conditions. To measure local cellular elasticity, scanning probe techniques have been increasingly employed. In particular, non-contact scanning ion conductance microscopy (SICM) has been used for this purpose; thanks to the application of a hydrostatic pressure via the SICM pipette. However, the measurement of sample deformations induced by weak pressures at a short distance has not yet been carried out. A direct quantification of the applied pressure has not been also achieved up to now. These two issues are highly relevant, especially when one addresses the investigation of thin cell regions. In this paper, we present an approach to solve these problems based on the use of a setup integrating SICM, atomic force microscopy, and optical microscopy. In particular, we describe how we can directly image the pipette aperture in situ. Additionally, we can measure the force induced by a constant hydrostatic pressure applied via the pipette over the entire probe-sample distance range from a remote point to contact. Then, we demonstrate that the sample deformation induced by an external pressure applied to the pipette can be indirectly and reliably evaluated from the analysis of the current-displacement curves. This method allows us to measure the linear relationship between indentation and applied pressure on uniformly deformable elastomers of known Young's modulus. Finally, we apply the method to murine fibroblasts and we show that it is sensitive to local and temporally induced variations of the cell surface elasticity.

  15. Evaluation of Fluorophores to Label SNAP-Tag Fused Proteins for Multicolor Single-Molecule Tracking Microscopy in Live Cells

    PubMed Central

    Bosch, Peter J.; Corrêa, Ivan R.; Sonntag, Michael H.; Ibach, Jenny; Brunsveld, Luc; Kanger, Johannes S.; Subramaniam, Vinod

    2014-01-01

    Single-molecule tracking has become a widely used technique for studying protein dynamics and their organization in the complex environment of the cell. In particular, the spatiotemporal distribution of membrane receptors is an active field of study due to its putative role in the regulation of signal transduction. The SNAP-tag is an intrinsically monovalent and highly specific genetic tag for attaching a fluorescent label to a protein of interest. Little information is currently available on the choice of optimal fluorescent dyes for single-molecule microscopy utilizing the SNAP-tag labeling system. We surveyed 6 green and 16 red excitable dyes for their suitability in single-molecule microscopy of SNAP-tag fusion proteins in live cells. We determined the nonspecific binding levels and photostability of these dye conjugates when bound to a SNAP-tag fused membrane protein in live cells. We found that only a limited subset of the dyes tested is suitable for single-molecule tracking microscopy. The results show that a careful choice of the dye to conjugate to the SNAP-substrate to label SNAP-tag fusion proteins is very important, as many dyes suffer from either rapid photobleaching or high nonspecific staining. These characteristics appear to be unpredictable, which motivated the need to perform the systematic survey presented here. We have developed a protocol for evaluating the best dyes, and for the conditions that we evaluated, we find that Dy 549 and CF 640 are the best choices tested for single-molecule tracking. Using an optimal dye pair, we also demonstrate the possibility of dual-color single-molecule imaging of SNAP-tag fusion proteins. This survey provides an overview of the photophysical and imaging properties of a range of SNAP-tag fluorescent substrates, enabling the selection of optimal dyes and conditions for single-molecule imaging of SNAP-tagged fusion proteins in eukaryotic cell lines. PMID:25140415

  16. Imaging of green fluorescent protein in live plant by scanning near-field optical microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Jianhua; Chen, Tao; Sun, Jialin; Guo, Jihua; Zhao, Jun

    2002-04-01

    An auxin/IAA induced in vivo green fluorescent protein (GFP) in a living plant Arabidopsis root has been studied by a scanning near-field microscope in transmission mode. The promising near-field images of the inducible GFPs at sub- surface of a plant cell suggest that they may locate proximity to the cell wall, i.e. both sides of and in the cytoplasm membrane. The clear and faint fluorescent spots with 1-3 micrometers showed that the proteins localized nearer and farther to the cell wall, respectively. All GFP molecules gathered together in a cell, and no individual GFP was observed in the experiment.

  17. Residual solvent content in conducting polymer-blend films mapped with scanning transmission x-ray microscopy

    NASA Astrophysics Data System (ADS)

    Meier, Robert; Schindler, Markus; Müller-Buschbaum, Peter; Watts, Benjamin

    2011-11-01

    Near-edge x-ray absorption fine-structure spectra prove the presence of solvent molecules in conducting polymer films and are used to calculate the absolute solvent uptake of, e.g., 5 vol.% in poly(vinylcarbazole) (PVK) films, which were prepared by solution casting with cyclohexanone as solvent. Nanoscale scanning transmission x-ray microscopy (STXM) reveals a thickness-independent solvent content in a PVK gradient sample due to the formation of an enrichment layer of residual solvent. In polymer-blend films of PVK and poly(3-hexylthiophene) (P3HT), STXM probes a lateral residual solvent uptake, which depends on the composition of the phase-separation domains. For all measurements, oxygen-containing solvent molecules in oxygen-free conducting polymer films are used as marker material, and a significant amount of residual solvent is found in all types of investigated samples.

  18. Nanoscale studies of switching behavior of ferroelectric thin films by using piezoresponse force microscopy

    NASA Astrophysics Data System (ADS)

    Wu, Dong

    The work presented in this dissertation is focused on the study of ferroelectric thin films using the method of Piezo-response Force Microscopy (PFM) with several modifications specific to ferroelectrics. In this research, the main motivation is the study of polarization-reversal mechanisms for different sizes of very small-scale (0.5-5 mum size) ferroelectric capacitors in possible applications to ferroelectric random-access-memory devices (FeRAM). In order to make such FeRAM devices more competitive with other types of nonvolatile memory technologies such as phase-change random access memory (PRAM) and magneto-resistive random access memory (MRAM), etc., it is necessary to increase the integration density and therefore reduce bit-cell size. This in turn requires a detailed understanding of (and therefore studies of) the switching properties of small-scale ferroelectric capacitors at the micrometer and sub-micrometer size scale. With traditional methods such as the polarization-hysteresis-loop measurement and the transient-switching-current measurement, such switching properties at the sub-micrometer or nanometer scale are difficult to obtain. This is due to the difficulty of electrically contacting each individual capacitor and also due to the drastically reduced electric signal at such a small scale. In addition, these methods do not provide needed spatially-resolved information about local switching. By using different experimental approaches based on PFM, all of these problems were solved and now one can directly study the switching behavior of these ferroelectric capacitors (as shown in this thesis) through observing and quantifying their PFM images. Also this thesis also presents a detailed description of PFM theory as well as the modified PFM experimental setup. In this thesis we present studies of ferroelectric thin films of two different types: polycrystalline and epitaxial. Each film has a different texture and therefore different interface defects which

  19. An introduction to the wound healing assay using live-cell microscopy

    PubMed Central

    Jonkman, James E. N.; Cathcart, Judith A.; Xu, Feng; Bartolini, Miria E.; Amon, Jennifer E.; Stevens, Katarzyna M.; Colarusso, Pina

    2014-01-01

    Abstract The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results. PMID:25482647

  20. An introduction to the wound healing assay using live-cell microscopy.

    PubMed

    Jonkman, James E N; Cathcart, Judith A; Xu, Feng; Bartolini, Miria E; Amon, Jennifer E; Stevens, Katarzyna M; Colarusso, Pina

    2014-01-01

    The wound healing assay is used in a range of disciplines to study the coordinated movement of a cell population. In this technical review, we describe the workflow of the wound healing assay as monitored by optical microscopy. Although the assay is straightforward, a lack of standardization in its application makes it difficult to compare results and reproduce experiments among researchers. We recommend general guidelines for consistency, including: (1) sample preparation including the creation of the gap, (2) microscope equipment requirements, (3) image acquisition, and (4) the use of image analysis to measure the gap size and its rate of closure over time. We also describe parameters that are specific to the particular research question, such as seeding density and matrix coatings. All of these parameters must be carefully controlled within a given set of experiments in order to achieve accurate and reproducible results.

  1. Lab-On-Chip Clinorotation System for Live-Cell Microscopy Under Simulated Microgravity

    NASA Technical Reports Server (NTRS)

    Yew, Alvin G.; Atencia, Javier; Chinn, Ben; Hsieh, Adam H.

    1980-01-01

    Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

  2. Lab-On-Chip Clinorotation System for Live-Cell Microscopy Under Simulated Microgravity

    NASA Technical Reports Server (NTRS)

    Yew, Alvin G.; Atencia, Javier; Chinn, Ben; Hsieh, Adam H.

    2013-01-01

    Cells in microgravity are subject to mechanical unloading and changes to the surrounding chemical environment. How these factors jointly influence cellular function is not well understood. We can investigate their role using ground-based analogues to spaceflight, where mechanical unloading is simulated through the time-averaged nullification of gravity. The prevailing method for cellular microgravity simulation is to use fluid-filled containers called clinostats. However, conventional clinostats are not designed for temporally tracking cell response, nor are they able to establish dynamic fluid environments. To address these needs, we developed a Clinorotation Time-lapse Microscopy (CTM) system that accommodates lab-on- chip cell culture devices for visualizing time-dependent alterations to cellular behavior. For the purpose of demonstrating CTM, we present preliminary results showing time-dependent differences in cell area between human mesenchymal stem cells (hMSCs) under modeled microgravity and normal gravity.

  3. [Frontiers in Live Bone Imaging Researches. Two-Photon Excitation Microscopy, principles and technologies].

    PubMed

    Oikawa, Yoshiro

    2015-06-01

    The "two photon absorption" phenomenon had been predicted by the American Physicist, Maria Ghöppert-Mayer in 1931. Denk and Webb group had proved it in 1990 and the first product had been launched in the market in 1996. But ever since the product became available, the number of users are not increased. Moreover, the system had been too difficult to use and the system sometimes stay not working in labs. But recently, the new easier-to-use products are released and the ultra short pulse IR laser became stable. And its applications are extending from neuro-science to oncology or immunology fields. Due to these reasons, the shipment of multi-photon microscope in Japan in 2013 is approximately 40 units which is 3 times bigger than in 2010. In this paper, I would like to discuss the principles of two-photon microscopy and some of the new technologies for the higher signal capture efficiency.

  4. Picosecond-hetero-FRET microscopy to probe protein-protein interactions in live cells.

    PubMed Central

    Tramier, Marc; Gautier, Isabelle; Piolot, Tristan; Ravalet, Sylvie; Kemnitz, Klaus; Coppey, Jacques; Durieux, Christiane; Mignotte, Vincent; Coppey-Moisan, Maïté

    2002-01-01

    By using a novel time- and space-correlated single-photon counting detector, we show that fluorescence resonance energy transfer (FRET) between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) fused to herpes simplex virus thymidine kinase (TK) monomers can be used to reveal homodimerization of TK in the nucleus and cytoplasm of live cells. However, the quantification of energy transfer was limited by the intrinsic biexponential fluorescence decay of the donor CFP (lifetimes of 1.3 +/- 0.2 ns and 3.8 +/- 0.4 ns) and by the possibility of homodimer formation between two TK-CFP. In contrast, the heterodimerization of the transcriptional factor NF-E2 in the nucleus of live cells was quantified from the analysis of the fluorescence decays of GFP in terms of 1) FRET efficiency between GFP and DsRed chromophores fused to p45 and MafG, respectively, the two subunits of NF-E2 (which corresponds to an interchromophoric distance of 39 +/- 1 A); and 2) fractions of GFP-p45 bound to DsRed-MafG (constant in the nucleus, varying in the range of 20% to 70% from cell to cell). The picosecond resolution of the fluorescence kinetics allowed us to discriminate between very short lifetimes of immature green species of DsRed-MafG and that of GFP-p45 involved in FRET with DsRed-MafG. PMID:12496124

  5. Deciphering the internal complexity of living cells with quantitative phase microscopy: a multiscale approach

    NASA Astrophysics Data System (ADS)

    Martinez-Torres, Cristina; Laperrousaz, Bastien; Berguiga, Lotfi; Boyer-Provera, Elise; Elezgaray, Juan; Nicolini, Franck E.; Maguer-Satta, Veronique; Arneodo, Alain; Argoul, Françoise

    2015-09-01

    The distribution of refractive indices (RIs) of a living cell contributes in a nonintuitive manner to its optical phase image and quite rarely can be inverted to recover its internal structure. The interpretation of the quantitative phase images of living cells remains a difficult task because (1) we still have very little knowledge on the impact of its internal macromolecular complexes on the local RI and (2) phase changes produced by light propagation through the sample are mixed with diffraction effects by the internal cell bodies. We propose to implement a two-dimensional wavelet-based contour chain detection method to distinguish internal boundaries based on their greatest optical path difference gradients. These contour chains correspond to the highest image phase contrast and follow the local RI inhomogeneities linked to the intracellular structural intricacy. Their statistics and spatial distribution are the morphological indicators suited for comparing cells of different origins and/or to follow their transformation in pathologic situations. We use this method to compare nonadherent blood cells from primary and laboratory culture origins and to assess the internal transformation of hematopoietic stem cells by the transduction of the BCR-ABL oncogene responsible for the chronic myelogenous leukemia.

  6. Measuring the Mechanical Properties of Living Cells Using Atomic Force Microscopy

    PubMed Central

    Thomas, Gawain; Burnham, Nancy A.; Camesano, Terri Anne; Wen, Qi

    2013-01-01

    Mechanical properties of cells and extracellular matrix (ECM) play important roles in many biological processes including stem cell differentiation, tumor formation, and wound healing. Changes in stiffness of cells and ECM are often signs of changes in cell physiology or diseases in tissues. Hence, cell stiffness is an index to evaluate the status of cell cultures. Among the multitude of methods applied to measure the stiffness of cells and tissues, micro-indentation using an Atomic Force Microscope (AFM) provides a way to reliably measure the stiffness of living cells. This method has been widely applied to characterize the micro-scale stiffness for a variety of materials ranging from metal surfaces to soft biological tissues and cells. The basic principle of this method is to indent a cell with an AFM tip of selected geometry and measure the applied force from the bending of the AFM cantilever. Fitting the force-indentation curve to the Hertz model for the corresponding tip geometry can give quantitative measurements of material stiffness. This paper demonstrates the procedure to characterize the stiffness of living cells using AFM. Key steps including the process of AFM calibration, force-curve acquisition, and data analysis using a MATLAB routine are demonstrated. Limitations of this method are also discussed. PMID:23851674

  7. Measuring Local Viscosities near Plasma Membranes of Living Cells with Photonic Force Microscopy

    PubMed Central

    Jünger, Felix; Kohler, Felix; Meinel, Andreas; Meyer, Tim; Nitschke, Roland; Erhard, Birgit; Rohrbach, Alexander

    2015-01-01

    The molecular processes of particle binding and endocytosis are influenced by the locally changing mobility of the particle nearby the plasma membrane of a living cell. However, it is unclear how the particle’s hydrodynamic drag and momentum vary locally and how they are mechanically transferred to the cell. We have measured the thermal fluctuations of a 1 μm-sized polystyrene sphere, which was placed in defined distances to plasma membranes of various cell types by using an optical trap and fast three-dimensional (3D) interferometric particle tracking. From the particle position fluctuations on a 30 μs timescale, we determined the distance-dependent change of the viscous drag in directions perpendicular and parallel to the cell membrane. Measurements on macrophages, adenocarcinoma cells, and epithelial cells revealed a significantly longer hydrodynamic coupling length of the particle to the membrane than those measured at giant unilamellar vesicles (GUVs) or a plane glass interface. In contrast to GUVs, there is also a strong increase in friction and in mean first passage time normal to the cell membrane. This hydrodynamic coupling transfers a different amount of momentum to the interior of living cells and might serve as an ultra-soft stimulus triggering further reactions. PMID:26331245

  8. Optical lock-in detection imaging microscopy for contrast-enhanced imaging in living cells.

    PubMed

    Marriott, Gerard; Mao, Shu; Sakata, Tomoyo; Ran, Jing; Jackson, David K; Petchprayoon, Chutima; Gomez, Timothy J; Warp, Erica; Tulyathan, Orapim; Aaron, Holly L; Isacoff, Ehud Y; Yan, Yuling

    2008-11-18

    One of the limitations on imaging fluorescent proteins within living cells is that they are usually present in small numbers and need to be detected over a large background. We have developed the means to isolate specific fluorescence signals from background by using lock-in detection of the modulated fluorescence of a class of optical probe termed "optical switches." This optical lock-in detection (OLID) approach involves modulating the fluorescence emission of the probe through deterministic, optical control of its fluorescent and nonfluorescent states, and subsequently applying a lock-in detection method to isolate the modulated signal of interest from nonmodulated background signals. Cross-correlation analysis provides a measure of correlation between the total fluorescence emission within single pixels of an image detected over several cycles of optical switching and a reference waveform detected within the same image over the same switching cycles. This approach to imaging provides a means to selectively detect the emission from optical switch probes among a larger population of conventional fluorescent probes and is compatible with conventional microscopes. OLID using nitrospirobenzopyran-based probes and the genetically encoded Dronpa fluorescent protein are shown to generate high-contrast images of specific structures and proteins in labeled cells in cultured and explanted neurons and in live Xenopus embryos and zebrafish larvae.

  9. 3D measurements of live cells via digital holographic microscopy and terahertz spectroscopy

    NASA Astrophysics Data System (ADS)

    Park, Jun Yong; Oser, Dorian; Iapozzuto, Peter; Norbury, Sean; Mahajan, Supriya; Khmaladze, Alexander; Sharikova, Anna

    2016-03-01

    This is a study of the central nervous system (CNS) cells, including brain micro vascular endothelial cells (BMV) that constitute the blood brain barrier, and C6 glial cells that are the predominant cell in the brain. The cells are exposed to various chemicals by non-invasive, label-free methods. Digital holographic microscopy (DHM) is a technique that records an interference pattern between an object and reference waves, so that the computationally reconstructed holographic image contains both amplitude and phase information, and 3D images are obtained. The measurement of cell cultures by digital holographic microscopy yields information about cell death mechanisms, since these processes are correlated with individual cell volume. Our in-house DHM combines a visible (red) laser source with a conventional microscope base, and LabVIEW-run data processing. Terahertz spectral signatures are associated with structural changes in molecules and provide complementary information about cells. Both CNS cells BMV and C6 cells are treated with the drug "Methamphetamine" (METH), which induces apoptosis in neuronal cells and exhibits decrease in cell volume, a characteristic of cells undergoing apoptosis (induced cell death). METH can cause CNS cell death by cross-talk between mitochondria-, endoplasmic reticulum-, and receptor-mediated apoptotic events, all of which results in drug induced changes in neuroplasticity and significant neuropathology. Doxorubicin (DOX), a popular anticancer drug, is used as a control. We observe that METH treatment resulted in more pronounced cell volume shrinkage in both the BMV and C6 cells, as compared to DOX-induced cell apoptosis.

  10. Filming the formation and fluctuation of skyrmion domains by cryo-Lorentz transmission electron microscopy

    PubMed Central

    Rajeswari, Jayaraman; Huang, Ping; Mancini, Giulia Fulvia; Murooka, Yoshie; Latychevskaia, Tatiana; McGrouther, Damien; Cantoni, Marco; Baldini, Edoardo; White, Jonathan Stuart; Magrez, Arnaud; Giamarchi, Thierry; Rønnow, Henrik Moodysson; Carbone, Fabrizio

    2015-01-01

    Magnetic skyrmions are promising candidates as information carriers in logic or storage devices thanks to their robustness, guaranteed by the topological protection, and their nanometric size. Currently, little is known about the influence of parameters such as disorder, defects, or external stimuli on the long-range spatial distribution and temporal evolution of the skyrmion lattice. Here, using a large (7.3×7.3 μm2) single-crystal nanoslice (150 nm thick) of Cu2OSeO3, we image up to 70,000 skyrmions by means of cryo-Lorentz transmission electron microscopy as a function of the applied magnetic field. The emergence of the skyrmion lattice from the helimagnetic phase is monitored, revealing the existence of a glassy skyrmion phase at the phase transition field, where patches of an octagonally distorted skyrmion lattice are also discovered. In the skyrmion phase, dislocations are shown to cause the emergence and switching between domains with different lattice orientations, and the temporal fluctuation of these domains is filmed. These results demonstrate the importance of direct-space and real-time imaging of skyrmion domains for addressing both their long-range topology and stability. PMID:26578765

  11. Nanoscale Photoconductivity Imaging of Thin-film Semiconductors by Laser-assisted Microwave Impedance Microscopy

    NASA Astrophysics Data System (ADS)

    Chu, Zhaodong; Wu, Di; Ren, Yuan; Yang, Seungcheol; Sun, Liuyang; Li, Xiaoqin; Lai, Keji

    The photo-response of semiconductors is usually studied by detecting the photocurrent across source-drain electrodes under light illumination. By integrating the microwave impedance microscopy (MIM) technique with focused-laser stimulation, we are able to perform the real-space photoconductivity mapping of photo-sensitive materials without the need of patterning contact electrodes. Here, we report the MIM results of various thin-film materials, such as In2Se3 nano-sheets and transition metal dichalcogenides (TMD) flakes, illuminated by laser beams of different wavelengths in the ambient condition. With no or below-gap illumination, the samples were highly resistive, as indicated by the low MIM signals. The MIM contrast emerges under above-gap light and increases as increasing laser intensity, which clearly demonstrates the local imaging of photoconductivity rather than the transport photocurrent. Interestingly, clear domain structures with mesoscopic length scales were seen in the data due to the coexistence of multiple phases in In2Se3. The unique combination of MIM and laser stimulation thus provides a new direction to explore the microscopic origin of various light-driven phenomena in complex systems. We gratefully acknowledge financial support from NSF.

  12. A Microperfusion and In-Bore Oxygenator System Designed for Magnetic Resonance Microscopy Studies on Living Tissue Explants

    PubMed Central

    Flint, Jeremy J.; Menon, Kannan; Hansen, Brian; Forder, John; Blackband, Stephen J.

    2015-01-01

    Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges—the most evident of which are spatial limitations—to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and—being designed almost exclusively for light microscopy or electrophysiology studies—seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker’s series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig’s continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development. PMID:26666980

  13. Vibrational imaging of glucose uptake activity in live cells and tissues by stimulated Raman scattering microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Hu, Fanghao; Chen, Zhixing; Zhang, Luyuan; Shen, Yihui; Wei, Lu; Min, Wei

    2016-03-01

    Glucose is consumed as an energy source by virtually all living organisms, from bacteria to humans. Its uptake activity closely reflects the cellular metabolic status in various pathophysiological transformations, such as diabetes and cancer. Extensive efforts such as positron emission tomography, magnetic resonance imaging and fluorescence microscopy have been made to specifically image glucose uptake activity but all with technical limitations. Here, we report a new platform to visualize glucose uptake activity in live cells and tissues with subcellular resolution and minimal perturbation. A novel glucose analogue with a small alkyne tag (carbon-carbon triple bond) is developed to mimic natural glucose for cellular uptake, which can be imaged with high sensitivity and specificity by targeting the strong and characteristic alkyne vibration on stimulated Raman scattering (SRS) microscope to generate a quantitative three dimensional concentration map. Cancer cells with differing metabolic characteristics can be distinguished. Heterogeneous uptake patterns are observed in tumor xenograft tissues, neuronal culture and mouse brain tissues with clear cell-cell variations. Therefore, by offering the distinct advantage of optical resolution but without the undesirable influence of bulky fluorophores, our method of coupling SRS with alkyne labeled glucose will be an attractive tool to study energy demands of living systems at the single cell level.

  14. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

    PubMed Central

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.

    2016-01-01

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics. PMID:27872481

  15. Microfluidic Approaches to Synchrotron Radiation-Based Fourier Transform Infrared (SR-FTIR) Spectral Microscopy of Living Biosystems

    PubMed Central

    Loutherback, Kevin; Birarda, Giovanni; Chen, Liang; Holman, Hoi-Ying N.

    2016-01-01

    A long-standing desire in biological and biomedical sciences is to be able to probe cellular chemistry as biological processes are happening inside living cells. Synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy is a label-free and nondestructive analytical technique that can provide spatiotemporal distributions and relative abundances of biomolecules of a specimen by their characteristic vibrational modes. Despite great progress in recent years, SR-FTIR imaging of living biological systems remains challenging because of the demanding requirements on environmental control and strong infrared absorption of water. To meet this challenge, microfluidic devices have emerged as a method to control the water thickness while providing a hospitable environment to measure cellular processes and responses over many hours or days. This paper will provide an overview of microfluidic device development for SR-FTIR imaging of living biological systems, provide contrast between the various techniques including closed and open-channel designs, and discuss future directions of development within this area. Even as the fundamental science and technological demonstrations develop, other ongoing issues must be addressed; for example, choosing applications whose experimental requirements closely match device capabilities, and developing strategies to efficiently complete the cycle of development. These will require imagination, ingenuity and collaboration. PMID:26732243

  16. A Microperfusion and In-Bore Oxygenator System Designed for Magnetic Resonance Microscopy Studies on Living Tissue Explants

    NASA Astrophysics Data System (ADS)

    Flint, Jeremy J.; Menon, Kannan; Hansen, Brian; Forder, John; Blackband, Stephen J.

    2015-12-01

    Spectrometers now offer the field strengths necessary to visualize mammalian cells but were not designed to accommodate imaging of live tissues. As such, spectrometers pose significant challenges—the most evident of which are spatial limitations—to conducting experiments in living tissue. This limitation becomes problematic upon trying to employ commercial perfusion equipment which is bulky and—being designed almost exclusively for light microscopy or electrophysiology studies—seldom includes MR-compatibility as a design criterion. To overcome problems exclusive to ultra-high magnetic field environments with limited spatial access, we have designed microperfusion and in-bore oxygenation systems capable of interfacing with Bruker’s series of micro surface-coils. These devices are designed for supporting cellular resolution imaging in MR studies of excised, living tissue. The combined system allows for precise control of both dissolved gas and pH levels in the perfusate thus demonstrating applicability for a wide range of tissue types. Its compactness, linear architecture, and MR-compatible material content are key design features intended to provide a versatile hardware interface compatible with any NMR spectrometer. Such attributes will ensure the microperfusion rig’s continued utility as it may be used with a multitude of contemporary NMR systems in addition to those which are currently in development.

  17. Imaging Cellular Dynamics with Spectral Relaxation Imaging Microscopy: Distinct Spectral Dynamics in Golgi Membranes of Living Cells

    NASA Astrophysics Data System (ADS)

    Lajevardipour, Alireza; Chon, James W. M.; Chattopadhyay, Amitabha; Clayton, Andrew H. A.

    2016-11-01

    Spectral relaxation from fluorescent probes is a useful technique for determining the dynamics of condensed phases. To this end, we have developed a method based on wide-field spectral fluorescence lifetime imaging microscopy to extract spectral relaxation correlation times of fluorescent probes in living cells. We show that measurement of the phase and modulation of fluorescence from two wavelengths permit the identification and determination of excited state lifetimes and spectral relaxation correlation times at a single modulation frequency. For NBD fluorescence in glycerol/water mixtures, the spectral relaxation correlation time determined by our approach exhibited good agreement with published dielectric relaxation measurements. We applied this method to determine the spectral relaxation dynamics in membranes of living cells. Measurements of the Golgi-specific C6-NBD-ceramide probe in living HeLa cells revealed sub-nanosecond spectral dynamics in the intracellular Golgi membrane and slower nanosecond spectral dynamics in the extracellular plasma membrane. We interpret the distinct spectral dynamics as a result of structural plasticity of the Golgi membrane relative to more rigid plasma membranes. To the best of our knowledge, these results constitute one of the first measurements of Golgi rotational dynamics.

  18. 3D myofibril imaging in live cardiomyocytes via hybrid SHG-TPEF microscopy

    NASA Astrophysics Data System (ADS)

    Shao, Yonghong; Liu, Honghai; Ye, Tong; Borg, Tom; Qu, Junle; Peng, Xiang; Niu, Hanben; Gao, Bruce

    2011-03-01

    We developed a hybrid SHG-TPEF polarization imaging system that allowed the excitation beam from an fs Ti:Sappire laser being bi-directionally raster scanned across the focal plane using a pair of orthogonal galvanometers. To implement high-speed scanning, the turning regions of the triangular waves were smoothed by a custom-designed waveform. The SHG and TPEF signals from samples were recorded by two PMTs in the forward and backward direction. Using this imaging system, we obtained 3D images of the sarcomere structure via SHG and DiO-stained lipid membrane via TPEF in live cardiomyocytes isolated from neonatal and adult rats. The results demonstrated the potential applications of SHG and TPEF in the research of myofibrillogensis.

  19. Direct Observation of the Outermost Surfaces of Mesoporous Silica Thin Films by High Resolution Ultralow Voltage Scanning Electron Microscopy.

    PubMed

    Kobayashi, Maho; Susuki, Kyoka; Otsuji, Haruo; Sakuda, Yusuke; Asahina, Shunsuke; Kikuchi, Naoki; Kanazawa, Toshiyuki; Kuroda, Yoshiyuki; Wada, Hiroaki; Shimojima, Atsushi; Kuroda, Kazuyuki

    2017-03-07

    The properties of the outermost surfaces of mesoporous silica thin films are critical in determining their functions. Obtaining information on the presence or absence of silica layers on the film surfaces and on the degree of mesopore opening is essential for applications of surface mesopores. In this study, the outermost surfaces of mesoporous silica thin films with 3-dimensional orthorhombic and 2-dimensional hexagonal structures were observed using ultralow voltage high resolution scanning electron microscopy (HR-SEM) with decelerating optics. SEM images of the surfaces before and after etching with NH4F were taken at various landing voltages. Comparing the images taken under different conditions indicated that the outermost surfaces of the nonetched mesoporous silica thin films are coated with a thin layer of silica. The images taken at an ultralow landing voltage (i.e., 80 V) showed that the presence or absence of surface silica layers depends on whether the film was etched with an aqueous solution of NH4F. The mesostructures of both the etched and nonetched films were visible in images taken at a conventional landing voltage (2 kV); hence, the ultralow landing voltage was more suitable for analyzing the outermost surfaces. The SEM observations provided detailed information about the surfaces of mesoporous silica thin films, such as the degree of pore opening and their homogeneities. AFM images of nonetched 2-dimensional hexagonal mesoporous silica thin films show that the shape of the silica layer on the surface of the films reflects the curvature of the top surface of the cylindrical mesochannels. SEM images taken at various landing voltages are discussed, with respect to the electron penetration range at each voltage. This study increases our understanding of the surfaces of mesoporous silica thin films, which may lead to potential applications utilizing the periodically arranged mesopores on these surfaces.

  20. Interface morphology studies of liquid phase epitaxy grown HgCdTe films by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Azoulay, M.; George, M. A.; Burger, A.; Collins, W. E.; Silberman, E.

    1994-04-01

    In this paper we report an investigation of the morphology of the interfaces of liquid phase epitaxy (LPE) grown HgCdTe thin films on CdTe and CdZnTe substrates by atomic force microscopy (AFM) on freshly cleaved (110) crystallographic planes. An empirical observation which may be linked to lattice mismatch was indicated by an angle between the cleavage steps of the substrate to those of the film. The precipitates with size ranging from 5 nm to 20 nm were found to be most apparent near the interface.

  1. Investigation of lipid homeostasis in living Drosophila by coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    Chien, Cheng-Hao; Chen, Wei-Wen; Wu, June-Tai; Chang, Ta-Chau

    2012-12-01

    To improve our understanding of lipid metabolism, Drosophila is used as a model animal, and its lipid homeostasis is monitored by coherent anti-Stokes Raman scattering microscopy. We are able to achieve in vivo imaging of larval fat body (analogous to adipose tissue in mammals) and oenocytes (analogous to hepatocytes) in Drosophila larvae at subcellular level without any labeling. By overexpressing two lipid regulatory proteins-Brummer lipase (Bmm) and lipid storage droplet-2 (Lsd-2)-we found different phenotypes and responses under fed and starved conditions. Comparing with the control larva, we observed more lipid droplet accumulation by ˜twofold in oenocytes of fat-body-Bmm-overexpressing (FB-Bmm-overexpressing) mutant under fed condition, and less lipid by ˜fourfold in oenocytes of fat-body-Lsd-2-overexpressing (FB-Lsd-2-overexpressing) mutant under starved condition. Moreover, together with reduced size of lipid droplets, the lipid content in the fat body of FB-Bmm-overexpressing mutant decreases much faster than that of the control and FB-Lsd-2-overexpressing mutant during starvation. From long-term starvation assay, we found FB-Bmm-overexpressing mutant has a shorter lifespan, which can be attributed to faster consumption of lipid in its fat body. Our results demonstrate in vivo observations of direct influences of Bmm and Lsd-2 on lipid homeostasis in Drosophila larvae.

  2. Readily Accessible Multiplane Microscopy: 3D Tracking the HIV-1 Genome in Living Cells.

    PubMed

    Itano, Michelle S; Bleck, Marina; Johnson, Daniel S; Simon, Sanford M

    2016-02-01

    Human immunodeficiency virus (HIV)-1 infection and the associated disease AIDS are a major cause of human death worldwide with no vaccine or cure available. The trafficking of HIV-1 RNAs from sites of synthesis in the nucleus, through the cytoplasm, to sites of assembly at the plasma membrane are critical steps in HIV-1 viral replication, but are not well characterized. Here we present a broadly accessible microscopy method that captures multiple focal planes simultaneously, which allows us to image the trafficking of HIV-1 genomic RNAs with high precision. This method utilizes a customization of a commercial multichannel emission splitter that enables high-resolution 3D imaging with single-macromolecule sensitivity. We show with high temporal and spatial resolution that HIV-1 genomic RNAs are most mobile in the cytosol, and undergo confined mobility at sites along the nuclear envelope and in the nucleus and nucleolus. These provide important insights regarding the mechanism by which the HIV-1 RNA genome is transported to the sites of assembly of nascent virions.

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

    PubMed Central

    Huebinger, Jan; Han, Hong-Mei

    2016-01-01

    Rapid cooling of aqueous solutions is a useful approach for two important biological applications: (I) cryopreservation of cells and tissues for long-term storage, and (II) cryofixation for ultrastructural investigations by electron and cryo-electron microscopy. Usually, both approaches are very different in methodology. Here we show that a novel, fast and easy to use cryofixation technique called self-pressurized rapid freezing (SPRF) is–after some adaptations–also a useful and versatile technique for cryopreservation. Sealed metal tubes with high thermal diffusivity containing the samples are plunged into liquid cryogen. Internal pressure builds up reducing ice crystal formation and therefore supporting reversible cryopreservation through vitrification of cells. After rapid rewarming of pressurized samples, viability rates of > 90% can be reached, comparable to best-performing of the established rapid cooling devices tested. In addition, the small SPRF tubes allow for space-saving sample storage and the sealed containers prevent contamination from or into the cryogen during freezing, storage, or thawing. PMID:27711254

  4. Laser phase microscopy and functional imaging of living human cancer cells during the cell cycle

    NASA Astrophysics Data System (ADS)

    Perevedentseva, Elena V.; Graschew, Georgi; Balanos, Evangelos; Dressler, Cathrin; Beuthan, Juergen; Schlag, Peter M.

    2000-05-01

    The purpose of the investigation was to elaborate a new method of functional imaging of living tumor cells. Human colon carcinoma cells HCT116 were investigated with a conventional light microscope, confocal laser scanning microscope and with a laser phase microscope (LPM). The LPM is a functional imaging technique providing information about cell morphology which is imposed by the physiological inhomogeneity of the refractive index. The phase of the light wave passing through an object contains quantitative information about the object thickness, the shape, and the spatial distribution of the refractive index varying with morphology and chemical composition inhomogeneity inside the object. The new method of investigation of the cells in different stages of the cell cycle is developed. Every phase image of the investigated cells has been compared with conventional light microscopic and confocal microscopic images of the same cell. the relation between the cell state, their morphological peculiarities and the phase characteristics of the measured cell is determined. Data thus acquired, quantitatively characterizing intra- and intercellular processes during the cell cycle, and the method of measurements can be used to investigate with high optic resolution the mechanisms of different physical, chemical and biomolecular interactions with the tumor cells.

  5. Imaging living hair cells within the cochlear epithelium of mice using two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Yuan, Tao; Gao, Simon S.; Saggau, Peter; Oghalai, John S.

    2009-02-01

    Mice are an excellent model for studying mammalian hearing and transgenic mouse models of human hearing loss are commonly available for research. However, the mouse cochlea is substantially smaller than other animal models routinely used to study cochlear physiology. This makes the study of their hair cells difficult. We developed a novel methodology to optically image calcium within living hair cells left undisturbed within the excised mouse cochlea. Fresh cochleae were harvested, left intact within their otic capsule bone, and glued upright in a recording chamber. The bone overlying the region of the cochlear epithelium to be studied was opened and Reissner's membrane was incised. A fluorescent indicator was applied to the preparation to image intracellular calcium. A custom-built upright two-photon microscope was used to image the preparation using three dimensional scanning. We were able to image about 1/3 of a cochlear turn simultaneously, in either the apical or basal regions. Within one hour of animal sacrifice, we found that outer hair cells demonstrated increased fluorescence compared with surrounding supporting cells. Thus, this methodology can be used to visualize hair cell calcium changes and mechanotransduction over a region of the epithelium. Because the epithelium is left within the cochlea, dissection trauma is minimized and artifactual changes in hair cell physiology are reduced.

  6. Analysis of protein mobilities and interactions in living cells by multifocal fluorescence fluctuation microscopy.

    PubMed

    Heuvelman, Gerrit; Erdel, Fabian; Wachsmuth, Malte; Rippe, Karsten

    2009-07-01

    The spatial and temporal fluctuation microscope (STFM) presented here extends the concept of a fluorescence confocal laser scanning microscope to illumination and detection along a line. The parallel multichannel acquisition of the fluorescence signal was accomplished by using a single line of an electron-multiplying charge-coupled device camera at 14 mus time resolution for detection of the fluorescence signal. The STFM system provided fast confocal imaging (30 images per second) and allowed for the spatially resolved detection of particle concentration fluctuations in fluorescence correlation spectroscopy experiments. For the application of the STFM, an approximated theoretical description of the beam geometry, the point-spread function, and the fluorescence auto- and cross-correlation functions were derived. The STFM was applied to studies of the dynamics of promyelocytic leukemia nuclear bodies, green fluorescent protein, and chromatin-remodeling complexes in living cells. The results demonstrate the unique capabilities of the STFM for characterizing the position-dependent translocations and interactions of proteins in the cell.

  7. Atomic Force Microscopy Measurements of the Mechanical Properties of Cell Walls on Living Bacterial Cells

    NASA Astrophysics Data System (ADS)

    Bailey, Richard; Mullin, Nic; Turner, Robert; Foster, Simon; Hobbs, Jamie

    2014-03-01

    Staphylococcus aureus is a major cause of infection in humans, including the Methicillin resistant strain, MRSA. However, very little is known about the mechanical properties of these cells. Our investigations use AFM to examine live S. aureus cells to quantify mechanical properties. These were explored using force spectroscopy with different trigger forces, allowing the properties to be extracted at different indentation depths. A value for the cell wall stiffness has been extracted, along with a second, higher value which is found upon indenting at higher forces. This higher value drops as the cells are exposed to high salt, sugar and detergent concentrations, implying that this measurement contains a contribution from the internal turgor pressure. We have monitored these properties as the cells progress through the cell cycle. Force maps were taken over the cells at different stages of the growth process to identify changes in the mechanics throughout the progression of growth and division. The effect of Oxacillin has also been studied, to better understand its mechanism of action. Finally mutant strains of S. aureus and a second species Bacillus subtilis have been used to link the mechanical properties of the cell walls with the chain lengths and substructures involved.

  8. Mapping power-law rheology of living cells using multi-frequency force modulation atomic force microscopy

    SciTech Connect

    Takahashi, Ryosuke; Okajima, Takaharu

    2015-10-26

    We present multi-frequency force modulation atomic force microscopy (AFM) for mapping the complex shear modulus G* of living cells as a function of frequency over the range of 50–500 Hz in the same measurement time as the single-frequency force modulation measurement. The AFM technique enables us to reconstruct image maps of rheological parameters, which exhibit a frequency-dependent power-law behavior with respect to G{sup *}. These quantitative rheological measurements reveal a large spatial variation in G* in this frequency range for single cells. Moreover, we find that the reconstructed images of the power-law rheological parameters are much different from those obtained in force-curve or single-frequency force modulation measurements. This indicates that the former provide information about intracellular mechanical structures of the cells that are usually not resolved with the conventional force measurement methods.

  9. Size effects and electron microscopy of thin metal films. M.S. Thesis

    NASA Technical Reports Server (NTRS)

    Hernandez, J. D.

    1978-01-01

    All films were deposited by resistive heated evaporation in an oil diffusion pumped vacuum system (ultimate approx. equal to 0.0000001 torr). The growth from nuclei to a continuous film is highly dependent on the deposition parameters, evaporation rate as well as substrate material and substrate temperature. The growth stages of a film and the dependence of grain size on various deposition and annealing parameters are shown. Resistivity measurements were taken on thin films to observe size effects.

  10. Plasticity mechanisms in ultrafine grained freestanding aluminum thin films revealed by in-situ transmission electron microscopy nanomechanical testing

    SciTech Connect

    Idrissi, Hosni; Kobler, Aaron; Amin-Ahmadi, Behnam; Schryvers, Dominique; Coulombier, Michael; Pardoen, Thomas; Galceran, Montserrat; Godet, Stéphane; Kübel, Christian

    2014-03-10

    In-situ bright field transmission electron microscopy (TEM) nanomechanical tensile testing and in-situ automated crystallographic orientation mapping in TEM were combined to unravel the elementary mechanisms controlling the plasticity of ultrafine grained Aluminum freestanding thin films. The characterizations demonstrate that deformation proceeds with a transition from grain rotation to intragranular dislocation glide and starvation plasticity mechanism at about 1% deformation. The grain rotation is not affected by the character of the grain boundaries. No grain growth or twinning is detected.

  11. Filmed versus Live Delivery of Full-Spectrum Home Training for Primary Enuresis: Presenting the Information Is Not Enough.

    ERIC Educational Resources Information Center

    Houts, Arthur C.; And Others

    1987-01-01

    Compared the effectiveness of live versus videotape delivery of a behavioral treatment package for enuresis. Outcome was superior for the live delivery. Pretreatment measures of family and child psychosocial adjustment failed to predict treatment response. Film delivery resulted in higher confidence in children of their parents, but lower…

  12. X-ray diffraction Microscopy of Bi2 Se3 thin film on graphene/SiC

    NASA Astrophysics Data System (ADS)

    Laanait, Nouamane; Zhang, Zhan; Fenter, Paul

    2014-03-01

    We present an x-ray diffraction microscopy study of a thin film of Bi2Se3 on epitaxial graphene/6H-SiC(001). The Bi2Se3 thin film, consisting of 30 quintuple layers (Se-Bi-Se-Bi-Se), is a topological insulator that was grown by molecular beam epitaxy. The x-ray microscope resolves the lateral distribution of the film thickness at the sub-100 nm scale with the contrast produced by the thin film diffraction signal. Utilizing the depth penetration of x-rays, we imaged the buried interfaces in this system, to probe the correlation between the structure and topography of the supporting interfaces and the growth of the thin film. We find that the Bi2Se3 thickness distribution closely follows the underlying substrate topography and is strongly affected by the inhomogeneous distribution of graphene near the steps of SiC, whereby nucleation induces the growth of a large number of carbon layers. High-resolution surface diffraction was also measured from this system to extract the atomic positions in the thin film to investigate the transition from graphene to Bi2Se3.

  13. Optical, ferroelectric, and piezoresponse force microscopy studies of pulsed laser deposited Aurivillius Bi₅FeTi₃O₁₅ thin films

    SciTech Connect

    Kooriyattil, Sudheendran; Pavunny, Shojan P. E-mail: shojanpp@gmail.com; Barrionuevo, Danilo; Katiyar, Ram S. E-mail: shojanpp@gmail.com

    2014-10-14

    Bi₅FeTi₃O₁₅ (BFTO) based Aurivillius ferroelectric thin films were fabricated on strontium ruthanate coated amorphous fused silica substrates using pulsed laser deposition technique. Optical, ferroelectric, and piezoresponse properties of these thin films were investigated. The estimated refractive index (n) and extinction coefficient (k) for these films were in the range from 2.40 to 2.59 and 0.012 to 0.19, respectively. The bandgap of the BFTO thin layers was estimated to be 2.88 eV. Domain switching and hysteresis loops of BFTO films were studied utilizing piezoresponse force microscopy (PFM). The measured apparent polarization (P{sub r}) and coercive field (E{sub c}) for the samples were 20 μC/cm² and 250 kV/cm, respectively. The amplitude and phase hysteresis curves obtained from PFM characterization reveal that these films can be switched below 5 V. These results suggest that BFTO in thin film form is a promising material for photo ferroelectric and optoelectronic devices applications.

  14. Diffusion properties of single FoF1-ATP synthases in a living bacterium unraveled by localization microscopy

    NASA Astrophysics Data System (ADS)

    Renz, Marc; Rendler, Torsten; Börsch, Michael

    2012-03-01

    FoF1-ATP synthases in Escherichia coli (E. coli) bacteria are membrane-bound enzymes which use an internal protondriven rotary double motor to catalyze the synthesis of adenosine triphosphate (ATP). According to the 'chemiosmotic hypothesis', a series of proton pumps generate the necessary pH difference plus an electric potential across the bacterial plasma membrane. These proton pumps are redox-coupled membrane enzymes which are possibly organized in supercomplexes, as shown for the related enzymes in the mitochondrial inner membrane. We report diffusion measurements of single fluorescent FoF1-ATP synthases in living E. coli by localization microscopy and single enzyme tracking to distinguish a monomeric enzyme from a supercomplex-associated form in the bacterial membrane. For quantitative mean square displacement (MSD) analysis, the limited size of the observation area in the membrane with a significant membrane curvature had to be considered. The E. coli cells had a diameter of about 500 nm and a length of about 2 to 3 μm. Because the surface coordinate system yielded different localization precision, we applied a sliding observation window approach to obtain the diffusion coefficient D = 0.072 μm2/s of FoF1-ATP synthase in living E. coli cells.

  15. A coral-on-a-chip microfluidic platform enabling live-imaging microscopy of reef-building corals

    PubMed Central

    Shapiro, Orr H.; Kramarsky-Winter, Esti; Gavish, Assaf R.; Stocker, Roman; Vardi, Assaf

    2016-01-01

    Coral reefs, and the unique ecosystems they support, are facing severe threats by human activities and climate change. Our understanding of these threats is hampered by the lack of robust approaches for studying the micro-scale interactions between corals and their environment. Here we present an experimental platform, coral-on-a-chip, combining micropropagation and microfluidics to allow direct microscopic study of live coral polyps. The small and transparent coral micropropagates are ideally suited for live-imaging microscopy, while the microfluidic platform facilitates long-term visualization under controlled environmental conditions. We demonstrate the usefulness of this approach by imaging coral micropropagates at previously unattainable spatio-temporal resolutions, providing new insights into several micro-scale processes including coral calcification, coral–pathogen interaction and the loss of algal symbionts (coral bleaching). Coral-on-a-chip thus provides a powerful method for studying coral physiology in vivo at the micro-scale, opening new vistas in coral biology. PMID:26940983

  16. Three-dimensional structural changes in living hippocampal neurons imaged using magnetic AC mode atomic force microscopy.

    PubMed

    Yunxu, Sun; Danying, Lin; Yanfang, Rui; Dong, Han; Wanyun, Ma

    2006-06-01

    We developed the magnetic AC (MAC) mode atomic force microscopy (AFM) to image the 3D ultrastructure of living hippocampal neurons under physiological conditions. Initially, the soma, the dendrites and the growth cones of hippocampal neurons were imaged. The imaging force was adjusted to a small value for the long-term observation. The neural spines were damaged when the tip produced a large force; the spines regenerated after the force was reduced. Subsequently, we explored the relationship between structural changes in hippocampal neurons and Alzheimer's disease by employing the new imaging technique. Time-lapse image acquisition (10 min intervals) showed that the growth cone collapsed after the addition amyloid peptide fragment beta(25-35), which is thought to initiate Alzheimer's disease. In addition, we found substantial changes in mechanical properties and in the volume of individual growth cone. This study suggested that MAC mode AFM may be a powerful tool for observing long-term structural changes in living neural cells under physiological conditions.

  17. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

    PubMed Central

    Jünger, Felix; Olshausen, Philipp v.; Rohrbach, Alexander

    2016-01-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes. PMID:27465033

  18. Fast, label-free super-resolution live-cell imaging using rotating coherent scattering (ROCS) microscopy

    NASA Astrophysics Data System (ADS)

    Jünger, Felix; Olshausen, Philipp V.; Rohrbach, Alexander

    2016-07-01

    Living cells are highly dynamic systems with cellular structures being often below the optical resolution limit. Super-resolution microscopes, usually based on fluorescence cell labelling, are usually too slow to resolve small, dynamic structures. We present a label-free microscopy technique, which can generate thousands of super-resolved, high contrast images at a frame rate of 100 Hertz and without any post-processing. The technique is based on oblique sample illumination with coherent light, an approach believed to be not applicable in life sciences because of too many interference artefacts. However, by circulating an incident laser beam by 360° during one image acquisition, relevant image information is amplified. By combining total internal reflection illumination with dark-field detection, structures as small as 150 nm become separable through local destructive interferences. The technique images local changes in refractive index through scattered laser light and is applied to living mouse macrophages and helical bacteria revealing unexpected dynamic processes.

  19. Monitor RNA synthesis in live cell nuclei by using two-photon excited fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Peng, Xiao; Lin, Danying; Wang, Yan; Qi, Jing; Yan, Wei; Qu, Junle

    2015-03-01

    Probing of local molecular environment in cells is of significant value in creating a fundamental understanding of cellular processes and molecular profiles of diseases, as well as studying drug cell interactions. In order to investigate the dynamically changing in subcellular environment during RNA synthesis, we applied two-photon excited fluorescence lifetime imaging microscopy (FLIM) method to monitor the green fluorescent protein (GFP) fused nuclear protein ASF/SF2. The fluorescence lifetime of fluorophore is known to be in inverse correlation with a local refractive index, and thus fluorescence lifetimes of GFP fusions provide real-time information of the molecular environment of ASF/SF2- GFP. The FLIM results showed continuous and significant fluctuations of fluorescence lifetimes of the fluorescent protein fusions in live HeLa cells under physiological conditions. The fluctuations of fluorescence lifetime values indicated the variations of activities of RNA polymerases. Moreover, treatment with pharmacological drugs inhibiting RNA polymerase activities led to irreversible decreases of fluorescence lifetime values. In summary, our study of FLIM imaging of GFP fusion proteins has provided a sensitive and real-time method to investigate RNA synthesis in live cell nuclei.

  20. Local elastic modulus of RF sputtered HfO{sub 2} thin film by atomic force acoustic microscopy

    SciTech Connect

    Jena, S. Tokas, R. B. Sarkar, P. Thakur, S.; Sahoo, N. K.; Misal, J. S.; Rao, K. D.

    2014-04-24

    Atomic force acoustic microscopy (AFAM) is a useful nondestructive technique for measurement of local elastic modulus of materials at nano-scale spatial resolution by measuring the contact resonance spectra for higher order modes of the AFM cantilever. The elastic modulus of RF sputtered HfO{sub 2} thin film has been measured quantitatively, using reference approach in which measurements are performed on the test and reference samples. Using AFAM, the measured elastic modulus of the HfO{sub 2} thin film is 223±27 GPa, which is in agreement with the literature value of 220±40 GPa for atomic layer deposited HfO{sub 2} thin film using nanoindentation technique.

  1. Motion picture imaging of a nanometer-thick liquid film dewetting by ellipsometric microscopy with a submicrometer lateral resolution.

    PubMed

    Fukuzawa, Kenji; Yoshida, Tomohiko; Itoh, Shintaro; Zhang, Hedong

    2008-10-21

    We visualized the detwetting of a nanometer-thick unstable liquid film on a nanotextured solid surface with a high lateral spatial resolution. The dewetting was imaged as a motion picture at a submicrometer spatial resolution and a frame rate of 4 frames/s, using ellipsometric microscopy in a vertical objective configuration. The observation revealed that the dewetting process significantly depends on the sign of the disjoining pressure Pi. When Pi is negative, the film rupture due to the spinodal dewetting proceeds to droplet formation in a single step, whereas, when Pi is positive, the film rupture due to the spinodal dewetting stops when the pressure of the thicker region balances with that of the thinner region, and then the heterogeneous grooves are nucleated and grow. The dewetting process dependence on the sign of Pi can be found in systems other than that reported here because the sign of Pi changes at the local maximum of the surface energy.

  2. Strongly compressed Bi (111) bilayer films on Bi{sub 2}Se{sub 3} studied by scanning tunneling microscopy

    SciTech Connect

    Zhang, K. F.; Yang, Fang; Song, Y. R.; Liu, Canhua; Qian, Dong; Gao, C. L.; Jia, Jin-Feng

    2015-09-21

    Ultra-thin Bi films show exotic electronic structure and novel quantum effects, especially the widely studied Bi (111) film. Using reflection high-energy electron diffraction and scanning tunneling microscopy, we studied the structure and morphology evolution of Bi (111) thin films grown on Bi{sub 2}Se{sub 3}. A strongly compressed, but quickly released in-plane lattice of Bi (111) is found in the first three bilayers. The first bilayer of Bi shows a fractal growth mode with flat surface, while the second and third bilayer show a periodic buckling due to the strong compression of the in-plane lattice. The lattice slowly changes to its bulk value with further deposition of Bi.

  3. Time-domain fluorescence lifetime imaging microscopy: a quantitative method to follow transient protein-protein interactions in living cells.

    PubMed

    Padilla-Parra, Sergi; Audugé, Nicolas; Tramier, Marc; Coppey-Moisan, Maïté

    2015-06-01

    Quantitative analysis in Förster resonance energy transfer (FRET) imaging studies of protein-protein interactions within live cells is still a challenging issue. Many cellular biology applications aim at the determination of the space and time variations of the relative amount of interacting fluorescently tagged proteins occurring in cells. This relevant quantitative parameter can be, at least partially, obtained at a pixel-level resolution by using fluorescence lifetime imaging microscopy (FLIM). Indeed, fluorescence decay analysis of a two-component system (FRET and no FRET donor species), leads to the intrinsic FRET efficiency value (E) and the fraction of the donor-tagged protein that undergoes FRET (fD). To simultaneously obtain fD and E values from a two-exponential fit, data must be acquired with a high number of photons, so that the statistics are robust enough to reduce fitting ambiguities. This is a time-consuming procedure. However, when fast-FLIM acquisitions are used to monitor dynamic changes in protein-protein interactions at high spatial and temporal resolutions in living cells, photon statistics and time resolution are limited. In this case, fitting procedures are unreliable, even for single lifetime donors. We introduce the concept of a minimal fraction of donor molecules involved in FRET (mfD), obtained from the mathematical minimization of fD. Here, we discuss different FLIM techniques and the compromises that must be made between precision and time invested in acquiring FLIM measurements. We show that mfD constitutes an interesting quantitative parameter for fast FLIM because it gives quantitative information about transient interactions in live cells.

  4. Mapping Cd²⁺-induced membrane permeability changes of single live cells by means of scanning electrochemical microscopy.

    PubMed

    Filice, Fraser P; Li, Michelle S M; Henderson, Jeffrey D; Ding, Zhifeng

    2016-02-18

    Scanning Electrochemical Microscopy (SECM) is a powerful, non-invasive, analytical methodology that can be used to investigate live cell membrane permeability. Depth scan SECM imaging allowed for the generation of 2D current maps of live cells relative to electrode position in the x-z or y-z plane. Depending on resolution, one depth scan image can contain hundreds of probe approach curves (PACs). Individual PACs were obtained by simply extracting vertical cross-sections from the 2D image. These experimental PACs were overlaid onto theoretically generated PACs simulated at specific geometry conditions. Simulations were carried out using 3D models in COMSOL Multiphysics to determine the cell membrane permeability coefficients at different locations on the surface of the cells. Common in literature, theoretical PACs are generated using a 2D axially symmetric geometry. This saves on both compute time and memory utilization. However, due to symmetry limitations of the model, only one experimental PAC right above the cell can be matched with simulated PAC data. Full 3D models in this article were developed for the SECM system of live cells, allowing all experimental PACs over the entire cell to become usable. Cd(2+)-induced membrane permeability changes of single human bladder (T24) cells were investigated at several positions above the cell, displaced from the central axis. The experimental T24 cells under study were incubated with Cd(2+) in varying concentrations. It is experimentally observed that 50 and 100 μM Cd(2+) caused a decrease in membrane permeability, which was uniform across all locations over the cell regardless of Cd(2+) concentration. The Cd(2+) was found to have detrimental effects on the cell, with cells shrinking in size and volume, and the membrane permeability decreasing. A mapping technique for the analysis of the cell membrane permeability under the Cd(2+) stress is realized by the methodology presented.

  5. Quantitative Spatiotemporal Chemical Profiling of Individual Lipid Droplets by Hyperspectral CARS Microscopy in Living Human Adipose-Derived Stem Cells.

    PubMed

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

    2016-04-05

    There is increasing evidence showing that cytosolic lipid droplets, present in all eukaryotic cells, play a key role in many cellular functions. Yet their composition at the individual droplet level and how it evolves over time in living cells is essentially unknown due to the lack of suitable quantitative nondestructive measurement techniques. In this work, we demonstrate the ability of label-free hyperspectral coherent anti-Stokes Raman scattering (CARS) microscopy, together with a quantitative image analysis algorithm developed by us, to quantify the lipid type and content in vol/vol concentration units of individual lipid droplets in living human adipose-derived stem cells during differentiation over 9 days in media supplemented with different fatty acids. Specifically, we investigated the addition of the polyunsaturated linoleic and alpha-linolenic fatty acids into the normal differentiation medium (mostly containing monounsaturated fatty acids). We observe a heterogeneous uptake which is droplet-size dependent, time dependent, and lipid dependent. Cells grown in linoleic-acid-supplemented medium show the largest distribution of lipid content across different droplets at all times during differentiation. When analyzing the average lipid content, we find that adding linoleic or alpha-linolenic fatty acids at day 0 results in uptake of the new lipid components with an exponential time constant of 22 ± 2 h. Conversely, switching lipids at day 3 results in an exponential time constant of 60 ± 5 h. These are unprecedented findings, exemplifying that the quantitative imaging method demonstrated here could open a radically new way of studying and understanding cytosolic lipid droplets in living cells.

  6. Potential and limitations of microscopy and Raman spectroscopy for live-cell analysis of 3D cell cultures.

    PubMed

    Charwat, Verena; Schütze, Karin; Holnthoner, Wolfgang; Lavrentieva, Antonina; Gangnus, Rainer; Hofbauer, Pablo; Hoffmann, Claudia; Angres, Brigitte; Kasper, Cornelia

    2015-07-10

    Today highly complex 3D cell culture formats that closely mimic the in vivo situation are increasingly available. Despite their wide use, the development of analytical methods and tools that can work within the depth of 3D-tissue constructs lags behind. In order to get the most information from a 3D cell sample, adequate and reliable assays are required. However, the majority of tools and methods used today have been originally designed for 2D cell cultures and translation to a 3D environment is in general not trivial. Ideally, an analytical method should be non-invasive and allow for repeated observation of living cells in order to detect dynamic changes in individual cells within the 3D cell culture. Although well-established laser confocal microscopy can be used for these purposes, this technique has serious limitations including penetration depth and availability. Focusing on two relevant analytical methods for live-cell monitoring, we discuss the current challenges of analyzing living 3D samples: microscopy, which is the most widely used technology to observe and examine cell cultures, has been successfully adapted for 3D samples by recording of so-called "z-stacks". However the required equipment is generally very expensive and therefore access is often limited. Consequently alternative and less advanced approaches are often applied that cannot capture the full structural complexity of a 3D sample. Similarly, image analysis tools for quantification of microscopic images range from highly specialized and costly to simplified and inexpensive. Depending on the actual sample composition and scientific question the best approach needs to be assessed individually. Another more recently introduced technology for non-invasive cell analysis is Raman micro-spectroscopy. It enables label-free identification of cellular metabolic changes with high sensitivity and has already been successful applied to 2D and 3D cell cultures. However, its future significance for cell

  7. Combined atomic force microscopy and spectroscopic ellipsometry applied to the analysis of lipid-protein thin films.

    PubMed

    Finot, Eric; Markey, Laurent; Hane, Francis; Amrein, Mathias; Leonenko, Zoya

    2013-04-01

    Pulmonary surfactant is a complex mixture of phospholipids and proteins and forms a thin film at the lung alveolar interface separating air from liquid environment. The film reduces the work of breathing during repeatable compressions of the alveoli which form a characteristic multilayer upon compression. In this work, we investigated the structure of bovine lipid extract surfactant (BLES). We analysed the BLES films by atomic force microscopy (AFM) and spectroscopic ellipsometry (SE) in order to provide combined characterization of both morphology and thickness of surfactant films. We show how the spectroscopic ellipsometry can be used to supplement the data obtained by AFM. We demonstrate that indium tin oxide (ITO) substrate used for spectroscopic ellipsometry is preferable over glass substrate to enhance the optical contrast. An optical model was proposed to account for non-uniform film morphology. We obtained good correlations between the multilayer surface coverage, determined by both AFM and SE. SE measures the thickness of the first uniform monolayer as 2.6 nm that cannot be achieved by AFM imaging alone.

  8. In-situ transmission electron microscopy crystallization studies of sol-gel-derived barium titanate thin films

    SciTech Connect

    Gust, M.C.; Mecartney, M.L.; Evans, N.D.; Momoda, L.A.

    1997-11-01

    Barium titanate (BaTiO{sub 3}) thin films that were derived from methoxypropoxide precursors were deposited onto (100) Si, Pt/Ti/SiO{sub 2}/(100) Si, and molecular-beam-epitaxy-grown (MBE-grown) (100) BaTiO{sub 3} on (100) Si substrates by spin coating. The crystallization behavior of the amorphous-gel films was characterized using in-situ transmission electron microscopy heating experiments, glancing-angle X-ray diffraction, and differential thermal analysis/thermogravimetric analysis. Amorphous-gel films crystallized at a temperature of {approximately}600 C to an intermediate nanoscale (5--10 nm) barium titanium carbonate phase, presumably BaTiO{sub 2}CO{sub 3}, that subsequently transformed to nanocrystalline (20--50 nm) BaTiO{sub 3}. Random nucleation in the bulk of the gel film was observed on all substrates. In addition, oriented growth of BaTiO{sub 3} was concurrently observed on MBE-grown BaTiO{sub 3} on (100) Si. High-temperature decomposition of the intermediate carbonate phase contributed to nanometer-scale residual porosity in the films. High concentrations of water of hydrolysis inhibited the formation of the intermediate carbonate phase; however, these sols precipitated and were not suitable for spin coating.

  9. Preliminary indications from atomic force microscopy of the presence of rapidly-formed nanoscale films on aquifer material surfaces.

    PubMed

    Gaebel, Claudia; Lead, Jamie R; Renshaw, Joanna C; Tellam, John H

    2009-08-11

    The objective of this study was to determine if there is a nanoscale surface film on aquifer-like materials exposed to deep groundwaters, as has previously been found on surfaces exposed to surface and soil waters. Such surface films will modify surface properties that are so important in determining the mobility of many groundwater pollutants. Muscovite mica was used because a) it is a good analogue for the main sorbing phases of many clastic aquifers and b) its cleavage planes are atomically flat allowing high resolution imaging. Freshly-cleaved muscovite plates were exposed to groundwater from a sandstone aquifer for 30 min, and surface properties (morphology, coverage, roughness and tip-substrate force interactions) were measured using atomic force microscopy (AFM). A patchy surface film of several nanometres in depth, incorporating larger separate particles, was found on the mica surface. This film was associated with significantly increased roughness values and AFM probe-sample interaction forces compared with pure water and inorganic (synthetic groundwater) solution controls. Although the results reported are preliminary in nature, if confirmed, such films are likely to affect sorption reactions, surface-facilitated redox interactions, non-aqueous phase liquid wetting angles, and colloid-pathogen-rock attachment, and will thus be of importance in understanding natural attenuation and migration of dissolved, non-aqueous and particulate phases in groundwaters.

  10. Preliminary indications from atomic force microscopy of the presence of rapidly-formed nanoscale films on aquifer material surfaces

    NASA Astrophysics Data System (ADS)

    Gaebel, Claudia; Lead, Jamie R.; Renshaw, Joanna C.; Tellam, John H.

    2009-08-01

    The objective of this study was to determine if there is a nanoscale surface film on aquifer-like materials exposed to deep groundwaters, as has previously been found on surfaces exposed to surface and soil waters. Such surface films will modify surface properties that are so important in determining the mobility of many groundwater pollutants. Muscovite mica was used because a) it is a good analogue for the main sorbing phases of many clastic aquifers and b) its cleavage planes are atomically flat allowing high resolution imaging. Freshly-cleaved muscovite plates were exposed to groundwater from a sandstone aquifer for 30 min, and surface properties (morphology, coverage, roughness and tip-substrate force interactions) were measured using atomic force microscopy (AFM). A patchy surface film of several nanometres in depth, incorporating larger separate particles, was found on the mica surface. This film was associated with significantly increased roughness values and AFM probe-sample interaction forces compared with pure water and inorganic (synthetic groundwater) solution controls. Although the results reported are preliminary in nature, if confirmed, such films are likely to affect sorption reactions, surface-facilitated redox interactions, non-aqueous phase liquid wetting angles, and colloid-pathogen-rock attachment, and will thus be of importance in understanding natural attenuation and migration of dissolved, non-aqueous and particulate phases in groundwaters.

  11. Imaging Live Cells at the Nanometer-Scale with Single-Molecule Microscopy: Obstacles and Achievements in Experiment Optimization for Microbiology

    PubMed Central

    Haas, Beth L.; Matson, Jyl S.; DiRita, Victor J.; Biteen, Julie S.

    2015-01-01

    Single-molecule fluorescence microscopy enables biological investigations inside living cells to achieve millisecond- and nanometer-scale resolution. Although single-molecule-based methods are becoming increasingly accessible to non-experts, optimizing new single-molecule experiments can be challenging, in particular when super-resolution imaging and tracking are applied to live cells. In this review, we summarize common obstacles to live-cell single-molecule microscopy and describe the methods we have developed and applied to overcome these challenges in live bacteria. We examine the choice of fluorophore and labeling scheme, approaches to achieving single-molecule levels of fluorescence, considerations for maintaining cell viability, and strategies for detecting single-molecule signals in the presence of noise and sample drift. We also discuss methods for analyzing single-molecule trajectories and the challenges presented by the finite size of a bacterial cell and the curvature of the bacterial membrane. PMID:25123183

  12. Imaging of Dynamic Secretory Vesicles in Living Pollen Tubes of Picea meyeri Using Evanescent Wave Microscopy1[W

    PubMed Central

    Wang, Xiaohua; Teng, Yan; Wang, Qinli; Li, Xiaojuan; Sheng, Xianyong; Zheng, Maozhong; Šamaj, Jozef; Baluška, František; Lin, Jinxing

    2006-01-01

    Evanescent wave excitation was used to visualize individual, FM4-64-labeled secretory vesicles in an optical slice proximal to the plasma membrane of Picea meyeri pollen tubes. A standard upright microscope was modified to accommodate the optics used to direct a laser beam at a variable angle. Under evanescent wave microscopy or total internal reflection fluorescence microscopy, fluorophores localized near the surface were excited with evanescent waves, which decay exponentially with distance from the interface. Evanescent waves with penetration depths of 60 to 400 nm were generated by varying the angle of incidence of the laser beam. Kinetic analysis of vesicle trafficking was made through an approximately 300-nm optical section beneath the plasma membrane using time-lapse evanescent wave imaging of individual fluorescently labeled vesicles. Two-dimensional trajectories of individual vesicles were obtained from the resulting time-resolved image stacks and were used to characterize the vesicles in terms of their average fluorescence and mobility, expressed here as the two-dimensional diffusion coefficient D2. The velocity and direction of vesicle motions, frame-to-frame displacement, and vesicle trajectories were also calculated. Analysis of individual vesicles revealed for the first time, to our knowledge, that two types of motion are present, and that vesicles in living pollen tubes exhibit complicated behaviors and oscillations that differ from the simple Brownian motion reported in previous investigations. Furthermore, disruption of the actin cytoskeleton had a much more pronounced effect on vesicle mobility than did disruption of the microtubules, suggesting that actin cytoskeleton plays a primary role in vesicle mobility. PMID:16798949

  13. Imaging the uptake of gold nanoshells in live cells using plasmon resonance enhanced four wave mixing microscopy.

    PubMed

    Garrett, Natalie; Whiteman, Matt; Moger, Julian

    2011-08-29

    Gold nanoshells (GNS) are novel metal nanoparticles exhibiting attractive optical properties which make them highly suitable for biophotonics applications. We present a novel investigation using plasmon-enhanced four wave mixing microscopy combined with coherent anti-Stokes Raman scattering (CARS) microscopy to visualize the distribution of 75 nm radius GNS within live cells. During a laser tolerance study we found that cells containing nanoshells could be exposed to < 2.5 mJ each with no photo-thermally induced necrosis detected, while cell death was linearly proportional to the power over this threshold. The majority of the GNS signal detected was from plasmon-enhanced four wave mixing (FWM) that we detected in the epi-direction with the incident lasers tuned to the silent region of the Raman spectrum. The cellular GNS distribution was visualized by combining the epi-detected signal with forwards-detected CARS at the CH2 resonance. The applicability of this technique to real-world nanoparticle dosing problems was demonstrated in a study of the effect of H2S on nanoshell uptake using two donor molecules, NaHS and GYY4137. As GYY4137 concentration was increased from 10 µM to 1 mM, the nanoshell pixel percentage as a function of cell volume (PPCV) increased from 2.15% to 3.77%. As NaHS concentration was increased over the same range, the nanoshell PPCV decreased from 12.67% to 11.47%. The most important factor affecting uptake in this study was found to be the rate of H2S release, with rapid-release from NaHS resulting in significantly greater uptake.

  14. pH and chloride recordings in living cells using two-photon fluorescence lifetime imaging microscopy

    NASA Astrophysics Data System (ADS)

    Lahn, Mattes; Hille, Carsten; Koberling, Felix; Kapusta, Peter; Dosche, Carsten

    2010-02-01

    Today fluorescence lifetime imaging microscopy (FLIM) has become an extremely powerful technique in life sciences. The independency of the fluorescence decay time on fluorescence dye concentration and emission intensity circumvents many artefacts arising from intensity based measurements. To minimize cell damage and improve scan depth, a combination with two-photon (2P) excitation is quite promising. Here, we describe the implementation of a 2P-FLIM setup for biological applications. For that we used a commercial fluorescence lifetime microscope system. 2P-excitation at 780nm was achieved by a non-tuneable, but inexpensive and easily manageable mode-locked fs-fiber laser. Time-resolved fluorescence image acquisition was performed by objective-scanning with the reversed time-correlated single photon counting (TCSPC) technique. We analyzed the suitability of the pH-sensitive dye BCECF and the chloride-sensitive dye MQAE for recordings in an insect tissue. Both parameters are quite important, since they affect a plethora of physiological processes in living tissues. We performed a straight forward in situ calibration method to link the fluorescence decay time with the respective ion concentration and carried out spatially resolved measurements under resting conditions. BCECF still offered only a limited dynamic range regarding fluorescence decay time changes under physiologically pH values. However, MQAE proofed to be well suited to record chloride concentrations in the physiologically relevant range. Subsequently, several chloride transport pathways underlying the intracellular chloride homeostasis were investigated pharmacologically. In conclusion, 2P-FLIM is well suited for ion detection in living tissues due to precise and reproducible decay time measurements in combination with reduced cell and dye damages.

  15. Development of quantitative microscopy-based assays for evaluating dynamics of living cultures of mouse spermatogonial stem/progenitor cells.

    PubMed

    Heim, Crystal N; Fanslow, Danielle A; Dann, Christina Tenenhaus

    2012-10-01

    Spermatogonial stem cell (SSC) self-renewal and differentiation are required for continuous production of spermatozoa and long-term fertility. Studying SSCs in vivo remains challenging because SSCs are rare cells and definitive molecular markers for their identification are lacking. The development of a method for propagating SSCs in vitro greatly facilitated analysis of SSCs. The cultured cells grow as clusters of a dynamic mixture of "true" stem cells and differentiating progenitor cells. Cells in the stem/progenitor culture system share many properties with spermatogonia in vivo; however, to fully exploit it as a model for spermatogonial development, new assays are needed that account for the dynamic heterogeneity inherent in the culture system. Here, assays were developed for quantifying dynamics of cultures of stem/progenitor cells that expressed histone-green fluorescent protein (GFP). First, we built on published results showing that cluster formation in vitro reliably predicts the relative number of SSCs. The GFP-based in vitro cluster assay allows quantification of SSCs with significantly fewer resources than a transplantation assay. Second, we compared the dynamics of differentiation in two experimental paradigms by imaging over a 17-day time frame. Finally, we performed short-term live imaging and observed cell migration, coordinated cell proliferation, and cell death resembling that of spermatogonia in the testes. The methods that we present provide a foundation for the use of fluorescent reporters in future microscopy-based high-throughput screens by using living spermatogonial stem/progenitor cultures applicable to toxicology, contraceptive discovery, and identification of regulators of self-renewal and differentiation.

  16. Molecular organization of cytokinesis nodes and contractile rings by super-resolution fluorescence microscopy of live fission yeast

    PubMed Central

    Laplante, Caroline; Huang, Fang; Tebbs, Irene R.; Bewersdorf, Joerg; Pollard, Thomas D.

    2016-01-01

    Cytokinesis in animals, fungi, and amoebas depends on the constriction of a contractile ring built from a common set of conserved proteins. Many fundamental questions remain about how these proteins organize to generate the necessary tension for cytokinesis. Using quantitative high-speed fluorescence photoactivation localization microscopy (FPALM), we probed this question in live fission yeast cells at unprecedented resolution. We show that nodes, protein assembly precursors to the contractile ring, are discrete structural units with stoichiometric ratios and distinct distributions of constituent proteins. Anillin Mid1p, Fes/CIP4 homology-Bin/amphiphysin/Rvs (F-BAR) Cdc15p, IQ motif containing GTPase-activating protein (IQGAP) Rng2p, and formin Cdc12p form the base of the node that anchors the ends of myosin II tails to the plasma membrane, with myosin II heads extending into the cytoplasm. This general node organization persists in the contractile ring where nodes move bidirectionally during constriction. We observed the dynamics of the actin network during cytokinesis, starting with the extension of short actin strands from nodes, which sometimes connected neighboring nodes. Later in cytokinesis, a broad network of thick bundles coalesced into a tight ring around the equator of the cell. The actin ring was ∼125 nm wide and ∼125 nm thick. These observations establish the organization of the proteins in the functional units of a cytokinetic contractile ring. PMID:27647921

  17. Dissecting the actin cortex density and membrane-cortex distance in living cells by super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Clausen, M. P.; Colin-York, H.; Schneider, F.; Eggeling, C.; Fritzsche, M.

    2017-02-01

    Nanoscale spacing between the plasma membrane and the underlying cortical actin cytoskeleton profoundly modulates cellular morphology, mechanics, and function. Measuring this distance has been a key challenge in cell biology. Current methods for dissecting the nanoscale spacing either limit themselves to complex survey design using fixed samples or rely on diffraction-limited fluorescence imaging whose spatial resolution is insufficient to quantify distances on the nanoscale. Using dual-color super-resolution STED (stimulated-emission-depletion) microscopy, we here overcome this challenge and accurately measure the density distribution of the cortical actin cytoskeleton and the distance between the actin cortex and the membrane in live Jurkat T-cells. We found an asymmetric cortical actin density distribution with a mean width of 230 (+105/-125) nm. The spatial distances measured between the maximum density peaks of the cortex and the membrane were bi-modally distributed with mean values of 50  ±  15 nm and 120  ±  40 nm, respectively. Taken together with the finite width of the cortex, our results suggest that in some regions the cortical actin is closer than 10 nm to the membrane and a maximum of 20 nm in others.

  18. An atomic force microscopy study of DNA hairpin probes monolabelled with gold nanoparticle: Grafting and hybridization on oxide thin films

    NASA Astrophysics Data System (ADS)

    Lavalley, V.; Chaudouët, P.; Stambouli, V.

    2007-12-01

    First and original results are reported regarding the surface evolution of two kinds of oxide film after covalent grafting and hybridization of hairpin oligonucleotide probes. These hairpin probes were monolabelled with a 1.4 nm gold nanoparticle. One kind of oxide film was rough Sb doped SnO 2 oxide film and the other kind was smooth SiO 2 film. Same process of covalent grafting, involving a silanization step, was performed on both oxide surfaces. Atomic force microscopy (AFM) was used to study the evolution of each oxide surface after different steps of the process: functionalization, probe grafting and hybridization. In the case of rough SnO 2 films, a slight decrease of the roughness was observed after each step whereas in the case of smooth SiO 2 films, a maximum of roughness was obtained after probe grafting. Step height measurements of grafted probes could be performed on SiO 2 leading to an apparent thickness of around 3.7 ± 1.0 nm. After hybridization, on the granular surface of SnO 2, by coupling AFM with SEM FEG analyses, dispersed and well-resolved groups of gold nanoparticles linked to DNA duplexes could be observed. Their density varied from 6.6 ± 0.3 × 10 10 to 2.3 ± 0.3 × 10 11 dots cm -2. On the contrary, on smooth SiO 2 surface, the DNA duplexes behave like a dense carpet of globular structures with a density of 2.9 ± 0.5 × 10 11 globular structures cm -2.

  19. Magnetic domain imaging of nano-magnetic films using magnetic force microscopy with polar and longitudinally magnetized tips.

    PubMed

    Chen, Sy-Hann; Chang, Yu-Hsiang; Su, Chiung-Wu; Tsay, Jyh-Shen

    2016-10-01

    Perpendicular or parallel magnetic fields are used to magnetize the tips used in magnetic force microscopy (MFM). In this process, perpendicular or parallel magnetic dipole moments are produced on the tip plane, thus leading to the formation of polar magnetized tips (PM-tips) or longitudinally magnetized tips (LM-tips), respectively. The resolution of an MFM image of a magneto-optic disk is used for calibration of these tips, and the saturated magnetic fields of the PM- and LM-tips are found to be 2720 Oe and 680 Oe, respectively. Because both tips can simultaneously magnetize the sample during the scanning process when measuring a Co thin film, clear MFM images are captured, which enable the identification of magnetizable regions and the distribution of the magnetic domains on the sample surface. These results will be useful for improving the manufacturing processes required for soft nano-magnetic film production.

  20. Retrieving spin textures on curved magnetic thin films with full-field soft X-ray microscopies

    DOE PAGES

    Streubel, Robert; Kronast, Florian; Fischer, Peter; ...

    2015-07-03

    X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue—magnetic X-ray tomography—is yet to be developed. We demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. In the 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. By using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomenamore » between windings of azimuthally and radially magnetized tubular objects. In conclusion, the present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape.« less

  1. Retrieving spin textures on curved magnetic thin films with full-field soft X-ray microscopies

    SciTech Connect

    Streubel, Robert; Kronast, Florian; Fischer, Peter; Parkinson, Dula; Schmidt, Oliver G.; Makarov, Denys

    2015-07-03

    X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue—magnetic X-ray tomography—is yet to be developed. We demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. In the 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. By using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomena between windings of azimuthally and radially magnetized tubular objects. In conclusion, the present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape.

  2. Retrieving spin textures on curved magnetic thin films with full-field soft X-ray microscopies

    PubMed Central

    Streubel, Robert; Kronast, Florian; Fischer, Peter; Parkinson, Dula; Schmidt, Oliver G.; Makarov, Denys

    2015-01-01

    X-ray tomography is a well-established technique to characterize 3D structures in material sciences and biology; its magnetic analogue—magnetic X-ray tomography—is yet to be developed. Here we demonstrate the visualization and reconstruction of magnetic domain structures in a 3D curved magnetic thin films with tubular shape by means of full-field soft X-ray microscopies. The 3D arrangement of the magnetization is retrieved from a set of 2D projections by analysing the evolution of the magnetic contrast with varying projection angle. Using reconstruction algorithms to analyse the angular evolution of 2D projections provides quantitative information about domain patterns and magnetic coupling phenomena between windings of azimuthally and radially magnetized tubular objects. The present approach represents a first milestone towards visualizing magnetization textures of 3D curved thin films with virtually arbitrary shape. PMID:26139445

  3. High resolution transmission electron microscopy characterization of fcc --> 9R transformation in nanocrystalline palladium films due to hydriding

    NASA Astrophysics Data System (ADS)

    Amin-Ahmadi, Behnam; Idrissi, Hosni; Delmelle, Renaud; Pardoen, Thomas; Proost, Joris; Schryvers, Dominique

    2013-02-01

    Sputtered nanocrystalline palladium thin films with nanoscale growth twins have been subjected to hydriding cycles. The evolution of the twin boundaries has been investigated using high resolution transmission electron microscopy. Surprisingly, the ∑3{112} incoherent twin boundaries dissociate after hydriding into two phase boundaries bounding a 9R phase. This phase which corresponds to single stacking faults located every three {111} planes in the fcc Pd structure was not expected because of the high stacking fault energy of Pd. This observation is connected to the influence of the Hydrogen on the stacking fault energy of palladium and the high compressive stresses building up during hydriding.

  4. Characterization of durable nanostructured thin film catalysts tested under transient conditions using analytical aberration-corrected electron microscopy

    SciTech Connect

    Cullen, David A; More, Karren Leslie; Reeves, Kimberly Shawn; Vernstrom, George; Atanasoska, Liliana; Haugen, Gregory; Atanasoski, Radoslav

    2011-01-01

    The stability of Ru0.1Ir0.9 oxidation evolution reaction (OER) catalysts deposited on Pt-coated nanostructured thin films (NSTFs) has been investigated by aberration-corrected electron microscopy. Accelerated stress tests showed that the OER catalysts significantly improved the durability of the Pt under cell reversal conditions. High-resolution images of the end-of-life NSTFs showed significant Ir loss from the whisker surfaces, while no Pt loss was observed, indicating that the OER catalysts had protected the catalyst coated whisker surfaces from degradation.

  5. Growth analysis of cadmium sulfide thin films by atomic force microscopy

    SciTech Connect

    Moutinho, H.R.; Dhere, R.G.; Ramanathan, K.

    1996-05-01

    CdS films have been deposited by solution growth on SnO{sub 2} and glass substrates. Nucleation on SnO{sub 2} occurs at early deposition times, and complete conformal coverage is observed at low thickness values. The average grain size of the CdS films is established at these early times. In films deposited on glass substrates, nucleation is slower and occurs through 3-dimensional islands that increase in size and number as deposition proceeds. Optical measurements show that the bandgap values of CdS films deposited on SnO{sub 2} depend mainly on substrate structure. Hydrogen heat treatment does not affect the surface morphology of the samples, but decreases bandgap values.

  6. Ex utero: live human fetal research and the films of Davenport Hooker.

    PubMed

    Wilson, Emily K

    2014-01-01

    Between 1932 and 1963 University of Pittsburgh anatomist Davenport Hooker, Ph.D., performed and filmed noninvasive studies of reflexive movement on more than 150 surgically aborted human fetuses. The resulting imagery and information would contribute substantially to new visual and biomedical conceptions of fetuses as baby-like, autonomous human entities that emerged in the 1960s and 1970s. Hooker's methods, though broadly conforming to contemporary research practices and views of fetuses, would not have been feasible later. But while Hooker and the 1930s medical and general public viewed live fetuses as acceptable materials for nontherapeutic research, they also shared a regard for fetuses as developing humans with some degree of social value. Hooker's research and the various reactions to his work demonstrate the varied and changing perspectives on fetuses and fetal experimentation, and the influence those views can have on biomedical research.

  7. In situ oxygen conditioning of /001/ MgO thin film substrates for film growth studies by electron microscopy

    NASA Technical Reports Server (NTRS)

    Moorhead, R. D.; Poppa, H.

    1979-01-01

    It was found that the in situ treatment of 001-plane single-crystal films of MgO (prepared by epitaxial growth from the vapor phase) at high temperatures with a jet of oxygen will produce a surface that is almost equivalent, for epitaxial studies, to surfaces with the same orientation prepared by vacuum cleavage of bulk single crystals. The effectiveness of the process is demonstrated by its impact on the epitaxy of silver.

  8. Femtosecond laser printing of living cells using absorbing film-assisted laser-induced forward transfer

    NASA Astrophysics Data System (ADS)

    Hopp, Béla; Smausz, Tomi; Szabó, Gábor; Kolozsvári, Lajos; Kafetzopoulos, Dimitris; Fotakis, Costas; Nógrádi, Antal

    2012-01-01

    The applicability of a femtosecond KrF laser in absorbing film-assisted, laser-induced forward transfer of living cells was studied. The absorbing materials were 50-nm-thick metal films and biomaterials (gelatine, Matrigel, each 50 μm thick, and polyhydroxybutyrate, 2 μm). The used cell types were human neuroblastoma, chronic myeloid leukemia, and osteogenic sarcoma cell lines, and primary astroglial rat cells. Pulses of a 500-fs KrF excimer laser focused onto the absorbing layer in a 250-μm diameter spot with 225 mJ/cm2 fluence were used to transfer the cells to the acceptor plate placed at 0.6 mm distance, which was a glass slide either pure or covered with biomaterials. While the low-absorptivity biomaterial absorbing layers proved to be ineffective in transfer of cells, when applied on the surface of acceptor plate, the wet gelatine and Matrigel layers successfully ameliorated the impact of the cells, which otherwise did not survive the arrival onto a hard surface. The best short- and long-term survival rate was between 65% and 70% for neuroblastoma and astroglial cells. The long-term survival of the transferred osteosarcoma cells was low, while the myeloid leukemia cells did not tolerate the procedure under the applied experimental conditions.

  9. Local Imaging of Optoelectronic Properties and Film Degradation in Polymer/Fullerene Solar Cells with Electrostatic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Cox, Phillip Alexander

    With power conversion efficiencies on the rise, organic photovoltaics (OPVs) hold promise as a next-generation thin-film solar technology. However, both device performance and stability are inextricably linked to local film structure. Methods capable of probing nanoscale electronic properties as a function of film structure are thus a crucial component of the rational design of efficient and robust devices. This dissertation describes the use of three scanning probe methods for studying local charge generation and photodegradation in polymer/fullerene solar cells. First, we show that time-resolved electrostatic force microscopy (trEFM) is capable of resolving local photocurrent from sub-bandgap excitation down to attoampere level currents, a result unattainable by traditional contact-mode methods. We find that the local charging rates measured with trEFM are proportional to external quantum efficiency (EQE) measurements made on completed devices, making trEFM images equivalent to local EQE maps across the entire solar spectrum. For both phase-segregated and well-mixed MDMO-PPV:PCBM film morphologies, we show that the local distribution of photocurrent is invariant to excitation wavelength, providing local evidence for the controversial result that the probability of generating separated charge carriers does not depend on whether excitons are formed at the singlet state or charge transfer state. Next, we describe how local dissipation imaging can be performed with commercially-available frequency-modulated electrostatic force microscopy (FM-EFM) and show that dissipation maps are highly sensitive to photo-oxidative effects in organic semiconductors. We show that photo-oxidation induced changes in cantilever energy dissipation are proportional to device performance losses. We further develop dissipation imaging by implementing ringdown imaging, which directly measures the quality factor of the cantilever, enabling quantitative dissipation mapping. Using organic

  10. Rh-V alloy formation in Rh-VOx thin films after high-temperature reduction studied by electron microscopy.

    PubMed

    Penner, S; Jenewein, B; Wang, D; Schlögl, R; Hayek, K

    2006-03-14

    Rh nanoparticles (mean size 10 and 15 nm), prepared by epitaxial growth on NaCl surfaces, were covered with layers of crystalline vanadium oxide (mean thickness 1.5 and 25 nm) by reactive deposition in 10(-2) mbar O2. The 1.5 nm film was further stabilized with a coating layer of 25 nm amorphous alumina. The so-obtained Rh/vanadia films, containing vanadium in the V3+ and V2+ state, were treated in 1 bar O2 at 673 K for 1 h and thereafter reduced in 1 bar H2 at increased temperatures, particularly between 723 and 873 K. The structural and morphological changes were followed by (high-resolution) transmission electron microscopy and selected area diffraction. Oxidation at 673 K transforms the purely vanadia-supported samples into Rh/V2O5, while in the alumina-supported films containing only small amounts of VOx, the formation of topotactic V2O3 is observed. The formation of Rh-V alloys during the subsequent reduction is strongly determined by the intimate contact and the structural and orientational relationship between Rh particles and the surrounding VOx phase. Reduction above 473 K transforms the support into substoichiometric vanadium oxides of composition VO and V2O. Analysis of high-resolution images and diffraction patterns reveals the presence of different alloy phases after reduction with increasing T (from 573 up to 823 K). In the alumina-supported film (low V/Rh ratio) the epitaxial alignment between the Rh particles and the surrounding V2O3 phase apparently favours the primary formation of defined alloys of type V3Rh and VRh3, followed by VRh at higher temperature. On the contrary, mainly V3Rh5 is formed in the purely VOx-supported Rh/films, due to different epitaxial relations in the initial state. Possible pathways of alloy formation are discussed.

  11. Probing the viscoelastic response of glassy polymer films using atomic force microscopy.

    PubMed

    Yang, Guanwen; Rao, Nanxia; Yin, Zejie; Zhu, Da-Ming

    2006-05-01

    The mechanical properties of glassy films and glass surfaces have been studied using an atomic force microscope (AFM) through various imaging modes and measuring methods. In this paper, we discuss the viscoelastic response of a glassy surface probed using an AFM. We analyzed the force-distance curves measured on a glassy film or a glassy surface at temperatures near the glass transition temperature, Tg, using a Burgers model. We found that the material's characteristics of reversible anelastic response and viscous creep can be extracted from a force-distance curve. Anelastic response shifts the repulsive force-distance curve while viscous creep strongly affects the slope of the repulsive force-distance curve. When coupled with capillary force, due to the condensation of a thin layer of liquid film at the tip-surface joint, the anelasticity and viscous creep can alter the curve significantly in the attractive region.

  12. Characterization of defect growth structures in ion plated films by scanning electron microscopy

    NASA Technical Reports Server (NTRS)

    Spalvins, T.

    1979-01-01

    Gold and copper films (0.2-2 micron thick) are ion plated on very smooth stainless steel 304 and mica surfaces. The deposited films are examined by SEM to identify the morphological growth of defects. Three types of coating defects are distinguished: nodular growth, abnormal or runaway growth, and spits. The potential nucleation sites for defect growth are analyzed to determine the cause of defect formation. It is found that nuclear growth is due to inherent surface microdefects, abnormal or runaway growth is due to external surface inclusions, and spits are due to nonuniform evaporation and ejection of droplets. All these defects have adverse effects on the coatings.

  13. Long-lived charge carrier generation in ordered films of a covalent perylenediimide–diketopyrrolopyrrole–perylenediimide molecule

    DOE PAGES

    Hartnett, Patrick E.; Dyar, Scott M.; Margulies, Eric A.; ...

    2015-07-31

    The photophysics of a covalently linked perylenediimide–diketopyrrolopyrrole–perylenediimide acceptor–donor–acceptor molecule (PDI–DPP–PDI, 1) were investigated and found to be markedly different in solution versus in unannealed and solvent annealed films. Photoexcitation of 1 in toluene results in quantitative charge separation in τ = 3.1 ± 0.2 ps, with charge recombination in τ = 340 ± 10 ps, while in unannealed/disordered films of 1, charge separation occurs in τ < 250 fs, while charge recombination displays a multiexponential decay in ~6 ns. The absence of long-lived, charge separation in the disordered film suggests that few free charge carriers are generated. In contrast, uponmore » CH₂Cl₂ vapor annealing films of 1, grazing-incidence X-ray scattering shows that the molecules form a more ordered structure. Photoexcitation of the ordered films results in initial formation of a spin-correlated radical ion pair (electron–hole pair) as indicated by magnetic field effects on the formation of free charge carriers which live for ~4 μs. This result has significant implications for the design of organic solar cells based on covalent donor–acceptor systems and shows that long-lived, charge-separated states can be achieved by controlling intramolecular charge separation dynamics in well-ordered systems.« less

  14. Long-lived charge carrier generation in ordered films of a covalent perylenediimide–diketopyrrolopyrrole–perylenediimide molecule

    SciTech Connect

    Hartnett, Patrick E.; Dyar, Scott M.; Margulies, Eric A.; Shoer, Leah E.; Cook, Andrew W.; Eaton, Samuel W.; Marks, Tobin J.; Wasielewski, Michael R.

    2015-07-31

    The photophysics of a covalently linked perylenediimide–diketopyrrolopyrrole–perylenediimide acceptor–donor–acceptor molecule (PDI–DPP–PDI, 1) were investigated and found to be markedly different in solution versus in unannealed and solvent annealed films. Photoexcitation of 1 in toluene results in quantitative charge separation in τ = 3.1 ± 0.2 ps, with charge recombination in τ = 340 ± 10 ps, while in unannealed/disordered films of 1, charge separation occurs in τ < 250 fs, while charge recombination displays a multiexponential decay in ~6 ns. The absence of long-lived, charge separation in the disordered film suggests that few free charge carriers are generated. In contrast, upon CH₂Cl₂ vapor annealing films of 1, grazing-incidence X-ray scattering shows that the molecules form a more ordered structure. Photoexcitation of the ordered films results in initial formation of a spin-correlated radical ion pair (electron–hole pair) as indicated by magnetic field effects on the formation of free charge carriers which live for ~4 μs. This result has significant implications for the design of organic solar cells based on covalent donor–acceptor systems and shows that long-lived, charge-separated states can be achieved by controlling intramolecular charge separation dynamics in well-ordered systems.

  15. Correlating Interfacial Structure and Magnetism in Thin-Film Oxide Heterostructures Using Transmission Electron Microscopy and Polarized Neutron Reflectometry

    NASA Astrophysics Data System (ADS)

    Spurgeon, Steven Richard

    Oxide thin-films have attracted considerable attention for a new generation of spintronics devices, where both electron charge and spin are used to transport information. However, a poor understanding of the local features that mediate magnetization and coupling in these materials has greatly limited their deployment into new information and communication technologies. This thesis describes direct, local measurements of structure-property relationships in ferrous thin-films and La1--xSrxMnO3 (LSMO) / Pb(ZrxTi1--x)O3 (PZT) thin-film heterostructures using spatially-resolved characterization techniques. In the first part of this thesis we explore the properties of ferrous spintronic thin-films. These films serve as a model system to establish a suite of interfacial characterization techniques for subsequent studies. We then study the static behavior of LSMO / PZT devices with polarization set by the underlying substrate. Using transmission electron microscopy and geometric phase analysis we reveal the presence of significant local strain gradients in these films for the first time. Electron energy loss spectroscopy mapping of the LSMO / PZT interface reveals Mn valence changes induced by charge-transfer screening. Bulk magnetometry and polarized neutron reflectometry indicate that these chemical and strain changes are associated with a graded magnetization across the LSMO layer. Density functional theory calculations are presented, which show that strain and charge-transfer screening act locally to suppress magnetization in the LSMO by changing the Mn orbital polarization. In the second half of this thesis, we explore asymmetric screening effects on magnetization LSMO / PZT composites. We find that the local ferroelectric polarization can vary widely and that this may be responsible for reduced charge-transfer effects, as well as magnetic phase gradients at interfaces. From this information and electron energy loss spectroscopy, we construct a map of the magnetic

  16. Polarized 3D Raman and nanoscale near-field optical microscopy of optically inscribed surface relief gratings: chromophore orientation in azo-doped polymer films.

    PubMed

    Di Florio, Giuseppe; Bründermann, Erik; Yadavalli, Nataraja Sekhar; Santer, Svetlana; Havenith, Martina

    2014-03-14

    We have used polarized confocal Raman microspectroscopy and scanning near-field optical microscopy with a resolution of 60 nm to characterize photoinscribed grating structures of azobenzene doped polymer films on a glass support. Polarized Raman microscopy allowed determining the reorientation of the chromophores as a function of the grating phase and penetration depth of the inscribing laser in three dimensions. We found periodic patterns, which are not restricted to the surface alone, but appear also well below the surface in the bulk of the material. Near-field optical microscopy with nanoscale resolution revealed lateral two-dimensional optical contrast, which is not observable by atomic force and Raman microscopy.

  17. Direct mapping of the electric permittivity of heterogeneous non-planar thin films at gigahertz frequencies by scanning microwave microscopy.

    PubMed

    Biagi, Maria Chiara; Badino, Giorgio; Fabregas, Rene; Gramse, Georg; Fumagalli, Laura; Gomila, Gabriel

    2017-02-01

    We obtained maps of electric permittivity at ∼19 GHz frequencies on non-planar thin film heterogeneous samples by means of combined atomic force-scanning microwave microscopy (AFM-SMM). We show that the electric permittivity maps can be obtained directly from the capacitance images acquired in contact mode, after removing the topographic cross-talk effects. This result demonstrates the possibility of identifying the electric permittivity of different materials in a thin film sample irrespectively of their thickness by just direct imaging and processing. We show, in addition, that quantitative maps of the electric permittivity can be obtained with no need for any theoretical calculation or complex quantification procedures when the electric permittivity of one of the materials is known. To achieve these results the use of contact mode imaging is a key factor. For non-contact imaging modes the effects of local sample thickness and of the imaging distance make the interpretation of the capacitance images in terms of the electric permittivity properties of the materials much more complex. The present results represent a substantial contribution to the field of nanoscale microwave dielectric characterization of thin film materials with important implications for the characterization of novel 3D electronic devices and 3D nanomaterials.

  18. Nuclear magnetic resonance force microscopy of (NH_4)_2SO4 crystal and PMMA thin film

    NASA Astrophysics Data System (ADS)

    Choi, Jae-Hyuk; Miller, Casey W.; Guchhait, Samaresh; Chabot, Michelle D.; Markert, John T.

    2004-03-01

    In preparation for scanning dynamical studies, we performed room temperature 1-D and 2-D nuclear magnetic resonance force microscopy (NMRFM) measurements on a (NH_4)_2SO4 (ammonium sulfate) single crystal and on PMMA thin films using `magnet-on-oscillator' scanning mode. A cantilever-frequency modulation of the ˜ 350-MHz rf field induced cyclic adiabatic inversion of a resonant slice of ^1H nuclear spins. 1-D scans through the surface of the ammonium sulfate crystal with a resonant-slice thickness (z resolution) as small as 150 nm were achieved using a permalloy thin-film magnet, 4.0 μm in diameter and 0.18 μm thick. For the 2D scans, ˜1 μm-thick PMMA films were patterned to provide a 2-D array of 5.0 μm-diameter polymer islands. Oscillator-detected NMR-induced forces around 1× 10-15 N were typical. These NMRFM imaging studies employed mechanical oscillators with sping constants k ≈ 2× 10-4 N/m, resonant frequencies near 4 kHz, and quality factors in the range Q = 1--6× 10^3.

  19. Spatial and temporal imaging of long-range charge transport in perovskite thin films by ultrafast microscopy

    PubMed Central

    Guo, Zhi; Manser, Joseph S.; Wan, Yan; Kamat, Prashant V.; Huang, Libai

    2015-01-01

    Charge carrier diffusion coefficient and length are important physical parameters for semiconducting materials. Long-range carrier diffusion in perovskite thin films has led to remarkable solar cell efficiencies; however, spatial and temporal mechanisms of charge transport remain unclear. Here we present a direct measurement of carrier transport in space and in time by mapping carrier density with simultaneous ultrafast time resolution and ∼50-nm spatial precision in perovskite thin films using transient absorption microscopy. These results directly visualize long-range carrier transport of ∼220 nm in 2 ns for solution-processed polycrystalline CH3NH3PbI3 thin films. Variations of the carrier diffusion coefficient at the μm length scale have been observed with values ranging between 0.05 and 0.08 cm2 s−1. The spatially and temporally resolved measurements reported here underscore the importance of the local morphology and establish an important first step towards discerning the underlying transport properties of perovskite materials. PMID:26101051

  20. Characterization of local dielectric breakdown in ultrathin SiO2 films using scanning tunneling microscopy and spectroscopy

    NASA Astrophysics Data System (ADS)

    Watanabe, Heiji; Baba, Toshio; Ichikawa, Masakazu

    1999-05-01

    Local dielectric breakdown of ultrathin SiO2 films grown on silicon substrates has been investigated by using scanning tunneling microscopy (STM) and scanning tunneling spectroscopy (STS). We found that STM observation can reveal individual quasibreakdown spots created by hot-electron injection into the oxide, as well as features of the topography such as atomic steps on the oxide surface. STS was used to study the local electrical properties of the oxide films before and after electrical stressing. We observed a leakage current at the quasibreakdown spots that passed through defect levels in the ultrathin oxide films. We also found that several tunneling spectra obtained from near leakage sites showed clear negative differential resistance. This phenomenon was attributed to the conductance change in the leakage path due to electron charging effects. Moreover, we confirmed the stressing polarity dependence of the leakage-site creation, and that atomic steps on the oxide and at the SiO2/Si interface did not cause any serous problem in the quasibreakdown process.

  1. Structure and chemistry of epitaxial ceria thin films on yttria-stabilized zirconia substrates, studied by high resolution electron microscopy.

    PubMed

    Sinclair, Robert; Lee, Sang Chul; Shi, Yezhou; Chueh, William C

    2017-01-06

    We have applied aberration-corrected transmission electron microscopy (TEM) imaging and electron energy loss spectroscopy (EELS) to study the structure and chemistry of epitaxial ceria thin films, grown by pulsed laser deposition onto (001) yttria-stabilized zirconia (YSZ) substrates. There are few observable defects apart from the expected mismatch interfacial dislocations and so the films would be expected to have good potential for applications. Under high electron beam dose rate (above about 6000 e(-)/Å(2)s) domains of an ordered structure appear and these are interpreted as being created by oxygen vacancy ordering. The ordered structure does not appear at lower lose rates (ca. 2600 e(-)/Å(2)s) and can be removed by imaging under 1 mbar oxygen gas in an environmental TEM. EELS confirms that there is both oxygen deficiency and the associated increase in Ce(3+) versus Ce(4+) cations in the ordered domains. In situ high resolution TEM recordings show the formation of the ordered domains as well as atomic migration along the ceria thin film (001) surface.

  2. a Study of SILVER/SILVER(100) Thin Film Growth with Scanning Tunneling Microscopy.

    NASA Astrophysics Data System (ADS)

    Wen, Jianming

    1995-01-01

    We used STM to study metal thin film growth processes. The following are main conclusions: (1) Large metal cluster diffuse on Ag(100) surface. The metal-metal system large adatom cluster diffusion is reported for the first time. The evaporation-condensation of adatoms dominating the vacancy cluster diffusion process is suggested. The diffusion coefficients is on the order of rm 10^ {-18} cm^2s^{-1}. (2) Metal film evolution behaviors at different coverage regime are examined. At low coverage, cluster diffusion dominates the coarsening process. At high submonolayer coverage, vacancy cluster repining dominates the process. At moderate coverage, edge-running dominates the process. (3) The vacancy cluster diffusion is also observed. The vacancy were formed by controlling the deposition flux and film coverage. The method is simple and offers similar advantages of the sputtering method. The diffusion coefficients is on the order of rm 10^{-18} cm^2s^{-1}, is smaller than that of adatom clusters, and has a scaling factor alpha roughly close to 0.5 in D ~ N^{-alpha }. The evaporation-condensation of vacancy monomers dominating the vacancy cluster diffusion process is suggested. (4) We have presented a number of observations of dynamical phenomena of Ag cluster on Ag (100) with UHV -STM. Ag cluster on Ag (100) at room temperature are mobile. The interaction between clusters and between clusters and surface steps are mediated via 2D dilute adatom gas on the terrace and different adatom concentrations around clusters and steps. Background gases and impurities on cluster edges can strongly affect film coarsening process. The former can enhance clusters dissolution, but the latter can stabilize the local structure and geometry of the Ag film.

  3. Properties of Ca-rich and Mg-rich carbonate films on dolomite: implications for compositional surface mapping with scanning force microscopy.

    PubMed

    Hu, Xiaoming; Cubillas, Pablo; Higgins, Steven R

    2010-04-06

    A self-limited monolayer grown on dolomite (CaMg(CO(3))(2)), showing distinct friction contrast with the substrate as reported earlier using lateral force microscopy, was investigated with in situ atomic force microscopy (AFM) adhesion mapping and force-modulation techniques. Force-modulation microscopy revealed lower stiffness on a Ca-rich film in comparison to that on the dolomite surface. The friction contrast therefore results from a larger tip-surface contact area when the AFM probe is in contact with the Ca-rich film as opposed to the contact area with dolomite. The Ca-rich film also exhibited a slightly higher adhesion than did the dolomite substrate; however, the critical shear stresses for the two tip-surface contacts were indistinguishable. A comparative study with a Mg-rich film did not yield noticeable force modulation contrast, indicating similar surface stiffness of the film and the dolomite surface. The similarity in these stiffness quantities was further corroborated by friction-load data that demonstrated similar friction forces on the two surfaces. The previously reported film strain in the Ca-rich system is likely linked to the lower stiffness observed, with both of these properties related to the Ca/Mg composition of the film.

  4. Ferromagnetic resonance imaging of Co films using magnetic resonance force microscopy

    SciTech Connect

    Suh, B.J.; Hammel, P.C.; Zhang, Z.; Midzor, M.M.; Roukes, M.L.; Childress, J.R.

    1998-07-01

    Magnetic resonance force microscope (MRFM) technique has been applied to the study of spatial imaging in thin Co ferromagnetic film. A novel approach is proposesd to improve spatial resolution in MRFM, which is limited by the broad width of Co ferromagnetic resonance (FMR) line. The authors introduce a selective local field with a small yittrium iron garnet (YIG) grain. They have performed MRFM detected FMR on a sample consisting of two sections of Co films laterally separated by {approximately}20 {micro}m. The experimental results demonstrate the scanning imaging capabilities of MRFM. The results can be understood qualitatively by means of the calculated magnetic field and field gradient profiles generated by the YIG shere.

  5. Scanning-tunneling-microscopy and spectroscopy studies of C70 thin films on gold substrates

    NASA Astrophysics Data System (ADS)

    Chen, T.; Howells, S.; Gallagher, M.; Sarid, D.; Lamb, L. D.; Huffman, D. R.; Workman, R. K.

    1992-06-01

    Thin films of high-purity C70 were deposited on polycrystalline gold surfaces and studied by scanning tunneling microscropy (STM) in ultrahigh vacuum. Topographic images reveal initial-stage growth patterns ranging from close packing with a twofold symmetry to random stacking. C70 molecules appear oval-shaped, in agreement with the proposed D5h structure of C70. Static orientational disorder is observed in both close-packed and random-stacked regions. The adsorbed C70 molecules show the highest STM contrast at bias voltages well below the HOMO-LUMO gap voltage. These and scanning-tunneling-spectroscopy results are discussed and compared with those obtained previously on similarly prepared C60 thin films.

  6. Photoemission Electron Microscopy Study of Ultrathin FeNi Alloy Films on Cu(111)

    NASA Astrophysics Data System (ADS)

    Sato, Yu; Johnson, Tracey; Giacomo, Jason; Chiang, Shirley; Zhu, Xiangdong; Land, Donald; Nolting, Frithjof; Scholl, Andreas

    2002-03-01

    We are studying the system of FeNi/Cu(111) to understand and control the surface/interface magnetism relevant to the application of the giant magnetoresistive effect to magnetic recording heads. We used the Photoemission Electron Microscope (PEEM2) at the Advanced Light Source to observe the domain structures of the alloy films. PEEM has the unique capability of imaging the film's magnetic structure with high spatial resolution and elemental specificity. At two different thicknesses, we have made sixteen samples and studied the dependence of magnetic structure on varying Fe concentration and substrate quality. Samples with higher Fe content were non-magnetic at room temperature. We speculate this is a structure-driven effect related to the "Invar effect" in the bulk alloy. The PEEM images clearly show that Fe and Ni form a good alloy and have the same domain structures with their magnetization aligned. Further, we find a strong thickness and concentration dependence of the magnetic domain structures.

  7. Characterization of coplanar poled electro optic polymer films for Si-photonic devices with multiphoton microscopy

    SciTech Connect

    Himmelhuber, R. Mehravar, S. S.; Herrera, O. D.; Demir, V.; Kieu, K.; Norwood, R. A.; Peyghambarian, N.; Luo, J.; Jen, A. K.-Y.

    2014-04-21

    We imaged coplanar poled electro optic (EO) polymer films on transparent substrates with a multiple-photon microscope in reflection and correlated the second-harmonic light intensity with the results of Pockels coefficient (r{sub 33}) measurements. This allowed us to make quantitative measurements of poled polymer films on non-transparent substrates like silicon, which are not accessible with traditional Pockels coefficient measurement techniques. Phase modulators consisting of silicon waveguide devices with EO polymer claddings with a known Pockels coefficient (from V{sub π} measurements) were used to validate the correlation between the second-harmonic signal and r{sub 33}. This also allowed us to locally map the r{sub 33} coefficient in the poled area.

  8. Nanoplough-constrictions on thin YBCO films made with atomic force microscopy.

    PubMed

    Elkaseh, A A O; Büttner, U; Meincken, M; Hardie, G L; Srinivasu, V V; Perold, W J

    2007-09-01

    Utilizing atomic force microscope (AFM) with a diamond tip, we were able to successfully plough nano-constrictions on epitaxially grown YBa2Cu3O(7-x) thin films deposited on MgO substrates. The thickness, width, and length of the obtained constrictions were in the range of a few 100 nm. Furthermore, we managed to produce a new S-type constriction, of which the dimensions are easier to control than for conventional constrictions.

  9. The In Situ Observation of Epitaxial Diamond Thin Film Nucleation and Growth Using Emission Electron Microscopy

    DTIC Science & Technology

    1993-12-01

    diamond carbon on diamond Measurements of CVD diamond grown directly on Mo TEM specimen grids were made through a collaboration with the Fritz Haber ...Hawaii, May 1993. 2. --- , University of Illinois at Chicago, March 1993. 3. --- , Fritz Haber Institute, Berlin, June 1993. 3.0 Appendix: 8 1 Real...University, Athens OH 45701 -2979 *Permanent address: Fritz Haber Institute, Berlin, Germany. Thin (1Onm) carbon films are found to adhere to Chemical Vapor

  10. Scanning tunneling microscopy/spectroscopy on perovskite oxide thin films deposited in situ.

    PubMed

    Hitosugi, Taro; Shimizu, Ryota; Ohsawa, Takeo; Iwaya, Katsuya

    2014-10-01

    Complex oxide surfaces and interfaces, consisting of two or more cations and oxygen anions, have attracted a great deal of attention because their properties are crucial factors in the performance of catalysts, fuel cells, and Li-ion batteries. However, atomic-scale investigations of these oxide surfaces have been hindered because of the difficulties in surface preparation. Here, we demonstrate atomic-scale surface studies of complex perovskite oxides and the initial growth processes in oxide epitaxial films deposited on (✓13 × ✓13)-R33.7° reconstructed SrTiO3 (001) substrates using a scanning tunneling microscope integrated with a pulsed laser deposition system. The atomically ordered, reconstructed SrTiO3 (001) surface is stable under the typical conditions necessary for the growth of oxide thin films, and hence is considered suitable for the study of the initial growth processes in oxide films. The atomic-scale microscopic/spectroscopic characterizations performed here shed light on the microscopic origin of electronic properties observed in complex oxides and their heterostructures.

  11. Measurement of apparent diffusion coefficients within ultrathin nafion Langmuir-Schaefer films: comparison of a novel scanning electrochemical microscopy approach with cyclic voltammetry.

    PubMed

    Bertoncello, Paolo; Ciani, Ilenia; Li, Fei; Unwin, Patrick R

    2006-12-05

    The use of scanning electrochemical microscopy (SECM) to evaluate the apparent diffusion coefficient, Dapp, of redox-active species in ultrathin Nafion films is described. In this technique, an ultramicroelectrode (UME) tip, positioned close to a film on a macroscopic electrode, is used to oxidize (or reduce) a species in bulk solution, causing the tip-generated oxidant (reductant) to diffuse to the film/solution interface. The oxidation (reduction) of film-confined species regenerates the reductant (oxidant) in solution, leading to feedback to the UME. A numerical model is developed that allows Dapp to be determined. For these studies, ultrathin films of Nafion were prepared using the Langmuir-Schaefer (LS) technique and loaded with an electroactive species, either the ferrocene derivative ferrocenyltrimethylammonium cation, FA+, or tris(2,2'-bipyridyl)ruthenium(II), Ru(bpy)32+. The morphology and the thickness of the Nafion LS films (1.5 +/- 0.2 nm per layer deposited) were evaluated using atomic force microscopy (AFM). For comparison with the SECM measurements, cyclic voltammetry (CV) was employed to evaluate the concentration of electroactive species within the Nafion LS films and to determine Dapp. The latter was found to be essentially invariant with film thickness, but the value for Ru(bpy)32+ was 1 order of magnitude larger than for FA+. CV and SECM measurements yield different values of Dapp, and the underlying reasons are discussed. In general, the Dapp values for these films are considerably smaller than for recast Nafion films, which can be attributed to the compactness of Nafion LS films. Nonetheless, the ultrathin nature of the films leads to fast response times, and we thus expect that these modified electrodes could find applications in sensing, electroanalysis, and electrocatalysis.

  12. In-situ microscopy of the first-order magnetic phase transition in FeRh thin films

    NASA Astrophysics Data System (ADS)

    Baldasseroni, Chloe

    Simple ferromagnetic (FM) and antiferromagnetic (AF) materials such as Fe and Cr become paramagnetic when heated above some critical temperature, in what is known as a second-order phase transition. Less usual magnetic transitions are found in the magnetic world, for example a first-order magnetic phase transition from AF to FM with increasing temperature. Equiatomic FeRh has been known to exhibit such a transition for over 50 years, with a transition temperature slightly above room temperature. Interest in this material has been renewed in the recent years due to its potential application for heat-assisted magnetic recording, as well as a test system for fundamental studies of the physics of magnetic phase transitions. Similarly to crystallization, this AF-FM transition is expected to proceed by nucleation of magnetic domains but the features of the first-order hysteretic transition have been difficult to study with macroscopic measurements and very few microscopic studies have been performed. In this work, FeRh thin films were synthesized by magnetron sputtering and structurally and magnetically characterized. A membrane-based heating device was designed to enable temperature-dependent microscopy measurements, providing a thermally uniform and well-controlled sample area. Synchrotron x-ray magnetic microscopy was used to study the temperature-driven AF-FM phase transition in epitaxial FeRh thin films in zero field. Using magnetic microscopy with x-ray magnetic circular dichroism, the different stages of nucleation, growth and coalescence of FM domains were observed across the transition and details of the nucleation were identified. The FM phase nucleates into single domain islands and the width of the transition of the individual nuclei upon heating is sharper than that of the macroscopic transition. Using magnetic microscopy with x-ray magnetic linear dichroism, the evolution of the AF phase was studied. Differences in the morphology of AF and FM phases were

  13. Nanosecond time-scale switching of permalloy thin film elements studied by wide-field time-resolved Kerr microscopy

    NASA Astrophysics Data System (ADS)

    Chumakov, Dmitry; McCord, Jeffrey; Schäfer, Rudolf; Schultz, Ludwig; Vinzelberg, Hartmut; Kaltofen, Rainer; Mönch, Ingolf

    2005-01-01

    The switching of extended Ni81Fe19 thin film elements with a thickness of 50nm and various shapes (squared, rectangular, pointed) has been studied by time-resolved stroboscopic Kerr microscopy based on a conventional wide-field optical polarization microscope. The elements are deposited on coplanar strip-lines that generate field pulses driven by electronic pulse generators. Time resolution is obtained by imaging with a gated and intensified charge-coupled device camera. The opening can be varied from 250ps to continuous exposure, allowing the comparison of fast magnetization processes and quasistatic switching in slowly varying fields. The latter is typically characterized by the formation of a concertina domain pattern that irreversibly decays in a multidomain ground state by the abrupt motion of vortices and domain walls. After excitation with fast field pulses similar blocked patterns are formed. They dissolve by spatially inhomogeneous rotational processes involving cross-tie-wall-like domain boundaries.

  14. Chitinase activity on amorphous chitin thin films: a quartz crystal microbalance with dissipation monitoring and atomic force microscopy study.

    PubMed

    Wang, Chao; Kittle, Joshua D; Qian, Chen; Roman, Maren; Esker, Alan R

    2013-08-12

    Chitinases are widely distributed in nature and have wide-ranging pharmaceutical and biotechnological applications. This work highlights a real-time and label-free method to assay Chitinase activity via a quartz crystal microbalance with dissipation monitoring (QCM-D) and atomic force microscopy (AFM). The chitin substrate was prepared by spincoating a trimethylsilyl chitin solution onto a silica substrate, followed by regeneration to amorphous chitin (RChi). The QCM-D and AFM results clearly showed that the hydrolysis rate of RChi films increased as Chitinase (from Streptomyces griseus) concentrations increased, and the optimal temperature and pH for Chitinase activity were around 37 °C and 6-8, respectively. The Chitinase showed greater activity on chitin substrates, having a high degree of acetylation, than on chitosan substrates, having a low degree of acetylation.

  15. Nanocrystal Diffusion in a Liquid Thin Film Observed by in situ Transmission Electron Microscopy

    SciTech Connect

    Zheng, Haimei; Claridge, Shelley A.; Minor, Andrew M.; Alivisatos, A. Paul; Dahmen, Ulrich

    2009-04-17

    We have directly observed motion of inorganic nanoparticles during fluid evaporation using a Transmission Electron Microscope. Tracking real-time diffusion of both spherical (5-15 nm) and rod-shaped (5x10 nm) gold nanocrystals in a thin-film of water-15percentglycerol reveals complex movements, such as rolling motions coupled to large-step movements and macroscopic violations of the Stokes-Einstein relation for diffusion. As drying patches form during the final stages of evaporation, particle motion is dominated by the nearby retracting liquid front.

  16. Imaging ac losses in superconducting films via scanning Hall probe microscopy

    NASA Astrophysics Data System (ADS)

    Dinner, Rafael B.; Moler, Kathryn A.; Feldmann, D. Matthew; Beasley, M. R.

    2007-04-01

    Various local probes have been applied to understanding current flow through superconducting films, which are often surprisingly inhomogeneous. Here, we show that magnetic imaging allows quantitative reconstruction of both current density J and electric field E resolved in time and space in a film carrying subcritical ac current. Current reconstruction entails inversion of the Biot-Savart law, while electric fields are reconstructed using Faraday’s law. We describe the corresponding numerical procedures, largely adapting existing work to the case of a strip carrying ac current, but including other methods of obtaining the complete electric field from the inductive portion determined by Faraday’s law. We also delineate the physical requirements behind the mathematical transformations. We then apply the procedures to images of a strip of YBa2Cu3O7-δ carrying an ac current at 400Hz . Our scanning Hall probe microscope produces a time series of magnetic images of the strip with 1μm spatial resolution and 25μs time resolution. Combining the reconstructed J and E , we obtain a complete characterization including local critical current density, E-J curves, and power losses. This analysis has a range of applications from fundamental studies of vortex dynamics to practical coated conductor development.

  17. High speed direct imaging of thin metal film ablation by movie-mode dynamic transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Hihath, Sahar; Santala, Melissa K.; Cen, Xi; Campbell, Geoffrey; van Benthem, Klaus

    2016-03-01

    Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combination of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Our results allow for improved safety during laser ablation in manufacturing and medical applications.

  18. High speed direct imaging of thin metal film ablation by movie-mode dynamic transmission electron microscopy.

    PubMed

    Hihath, Sahar; Santala, Melissa K; Cen, Xi; Campbell, Geoffrey; van Benthem, Klaus

    2016-03-11

    Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combination of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Our results allow for improved safety during laser ablation in manufacturing and medical applications.

  19. High speed direct imaging of thin metal film ablation by movie-mode dynamic transmission electron microscopy

    DOE PAGES

    Hihath, Sahar; Santala, Melissa K.; Cen, Xi; ...

    2016-03-11

    Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combinationmore » of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Ultimately, our results allow for improved safety during laser ablation in manufacturing and medical applications.« less

  20. High speed direct imaging of thin metal film ablation by movie-mode dynamic transmission electron microscopy

    SciTech Connect

    Hihath, Sahar; Santala, Melissa K.; Cen, Xi; Campbell, Geoffrey; van Benthem, Klaus

    2016-03-11

    Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combination of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Ultimately, our results allow for improved safety during laser ablation in manufacturing and medical applications.

  1. High speed direct imaging of thin metal film ablation by movie-mode dynamic transmission electron microscopy

    PubMed Central

    Hihath, Sahar; Santala, Melissa K.; Cen, Xi; Campbell, Geoffrey; van Benthem, Klaus

    2016-01-01

    Obliteration of matter by pulsed laser beams is not only prevalent in science fiction movies, but finds numerous technological applications ranging from additive manufacturing over machining of micro- and nanostructured features to health care. Pulse lengths ranging from femtoseconds to nanoseconds are utilized at varying laser beam energies and pulse lengths, and enable the removal of nanometric volumes of material. While the mechanisms for removal of material by laser irradiation, i.e., laser ablation, are well understood on the micrometer length scale, it was previously impossible to directly observe obliteration processes on smaller scales due to experimental limitations for the combination of nanometer spatial and nanosecond temporal resolution. Here, we report the direct observation of metal thin film ablation from a solid substrate through dynamic transmission electron microscopy. Quantitative analysis reveals liquid-phase dewetting of the thin-film, followed by hydrodynamic sputtering of nano- to submicron sized metal droplets. We discovered unexpected fracturing of the substrate due to evolving thermal stresses. This study confirms that hydrodynamic sputtering remains a valid mechanism for droplet expulsion on the nanoscale, while irradiation induced stress fields represent limit laser processing of nanostructured materials. Our results allow for improved safety during laser ablation in manufacturing and medical applications. PMID:26965073

  2. Scanning tunneling microscopy study of thin PTCDI films on Ag/Si(111)-√3 × √3

    NASA Astrophysics Data System (ADS)

    Emanuelsson, C.; Zhang, H. M.; Moons, E.; Johansson, L. S. O.

    2017-03-01

    3,4,9,10-perylene tetracarboxylic diimide molecules were evaporated onto a Ag/Si(111)-√3 × √3 surface and studied by scanning tunneling microscopy/spectroscopy and low energy electron diffraction (LEED). The growth mode was characterized as layer-by-layer growth with a single molecular unit cell in a short range order. The growth of the first two monolayers involves a molecule/substrate superstructure and a molecule/molecule superstructure. At higher coverages, the molecules in each layer were found to align so that unit cells are on top of each other. The experimentally obtained LEED pattern is described as a combination of patterns from the molecular unit cell and the molecule/substrate superstructure. The electronic structure was found to be strongly dependent on the film thickness for the first few layers: Several extra states are found at low coverages compared to higher coverages resulting in a very small pseudo gap of 0.9 eV for the first layer, which widens up to 4.0 eV for thicker films.

  3. Molecular resolution friction microscopy of Cu phthalocyanine thin films on dolomite (104) in water.

    PubMed

    Nita, Paweł; Pimentel, Carlos; Luo, Feng; Milián-Medina, Begoña; Gierschner, Johannes; Pina, Carlos M; Gnecco, Enrico

    2014-07-21

    The reliability of ultrathin organic layers as active components for molecular electronic devices depends ultimately on an accurate characterization of the layer morphology and ability to withstand mechanical stresses on the nanoscale. To this end, since the molecular layers need to be electrically decoupled using thick insulating substrates, the use of AFM becomes mandatory. Here, we show how friction force microscopy (FFM) in water allows us to identify the orientation of copper(ii)phthalocyanine (CuPc) molecules previously self-assembled on a dolomite (104) mineral surface in ultra-high vacuum. The molecular features observed in the friction images show that the CuPc molecules are stacked in parallel rows with no preferential orientation with respect to the dolomite lattice, while the stacking features resemble well the single CuPc crystal structure. This proves that the substrate induction is low and makes friction force microscopy in water a suitable alternative to more demanding dynamic AFM techniques in ultra-high vacuum.

  4. Transmission electron microscopy analysis of phase separation in GaInAsSb films grown on GaSb substrate.

    PubMed

    Szczeszek, P; Amariei, A; Schöne, J; Zoulis, G; Vouroutzis, N; Polychroniadis, E K; Stróz, D

    2006-10-01

    The GaSb-based quaternary alloys are a good choice for thermophotovoltaic applications. The thermophotovoltaic cell converts infrared radiation to electricity, using the same principles as photovoltaic devices. The aim of the present work was the microstructural study of such an alloy, namely Ga(0.84)In(0.16)As(0.12)Sb(0.88). A thin film of the material was grown by metal organic vapour phase epitaxy on a (100)alpha-->[111]B (alpha = 2 degrees, 4 degrees, 6 degrees) GaSb substrate. The GaInAsSb alloy has an appropriate band gap, but suffers from a phase separation consisting of GaAs-rich and InSb-rich regions that is disadvantageous for cell efficiency. In this work, we employed a morphological approach to phase separation, with the use of conventional transmission electron microscopy and atomic force microscopy. The phase separation occurs in two different orientations: parallel to the growth direction (vertical) and inclined (lateral). After application of fast Fourier transformation filtering, the vertical periodicity was found to be lambda = 5 nm for the pair (black and white) of layers independently of the cut-off angle, whereas the lateral periodicity was related to it.

  5. Atomic probe microscopy of 3C SiC films grown on 6H SiC substrates

    NASA Technical Reports Server (NTRS)

    Steckl, A. J.; Roth, M. D.; Powell, J. A.; Larkin, D. J.

    1993-01-01

    The surface of 3C SiC films grown on 6H SiC substrates has been studied by atomic probe microscopy in air. Atomic-scale images of the 3C SiC surface have been obtained by STM which confirm the 111 line type orientation of the cubic 3C layer grown on the 0001 plane type surface of the hexagonal 6H substrate. The nearest-neighbor atomic spacing for the 3C layer has been measured to be 3.29 +/- 0.2 A, which is within 7 percent of the bulk value. Shallow terraces in the 3C layer have been observed by STM to separate regions of very smooth growth in the vicinity of the 3C nucleation point from considerably rougher 3C surface regions. These terraces are oriented at right angles to the growth direction. Atomic force microscopy has been used to study etch pits present on the 6H substrate due to high temperature HCl cleaning prior to CVD growth of the 3C layer. The etch pits have hexagonal symmetry and vary in depth from 50 nm to 1 micron.

  6. Atomic force microscopy identification of Al-sites on ultrathin aluminum oxide film on NiAl(110).

    PubMed

    Li, Yan Jun; Brndiar, J; Naitoh, Y; Sugawara, Y; Štich, I

    2015-12-18

    Ultrathin alumina film formed by oxidation of NiAl(110) was studied by non-contact atomic force microscopy in an ultra high vacuum at room temperature with the quest to provide the ultimate understanding of structure and bonding of this complicated interface. Using a very stiff Si cantilever with significantly improved resolution, we have obtained images of this system with unprecedented resolution, surpassing all the previous results. In particular, we were able to unambiguously resolve all the differently coordinated aluminum atoms. This is of importance as the previous images provide very different image patterns, which cannot easily be reconciled with the existing structural models. Experiments are supported by extensive density functional theory modeling. We find that the system is strongly ionic and the atomic force microscopy images can reliably be understood from the electrostatic potential which provides an image model in excellent agreement with the experiments. However, in order to resolve the finer contrast features we have proposed a more sophisticated model based on more realistic approximants to the incommensurable alumina interface.

  7. Thickness measurement of soft thin films on periodically patterned magnetic substrates by phase difference magnetic force microscopy.

    PubMed

    Passeri, D; Dong, C; Angeloni, L; Pantanella, F; Natalizi, T; Berlutti, F; Marianecci, C; Ciccarello, F; Rossi, M

    2014-01-01

    The need for accurate measurement of the thickness of soft thin films is continuously encouraging the development of techniques suitable for this purpose. We propose a method through which the thickness of the film is deduced from the quantitative measurement of the contrast in the phase images of the sample surface acquired by magnetic force microscopy, provided that the film is deposited on a periodically patterned magnetic substrate. The technique is demonstrated by means of magnetic substrates obtained from standard floppy disks. Colonies of Staphylococcus aureus adherent to such substrates were used to obtain soft layers with limited lateral (a few microns) and vertical (hundreds of nanometers) size. The technique is described and its specific merits, limitations and potentialities in terms of accuracy and measurable thickness range are discussed. These parameters depend on the characteristics of the sensing tip/cantilever as well as of the substrates, the latter in terms of spatial period and homogeneity of the magnetic domains. In particular, with the substrates used in this work we evaluated an uncertainty of about 10%, a limit of detection of 50-100 nm and an upper detection limit (maximum measurable thickness) of 1 μm, all obtained with standard lift height values (50-100 nm). Nonetheless, these parameters can be easily optimized by selecting/realizing substrates with suitable spacing and homogeneity of the magnetic domains. For example, the upper detection limit can be increased up to 25-50 μm while the limit of detection can be reduced to a few tens of nanometers or a few nanometers.

  8. Scanning electrochemical microscopy of graphene/polymer hybrid thin films as supercapacitors: Physical-chemical interfacial processes

    SciTech Connect

    Gupta, Sanju Price, Carson

    2015-10-15

    Hybrid electrode comprising an electric double-layer capacitor of graphene nanosheets and a pseudocapacitor of the electrically conducting polymers namely, polyaniline; PAni and polypyrrole; PPy are constructed that exhibited synergistic effect with excellent electrochemical performance as thin film supercapacitors for alternative energy. The hybrid supercapacitors were prepared by layer-by-layer (LbL) assembly based on controlled electrochemical polymerization followed by reduction of graphene oxide electrochemically producing ErGO, for establishing intimate electronic contact through nanoscale architecture and chemical stability, producing a single bilayer of (PAni/ErGO){sub 1}, (PPy/ErGO){sub 1}, (PAni/GO){sub 1} and (PPy/GO){sub 1}. The rationale design is to create thin films that possess interconnected graphene nanosheets (GNS) with polymer nanostructures forming well-defined tailored interfaces allowing sufficient surface adsorption and faster ion transport due to short diffusion distances. We investigated their electrochemical properties and performance in terms of gravimetric specific capacitance, C{sub s}, from cyclic voltammograms. The LbL-assembled bilayer films exhibited an excellent C{sub s} of ≥350 F g{sup −1} as compared with constituents (∼70 F g{sup −1}) at discharge current density of 0.3 A g{sup −1} that outperformed many other hybrid supercapacitors. To gain deeper insights into the physical-chemical interfacial processes occurring at the electrode/electrolyte interface that govern their operation, we have used scanning electrochemical microscopy (SECM) technique in feedback and probe approach modes. We present our findings from viewpoint of reinforcing the role played by heterogeneous electrode surface composed of nanoscale graphene sheets (conducting) and conducting polymers (semiconducting) backbone with ordered polymer chains via higher/lower probe current distribution maps. Also targeted is SECM imaging that allowed to determine

  9. Scanning electrochemical microscopy of graphene/polymer hybrid thin films as supercapacitors: Physical-chemical interfacial processes

    NASA Astrophysics Data System (ADS)

    Gupta, Sanju; Price, Carson

    2015-10-01

    Hybrid electrode comprising an electric double-layer capacitor of graphene nanosheets and a pseudocapacitor of the electrically conducting polymers namely, polyaniline; PAni and polypyrrole; PPy are constructed that exhibited synergistic effect with excellent electrochemical performance as thin film supercapacitors for alternative energy. The hybrid supercapacitors were prepared by layer-by-layer (LbL) assembly based on controlled electrochemical polymerization followed by reduction of graphene oxide electrochemically producing ErGO, for establishing intimate electronic contact through nanoscale architecture and chemical stability, producing a single bilayer of (PAni/ErGO)1, (PPy/ErGO)1, (PAni/GO)1 and (PPy/GO)1. The rationale design is to create thin films that possess interconnected graphene nanosheets (GNS) with polymer nanostructures forming well-defined tailored interfaces allowing sufficient surface adsorption and faster ion transport due to short diffusion distances. We investigated their electrochemical properties and performance in terms of gravimetric specific capacitance, Cs, from cyclic voltammograms. The LbL-assembled bilayer films exhibited an excellent Cs of ≥350 F g-1 as compared with constituents (˜70 F g-1) at discharge current density of 0.3 A g-1 that outperformed many other hybrid supercapacitors. To gain deeper insights into the physical-chemical interfacial processes occurring at the electrode/electrolyte interface that govern their operation, we have used scanning electrochemical microscopy (SECM) technique in feedback and probe approach modes. We present our findings from viewpoint of reinforcing the role played by heterogeneous electrode surface composed of nanoscale graphene sheets (conducting) and conducting polymers (semiconducting) backbone with ordered polymer chains via higher/lower probe current distribution maps. Also targeted is SECM imaging that allowed to determine electrochemical (re)activity of surface ion adsorption sites

  10. Potential variations around grain boundaries in impurity-doped BaSi₂ epitaxial films evaluated by Kelvin probe force microscopy

    SciTech Connect

    Tsukahara, D.; Baba, M.; Honda, S.; Toko, K.; Imai, Y.; Hara, K. O.; Usami, N.; Werner, J. H.; Suemasu, T.

    2014-09-28

    Potential variations around the grain boundaries (GBs) in antimony (Sb)-doped n-type and boron (B)-doped p-type BaSi₂ epitaxial films on Si(111) were evaluated by Kelvin probe force microscopy. Sb-doped n-BaSi₂ films exhibited positively charged GBs with a downward band bending at the GBs. The average barrier height for holes was approximately 10 meV for an electron concentration n ≈ 10¹⁷ cm⁻³. This downward band bending changed to upward band bending when n was increased to n = 1.8 × 10¹⁸cm⁻³. In the B-doped p-BaSi₂ films, the upward band bending was observed for a hole concentration p ≈ 10¹⁸cm⁻³. The average barrier height for electrons decreased from approximately 25 to 15 meV when p was increased from p = 2.7 × 10¹⁸ to p = 4.0 × 10¹⁸ cm⁻³. These results are explained under the assumption that the position of the Fermi level E{sub f} at GBs depends on the degree of occupancy of defect states at the GBs, while E{sub f} approached the bottom of the conduction band or the top of the valence band in the BaSi₂ grain interiors with increasing impurity concentrations. In both cases, such small barrier heights may not deteriorate the carrier transport properties. The electronic structures of impurity-doped BaSi₂ are also discussed using first-principles pseudopotential method to discuss the insertion sites of impurity atoms and clarify the reason for the observed n-type conduction in the Sb-doped BaSi₂ and p-type conduction in the B-doped BaSi₂.

  11. Nanoscale probing of electronic band gap and topography of VO2 thin film surfaces by scanning tunneling microscopy

    NASA Astrophysics Data System (ADS)

    Yin, W.; Wolf, S.; Ko, C.; Ramanathan, S.; Reinke, P.

    2011-01-01

    The metal-insulator transition (MIT) in vanadium dioxide in the vicinity of room temperature makes it one of the most interesting materials for novel switching device applications. It is therefore essential to have a fundamental understanding of the VO2 surface when it is incorporated into multilayer structures or nanodevices. This study focuses on the surface modification of VO2 in response to the thermal treatment during phase transition. Vacuum annealing at temperatures in the vicinity of the MIT triggers a partial reduction in the surface, and thus initiates a chemical phase transition. Scanning tunneling microscopy and spectroscopy are used to investigate the electronic properties and surface structure of the VO2 thin film on (0001) sapphire substrates. Band gap maps with a high spatial resolution and single point spectroscopy I-V curves are measured as the sample is cycled through the MIT, and thus provide a direct observation of the surface phase transition at the nanoscale. The VO2 surface exhibits a homogeneous insulating behavior with a typical band gap of ˜0.5 eV at room temperature, and the surface becomes more metallic and spatially inhomogeneous in conductivity during MIT, and wide range of surface oxides can be identified. The surface still remains partially metallic after cooling down from a long period anneal, and such irreversible surface electrical change is attributed to the loss of oxygen. The location of metallic islands after thermal cycling is strongly coupled to the topography of the film, and relaxation processes and continued modification of the spatial distribution of the metallic regions are recognized on a longer timescale. The impact of film morphology, strain, surface chemistry, and structural phase transition on the electronic characteristics of VO2 surfaces are discussed.

  12. Electric transport through nanometric CoFe{sub 2}O{sub 4} thin films investigated by conducting atomic force microscopy

    SciTech Connect

    Foerster, M.; Gutierrez, D. F.; Rigato, F.; Fontcuberta, J.; Rebled, J. M.; Peiro, F.

    2012-01-01

    A systematic study of electric transport through thin (2-8 nm) CoFe{sub 2}O{sub 4} films deposited on epitaxial SrRuO{sub 3} bottom electrodes was performed by conducting atomic force microscopy (CAFM). Experimental procedures to investigate transport through thin insulating films by CAFM are critically revised, and the potential of CoFe{sub 2}O{sub 4} films for the use as spin-filtering barriers is assessed. It is concluded that, at room-temperature, a non-tunnel channel significantly contributes to the electric transport, thus limiting the spin-filtering efficiency.

  13. Non-invasive in vivo measurement of the tear film using spatial autocorrelation in a live mammal model

    PubMed Central

    Azartash, Kaveh; Shy, Chyong-jy Nein; Flynn, Kevin; Jester, James V.; Gratton, Enrico

    2010-01-01

    Tear film stability and its interaction with the corneal surface play an important role in maintaining ocular surface integrity and quality of vision. We present a non-invasive technique to quantify the pre-corneal tear film thickness. A cMOS camera is used to record the interference pattern produced by the reflections from multiple layers of the tear film Principles of spatial autocorrelation are applied to extract the frequency of the periodic patterns in the images. A mathematical model is developed to obtain the thickness of the tear film from the spatial autocorrelation image. The technique is validated using micro-fabricated thin parylene films. We obtained repeatable and precise measurement on a live rabbit model (N = 6). We obtained an average value of 10.2µm and standard deviation of, SD = 0.3 (N = 4). We measured one rabbit infected with HSV-1 virus that had a baseline tear film thickness of 4.7µm. PMID:21258535

  14. Exploring the limits of optical microscopy: live cell and superresolution fluorescence microscopy of HIV-1 Transfer Between T lymphocytes Across the Virological Synapse

    NASA Astrophysics Data System (ADS)

    McNerney, Gregory Paul

    Human immunodeficiency virus 1 (HIV-1) is a human retrovirus that efficiently, albeit gradually, overruns the immune system. An already infected T lymphocyte can latch onto another T lymphocyte whereby creating a virological synapse (VS); this junction drives viral assembly and transfer to the target cell in batches in an efficient, protective manor. My Ph.D. doctoral thesis focused on studying this transmission mechanism using advanced optical imaging modalities and the fully infectious fluorescent clone HIV Gag-iGFP. T lymphocytes are non-adherent cells (˜10 um thick) and the viral transmission process is fairly dynamic, hence we employed a custom spinning disk confocal microscope that revealed many interesting characteristics of this cooperative event. This methodology has low throughput as cell contact and transfer is at random. Optical tweezers was then added to the microscope to directly initiate cell contact at will. To assess when viral maturation occurs post-transfer, an optical assay based off of Forster resonance energy transfer was developed to monitor maturation. Structured illumination microscopy was further used to image the process at higher resolution and it showed that viral particles are not entering existing degradative compartments. Non-HIV-1 applications of the optical technologies are also reviewed.

  15. Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica: surface plasmon resonance and atomic force microscopy study.

    PubMed

    Shinozaki, Yukiko; Kikkawa, Yoshihiro; Sato, Shun; Fukuoka, Tokuma; Watanabe, Takashi; Yoshida, Shigenobu; Nakajima-Kambe, Toshiaki; Kitamoto, Hiroko K

    2013-10-01

    Enzymatic degradation of polyester films by a cutinase-like enzyme from Pseudozyma antarctica JCM10317 (PaE) was analyzed by surface plasmon resonance (SPR). The adsorption of PaE and the degradation rate for polyester films were quantitatively monitored by a positive and negative SPR signal shifts, respectively. The decrease in SPR signal and the erosion depth of amorphous poly(L-lactide) (a-PLLA) film measured by atomic force microscopy (AFM) had a linear relationship, and the weight loss was estimated from the AFM data combined with a density of a-PLLA film. Furthermore, SPR sensorgrams for various polyester films showed that degradation rate of poly(ε-caprolactone) and poly(butylene succinate-co-adipate) which contain C6 units was higher than that of other polyesters such as poly(butylene succinate) and a-PLLA. These results suggest that C6 is the preferred chain length as substrates for PaE.

  16. Molecular beam epitaxy growth and post-growth annealing of FeSe films on SrTiO3: a scanning tunneling microscopy study.

    PubMed

    Li, Zhi; Peng, Jun-Ping; Zhang, Hui-Min; Zhang, Wen-Hao; Ding, Hao; Deng, Peng; Chang, Kai; Song, Can-Li; Ji, Shuai-Hua; Wang, Lili; He, Ke; Chen, Xi; Xue, Qi-Kun; Ma, Xu-Cun

    2014-07-02

    Low temperature scanning tunneling microscopy and spectroscopy are used to investigate the atomic and electronic structure evolution of FeSe films grown on SrTiO3 as a function of post-growth annealing. Single unit cell FeSe films are found to bond strongly with the underlying substrate, and become superconductive with diminishing chemical bond disorders at the interface via post-annealing. For thicker FeSe films, post-annealing removes excess Se in the films and leads to a transition from semiconductor into metallic behaviors. In double and multilayer films, strain-induced complex textures are observed and suggested to be the main cause for the absent superconductivity.

  17. Spin polarized low energy electron microscopy of quantum well resonances in Fe films on the Cu-covered W(110) surface.

    PubMed

    Wu, Qiang; Altman, M S

    2013-07-01

    Spin polarized low energy electron microscopy has been used to investigate the quantum size effect (QSE) in electron reflectivity from Fe films grown on a pseudomorphic Cu layer on a W(110) surface. Intensity oscillations caused by the QSE as functions of Fe film thickness and incident electron energy identify quantum well resonance conditions in the film. Evaluation of these intensity oscillations using the phase accumulation model provides information on the unoccupied spin polarized band structure in the Fe film above the vacuum level. We also find evidence that the presence of the non-magnetic Cu layer shifts spin polarized quantum well resonances in the Fe layer uniformly downward in energy by 1.1eV compared to Fe/W(110) films without an interface Cu layer, suggesting that the Cu layer gives a small degree of control over the quantum well resonances.

  18. Electron microscopy study of Ni induced crystallization in amorphous Si thin films

    SciTech Connect

    Radnóczi, G. Z.; Battistig, G.; Pécz, B.; Dodony, E.; Vouroutzis, N.; Stoemenos, J.; Frangis, N.; Kovács, A.

    2015-02-17

    The crystallization of amorphous silicon is studied by transmission electron microscopy. The effect of Ni on the crystallization is studied in a wide temperature range heating thinned samples in-situ inside the microscope. Two cases of limited Ni source and unlimited Ni source are studied and compared. NiSi{sub 2} phase started to form at a temperature as low as 250°C in the limited Ni source case. In-situ observation gives a clear view on the crystallization of silicon through small NiSi{sub 2} grain formation. The same phase is observed at the crystallization front in the unlimited Ni source case, where a second region is also observed with large grains of Ni{sub 3}Si{sub 2}. Low temperature experiments show, that long annealing of amorphous silicon at 410 °C already results in large crystallized Si regions due to the Ni induced crystallization.

  19. Identifying dislocations and stacking faults in GaN films by scanning transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Su, X. J.; Niu, M. T.; Zeng, X. H.; Huang, J.; Zhang, J. C.; Zhang, J. P.; Wang, J. F.; Xu, K.

    2016-08-01

    The application of annular bright field (ABF) and medium-angle annular dark field (MAADF) scanning transmission electron microscopy (STEM) imaging to crystalline defect analysis has been extended to dislocations and stacking faults (SFs). Dislocations and SFs have been imaged under zone-axis and two-beam diffraction conditions. Comparing to conventional two-beam diffraction contrast images, the ABF and MAADF images of dislocations and SFs not only are complementary and symmetrical with their peaks at dislocation core and SFs plane, but also show similar extinction phenomenon. It is demonstrated that conventional TEM rules for diffraction contrast, i.e. g · b and g · R invisibility criteria remain applicable. The contrast mechanism and extinction of dislocation and SFs in ABF and MAADF STEM are illuminated by zero-order Laue zone Kikuchi diffraction.

  20. Detection of living Sarcoptes scabiei larvae by reflectance mode confocal microscopy in the skin of a patient with crusted scabies

    NASA Astrophysics Data System (ADS)

    Levi, Assi; Mumcuoglu, Kosta Y.; Ingber, Arieh; Enk, Claes D.

    2012-06-01

    Scabies is an intensely pruritic disorder induced by a delayed type hypersensitivity reaction to infestation of the skin by the mite Sarcoptes scabiei. The diagnosis of scabies is established clinically and confirmed by identifying mites or eggs by microscopic examination of scrapings from the skin or by surface microscopy using a dermatoscope. Reflectance-mode confocal microscopy is a novel technique used for noninvasive imaging of skin structures and lesions at a resolution compatible to that of conventional histology. Recently, the technique was employed for the confirmation of the clinical diagnosis of scabies. We demonstrate the first ever documentation of a larva moving freely inside the skin of a patient infected with scabies.

  1. Exploring diffusion of ultrasonically consolidated aluminum and copper films through scanning and transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Sietins, Jennifer Mueller

    Ultrasonic consolidation (UC) is a promising manufacturing method for metal matrix composite pre-preg tapes or foils that utilizes a layer build-up technique. The process involves three main variables: applied load, oscillation amplitude, and rolling speed. A main advantage of this process is the ability to manufacture multi-material parts at lower processing temperatures compared to other metal matrix composites processes. A major disadvantage, however, is a lack of understanding of diffusion during the ultrasonic consolidation process, which is expected to affect the microstructure, bond quality, and strength within the interface region. The role of diffusion during the low temperature, short duration ultrasonic consolidation process was explored. First, scanning electron microscopy (SEM) x-ray energy dispersive spectroscopy (XEDS) was used to measure concentration profiles of ultrasonically consolidated high purity aluminum and copper through which the interdiffusion coefficients were calculated. It was found that the experimental accelerating voltage had a significant impact on the measurement of the concentration profiles, and associated interdiffusion coefficients, due to the interaction volume interference. The effect of the interaction volume on the concentration profiles was confirmed through Monte Carlo simulations of electron trajectories, and the error due the interaction volume was quantified. The results showed the diffusion distance was too small for accurate measurements with SEM XEDS even at low accelerating voltages. To significantly reduce the error due to the interaction volume, transmission electron microscopy (TEM) samples were prepared using a focused ion beam (FIB) to ensure a uniform thickness. The TEM XEDS concentration profile and images revealed intermetallic phase transformations that occurred during the welding process. TEM images also showed dislocation pile-up located at the subgrain/bulk aluminum interface. This microstructural

  2. Electron transport in ultra-thin films and ballistic electron emission microscopy

    NASA Astrophysics Data System (ADS)

    Claveau, Y.; Di Matteo, S.; de Andres, P. L.; Flores, F.

    2017-03-01

    We have developed a calculation scheme for the elastic electron current in ultra-thin epitaxial heterostructures. Our model uses a Keldysh’s non-equilibrium Green’s function formalism and a layer-by-layer construction of the epitaxial film. Such an approach is appropriate to describe the current in a ballistic electron emission microscope (BEEM) where the metal base layer is ultra-thin and generalizes a previous one based on a decimation technique appropriated for thick slabs. This formalism allows a full quantum mechanical description of the transmission across the epitaxial heterostructure interface, including multiple scattering via the Dyson equation, which is deemed a crucial ingredient to describe interfaces of ultra-thin layers properly in the future. We introduce a theoretical formulation needed for ultra-thin layers and we compare with results obtained for thick Au(1 1 1) metal layers. An interesting effect takes place for a width of about ten layers: a BEEM current can propagate via the center of the reciprocal space (\\overlineΓ ) along the Au(1 1 1) direction. We associate this current to a coherent interference finite-width effect that cannot be found using a decimation technique. Finally, we have tested the validity of the handy semiclassical formalism to describe the BEEM current.

  3. Electron transport in ultra-thin films and ballistic electron emission microscopy.

    PubMed

    Claveau, Y; Di Matteo, S; de Andres, P L; Flores, F

    2017-03-22

    We have developed a calculation scheme for the elastic electron current in ultra-thin epitaxial heterostructures. Our model uses a Keldysh's non-equilibrium Green's function formalism and a layer-by-layer construction of the epitaxial film. Such an approach is appropriate to describe the current in a ballistic electron emission microscope (BEEM) where the metal base layer is ultra-thin and generalizes a previous one based on a decimation technique appropriated for thick slabs. This formalism allows a full quantum mechanical description of the transmission across the epitaxial heterostructure interface, including multiple scattering via the Dyson equation, which is deemed a crucial ingredient to describe interfaces of ultra-thin layers properly in the future. We introduce a theoretical formulation needed for ultra-thin layers and we compare with results obtained for thick Au(1 1 1) metal layers. An interesting effect takes place for a width of about ten layers: a BEEM current can propagate via the center of the reciprocal space ([Formula: see text]) along the Au(1 1 1) direction. We associate this current to a coherent interference finite-width effect that cannot be found using a decimation technique. Finally, we have tested the validity of the handy semiclassical formalism to describe the BEEM current.

  4. Vacuum-Deposited Porphyrin Protective Films on Graphite: Electrochemical Atomic Force Microscopy Investigation during Anion Intercalation.

    PubMed

    Yivlialin, Rossella; Bussetti, Gianlorenzo; Penconi, Marta; Bossi, Alberto; Ciccacci, Franco; Finazzi, Marco; Duò, Lamberto

    2017-02-01

    The development of graphene products promotes a renewed interest toward the use of graphite in addition to the historical one for its proven viability as battery electrode. However, when exposed to harsh conditions, the graphite surface ages in ways that still need to be fully characterized. In applications to batteries, to optimize the electrode performances in acid solutions, different surface functionalizations have been studied. Among them, aromatic molecules have been recently proposed. In this communication, we report on the protective effect exerted by a physical-vapor-deposited porphyrin layer. Metal-free tetra-phenyl-porphyrins were deposited on a highly oriented pyrolytic graphite crystal to study the modifications that occur during anion intercalation in graphite. The graphite electrode was plunged in an electrolyte solution of 1 M sulfuric acid and subjected to cyclic voltammetry. The results indicate that blister formation, the characteristic swelling of graphite surface induced by anion intercalation, is significantly perturbed by the porphyrin overlayer; the process is inhibited in those areas where the protective porphyrin film is present. We ascribe the inhibition of the anion intercalation to the protective porphyrin wetting layer.

  5. Digital simulation of scanning electrochemical microscopy approach curves to enzyme films with Michaelis-Menten kinetics.

    PubMed

    Burchardt, Malte; Träuble, Markus; Wittstock, Gunther

    2009-06-15

    The formalism for simulating scanning electrochemical microscopy (SECM) experiments by boundary element methods in three space coordinates has been extended to allow consideration of nonlinear boundary conditions. This is achieved by iteratively refining the boundary conditions that are encoded in a boundary condition matrix. As an example, the simulations are compared to experimental approach curves in the SECM feedback mode toward samples modified with glucose oxidase (GOx). The GOx layer was prepared by the layer-by-layer assembly of polyelectrolytes using glucose oxidase as one of the polyelectrolytes. The comparison of the simulated and experimental curves showed that under a wide range of experimentally accessible conditions approximations of the kinetics at the sample by first order models yield misleading results. The approach curves differ also qualitatively from curves calculated with first order models. As a consequence, this may lead to severe deviations when such curves are fitted to first order kinetic models. The use of linear approximations to describe the enzymatic reaction in SECM feedback experiments is justified only if the ratio of the mediator and Michaelis-Menten constant is equal to or smaller than 0.1 (deviation less than 10%).

  6. Dumbbell Defects in FeSe Films: A Scanning Tunneling Microscopy and First-Principles Investigation.

    PubMed

    Huang, Dennis; Webb, Tatiana A; Song, Can-Li; Chang, Cui-Zu; Moodera, Jagadeesh S; Kaxiras, Efthimios; Hoffman, Jennifer E

    2016-07-13

    The properties of iron-based superconductors (Fe-SCs) can be varied dramatically with the introduction of dopants and atomic defects. As a pressing example, FeSe, parent phase of the highest-Tc Fe-SC, exhibits prevalent defects with atomic-scale "dumbbell" signatures as imaged by scanning tunneling microscopy (STM). These defects spoil superconductivity when their concentration exceeds 2.5%. Resolving their chemical identity is a prerequisite to applications such as nanoscale patterning of superconducting/nonsuperconducting regions in FeSe as well as fundamental questions such as the mechanism of superconductivity and the path by which the defects destroy it. We use STM and density functional theory to characterize and identify the dumbbell defects. In contrast to previous speculations about Se adsorbates or substitutions, we find that an Fe-site vacancy is the most energetically favorable defect in Se-rich conditions and reproduces our observed STM signature. Our calculations shed light more generally on the nature of Se capping, the removal of Fe vacancies via annealing, and their ordering into a √5 × √5 superstructure in FeSe and related alkali-doped compounds.

  7. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

    PubMed

    Turnbull, Lynne; Strauss, Michael P; Liew, Andrew T F; Monahan, Leigh G; Whitchurch, Cynthia B; Harry, Elizabeth J

    2014-09-29

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

  8. Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

    PubMed Central

    Liew, Andrew T. F.; Monahan, Leigh G.; Whitchurch, Cynthia B.; Harry, Elizabeth J.

    2014-01-01

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide. PMID:25286090

  9. Electron microscopy study of thermoelectric n-type Bi2(Te0.9Se0.1)3 film deposited by dc sputtering

    NASA Astrophysics Data System (ADS)

    Yildiz, Koksal; Akgul, Unal; Leipner, Hartmut S.; Atici, Yusuf

    2013-06-01

    Thermoelectric +90 film of the ternary compound was deposited by dc. sputtering from n-type Bi2Te2.7Se0.3 target on polyimide foil substrate at 200 °C. The surface morphology and elemental composition of the deposited film was characterised by scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDX). SEM images and EDX spectra showed that the surface morphology of thermoelectric film exists in the form of big grains with small grains, rough surface and expected structure. The percentage of deviation from stoichiometry was calculated as ±3.599% for Bi2(Te0.9Se0.1)3. Therefore, it was found that the composition of the film stoichiometry was close to the sputtering target stoichiometry. It was seen from transmission electron microscopy (TEM) images and selected area diffraction (SAD) patterns that the thermoelectric film has poly crystalline structures with nano-sized and different orientations. It was also observed that the film have no second phase, precipitate and separation between the layers. Study of lattice images for the sample was done by high-resolution TEM. dhkl values of grains with different orientations were measured and some deformed areas were observed. In addition to, a structural modulation structure was monitorized in the sample. These modulations have a periodicity of approximate 1 nm.

  10. Detection of charge storage on molecular thin films of tris(8-hydroxyquinoline) aluminum (Alq3) by Kelvin force microscopy: a candidate system for high storage capacity memory cells.

    PubMed

    Paydavosi, Sarah; Aidala, Katherine E; Brown, Patrick R; Hashemi, Pouya; Supran, Geoffrey J; Osedach, Timothy P; Hoyt, Judy L; Bulović, Vladimir

    2012-03-14

    Retention and diffusion of charge in tris(8-hydroxyquinoline) aluminum (Alq(3)) molecular thin films are investigated by injecting electrons and holes via a biased conductive atomic force microscopy tip into the Alq(3) films. After the charge injection, Kelvin force microscopy measurements reveal minimal changes with time in the spatial extent of the trapped charge domains within Alq(3) films, even for high hole and electron densities of >10(12) cm(-2). We show that this finding is consistent with the very low mobility of charge carriers in Alq(3) thin films (<10(-7) cm(2)/(Vs)) and that it can benefit from the use of Alq(3) films as nanosegmented floating gates in flash memory cells. Memory capacitors using Alq(3) molecules as the floating gate are fabricated and measured, showing durability over more than 10(4) program/erase cycles and the hysteresis window of up to 7.8 V, corresponding to stored charge densities as high as 5.4 × 10(13) cm(-2). These results demonstrate the potential for use of molecular films in high storage capacity nonvolatile memory cells.

  11. Direct observation of electron emission from the grain boundaries of chemical vapour deposition diamond films by tunneling atomic force microscopy

    SciTech Connect

    Chatterjee, Vijay; Harniman, Robert; May, Paul W.; Barhai, P. K.

    2014-04-28

    The emission of electrons from diamond in vacuum occurs readily as a result of the negative electron affinity of the hydrogenated surface due to features with nanoscale dimensions, which can concentrate electric fields high enough to induce electron emission from them. Electrons can be emitted as a result of an applied electric field (field emission) with possible uses in displays or cold-cathode devices. Alternatively, electrons can be emitted simply by heating the diamond in vacuum to temperatures as low as 350 °C (thermionic emission), and this may find applications in solar energy generation or energy harvesting devices. Electron emission studies usually use doped polycrystalline diamond films deposited onto Si or metallic substrates by chemical vapor deposition, and these films have a rough, faceted morphology on the micron or nanometer scale. Electron emission is often improved by patterning the diamond surface into sharp points or needles, the idea being that the field lines concentrate at the points lowering the barrier for electron emission. However, there is little direct evidence that electrons are emitted from these sharp tips. The few reports in the literature that have studied the emission sites suggested that emission came from the grain boundaries and not the protruding regions. We now present direct observation of the emission sites over a large area of polycrystalline diamond using tunneling atomic force microscopy. We confirm that the emission current comes mostly from the grain boundaries, which is consistent with a model for emission in which the non-diamond phase is the source of electrons with a threshold that is determined by the surrounding hydrogenated diamond surface.

  12. Studies of local structural distortions in strained ultrathin BaTiO3 films using scanning transmission electron microscopy.

    PubMed

    Park, Daesung; Herpers, Anja; Menke, Tobias; Heidelmann, Markus; Houben, Lothar; Dittmann, Regina; Mayer, Joachim

    2014-06-01

    Ultrathin ferroelectric heterostructures (SrTiO3/BaTiO3/BaRuO3/SrRuO3) were studied by scanning transmission electron microscopy (STEM) in terms of structural distortions and atomic displacements. The TiO2-termination at the top interface of the BaTiO3 layer was changed into a BaO-termination by adding an additional BaRuO3 layer. High-angle annular dark-field (HAADF) imaging by aberration-corrected STEM revealed that an artificially introduced BaO-termination can be achieved by this interface engineering. By using fast sequential imaging and frame-by-frame drift correction, the effect of the specimen drift was significantly reduced and the signal-to-noise ratio of the HAADF images was improved. Thus, a quantitative analysis of the HAADF images was feasible, and an in-plane and out-of-plane lattice spacing of the BaTiO3 layer of 3.90 and 4.22 Å were determined. A 25 pm shift of the Ti columns from the center of the unit cell of BaTiO3 along the c-axis was observed. By spatially resolved electron energy-loss spectroscopy studies, a reduction of the crystal field splitting (CFS, ΔL3=1.93 eV) and an asymmetric broadening of the eg peak were observed in the BaTiO3 film. These results verify the presence of a ferroelectric polarization in the ultrathin BaTiO3 film.

  13. Ultrastructural imaging and molecular modeling of live bacteria using soft x-ray contact microscopy with nanoseconds laser-plasma radiation

    NASA Astrophysics Data System (ADS)

    Kado, Masataka; Richardson, Martin C.; Gaebel, Kai; Torres, David S.; Rajyaguru, Jayshree; Muszynski, Michael J.

    1995-09-01

    X-ray images of the various live bacteria, such as Staphylococcus and Streptococcus, and micromolecule such as chromosomal DNA from Escherichis coli, and Lipopolysacchride from Burkholderia cepacia, are obtained with soft x-ray contact microscopy. A compact tabletop type glass laser system is used to produce x-rays from Al, Si, and Au targets. The PMMA photoresists are used to record x-ray images. An AFM (atomic force microscope) is used to reproduce the x-ray images from the developed photoresists. The performance of the 50nm spatial resolutions are achieved and images are able to be discussed on the biological view.

  14. The use of high energy laser-plasma sources in soft X-ray contact microscopy of living biological samples

    NASA Astrophysics Data System (ADS)

    Batani, D.; Botto, C.; Moret, M.; Milani, M.; Lucchini, G.; Eidmann, K.; Cotelli, F.; Lora Lamia Donin, C.; Poletti, G.; Ford, T.; Stead, A.

    2002-11-01

    In this paper the results of an experiment on soft X-ray contact microscopy using a laser-plasma source are presented. A resolution of 50 nm has been achieved imaging pig sperm cells, while other specimens, such as algae and yeast cells, showed internal details, proving the technique to be a powerful tool for biological investigations. Original biological information has been obtained and the conditions for optimal image formation have been studied.

  15. Inventing atomic resolution scanning dielectric microscopy to see a single protein complex operation live at resonance in a neuron without touching or adulterating the cell.

    PubMed

    Agrawal, Lokesh; Sahu, Satyajit; Ghosh, Subrata; Shiga, Takashi; Fujita, Daisuke; Bandyopadhyay, Anirban

    2016-12-01

    A substantial ion flow in a normally wet protein masks any other forms of signal transmission. We use hysteresis and linear conduction (both are artifacts) as a marker to precisely wet a protein, which restricts the ionic conduction (hysteresis disappears), and at the same time, it is not denatured (quantized conductance and Raman spectra are intact). Pure electric visualization of proteins at work by eliminating the screening of ions, electrons, would change the way we study biology. Here we discuss the technical challenges resolved for imaging a protein or live cell using nonlinear dielectric response (spatial distribution of conductance, capacitance and phase, GCP trio). We electromagnetically triggered electrical, mechanical, thermal and ionic resonant vibrations in a protein. During resonant oscillations, we imaged the protein using resonant scanning tunneling microscopy of biomaterials (Brestum) and during ionic firing we imaged live what happens inside an axon core of a neuron by using our atomic scale scanning dielectric microscopy (Asadim). Both Asadim and Brestum are housed in a homebuilt scanning tunneling microscope (bio-STM) and a special micro-grid developed by us (patent JP-5187804) for fractal supercomputing. We found the trick to turn a membrane transparent and see inside without making any physical contact. We image live that a protein molecule adopts a unique configuration for each resonance frequency, - thus far unknown to biology. "Membrane alone fires" is found to be wrong after a century, micro-neuro-filaments communicate prior to firing to decide its necessity and then regulate it suitably. We introduce a series of technologies e.g., fractal grid, point contact, micro THz antenna, to discover that from atomic structure to a living cell, the biomaterials vibrate collectively.

  16. Microstructure of highly strained BiFeO{sub 3} thin films: Transmission electron microscopy and electron-energy loss spectroscopy studies

    SciTech Connect

    Heon Kim, Young; Bhatnagar, Akash; Pippel, Eckhard; Hesse, Dietrich; Alexe, Marin

    2014-01-28

    Microstructure and electronic structure of highly strained bismuth ferrite (BiFeO{sub 3}) thin films grown on lanthanum aluminate substrates are studied using high-resolution transmission and scanning transmission electron microscopies and electron energy loss spectroscopy (EELS). Monoclinic and tetragonal phases were observed in films grown at different temperatures, and a mix of both phases was detected in a film grown at intermediate temperature. In this film, a smooth transition of the microstructure was found between the monoclinic and the tetragonal phases. A considerable increase in the c-axis parameters was observed in both phases compared with the rhombohedral bulk phase. The off-center displacement of iron (Fe) ions was increased in the monoclinic phase as compared with the tetragonal phase. EEL spectra show different electronic structures in the monoclinic and the tetragonal phases. These experimental observations are well consistent with the results of theoretical first-principle calculations performed.

  17. Microstructure of highly strained BiFeO3 thin films: Transmission electron microscopy and electron-energy loss spectroscopy studies

    NASA Astrophysics Data System (ADS)

    Heon Kim, Young; Bhatnagar, Akash; Pippel, Eckhard; Alexe, Marin; Hesse, Dietrich

    2014-01-01

    Microstructure and electronic structure of highly strained bismuth ferrite (BiFeO3) thin films grown on lanthanum aluminate substrates are studied using high-resolution transmission and scanning transmission electron microscopies and electron energy loss spectroscopy (EELS). Monoclinic and tetragonal phases were observed in films grown at different temperatures, and a mix of both phases was detected in a film grown at intermediate temperature. In this film, a smooth transition of the microstructure was found between the monoclinic and the tetragonal phases. A considerable increase in the c-axis parameters was observed in both phases compared with the rhombohedral bulk phase. The off-center displacement of iron (Fe) ions was increased in the monoclinic phase as compared with the tetragonal phase. EEL spectra show different electronic structures in the monoclinic and the tetragonal phases. These experimental observations are well consistent with the results of theoretical first-principle calculations performed.

  18. Piezoelectric force microscopy studies of PbTiO{sub 3} thin films grown via layer-by-layer gas phase reaction.

    SciTech Connect

    Park, M.; Hong, S.; Kim, J.; Kim, Y.; Buehlmann, S.; Kim, Y. K.; No, K.; Materials Science Division; Korea Advanced Inst. of Science and Technology; Imperial Col.; Samsung Electronics

    2009-01-01

    We fabricated 20 nm thick PbTiO{sub 3} films via reactive magnetron sputtering and studied the domain switching phenomena and retention properties using piezoresponse force microscopy. We found that multistep deposited PbTiO{sub 3} thin films showed 29% smaller rms roughness (2.5 versus 3.5 nm), 28% smaller coercive voltage (1.68 versus 2.32 V), 100% higher d{sub 33} value, and improved retention characteristic (89% versus 52% of remained poled domain area in 1280 min after poling) than single-step deposited PbTiO{sub 3} thin films. We attribute the improvement to the more complete chemical reaction between PbO and TiO{sub 2} during the film growth.

  19. Study of acetowhitening mechanisms in live mammalian cells with label-free subcellular-level multimodal nonlinear optical microscopy

    NASA Astrophysics Data System (ADS)

    Lin, Jian; Teh, Sengkhoon; Zheng, Wei; Wang, Zi; Huang, Zhiwei

    2015-03-01

    The tissue acetowhitening effect in acetic acid instillation procedure is a simple and economic method for neoplasia detection and has been clinically utilized since 1925. It is suspected that the optical property (e.g. scattering) change in acetowhitening is due to coagulation of intracellular proteins, but no experimental proof has been reported yet. In this work, we use third-harmonic generation (THG) and two-photon excited fluorescence (TPEF) to investigate the acetowhitening phenomenon induced by acidic acid in live mammalian cells without labeling. We studied the acetowhitening effect with different acetic acid concentrations and the co-localized TPEF and THG imaging on tryptophan and NADH at subcellular-level reveals that the acetowhitening phenomenon is highly related with proteins involved in metabolic pathways in the nucleus and cytoplasm in live cells.

  20. Video-Rate Confocal Microscopy for Single-Molecule Imaging in Live Cells and Superresolution Fluorescence Imaging

    PubMed Central

    Lee, Jinwoo; Miyanaga, Yukihiro; Ueda, Masahiro; Hohng, Sungchul

    2012-01-01

    There is no confocal microscope optimized for single-molecule imaging in live cells and superresolution fluorescence imaging. By combining the swiftness of the line-scanning method and the high sensitivity of wide-field detection, we have developed a, to our knowledge, novel confocal fluorescence microscope with a good optical-sectioning capability (1.0 μm), fast frame rates (<33 fps), and superior fluorescence detection efficiency. Full compatibility of the microscope with conventional cell-imaging techniques allowed us to do single-molecule imaging with a great ease at arbitrary depths of live cells. With the new microscope, we monitored diffusion motion of fluorescently labeled cAMP receptors of Dictyostelium discoideum at both the basal and apical surfaces and obtained superresolution fluorescence images of microtubules of COS-7 cells at depths in the range 0–85 μm from the surface of a coverglass. PMID:23083712

  1. Quantitative phase imaging of live cells with near on-axis digital holographic microscopy using constrained optimization approach

    NASA Astrophysics Data System (ADS)

    Pandiyan, Vimal Prabhu; Khare, Kedar; John, Renu

    2016-10-01

    We demonstrate a single-shot near on-axis digital holographic microscope that uses a constrained optimization approach for retrieval of the complex object function in the hologram plane. The recovered complex object is back-propagated from the hologram plane to image plane using the Fresnel back-propagation algorithm. A numerical aberration compensation algorithm is employed for correcting the aberrations in the object beam. The reference beam angle is calculated automatically using the modulation property of Fourier transform without any additional recording. We demonstrate this approach using a United States Air Force (USAF) resolution target as an object on our digital holographic microscope. We also demonstrate this approach by recovering the quantitative phase images of live yeast cells, red blood cells and dynamics of live dividing yeast cells.

  2. Lattice reorientation in tetragonal PMN-PT thin film induced by focused ion beam preparation for transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Denneulin, Thibaud; Maeng, Wanjoo; Eom, Chang-Beom; Hÿtch, Martin

    2017-02-01

    Focused ion beam sample preparation for transmission electron microscopy (TEM) can induce relaxation mechanisms in epitaxial thin films. Here, we describe a relaxation mechanism that can occur in materials having a tetragonal structure. We investigated the lattice structure of a 600 nm thick 0.4 [ Pb ( Mg 1 / 3 Nb 2 / 3 ) O 3 ] - 0.6 [ PbTiO 3 ] layer grown by epitaxy on (110) GdScO3 substrate using geometrical phase analysis applied to high resolution TEM images. The lattice mismatch at the interface is expected to favor the formation of c-domains. However, it was measured that the out-of-plane lattice parameter can decrease abruptly along the growth direction and the transition depends on the thickness of the TEM lamella. Different observations indicate that the crystal flipped by 90° following the preparation of the sample, so that the c-axis is oriented in the thinning direction. Such a mechanism can easily lead to misinterpretations and might happen in other materials with a similar structure.

  3. Scanning transmission X-ray microscopy of nano structured thin film catalysts for proton-exchange-membrane fuel cells

    NASA Astrophysics Data System (ADS)

    Lee, Vincent; Berejnov, Viatcheslav; West, Marcia; Kundu, Sumit; Susac, Darija; Stumper, Jürgen; Atanasoski, Radoslav T.; Debe, Mark; Hitchcock, Adam P.

    2014-10-01

    Scanning transmission X-ray microscopy (STXM) has been applied to characterize nano structured thin film (NSTF) catalysts implemented as electrode materials in proton-exchange-membrane (PEM) fuel cells. STXM is used to study all chemical constituents at various stages in the fabrication process, from the perylene red (PR149) starting material, through the formation of the uncoated perylene whiskers, their coated form with Pt-based catalyst, and toward the NSTF anode fully integrated into the catalyst coated membrane (CCM). CCM samples were examined prior to operational testing and after several different accelerated testing protocols: start-up/shut-down (SU/SD), and reversal tests. It was found that, while the perylene support material is present in the pre-test samples, it was completely absent in the post-test samples. We attribute this loss of perylene material to the presence of cracks in the catalyst combined with intensive hydrogenation processes happening at the anode during operation. Despite the loss of the perylene support, the platinum shells forming the NSTF anode catalyst layer performed well during the tests.

  4. Contact resistance asymmetry of amorphous indium-gallium-zinc-oxide thin-film transistors by scanning Kelvin probe microscopy

    NASA Astrophysics Data System (ADS)

    Chen-Fei, Wu; Yun-Feng, Chen; Hai, Lu; Xiao-Ming, Huang; Fang-Fang, Ren; Dun-Jun, Chen; Rong, Zhang; You-Dou, Zheng

    2016-05-01

    In this work, a method based on scanning Kelvin probe microscopy is proposed to separately extract source/drain (S/D) series resistance in operating amorphous indium-gallium-zinc-oxide (a-IGZO) thin-film transistors. The asymmetry behavior of S/D contact resistance is deduced and the underlying physics is discussed. The present results suggest that the asymmetry of S/D contact resistance is caused by the difference in bias conditions of the Schottky-like junction at the contact interface induced by the parasitic reaction between contact metal and a-IGZO. The overall contact resistance should be determined by both the bulk channel resistance of the contact region and the interface properties of the metal-semiconductor junction. Project supported by the Key Industrial R&D Program of Jiangsu Province, China (Grant No. BE2015155), the Priority Academic Program Development of Higher Education Institutions of Jiangsu Province, China, and the Fundamental Research Funds for the Central Universities, China (Grant No. 021014380033).

  5. Studying the Polarization Switching in Polycrystalline BiFeO3 Films by 2D Piezoresponse Force Microscopy

    PubMed Central

    Jin, Yaming; Lu, Xiaomei; Zhang, Junting; Kan, Yi; Bo, Huifeng; Huang, Fengzhen; Xu, Tingting; Du, Yingchao; Xiao, Shuyu; Zhu, Jinsong

    2015-01-01

    For rhombohedral multiferroelectrics, non-180° ferroelectric domain switching may induce ferroelastic and/or (anti-)ferromagnetic effect. So the determination and control of ferroelectric domain switching angles is crucial for nonvolatile information storage and exchange-coupled magnetoelectric devices. We try to study the intrinsic characters of polarization switching in BiFeO3 by introducing a special data processing method to determine the switching angle from 2D PFM (Piezoresponse Force Microscopy) images of randomly oriented samples. The response surface of BiFeO3 is first plotted using the piezoelectric tensor got from first principles calculations. Then from the normalized 2D PFM signals before and after switching, the switching angles of randomly oriented BiFeO3 grains can be determined through numerical calculations. In the polycrystalline BiFeO3 films, up to 34% of all switched area is that with original out-of-plane (OP) polarization parallel to the poling field. 71° polarization switching is more favorable, with the area percentages of 71°, 109° and 180° domain switching being about 42%, 29% and 29%, respectively. Our analysis further reveals that IP stress and charge migration have comparable effect on switching, and they are sensitive to the geometric arrangements. This work helps exploring a route to control polarization switching in BiFeO3, so as to realize desirable magnetoelectric coupling. PMID:26192555

  6. TIRET microscopy: monitoring protein (amyloid precursor protein and beta-secretase) interaction on the surface of living cells

    NASA Astrophysics Data System (ADS)

    von Arnim, Christine; Wagner, Michael; Weber, Petra; Schneckenburger, Herbert

    2007-02-01

    Total internal reflection fluorescence microscopy (TIRFM) and non-radiative energy transfer (FRET) measurements have been combined in order to examine co-localization of the amyloid precursor protein (APP) and the β-site APPcleaving enzyme (BACE) in human glioblastoma cells. So far, these proteins have been co-localized within whole cells (depending on the intracellular amount of cholesterol) and in some cases also within their plasma membranes. This supports the present hypothesis of localization within lipid domains on the cell surface and co-internalization via endocytosis.

  7. Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

    PubMed Central

    Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

    2016-01-01

    Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level. PMID:27795878

  8. In vivo microdissection and live embryo imaging by two-photon microscopy to study Drosophila melanogaster early development

    NASA Astrophysics Data System (ADS)

    Supatto, Willy; Brouzes, Eric; Farge, Emmanuel; Beaurepaire, Emmanuel

    2004-09-01

    Animal embryo development exhibits a complex choreography of cell movements highly regulated both in time and space. This sequence of morphogenetic movements is initiated at gastrulation and is tightly controlled by a cascade of developmental gene expression. We have recently reported that developmental gene expression can in turn be mechanically regulated by morphogenetic movements during Drosophila melanogaster early development. In order to study this phenomenon of mechanically induced gene expression, it is necessary to develop new techniques of in vivo investigation. We show that the combination of femtosecond pulse intratissue surgery and two-photon-excitation fluorescence (2PEF) microscopy is a powerful tool for (i) disrupting natural morphogenetic movements and (ii) imaging native and disrupted morphogenetic movements during Drosophila development. (i) First, non-linear-absorption-mediated photo-disruption makes it possible to perform controlled intra-vital micro-dissections resulting in the modulation of morphogenetic movements and subsequent mechano-sensitive gene expression. (ii) Second, in vivo 2PEF microscopy of transgenic GFP systems appears to be an excellent technique for long-term in vivo imaging of the complex morphogenetic movements involved in normal or perturbed Drosophila gastrulation. Together, these two techniques provide a powerful novel approach to study embryo development.

  9. Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size.

    PubMed

    Puah, Wee Choo; Chinta, Rambabu; Wasser, Martin

    2017-03-15

    Time-lapse microscopy is a powerful tool to investigate cellular and developmental dynamics. In Drosophila melanogaster, it can be used to study division cycles in embryogenesis. To obtain quantitative information from 3D time-lapse data and track proliferating nuclei from the syncytial stage until gastrulation, we developed an image analysis pipeline consisting of nuclear segmentation, tracking, annotation and quantification. Image analysis of maternal-haploid (mh) embryos revealed that a fraction of haploid syncytial nuclei fused to give rise to nuclei of higher ploidy (2n, 3n, 4n). Moreover, nuclear densities in mh embryos at the mid-blastula transition varied over threefold. By tracking synchronized nuclei of different karyotypes side-by-side, we show that DNA content determines nuclear growth rate and size in early interphase, while the nuclear to cytoplasmic ratio constrains nuclear growth during late interphase. mh encodes the Drosophila ortholog of human Spartan, a protein involved in DNA damage tolerance. To explore the link between mh and chromosome instability, we fluorescently tagged Mh protein to study its subcellular localization. We show Mh-mKO2 localizes to nuclear speckles that increase in numbers as nuclei expand in interphase. In summary, quantitative microscopy can provide new insights into well-studied genes and biological processes.

  10. Live, video-rate super-resolution microscopy using structured illumination and rapid GPU-based parallel processing.

    PubMed

    Lefman, Jonathan; Scott, Keana; Stranick, Stephan

    2011-04-01

    Structured illumination fluorescence microscopy is a powerful super-resolution method that is capable of achieving a resolution below 100 nm. Each super-resolution image is computationally constructed from a set of differentially illuminated images. However, real-time application of structured illumination microscopy (SIM) has generally been limited due to the computational overhead needed to generate super-resolution images. Here, we have developed a real-time SIM system that incorporates graphic processing unit (GPU) based in-line parallel processing of raw/differentially illuminated images. By using GPU processing, the system has achieved a 90-fold increase in processing speed compared to performing equivalent operations on a multiprocessor computer--the total throughput of the system is limited by data acquisition speed, but not by image processing. Overall, more than 350 raw images (16-bit depth, 512 × 512 pixels) can be processed per second, resulting in a maximum frame rate of 39 super-resolution images per second. This ultrafast processing capability is used to provide immediate feedback of super-resolution images for real-time display. These developments are increasing the potential for sophisticated super-resolution imaging applications.

  11. Quantitative microscopy uncovers ploidy changes during mitosis in live Drosophila embryos and their effect on nuclear size

    PubMed Central

    Puah, Wee Choo; Chinta, Rambabu

    2017-01-01

    ABSTRACT Time-lapse microscopy is a powerful tool to investigate cellular and developmental dynamics. In Drosophila melanogaster, it can be used to study division cycles in embryogenesis. To obtain quantitative information from 3D time-lapse data and track proliferating nuclei from the syncytial stage until gastrulation, we developed an image analysis pipeline consisting of nuclear segmentation, tracking, annotation and quantification. Image analysis of maternal-haploid (mh) embryos revealed that a fraction of haploid syncytial nuclei fused to give rise to nuclei of higher ploidy (2n, 3n, 4n). Moreover, nuclear densities in mh embryos at the mid-blastula transition varied over threefold. By tracking synchronized nuclei of different karyotypes side-by-side, we show that DNA content determines nuclear growth rate and size in early interphase, while the nuclear to cytoplasmic ratio constrains nuclear growth during late interphase. mh encodes the Drosophila ortholog of human Spartan, a protein involved in DNA damage tolerance. To explore the link between mh and chromosome instability, we fluorescently tagged Mh protein to study its subcellular localization. We show Mh-mKO2 localizes to nuclear speckles that increase in numbers as nuclei expand in interphase. In summary, quantitative microscopy can provide new insights into well-studied genes and biological processes. PMID:28108477

  12. Hard x-ray contact microscopy with 250 nm spatial resolution using a LiF film detector and a tabletop microsource

    NASA Astrophysics Data System (ADS)

    Almaviva, S.; Bonfigli, F.; Franzini, I.; Lai, A.; Montereali, R. M.; Pelliccia, D.; Cedola, A.; Lagomarsino, S.

    2006-07-01

    An innovative route for deep-submicrometer spatial resolution hard x-ray microscopy with tabletop x-ray source is proposed. A film of lithium fluoride (LiF) was used as imaging detector in contact mode. We present here the x-ray images recorded on LiF films of a Fresnel zone plate with submicrometer gold structures and of an onion cataphyll. The images were read with an optical confocal microscope in fluorescence mode. The measured spatial resolution was about 250nm, i.e., close to the resolution limit of the confocal microscope. The advantages and drawbacks, and the possible improvements, of this route are discussed.

  13. Poled polymer thin-film gratings studied with far-field optical diffraction and second-harmonic near-field microscopy.

    PubMed

    Schaller, R D; Saykally, R J; Shen, Y R; Lagugné-Labarthet, F

    2003-08-01

    Electrical poling induces polar ordering of molecules in a grating that has been holographically inscribed on a thin film of polymer with azobenzene side chains. The resulting chi2 grating, seen by second-harmonic-generation (SHG) near-field scanning optical microscopy, can have a periodic structure that is significantly different from the topographical image. The far-field linear and SHG diffration patterns correlate well with the grating structures. Poling of the thin-film grating, which presumably has photodriven nonuniform material properties within each period, leads to the more complex structure of the chi2 grating.

  14. Hard x-ray contact microscopy with 250 nm spatial resolution using a LiF film detector and a tabletop microsource

    SciTech Connect

    Almaviva, S.; Bonfigli, F.; Franzini, I.; Lai, A.; Montereali, R. M.; Pelliccia, D.; Cedola, A.; Lagomarsino, S.

    2006-07-31

    An innovative route for deep-submicrometer spatial resolution hard x-ray microscopy with tabletop x-ray source is proposed. A film of lithium fluoride (LiF) was used as imaging detector in contact mode. We present here the x-ray images recorded on LiF films of a Fresnel zone plate with submicrometer gold structures and of an onion cataphyll. The images were read with an optical confocal microscope in fluorescence mode. The measured spatial resolution was about 250 nm, i.e., close to the resolution limit of the confocal microscope. The advantages and drawbacks, and the possible improvements, of this route are discussed.

  15. Ferroelectric domains in epitaxial PbxSr1-xTiO3 thin films investigated using X-ray diffraction and piezoresponse force microscopy

    NASA Astrophysics Data System (ADS)

    Fernandez-Peña, S.; Lichtensteiger, C.; Zubko, P.; Weymann, C.; Gariglio, S.; Triscone, J.-M.

    2016-08-01

    We present a detailed study of compressively strained PbxSr1-xTiO3 thin films grown by off-axis radio frequency magnetron sputtering on (001)-oriented Nb-doped SrTiO3 substrates. Film tetragonality and the ferroelectric critical temperatures are measured for samples of different composition and thickness and compared with a phenomenological Landau-Devonshire model. 180∘ ferroelectric domains are observed using both X-ray diffraction and piezoresponse force microscopy and domain sizes obtained by the two techniques are compared and discussed.

  16. Origin of surface potential change during ferroelectric switching in epitaxial PbTiO{sub 3} thin films studied by scanning force microscopy.

    SciTech Connect

    Kim, Y.; Bae, C.; Ryu, K.; Ko, H.; Kim, Y. K.; Hong, S.; Shin, H.; Materials Science Division; Kookmin Univ.; Korea Advanced Inst. of Science and Technology; Samsung Advanced Inst. Science and Technology

    2009-01-01

    We investigated the surface potential of the ferroelectric domains of the epitaxial PbTiO{sub 3} (PTO) films using both Kelvin probe and piezoresponse force microscopy. The surface potential changes as a function of applied biases suggested that the amount and sign of surface potentials depend on the correlation between polarization and screen charges. It also suggested that the trapped negative charges exist on the as-deposited PTO surfaces. Injected charges and their resultant surface potentials are investigated by grounded tip scans. The results unveiled the origin of surface potential changes during ferroelectric switching in the epitaxial PTO films.

  17. From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

    PubMed Central

    Spiegelhalter, Coralie; Tosch, Valérie; Hentsch, Didier; Koch, Marc; Kessler, Pascal; Schwab, Yannick; Laporte, Jocelyn

    2010-01-01

    Background In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). Methodology To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. Conclusion Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM. PMID:20140253

  18. Local Viscoelastic Properties of Live Cells Investigated Using Dynamic and Quasi-Static Atomic Force Microscopy Methods

    PubMed Central

    Cartagena, Alexander; Raman, Arvind

    2014-01-01

    The measurement of viscoelasticity of cells in physiological environments with high spatio-temporal resolution is a key goal in cell mechanobiology. Traditionally only the elastic properties have been measured from quasi-static force-distance curves using the atomic force microscope (AFM). Recently, dynamic AFM-based methods have been proposed to map the local in vitro viscoelastic properties of living cells with nanoscale resolution. However, the differences in viscoelastic properties estimated from such dynamic and traditional quasi-static techniques are poorly understood. In this work we quantitatively reconstruct the local force and dissipation gradients (viscoelasticity) on live fibroblast cells in buffer solutions using Lorentz force excited cantilevers and present a careful comparison between mechanical properties (local stiffness and damping) extracted using dynamic and quasi-static force spectroscopy methods. The results highlight the dependence of measured viscoelastic properties on both the frequency at which the chosen technique operates as well as the interactions with subcellular components beyond certain indentation depth, both of which are responsible for differences between the viscoelasticity property maps acquired using the dynamic AFM method against the quasi-static measurements. PMID:24606928

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

    PubMed Central

    Okada, Tomoko; Ogura, Toshihiko

    2017-01-01

    Nanometre-scale-resolution imaging technologies for liquid-phase specimens are indispensable tools in various scientific fields. In biology, observing untreated living cells in a medium is essential for analysing cellular functions. However, nanoparticles that bind living cells in a medium are hard to detect directly using traditional optical or electron microscopy. Therefore, we previously developed a novel scanning electron-assisted dielectric microscope (SE-ADM) capable of nanoscale observations. This method enables observation of intact cells in aqueous conditions. Here, we use this SE-ADM system to clearly observe antibody-binding nanobeads in liquid-phase. We also report the successful direct detection of streptavidin-conjugated nanobeads binding to untreated cells in a medium via a biotin-conjugated anti-CD44 antibody. Our system is capable of obtaining clear images of cellular organelles and beads on the cells at the same time. The direct observation of living cells with nanoparticles in a medium allowed by our system may contribute the development of carriers for drug delivery systems (DDS). PMID:28230204

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

    NASA Astrophysics Data System (ADS)

    Okada, Tomoko; Ogura, Toshihiko

    2017-02-01

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

  1. Films made of cellulose nanofibrils: surface modification by adsorption of a cationic surfactant and characterization by computer-assisted electron microscopy

    NASA Astrophysics Data System (ADS)

    Syverud, K.; Xhanari, K.; Chinga-Carrasco, G.; Yu, Y.; Stenius, P.

    2011-02-01

    Films made of nanofibrils were modified by adsorption of a cationic surfactant directly on the film surfaces. The nanofibrils were prepared by 2,2,6,6-tetramethylpiperidinyl-1-oxyl (TEMPO)-mediated oxidation and mechanical fibrillation, and were relatively homogeneous in size. The average nanofibril diameter and surface porosity was quantified based on computer-assisted field-emission scanning electron microscopy (FE-SEM). The cationic surfactant used in the adsorption was n-hexadecyl trimethylammonium bromide (cetyltrimethylammonium bromide, CTAB). The adsorption of CTAB was confirmed by Fourier transform infrared (FTIR) spectroscopy and high-resolution transmission electron microscopy (HRTEM) analyses. It was shown that the adsorbed layer of CTAB increased the hydrophobicity, without affecting the tensile index significantly. This capability, combined with the antiseptic properties of CTAB, may be a major advantage for several applications.

  2. Atomic force microscopy study of surface topography of films of cholesteric oligomer- and polymer-based mixtures with photovariable helix pitch

    NASA Astrophysics Data System (ADS)

    Bobrovsky, Alexey; Sinitsyna, Olga; Abramchuk, Sergei; Yaminsky, Igor; Shibaev, Valery

    2013-01-01

    The surface topography of glass-forming polymers and oligomer cholesteric systems with a phototunable helix pitch was studied. For this purpose several mixtures based on nematic polyacrylate and cholesteric cyclosiloxanes doped with chiral-photochromic dopant were prepared and investigated. The molecules of chiral-photochromic dopant consist of isosorbide chiral moiety and cinnamic C=C double bonds capable of E-Z photoisomerizing. UV irradiation of the planarly oriented films of mixtures leads to dopant photoisomerization and changes of its helical twisting power. During this process irreversible changes of helix pitch values take place, which allows one to obtain the same cholesteric systems with different helix pitch values. The films of the annealed mixtures were studied by atomic force microscopy and transmission electron microscopy. The correlations between the features of surface topography and helix pitch of cholesteric supramolecular structure were found and discussed.

  3. Spectrally resolved phase-shifting interference microscopy: technique based on optical coherence tomography for profiling a transparent film on a patterned substrate

    SciTech Connect

    Debnath, Sanjit K.; Kim, Seung-Woo; Kothiyal, Mahendra P.; Hariharan, Parameswaran

    2010-12-01

    Spectrally resolved white-light phase-shifting interference microscopy has been used for measurements of the thickness profile of a transparent thin-film layer deposited upon a patterned structure exhibiting steps and discontinuities. We describe a simple technique, using an approach based on spectrally resolved optical coherence tomography, that makes it possible to obtain directly a thickness profile along a line by inverse Fourier transformation of the complex spectral interference function.

  4. Spectrally resolved phase-shifting interference microscopy: technique based on optical coherence tomography for profiling a transparent film on a patterned substrate.

    PubMed

    Debnath, Sanjit K; Kim, Seung-Woo; Kothiyal, Mahendra P; Hariharan, Parameswaran

    2010-12-01

    Spectrally resolved white-light phase-shifting interference microscopy has been used for measurements of the thickness profile of a transparent thin-film layer deposited upon a patterned structure exhibiting steps and discontinuities. We describe a simple technique, using an approach based on spectrally resolved optical coherence tomography, that makes it possible to obtain directly a thickness profile along a line by inverse Fourier transformation of the complex spectral interference function.

  5. Spectrally resolved white-light phase-shifting interference microscopy for thickness-profile measurements of transparent thin film layers on patterned substrates.

    PubMed

    Debnath, Sanjit K; Kothiyal, Mahendra P; Schmit, Joanna; Hariharan, Parameswaran

    2006-05-29

    We describe how spectrally-resolved white-light phase-shifting interference microscopy with a windowed 8-step algorithm can be used for rapid and accurate measurements of the thickness profile of transparent thin film layers with a wide range of thicknesses deposited upon patterned structures exhibiting steps and discontinuities. An advantage of this technique is that it can be implemented with readily available hardware.

  6. Imaging of mucus clearance in the airways of living spontaneously breathing mice by optical coherence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Pieper, Mario; Schulz-Hildebrandt, Hinnerk; Hüttmann, Gereon; König, Peter

    2016-03-01

    Mucus transport is essential to remove inhaled particles and pathogens from the lung. Impaired removal of mucus often results in worsening of lung diseases. To understand the mechanisms of mucus transport and to monitor the impact of therapeutic strategies, it is essential to visualize airways and mucus in living animals without disturbing transport processes by intubation or surgically opening the airways. We developed a custom-built optical coherence microscope (OCM) providing a lateral and axial resolution of approximately 1.5 µm with a field of view of 2 mm at up to 150 images/s. Images of the intact trachea and its mucus transport were recorded in anesthetized spontaneously breathing mice. NaCl solution (0.9% and 7%) or Lipopolysaccharide were applied intranasally. OCM resolved detailed structure of the trachea and enabled measuring the airway surface liquid (ASL) thickness through the tracheal wall. Without stimulation, the amount of ASL was only a few µm above the epithelium and remained constant. After intranasal application of 30 µl saline at different concentrations, an early fast cough-like fluid removal with velocities higher than 1 mm/s was observed that removed a high amount of liquid. The ASL thickness increased transiently and quickly returned to levels before stimulation. In contrast to saline, application of Lipopolysaccharide induced substantial mucus release and an additional slow mucus transport by ciliary beating (around 100 µm/s) towards the larynx was observed. In conclusion, OCM is appropriate unique tool to study mechanisms of mucus transport in the airways and effects of therapeutic interventions in living animals.

  7. Optical coherence microscopy of living cells and bioengineered tissue dynamics in high-resolution cross-section.

    PubMed

    Hasegawa, Akiyuki; Haraguchi, Yuji; Oikaze, Hirotoshi; Kabetani, Yasuhiro; Sakaguchi, Katsuhisa; Shimizu, Tatsuya

    2017-04-01

    Optical coherence tomography (OCT) is a valuable tool in the cross-sectional observation/analysis of three-dimensional (3-D) biological tissues, and that histological observation is important clinically. However, the resolution of the technology is approximately 10-20 μm. In this study, optical coherence microscopy (OCM), a tomographic system combining OCT technology with a microscopic technique, was constructed for observing cells individually with a resolution at the submicrometer level. Cells and 3-D tissues fabricated by cell sheet technology were observed by OCM. Importantly, the cell nuclei and cytoplasm could be clearly distinguished, and the time-dependent dynamics of cell-sheet tissues could be observed in detail. Additionally, the 3-D migration of cells in the bioengineered tissue was also detected using OCM and metal-labeled cells. Bovine aortic endothelial cells, but not NIH3T3 murine embryonic skin fibroblasts, actively migrated within the 3-D tissues. This study showed that the OCM system would be a valuable tool in the fields of cell biology, tissue engineering, and regenerative medicine. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 481-488, 2017.

  8. Growth and structure of water on SiO2 films on Si investigated byKelvin probe microscopy and in situ X-ray Spectroscopies

    SciTech Connect

    Verdaguer, A.; Weis, C.; Oncins, G.; Ketteler, G.; Bluhm, H.; Salmeron, M.

    2007-06-14

    The growth of water on thin SiO{sub 2} films on Si wafers at vapor pressures between 1.5 and 4 torr and temperatures between -10 and 21 C has been studied in situ using Kelvin Probe Microscopy and X-ray photoemission and absorption spectroscopies. From 0 to 75% relative humidity (RH) water adsorbs forming a uniform film 4-5 layers thick. The surface potential increases in that RH range by about 400 mV and remains constant upon further increase of the RH. Above 75% RH the water film grows rapidly, reaching 6-7 monolayers at around 90% RH and forming a macroscopic drop near 100%. The O K-edge near-edge X-ray absorption spectrum around 75% RH is similar to that of liquid water (imperfect H-bonding coordination) at temperatures above 0 C and ice-like below 0 C.

  9. FeMn/Fe/Co/Cu(1,1,10) films studied using the magneto-optic Kerr effect and photoemission electron microscopy

    SciTech Connect

    Meng, Y.; Li, J.; Tan, A.; Park, J.; Jin, E.; Son, H.; Doran, A.; Scholl, A.; Arenholz, E.; Zhao, H. W.; Hwang, Chanyong; Qiu, Z. Q.

    2011-07-31

    FeMn/Fe/Co/Cu(1,1,10) films were grown epitaxially and investigated using the magneto-optic Kerr effect and photoemission electron microscopy. We found that FeMn/Fe/Co/Cu(1,1,10) exhibits the same properties as FeMn/Co/Cu(1,1,10) for the ferromagnetic phase of the face centered cubic (fcc) Fe film but a different property for the non-ferromagnetic phase of the fcc Fe film. This result indicates that the characteristic property reported in the literature for FeMn/Co/Cu(001) comes from the FeMn spin structure and is independent of the ferromagnetic layer.

  10. Microinterferometry: Three-Dimensional Reconstruction of Surface Microtopography for Thin-Film and Wetting Studies by Reflection Interference Contrast Microscopy (RICM).

    PubMed

    Wiegand, G; Neumaier, K R; Sackmann, E

    1998-10-10

    We present an improved theory of image formation by reflection interference contrast microscopy (RICM) for structural studies of stratified films on planar substrates and propose a new theoretical approach to analyzing the surface profile of nonplanar films. We demonstrate the validity of the new approach by analyzing the fringe patterns of RICM images from wedge-shaped liquid films and spherical probes. By simulation of various scenarios, we study the effect of finite-aperture illumination and the shape of the nonplanar interface on the interference fringe pattern of RICM images. We show how the reconstruction of the microscopic topography of the sample from the fringe spacing is corrected by angular and curvature correction terms. We discuss the variation of the mean intensity of the fringe patterns and the decay in the fringe amplitude with increasing fringe order that is caused by nonplanar interfaces of different slope.

  11. The effect of oxidation on charge carrier motion in PbS quantum dot thin films studied with Kelvin Probe Microscopy

    NASA Astrophysics Data System (ADS)

    Nguyen Hoang, Lan Phuong; Williams, Pheona; Moscatello, Jason; Aidala, Katherine E.; Aidala Group Team

    We developed a technique that uses scanning probe microscopy (SPM) to study the real-time injection and extraction of charge carriers in thin film devices. We investigate the effects of oxidation on thin films of Lead Sulfide (PbS) quantum dots with tetrabutyl-ammonium-iodide (TBAI) ligands in an inverted field effect transistor geometry with gold electrodes. By positioning the SPM tip at an individual location and using Kelvin Probe Force Microscopy (KPFM) to measure the potential over time, we can record how the charge carriers respond to changing the backgate voltage with grounded source and drain electrodes. We see relatively fast screening for negative backgate voltages because holes are quickly injected into the PbS film. The screening is slower for positive gate voltages, because some of these holes are trapped and therefore less mobile. We probe these trapped holes by applying different gate voltages and recording the change in potential at the surface. There are mixed reports about the effect of air exposure on thin films of PbS quantum dots, with initial exposure appearing to be beneficial to device characteristics. We study the change in current, mobility, and charge injection and extraction as measured by KPFM over hours and days of exposure to air. This work is supported by NSF Grant DMR-0955348, and the Center for Heirarchical Manufacturing at the University of Massachusetts, Amherst (NSF CMMI-1025020).

  12. Some aspects of pulsed laser deposited nanocrystalline LaB(6) film: atomic force microscopy, constant force current imaging and field emission investigations.

    PubMed

    Late, Dattatray J; Date, Kalyani S; More, Mahendra A; Misra, Pankaj; Singh, B N; Kukreja, Lalit M; Dharmadhikari, C V; Joag, Dilip S

    2008-07-02

    Nanocrystalline lanthanum hexaboride (LaB(6)) films have been deposited on molybdenum foil by the pulsed laser deposition (PLD) technique. The as-deposited films were characterized by x-ray diffraction (XRD), scanning electron microscopy (SEM) and atomic force microscopy (AFM). The XRD pattern shows the cubic crystallinity of the LaB(6) film. The AFM studies reveal that the conical shaped LaB(6) nanostructures have height 60 nm, base 800 nm, and a typical radius of curvature ∼20 nm. A comparison of force and in situ current imaging AFM studies reveals that current contrast does not originate from the surface topography of the LaB(6) film. Field emission studies have been performed in the planar diode configuration. A current density of 4.4 × 10(-2) A cm(-2) is drawn from the actual emitting area. The Fowler-Nordheim plot is found to be linear, in accordance with the quantum mechanical tunneling phenomenon. The field enhancement factor is estimated to be 3585, indicating that the field emission is from LaB(6) nanocrystallites present on the emitter surface, as confirmed by the AFM. The emission current-time plots show current stability to the extent of 5% fluctuation about the average current over a period of 3 h.

  13. "Un-annealed and Annealed Pd Ultra-Thin Film on SiC Characterized by Scanning Probe Microscopy and X-ray Photoelectron Spectroscopy"

    NASA Technical Reports Server (NTRS)

    Lu, W. J.; Shi, D. T.; Elshot, K.; Bryant, E.; Lafate, K.; Chen, H.; Burger, A.; Collins, W. E.

    1998-01-01

    Pd/SiC has been used as a hydrogen and a hydrocarbon gas sensor operated at high temperature. UHV (Ultra High Vacuum)-Scanning Tunneling Microscopy (STM), Atomic Force Microscopy (AFM) and X-ray Photoelectron Spectroscopy (XPS) techniques were applied to study the relationship between the morphology and chemical compositions for Pd ultra-thin films on SiC (less than 30 angstroms) at different annealing temperatures. Pd ultra-thin film on 6H-SiC was prepared by the RF sputtering method. The morphology from UHV-STM and AFM shows that the Pd thin film was well deposited on SiC substrate, and the Pd was partially aggregated to round shaped participates at an annealing temperature of 300 C. At 400 C, the amount of surface participates decreases, and some strap shape participates appear. From XPS, Pd2Si was formed on the surface after annealing at 300 C, and all Pd reacted with SiC to form Pd2Si after annealing at 400 C. The intensity of the XPS Pd peak decreases enormously at 400 C. The Pd film diffused into SiC, and the Schottky barrier height has almost no changes. The work shows the Pd sicilides/SiC have the same electronic properties with Pd/SiC, and explains why the Pd/SiC sensor still responds to hydrogen at high operating temperatures.

  14. Electron microscopy study of MOCVD-grown TiO sub 2 thin films and TiO sub 2 /Al sub 2 O sub 3 interfaces

    SciTech Connect

    Gao, Y.; Merkle, K.L.; Chang, H.L.M.; Zhang, T.J.; Lam, D. J.

    1990-11-01

    TiO{sub 2} thin films grown on (11{bar 2}0) sapphire at 800{degree}C by the MOCVD technique have been characterized by transmission electron microscopy. The TiO{sub 2} thin films are single crystalline and have the rutile structure. The epitaxial orientation relationship between the TiO{sub 2} thin films (R) and the substrate (S) has been found to be: (101)(0{bar 1}0){sub R}{parallel}(11{bar 2}0)(0001){sub S}. Growth twins in the films are commonly observed with the twin plane {l brace}101{r brace} and twinning direction {l angle}011{r angle}. Detailed atomic structures of the twin boundaries and TiO{sub 2}/{alpha}-Al{sub 2}O{sub 3} interfaces have been investigated by high-resolution electron microscopy (HREM). When the interfaces are viewed in the direction of (0{bar 1}0){sub R}/(0001){sub S}, the interfaces are found to be structurally coherent in the direction of ({bar 1}01){sub R}/(1{bar 1}00){sub S}, in which the lattice mismatch at the interfaces is about 0.5%. 8 refs., 4 figs.

  15. Long-lived ultrafast spin precession in manganese alloys films with a large perpendicular magnetic anisotropy.

    PubMed

    Mizukami, S; Wu, F; Sakuma, A; Walowski, J; Watanabe, D; Kubota, T; Zhang, X; Naganuma, H; Oogane, M; Ando, Y; Miyazaki, T

    2011-03-18

    Spin precession with frequencies up to 280 GHz is observed in Mn(3-δ)Ga alloy films with a perpendicular magnetic anisotropy constant K(u)∼15  M erg/cm(3). The damping constant α, characterizing macroscopic spin relaxation and being a key factor in spin-transfer-torque systems, is not larger than 0.008 (0.015) for the δ=1.46 (0.88) film. Those are about one-tenth of α values for known materials with large K(u). First-principles calculations well describe both low α and large K(u) for these alloys.

  16. Covisualization by computational optical-sectioning microscopy of integrin and associated proteins at the cell membrane of living onion protoplasts

    NASA Technical Reports Server (NTRS)

    Gens, J. S.; Reuzeau, C.; Doolittle, K. W.; McNally, J. G.; Pickard, B. G.; Evans, M. L. (Principal Investigator)

    1996-01-01

    Using higher-resolution wide-field computational optical-sectioning fluorescence microscopy, the distribution of antigens recognized by antibodies against animal beta 1 integrin, fibronectin, and vitronectin has been visualized at the outer surface of enzymatically protoplasted onion epidermis cells and in depectinated cell wall fragments. On the protoplast all three antigens are colocalized in an array of small spots, as seen in raw images, in Gaussian filtered images, and in images restored by two different algorithms. Fibronectin and vitronectin but not beta 1 integrin antigenicities colocalize as puncta in comparably prepared and processed images of the wall fragments. Several control visualizations suggest considerable specifity of antibody recognition. Affinity purification of onion cell extract with the same anti-integrin used for visualization has yielded protein that separates in SDS-PAGE into two bands of about 105-110 and 115-125 kDa. These bands are again recognized by the visualization antibody, which was raised against the extracellular domain of chicken beta 1 integrin, and are also recognized by an antibody against the intracellular domain of chicken beta 1 integrin. Because beta 1 integrin is a key protein in numerous animal adhesion sites, it appears that the punctate distribution of this protein in the cell membranes of onion epidermis represents the adhesion sites long known to occur in cells of this tissue. Because vitronectin and fibronection are matrix proteins that bind to integrin in animals, the punctate occurrence of antigenically similar proteins both in the wall (matrix) and on enzymatically prepared protoplasts reinforces the concept that onion cells have adhesion sites with some similarity to certain kinds of adhesion sites in animals.

  17. Hyperspectral imaging and characterization of live cells by broadband coherent anti-Stokes Raman scattering (CARS) microscopy with singular value decomposition (SVD) analysis.

    PubMed

    Khmaladze, Alexander; Jasensky, Joshua; Price, Erika; Zhang, Chi; Boughton, Andrew; Han, Xiaofeng; Seeley, Emily; Liu, Xinran; Banaszak Holl, Mark M; Chen, Zhan

    2014-01-01

    Coherent anti-Stokes Raman scattering (CARS) microscopy can be used as a powerful imaging technique to identify chemical compositions of complex samples in biology, biophysics, medicine, and materials science. In this work we developed a CARS microscopic system capable of hyperspectral imaging. By employing an ultrafast laser source, a photonic crystal fiber, and a scanning laser microscope together with spectral detection by a highly sensitive back-illuminated cooled charge-coupled device (CCD) camera, we were able to rapidly acquire and process hyperspectral images of live cells with chemical selectivity. We discuss various aspects of hyperspectral CARS image analysis and demonstrate the use of singular value decomposition methods to characterize the cellular lipid content.

  18. Simultaneous mechanical stiffness and electrical potential measurements of living vascular endothelial cells using combined atomic force and epifluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Callies, Chiara; Schön, Peter; Liashkovich, Ivan; Stock, Christian; Kusche-Vihrog, Kristina; Fels, Johannes; Sträter, Alexandra S.; Oberleithner, Hans

    2009-04-01

    The degree of mechanical stiffness of vascular endothelial cells determines the endogenous production of the vasodilating gas nitric oxide (NO). However, the underlying mechanisms are not yet understood. Experiments on vascular endothelial cells suggest that the electrical plasma membrane potential is involved in this regulatory process. To test this hypothesis we developed a technique that simultaneously measures the electrical membrane potential and stiffness of vascular endothelial cells (GM7373 cell line derived from bovine aortic endothelium) under continuous perfusion with physiological electrolyte solution. The cellular stiffness was determined by nano-indentation using an atomic force microscope (AFM) while the electrical membrane potential was measured with bis-oxonol, a voltage-reporting fluorescent dye. These two methods were combined using an AFM attached to an epifluorescence microscope. The electrical membrane potential and mechanical stiffness of the same cell were continuously recorded for a time span of 5 min. Fast fluctuations (in the range of seconds) of both the electrical membrane potential and mechanical stiffness could be observed that were not related to each other. In contrast, slow cell depolarizations (in the range of minutes) were paralleled by significant increases in mechanical stiffness. In conclusion, using the combined AFM-fluorescence technique we monitored for the first time simultaneously the electrical plasma membrane potential and mechanical stiffness in a living cell. Vascular endothelial cells exhibit oscillatory non-synchronized waves of electrical potential and mechanical stiffness. The sustained membrane depolarization, however, is paralleled by a concomitant increase of cell stiffness. The described method is applicable for any fluorophore, which opens new perspectives in biomedical research.

  19. Evidence for Homodimerization of the c-Fos Transcription Factor in Live Cells Revealed by Fluorescence Microscopy and Computer Modeling.

    PubMed

    Szalóki, Nikoletta; Krieger, Jan Wolfgang; Komáromi, István; Tóth, Katalin; Vámosi, György

    2015-11-01

    The c-Fos and c-Jun transcription factors, members of the activator protein 1 (AP-1) complex, form heterodimers and bind to DNA via a basic leucine zipper and regulate the cell cycle, apoptosis, differentiation, etc. Purified c-Jun leucine zipper fragments could also form stable homodimers, whereas c-Fos leucine zipper homodimers were found to be much less stable in earlier in vitro studies. The importance of c-Fos overexpression in tumors and the controversy in the literature concerning c-Fos homodimerization prompted us to investigate Fos homodimerization. Förster resonance energy transfer (FRET) and molecular brightness analysis of fluorescence correlation spectroscopy data from live HeLa cells transfected with fluorescent-protein-tagged c-Fos indicated that c-Fos formed homodimers. We developed a method to determine the absolute concentrations of transfected and endogenous c-Fos and c-Jun, which allowed us to determine dissociation constants of c-Fos homodimers (Kd = 6.7 ± 1.7 μM) and c-Fos-c-Jun heterodimers (on the order of 10 to 100 nM) from FRET titrations. Imaging fluorescence cross-correlation spectroscopy (SPIM-FCCS) and molecular dynamics modeling confirmed that c-Fos homodimers were stably associated and could bind to the chromatin. Our results establish c-Fos homodimers as a novel form of the AP-1 complex that may be an autonomous transcription factor in c-Fos-overexpressing tissues and could contribute to tumor development.

  20. Visualization of living terminal hypertrophic chondrocytes of growth plate cartilage in situ by differential interference contrast microscopy and time-lapse cinematography.

    PubMed

    Farnum, C E; Turgai, J; Wilsman, N J

    1990-09-01

    The functional unit within the growth plate consists of a column of chondrocytes that passes through a sequence of phases including proliferation, hypertrophy, and death. It is important to our understanding of the biology of the growth plate to determine if distal hypertrophic cells are viable, highly differentiated cells with the potential of actively controlling terminal events of endochondral ossification prior to their death at the chondro-osseous junction. This study for the first time reports on the visualization of living hypertrophic chondrocytes in situ, including the terminal hypertrophic chondrocyte. Chondrocytes in growth plate explants are visualized using rectified differential interference contrast microscopy. We record and measure, using time-lapse cinematography, the rate of movement of subcellular organelles at the limit of resolution of this light microscopy system. Control experiments to assess viability of hypertrophic chondrocytes include coincubating organ cultures with the intravital dye fluorescein diacetate to assess the integrity of the plasma membrane and cytoplasmic esterases. In this system, all hypertrophic chondrocytes, including the very terminal chondrocyte, exist as rounded, fully hydrated cells. By the criteria of intravital dye staining and organelle movement, distal hypertrophic chondrocytes are identical to chondrocytes in the proliferative and early hypertrophic cell zones.

  1. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy

    PubMed Central

    2015-01-01

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  2. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

    PubMed

    Hammers, Matthew D; Taormina, Michael J; Cerda, Matthew M; Montoya, Leticia A; Seidenkranz, Daniel T; Parthasarathy, Raghuveer; Pluth, Michael D

    2015-08-19

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems.

  3. Atomic force microscopy stiffness tomography on living Arabidopsis thaliana cells reveals the mechanical properties of surface and deep cell-wall layers during growth.

    PubMed

    Radotić, Ksenija; Roduit, Charles; Simonović, Jasna; Hornitschek, Patricia; Fankhauser, Christian; Mutavdžić, Dragosav; Steinbach, Gabor; Dietler, Giovanni; Kasas, Sandor

    2012-08-08

    Cell-wall mechanical properties play a key role in the growth and the protection of plants. However, little is known about genuine wall mechanical properties and their growth-related dynamics at subcellular resolution and in living cells. Here, we used atomic force microscopy (AFM) stiffness tomography to explore stiffness distribution in the cell wall of suspension-cultured Arabidopsis thaliana as a model of primary, growing cell wall. For the first time that we know of, this new imaging technique was performed on living single cells of a higher plant, permitting monitoring of the stiffness distribution in cell-wall layers as a function of the depth and its evolution during the different growth phases. The mechanical measurements were correlated with changes in the composition of the cell wall, which were revealed by Fourier-transform infrared (FTIR) spectroscopy. In the beginning and end of cell growth, the average stiffness of the cell wall was low and the wall was mechanically homogenous, whereas in the exponential growth phase, the average wall stiffness increased, with increasing heterogeneity. In this phase, the difference between the superficial and deep wall stiffness was highest. FTIR spectra revealed a relative increase in the polysaccharide/lignin content.

  4. Epitaxial BaTiO3(100) films on Pt(100): a low-energy electron diffraction, scanning tunneling microscopy, and x-ray photoelectron spectroscopy study.

    PubMed

    Förster, Stefan; Huth, Michael; Schindler, Karl-Michael; Widdra, Wolf

    2011-09-14

    The growth of epitaxial ultrathin BaTiO(3) films on a Pt(100) substrate has been studied by scanning tunneling microscopy (STM), low-energy electron diffraction (LEED), and x-ray photoelectron spectroscopy (XPS). The films have been prepared by radio-frequency-assisted magnetron sputter deposition at room temperature and develop a long-range order upon annealing at 900 K in O(2). By adjusting the Ar and O(2) partial pressures of the sputter gas, the stoichiometry was tuned to match that of a BaTiO(3)(100) single crystal as determined by XPS. STM reveals the growth of continuous BaTiO(3) films with unit cell high islands on top. With LEED already for monolayer thicknesses, the formation of a BaTiO(3)(100)-(1 × 1) structure has been observed. Films of 2-3 unit cell thickness show a brilliant (1 × 1) LEED pattern for which an extended set of LEED I-V data has been acquired. At temperatures above 1050 K the BaTiO(3) thin film starts to decay by formation of vacancy islands. In addition (4 × 4) and (3 × 3) surface reconstructions develop upon prolonged heating.

  5. Living Composites of Bacteria and Polymers as Biomimetic Films for Metal Sequestration and Bioremediation.

    PubMed

    Knierim, Christian; Enzeroth, Michaela; Kaiser, Patrick; Dams, Christian; Nette, David; Seubert, Andreas; Klingl, Andreas; Greenblatt, Charles L; Jérôme, Valérie; Agarwal, Seema; Freitag, Ruth; Greiner, Andreas

    2015-08-01

    Herein, we report on composite materials of biologically active microorganisms placed in a synthetic polymer matrix. These so-called "living composites" were utilized for gold sequestration (Micrococcus luteus) and bioremediation of nitrite (Nitrobacter winogradskyi) to demonstrate functionality. For the preparation of the living composites the bacteria were first encased in a water-soluble polymer fiber (poly(vinyl alcohol), PVA) followed by coating the fibers with a shell of hydrophobic poly(p-xylylene) (PPX) by chemical vapor deposition (CVD). The combination of bacteria with polymer materials assured the stability and biologically activity of the bacteria in an aqueous environment for several weeks.

  6. Tal Como Somos/just as we are: an educational film to reduce stigma toward gay and bisexual men, transgender individuals, and persons living with HIV/AIDS.

    PubMed

    Ramirez-Valles, Jesus; Kuhns, Lisa M; Manjarrez, Dianna

    2014-04-01

    In this article, the authors describe the development and dissemination of a film-based educational intervention to reduce negative attitudes toward gay and bisexual men, transgender women, and people living with HIV/AIDS in Latino communities, with a focus on youth. The intervention, Tal Como Somos/Just as We Are, is based on stigma and attribution theories, extensive formative research, and community input. Evaluation findings among educators and school youth suggest the film has the potential to effectively influence attitudes toward gay and bisexual men, transgender women, and people living with HIV/AIDS. The film and intervention are being disseminated using diffusion of innovations theory through community-based organizations, schools, television broadcasting, and film festivals.

  7. Surface charge and carbon contamination on an electron-beam-irradiated hydroxyapatite thin film investigated by photoluminescence and phase imaging in atomic force microscopy.

    PubMed

    Hristu, Radu; Tranca, Denis E; Stanciu, Stefan G; Gregor, Maros; Plecenik, Tomas; Truchly, Martin; Roch, Tomas; Tofail, Syed A M; Stanciu, George A

    2014-04-01

    The surface properties of hydroxyapatite, including electric charge, can influence the biological response, tissue compatibility, and adhesion of biological cells and biomolecules. Results reported here help in understanding this influence by creating charged domains on hydroxyapatite thin films deposited on silicon using electron beam irradiation and investigating their shape, properties, and carbon contamination for different doses of incident injected charge by two methods. Photoluminescence laser scanning microscopy was used to image electrostatic charge trapped at pre-existing and irradiation-induced defects within these domains, while phase imaging in atomic force microscopy was used to image the carbon contamination. Scanning Auger electron spectroscopy and Kelvin probe force microscopy were used as a reference for the atomic force microscopy phase contrast and photoluminescence laser scanning microscopy measurements. Our experiment shows that by combining the two imaging techniques the effects of trapped charge and carbon contamination can be separated. Such separation yields new possibilities for advancing the current understanding of how surface charge influences mediation of cellular and protein interactions in biomaterials.

  8. Surface chemical properties of nanoscale domains on UV-treated polystyrene-poly(methyl methacrylate) diblock copolymer films studied using scanning force microscopy.

    PubMed

    Ibrahim, Shaida; Ito, Takashi

    2010-02-02

    This paper reports the surface chemical properties of ca. 20 nm wide domains on a UV-treated thin film of a polystyrene-poly(methyl methacrylate) diblock copolymer (PS-b-PMMA; 0.3 as the PMMA volume fraction). UV irradiation and subsequent acetic acid (AcOH) treatment were used for selectively etching horizontally aligned PMMA domains on a thin PS-b-PMMA film to obtain nanoscale trenches and ridges. The surface charge and hydrophilicity of the trenches (etched PMMA domains) and ridges (PS domains) were investigated using three approaches based on scanning force microscopy. Chemical force titration data with a COOH-terminated tip showed a prominent decrease in adhesion force from pH 3 to 4.5 due to electrostatic repulsion between negatively charged functional groups on the tip and film surface but could not clarify the difference in chemical properties between the two nanoscale domains. Friction force images in n-dodecane showed higher friction over etched PMMA and PS domains with an OH-terminated tip and a CH(3)-terminated tip, respectively, exhibiting higher hydrophilicity of the etched PMMA domains. In an atomic force microscopy image of a UV/AcOH-treated PS-b-PMMA film upon immersion in a ferritin solution, approximately 80% of the ferritin deposited on the film was found on the PS domains. The preferential deposition of ferritin on the PS domains was probably due to the electrostatic repulsion between negatively charged ferritin and negatively charged etched PMMA surface in addition to the hydrophobic interaction between ferritin and the PS surface. These results indicated that the etched PMMA domains were more hydrophilic than the PS domains due to the presence of acidic functional groups (e.g., -COOH groups) at a higher density.

  9. Atomic force microscopy investigation of the interaction of low-level laser irradiation of collagen thin films in correlation with fibroblast response.

    PubMed

    Stylianou, Andreas; Yova, Dido

    2015-12-01

    Low-level red laser (LLRL)-tissue interactions have a wide range of medical applications and are garnering increased attention. Although the positive effects of low-level laser therapy (LLLT) have frequently been reported and enhanced collagen accumulation has been identified as one of the most important mechanisms involved, little is known about LLRL-collagen interactions. In this study, we aimed to investigate the influence of LLRL irradiation on collagen, in correlation with fibroblast response. Atomic force microscopy (AFM) and fluorescence spectroscopy were used to characterize surfaces and identify conformational changes in collagen before and after LLRL irradiation. Irradiated and non-irradiated collagen thin films were used as culturing substrates to investigate fibroblast response with fluorescence microscopy. The results demonstrated that LLRL induced small alterations in fluorescence emission and had a negligible effect on the topography of collagen thin films. However, fibroblasts cultured on LLRL-irradiated collagen thin films responded to LRLL. The results of this study show for the first time the effect of LLRL irradiation on pure collagen. Although irradiation did not affect the nanotopography of collagen, it influenced cell behavior. The role of collagen appears to be crucial in the LLLT mechanism, and our results demonstrated that LLRL directly affects collagen and indirectly affects cell behavior.

  10. Morphological changes in adsorbed protein films at the air-water interface subjected to large area variations, as observed by brewster angle microscopy.

    PubMed

    Xu, Rong; Dickinson, Eric; Murray, Brent S

    2007-04-24

    Adsorbed films of proteins at the air-water interface have been imaged using Brewster angle microscopy (BAM). The proteins beta-lactoglobulin (beta-L) and ovalbumin (OA) were studied at a range of protein concentrations and surface ages at 25.0 degrees C and two pH values (7 and 5) in a Langmuir trough. The adsorbed films were periodically subjected to compression and expansion cycles such that the film area was typically varied between 125% and 50% of the original film area. With beta-L on its own, no structural changes were observable at pH 7. When a low-area fraction (less than 0.01%) of 20 mum polystyrene latex particles was spread at the interface before adsorption of beta-L, the particles became randomly distributed throughout the interface, but after protein adsorption and compression/expansion, the particles highlighted notable structural features not visible in their absence. Such features included the appearance of long (several hundred micrometers or more) folds and cracks in the films, generally oriented at right angles to the direction of compression, and also aggregates of protein and/or particles. Such structuring was more visible the longer the film was aged or at higher initial protein concentrations for shorter adsorption times. At pH 5, close to the isoelectric pH of beta-L, such features were just noticeable in the absence of particles but were much more pronounced than at pH 7 in the presence of particles. Similar experiments with OA revealed even more pronounced structural features, both in the absence and presence of particles, particularly at pH 5 (close to the isoelectric pH of OA also), producing striking stripelike and meshlike domains. Changes in the dilatational elasticity of the films could be correlated with the variations in the structural integrity of the films as observed via BAM. The results indicate that interfacial area changes of this type, typical of those that occur in food colloid processing, will lead to highly

  11. Correlative microscopy.

    PubMed

    Loussert Fonta, Céline; Humbel, Bruno M

    2015-09-01

    In recent years correlative microscopy, combining the power and advantages of different imaging system, e.g., light, electrons, X-ray, NMR, etc., has become an important tool for biomedical research. Among all the possible combinations of techniques, light and electron microscopy, have made an especially big step forward and are being implemented in more and more research labs. Electron microscopy profits from the high spatial resolution, the direct recognition of the cellular ultrastructure and identification of the organelles. It, however, has two severe limitations: the restricted field of view and the fact that no live imaging can be done. On the other hand light microscopy has the advantage of live imaging, following a fluorescently tagged molecule in real time and at lower magnifications the large field of view facilitates the identification and location of sparse individual cells in a large context, e.g., tissue. The combination of these two imaging techniques appears to be a valuable approach to dissect biological events at a submicrometer level. Light microscopy can be used to follow a labelled protein of interest, or a visible organelle such as mitochondria, in time, then the sample is fixed and the exactly same region is investigated by electron microscopy. The time resolution is dependent on the speed of penetration and fixation when chemical fixatives are used and on the reaction time of the operator for cryo-fixation. Light microscopy can also be used to identify cells of interest, e.g., a special cell type in tissue or cells that have been modified by either transfections or RNAi, in a large population of non-modified cells. A further application is to find fluorescence labels in cells on a large section to reduce searching time in the electron microscope. Multiple fluorescence labelling of a series of sections can be correlated with the ultrastructure of the individual sections to get 3D information of the distribution of the marked proteins: array

  12. Bias assisted scanning probe microscopy direct write lithography enables local oxygen enrichment of lanthanum cuprates thin films

    SciTech Connect

    Lavini, Francesco; Yang, Nan; Vasudevan, Rama K.; Strelcov, Evgheni; Jesse, Stephen; Okatan, Mahmut Baris; Kravchenko, Ivan I.; Di Castro, Daniele; Kalinin, Sergei V.; Balestrino, Giuseppe; Foglietti, Vittorio; Aruta, Carmela

    2015-01-01

    Here, scanning probe bias techniques have been used as a method to locally dope thin epitaxial films of La2CuO4 (LCO) fabricated by pulsed laser deposition. The local electrochemical oxidation of LCO very efficiently introduces interstitial oxygen defects in the thin film. Details on the influence of the tip voltage bias and environmental conditions on the surface morphology have been investigated. The results show that a local uptake of oxygen occurs in the oxidized films.

  13. Investigation of MBE-grown high T c films by RHEED, atomic force microscopy and X-ray diffraction

    NASA Astrophysics Data System (ADS)

    Wang, H. S.; Eissler, D.; Dietsche, W.; Fischer, A.; Ploog, K.

    1993-02-01

    Results on the preparation of the molecular beam epitaxial (MBE) growth and on structural investigations of high Tc DyBa 2Cu 3O 7- y (DBCO) superconducting thin films are presented. We prepared high quality DBCO thin films on SrTiO 3, MgO, LaAlO 3 and NdGaO 3 substrates in situ with high reproductivity. We also grew DBCO/Dy 2O 3/DBCO/SrTiO 3 multilayer structures. The structure and morphology of the films were studied by RHEED, STM, AFM, XRD and X-ray Weissenberg camera techniques.

  14. SU-E-T-30: Absorbed Doses Determined by Texture Analysis of Gafchromic EBT3 Films Using Scanning Electron Microscopy: A Feasibility Study

    SciTech Connect

    Park, S; Kim, H; Ye, S

    2014-06-01

    Purpose: The texture analysis method is useful to estimate structural features of images as color, size, and shape. The study aims to determine a dose-response curve by texture analysis of Gafchromic EBT3 film images using scanning electron microscopy (SEM). Methods: The uncoated Gafchromic EBT3 films were prepared to directly scan over the active surface layer of EBT3 film using SEM. The EBT3 films were exposed at a dose range of 0 to 10 Gy using a 6 MV photon beam. The exposed film samples were SEM-scanned at 100X, 1000X, and 3000X magnifications. The four texture features (Homogeneity, Correlation, Contrast, and Energy) were calculated based on the gray level co-occurrence matrix (GLCM) derived from the SEM images at each dose. To validate a correlation between delivered doses and texture features, an R-squared value in linear regression was tested. Results: The results showed that the Correlation index was more suitable as dose indices than the other three texture features due to higher linearity and sensitivity of the dose response curves. Further the Correlation index of 3000X magnified SEM images with 9 pixel offsets had an R-squared value of 0.964. The differences between the delivered doses and the doses measured by this method were 0.9, 1.2, 0.2, and 0.2 Gy at 5, 10, 15, and 20 Gy, respectively. Conclusion: It seems to be feasible to convert micro-scale structural features of {sub χ}t{sub χχχ}he EBT3 films to absorbed doses using the texture analysis method.

  15. 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.

  16. Element-specific study of epitaxial NiO/Ag/CoO/Fe films grown on vicinal Ag(001) using photoemission electron microscopy

    SciTech Connect

    Meng, Y.; Li, J.; Tan, A.; Jin, E.; Son, J.; Park, J. S.; Doran, A.; Young, A. T.; Scholl, A.; Arenholz, E.; Wu, J.; Hwang, C.; Zhao, H. W.; Qiu, Z. Q.

    2011-01-10

    NiO/Ag/CoO/Fe single crystalline films are grown epitaxially on a vicinal Ag(001) substrate using molecular beam epitaxy and investigated by photoemission electron microscopy. We find that after zero-field cooling, the in-plane Fe magnetization switches from parallel to perpendicular direction of the atomic steps of the vicinal surface at thinner CoO thickness but remains in its original direction parallel to the steps at thicker CoO thickness. CoO and NiO domain imaging result shows that both CoO/Fe and NiO/CoO spins are perpendicularly coupled, suggesting that the Fe magnetization switching may be associated with the rotatable-frozen spin transition of the CoO film.

  17. A simple calibration approach based on film-casting for confocal Raman microscopy to support the development of a hot-melt extrusion process.

    PubMed

    Netchacovitch, L; Thiry, J; De Bleye, C; Dumont, E; Dispas, A; Hubert, C; Krier, F; Sacré, P-Y; Evrard, B; Hubert, Ph; Ziemons, E

    2016-07-01

    When developing a new formulation, the development, calibration and validation steps of analytical methods based on vibrational spectroscopy are time-consuming. For each new formulation, real samples must be produced and a "reference method" must be used in order to determine the Active Pharmaceutical Ingredient (API) content of each sample. To circumvent this issue, the paper presents a simple approach based on the film-casting technique used as a calibration tool in the framework of hot-melt extrusion process. Confocal Raman microscopic method was successfully validated for the determination of itraconazole content in film-casting samples. Then, hot-melt extrusion was carried out to produce real samples in order to confront the results obtained with confocal Raman microscopy and Ultra High Performance Liquid Chromatography (UHPLC). The agreement between both methods was demonstrated using a comparison study based on the Bland and Altman's plot.

  18. Transmission electron microscopy study of GaInNAs(Sb) thin films grown by atomic hydrogen-assisted molecular beam epitaxy

    NASA Astrophysics Data System (ADS)

    Oshima, R.; Huang, J. Y.; Miyashita, N.; Matsubara, K.; Okada, Y.; Ponce, F. A.

    2011-11-01

    The quaternary GaInNAs is a promising material system for use in next generation multijunction photovoltaic devices. We have investigated the effect of introducing antimony on the growth by using transmission electron microscopy and energy dispersive x-ray (EDX) spectroscopy. Two-dimensional growth was observed in GaInNAs films with striation features associated with compositional fluctuation and nanometer scale elemental segregation on the growth front. On the contrary, GaInNAsSb films exhibit uniform contrast throughout. EDX profile indicates uniform compositional distribution, as antimony atoms suppress the surface mobilites of adatoms resulting in a lower probability to generate the favored bonds, such as Ga-N and In-As.

  19. X-ray and transmission electron microscopy characterization of twinned CdO thin films grown on a-plane sapphire by metalorganic vapour phase epitaxy

    NASA Astrophysics Data System (ADS)

    Martínez-Tomás, M. C.; Zúñiga-Pérez, J.; Vennéguès, P.; Tottereau, O.; Muñoz-Sanjosé, V.

    2007-07-01

    In the frame of studying II VI oxides of interest in optoelectronic technologies, the structural properties of CdO films grown by metalorganic vapour phase epitaxy on a-plane sapphire substrates have been analysed. The study has been performed by means of X-ray diffraction and cross-sectional transmission electron microscopy measurements. CdO films have been found to grow along [111] with the presence of twinned domains. Asymmetrical reflections have been used to study the crystalline quality of the twinned domains, independent of each other, as well as to determine their relative population. The analysis has been made as a function of growth conditions: VI/II precursors molar ratio and growth temperature.

  20. Direct observation of fatigue in epitaxially grown Pb(Zr,Ti)O3 thin films using second harmonic piezoresponse force microscopy

    SciTech Connect

    Murari, Nishit M; Hong, Seungbum; Lee, Ho Nyung; Katiyar, Ram S.

    2011-01-01

    Here, we present a direct observation of fatigue phenomena in epitaxially grown Pb(Zr{sub 0.2}Ti{sub 0.8})O{sub 3} (PZT) thin films using second harmonic piezoresponse force microscopy (SH-PFM). We observed strong correlation between the SH-PFM amplitude and phase signals with the remnant piezoresponse at different switching cycles. The SH-PFM results indicate that the average fraction of switchable domains decreases globally and the phase delays of polarization switching differ locally. In addition, we found that the fatigue developed uniformly over the whole area without developing region-by-region suppression of switchable polarization as in polycrystalline PZT thin films.

  1. Simplification of femtosecond transient absorption microscopy data from CH3NH3PbI3 perovskite thin films into decay associated amplitude maps

    DOE PAGES

    Doughty, Benjamin; Simpson, Mary Jane; Yang, Bin; ...

    2016-02-16

    Our work aims to simplify multi-dimensional femtosecond transient absorption microscopy (TAM) data into decay associated amplitude maps that describe the spatial distributions of dynamical processes occurring on various characteristic timescales. Application of this method to TAM data obtained from a model methyl-ammonium lead iodide (CH3NH3PbI3) perovskite thin film allows us to simplify the dataset consisting of a 68 time-resolved images into 4 decay associated amplitude maps. Furthermore, these maps provide a simple means to visualize the complex electronic excited-state dynamics in this system by separating distinct dynamical processes evolving on characteristic timescales into individual spatial images. This approach provides newmore » insight into subtle aspects of ultrafast relaxation dynamics associated with excitons and charge carriers in the perovskite thin film, which have recently been found to coexist at spatially distinct locations.« less

  2. Tunneling spectra and superconducting gaps observed by scanning tunneling microscopy near the grain boundaries of FeSe0.3Te0.7 films

    NASA Astrophysics Data System (ADS)

    Lin, K. C.; Li, Y. S.; Shen, Y. T.; Wu, M. K.; Chi, C. C.

    2013-12-01

    We used scanning tunneling microscopy (STM) to study the tunneling spectra of FeSe0.3Te0.7 films with two orientations of the ab-planes and a connection ramp between them. We discovered that by pulsed laser deposition (PLD) method, the a- and b-axis of the FeSe0.3Te0.7 film deposited on an Ar-ion-milled magnesium oxide (MgO) substrate were rotated 45° with respect to those of MgO, whereas the a- and b-axis of the film grown on a pristine MgO substrate were parallel to those of MgO. With photolithography and this technique, we can prepare FeSe0.3Te0.7 films with two orientations on the same MgO substrate so that the connection between them forms a ramp at an angle of about 25° to the substrate plane. In the planar region, for either the 0° or 45° orientation, we observed tunneling spectra with a superconducting gap of about 5 meV and 1.78 meV, respectively. However, a much larger gap at about 18 meV was observed in the ramp region. Furthermore, we observed a small zero-bias conductance peak (ZBCP) inside the large gap at T = 4.3 K. The ZBCP becomes smaller with increasing temperature and disappeared at temperature above 7 K.

  3. Quantitative analysis of the magnetic domain structure in polycrystalline La(0.7)Sr(0.3)MnO3 thin films by magnetic force microscopy.

    PubMed

    Li, Zhenghua; Wei, Fulin; Yoshimura, Satoru; Li, Guoqing; Asano, Hidefumi; Saito, Hitoshi

    2013-01-14

    The nanoscale magnetic domain structure of the polycrystalline La(0.7)Sr(0.3)MnO(3) granular thin films was imaged with a developed magnetic force microscopy technique by simultaneously detecting both the perpendicular and in-plane components of magnetic field gradients during the same scan of the tip oscillation. The characteristics of both the perpendicular and in-plane magnetic field gradient at the grain edges or the nonmagnetic grain boundary phase for LSMO films were demonstrated and can be used to evaluate the magnetic domain structure and magnetic isolation between neighboring grains. A two dimensional signal transformation algorithm to reconstruct the in-plane magnetization distribution of the polycrystalline LSMO thin films from the measured raw MFM images with the aid of the deconvolution technique was presented. The comparison between the experimental and simulated MFM images indicates that the magnetic grains or clusters are in the single domain (SD) or multi-domain (MD) state with the magnetic moments parallel or anti-parallel to the effective magnetic field of each grain, possibly due to the need for minimizing the total energy. The quantitative interpretation of the magnetic domain structure indicates that the large magnetoresistance in the studied LSMO films is mainly due to tunnel effect and scattering of conducted electrons at the nonmagnetic grain boundary phase related to the different configurations of magnetic domain states between neighboring grains.

  4. Peptide isolated from Cry1Ab16 toxin present in Bacillus thuringiensis: Synthesis and morphology data for layer-by-layer films studied by atomic force microscopy.

    PubMed

    Plácido, Alexandra; de Oliveira Farias, Emanuel Airton; Marani, Mariela M; Gomes Vasconcelos, Andreanne; Leite, José R S A; Delerue-Matos, Cristina

    2016-09-01

    The peptide PcL342-354C was obtained from the Cry1Ab16 toxin present in Bacillus thuringiensis ("Computational Modeling Deduced Three Dimensional Structure of Cry1Ab16 Toxin from B. thuringiensis AC11" (Kashyap, 2012) [1]). In this data article, we report the synthesis and characterization of the PcL342-354C peptide by MALDI-TOF/TOF mass spectrometry. In addition, the preparation of layer-by-layer films is shown based on interspersion of this peptide with both polyethylenimine (PEI) and poly(sodium 4-styrenesulfonate) (PSS), self-assembled on ITO (indium tin oxide) electrodes. The morphology of the ITO/PEI/PSS/PcL342-354C film was analyzed using atomic force microscopy (AFM). We also evaluated the effect of the number of bilayers in ITO/PEI/(PSS/PcL342-354C) n on the morphology of the film using AFM amplitude images. Further details about this study were published elsewhere, "Layer-by-layer films containing peptides of the Cry1Ab16 toxin from B. thuringiensis for potential biotechnological applications," (Plácido et al., 2016) [2].

  5. Chemical imaging of the surface of self-assembled polystyrene-b-poly(methyl methacrylate) diblock copolymer films using apertureless near-field IR microscopy.

    PubMed

    Mueller, Kerstin; Yang, Xiujuan; Paulite, Melissa; Fakhraai, Zahra; Gunari, Nikhil; Walker, Gilbert C

    2008-06-01

    The nanoscale chemical composition variations of the surfaces of thin films of polystyrene- b-poly(methyl methacrylate) (PS- b-PMMA) diblock copolymers are investigated using apertureless near-field IR microscopy. The scattering of the incident infrared beam from a modulated atomic force microscopy (AFM) tip is probed using homodyne detection and demodulation at the tip oscillation frequency. An increase in the IR attenuation is observed in the PMMA-rich domains with a wavenumber dependence that is consistent with the bulk absorption spectrum. The results indicate that even though a small topography-induced artifact can be observed in the near-field images, the chemical signature of the sample is detected clearly.

  6. Rational design, synthesis and characterization of highly fluorescent optical switches for high-contrast optical lock-in detection (OLID) imaging microscopy in living cells

    PubMed Central

    Petchprayoon, Chutima; Yan, Yuling; Mao, Shu; Marriott, Gerard

    2010-01-01

    A major challenge in cell biology is to elucidate molecular mechanisms that underlie the spatio-temporal control of cellular processes. These studies require microscope imaging techniques and associated optical probes that provide high-contrast and high-resolution images of specific proteins and their complexes. Auto-fluorescence however, can severely compromise image contrast and represents a fundamental limitation for imaging proteins within living cells. We have previously shown that optical switch probes and optical lock-in detection (OLID) image microscopy improve image contrast in high background environments. Here, we present the design, synthesis and characterization of amino- reactive and cell permeable optical switches that integrate the highly fluorescent fluorophore, tetramethylrhodamine (TMR) and spironaphthoxazine (NISO), a highly efficient optical switch. The NISO moiety in TMR-NISO undergoes rapid and reversible, excited-state driven transitions between a colorless spiro (SP)-state and a colored merocyanine (MC)-state in response to irradiation with 365 nm and >530 nm light. In the MC-state, the TMR (donor) emission is almost completely extinguished by Förster resonance energy transfer (FRET) to the MC probe (acceptor), whereas in the colorless SP-state, the quantum yield for TMR fluorescence is maximal. Irradiation of TMR-NISO with a defined sequence of 365 nm and 546 nm manipulates the levels of SP and MC with concomitant modulation of FRET efficiency and the TMR fluorescence signal. High fidelity optical switching of TMR fluorescence is shown for TMR-NISO probes in vitro and for membrane permeable TMR-NISO within living cells. PMID:20674372

  7. Molecularly imprinted protein recognition thin films constructed by controlled/living radical polymerization.

    PubMed

    Sasaki, Shogo; Ooya, Tooru; Kitayama, Yukiya; Takeuchi, Toshifumi

    2015-02-01

    We demonstrated the synthesis of molecularly imprinted polymers (MIPs) with binding affinity toward a target protein, ribonuclease A (RNase) by atom transfer radical polymerization (ATRP) of acrylic acid, acrylamide, and N,N'-methylenebisacrylamide in the presence of RNase. The binding activity of the MIPs was evaluated by surface plasmon resonance (SPR) of the MIP thin layers prepared on the gold-coated sensor chips. The MIPs prepared by ATRP (MIP-ATRP) had a binding affinity toward RNase with larger binding amount compared to MIPs prepared by conventional free radical polymerization methods (MIP-RP). Moreover, protein selectivity was evaluated using reference proteins (cytochrome c, myoglobin, and α-lactalbumin) and was confirmed in MIP-ATRP of optimum film thickness determined experimentally to be 15-30 nm; however, protein selectivity was not achieved in all MIP-RP. We have shown that ATRP is powerful technique for preparing protein recognition materials by molecular imprinting.

  8. Redescription of the Tintinnid Stenosemella pacifica Kofoid and Campbell, 1929 (Ciliophora, Spirotricha) Based on Live Observation, Protargol Impregnation, and Scanning Electron Microscopy

    PubMed Central

    AGATHA, SABINE; TSAI, SHENG-FANG

    2010-01-01

    The tintinnid ciliate Stenosemella pacifica Kofoid and Campbell, 1929 was occasionally recorded from the pelagial of temperate, subtropical, and tropical neritic waters. Since its cytological features were unknown, the species is redescribed from material collected in the pelagial of the Irish Sea, using live observation, protargol impregnation, and scanning electron microscopy. Furthermore, the species diagnosis is improved to include new characteristics, e.g. the somatic ciliary pattern comprising a ventral, dorsal, and posterior kinety as well as a right, left, and lateral ciliary field. The stomatogenesis of S. pacifica is typical for species with such a complex somatic ciliary pattern: the oral primordium develops hypoapokinetally posterior to the lateral ciliary field. The presence of windows in the lorica collar of Stenosemella ventricosa, the type of the genus, necessitates (i) an improved genus diagnosis, (ii) a synonymization of the genus Luminella Kofoid and Campbell, 1939, and (iii) a transfer of the Luminella species to the genus Stenosemella, including Luminella neocalifornica, which becomes Stenosemella neocalifornica nov. comb. Owing to the lack of a description, Stenosemella crateri is considered a nomen nudum. PMID:18318859

  9. High-throughput characterization of stresses in thin film materials libraries using Si cantilever array wafers and digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Lai, Y. W.; Hamann, S.; Ehmann, M.; Ludwig, A.

    2011-06-01

    We report the development of an advanced high-throughput stress characterization method for thin film materials libraries sputter-deposited on micro-machined cantilever arrays consisting of around 1500 cantilevers on 4-inch silicon-on-insulator wafers. A low-cost custom-designed digital holographic microscope (DHM) is employed to simultaneously monitor the thin film thickness, the surface topography and the curvature of each of the cantilevers before and after deposition. The variation in stress state across the thin film materials library is then calculated by Stoney's equation based on the obtained radii of curvature of the cantilevers and film thicknesses. DHM with nanometer-scale out-of-plane resolution allows stress measurements in a wide range, at least from several MPa to several GPa. By using an automatic x-y translation stage, the local stresses within a 4-inch materials library are mapped with high accuracy within 10 min. The speed of measurement is greatly improved compared with the prior laser scanning approach that needs more than an hour of measuring time. A high-throughput stress measurement of an as-deposited Fe-Pd-W materials library was evaluated for demonstration. The fast characterization method is expected to accelerate the development of (functional) thin films, e.g., (magnetic) shape memory materials, whose functionality is greatly stress dependent.

  10. High-throughput characterization of stresses in thin film materials libraries using Si cantilever array wafers and digital holographic microscopy.

    PubMed

    Lai, Y W; Hamann, S; Ehmann, M; Ludwig, A

    2011-06-01

    We report the development of an advanced high-throughput stress characterization method for thin film materials libraries sputter-deposited on micro-machined cantilever arrays consisting of around 1500 cantilevers on 4-inch silicon-on-insulator wafers. A low-cost custom-designed digital holographic microscope (DHM) is employed to simultaneously monitor the thin film thickness, the surface topography and the curvature of each of the cantilevers before and after deposition. The variation in stress state across the thin film materials library is then calculated by Stoney's equation based on the obtained radii of curvature of the cantilevers and film thicknesses. DHM with nanometer-scale out-of-plane resolution allows stress measurements in a wide range, at least from several MPa to several GPa. By using an automatic x-y translation stage, the local stresses within a 4-inch materials library are mapped with high accuracy within 10 min. The speed of measurement is greatly improved compared with the prior laser scanning approach that needs more than an hour of measuring time. A high-throughput stress measurement of an as-deposited Fe-Pd-W materials library was evaluated for demonstration. The fast characterization method is expected to accelerate the development of (functional) thin films, e.g., (magnetic) shape memory materials, whose functionality is greatly stress dependent.

  11. Tal Como Somos/Just As We Are: An Educational Film to Reduce Stigma towards Gay and Bisexual Men, Transgender Individuals & Persons Living with HIV/AIDS

    PubMed Central

    Ramirez-Valles, Jesus; Kuhns, Lisa M.; Manjarrez, Dianna

    2013-01-01

    In this paper we describe the development and dissemination of a film-based educational intervention to reduce negative attitudes towards gay and bisexual men and transgender women (GBT) and people living with HIV/AIDS (PLWHA) in Latino communities, with a focus on youth. The intervention, Tal Como Somos/Just as We Are, is based on stigma and attribution theories, extensive formative research, and community input. Evaluation findings among educators and school youth suggest the film has the potential to effectively impact attitudes towards GBT and PLWHA. The film and intervention are being disseminated using diffusion of innovations theory through community-based organizations, schools, television broadcasting and film festivals. PMID:24377496

  12. Spin-Relaxation Dynamics of E' Centers at High Density in SiO2 Thin Films for Single-Spin Tunneling Force Microscopy

    NASA Astrophysics Data System (ADS)

    Ambal, K.; Payne, A.; Waters, D. P.; Williams, C. C.; Boehme, C.

    2015-08-01

    The suitability of the spin dynamics of paramagnetic silicon dangling bonds (E' centers) in high-E'-density amorphous silicon dioxide (SiO2 ) as probe spins for single-spin tunneling force microscopy (SSTFM) is studied. SSTFM is a spin-selection-rule-based scanning-probe single-spin readout concept. Following the synthesis of SiO2 thin films on (111)-oriented crystalline-silicon substrates with room-temperature stable densities of [E'] >5 ×1018 cm-3 throughout the 60-nm thin film, pulsed electron paramagnetic resonance spectroscopy is conducted on the E' centers at temperatures between T =5 K and T =70 K . The measurements reveal that the spin coherence (the transverse spin-relaxation time T2) of these centers is significantly shortened compared to low-E'-density SiO2 films and within error margins not dependent on temperature. In contrast, the spin-flip times (the longitudinal relaxation times T1) are dependent on the temperature but with much weaker dependence than low-density SiO2 , with the greatest deviations from low-density SiO2 seen for T =5 K . These results, discussed in the context of the spin-relaxation dynamics of dangling-bond states of other silicon-based disordered solids, indicate the suitability of E' centers in high-density SiO2 as probe spins for SSTFM.

  13. Current mapping of nonpolar a-plane and polar c-plane GaN films by conductive atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Shengrui; Jiang, Teng; Lin, Zhiyu; Zhao, Ying; Yang, Linan; Zhang, Jincheng; Li, Peixian; Hao, Yue

    2016-10-01

    Nonpolar (11-20) a-plane GaN and polar (0001) c-plane GaN films have been grown by metal organic chemical vapor deposition on r-plane (1-102) and c-plane (0001) sapphire substrates, respectively. Conductive atomic force microscopy (C-AFM) has been used to investigate the local conductivity of the films. C-AFM shows enhanced current conduction within the etch pits of c-plane GaN and triangular pits of a-plane GaN. The results indicate that the off-axis planes are more electrically active than c-plane and a-plane. Surprisingly, the C-AFM values in triangular pit of the a-plane GaN are much smaller than that in etch pits of the c-plane GaN. The dislocations type related current leakage mechanism is revealed for polar c-plane and nonpolar a-plane GaN films.

  14. Films.

    ERIC Educational Resources Information Center

    Philadelphia Board of Education, PA. Div. of Instructional Materials.

    The Affective Curriculum Research Project produced five films and two records during a series of experimental summer programs. The films and records form part of a curriculum designed to teach to the concerns of students. The films were an effort to describe the Philadelphia Cooperative Schools Program, to explain its importance, and to…

  15. Role of ICAM-1 polymorphisms (G241R, K469E) in mediating its single-molecule binding ability: Atomic force microscopy measurements on living cells

    SciTech Connect

    Bai, Rui; Yi, Shaoqiong; Zhang, Xuejie; Liu, Huiliang; Fang, Xiaohong

    2014-06-13

    Highlights: • We evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations. • AFM was used to measure single-molecule binding ability on living cells. • The SNP of ICAM-1 may induce changes in expressions rather than single-molecule binding ability. - Abstract: Atherosclerosis (As) is characterized by chronic inflammation and is a major cause of human mortality. ICAM-1-mediated adhesion of leukocytes in vessel walls plays an important role in the pathogenesis of atherosclerosis. Two single nucleotide polymorphisms (SNPs) of human intercellular adhesion molecule-1 (ICAM-1), G241R and K469E, are associated with a number of inflammatory diseases. SNP induced changes in ICAM-1 function rely not only on the expression level but also on the single-molecule binding ability which may be affected by single molecule conformation variations such as protein splicing and folding. Previous studies have shown associations between G241R/K469E polymorphisms and ICAM-1 gene expression. Nevertheless, few studies have been done that focus on the single-molecule forces of the above SNPs and their ligands. In the current study, we evaluated both single molecule binding ability and expression level of 4 ICAM-1 mutations – GK (G241/K469), GE (G241/E469), RK (R241/K469) and RE (R241/E469). No difference in adhesion ability was observed via cell adhesion assay or atomic force microscopy (AFM) measurement when comparing the GK, GE, RK, or RE genotypes of ICAM-1 to each other. On the other hand, flow cytometry suggested that there was significantly higher expression of GE genotype of ICAM-1 on transfected CHO cells. Thus, we concluded that genetic susceptibility to diseases related to ICAM-1 polymorphisms, G241R or K469E, might be due to the different expressions of ICAM-1 variants rather than to the single-molecule binding ability of ICAM-1.

  16. Direct electrocaloric measurement of 0.9Pb(Mg1/3Nb2/3)O3-0.1PbTiO3 films using scanning thermal microscopy

    NASA Astrophysics Data System (ADS)

    Crossley, S.; Usui, T.; Nair, B.; Kar-Narayan, S.; Moya, X.; Hirose, S.; Ando, A.; Mathur, N. D.

    2016-01-01

    We show that scanning thermal microscopy can measure reversible electrocaloric (EC) effects in <40 μm-thick ceramic films of the relaxor ferroelectric 0.9Pb(Mg1/3Nb2/3)O3-0.1PbTiO3, with the substrate present. We recorded roughly the same non-adiabatic temperature change (±0.23 K) for a thinner film that was driven harder than a thicker film (±31 V μm-1 across 13 μm versus ±11 V μm-1 across 38 μm), because the thicker film lay relatively closer to the substantially larger adiabatic values that we predicted by thermodynamic analysis of electrical data. Film preparation was compatible with the fabrication of EC multilayer capacitors, and therefore our measurement method may be exploited for rapid characterisation of candidate films for cooling applications.

  17. Use of Ultrasonic Force Microscopy to Image the Interior Nanoparticles in YBa2Cu3O7-x Films (Postprint)

    DTIC Science & Technology

    2012-02-01

    of the screw dislocation induced terraces present in the films. Index Terms—Fluxpinning, nanoparticles, nondestructive testing , ultrasonic force...superconductors. A nondestructive technique, such as an ultrasonic force mi- croscope (UFM) can be very useful for the development of HTS coated conductor if it... ultrasonic frequencies in the range of 300–500 kHz were optimized for each sample such that the nanometer sized particles on the surface as well as

  18. Surface potential measurement on contact resistance of amorphous-InGaZnO thin film transistors by Kelvin probe force microscopy

    NASA Astrophysics Data System (ADS)

    Han, Zhiheng; Xu, Guangwei; Wang, Wei; Lu, Congyan; Lu, Nianduan; Ji, Zhuoyu; Li, Ling; Liu, Ming

    2016-07-01

    Contact resistance plays an important role in amorphous InGaZnO (a-IGZO) thin film transistors (TFTs). In this paper, the surface potential distributions along the channel have been measured by using Kelvin probe force microscopy (KPFM) on operating a-IGZO TFTs, and sharp potential drops at the edges of source and drain were observed. The source and drain contact resistances can be extracted by dividing sharp potential drops with the corresponding drain to source current. It is found that the contact resistances could not be neglected compared with the whole channel resistances in the a-IGZO TFT, and the contact resistances decrease remarkably with increasing gate biased voltage. Our results suggest that the contact resistances can be controlled by tuning the gate biased voltage. Moreover, a transition from gradual channel approximation to space charge region was observed through the surface potential map directly when TFT operating from linear regime to saturation regime.

  19. Analysis of low-field isotropic vortex glass containing vortex groups in YBa2Cu3O7−x thin films visualized by scanning SQUID microscopy

    PubMed Central

    Wells, Frederick S.; Pan, Alexey V.; Wang, X. Renshaw; Fedoseev, Sergey A.; Hilgenkamp, Hans

    2015-01-01

    The glass-like vortex distribution in pulsed laser deposited YBa2Cu3O7 − x thin films is observed by scanning superconducting quantum interference device microscopy and analysed for ordering after cooling in magnetic fields significantly smaller than the Earth's field. Autocorrelation calculations on this distribution show a weak short-range positional order, while Delaunay triangulation shows a near-complete lack of orientational order. The distribution of these vortices is finally characterised as an isotropic vortex glass. Abnormally closely spaced groups of vortices, which are statistically unlikely to occur, are observed above a threshold magnetic field. The origin of these groups is discussed, but will require further investigation. PMID:25728772

  20. In situ high-resolution transmission electron microscopy study of interfacial reactions of Cu thin films on amorphous silicon

    NASA Astrophysics Data System (ADS)

    Lee, Sung Bo; Choi, Duck-Kyun; Phillipp, Fritz; Jeon, Kyung-Sook; Kim, Chang Kyung

    2006-02-01

    Interfacial reactions of Cu with amorphous silicon (a-Si) in the Cu /a-Si/glass system are studied by in situ high-resolution transmission electron microscopy at 550°C. Various Cu silicides, such as η-Cu3Si, Cu15Si4, and Cu5Si, and Cu particles are observed. The formation of the Cu particles can be attributed to a heating effect from electron beam irradiation. Around the Cu silicides, crystallization of a-Si occurs. Around the Cu particles, however, crystallization does not occur. Crystallization appears to be enhanced by Cu dissolved in a-Si.

  1. Specific anchoring modes of two distinct dystrophin rod sub-domains interacting in phospholipid Langmuir films studied by atomic force microscopy and PM-IRRAS.

    PubMed

    Vié, V; Legardinier, S; Chieze, L; Le Bihan, O; Qin, Y; Sarkis, J; Hubert, J-F; Renault, A; Desbat, B; Le Rumeur, E

    2010-08-01

    Dystrophin rod repeats 1-3 sub-domain binds to acidic phosphatidylserine in a small vesicle binding assay, while the repeats 20-24 sub-domain does not. In the present work, we studied the adsorption behaviour of both sub-domains at the air/liquid interface and at the air/lipid interface in a Langmuir trough in order to highlight differences in interfacial properties. The adsorption behaviour of the two proteins at the air/liquid interface shows that they display surface activity while maintaining their alpha-helical secondary structure as shown by PM-IRRAS. Strikingly, R20-24 needs to be highly hydrated even at the interface, while this is not the case for R1-3, indicating that the surface activity is dramatically higher for R1-3 than R20-24. Surface-pressure measurements, atomic force microscopy and PM-IRRAS are used in a Langmuir experiment with DOPC-DOPS monolayers at two different surface pressures, 20 mN/m and 30 mN/m. At the lower surface pressure, the proteins are adsorbed at the lipid film interface while maintaining its alpha-helical structure. After an increase of the surface pressure, R1-3 subsequently produces a stable film, while R20-24 induces a reorganization of the lipid film with a subsequent decrease of the surface pressure close to the initial value. AFM and PM-IRRAS show that R1-3 is present in high amounts at the interface, being arranged in clusters representing 3.3% of the surface at low pressure. By contrast, R20-24 is present at the interface in small amounts bound only by a few electrostatic residues to the lipid film while the major part of the molecule remains floating in the sub-phase. Then for R1-3, the electrostatic interaction between the proteins and the film is enhanced by hydrophobic interactions. At higher surface pressure, the number of protein clusters increases and becomes closer in both cases implying the electrostatic character of the binding. These results indicate that even if the repeats exhibit large structural

  2. Microstructural characterization of GdBa2Cu3O7-δ superconductor films with BaHfO3 artificial pinning centers by scanning transmission electron microscopy.

    PubMed

    Yamada, Kazuhiro; Nishiyama, Takeshi; Kaneko, Kenji; Sato, Yukio; Teranishi, Ryo; Kato, Takeharu; Ibi, Akira; Yoshizumi, Masateru; Izumi, Teruo; Shiohara, Yuh

    2014-11-01

    Critical current (IC) of superconductor films under magnetic field is strongly influenced by dispersions and morphologies of artificial pinning centers (APCs) in general [1]. BaHfO3 (BHO) is acknowledged as the best candidates of APCs for REBCO films, which shows utmost thickness dependence and isotropic angular dependence of IC values for REBCO films [2]. Moreover, several researchers have focused on the nanostrains caused by the lattice mismatch at the interface between APCs and REBCO matrix, which are also the source for enhanced vortex pinning of the REBCO films [3]. In this study, we investigated to examine the nanostrain at the interface using spherical aberration (CS) corrected scanning transmission electron microscopy (STEM).BHO introduced GdBa2Cu3O7-δ (GdBCO) film was fabricated by pulsed laser deposition (PLD) method. TEM samples were prepared by focused ion beam (FIB; Quanta 3D 200i, FEI) method followed by Ar ion thinning (NanoMill, Fischione) method. Atomic scale imaging was performed by spherical aberration corrected STEM (JEM-ARM200F, JEOL), then microstructures of BHO/GdBCO interface was then examined by Fourier transformation (FFT).BHO nanorods and nanoparticles were found dispersed in the GdBCO matrix, where {100} and {110} facets were present at BHO/GdBCO interfaces, as shown in Fig. 1. In the case of PLD process, most favorable growth direction of BHO is [001] direction, so that the regular quadrangular prism shaped BHO with {100} facets would be grown along [001] direction of GdBCO matrix [4]. {110} facets of BHO were formed to maintain the minimum surface area at BHO/GdBCO to reduce the interfacial energy.jmicro;63/suppl_1/i27-a/DFU082F1F1DFU082F1Fig. 1.Plan view HAADF-STEM image and FFT image showing facets at BHO/GdBCO interfaces. This work was supported by the Ministry of Economy, Trade and Industry (METI) as "Development of Fundamental Technologies for HTS Coils" and the JSPS KAKENHI (26600046).

  3. Application of colloid probe atomic force microscopy to the adhesion of thin films of viscous and viscoelastic silicone fluids.

    PubMed

    Bowen, James; Cheneler, David; Andrews, James W; Avery, Andrew R; Zhang, Zhibing; Ward, Michael C L; Adams, Michael J

    2011-09-20

    The adhesive characteristics of thin films (0.2-2 μm) of linear poly(dimethylsiloxane) (PDMS) liquids with a wide range of molecular weights have been measured using an atomic force microscope with a colloid probe (diameters 5 and 12 μm) for different separation velocities. The data were consistent with a residual film in the contact region having a thickness of ∼6 nm following an extended dwell time before separation of the probe. It was possible to estimate the maximum adhesive force as a function of the capillary number, Ca, by applying existing theoretical models based on capillary interactions and viscous flow except at large values of Ca in the case of viscoelastic fluids, for which it was necessary to develop a nonlinear viscoelastic model. The compliance of the atomic force microscope colloid beam was an important factor in governing the retraction velocity of the probe and therefore the value of the adhesive force, but the inertia of the beam and viscoelastic stress overshoot effects were not significant in the range of separation velocities investigated.

  4. Tracking the Evolution of Polymer Interface Films during the Process of Thermal Annealing at the Domain and Single Molecular Levels using Scanning Tunneling Microscopy.

    PubMed

    Duan, Xiao-Ling; Chen, Hua-Jie; Huang, Jian-Yao; Liu, Zhi-Fei; Li, Jin-Kuo; Yang, Zhi-Yong; Zhang, Wei-Feng; Yu, Gui

    2016-09-20

    Structural evolution of polymer (NTZ12) interface films during the process of annealing is revealed at the domain and single molecular levels using the statistical data measured from scanning tunneling microscopy images and through theoretical calculations. First, common features of the interface films are examined. Then, mean values of surface-occupied ratio, size and density of the domain are used to reveal the intrinsic derivation of the respective stages. Formation of new domains is triggered at 70 °C, but domain ripening is not activated. At 110 °C, the speed of formation of new domains is almost balanced by the consumption due to the ripening process. However, formation of new domains is reduced heavily at 150 °C but restarted at 190 °C. At the single molecular level, the ratio of the average length of linear to curved backbones is increased during annealing, whereas the ratios of the total length and the total number of linear to curved skeletons reaches a peak value at 150 °C. The two major conformations of curved backbones for all samples are 120° and 180° bending, but the ripening at 150 °C reduces 180° folding dramatically. Molecular dynamic simulations disclose the fast relaxing process of curved skeletons at high temperature.

  5. Mn Doping Effects on the Electronic Band Structure of PbS Quantum Dot Thin Films: A Scanning Tunneling Microscopy Analysis

    NASA Astrophysics Data System (ADS)

    Yost, Andrew J.; Rimal, Gaurab; Tang, Jinke; Chien, Teyu

    A thorough understanding of the phenomena associated with doping of transition metals in semiconductors is important for the development of semiconducting electronic technologies such as semiconducting quantum dot sensitized solar cells (QDSSC). Manganese doping is of particular interest in a PbS QD as it is potentially capable of increasing overall QDSSC performance. Here we present scanning tunneling microscopy and spectroscopy studies about the effects of Manganese doping on the energy band structures of PbS semiconducting QD thin films, grown using pulsed laser deposition. As a result of Manganese doping in the PbS QD thin films, a widening of the electronic band gap was observed, which is responsible for the observed increase in resistivity. Furthermore, a loss of long range periodicity observed by XRD, upon incorporation of Manganese, indicates that the Manganese dopants also induce a large amount of grain boundaries. This work was supported by the following: U.S. Department of Energy, Office of Basic Energy Sciences, Division of Materials Science and Engineering, DEFG02-10ER46728 and the National Science Foundation Grant #0948027.

  6. Frequency modulated atomic force microscopy on MgO(001) thin films: interpretation of atomic image resolution and distance dependence of tip-sample interaction.

    PubMed

    Heyde, M; Sterrer, M; Rust, H-P; Freund, H-J

    2006-04-14

    Atomically resolved images on a MgO(001) thin film deposited on Ag(001) obtained in ultrahigh vacuum by frequency modulated atomic force microscopy at low temperature are presented and analysed. Images obtained in the attractive regime show a different type of contrast formation from those acquired in the repulsive regime. For the interpretation of the image contrast we have investigated the tip-sample interaction. Force and energy were recovered from frequency shift versus distance curves. The derived force curves have been compared to the force laws of long-range, short-range and contact forces. In the attractive regime close to the minimum of the force-distance curve elastic deformations have been confirmed. The recovered energy curve has been scaled to the universal Rydberg model, yielding a decay length of l = 0.3 nm and ΔE = 4.2 aJ (26 eV) for the maximum adhesion energy. A universal binding-energy-distance relation is confirmed for the MgO(001) thin film.

  7. Cathodoluminescence and Cross-sectional Transmission Electron Microscopy Studies for Deformation Behaviors of GaN Thin Films Under Berkovich Nanoindentation

    PubMed Central

    2008-01-01

    In this study, details of Berkovich nanoindentation-induced mechanical deformation mechanisms of metal-organic chemical-vapor deposition-derived GaN thin films have been systematic investigated with the aid of the cathodoluminescence (CL) and the cross-sectional transmission electron microscopy (XTEM) techniques. The multiple “pop-in” events were observed in the load-displacement (P–h) curve and appeared to occur randomly by increasing the indentation load. These instabilities are attributed to the dislocation nucleation and propagation. The CL images of nanoindentation show very well-defined rosette structures with the hexagonal system and, clearly display the distribution of deformation-induced extended defects/dislocations which affect CL emission. By using focused ion beam milling to accurately position the cross-section of an indented area, XTEM results demonstrate that the major plastic deformation is taking place through the propagation of dislocations. The present observations are in support to the massive dislocations activities occurring underneath the indenter during the loading cycle. No evidence of either phase transformation or formation of micro-cracking was observed by means of scanning electron microscopy and XTEM observations. We also discuss how these features correlate with Berkovich nanoindentation produced defects/dislocations structures.

  8. Traceable Quantitative Raman Microscopy and X-ray Fluorescence Analysis as Nondestructive Methods for the Characterization of Cu(In,Ga)Se2 Absorber Films.

    PubMed

    Zakel, Sabine; Pollakowski, Beatrix; Streeck, Cornelia; Wundrack, Stefan; Weber, Alfons; Brunken, Stefan; Mainz, Roland; Beckhoff, Burckhardt; Stosch, Rainer

    2016-02-01

    The traceability of measured quantities is an essential condition when linking process control parameters to guaranteed physical properties of a product. Using Raman spectroscopy as an analytical tool for monitoring the production of Cu(In1-xGax)Se2 thin-film solar cells, proper calibration with regard to chemical composition and lateral dimensions is a key prerequisite. This study shows how the multiple requirements of calibration in Raman microscopy might be addressed. The surface elemental composition as well as the integral elemental composition of the samples is traced back by reference-free X-ray fluorescence analysis. Reference Raman spectra are then generated for the relevant Cu(In1-xGax)Se2 related compounds. The lateral dimensions are calibrated with the help of a novel dimensional standard whose regular structures have been traced back to the International System of Units by metrological scanning force microscopy. On this basis, an approach for the quantitative determination of surface coverage values from lateral Raman mappings is developed together with a complete uncertainty budget. Raman and X-ray spectrometry have here been proven as complementary nondestructive methods combining surface sensitivity and in-depth information on elemental and species distribution for the reliable quality control of Cu(In1-xGax)Se2 absorbers and Cu(In1-xGax)3Se5 surface layer formation.

  9. Detailed Study of BSA Adsorption on Micro- and Nanocrystalline Diamond/β-SiC Composite Gradient Films by Time-Resolved Fluorescence Microscopy.

    PubMed

    Handschuh-Wang, Stephan; Wang, Tao; Druzhinin, Sergey I; Wesner, Daniel; Jiang, Xin; Schönherr, Holger

    2017-01-24

    The adsorption of bovine serum albumin (BSA) on micro- and nanocrystalline diamond/β-SiC composite films synthesized using the hot filament chemical vapor deposition (HFCVD) technique has been investigated by confocal fluorescence lifetime imaging microscopy. BSA labeled with fluorescein isothiocyanate (FITC) was employed as a probe. The BSA(FITC) conjugate was found to preferentially adsorb on both O-/OH-terminated microcrystalline and nanocrystalline diamond compared to the OH-terminated β-SiC, resulting in an increasing amount of BSA adsorbed to the gradient surfaces with an increasing diamond/β-SiC ratio. The different strength of adsorption (>30 times for diamond with a grain size of 570 nm) coincides with different surface energy parameters and differing conformational changes upon adsorption. Fluorescence data of the adsorbed BSA(FITC) on the gradient film with different diamond coverage show a four-exponential decay with decay times of 3.71, 2.54, 0.66, and 0.13 ns for a grain size of 570 nm. The different decay times are attributed to the fluorescence of thiourea fluorescein residuals of linked FITC distributed in BSA with different dye-dye and dye-surface distances. The longest decay time was found to correlate linearly with the diamond grain size. The fluorescence of BSA(FITC) undergoes external dynamic fluorescence quenching on the diamond surface by H- and/or sp(2)-defects and/or by amorphous carbon or graphite phases. An acceleration of the internal fluorescence concentration quenching in BSA(FITC) because of structural changes of albumin due to adsorption, is concluded to be a secondary contributor. These results suggest that the micro- and nanocrystalline diamond/β-SiC composite gradient films can be utilized to spatially control protein adsorption and diamond crystallite size, which facilitates systematic studies at these interesting (bio)interfaces.

  10. Films

    NASA Astrophysics Data System (ADS)

    Li, Ming; Zhang, Yang; Shao, Yayun; Zeng, Min; Zhang, Zhang; Gao, Xingsen; Lu, Xubing; Liu, J.-M.; Ishiwara, Hiroshi

    2014-09-01

    In this paper, we investigated the microstructure and electrical properties of Bi2SiO5 (BSO) doped SrBi2Ta2O9 (SBT) films deposited by chemical solution deposition. X-ray diffraction observation indicated that the crystalline structures of all the BSO-doped SBT films are nearly the same as those of a pure SBT film. Through BSO doping, the 2Pr and 2Ec values of SBT films were changed from 15.3 μC/cm2 and 138 kV/cm of pure SBT to 1.45 μC/cm2 and 74 kV/cm of 10 wt.% BSO-doped SBT. The dielectric constant at 1 MHz for SBT varied from 199 of pure SBT to 96 of 10 wt.% BSO-doped SBT. The doped SBT films exhibited higher leakage current than that of non-doped SBT films. Nevertheless, all the doped SBT films still had small dielectric loss and low leakage current. Our present work will provide useful insights into the BSO doping effects to the SBT films, and it will be helpful for the material design in the future nonvolatile ferroelectric memories.

  11. Surface Roughness and Critical Exponent Analyses of Boron-Doped Diamond Films Using Atomic Force Microscopy Imaging: Application of Autocorrelation and Power Spectral Density Functions

    NASA Astrophysics Data System (ADS)

    Gupta, S.; Vierkant, G. P.

    2014-09-01

    The evolution of the surface roughness of growing metal or semiconductor thin films provides much needed information about their growth kinetics and corresponding mechanism. While some systems show stages of nucleation, coalescence, and growth, others exhibit varying microstructures for different process conditions. In view of these classifications, we report herein detailed analyses based on atomic force microscopy (AFM) characterization to extract the surface roughness and growth kinetics exponents of relatively low boron-doped diamond (BDD) films by utilizing the analytical power spectral density (PSD) and autocorrelation function (ACF) as mathematical tools. The machining industry has applied PSD for a number of years for tool design and analysis of wear and machined surface quality. Herein, we present similar analyses at the mesoscale to study the surface morphology as well as quality of BDD films grown using the microwave plasma-assisted chemical vapor deposition technique. PSD spectra as a function of boron concentration (in gaseous phase) are compared with those for samples grown without boron. We find that relatively higher boron concentration yields higher amplitudes of the longer-wavelength power spectral lines, with amplitudes decreasing in an exponential or power-law fashion towards shorter wavelengths, determining the roughness exponent ( α ≈ 0.16 ± 0.03) and growth exponent ( β ≈ 0.54), albeit indirectly. A unique application of the ACF, which is widely used in signal processing, was also applied to one-dimensional or line analyses (i.e., along the x- and y-axes) of AFM images, revealing surface topology datasets with varying boron concentration. Here, the ACF was used to cancel random surface "noise" and identify any spatial periodicity via repetitive ACF peaks or spatially correlated noise. Periodicity at shorter spatial wavelengths was observed for no doping and low doping levels, while smaller correlations were observed for relatively

  12. Revealing nanoscale optical properties and morphology in perfluoropentacene films by confocal and tip-enhanced near-field optical microscopy and spectroscopy.

    PubMed

    Wang, Xiao; Broch, Katharina; Schreiber, Frank; Meixner, Alfred J; Zhang, Dai

    2016-06-21

    Combining high resolution optical microscopy and spectroscopy, we propose a novel, generally applicable and highly sensitive method for determining the local morphology in organic semiconductor thin films (e.g. perfluoropentacene (PFP)). An azimuthally or radially polarized doughnut mode (APDM or RPDM) laser beam is focused by a high numerical aperture parabolic-mirror to excite a diffraction limited volume of the PFP film with an electric field polarized either exclusively in-plane or dominantly out-of-plane (relative to the substrate). We find two distinct morphologies of thin PFP films: molecular aggregates and crystalline terraces. The well-defined dipole emission patterns observed from the molecular aggregates strongly suggest the presence of localized excitations. For both laser modes, we observe that for the PFP aggregates, the photoluminescence (PL) emission from the main electronic transition is blue-shifted by about 10 meV, as compared to that from the molecular terraces. For the C-C bending modes, the B3g at 1581 cm(-1) (ν1) and the Ag at 1316 cm(-1) (ν0), we observe a decrease of the intensity ratio (Iν1/Iν0) from 0.6 (terrace) to 0.15 (aggregate). Furthermore, the intensity ratios (IAPDM/IRPDM) of ν1 excited by different polarizations increase from 0.12 (terrace) to 0.73 (aggregate). These results indicate that the PFP molecules orient rather parallel to the substrate in the aggregates, whilst more upright in the terraces. Benefiting from the nanometer scale optical resolution offered by the tip-enhanced near-field optical method, we observe clear optical contrasts between the molecular aggregate and the terrace as well as individual layers within a terrace. Tip-enhanced optical spectra locally taken from the molecular terrace and the aggregate show similar blue-shift of the main PL peak and change in the Raman intensity with different polarizations as from the far-field assemble-measurements, which further confirms the different molecular

  13. An In Situ Electric Field Study of Magnetoelectric Coupling in PZT-LSMO Thin Film Heterostructures Using Polarized Neutron Reflectometry and Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Spurgeon, Steven; Sloppy, Jennifer; Huang, Esther; Vasudevan, Rama; Lofland, Samuel; Lauter, Valeria; Valanoor, Nagarajan; Taheri, Mitra

    2013-03-01

    The development of ``spintronics'' devices based on charge and spin transport has signaled a paradigm shift in the design of data storage and computing technologies. Magnetoelectric materials, which exhibit intrinsic coupling between electronic and magnetic order, are ideal for these applications. Unfortunately, single-phase magnetoelectrics are exceedingly rare in nature and attention has turned to composite heterostructures that display coupled functionalities at interfaces. A promising system in which to explore this coupling is a thin film oxide heterostructure of the piezoelectric Pb(Zr0.2Ti0.8)O3 (PZT) and the half-metal La0.7Sr0.3MnO3 (LSMO). We show that it is possible to construct a capacitor-type device structure from these materials that may form the basis for an electrically-switched magnetic memory. We conduct polarized neutron reflectometry (PNR) measurements and measure changes in the magnetization depth profile throughout the composite under the reversal of an in situ electric field. We then correlate these PNR results to local strain and chemistry using transmission electron microscopy (TEM). We find that a combination of charge doping and strain mechanisms governs coupling in this system.

  14. Microstructural investigation of the oxidation behavior of Cu in Ag-coated Cu films using a focused ion beam transmission electron microscopy technique

    NASA Astrophysics Data System (ADS)

    Kim, Ji Hwan; Lee, Jong-Hyun

    2016-06-01

    With the aim of elucidating a detailed mechanism for the oxidation behavior in submicron Cu particles coated with a thin Ag layer, the dewetting of Ag and the oxidation behavior of Cu in Ag-coated Cu films upon heating were investigated with a focused ion beam transmission electron microscopy technique. A slight dewetting of the Ag layer began at approximately 200 °C and aggregates of Cu2O particles were formed on the Ag layer, indicating that the initial Cu2O phase was formed on the thin Ag layer. Voids were formed in the Cu layer because of Cu atoms diffusing through the thin Ag layer to be oxidized in the upper Cu2O aggregates. After being heated to 250 °C, the Ag layer became more irregular, and in some regions, it disappeared because of intensive dewetting. The number and average size of the voids also increased. At 300 °C, a hollow structure with a Cu2O shell was formed. Pillar-like structures of unoxidized Cu and large voids were found under the Cu2O layer.

  15. Quantitative investigation of the photodegradation of polyethylene terephthalate film by friction force microscopy, contact-angle goniometry, and X-ray photoelectron spectroscopy.

    PubMed

    Hurley, Claire R; Leggett, Graham J

    2009-08-01

    Studies of the UV-induced photodegradation of poly(ethylene terephthalate) (PET) have been carried out using contact-angle goniometry, X-ray photoelectron spectroscopy (XPS), and friction force microscopy (FFM). The advancing contact angle of water, theta, decreased following exposure of free-standing PET films to UV light. Measurements of surface friction by FFM showed that the coefficient of friction mu increased as the degradation proceeded, reaching a limiting value after ca 200 min, in agreement with the contact angle data. Using a modified form of the Cassie equation, a quantitative analysis of the extent of modification could be carried out. There was a very close correlation between the coefficient of friction determined by FFM and the value of cos theta. XPS provided more detailed information on surface bonding that also correlated closely with the FFM data. Although FFM provides quantitative data on surface modification with nanometer-scale spatial resolution, it does not provide detailed structural information such as is provided by XPS. The oxygen content at the surface was found to increase as photo-generated radicals within the PET reacted with atmospheric oxygen. Increases in both ester and carbonyl contributions within XPS data accompanied this increase. It was concluded that the photodegradation process follows mainly Norrish type I reaction pathways, following previous work by Fechine et al and Grosstete et al.

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

    NASA Astrophysics Data System (ADS)

    Hassanzadeh, Abdollah

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

  17. A Rotational BODIPY Nucleotide: An Environment-Sensitive Fluorescence-Lifetime Probe for DNA Interactions and Applications in Live-Cell Microscopy.

    PubMed

    Dziuba, Dmytro; Jurkiewicz, Piotr; Cebecauer, Marek; Hof, Martin; Hocek, Michal

    2016-01-04

    Fluorescent probes for detecting the physical properties of cellular structures have become valuable tools in life sciences. The fluorescence lifetime of molecular rotors can be used to report on variations in local molecular packing or viscosity. We used a nucleoside linked to a meso-substituted BODIPY fluorescent molecular rotor (dC(bdp)) to sense changes in DNA microenvironment both in vitro and in living cells. DNA incorporating dC(bdp) can respond to interactions with DNA-binding proteins and lipids by changes in the fluorescence lifetimes in the range 0.5-2.2 ns. We can directly visualize changes in the local environment of exogenous DNA during transfection of living cells. Relatively long fluorescence lifetimes and extensive contrast for detecting changes in the microenvironment together with good photostability and versatility for DNA synthesis make this probe suitable for analysis of DNA-associated processes, cellular structures, and also DNA-based nanomaterials.

  18. Investigation of subcellular localization and dynamics of membrane proteins in living bacteria by combining optical micromanipulation and high-resolution microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Barroso Peña, Álvaro; Nieves, Marcos; Teper, Konrad; Wedlich-Soldner, Roland; Denz, Cornelia

    2016-09-01

    The plasma membrane serves as protective interface between cells and their environment. It also constitutes a hub for selective nutrient uptake and signal transduction. Increasing evidence over the last years indicates that, similar to eukaryotic cells, lateral membrane organization plays an important role in the regulation of prokaryotic signaling pathways. However, the mechanisms underlying this phenomenon are still poorly understood. Spatiotemporal characterization of bacterial signal transduction demands very sensitive high-resolution microscopy techniques due to the low expression levels of most signaling proteins and the small size of bacterial cells. In addition, direct study of subcellular confinement and dynamics of bacterial signaling proteins during the different stages of the signal transduction also requires immobilization in order to avoid cell displacement caused by Brownian motion, local fluid flows and bacterial self-propulsion. In this work we present a novel approach based on the combination of high resolution imaging and optical manipulation that enables the investigation of the distribution and dynamics of proteins at the bacterial plasma membrane. For this purpose, we combine the versatility of holographic optical tweezers (HOT) with the sensitivity and resolution of total internal reflection fluorescence (TIRF) microscopy. Furthermore, we discuss the implementation of microfluidic devices in our integrated HOT+TIRF system for the control of growth conditions of bacterial cells. The capabilities of our workstation provides thus new valuable insights into the fundamental cellular and physical mechanisms underlying the regulation of bacterial signal transduction.

  19. Direct electrocaloric measurement of 0.9Pb(Mg{sub 1/3}Nb{sub 2/3})O{sub 3}-0.1PbTiO{sub 3} films using scanning thermal microscopy

    SciTech Connect

    Crossley, S.; Nair, B.; Kar-Narayan, S.; Moya, X.; Mathur, N. D.; Usui, T.; Hirose, S.; Ando, A.

    2016-01-18

    We show that scanning thermal microscopy can measure reversible electrocaloric (EC) effects in <40 μm-thick ceramic films of the relaxor ferroelectric 0.9Pb(Mg{sub 1/3}Nb{sub 2/3})O{sub 3}-0.1PbTiO{sub 3}, with the substrate present. We recorded roughly the same non-adiabatic temperature change (±0.23 K) for a thinner film that was driven harder than a thicker film (±31 V μm{sup −1} across 13 μm versus ±11 V μm{sup −1} across 38 μm), because the thicker film lay relatively closer to the substantially larger adiabatic values that we predicted by thermodynamic analysis of electrical data. Film preparation was compatible with the fabrication of EC multilayer capacitors, and therefore our measurement method may be exploited for rapid characterisation of candidate films for cooling applications.

  20. Use of Optical Microscopy to Examine Crystallite Nucleation and Growth in Thermally Annealed Plasma Enhanced Chemical Vapor Deposition and Hot Wire Chemical Vapor Deposition a-Si:H Films

    SciTech Connect

    Mahan, A. H.; Dabney, M. S.; Reedy, Jr R. C.; Molina, D.; Ginley, D. S.

    2012-05-15

    We report a simple method to investigate crystallite nucleation and growth in stepwise, thermally annealed plasma enhanced chemical vapor deposition and hot wire chemical vapor deposition a-Si:H films. By confining film thicknesses to the range 500-4000 {angstrom}, optical microscopy in the reflection mode can be used to readily detect crystallites in the thermally annealed a-Si:H lattice. Measurements of the crystallite density versus annealing time for identically prepared films of different thickness show that the crystallite nucleation rate is smaller for thinner films, suggesting that crystallite nucleation is homogeneous, in agreement with previous results. A comparison of film nucleation rates with those obtained by other methods on identically prepared films shows excellent agreement, thus establishing the validity of the current technique. The potential effect of impurity (oxygen) incorporation during the stepwise annealing in air is shown not to affect crystallite nucleation and growth, in that SIMS oxygen profiles for stepwise versus continuous annealing show not only similar impurity profiles but also similar bulk impurity densities.