Science.gov

Sample records for live microscopy film

  1. Filming a live cell by scanning electrochemical microscopy: label-free imaging of the dynamic morphology in real time.

    PubMed

    Zhang, Michelle Meng-Ni; Long, Yi-Tao; Ding, Zhifeng

    2012-03-21

    The morphology of a live cell reflects the organization of the cytoskeleton and the healthy status of the cell. We established a label-free platform for monitoring the changing morphology of live cells in real time based on scanning electrochemical microscopy (SECM). The dynamic morphology of a live human bladder cancer cell (T24) was revealed by time-lapse SECM with dissolved oxygen in the medium solution as the redox mediator. Detailed local movements of cell membrane were presented by time-lapse cross section lines extracted from time-lapse SECM. Vivid dynamic morphology is presented by a movie made of time-lapse SECM images. The morphological change of the T24 cell by non-physiological temperature is in consistence with the morphological feature of early apoptosis. To obtain dynamic cellular morphology with other methods is difficult. The non-invasive nature of SECM combined with high resolution realized filming the movements of live cells.

  2. Confocal diffraction phase microscopy of live cells.

    PubMed

    Lue, Niyom; Choi, Wonshik; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S; Popescu, Gabriel

    2008-09-15

    We present a new quantitative phase microscopy technique, confocal diffraction phase microscopy, which provides quantitative phase measurements from localized sites on a sample with high sensitivity. The technique combines common-path interferometry with confocal microscopy in a transmission geometry. The capability of the technique for static imaging is demonstrated by imaging polystyrene microspheres and live HT29 cells, while dynamic imaging is demonstrated by quantifying the nanometer scale fluctuations of red blood cell membranes.

  3. 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 (

  4. 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

  5. Spectro-microscopy of living plant cells.

    PubMed

    Harter, Klaus; Meixner, Alfred J; Schleifenbaum, Frank

    2012-01-01

    Spectro-microscopy, a combination of fluorescence microscopy with spatially resolved spectroscopic techniques, provides new and exciting tools for functional cell biology in living organisms. This review focuses on recent developments in spectro-microscopic applications for the investigation of living plant cells in their native tissue context. The application of spectro-microscopic methods led to the recent discovery of a fast signal response pathway for the brassinosteroide receptor BRI1 in the plasma membrane of living plant cells. Moreover, the competence of different plant cell types to respond to environmental or endogenous stimuli was determined in vivo by correlation analysis of different optical and spectroscopic readouts such as fluorescence lifetime (FLT). Furthermore, a new spectro-microscopic technique, fluorescence intensity decay shape analysis microscopy (FIDSAM), has been developed. FIDSAM is capable of imaging low-expressed fluorophore-tagged proteins at high spatial resolution and precludes the misinterpretation of autofluorescence artifacts. In addition, FIDSAM provides a very effective and sensitive tool on the basis of Förster resonance energy transfer (FRET) for the qualitative and quantitative determination of protein-protein interaction. Finally, we report on the quantitative analysis of the photosystem I and II (PSI/PSII) ratio in the chloroplasts of living Arabidopsis plants at room temperature, using high-resolution, spatially resolved fluorescence spectroscopy. With this technique, it was not only possible to measure PSI/PSII ratios, but also to demonstrate the differential competence of wild-type and carbohydrate-deficient plants to adapt the PSI/PSII ratio to different light conditions. In summary, the information content of standard microscopic images is extended by several dimensions by the use of spectro-microscopic approaches. Therefore, novel cell physiological and molecular topics can be addressed and valuable insights into

  6. 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.

  7. 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

  8. 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.

  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. 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

  11. Analytical electron microscopy of thin films

    NASA Astrophysics Data System (ADS)

    Malac, Marek

    An analytical transmission electron microscope (ATEM) yields an impressive amount of information from a single instrument. The chemical composition of small areas of a sample is often obtained by energy dispersive x-ray microanalysis (EDX). EDX is routinely used both in research and industry to obtain fractions of heavier elements (Z > 11). To allow quantitative EDX analysis of samples containing light elements (B, C, N, O, F, Mg and Si) we developed, fabricated and characterized a set of three calibration samples. These calibration specimens allow users to obtain experimental Cliff-Lorimer factors with 10% to 15% accuracy and are sufficiently stable during storage, as well as under electron beam irradiation. Quantitative electron energy-loss spectroscopy (EELS) was employed to characterize these samples. The good light-element sensitivity of EELS makes it a suitable method for chemical analysis of biological samples in ATEM. It is desirable to probe the detection limits of EELS and energy filtering transmission electron microscopy (EFTEM) as well as determine what physical processes underlying these limits. We find that a TEM/EELS system is capable quantifying of 2000 ppm of boron with about 10% accuracy and 1 mum resolution. EFTEM mapping using Gatan Image filter is capable of mapping 5000 ppm of boron with 66 nm pixel size. The minimum detectable fraction (MDF) was limited by detector gain-variations and beam-shot noise. Spatial (EFTEM or TEM/EELS) mapping of low boron concentrations is important for boron-neutron capture therapy (BNCT), a method of cancer treatment. The high spatial resolution of TEM imaging and chemical analysis was applied to study microscopic mechanism of growth of thin films deposited onto oblique (rotating) substrate. The structure of these films can vary between arrays of columns (stationary substrate), helices (slowly-rotated substrate) or pillars (fast-rotated substrate). These structures (columns, pillars, helices) are composed of many

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. Atomic-force microscopy of submicron films of electroactive polymer

    NASA Astrophysics Data System (ADS)

    Karamov, D. D.; Kornilov, V. M.; Lachinov, A. N.; Kraikin, V. A.; Ionova, I. A.

    2016-07-01

    Atomic-force microscopy is used to study the supramolecular structure of submicron films of electroactive thermally stable polymer (polydiphenylenephthalide (PDP)). It has been demonstrated that PDP films produced using centrifuging are solid homogeneous films with thicknesses down to several nanometers, which correspond to two or three monomolecular layers. The film volume is structurized at thicknesses greater than 100 nm. The study of the rheological properties of solutions used for film production yields a crossover point that separates the domains of strongly diluted and semidiluted solutions. A transition from the globular structure to the associate structure is observed in films that are produced using solutions with a boundary concentration. A model of the formation of polymer film that involves the presence of associates in the original solution is discussed.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

  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. Fast live simultaneous multiwavelength four-dimensional optical microscopy

    PubMed Central

    Carlton, Peter M.; Boulanger, Jérôme; Kervrann, Charles; Sibarita, Jean-Baptiste; Salamero, Jean; Gordon-Messer, Susannah; Bressan, Debra; Haber, James E.; Haase, Sebastian; Shao, Lin; Winoto, Lukman; Matsuda, Atsushi; Kner, Peter; Uzawa, Satoru; Gustafsson, Mats; Kam, Zvi; Agard, David A.; Sedat, John W.

    2010-01-01

    Live fluorescence microscopy has the unique capability to probe dynamic processes, linking molecular components and their localization with function. A key goal of microscopy is to increase spatial and temporal resolution while simultaneously permitting identification of multiple specific components. We demonstrate a new microscope platform, OMX, that enables subsecond, multicolor four-dimensional data acquisition and also provides access to subdiffraction structured illumination imaging. Using this platform to image chromosome movement during a complete yeast cell cycle at one 3D image stack per second reveals an unexpected degree of photosensitivity of fluorophore-containing cells. To avoid perturbation of cell division, excitation levels had to be attenuated between 100 and 10,000× below the level normally used for imaging. We show that an image denoising algorithm that exploits redundancy in the image sequence over space and time allows recovery of biological information from the low light level noisy images while maintaining full cell viability with no fading. PMID:20705899

  4. 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.

  5. Single-Molecule Fluorescence Microscopy in Living Caenorhabditis elegans.

    PubMed

    van Krugten, Jaap; Peterman, Erwin J G

    2018-01-01

    Transportation of organelles and biomolecules is vital for many cellular processes. Single-molecule (SM) fluorescence microscopy can expose molecular aspects of the dynamics that remain unresolved in ensemble experiments. For example, trajectories of individual, moving biomolecules can reveal velocity and changes therein, including pauses. We use SM imaging to study the dynamics of motor proteins and their cargo in the cilia of living C. elegans. To this end, we employ standard fluorescent proteins, an epi-illuminated, wide-field fluorescence microscope and mostly open-source software. This chapter describes the setup we use, the preparation of samples, a protocol for single-molecule imaging in C. elegans and data analysis.

  6. 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

  7. Live cell refractometry using Hilbert phase microscopy and confocal reflectance microscopy.

    PubMed

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R; Feld, Michael S

    2009-11-26

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ.

  8. 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.

  9. 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.

  10. Imaging non-fluorescent nanoparticles in living cells with wavelength-dependent differential interference contrast microscopy and planar illumination microscopy.

    PubMed

    Sun, Wei; Xiao, Lehui; Fang, Ning

    2013-01-01

    Optical microscopy is a simple yet robust strategy to study live cellular processes. By changing the wavelength of the illumination light, different non-fluorescent nanoparticle probes can be identified and tracked dynamically inside crowded living cells with either differential interference contrast (DIC) microscopy or planar illumination microscopy (PIM). The translational and rotational dynamics of anisotropic nanoparticles can be readily extracted via the modified DIC microscope and the home-built PIM. In this protocol, the optimization procedures for DIC microscopy and PIM imaging are explained, and the sample preparation procedures to image non-fluorescent nanoparticles in living cells are described.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. 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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-05-01

    The structure of Fe1 + δTe1 - x Se x films ( x = 0; 0.05) grown on single-crystal MgO and LaAlO3 substrates has been investigated by transmission and scanning transmission electron microscopy. The study of Fe1.11Te/MgO structures has revealed two crystallographic orientation relationships between the film and substrate. It is shown that the lattice mismatch between the film and substrate is compensated for by the formation of misfit dislocations. The Burgers vector projection is determined. The stresses in the film can partially be compensated for due to the formation of an intermediate disordered layer. It is shown that a FeTe0.5Se0.5 film grown on a LaAlO3 substrate is single-crystal and that the FeTe0.5Se0.5/LaAlO3 interface in a selected region is coherent. The orientation relationships between the film and substrate are also determined for this case.

  20. 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.

  1. 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

  2. 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.

  3. 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

  4. 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.

  5. Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

    PubMed Central

    Lewis, Leanne E.; Bain, Judith M.; Okai, Blessing; Gow, Neil A.R.; Erwig, Lars Peter

    2013-01-01

    Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10. In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a

  6. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    PubMed Central

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-01-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution. PMID:26390855

  7. Simultaneous Fluorescence and Phosphorescence Lifetime Imaging Microscopy in Living Cells

    NASA Astrophysics Data System (ADS)

    Jahn, Karolina; Buschmann, Volker; Hille, Carsten

    2015-09-01

    In living cells, there are always a plethora of processes taking place at the same time. Their precise regulation is the basis of cellular functions, since small failures can lead to severe dysfunctions. For a comprehensive understanding of intracellular homeostasis, simultaneous multiparameter detection is a versatile tool for revealing the spatial and temporal interactions of intracellular parameters. Here, a recently developed time-correlated single-photon counting (TCSPC) board was evaluated for simultaneous fluorescence and phosphorescence lifetime imaging microscopy (FLIM/PLIM). Therefore, the metabolic activity in insect salivary glands was investigated by recording ns-decaying intrinsic cellular fluorescence, mainly related to oxidized flavin adenine dinucleotide (FAD) and the μs-decaying phosphorescence of the oxygen-sensitive ruthenium-complex Kr341. Due to dopamine stimulation, the metabolic activity of salivary glands increased, causing a higher pericellular oxygen consumption and a resulting increase in Kr341 phosphorescence decay time. Furthermore, FAD fluorescence decay time decreased, presumably due to protein binding, thus inducing a quenching of FAD fluorescence decay time. Through application of the metabolic drugs antimycin and FCCP, the recorded signals could be assigned to a mitochondrial origin. The dopamine-induced changes could be observed in sequential FLIM and PLIM recordings, as well as in simultaneous FLIM/PLIM recordings using an intermediate TCSPC timing resolution.

  8. 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. © 2013 International Society for Neurochemistry.

  9. 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.

  10. 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.

  11. Thin-film tunable filters for hyperspectral fluorescence microscopy

    PubMed Central

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

    2013-01-01

    Abstract. 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. PMID:24077519

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  18. Detecting RNA viruses in living mammalian cells by fluorescence microscopy.

    PubMed

    Sivaraman, Divya; Biswas, Payal; Cella, Lakshmi N; Yates, Marylynn V; Chen, Wilfred

    2011-07-01

    Traditional methods that rely on viral isolation and culture techniques continue to be the gold standards used for detection of infectious viral particles. However, new techniques that rely on visualization of live cells can shed light on understanding virus-host interaction for early stage detection and potential drug discovery. Live-cell imaging techniques that incorporate fluorescent probes into viral components provide opportunities for understanding mRNA expression, interaction, and virus movement and localization. Other viral replication events inside a host cell can be exploited for non-invasive detection, such as single-virus tracking, which does not inhibit viral infectivity or cellular function. This review highlights some of the recent advances made using these novel approaches for visualization of viral entry and replication in live cells. Copyright © 2011 Elsevier Ltd. All rights reserved.

  19. Physical chemistry in a single live cell: confocal microscopy.

    PubMed

    Amin, Md Asif; Nandi, Somen; Mondal, Prasenjit; Mahata, Tanushree; Ghosh, Surajit; Bhattacharyya, Kankan

    2017-05-24

    A live cell is a complex, yet extremely important container. Understanding the dynamics in a selected intracellular component is a challenging task. We have recently made significant progress in this direction using a confocal microscope as a tool. The smallest size of the focused spot in a confocal microscope is ∼0.2 μm (200 nm). This is nearly one hundred times smaller than the size of a live cell. Thus, one can selectively study different intracellular components/organelles in a live cell. In this paper, we discuss how one can image different intracellular components/organelles, record fluorescence spectra and decay at different locations, ascertain local polarity and viscosity, and monitor the dynamics of solvation, proton transfer, red-ox and other phenomena at specified locations/organelles inside a cell. We will highlight how this knowledge enriched us in differentiating between cancer and non-cancer cells, 3D tumor spheroids and towards drug delivery.

  20. 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.

  1. Nanoscale observation of organic thin film by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Mochizuki, Shota; Uruma, Takeshi; Satoh, Nobuo; Saravanan, Shanmugam; Soga, Tetsuo

    2017-08-01

    Organic photovoltaics (OPVs) fabricated using organic semiconductors and hybrid solar cells (HSCs) based on organic semiconductors/quantum dots (QDs) have been attracting significant attention owing to their potential use in low-cost solar energy-harvesting applications and flexible, light-weight, colorful, large-area devices. In this study, we observed and evaluated the surface of a photoelectric conversion layer (active layer) of the OPVs and HSCs based on phenyl-C61-butyric acid methyl ester (PCBM), poly(3-hexylthiophene) (P3HT), and zinc oxide (ZnO) nanoparticles. The experiment was performed using atomic force microscopy (AFM) combined with a frequency modulation detector (FM detector) and a contact potential difference (CPD) detection circuit. We experimentally confirmed the changes in film thickness and surface potential, as affected by the ZnO nanoparticle concentration. From the experimental results, we confirmed that ZnO nanoparticles possibly affect the structures of PCBM and P3HT. Also, we prepared an energy band diagram on the basis of the observation results, and analyzed the energy distribution inside the active layer.

  2. 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.

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. In Situ Probing the Relaxation Properties of Ultrathin Polystyrene Films by Using Electric Force Microscopy

    NASA Astrophysics Data System (ADS)

    Qian, Xiaoqin; Lin, Zihong; Guan, Li; Li, Qiang; Wang, Yapei; Zhang, Meining; Dong, Mingdong

    2017-04-01

    The rapid development of nanoscience and nanotechnology involves polymer films with thickness down to nanometer scale. However, the properties of ultrathin polymer films are extremely different from that of bulk matrix or thin films. It is challenging to distinguish the changes of physical properties in ultrathin films using conventional techniques especially when it locates near the glass transition temperature ( T g). In this work, we successfully evaluated a series of physical properties of ultrathin polystyrene (PS) films by in situ characterizing the discharging behavior of the patterned charges using electric force microscopy. By monitoring the surface potential in real time, we found that the T g of ultrathin PS films is clearly independent of film thickness, which are greatly different from that of thin PS films (film thickness larger than 10 nm).

  8. Live correlative light-electron microscopy to observe molecular dynamics in high resolution.

    PubMed

    Kobayashi, Shouhei; Iwamoto, Masaaki; Haraguchi, Tokuko

    2016-08-01

    Fluorescence microscopy (FM) is a powerful tool for observing specific molecular components in living cells, but its spatial resolution is relatively low. In contrast, electron microscopy (EM) provides high-resolution information about cellular structures, but it cannot provide temporal information in living cells. To achieve molecular selectivity in imaging at high resolution, a method combining EM imaging with live-cell fluorescence imaging, known as live correlative light-EM (CLEM), has been developed. In this method, living cells are first observed by FM, fixed in situ during the live observation and then subjected to EM observation. Various fluorescence techniques and tools can be applied for FM, resulting in the generation of various modified methods that are useful for understanding cellular structure in high resolution. Here, we review the methods of CLEM and live-cell imaging associated with CLEM (live CLEM). Such methods can greatly advance the understanding of the function of cellular structures on a molecular level, and thus are useful for medical fields as well as for basic biology. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    PubMed

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

    2016-08-01

    Cell theory has officially reached 350 years of age as the first use of the word 'cell' in a biological context can be traced to a description of plant material by Robert Hooke in his historic publication 'Micrographia: or some physiological definitions of minute bodies'. The 2015 Royal Microscopical Society Botanical Microscopy meeting was a celebration of the streams of investigation initiated by Hooke to understand at the subcellular scale how plant cell function and form arises. Much of the work presented, and Honorary Fellowships awarded, reflected the advanced application of bioimaging informatics to extract quantitative data from micrographs that reveal dynamic molecular processes driving cell growth and physiology. The field has progressed from collecting many pixels in multiple modes to associating these measurements with objects or features that are meaningful biologically. The additional complexity involves object identification that draws on a different type of expertise from computer science and statistics that is often impenetrable to biologists. There are many useful tools and approaches being developed, but we now need more interdisciplinary exchange to use them effectively. In this review we show how this quiet revolution has provided tools available to any personal computer user. We also discuss the oft-neglected issue of quantifying algorithm robustness and the exciting possibilities offered through the integration of physiological information generated by biosensors with object detection and tracking. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  10. Magnetic anisotropy and microscopy studies in magnetostrictive Tb-(Fe,Co) thin films

    NASA Astrophysics Data System (ADS)

    Umadevi, K.; Talapatra, A.; Arout Chelvane, J.; Palit, Mithun; Mohanty, J.; Jayalakshmi, V.

    2017-08-01

    This paper reports the effect of the film thickness on the magnetostrictive behavior of (Fe,Co) rich Tb-(Fe,Co) films grown on Si ⟨100⟩ by electron beam evaporation. Magnetostriction was found to decrease with an increase in film thicknesses. To understand the variation of magnetostriction with the film thickness, detailed structural, microstructural, magnetization, and magnetic microscopy studies were carried out. X-ray diffraction studies indicated the presence of two phases, viz., Tb2 (Fe, Co)17 and Fe-Co phases, for all the films. With the increase in the film thickness, the peak intensity of the Tb2 (Fe, Co)17 phase decreased and that of the Fe-Co phase increased. Magnetization studies showed the presence of strong in-plane anisotropy for all the films. In addition to this, the presence of the out-of-plane component of magnetization was also observed for the films grown with higher thicknesses. This anisotropic behavior was also validated through magnetic microscopy studies carried out along the in-plane and out-of-plane directions employing magneto-optic Kerr microscopy and magnetic force microscopy, respectively. The decrease in magnetostriction was explained on the basis of dual phase formation and complex interplay between the in-plane and out-of-plane magnetic anisotropies present in the film.

  11. Transmission electron microscopy of undermined passive films on stainless steel

    SciTech Connect

    Isaacs, H.S.; Zhu, Y.; Sabatini, R.L.; Ryan, M.P.

    1999-06-01

    A study has been made of the passive film remaining over pits on stainless steel using a high resolution transmission electron microscope. Type 305 stainless steel was passivated in a borate buffer solution and pitted in ferric chloride. Passive films formed at 0.2 V relative to a saturated calomel electrode were found to be amorphous. Films formed at higher potentials showed only broad diffraction rings. The passive film was found to cover a remnant lacy structure formed over pits passivated at 0.8 V. The metallic strands of the lace were roughly hemitubular in shape with the curved surface facing the center of the pit.

  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. Correlation of live-cell imaging with volume scanning electron microscopy.

    PubMed

    Lucas, Miriam S; Günthert, Maja; Bittermann, Anne Greet; de Marco, Alex; Wepf, Roger

    2017-01-01

    Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. 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

  15. 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.

  16. Benzofurazan Sulfides for Thiol Imaging and Quantification in Live Cells through Fluorescence Microscopy

    PubMed Central

    Li, Yinghong; Yang, Yang; Guan, Xiangming

    2012-01-01

    Thiol groups play a significant role in various cellular functions. Cellular thiol concentrations can be affected by various physiological or pathological factors. A fluorescence imaging agent that can effectively and specifically image thiols in live cells through fluorescence microscopy is desirable for live cell thiol monitoring. Benzofurazan sulfides 1a–e were synthesized and found to be thiol specific fluorogenic agents except 1d. They are not fluorescent but form strong fluorescent thiol adducts after reacting with thiols through a sulfide-thiol exchange reaction. On the other hand, they exhibit no reaction with other biologically relevant nucleophilic functional groups such as -NH2, -OH, or -COOH revealing the specificity for the detection of thiols. Sulfide 1a was selected to confirm its ability to image cellular thiols through fluorescence microscopy. The compound was demonstrated to effectively image and quantify thiol changes in live cells through fluorescence microscopy using 430 nm and 520 nm as the excitation and emission wavelengths respectively. The quantification results of total thiol in live cells obtained from fluorescence microscopy were validated by an HPLC/UV total thiol assay method. The reagents and method will be of a great value to thiol redox-related research. PMID:22794193

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

    PubMed

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

    2013-06-07

    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.

  18. Correlation of histology and linear and nonlinear microscopy of the living human cornea.

    PubMed

    Masters, Barry R

    2009-03-01

    The morphology and the function of cellular and non-cellular structures in the living human cornea can be determined with modern correlative linear and nonlinear optical microscopic techniques and histology. Correlative microscopy is based on the use of different optical techniques to study the same specimen, ideally at the same location within the specimen, in order to increase the functional and/or morphological understanding of the specimen. A case study to assess the effect of overnight lid-closure on in vivo human corneal morphology is presented to illustrate correlative linear microscopy and optical low-coherence reflectometry. Nonlinear multiphoton excitation microscopy provides functional information on cellular metabolism based on the intrinsic fluorescence from the reduced pyridine nucleotides and the oxidized flavoproteins. Second-harmonic generation microscopy, a scattering process that does not deposit net energy into the tissue, provides structural information on corneal collagen organization. Molecular third-harmonic generation microscopy generates a signal in all materials and it an emerging technique. Coherent anti-Stokes Raman scattering microscopy provides chemical imaging for biology and medicine. The comparison and limitations of these microscopic modalities, linear and nonlinear microscopy applied to the cornea, and a review of some key findings is analyzed. A correlative integration and correlation of linear and nonlinear microscopies to study corneal function and structure is proposed to validate the clinical interpretation of microscopic images of the cornea.

  19. Latex film morphology and electrical potential pattern dependence on serum components: a scanning probe microscopy study.

    PubMed

    Keslarek, Amauri José; Costa, Carlos Alberto Rodrigues; Galembeck, Fernando

    2002-11-01

    Dry films formed by surfactant-stabilized, peroxide-initiated styrene-butyl acrylate latex were examined by atomic and electric force microscopy (AFM and EFM). The effects of latex serum components on the films were observed by subjecting the latex to extensive dialysis prior to film formation, and comparing the results to as-prepared latex. The films formed with the dialyzed latex are smoother (as evidenced by roughness and fractal dimension measurements) than the films from the as-prepared latex, but they display large electric force gradients between neighboring domains. The films made with the as-prepared latex have the highest electric uniformity, with a maximum potential variation lower than 80 mV, while this reaches 200 mV in the dialyzed latex film.

  20. 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.

  1. Characterization of MSB Synapses in Dissociated Hippocampal Culture with Simultaneous Pre- and Postsynaptic Live Microscopy

    PubMed Central

    Reilly, James E.; Hanson, Hugo H.; Fernández-Monreal, Mónica; 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. PMID:22028887

  2. 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.

  3. 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.

  4. 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.

  5. Molecular positional order in langmuir-blodgett films by atomic force microscopy.

    PubMed

    Bourdieu, L; Ronsin, O; Chatenay, D

    1993-02-05

    Langmuir-Blodgett films of barium arachidate have been studied on both macroscopic and microscopic scales by atomic force microscopy. As prepared, the films exhibit a disordered hexagonal structure; molecularly resolved images in direct space establish a connection between the extent of the positional order and the presence of defects such as dislocations. Upon heating, the films reorganize into a more condensed state with a centered rectangular crystallographic arrangement; in this new state the films exhibit long-range positional order and unusual structural features, such as a height modulation of the arachidic acid molecules.

  6. Molecular Positional Order in Langmuir-Blodgett Films by Atomic Force Microscopy

    NASA Astrophysics Data System (ADS)

    Bourdieu, L.; Ronsin, O.; Chatenay, D.

    1993-02-01

    Langmuir-Blodgett films of barium arachidate have been studied on both macroscopic and microscopic scales by atomic force microscopy. As prepared, the films exhibit a disordered hexagonal structure; molecularly resolved images in direct space establish a connection between the extent of the positional order and the presence of defects such as dislocations. Upon heating, the films reorganize into a more condensed state with a centered rectangular crystallographic arrangement; in this new state the films exhibit long-range positional order and unusual structural features, such as a height modulation of the arachidic acid molecules.

  7. 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.

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed

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

    2011-12-01

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

  10. 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.

  11. 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

  12. 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.

  13. Microscopy of thin polymer blend films of polystyrene and poly-n-butyl-methacrylate

    NASA Astrophysics Data System (ADS)

    Schmitt, T.; Guttmann, P.; Schmidt, O.; Müller-Buschbaum, P.; Stamm, M.; Schönhense, G.; Schmahl, G.

    2000-05-01

    The structure of thin polymer blend films of polystyrene (PS) and poly-n-butyl-methacrylate (PnBMA) was examined with Transmission X-ray Microscopy (TXM), Scanning Force Microscopy (SFM), X-Ray Photoemission Electron Microscopy (X-PEEM) and Optical Microscopy (OM). Thin films were prepared by spin casting of a toluene solution of the polymer mixture onto silicon wafers retaining the native oxide. Depending on blend composition and annealing conditions smooth films with and without holes or films with well pronounced surface features (ribbons or islands) were produced. By TXM measurements a high lateral resolution study of the as cast and the annealed polymer blend samples was performed. The contrast in TXM is due to different absorption of x-radiation of the used polymers and due to variation in thickness. With X-PEEM the lateral distribution of the two polymers near the surface was mapped by employing the characteristic Near Edge X-ray Absorption Fine Structure (NEXAFS) spectra of the polymers. The TXM technique is a microscopic method integrating over the total film thickness, whereas the X-PEEM technique is a highly surface sensitive method. TXM and X-PEEM are therefore complementary methods which provide important information on the structure of thin polymer blend films additional to the standard techniques SFM and OM.

  14. 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…

  15. 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…

  16. 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.

  17. 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.

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

    PubMed

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

    2016-06-15

    Mammalian oocytes contain lipid droplets that are a store of fatty acids, whose metabolism plays a substantial role in pre-implantation development. Fluorescent staining has previously been used to image lipid droplets in mammalian oocytes and embryos, but this method is not quantitative and often incompatible with live cell imaging and subsequent development. Here we have applied chemically specific, label-free coherent anti-Stokes Raman scattering (CARS) microscopy to mouse oocytes and pre-implantation embryos. We show that CARS imaging can quantify the size, number and spatial distribution of lipid droplets in living mouse oocytes and embryos up to the blastocyst stage. Notably, it can be used in a way that does not compromise oocyte maturation or embryo development. We have also correlated CARS with two-photon fluorescence microscopy simultaneously acquired using fluorescent lipid probes on fixed samples, and found only a partial degree of correlation, depending on the lipid probe, clearly exemplifying the limitation of lipid labelling. In addition, we show that differences in the chemical composition of lipid droplets in living oocytes matured in media supplemented with different saturated and unsaturated fatty acids can be detected using CARS hyperspectral imaging. These results demonstrate that CARS microscopy provides a novel non-invasive method of quantifying lipid content, type and spatial distribution with sub-micron resolution in living mammalian oocytes and embryos. © 2016. Published by The Company of Biologists Ltd.

  19. 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

  20. 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

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

    PubMed

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

    2016-09-13

    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/cm(2)) live-cell SR imaging at ∼60-nm resolution at subsecond acquisition times for tens of time points over broad field of view.

  2. 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.

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

    PubMed Central

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

    2013-01-01

    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. PMID:23798771

  4. 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.

  5. 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.

  6. 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.

  7. Localizing Proteins in Fixed Giardia lamblia and Live Cultured Mammalian Cells by Confocal Fluorescence Microscopy.

    PubMed

    Nyindodo-Ogari, Lilian; Schwartzbach, Steven D; Skalli, Omar; Estraño, Carlos E

    2016-01-01

    Confocal fluorescence microscopy and electron microscopy (EM) are complementary methods for studying the intracellular localization of proteins. Confocal fluorescence microscopy provides a rapid and technically simple method to identify the organelle in which a protein localizes but only EM can identify the suborganellular compartment in which that protein is present. Confocal fluorescence microscopy, however, can provide information not obtainable by EM but required to understand the dynamics and interactions of specific proteins. In addition, confocal fluorescence microscopy of cells transfected with a construct encoding a protein of interest fused to a fluorescent protein tag allows live cell studies of the subcellular localization of that protein and the monitoring in real time of its trafficking. Immunostaining methods for confocal fluorescence microscopy are also faster and less involved than those for EM allowing rapid optimization of the antibody dilution needed and a determination of whether protein antigenicity is maintained under fixation conditions used for EM immunogold labeling. This chapter details a method to determine by confocal fluorescence microscopy the intracellular localization of a protein by transfecting the organism of interest, in this case Giardia lamblia, with the cDNA encoding the protein of interest and then processing these organisms for double label immunofluorescence staining after chemical fixation. Also presented is a method to identify the organelle targeting information in the presequence of a precursor protein, in this case the presequence of the precursor to the Euglena light harvesting chlorophyll a/b binding protein of photosystem II precursor (pLHCPII), using live cell imaging of mammalian COS7 cells transiently transfected with a plasmid encoding a pLHCPII presequence fluorescent protein fusion and stained with organelle-specific fluorescent dyes.

  8. 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.

  9. 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.

  10. 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.

  11. Finite-size effects and analytical modeling of electrostatic force microscopy applied to dielectric films.

    PubMed

    Gomila, G; Gramse, G; Fumagalli, L

    2014-06-27

    A numerical analysis of the polarization force between a sharp conducting probe and a dielectric film of finite lateral dimensions on a metallic substrate is presented with the double objective of (i) determining the conditions under which the film can be approximated by a laterally infinite film and (ii) proposing an analytical model valid in this limit. We show that, for a given dielectric film, the critical diameter above which the film can be modeled as laterally infinite depends not only on the probe geometry, as expected, but mainly on the film thickness. In particular, for films with intermediate to large thicknesses (>100 nm), the critical diameter is nearly independent from the probe geometry and essentially depends on the film thickness and dielectric constant following a relatively simple phenomenological expression. For films that can be considered as laterally infinite, we propose a generalized analytical model valid in the thin-ultrathin limit (<20-50 nm) that reproduces the numerical calculations and the experimental data. Present results provide a general framework under which accurate quantification of electrostatic force microscopy measurements on dielectric films on metallic substrates can be achieved.

  12. 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.

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. Magnetic properties of thin MnGe films investigated by magnetic force microscopy

    NASA Astrophysics Data System (ADS)

    Castrucci, P.; Pinto, N.; Morresi, L.; Gunnella, R.; Murri, R.; Scarselli, M.; de Crescenzi, M.

    2004-05-01

    Magnetic force microscopy has been used to study MnxGe1-x/Ge(100) films grown at 433K as a function of Mn concentration. Imaging has been performed at room temperature and in zero magnetic field. Magnetic domains with a magnetization direction normal to the surface have been detected for x=0.027 and 0.044. No clearcut magnetic effect has been observed for x=0.053. Scanning tunneling microscopy has been used to investigate their morphology.

  18. Friction force microscopy study of annealed diamond-like carbon film

    SciTech Connect

    Choi, Won Seok; Joung, Yeun-Ho; Heo, Jinhee; Hong, Byungyou

    2012-10-15

    In this paper we introduce mechanical and structural characteristics of diamond-like carbon (DLC) films which were prepared on silicon substrates by radio frequency (RF) plasma enhanced chemical vapor deposition (PECVD) method using methane (CH{sub 4}) and hydrogen (H{sub 2}) gas. The films were annealed at various temperatures ranging from 300 to 900 °C in steps of 200 °C using rapid thermal processor (RTP) in nitrogen ambient. Tribological properties of the DLC films were investigated by atomic force microscopy (AFM) in friction force microscopy (FFM) mode. The structural properties of the films were obtained by high resolution transmission electron microscopy (TEM) and X-ray photoelectron spectroscopy (XPS). The wettability of the films was obtained using contact angle measurement. XPS analysis showed that the sp{sup 3} content is decreased from 75.2% to 24.1% while the sp{sup 2} content is increased from 24.8% to 75.9% when the temperature is changed from 300 to 900 °C. The contact angles of DLC films were higher than 70°. The FFM measurement results show that the highest friction coefficient value was achieved at 900 °C annealing temperature.

  19. 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.

  20. Polarized Fluorescence Microscopy to Study Cytoskeleton Assembly and Organization in Live Cells.

    PubMed

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

    2015-06-01

    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 (http://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. Copyright © 2015 John Wiley & Sons, Inc.

  1. 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

  2. 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.

  3. 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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Crystallographic mapping of ferroelectric thin films using piezoresponse force microscopy and electron backscatter diffraction

    NASA Astrophysics Data System (ADS)

    Lowe, M.; Hegarty, T.; Mingard, K.; Li, J.; Cain, M.

    2008-08-01

    Ferroelectric lead zirconate titanate (PZT) thin films have been analysed using electron backscatter diffraction (EBSD). Grain orientation mapping has been demonstrated, showing that features smaller than 100 nm may be successfully indexed. In conjunction with piezoresponse force microscopy (PFM), which was used to map and quantify the piezoelectric response from the same region of the films with a resolution of 10 nm, an analysis of the effects of grain orientation on the measured response at the nanoscale was possible. The microtexture of the film showed the presence of both mono- and multi-domains within grains exhibiting sizes of hundreds of nanometres.

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. High-speed atomic force microscopy imaging of live mammalian cells

    PubMed Central

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

    2017-01-01

    Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons. PMID:28900590

  17. Investigation of nucleation and growth processes of diamond films by atomic force microscopy

    NASA Technical Reports Server (NTRS)

    George, M. A.; Burger, A.; Collins, W. E.; Davidson, J. L.; Barnes, A. V.; Tolk, N. H.

    1994-01-01

    The nucleation and growth of plasma-enhanced chemical-vapor deposited polycrystalline diamond films were studied using atomic force microscopy (AFM). AFM images were obtained for (1) nucleated diamond films produced from depositions that were terminated during the initial stages of growth, (2) the silicon substrate-diamond film interface side of diamond films (1-4 micrometers thick) removed from the original surface of the substrate, and (3) the cross-sectional fracture surface of the film, including the Si/diamond interface. Pronounced tip effects were observed for early-stage diamond nucleation attributed to tip convolution in the AFM images. AFM images of the film's cross section and interface, however, were not highly affected by tip convolution, and the images indicate that the surface of the silicon substrate is initially covered by a small grained polycrystalline-like film and the formation of this precursor film is followed by nucleation of the diamond film on top of this layer. X-ray photoelectron spectroscopy spectra indicate that some silicon carbide is present in the precursor layer.

  18. Investigation of nucleation and growth processes of diamond films by atomic force microscopy

    NASA Technical Reports Server (NTRS)

    George, M. A.; Burger, A.; Collins, Warren E.; Hu, Z.

    1995-01-01

    The nucleation and growth of plasma enhanced chemical vapor deposited (PECVD) polycrystalline diamond films were studied using atomic force microscopy (AFM). AFM images were obtained for: (1) nucleated diamond films produced from depositions that were terminated during the initial stages of growth, (2) the silicon substrate-diamond film interface side of diamond films (1-4 micrometers thick) removed from the original surface of the substrate, and (3) cross-sectional fracture surface of the film, including the Si/diamond interface. Pronounced tip effects were observed for early-stage diamond nucleation attributed to tip convolution in the AFM images. AFM images of the films cross-section and interface however were not affected by tip convolution, and the images indicate that the surface of the silicon substrate is initially covered by small grained polycrystalline-like film and the formation of this precursor film is followed by nucleation of the diamond film on top of this layer. X-ray photoelectron spectroscoy (XPS) spectra indicates that some silicon carbide is present in the precursor layer.

  19. Live Cell Refractometry Using Hilbert Phase Microscopy and Confocal Reflectance Microscopy†

    PubMed Central

    Lue, Niyom; Choi, Wonshik; Popescu, Gabriel; Yaqoob, Zahid; Badizadegan, Kamran; Dasari, Ramachandra R.; Feld, Michael S.

    2010-01-01

    Quantitative chemical analysis has served as a useful tool for understanding cellular metabolisms in biology. Among many physical properties used in chemical analysis, refractive index in particular has provided molecular concentration that is an important indicator for biological activities. In this report, we present a method of extracting full-field refractive index maps of live cells in their native states. We first record full-field optical thickness maps of living cells by Hilbert phase microscopy and then acquire physical thickness maps of the same cells using a custom-built confocal reflectance microscope. Full-field and axially averaged refractive index maps are acquired from the ratio of optical thickness to physical thickness. The accuracy of the axially averaged index measurement is 0.002. This approach can provide novel biological assays of label-free living cells in situ. PMID:19803506

  20. 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

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Yield strength of glued Langmuir-Blodgett films determined by friction force microscopy.

    PubMed

    Wagner, Kyle C; Wang, Yao; Regen, Steven L; Vezenov, Dmitri V

    2013-09-07

    We used friction force microscopy measurements to determine the yield strength of several structurally similar Langmuir-Blodgett (LB) bilayer films deposited on a hydrophobic substrate. Film failure was initiated by increasing the load applied by the probe of the atomic force microscope in the course of continuous scanning at nominally the same location on the sample. This film failure was readily detected in friction versus load curves, as well as by imaging of trenches created due to removal of the film. The depths of the trenches formed in the course of these yield strength experiments were consistent with complete removal of these bilayer films, as evidenced by comparisons to film thicknesses measured by ellipsometry. The structure of the LB bilayer was modified by replacing a tetra-chain amphiphile bearing four quaternary ammonium groups with a polymeric surfactant resulting in little change in the yield strength. On the other hand, the addition of a polyanionic gluing layer at the central interface of the bilayers almost doubled the yield strength of the films. To uncover any possible structural effects created by changes in the terminal functionality, the hydrocarbon top layer of the bilayer was replaced with a perfluorinated capping layer. In spite of the changes in frictional properties, the yield strength of this film also appeared to be dominated by the presence of the glued interface.

  6. Quantitative phase microscopy: a new tool for investigating the structure and function of unstained live cells.

    PubMed

    Curl, Claire L; Bellair, Catherine J; Harris, Peter J; Allman, Brendan E; Roberts, Ann; Nugent, Keith A; Delbridge, Lea M D

    2004-12-01

    1. The optical transparency of unstained live cell specimens limits the extent to which information can be recovered from bright-field microscopic images because these specimens generally lack visible amplitude-modulating components. However, visualization of the phase modulation that occurs when light traverses these specimens can provide additional information. 2. Optical phase microscopy and derivatives of this technique, such as differential interference contrast (DIC) and Hoffman modulation contrast (HMC), have been used widely in the study of cellular materials. With these techniques, enhanced contrast is achieved, which is useful in viewing specimens, but does not allow quantitative information to be extracted from the phase content available in the images. 3. An innovative computational approach to phase microscopy, which provides mathematically derived information about specimen phase-modulating characteristics, has been described recently. Known as quantitative phase microscopy (QPM), this method derives quantitative phase measurements from images captured using a bright-field microscope without phase- or interference-contrast optics. 4. The phase map generated from the bright-field images by the QPM method can be used to emulate other contrast image modes (including DIC and HMC) for qualitative viewing. Quantitative phase microscopy achieves improved discrimination of cellular detail, which permits more rigorous image analysis procedures to be undertaken compared with conventional optical methods. 5. The phase map contains information about cell thickness and refractive index and can allow quantification of cellular morphology under experimental conditions. As an example, the proliferative properties of smooth muscle cells have been evaluated using QPM to track growth and confluency of cell cultures. Quantitative phase microscopy has also been used to investigate erythrocyte cell volume and morphology in different osmotic environments. 6. Quantitative

  7. 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.

  8. 3D live fluorescence imaging of cellular dynamics using Bessel beam plane illumination microscopy.

    PubMed

    Gao, Liang; Shao, Lin; Chen, Bi-Chang; Betzig, Eric

    2014-05-01

    3D live imaging is important for a better understanding of biological processes, but it is challenging with current techniques such as spinning-disk confocal microscopy. Bessel beam plane illumination microscopy allows high-speed 3D live fluorescence imaging of living cellular and multicellular specimens with nearly isotropic spatial resolution, low photobleaching and low photodamage. Unlike conventional fluorescence imaging techniques that usually have a unique operation mode, Bessel plane illumination has several modes that offer different performance with different imaging metrics. To achieve optimal results from this technique, the appropriate operation mode needs to be selected and the experimental setting must be optimized for the specific application and associated sample properties. Here we explain the fundamental working principles of this technique, discuss the pros and cons of each operational mode and show through examples how to optimize experimental parameters. We also describe the procedures needed to construct, align and operate a Bessel beam plane illumination microscope by using our previously reported system as an example, and we list the necessary equipment to build such a microscope. Assuming all components are readily available, it would take a person skilled in optical instrumentation ∼1 month to assemble and operate a microscope according to this protocol.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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.

  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. Enhancement of local piezoelectric properties of a perforated ferroelectric thin film visualized via piezoresponse force microscopy

    NASA Astrophysics Data System (ADS)

    Ivanov, M. S.; Sherstyuk, N. E.; Mishina, E. D.; Khomchenko, V. A.; Tselev, A.; Mukhortov, V. M.; Paixão, J. A.; Kholkin, A. L.

    2017-10-01

    The local piezoresponse in a Ba0.8Sr0.2TiO3 epitaxial ferroelectric film perforated by cylindrical channels has been investigated experimentally by means of piezoresponse force microscopy (PFM). A large enhancement of the effective values for both lateral and vertical components of piezoelectric tensor was experimentally detected in the perforated film as compared to non-perforated structure—by a factor of 8 for the lateral and by a factor 2 for the vertical piezoresponse. This result is consistent with the previously reported enhancement of the optical second harmonic generation over perforated films observed in macroscopic experiments. We assume that a possible mechanism for the increased PFM response is due to reduction of stress and clamping in the film imposed by the substrate. The obtained insight is critical for understanding nanoscale piezo- and ferroelectric responses in photonic crystals fabricated by focused ion beam milling.

  20. 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.

  1. 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.

  2. Label-free detection of anticancer drug paclitaxel in living cells by confocal Raman microscopy

    NASA Astrophysics Data System (ADS)

    Salehi, H.; Derely, L.; Vegh, A.-G.; Durand, J.-C.; Gergely, C.; Larroque, C.; Fauroux, M.-A.; Cuisinier, F. J. G.

    2013-03-01

    Confocal Raman microscopy, a non-invasive, label-free, and high spatial resolution imaging technique is employed to trace the anticancer drug paclitaxel in living Michigan Cancer Foundation-7 (MCF-7) cells. The Raman images were treated by K-mean cluster analysis to detect the drug in cells. Distribution of paclitaxel in cells is verified by calculating the correlation coefficient between the reference spectrum of the drug and the whole Raman image spectra. A time dependent gradual diffusion of paclitaxel all over the cell is observed suggesting a complementary picture of the pharmaceutical action of this drug based on rapid binding of free tubulin to crystallized paclitaxel.

  3. 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.

  4. 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.

  5. In-Situ Imaging of Langmuir Films using Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Miller, Aline

    2002-03-01

    Environmental scanning electron microscopy (ESEM) has been applied to image the organization of spread films at the air-water interface directly. This recent extension of conventional scanning electron microscopy permits the resolution of electron microscopy to be applied to insulated and hydrated samples maintained in their natural state. We demonstrate the great potential of this technique by in-situ imaging of both synthetic and natural polymer spread films at the air-water interface. A brief synopsis of the background to ESEM will be given followed by a discussion of the pertinent experimental conditions for the study of Langmuir layers. Experimental results for two systems will be presented. Firstly results for a nylon 6 6 polymer film will be given and discussed in relation to external reflection FTIR studies and those using the more conventional analytical techniques of surface pressure isotherms and optical microscopy. Secondly the kinetics of the interaction of a cationic intercalator amphiphile with double stranded DNA at the air-water interface will be presented and the structure of the densely packed two-dimensional arrays formed will be discussed.

  6. 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.

  7. 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

  8. Motion analysis of live objects by super-resolution fluorescence microscopy.

    PubMed

    Yao, Chunyan; Zhang, Jianwei; Wu, Guang; Zhang, Houxiang

    2012-01-01

    Motion analysis plays an important role in studing activities or behaviors of live objects in medicine, biotechnology, chemistry, physics, spectroscopy, nanotechnology, enzymology, and biological engineering. This paper briefly reviews the developments in this area mostly in the recent three years, especially for cellular analysis in fluorescence microscopy. The topic has received much attention with the increasing demands in biomedical applications. The tasks of motion analysis include detection and tracking of objects, as well as analysis of motion behavior, living activity, events, motion statistics, and so forth. In the last decades, hundreds of papers have been published in this research topic. They cover a wide area, such as investigation of cell, cancer, virus, sperm, microbe, karyogram, and so forth. These contributions are summarized in this review. Developed methods and practical examples are also introduced. The review is useful to people in the related field for easy referral of the state of the art.

  9. Modeling of the Deformation of Living Cells Induced by Atomic Force Microscopy

    SciTech Connect

    Rudd, R E; McElfresh, M; Baesu, E; Balhorn, R; Allen, M J; Belak, J

    2001-12-21

    We describe finite element modeling of the deformation of living cells by atomic force microscopy (AFM). Cells are soft systems, susceptible to large deformations in the course of an AFM measurement. Often the local properties, the subject of the measurement, are obscured by the response of the cell as a whole. The Lagrangian finite deformation model we have developed and implemented in finite elements analysis offers a solution to this problem. The effect of the gross deformation of the cell can be subtracted from the experimentally measured data in order to give a reproducible value for local properties. This facilitates concurrent experimental efforts to measure the mechanical properties at specific receptor sites on the membrane of a living cell.

  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. 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.

  12. 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.

  13. 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.

  14. 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

  15. Imaging complex protein metabolism in live organisms by stimulated Raman scattering microscopy with isotope labeling.

    PubMed

    Wei, Lu; Shen, Yihui; Xu, Fang; Hu, Fanghao; Harrington, Jamie K; Targoff, Kimara L; Min, Wei

    2015-03-20

    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.

  16. Diabetes increases stiffness of live cardiomyocytes measured by atomic force microscopy nanoindentation.

    PubMed

    Benech, Juan C; Benech, Nicolás; Zambrana, Ana I; Rauschert, Inés; Bervejillo, Verónica; Oddone, Natalia; Damián, Juan P

    2014-11-15

    Stiffness of live cardiomyocytes isolated from control and diabetic mice was measured using the atomic force microscopy nanoindentation method. Type 1 diabetes was induced in mice by streptozotocin administration. Histological images of myocardium from mice that were diabetic for 3 mo showed disorderly lineup of myocardial cells, irregularly sized cell nuclei, and fragmented and disordered myocardial fibers with interstitial collagen accumulation. Phalloidin-stained cardiomyocytes isolated from diabetic mice showed altered (i.e., more irregular and diffuse) actin filament organization compared with cardiomyocytes from control mice. Sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA2a) pump expression was reduced in homogenates obtained from the left ventricle of diabetic animals compared with age-matched controls. The apparent elastic modulus (AEM) for live control or diabetic isolated cardiomyocytes was measured using the atomic force microscopy nanoindentation method in Tyrode buffer solution containing 1.8 mM Ca(2+) and 5.4 mM KCl (physiological condition), 100 nM Ca(2+) and 5.4 mM KCl (low extracellular Ca(2+) condition), or 1.8 mM Ca(2+) and 140 mM KCl (contraction condition). In the physiological condition, the mean AEM was 112% higher for live diabetic than control isolated cardiomyocytes (91 ± 14 vs. 43 ± 7 kPa). The AEM was also significantly higher in diabetic than control cardiomyocytes in the low extracellular Ca(2+) and contraction conditions. These findings suggest that the material properties of live cardiomyocytes were affected by diabetes, resulting in stiffer cells, which very likely contribute to high diastolic LV stiffness, which has been observed in vivo in some diabetes mellitus patients. Copyright © 2014 the American Physiological Society.

  17. Chiral Nematic Structure of Cellulose Nanocrystal Suspensions and Films; Polarized Light and Atomic Force Microscopy

    PubMed Central

    Gray, Derek G.; Mu, Xiaoyue

    2015-01-01

    Cellulosic liquid crystalline solutions and suspensions form chiral nematic phases that show a rich variety of optical textures in the liquid crystalline state. These ordered structures may be preserved in solid films prepared by evaporation of solvent or suspending medium. Film formation from aqueous suspensions of cellulose nanocrystals (CNC) was investigated by polarized light microscopy, optical profilometry and atomic force microscopy (AFM). An attempt is made to interpret qualitatively the observed textures in terms of the orientation of the cellulose nanocrystals in the suspensions and films, and the changes in orientation caused by the evaporative process. Mass transfer within the evaporating droplet resulted in the formation of raised rings whose magnitude depended on the degree of pinning of the receding contact line. AFM of dry films at short length scales showed a radial orientation of the CNC at the free surface of the film, along with a radial height variation with a period of approximately P/2, ascribed to the anisotropic shrinkage of the chiral nematic structure. PMID:28793684

  18. 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.

  19. 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.

  20. 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.

  1. Nanosecond switching in GeSe phase change memory films by atomic force microscopy

    SciTech Connect

    Bosse, James L.; Huey, Bryan D.; Grishin, Ilya; Kolosov, Oleg V.; Gyu Choi, Yong; Cheong, Byung-ki; Lee, Suyoun

    2014-02-03

    Nanosecond scale threshold switching is investigated with conducting atomic force microscopy (AFM) for an amorphous GeSe film. Switched bits exhibit 2–3 orders of magnitude variations in conductivity, as demonstrated in phase change based memory devices. Through the nm-scale AFM probe, this crystallization was achieved with pulse durations of as low as 15 ns, the fastest reported with scanning probe based methods. Conductance AFM imaging of the switched bits further reveals correlations between the switched volume, pulse amplitude, and pulse duration. The influence of film heterogeneities on switching is also directly detected, which is of tremendous importance for optimal device performance.

  2. Thin films of metal oxides on metal single crystals: Structure and growth by scanning tunneling microscopy

    SciTech Connect

    Galloway, Heather Claire

    1995-12-01

    Detailed studies of the growth and structure of thin films of metal oxides grown on metal single crystal surfaces using Scanning Tunneling Microscopy (STM) are presented. The oxide overlayer systems studied are iron oxide and titanium oxide on the Pt(III) surface. The complexity of the metal oxides and large lattice mismatches often lead to surface structures with large unit cells. These are particularly suited to a local real space technique such as scanning tunneling microscopy. In particular, the symmetry that is directly observed with the STM elucidates the relationship of the oxide overlayers to the substrate as well as distinguishing, the structures of different oxides.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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

  8. Non-invasive monitoring for living cell culture with lensless Fourier transform digital holography microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Yunxin; Wang, Dayong; Zhao, Jie; Li, Yan; Meng, Puhui; Wan, Yuhong; Jiang, Zhuqing

    2010-08-01

    The number of cells is commonly employed to describe the cell viability and the status of cell culture in a certain extent. An automatic and non-invasive detecting method for the status analysis of cell culture is developed based on digital holography microscopy (DHM) technology. Digital holographic imaging can retrieve quantitative information of object wavefront by the numerical reconstruction from a single digital hologram recorded by a detector such as CCD or CMOS camera, which is especially suitable for the morphology detection of the transparent or semi-transparent cells. In this contribution, the lensless Fourier transform (LFT) based holography configuration is designed for cell imaging without prestaining, and the amplitude and phase of living cells can be reconstructed by digital reconstruction and phase unwrapped algorithms. Then the image filtering and segmentation are combined for the automatic evaluation of the level of confluency. In imaging experiments, the culture status of the cervical cancer cell TZMbl is detected, and the results demonstrate that digital holography microscopy provides a feasible non-invasive method for monitoring the living cell culture.

  9. Coherent Raman Imaging of Live Muscle Sarcomeres Assisted by SFG Microscopy.

    PubMed

    Kim, Hyunmin; Kim, Do-Young; Joo, Kyung-Il; Kim, Jung-Hye; Jeong, Soon Moon; Lee, Eun Seong; Hahm, Jeong-Hoon; Kim, Kyuhyung; Moon, Dae Woon

    2017-08-23

    In this study, we used spectrally focused coherent anti-Stokes Raman scattering (spCARS) microscopy assisted by sum-frequency generation (SFG) to monitor the variations in the structural morphology and molecular vibrations of a live muscle of Caenorhabditis elegans. The subunits of the muscle sarcomeres, such as the M-line, myosin, dense body, and α-actinin, were alternatively observed using spCARS microscopy for different sample orientations, with the guidance of a myosin positional marker captured by SFG microscopy. Interestingly enough, the beam polarization dependence of the spCARS contrasts for two parallel subunits (dense body and myosin) showed a ~90° phase difference. The chemically sensitive spCARS spectra induced by the time-varying overlap of two pulses allowed (after a robust subtraction of the non-resonant background using a modified Kramers-Krönig transformation method) high-fidelity detection of various genetically modified muscle sarcomeres tuned to the C-H vibration (2800-3100 cm(-1)). Conversely, SFG image mapping assisted by phase-retrieved spCARS spectra also facilitated label-free monitoring of the changes in the muscle content of C. elegans that are associated with aging, based on the hypothesis that the C-H vibrational modes could serve as qualitative chemical markers sensitive to the amount and/or structural modulation of the muscle.

  10. 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.

  11. Kelvin probe force microscopy for conducting nanobits of NiO thin films

    NASA Astrophysics Data System (ADS)

    Son, J. Y.; Shin, Y.-H.; Kim, H.; Cho, J. H.; Jang, H.

    2010-05-01

    We demonstrated the writing and reading of conducting nanobits on a NiO thin film deposited on Pt/TiO2/SiO2/Si substrates for a resistive random access memory (RRAM) application using conducting atomic force microscopy (CAFM) and Kelvin probe force microscopy (KFM). A Pt/NiO/Pt RRAM capacitor showed a typical unipolar switching behavior with bistable resistances. Conducting nanobits with diameters of 22 nm written with a bias of 3.0 V on the NiO thin film were observed with CAFM. The conducting nanobits observed by KFM exhibited negative potentials relative to the insulating regions when there was no bias at the Pt bottom electrode because image charges were induced by charges formed at the end of the KFM tip by the reference AC bias. Enhancement of the KFM signals for conducting nanobits was achieved using specific biases at the Pt bottom electrode, which provided clear KFM images for conducting nanobits.

  12. Kelvin probe force microscopy for conducting nanobits of NiO thin films.

    PubMed

    Son, J Y; Shin, Y-H; Kim, H; Cho, J H; Jang, H

    2010-05-28

    We demonstrated the writing and reading of conducting nanobits on a NiO thin film deposited on Pt/TiO(2)/SiO(2)/Si substrates for a resistive random access memory (RRAM) application using conducting atomic force microscopy (CAFM) and Kelvin probe force microscopy (KFM). A Pt/NiO/Pt RRAM capacitor showed a typical unipolar switching behavior with bistable resistances. Conducting nanobits with diameters of 22 nm written with a bias of 3.0 V on the NiO thin film were observed with CAFM. The conducting nanobits observed by KFM exhibited negative potentials relative to the insulating regions when there was no bias at the Pt bottom electrode because image charges were induced by charges formed at the end of the KFM tip by the reference AC bias. Enhancement of the KFM signals for conducting nanobits was achieved using specific biases at the Pt bottom electrode, which provided clear KFM images for conducting nanobits.

  13. 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.

  14. Investigation into the correlation factor of substrate and multilayer film surfaces by atomic force microscopy

    SciTech Connect

    Grishchenko, J. V. Zanaveskin, M. L.

    2013-05-15

    A method for studying the correlations between substrate and film-coating profiles by atomic force microscopy, which makes it possible to calculate the correlation factor (a function of spatial frequency), has been developed. The spatial-frequency range in which the correlation factor can be reliably calculated is established. The method proposed is used to calculate the dependence of the correlation factor on spatial frequency for multilayer interference mirror elements.

  15. 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.}

  16. 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.

  17. Quantitative measurement of hydrophilicity/hydrophobicity of the plasma-polymerized naphthalene film (Super Support Film) and other support films and grids in electron microscopy.

    PubMed

    Yamaguchi, Masashi; Aoyama, Toshihiro; Yamada, Norihiro; Chibana, Hiroji

    2016-10-01

    Hydrophilicity/hydrophobicity of the surfaces of plasma-polymerized naphthalene film (Super Support Film, Nisshin EM Co. Ltd., Tokyo), carbon and formvar support films, and copper and nickel grids were quantitatively estimated by contact angles measured from diameters and heights of water droplets placed on various specimens. With treatment of glow discharge, the surfaces of plasma-polymerized naphthalene and carbon support films became fully hydrophilic in 20 s. They remained hydrophilic for 6 h. The surfaces of copper and nickel grids became fully hydrophilic with 60 s of glow discharge treatment. They remained hydrophilic for only 1 h. This information is useful for negative staining, ultrathin sectioning and rapid freezing of biological specimens using the sandwich method. © The Author 2016. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved.For permissions, please e-mail: journals.permissions@oup.com.

  18. 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.

  19. 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.

  20. 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.

  1. Intracellular imaging of docosanol in living cells by coherent anti-Stokes Raman scattering microscopy

    NASA Astrophysics Data System (ADS)

    You, Sixian; Liu, Yuan; Arp, Zane; Zhao, Youbo; Chaney, Eric J.; Marjanovic, Marina; Boppart, Stephen A.

    2017-07-01

    Docosanol is an over-the-counter topical agent that has proved to be one of the most effective therapies for treating herpes simplex labialis. However, the mechanism by which docosanol suppresses lesion formation remains poorly understood. To elucidate its mechanism of action, we investigated the uptake of docosanol in living cells using coherent anti-Stokes Raman scattering microscopy. Based on direct visualization of the deuterated docosanol, we observed highly concentrated docosanol inside living cells 24 h after drug treatment. In addition, different spatial patterns of drug accumulation were observed in different cell lines. In keratinocytes, which are the targeted cells of docosanol, the drug molecules appeared to be docking at the periphery of the cell membrane. In contrast, the drug molecules in fibroblasts appeared to accumulate in densely packed punctate regions throughout the cytoplasm. These results suggest that this molecular imaging approach is suitable for the longitudinal tracking of drug molecules in living cells to identify cell-specific trafficking and may also have implications for elucidating the mechanism by which docosanol suppresses lesion formation.

  2. 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.

  3. 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

  4. Bright Lu2 O3 :Eu Thin-Film Scintillators for High-Resolution Radioluminescence Microscopy.

    PubMed

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

    2015-07-16

    The performance of a new thin-film Lu2 O3 :Eu scintillator for single-cell radionuclide imaging is investigated. Imaging the metabolic properties of heterogeneous cell populations in real time is an important challenge with clinical implications. An innovative technique called radioluminescence microscopy has been developed 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, that is, the material used and its geometrical configuration. Scintillators fabricated using conventional methods are relatively thick and therefore do not provide optimal spatial resolution. A thin-film Lu2 O3 :Eu scintillator is compared to a conventional 500 μm thick CdWO4 scintillator for radioluminescence imaging. Despite its thinness, the unique scintillation properties of the Lu2 O3 :Eu scintillator allow us to capture single-positron decays with fourfold higher sensitivity, which is a significant achievement. The thin-film Lu2 O3 :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. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy

    PubMed Central

    Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C.; Min, Wei

    2013-01-01

    Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial–temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon–deuterium bonds (C–D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo. PMID:23798434

  11. Vibrational imaging of newly synthesized proteins in live cells by stimulated Raman scattering microscopy.

    PubMed

    Wei, Lu; Yu, Yong; Shen, Yihui; Wang, Meng C; Min, Wei

    2013-07-09

    Synthesis of new proteins, a key step in the central dogma of molecular biology, has been a major biological process by which cells respond rapidly to environmental cues in both physiological and pathological conditions. However, the selective visualization of a newly synthesized proteome in living systems with subcellular resolution has proven to be rather challenging, despite the extensive efforts along the lines of fluorescence staining, autoradiography, and mass spectrometry. Herein, we report an imaging technique to visualize nascent proteins by harnessing the emerging stimulated Raman scattering (SRS) microscopy coupled with metabolic incorporation of deuterium-labeled amino acids. As a first demonstration, we imaged newly synthesized proteins in live mammalian cells with high spatial-temporal resolution without fixation or staining. Subcellular compartments with fast protein turnover in HeLa and HEK293T cells, and newly grown neurites in differentiating neuron-like N2A cells, are clearly identified via this imaging technique. Technically, incorporation of deuterium-labeled amino acids is minimally perturbative to live cells, whereas SRS imaging of exogenous carbon-deuterium bonds (C-D) in the cell-silent Raman region is highly sensitive, specific, and compatible with living systems. Moreover, coupled with label-free SRS imaging of the total proteome, our method can readily generate spatial maps of the quantitative ratio between new and total proteomes. Thus, this technique of nonlinear vibrational imaging of stable isotope incorporation will be a valuable tool to advance our understanding of the complex spatial and temporal dynamics of newly synthesized proteome in vivo.

  12. 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. Copyright 2003, Elsevier Science (USA). All rights reserved.

  13. Microscopy

    Treesearch

    Patricia A. Moss; Les Groom

    2001-01-01

    Microscopy is the study and interpretation of images produced by a microscope. "Interpretation" is the keyword, because the microscope enables one to see structures that are too small or too close together to be resolved by the unaided eye. (The human eye cannot separate two points or lines that are closer together than 0.1 mm.) it is important to...

  14. 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

  15. Visualizing lipid structure and raft domains in living cells with two-photon microscopy

    PubMed Central

    Gaus, Katharina; Gratton, Enrico; Kable, Eleanor P. W.; Jones, Allan S.; Gelissen, Ingrid; Kritharides, Leonard; Jessup, Wendy

    2003-01-01

    The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell–cell contacts and cover 10–15% of the cell surface at 37°C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 μm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis. PMID:14673117

  16. 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

  17. Living specimen tomography by digital holographic microscopy: morphometry of testate amoeba

    NASA Astrophysics Data System (ADS)

    Charrière, Florian; Pavillon, Nicolas; Colomb, Tristan; Depeursinge, Christian; Heger, Thierry J.; Mitchell, Edward A. D.; Marquet, Pierre; Rappaz, Benjamin

    2006-08-01

    This paper presents an optical diffraction tomography technique based on digital holographic microscopy. Quantitative 2-dimensional phase images are acquired for regularly-spaced angular positions of the specimen covering a total angle of π, allowing to built 3-dimensional quantitative refractive index distributions by an inverse Radon transform. A 20x magnification allows a resolution better than 3 μm in all three dimensions, with accuracy better than 0.01 for the refractive index measurements. This technique is for the first time to our knowledge applied to living specimen (testate amoeba, Protista). Morphometric measurements are extracted from the tomographic reconstructions, showing that the commonly used method for testate amoeba biovolume evaluation leads to systematic under evaluations by about 50%.

  18. 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.

  19. 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

  20. Label-free high-resolution imaging of live cells with deconvolved spatial light interference microscopy.

    PubMed

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

    2010-01-01

    Spatial light interference microscopy (SLIM) is a powerful new quantitative phase optical imaging technique that can be used for studying live cells without the need for exogenous contrast agents. This paper proposes a novel deconvolution-based approach to reconstructing SLIM data, which dramatically improves the visual quality of the images. The proposed deconvolution formulation is tailored to the physics of SLIM imaging of biological samples, and a new fast algorithm is designed for computationally-efficient image reconstruction in this setting. Simulation and experimental results demonstrate that deconvolution can reduce the width of the point-spread function by at least 20%, and can significantly improve the contrast of high-resolution features. Temporally-resolved SLIM imaging with the high spatial resolution enabled by deconvolution provides new opportunities for studying the dynamics of cellular and sub-cellular processes.

  1. 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. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  2. Dual photon excitation microscopy and image threshold segmentation in live cell imaging during compression testing.

    PubMed

    Moo, Eng Kuan; Abusara, Ziad; Abu Osman, Noor Azuan; Pingguan-Murphy, Belinda; Herzog, Walter

    2013-08-09

    Morphological studies of live connective tissue cells are imperative to helping understand cellular responses to mechanical stimuli. However, photobleaching is a constant problem to accurate and reliable live cell fluorescent imaging, and various image thresholding methods have been adopted to account for photobleaching effects. Previous studies showed that dual photon excitation (DPE) techniques are superior over conventional one photon excitation (OPE) confocal techniques in minimizing photobleaching. In this study, we investigated the effects of photobleaching resulting from OPE and DPE on morphology of in situ articular cartilage chondrocytes across repeat laser exposures. Additionally, we compared the effectiveness of three commonly-used image thresholding methods in accounting for photobleaching effects, with and without tissue loading through compression. In general, photobleaching leads to an apparent volume reduction for subsequent image scans. Performing seven consecutive scans of chondrocytes in unloaded cartilage, we found that the apparent cell volume loss caused by DPE microscopy is much smaller than that observed using OPE microscopy. Applying scan-specific image thresholds did not prevent the photobleaching-induced volume loss, and volume reductions were non-uniform over the seven repeat scans. During cartilage loading through compression, cell fluorescence increased and, depending on the thresholding method used, led to different volume changes. Therefore, different conclusions on cell volume changes may be drawn during tissue compression, depending on the image thresholding methods used. In conclusion, our findings confirm that photobleaching directly affects cell morphology measurements, and that DPE causes less photobleaching artifacts than OPE for uncompressed cells. When cells are compressed during tissue loading, a complicated interplay between photobleaching effects and compression-induced fluorescence increase may lead to interpretations in

  3. 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.

  4. 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.

  5. 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.

  6. Adhesion of living cells revealed by variable-angle total internal reflection fluorescence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Cardoso Dos Santos, Marcelina; Vézy, Cyrille; Jaffiol, Rodolphe

    2016-02-01

    Total Internal Reflection Fluorescence Microscopy (TIRFM) is a widespread technique to study cellular process occurring near the contact region with the glass substrate. In this field, determination of the accurate distance from the surface to the plasma membrane constitutes a crucial issue to investigate the physical basis of cellular adhesion process. However, quantitative interpretation of TIRF pictures regarding the distance z between a labeled membrane and the substrate is not trivial. Indeed, the contrast of TIRF images depends on several parameters more and less well known (local concentration of dyes, absorption cross section, angular emission pattern…). The strategy to get around this problem is to exploit a series of TIRF pictures recorded at different incident angles in evanescent regime. This technique called variable-angle TIRF microscopy (vaTIRFM), allowing to map the membrane-substrate separation distance with a nanometric resolution (10-20 nm). vaTIRFM was developed by Burmeister, Truskey and Reichert in the early 1990s with a prism-based TIRF setup [Journal of Microscopy 173, 39-51 (1994)]. We propose a more convenient prismless setup, which uses only a rotatable mirror to adjust precisely the laser beam on the back focal plane of the oil immersion objective (no azimuthal scanning is needed). The series of TIRF images permit us to calculate accurately membrane-surface distances in each pixel. We demonstrate that vaTIRFM are useful to quantify the adhesion of living cells for specific and unspecific membrane-surface interactions, achieved on various functionalized substrates with polymers (BSA, poly-L-lysin) or extracellular matrix proteins (collagen and fibronectin).

  7. 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

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. Real-time intravital microscopy of individual nanoparticle dynamics in liver and tumors of live mice

    PubMed Central

    van de Ven, Anne L; Kim, Pilhan; Ferrari, Mauro; Yun, Seok Hyun

    2013-01-01

    Intravital microscopy is emerging as an important experimental tool for the research and development of multi-functional therapeutic nanoconstructs. The direct visualization of nanoparticle dynamics within live animals provides invaluable insights into the mechanisms that regulate nanotherapeutics transport and cell-particle interactions. Here we present a protocol to image the dynamics of nanoparticles within the liver and tumors of live mice immediately following systemic injection using a high-speed (30-400 fps) confocal or multi-photon laser-scanning fluorescence microscope. Techniques for quantifying the real-time accumulation and cellular association of individual particles with a size ranging from several tens of nanometers to micrometers are described, as well as an experimental strategy for labeling Kupffer cells in the liver in vivo. Experimental design considerations and controls are provided, as well as minimum equipment requirements. The entire protocol takes approximately 4-8 hours and yields quantitative information. These techniques can serve to study a wide range of kinetic parameters that drive nanotherapeutics delivery, uptake, and treatment response. PMID:25383179

  15. 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.

  16. Resolution doubling in live, multicellular organisms via multifocal structured illumination microscopy.

    PubMed

    York, Andrew G; Parekh, Sapun H; Dalle Nogare, Damian; Fischer, Robert S; Temprine, Kelsey; Mione, Marina; Chitnis, Ajay B; Combs, Christian A; Shroff, Hari

    2012-05-13

    We demonstrate three-dimensional (3D) super-resolution in live multicellular organisms using structured illumination microscopy (SIM). Sparse multifocal illumination patterns generated by a digital micromirror device (DMD) allowed us to physically reject out-of-focus light, enabling 3D subdiffractive imaging in samples eightfold thicker than had been previously imaged with SIM. We imaged samples at one 2D image per second, at resolutions as low as 145 nm laterally and 400 nm axially. In addition to dual-labeled, whole fixed cells, we imaged GFP-labeled microtubules in live transgenic zebrafish embryos at depths >45 μm. We captured dynamic changes in the zebrafish lateral line primordium and observed interactions between myosin IIA and F-actin in cells encapsulated in collagen gels, obtaining two-color 4D super-resolution data sets spanning tens of time points and minutes without apparent phototoxicity. Our method uses commercially available parts and open-source software and is simpler than existing SIM implementations, allowing easy integration with wide-field microscopes.

  17. 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.

  18. 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

  19. Cross-sectional electrostatic force microscopy of thin-film solar cells

    NASA Astrophysics Data System (ADS)

    Ballif, C.; Moutinho, H. R.; Al-Jassim, M. M.

    2001-01-01

    In a recent work, we showed that atomic force microscopy (AFM) is a powerful technique to image cross sections of polycrystalline thin films. In this work, we apply a modification of AFM, namely, electrostatic force microscopy (EFM), to investigate the electronic properties of cleaved II-VI and multijunction thin-film solar cells. We cleave the devices in such a way that they are still working with their nominal photovoltaic efficiencies and can be polarized for the measurements. This allows us to differentiate between surface effects (work function and surface band bending) and bulk device properties. In the case of polycrystalline CdTe/CdS/SnO2/glass solar cells, we find a drop of the EFM signal in the area of the CdTe/CdS interface (±50 nm). This drop varies in amplitude and sign according to the applied external bias and is compatible with an n-CdS/p-CdTe heterojunction model, thereby invalidating the possibility of a deeply buried n-p CdTe homojunction. In the case of a triple-junction GaInP/GaAs/Ge device, we observe a variation of the EFM signal linked to both the material work-function differences and to the voltage bias applied to the cell. We attempt a qualitative explanation of the results and discuss the implications and difficulties of the EFM technique for the study of such thin-film devices.

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

    PubMed

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

    2012-09-01

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

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

    PubMed Central

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

    2012-01-01

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

  2. 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

  3. 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).

  4. A device for real-time live-cell microscopy during dynamic dual-modal mechanostimulation

    NASA Astrophysics Data System (ADS)

    Lorusso, D.; Nikolov, H. N.; Chmiel, T.; Beach, R. J.; Sims, S. M.; Dixon, S. J.; Holdsworth, D. W.

    2017-03-01

    Mechanotransduction - the process by which cells sense and respond to mechanical stimuli - is essential for several physiological processes including skeletal homeostasis. Mammalian cells are thought to be sensitive to different modes of mechanical stimuli, including vibration and fluid shear. To better understand the mechanisms underlying the early stages of mechanotransduction, we describe the development of devices for mechanostimulation (by vibration and fluid shear) of live cells that can be integrated with real-time optical microscopy. The integrated system can deliver up to 3 Pa of fluid shear simultaneous with high-frequency sinusoidal vibrations up to 1 g. Stimuli can be applied simultaneously or independently to cells during real-time microscopic imaging. A custom microfluidic chamber was prepared from polydimethylsiloxane on a glass-bottom cell culture dish. Fluid flow was applied with a syringe pump to induce shear stress. This device is compatible with a custom-designed motion control vibration system. A voice coil actuates the system that is suspended on linear air bushings. Accelerations produced by the system were monitored with an on-board accelerometer. Displacement was validated optically using particle tracking digital high-speed imaging (1200 frames per second). During operation at nominally 45 Hz and 0.3 g, displacements were observed to be within 3.56% of the expected value. MC3T3-E1 osteoblast like cells were seeded into the microfluidic device and loaded with the calcium sensitive fluorescent probe fura-2, then mounted onto the dual-modal mechanostimulation platform. Cells were then imaged and monitored for fluorescence emission. In summary, we have developed a system to deliver physiologically relevant vibrations and fluid shear to live cells during real-time imaging and photometry. Monitoring the behavior of live cells loaded with appropriate fluorescent probes will enable characterization of the signals activated during the initial

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

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

    SciTech Connect

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

    2016-05-06

    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.

  14. Field emission property of hydrogenated chemical vapor deposited diamond films studied by scanning tunneling microscopy.

    PubMed

    Liu, Fengbin; Wang, Jiadao; Chen, Darong

    2010-11-01

    By using scanning tunneling microscopy, the plots of tunneling current versus applied voltage, at the local points for hydrogenated and oxygenated chemical vapor deposited diamond films, were investigated. For comparison, the measurement points were adopted on the centers of the crystalline grains and at the grain boundaries, respectively. The results indicated that, for the hydrogenated chemical vapor deposited diamond, the field emission character is much better on the center of the crystalline grains than at the grain boundary. In contrast, for the oxygenated samples, the crystalline grains show a poor field emission character. The two diamond surfaces exhibit similar field emission characters at the grain boundaries. The surface emission mechanisms of the hydrogenated chemical vapor deposited diamond films were also discussed.

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

    PubMed Central

    Mudanyali, Onur; Bishara, Waheb; Ozcan, Aydogan

    2011-01-01

    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. PMID:21935102

  16. 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.

  17. 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.}

  18. 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

  19. 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.

  20. Magnetic flux dynamics in superconducting films studied by scanning Hall probe microscopy

    NASA Astrophysics Data System (ADS)

    Stan, Gheorghe

    In this thesis we address two different issues in the field of flux dynamics in superconductors with constricted geometry. In our experiments we used scanning Hall probe microscopy to investigate the magnetic field profile above the samples' surface. In the first experiment, Vortex Nucleation in Narrow Thin-Film Strips, we studied the magnetic flux nucleation in type-II superconducting thin-film strips of mesoscopic width. The maximum magnetic field below which vortices are completely expelled from niobium narrow thin-film strips was measured for different widths. Above this threshold field we examined the field dependence of the vortex density for the studied strips. In the second experiment, The Superconducting Dripping Faucet , we analyzed, in microbridge geometry, the dynamics of the magnetic flux nucleation in a one-dimensional type-I superconducting channel. For this experiment we have developed a novel high-bandwidth Hall probe to detect in real time the nucleation and subsequent motion of the magnetic flux tubes along a fabricated one-dimensional channel in a lead film. The complex dynamics exhibited by the flux tubes nucleating from one end of the channel shares many characteristics of the well-known dripping faucet experiment. Nonlinear time series analysis was used to investigate the dynamics of the flux tubes in our experiment.

  1. 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.

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Magnetic force microscopy of conducting nanodots in NiO thin films

    NASA Astrophysics Data System (ADS)

    Meang, Wan Joo; Seo, Jeongdae; Ahn, Yoonho; Son, J. Y.

    2016-03-01

    We report a nanoscale magnetic conducting filament in a resistive random access memory (RRAM) device by the direct investigation of conducting nanobits in NiO thin films using magnetic force microscopy. The conducting nanobit in a NiO RRAM capacitor formed by CAFM and KFM exhibited a typical bistable resistive switching characteristic. The magnetizations of the conducting nanobit were measured as a function of the set-reset switching cycle and as the switching cycles were increased, a strong ferromagnetic signal was observed. The metallic Ni formation in the nanoscale magnetic conducting filament could be a possible reason for the origin of the magnetism. [Figure not available: see fulltext.

  7. 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

  8. CARS microscopy for the monitoring of fat deposition mechanisms in a living organism

    NASA Astrophysics Data System (ADS)

    Enejder, Annika; Hellerer, Thomas; Hillertz, Per; Brackmann, Christian; Axäng, Claes; Pilon, Marc

    2006-02-01

    We introduce near-infrared Coherent Anti-Stokes Raman Scattering (CARS) microscopy as a method for the monitoring of fat deposition in a living organism by directly probing the CH II vibration of the lipids without the need for staining or labeling. This study nicely brings forward all the advantages of the technique: deep probe depth, low excitation powers, high 3-dimensional resolution, and visualization without the interference of exogenous label molecules, or fixation and staining procedures. Differences in fat deposition during the life cycle of the nematode Caenorhabditis elegans were evaluated quantitatively from the CARS microscopy images, showing that the technique can be used to study mechanisms that regulate lipid storage. Beside the wild type nematode, the feeding-deficient mutant pha-3 was studied. It was shown that the embryonal accumulation of energy stores is enough for the development of a full-sized pre-adult larva, being possible also for the mutant. However, the volume density of lipid stores at the fourth and last pre-adult development stage seems to determine its adult body size. Whereas the wild type larva maintains its size when becoming adult, though at the cost of reduced lipid density, the feeding deficient mutant instead has to reduce its body size in order to reach the same volume density of lipid stores. Both strains start off their adult life with a volume fraction of lipid stores corresponding to 6-7%; the wild type with a radius of 24+/-2 µm and the pha-3 mutant with a significantly smaller radius of 16+/-3 μm.

  9. 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

  10. A study of growth and thermal dewetting behavior of ultra-thin gold films using transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Sudheer, Mondal, Puspen; Rai, V. N.; Srivastava, A. K.

    2017-07-01

    The growth and solid-state dewetting behavior of Au thin films (0.7 to 8.4 nm) deposited on the formvar film (substrate) by sputtering technique have been studied using transmission electron microscopy. The size and number density of the Au nanoparticles (NPs) change with an increase in the film thickness (0.7 to 2.8 nm). Nearly spherical Au NPs are obtained for <3 nm thickness films whereas percolated nanostructures are observed for ≥3 nm thickness films as a consequence of the interfacial interaction of Au and formvar film. The covered area fraction (CAF) increases from ˜13 to 75 % with the change in film thickness from 0.7 to 8.4 nm. In-situ annealing of ≤3 nm film produces comparatively bigger size and better sphericity Au NPs along with their narrow distributions, whereas just percolated film produces broad distribution in size having spherical as well as elongated Au NPs. The films with thickness ≤3 nm show excellent thermal stability. The films having thickness >6 nm show capability to be used as an irreversible temperature sensor with a sensitivity of ˜0.1 CAF/°C. It is observed that annealing affects the crystallinity of the Au grains in the films. The electron diffraction measurement also shows annealing induced morphological evolution in the percolated Au thin films (≥3 nm) during solid-state dewetting and recrystallization of the grains.

  11. Identification of fluorescent compounds with non-specific binding property via high throughput live cell microscopy.

    PubMed

    Nath, Sangeeta; Spencer, Virginia A; Han, Ju; Chang, Hang; Zhang, Kai; Fontenay, Gerald V; Anderson, Charles; Hyman, Joel M; Nilsen-Hamilton, Marit; Chang, Young-Tae; Parvin, Bahram

    2012-01-01

    Compounds exhibiting low non-specific intracellular binding or non-stickiness are concomitant with rapid clearing and in high demand for live-cell imaging assays because they allow for intracellular receptor localization with a high signal/noise ratio. The non-stickiness property is particularly important for imaging intracellular receptors due to the equilibria involved. Three mammalian cell lines with diverse genetic backgrounds were used to screen a combinatorial fluorescence library via high throughput live cell microscopy for potential ligands with high in- and out-flux properties. The binding properties of ligands identified from the first screen were subsequently validated on plant root hair. A correlative analysis was then performed between each ligand and its corresponding physiochemical and structural properties. The non-stickiness property of each ligand was quantified as a function of the temporal uptake and retention on a cell-by-cell basis. Our data shows that (i) mammalian systems can serve as a pre-screening tool for complex plant species that are not amenable to high-throughput imaging; (ii) retention and spatial localization of chemical compounds vary within and between each cell line; and (iii) the structural similarities of compounds can infer their non-specific binding properties. We have validated a protocol for identifying chemical compounds with non-specific binding properties that is testable across diverse species. Further analysis reveals an overlap between the non-stickiness property and the structural similarity of compounds. The net result is a more robust screening assay for identifying desirable ligands that can be used to monitor intracellular localization. Several new applications of the screening protocol and results are also presented.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy.

    PubMed

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Boccara, A Claude; Bourdieu, Laurent

    2011-11-01

    Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

  20. Single myelin fiber imaging in living rodents without labeling by deep optical coherence microscopy

    NASA Astrophysics Data System (ADS)

    Ben Arous, Juliette; Binding, Jonas; Léger, Jean-François; Casado, Mariano; Topilko, Piotr; Gigan, Sylvain; Claude Boccara, A.; Bourdieu, Laurent

    2011-11-01

    Myelin sheath disruption is responsible for multiple neuropathies in the central and peripheral nervous system. Myelin imaging has thus become an important diagnosis tool. However, in vivo imaging has been limited to either low-resolution techniques unable to resolve individual fibers or to low-penetration imaging of single fibers, which cannot provide quantitative information about large volumes of tissue, as required for diagnostic purposes. Here, we perform myelin imaging without labeling and at micron-scale resolution with >300-μm penetration depth on living rodents. This was achieved with a prototype [termed deep optical coherence microscopy (deep-OCM)] of a high-numerical aperture infrared full-field optical coherence microscope, which includes aberration correction for the compensation of refractive index mismatch and high-frame-rate interferometric measurements. We were able to measure the density of individual myelinated fibers in the rat cortex over a large volume of gray matter. In the peripheral nervous system, deep-OCM allows, after minor surgery, in situ imaging of single myelinated fibers over a large fraction of the sciatic nerve. This allows quantitative comparison of normal and Krox20 mutant mice, in which myelination in the peripheral nervous system is impaired. This opens promising perspectives for myelin chronic imaging in demyelinating diseases and for minimally invasive medical diagnosis.

  1. 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

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Impaired Intracellular Ca2+ Dynamics in Live Cardiomyocytes Revealed by Rapid Line Scan Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Plank, David M.; Sussman, Mark A.

    2005-06-01

    Altered intracellular Ca2+ dynamics are characteristically observed in cardiomyocytes from failing hearts. Studies of Ca2+ handling in myocytes predominantly use Fluo-3 AM, a visible light excitable Ca2+ chelating fluorescent dye in conjunction with rapid line-scanning confocal microscopy. However, Fluo-3 AM does not allow for traditional ratiometric determination of intracellular Ca2+ concentration and has required the use of mathematic correction factors with values obtained from separate procedures to convert Fluo-3 AM fluorescence to appropriate Ca2+ concentrations. This study describes methodology to directly measure intracellular Ca2+ levels using inactivated, Fluo-3-AM-loaded cardiomyocytes equilibrated with Ca2+ concentration standards. Titration of Ca2+ concentration exhibits a linear relationship to increasing Fluo-3 AM fluorescence intensity. Images obtained from individual myocyte confocal scans were recorded, average pixel intensity values were calculated, and a plot is generated relating the average pixel intensity to known Ca2+ concentrations. These standard plots can be used to convert transient Ca2+ fluorescence obtained with experimental cells to Ca2+ concentrations by linear regression analysis. Standards are determined on the same microscope used for acquisition of unknown Ca2+ concentrations, simplifying data interpretation and assuring accuracy of conversion values. This procedure eliminates additional equipment, ratiometric imaging, and mathematic correction factors and should be useful to investigators requiring a straightforward method for measuring Ca2+ concentrations in live cells using Ca2+-chelating dyes exhibiting variable fluorescence intensity.

  7. 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.

  8. 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.

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues

    PubMed Central

    Benninger, Richard K.P.; Piston, David W.

    2013-01-01

    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. PMID:23728746

  15. 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.

  16. Distinctive behaviour of live biopsy-derived carcinoma cells unveiled using coherence-controlled holographic microscopy

    PubMed Central

    2017-01-01

    Head and neck squamous cell carcinoma is one of the most aggressive tumours and is typically diagnosed too late. Late diagnosis requires an urgent decision on an effective therapy. An individualized test of chemosensitivity should quickly indicate the suitability of chemotherapy and radiotherapy. No ex vivo chemosensitivity assessment developed thus far has become a part of general clinical practice. Therefore, we attempted to explore the new technique of coherence-controlled holographic microscopy to investigate the motility and growth of live cells from a head and neck squamous cell carcinoma biopsy. We expected to reveal behavioural patterns characteristic for malignant cells that can be used to imrove future predictive evaluation of chemotherapy. We managed to cultivate primary SACR2 carcinoma cells from head and neck squamous cell carcinoma biopsy verified through histopathology. The cells grew as a cohesive sheet of suspected carcinoma origin, and western blots showed positivity for the tumour marker p63 confirming cancerous origin. Unlike the roundish colonies of the established FaDu carcinoma cell line, the SACR2 cells formed irregularly shaped colonies, eliciting the impression of the collective invasion of carcinoma cells. Time-lapse recordings of the cohesive sheet activity revealed the rapid migration and high plasticity of these epithelial-like cells. Individual cells frequently abandoned the swiftly migrating crowd by moving aside and crawling faster. The increasing mass of fast migrating epithelial-like cells before and after mitosis confirmed the continuation of the cell cycle. In immunofluorescence, analogously shaped cells expressed the p63 tumour marker, considered proof of their origin from a carcinoma. These behavioural traits indicate the feasible identification of carcinoma cells in culture according to the proposed concept of the carcinoma cell dynamic phenotype. If further developed, this approach could later serve in a new functional

  17. 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

  18. 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

  19. 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.

  20. Direct observations of polarization reversal process in ferroelectric thin films using high speed piezoresponse force microscopy

    NASA Astrophysics Data System (ADS)

    Premnath, Ramesh Nath

    Ferroelectric thin films have been widely implicated for use in future ultra-high-density memory devices, using atomic force microscopy (AFM) related techniques for the read/write operations of ferroelectric memory media where formation of a single domain structure with a defined polarization direction acts as a distinct memory bit. Therefore, it is important to understand the mechanisms and kinetics involved in the polarization switching process, which includes the nucleation and growth of ferroelectric domains at the nanoscale. In recent years, the piezoresponse force microscopy (PFM) technique has been widely been used to image, characterize and modify the domain structures in ferroelectric films with this spatial resolution. However, operating speeds for PFM (and AFM in general) remain a continuing limitation for imaging dynamic processes such as domain switching and read/write operations. A simple method to increase PFM characterization speeds by several orders of magnitude is presented here based on standard commercial equipment and AFM probes. Essentially, an AC voltage at a high resonance frequency is applied to a conducting AFM tip, which is in contact with a ferroelectric surface. The tip is rapidly rastered across a surface without force feedback, while the amplitude and phase of the high frequency resonances are detected with a lock-in amplifier. Although the topography is not reliably recorded, stable contrast related to ferroelectric properties is accurately mapped due to the relative insensitivity of many dynamic AFM contrast mechanisms to variations in repulsive contact forces. Images with nanoscale contrast of ferroelectric domains are presented, acquired at complete frame rates as low as 6 seconds per 256x256 pixel image. The mechanism and kinetics involved in the dynamic domain switching process of PZT thin films are therefore uniquely presented with nanoscale and nanosecond resolution. It was found that domain dynamics processes are governed by

  1. Surface potential measurement of n-type organic semiconductor thin films by mist deposition via Kelvin probe microscopy

    NASA Astrophysics Data System (ADS)

    Odaka, Akihiro; Satoh, Nobuo; Katori, Shigetaka

    2017-08-01

    We partially deposited fullerene (C60) and phenyl-C61-butyric acid methyl ester thin films that are typical n-type semiconductor materials on indium-tin oxide by mist deposition at various substrate temperatures. The topographic and surface potential images were observed via dynamic force microscopy/Kelvin probe force microscopy with the frequency modulation detection method. We proved that the area where a thin film is deposited depends on the substrate temperature during deposition from the topographic images. It was also found that the surface potential depends on the substrate temperature from the surface potential images.

  2. In vivo mouse and live cell STED microscopy of neuronal actin plasticity using far-red emitting fluorescent proteins.

    PubMed

    Wegner, Waja; Ilgen, Peter; Gregor, Carola; van Dort, Joris; Mott, Alexander C; Steffens, Heinz; Willig, Katrin I

    2017-09-18

    The study of proteins in dendritic processes within the living brain is mainly hampered by the diffraction limit of light. STED microscopy is so far the only far-field light microscopy technique to overcome the diffraction limit and resolve dendritic spine plasticity at superresolution (nanoscopy) in the living mouse. After having tested several far-red fluorescent proteins in cell culture we report here STED microscopy of the far-red fluorescent protein mNeptune2, which showed best results for our application to superresolve actin filaments at a resolution of ~80 nm, and to observe morphological changes of actin in the cortex of a living mouse. We illustrate in vivo far-red neuronal actin imaging in the living mouse brain with superresolution for time periods of up to one hour. Actin was visualized by fusing mNeptune2 to the actin labels Lifeact or Actin-Chromobody. We evaluated the concentration dependent influence of both actin labels on the appearance of dendritic spines; spine number was significantly reduced at high expression levels whereas spine morphology was normal at low expression.

  3. Second-harmonic microscopy of unstained living cardiac myocytes: measurements of sarcomere length with 20-nm accuracy

    NASA Astrophysics Data System (ADS)

    Boulesteix, Thierry; Beaurepaire, Emmanuel; Sauviat, Martin-Pierre; Schanne-Klein, Marie-Claire

    2004-09-01

    We extend second-harmonic generation (SHG) microscopy to the measurement of sarcomere length in unstained living cardiac myocytes with 20-nm accuracy. We quantify individual sarcomere shortening in the presence of saxitoxin and find that it is in agreement with mechanical measurements of atrial tissue contracture. This functional application of SHG microscopy is generally applicable to quantify the physiological effects of drugs on contractile tissue. Our data also suggest that packed myosin heads in sarcomere thick filaments are responsible for the large second-harmonic endogenous signal in muscle tissue.

  4. 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

  5. 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.

  6. 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.

  7. Compact surface plasmon holographic microscopy for near-field film mapping.

    PubMed

    Zhang, Jiwei; Dai, Siqing; Ma, Chaojie; Di, Jianglei; Zhao, Jianlin

    2017-09-01

    We develop a compact objective-coupling surface plasmon holographic microscopy with a common-path configuration by introducing a Wollaston prism. Through off-axis hologram recording and numerical reconstruction, amplitude- and phase-contrast surface plasmon resonance (SPR) images can be obtained simultaneously. Based on the four-layer SPR model, the thin film thickness distribution in near field can be mapped unambiguously using a novel demodulation method without a priori knowledge. The technique demonstrates nondestructive and full-field measurement capabilities with sub-nanometer resolution theoretically. Furthermore, owing to the high temporal stability, the recommended system shows great potential for dynamic measurement of near-field tiny refractive index or thickness variation in fields such as chemistry and biomedicine, etc.

  8. X-Ray Diffraction and Electron Microscopy Study of Cr/Sb Multilayered Films

    NASA Astrophysics Data System (ADS)

    Dohnomae, Hitoshi

    1994-03-01

    Structures of [Cr(2 Å)/Sb(50 Å)] n multilayered films have been investigated by X-ray diffraction and transmission electron microscopy (TEM) of cross sections. When the substrate temperature (T s) was 90° C, an epitaxial structure with a coherent stacking of Sb and compound (CrSb) layers was formed by the interfacial reaction. On the other hand, at T s=-50° C, a non-epitaxial structure composed of crystalline Sb layers and amorphous Cr metal layers was obtained. Interfaces of multilayers observed by TEM are very flat for both samples. The structures of very thin Cr layers depend on the reactivity of interfaces and greatly affect on the orientations of Sb layers.

  9. 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.

  10. Identification of nodal tissue in the living heart using rapid scanning fiber-optics confocal microscopy and extracellular fluorophores.

    PubMed

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

    2013-09-01

    Risks associated with pediatric reconstructive heart surgery include injury of the sinoatrial node (SAN) and atrioventricular node (AVN), requiring cardiac rhythm management using implantable pacemakers. These injuries are the result of difficulties in identifying nodal tissues intraoperatively. Here we describe an approach based on confocal microscopy and extracellular fluorophores to quantify tissue microstructure and identify nodal tissue. Using conventional 3-dimensional confocal microscopy we investigated the microstructural arrangement of SAN, AVN, and atrial working myocardium (AWM) in fixed rat heart. AWM exhibited a regular striated arrangement of the extracellular space. In contrast, SAN and AVN had an irregular, reticulated arrangement. AWM, SAN, and AVN tissues were beneath a thin surface layer of tissue that did not obstruct confocal microscopic imaging. Subsequently, we imaged tissues in living rat hearts with real-time fiber-optics confocal microscopy. Fiber-optics confocal microscopy images resembled images acquired with conventional confocal microscopy. We investigated spatial regularity of tissue microstructure from Fourier analysis and second-order image moments. Fourier analysis of fiber-optics confocal microscopy images showed that the spatial regularity of AWM was greater than that of nodal tissues (37.5 ± 5.0% versus 24.3 ± 3.9% for SAN and 23.8 ± 3.7% for AVN; P<0.05). Similar differences of spatial regularities were revealed from second-order image moments (50.0 ± 7.3% for AWM versus 29.3 ± 6.7% for SAN and 27.3 ± 5.5% for AVN; P<0.05). The study demonstrates feasibility of identifying nodal tissue in living heart using extracellular fluorophores and fiber-optics confocal microscopy. Application of the approach in pediatric reconstructive heart surgery may reduce risks of injuring nodal tissues.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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.

  16. High sensitivity electric force microscopy (EFM) for organic polymer thin-films

    NASA Astrophysics Data System (ADS)

    Silveira, William R.; Muller, Erik; Jenkins, Neil; To, Brian; Marohn, John A.

    2003-03-01

    We are investigating the electric force microscope as a tool for studying and imaging electronic properties of organic materials. We have constructed a high sensitivity homebuilt microscope that operates in high vacuum and at cryogenic as well as room temperatures. Working in vacuum, we can measure work function very sensitively by recording cantilever frequency shifts as small as a few millihertz. We find that electric force microscopy (EFM) is a powerful tool to study and image work function in films of the hopping conductor triphenyldiamine-doped polycarbonate (TPD-PC), a molecularly doped polymer system used in xerography. Work function and capacitance measurements suggest that the surface potential of 1-nm-rms uniform thin films strongly depend on the properties of the underlying gold substrate. Surprisingly, we are finding that interpretation of EFM images is challenging for low mobility hopping conductors because few of the usual assumptions used to analyze EFM measurements apply. Progress towards developing and testing an adequate theory will be presented. Here, TPD-PC is a powerful test system because the mobility is tunable by varying TPD concentration. Finally, we have incorporated ultra-soft force sensors into the microscope for use in electromechanical dissipation force measurements that offer the exciting possibility of quantifying and imaging in-plane conductivity.

  17. 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.

  18. Thin-film Electrochemistry of Single Prussian Blue Nanoparticles Revealed by Surface Plasmon Resonance Microscopy.

    PubMed

    Jiang, Dan; Sun, Linlin; Liu, Tao; Wang, Wei

    2017-10-06

    Electrochemical behaviors of Prussian blue (PB) have been intensively studied for decades because it not only serves as a model electro-active nanomaterial in fundamental electrochemistry, but also a promising metal-ion storage electrode material for developing rechargeable batteries. Traditional electrochemical studies are mostly based on bulk materials, leading to an averaged property of billions of PB nanoparticles. In the present work, we employed surface plasmon resonance microscopy (SPRM) to resolve the optical cyclic voltammograms of single PB nanoparticles during electrochemical cycling. It was found that the electrochemical behavior of single PB nanoparticles nicely followed a classical thin-film electrochemistry theory. While kinetic controlled electron transfer was observed at slower scan rates, intra-particle diffusion of K+ ions began to take effect when the scan rate was higher than 60 mV/s. We further found that the electrochemical activity among individual PB nanoparticles was very heterogeneous and such a phenomenon has not been previously observed in the bulk measurements. The present work not only demonstrates the thin-film electrochemical feature of single electro-active nanomaterials for the first time, it also validates the applicability of SPRM technique to investigate a variety of metal ion-storage battery materials, with implications in both fundamental nano-electrochemistry and electro-active materials for sensing and battery applications.

  19. Quantitative model for near-field scanning microwave microscopy: application to metrology of thin film dielectrics.

    PubMed

    Reznik, Alexander N; Talanov, Vladimir V

    2008-11-01

    We have experimentally verified a recently proposed theoretical model for near-field microwave microscopy of multilayer media. The model addresses a near-field microwave probe as an electrically small antenna with a Gaussian-like current distribution that has a single characteristic length scale on the order of the probe size. Electrodynamic response of an antenna is calculated using Green functions in the form of integral transforms for electric and magnetic fields (both quasistatic and propagating), which are generated by a pointlike dipole. Experimental data were obtained at 4 GHz using a near-field scanning microwave microscope with aperture size of approximately 5 microm for a set of six SiO(2) films with thickness ranging from 0.1 to 1.5 microm. For each sample the probe resonant frequency was both measured and simulated as a function of the tip-sample distance, and good agreement between the theory and experimental data was observed. It was found that the model is capable of determining thin film dielectric constant with accuracy of approximately 5%-7%.

  20. 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

  1. 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…

  2. 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…

  3. Homogeneous fluorescent thin films as long-term stable microscopy reference layers

    NASA Astrophysics Data System (ADS)

    Brülisauer, Martina; ćaǧin, Emine; Bertsch, Dietmar; Lüthi, Stefan; Dietrich, Klaus; Heeb, Peter; Stärker, Ulrich; Bernard, André

    2017-05-01

    Calibration and validation of fluorescence microscopy devices and components require a high level of stability and repeatability in their fluorescent properties, both spatially and temporally. In order to establish a dependable reference point, from which all variations within the microscope and peripheral devices can be tested, an exceedingly homogeneous fluorescence response must be provided through a calibration tool. We present material system optimization and microfabrication process development, as well as long-term stability considerations for such a calibration tool. Stringent specifications for film thickness (< 1μm +/- 0.1% over 1.5x1.5 mm) and for fluorescence response distribution (within 1%) apply, and should hold for up to 100 hours under continuous white irradiation. Low conversion efficiency demands high pick up efficiency and therefor reduces focal depth by high NA of applied fluorescence microscope lens. High spatial resolutions demands use of high quality lenses that typically show low field curvatures and good chromatic corrections. Therefore, the focal plane is flat and well defined in the z-plane. Fluorescent, ligand capped core-shell quantum dots (SMQDs) were embedded in diluted PMMA at low concentrations. The formulations were spin-coated on silicon and glass wafers to obtain films with thicknesses under 1 μm and low variations on a 100 mm wafer. Fluorescence properties of the SMQD were preserved in the matrix material, and agglomerations were not detectable in the fluorescence response nor in SEM images. Gradual degradation of the fluorescence response due to film aging was managed through robust packaging solutions.

  4. 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

  5. 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.

  6. 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.

  7. Limits of single-molecule super-resolution microscopy in thin polymer films

    NASA Astrophysics Data System (ADS)

    Wang, Muzhou; Davanco, Marcelo; Marr, James M.; Liddle, J. Alexander; Gilman, Jeffrey W.

    Structural characterization by super-resolution microscopy has become increasingly widespread, particularly in the biological community. The technique is powerful because it can produce real-space images with resolutions of tens of nanometers, while sample preparation is relatively non-invasive. Previous studies have applied these techniques to important scientific problems in the life sciences, but relatively little work has explored the attainable limit of resolution using samples of known structure. In this work, we apply photo-activated localization microscopy (PALM) to polymer films that have been nanopatterned using electron-beam lithography. Trace amounts of a rhodamine spiroamide dye are dispersed into nanostructured poly(methyl methacrylate), and UV-induced switching of the fluorophores enables nanoscale localization of single molecules to generate a final composite super-resolution image. Features as small as 50 nm are clearly resolvable. To determine the ultimate resolution limit, we investigate sources of error in the system, particularly from systematic mislocalizations due to the effect of fluorophore orientation on the single-molecule point-spread function.

  8. 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.

  9. 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.

  10. 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.

  11. Spinning disk confocal microscopy of live, intraerythrocytic malarial parasites. 1. Quantification of hemozoin development for drug sensitive versus resistant malaria.

    PubMed

    Gligorijevic, Bojana; McAllister, Ryan; Urbach, Jeffrey S; Roepe, Paul D

    2006-10-17

    We have customized a Nipkow spinning disk confocal microscope (SDCM) to acquire three-dimensional (3D) versus time data for live, intraerythrocytic malarial parasites. Since live parasites wiggle within red blood cells, conventional laser scanning confocal microscopy produces blurred 3D images after reconstruction of z stack data. In contrast, since SDCM data sets at high x, y, and z resolution can be acquired in hundreds of milliseconds, key aspects of live parasite cellular biochemistry can be much better resolved on physiologically meaningful times scales. In this paper, we present the first 3D DIC transmittance "z stack" images of live malarial parasites and use those to quantify hemozoin (Hz) produced within the living parasite digestive vacuole, under physiologic conditions. Using live synchronized cultures and voxel analysis of sharpened DIC z stacks, we present the first quantitative in vivo analysis of the rate of Hz growth for chloroquine sensitive (CQS) versus resistant (CQR) malarial parasites. We present data for laboratory strains, as well as pfcrt transfectants expressing a CQR conferring mutant pfcrt gene. We also analyze the rate of Hz growth in the presence and absence of physiologically relevant doses of chloroquine (CQ) and verapamil (VPL) and thereby present the first in vivo quantification of key predictions from the well-known Fitch hypothesis for CQ pharmacology. In the following paper [Gligorijevic, B., et al. (2006) Biochemistry 45, pp 12411-12423], we acquire fluorescent images of live parasite DV via SDCM and use those to quantify DV volume for CQS versus CQR parasites.

  12. 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

  13. Development of an automated fluorescence microscopy system for photomanipulation of genetically encoded photoactivatable proteins (optogenetics) in live cells.

    PubMed

    Araki, Nobukazu; Ikeda, Yuka; Kato, Takuma; Kawai, Katsuhisa; Egami, Youhei; Miyake, Katsuya; Tsurumaki, Nobuhide; Yamaguchi, Mitsunari

    2014-06-01

    Photomanipulation of genetically encoded light-sensitive protein activity, also known as optogenetics, is one of the most innovative recent microscopy techniques in the fields of cell biology and neurobiology. Although photomanipulation is usually performed by diverting the photobleaching mode of a confocal laser microscope, photobleaching by the laser scanning unit is not always suitable for photoactivation. We have developed a simple automated wide-field fluorescence microscopy system for the photomanipulation of genetically encoded photoactivatable proteins in live cells. An electrically automated fluorescence microscope can be controlled through MetaMorph imaging software, making it possible to acquire time-lapse, multiwavelength images of live cells. Using the journal (macro recording) function of MetaMorph, we wrote a macro program to change the excitation filter for photoactivation and illumination area during the intervals of image acquisition. When this program was run on the wide-field fluorescence microscope, cells expressing genetically encoded photoactivatable Rac1, which is activated under blue light, showed morphological changes such as lamellipodial extension and cell surface ruffling in the illuminated region. Using software-based development, we successfully constructed a fully automated photoactivation microscopy system for a mercury lamp-based fluorescence microscope. © The Author 2014. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  14. 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

  15. 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

  16. Super-resolution fluorescence imaging of directly labelled DNA: from microscopy standards to living cells.

    PubMed

    Flors, C

    2013-07-01

    Super-resolution fluorescence microscopy is ideally suited to study the complex organization of cell DNA in the 10-100 nm range. Novel methods to image directly labelled DNA, instead of DNA-associated proteins, are being developed and refined. This minireview provides an update of recent progress in super-resolution fluorescence imaging methods for DNA. These developments should allow a deeper understanding of chromatin structure and widen the scope of biological processes that may be investigated with super-resolution fluorescence microscopy. © 2013 The Author Journal of Microscopy © 2013 Royal Microscopical Society.

  17. 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.

  18. 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

  19. Absolute 3D reconstruction of thin films topography in microfluidic channels by interference reflection microscopy.

    PubMed

    Huerre, A; Jullien, M-C; Theodoly, O; Valignat, M-P

    2016-03-07

    The travel of droplets, bubbles, vesicles, capsules, living cells or small organisms in microchannels is a hallmark in microfluidics applications. A full description of the dynamics of such objects requires a quantitative understanding of the complex hydrodynamic and interfacial interactions between objects and channel walls. In this paper, we present an interferometric method that allows absolute topographic reconstruction of the interspace between an object and channel walls for objects confined in microfluidic channels. Wide field microscopic imaging in reflection interference contrast mode (RICM) is directly performed at the bottom wall of microfluidic chips. Importantly, we show that the reflections at both the lower and upper surface of the microchannel have to be considered in the quantitative analysis of the optical signal. More precisely, the contribution of the reflection at the upper surface is weighted depending on the light coherence length and channel height. Using several wavelengths and illumination apertures, our method allows reconstructing the topography of thin films on channel walls in a range of 0-500 nm, with a precision as accurate as 2 nm for the thinnest films. A complete description of the protocol is exemplified for oil in water droplets travelling in channels of height 10-400 μm at a speed up to 5 mm s(-1).

  20. Organic Molecular Thin Films: Growth, Structure, and Manipulation Studied by Scanning Tunneling Microscopy

    NASA Astrophysics Data System (ADS)

    Stock, Taylor J. Z.

    Room temperature scanning tunneling microscopy (RT-STM) has been used to observe the growth modes, morphologies and crystal structures of sub-monolayer (ML) to multilayer thin films of phthalocyanines (H2Pc and CuPc), C60 fullerenes, and CuPc:C60 composites, grown on the Cu(111) surface. In addition to imaging these films, STM has been used to manipulate the various molecules via hot tunneling electron injection. At sub-ML coverage the phthalocyanines are mobile on the Cu(111) and form a diffuse 2D gas. Molecules in this mobile phase can be immobilized on the substrate through exposure to tunneling electrons at a bias voltage exceeding a threshold value. The bias threshold value and strength of the induced molecular immobilization is dependent on the particular phthalocyanine molecule/substrate combination. At approximately one ML coverage the phthalocyanine molecules become sterically confined and lie flat on the Cu(111), forming an ordered 2D lattice. As coverage is increased beyond 1ML in the Cu(111)-CuPc system, the molecule-substrate interaction diminishes in strength and the intermolecular interaction becomes dominant, causing the molecular crystal lattice parameters to evolve towards the bulk α-phase. This trend continues for the layer-by-layer growth of three complete ML, and then gives way to 3D island growth at a coverage of 4 ML. The 3D island growth mode of the pure CuPc films is dramatically suppressed by the inclusion of C60 during deposition. X-ray diffraction (XRD) and STM studies reveal that the CuPc molecular packing is altered upon C 60 inclusion, producing disordered CuPc-C60 interfaces. The ordered molecular stacking of CuPc is found to be disrupted completely when C60 concentration reaches 30 wt.%. This disorder in the CuPc:C 60 composites is explained in terms of the relative strengths of the intermolecular interactions. Furthermore, an understanding of these relative interaction strengths is exploited to grow ordered composite films

  1. 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.

  2. 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.

  3. Digital Correction of Motion Artifacts in Microscopy Image Sequences Collected from Living Animals Using Rigid and Non-Rigid Registration

    PubMed Central

    Lorenz, Kevin S.; Salama, Paul; Dunn, Kenneth W.; Delp, Edward J.

    2013-01-01

    Digital image analysis is a fundamental component of quantitative microscopy. However, intravital microscopy presents many challenges for digital image analysis. In general, microscopy volumes are inherently anisotropic, suffer from decreasing contrast with tissue depth, lack object edge detail, and characteristically have low signal levels. Intravital microscopy introduces the additional problem of motion artifacts, resulting from respiratory motion and heartbeat from specimens imaged in vivo. This paper describes an image registration technique for use with sequences of intravital microscopy images collected in time-series or in 3D volumes. Our registration method involves both rigid and non-rigid components. The rigid registration component corrects global image translations, while the non-rigid component manipulates a uniform grid of control points defined by B-splines. Each control point is optimized by minimizing a cost function consisting of two parts: a term to define image similarity, and a term to ensure deformation grid smoothness. Experimental results indicate that this approach is promising based on the analysis of several image volumes collected from the kidney, lung, and salivary gland of living rodents. PMID:22092443

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. 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.

  9. 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

  10. Localization of protein-protein interactions among three fluorescent proteins in a single living cell: three-color FRET microscopy

    NASA Astrophysics Data System (ADS)

    Sun, Yuansheng; Booker, Cynthia F.; Day, Richard N.; Periasamy, Ammasi

    2009-02-01

    Förster resonance energy transfer (FRET) methodology has been used for over 30 years to localize protein-protein interactions in living specimens. The cloning and modification of various visible fluorescent proteins (FPs) has generated a variety of new probes that can be used as FRET pairs to investigate the protein associations in living cells. However, the spectral cross-talk between FRET donor and acceptor channels has been a major limitation to FRET microscopy. Many investigators have developed different ways to eliminate the bleedthrough signals in the FRET channel for one donor and one acceptor. We developed a novel FRET microscopy method for studying interactions among three chromophores: three-color FRET microscopy. We generated a genetic construct that directly links the three FPs - monomeric teal FP (mTFP), Venus and tandem dimer Tomato (tdTomato), and demonstrated the occurrence of mutually dependent energy transfers among the three FPs. When expressed in cells and excited with the 458 nm laser line, the mTFP-Venus-tdTomato fusion proteins yielded parallel (mTFP to Venus and mTFP to tdTomato) and sequential (mTFP to Venus and then to tdTomato) energy transfer signals. To quantify the FRET signals in the three-FP system in a single living cell, we developed an algorithm to remove all the spectral cross-talk components and also to separate different FRET signals at a same emission channel using the laser scanning spectral imaging and linear unmixing techniques on the Zeiss510 META system. Our results were confirmed with fluorescence lifetime measurements and using acceptor photobleaching FRET microscopy.

  11. Transmission electron microscopy analysis of ``black belt:'' The masking film of white ribbon of Kooi effect in the local oxidation of silicon process

    NASA Astrophysics Data System (ADS)

    Sheng, Tan-Tsu; Lu, Chih-Yuan; Chang, Ruey-Dar; Chiang, Song-Tsan

    1994-04-01

    The first direct observation of the top view of micromasking film on the Si surface to gate oxidation in the local oxidation of silicon process with transmission electron microscopy (TEM) has been made. The micromasking film looks like an ``eyelash'' in cross-sectional transmission electron microscopy and like a ``black belt'' in top view TEM. In addition, direct and sequential TEM observations on the removal of the micromasking film by sacrificial oxidation were presented.

  12. 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.

  13. Transient absorption microscopy of gold nanorods as spectrally orthogonal labels in live cells†

    PubMed Central

    Chen, Tao; Chen, Shouhui; Zhou, Jihan; Liang, Dehai

    2015-01-01

    Gold nanorods (AuNRs) have shown great potential as bio-compatible imaging probes in various biological applications. Probing nanomaterials in live cells is essential to reveal the interaction between them. In this study, we used a transient absorption microscope to selectively image AuNRs in live cells. The transient absorption signals were monitored through lock-in amplification. This provides a new way of observing AuNRs with no interference from background autofluorescence. PMID:25098209

  14. Super-Resolution Microscopy and Single-Protein Tracking in Live Bacteria Using a Genetically Encoded, Photostable Fluoromodule.

    PubMed

    Saurabh, Saumya; Perez, Adam M; Comerci, Colin J; Shapiro, Lucy; Moerner, W E

    2017-06-19

    Visualization of dynamic protein structures in live cells is crucial for understanding the mechanisms governing biological processes. Fluorescence microscopy is a sensitive tool for this purpose. In order to image proteins in live bacteria using fluorescence microscopy, one typically genetically fuses the protein of interest to a photostable fluorescent tag. Several labeling schemes are available to accomplish this. Particularly, hybrid tags that combine a fluorescent or fluorogenic dye with a genetically encoded protein (such as enzymatic labels) have been used successfully in multiple cell types. However, their use in bacteria has been limited due to challenges imposed by a complex bacterial cell wall. Here, we describe the use of a genetically encoded photostable fluoromodule that can be targeted to cytosolic and membrane proteins in the Gram negative bacterium Caulobacter crescentus. Additionally, we summarize methods to use this fluoromodule for single protein imaging and super-resolution microscopy using stimulated emission depletion. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  15. 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

  16. 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.

  17. 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

  18. Higher-order electromechanical response of thin films by contact resonance piezoresponse force microscopy.

    PubMed

    Harnagea, Catalin; Pignolet, Alain; Alexe, Marin; Hesse, Dietrich

    2006-12-01

    Piezoresponse scanning force microscopy (PFM) has turned into an established technique for imaging ferroelectric domains in ferroelectric thin films. At least for soft cantilevers, the piezoresponse signal is not only dependent on the elastic properties of the material under investigation but also on the elastic properties of the cantilever. Due to this dependency, the cantilever response and, therefore, the measured properties depend on the frequency of the small alternating current (AC) testing voltage. At the contact resonance, the cantilever response is maximum, and this increased sensitivity can be used to detect very small signals or to decrease the voltage applied to the sample. We have shown that by using the hysteretic ferroelectric switching, it is possible to separate the signal into its components (viz. electromechanical and electrostatic contributions). Additionally, the measurement frequency can be tuned such that the second and third harmonics of the electromechanical response can be detected at the cantilever resonance, providing information about the higher-order electromechanical coefficients. We assume that this nonlinear behavior seen in local and macroscopic measurements is rooted in the nonlinearity of the dielectric permittivity. Our results are of crucial importance for the study of ferroelectric and electromechanical properties of nanostructures.

  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. 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.

  1. 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.

  2. Intracellular accumulation and dissolution of silver nanoparticles in L-929 fibroblast cells using live cell time-lapse microscopy.

    PubMed

    Wildt, Bridget E; Celedon, Alfredo; Maurer, Elizabeth I; Casey, Brendan J; Nagy, Amber M; Hussain, Saber M; Goering, Peter L

    2016-08-01

    Cytotoxicity assessments of nanomaterials, such as silver nanoparticles, are challenging due to interferences with test reagents and indicators as well uncertainties in dosing as a result of the complex nature of nanoparticle intracellular accumulation. Furthermore, current theories suggest that silver nanoparticle cytotoxicity is a result of silver nanoparticle dissolution and subsequent ion release. This study introduces a novel technique, nanoparticle associated cytotoxicity microscopy analysis (NACMA), which combines fluorescence microscopy detection using ethidium homodimer-1, a cell permeability marker that binds to DNA after a cell membrane is compromised (a classical dead-cell indicator dye), with live cell time-lapse microscopy and image analysis to simultaneously investigate silver nanoparticle accumulation and cytotoxicity in L-929 fibroblast cells. Results of this method are consistent with traditional methods of assessing cytotoxicity and nanoparticle accumulation. Studies conducted on 10, 50, 100 and 200 nm silver nanoparticles reveal size dependent cytotoxicity with particularly high cytotoxicity from 10 nm particles. In addition, NACMA results, when combined with transmission electron microscopy imaging, reveal direct evidence of intracellular silver ion dissolution and possible nanoparticle reformation within cells for all silver nanoparticle sizes.

  3. 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.

  4. 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.

  5. 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

  6. The experience of African American women living with HIV: creating a prevention film for teens.

    PubMed

    Norris, Anne E; DeMarco, Rosanna

    2005-01-01

    The personal and social costs of HIV are well documented. What remains unknown is the effect of public disclosure of HIV status on the individual who is doing the disclosing. This study describes the experience of four African American women living with HIV who participated in the development of an intergenerational education intervention for African American adolescent girls. These women suggested that they be filmed discussing the "dark side" of HIV in an effort to create an intergenerational education intervention that would alter the risk-taking behavior that they observed in young women in their community. After a rough cut of the film was completed, these women viewed the film and participated in a focus group during which they discussed what it was like to reveal and revisit their own painful experiences associated with becoming infected and then living with HIV. Findings from content analysis of transcribed dialogue included the following positive themes: (a) self-acceptance by telling one's own story and hearing the stories of the other women, (b) a sense of liberation by disclosing publicly one's image and message and letting go of others' judgments, (c) feeling supported by meeting other women who share the same experience, (d) value of using the film to impact or save young people from the pain one has experienced. A negative theme emerged related to personal pain in reliving the individual's history with HIV.

  7. 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.

  8. 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

  9. Dose limited fluorescence microscopy of 5-aminolevulinic acid induced protoporphyrin IX in living cells

    NASA Astrophysics Data System (ADS)

    Schneckenburger, H.; Weber, P.; Wagner, M.; Schickinger, S.; Bruns, T.; Strauss, W. S. L.

    2009-02-01

    Intracellular accumulation and location of photosensitizers, e.g. 5-ALA induced protoporphyrin IX, are crucial parameters for the efficiency of photodynamic therapy (PDT). Fluorescence microscopy has proved to be a powerful technique to assess these parameters, however, even at low light doses around or below 1 J/cm² cells may be irreversibly damaged. Therefore, prior to microscopic experiments non-phototoxic light doses were determined, and experimental conditions of laser scanning (LSM) and wide field microscopy were adapted to these doses. Wide field images appeared more brilliant than LSM images, thus demonstrating some advantage of simultaneous over sequential detection. In addition, human glioblastoma cells appeared less sensitive towards illumination by an evanescent electromagnetic field than towards epi-illumination, since only their plasma membranes and adjacent parts were exposed to light.

  10. Nonstoichiometry accommodation in SrTiO3 thin films studied by positron annihilation and electron microscopy

    NASA Astrophysics Data System (ADS)

    Keeble, D. J.; Wicklein, S.; Jin, L.; Jia, C. L.; Egger, W.; Dittmann, R.

    2013-05-01

    Accommodation of nonstoichiometry in SrTiO3 pulsed laser deposited (PLD) films was investigated using positron annihilation lifetime spectroscopy and (scanning) transmission electron microscopy. Increasing PLD laser fluence changed the stoichiometry from Ti to Sr deficient. Cation vacancy defects were detected, and the concentration ratio of Sr to Ti vacancies, [VSr]/[VTi], was observed to increase systematically in the Sr-deficient region, although no change in the electron microscopy lattice images was detected. Increasing Ti deficiency resulted in the accommodation of SrO layers in planar defects, and in the formation of vacancy cluster defects. A change from VTi to VSr defect positron trapping was also detected.

  11. 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

  12. Filmed v. live social contact interventions to reduce stigma: randomised controlled trial.

    PubMed

    Clement, Sarah; van Nieuwenhuizen, Adrienne; Kassam, Aliya; Flach, Clare; Lazarus, Anisha; de Castro, Melanie; McCrone, Paul; Norman, Ian; Thornicroft, Graham

    2012-07-01

    Direct social contact interventions are known to reduce mental health stigma. Filmed social contact may be equally effective and have practical and cost advantages. To compare the effectiveness of a DVD, a live intervention and a lecture control, in reducing stigma, testing the hypotheses that: (a) DVD and live interventions will be equally effective; and (b) the interventions with social contact (DVD/live) will be more effective than the lecture. Cost-effectiveness, process and acceptability are also assessed. Student nurses were randomised to: (a) watch a DVD of service users/informal carers talking about their experiences, (b) watch a similar live presentation, or (c) attend a lecture. Primary outcomes were changes in attitudes (using the Mental Illness: Clinicians Attitudes Scale, MICA), emotional reactions (using the Emotional Reactions to Mental Illness Scale, ERMIS), intended proximity (using the Reported and Intended Behaviour Scale, RIBS), and knowledge (using the Social Contact Intended Learning Outcomes, SCILO), immediately after the intervention and at 4-month follow-up. For the 216 participants, there were no differences between the DVD and live groups on MICA, ERMIS or RIBS scores. The DVD group had higher SCILO (knowledge) scores. The combined social contact group (DVD/live) had better MICA and RIBS scores than the lecture group, the latter difference maintained at 4 months. The DVD was the most cost-effective of the interventions, and the live session the most popular. Our hypotheses were confirmed. This study supports the wider use of filmed social contact interventions to reduce stigma about mental illness.

  13. 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.

  14. Plasma-deposited fluorocarbon films: insulation material for microelectrodes and combined atomic force microscopy-scanning electrochemical microscopy probes.

    PubMed

    Wiedemair, Justyna; Balu, Balamurali; Moon, Jong-Seok; Hess, Dennis W; Mizaikoff, Boris; Kranz, Christine

    2008-07-01

    Pinhole-free insulation of micro- and nanoelectrodes is the key to successful microelectrochemical experiments performed in vivo or in combination with scanning probe experiments. A novel insulation technique based on fluorocarbon insulation layers deposited from pentafluoroethane (PFE, CF3CHF2) plasmas is presented as a promising electrical insulation approach for microelectrodes and combined atomic force microscopy-scanning electrochemical microscopy (AFM-SECM) probes. The deposition allows reproducible and uniform coating, which is essential for many analytical applications of micro- and nanoelectrodes such as, e.g., in vivo experiments and SECM experiments. Disk-shaped microelectrodes and frame-shaped AFM tip-integrated electrodes have been fabricated by postinsulation focused ion beam (FIB) milling. The thin insulation layer for combined AFM-SECM probes renders this fabrication technique particularly useful for submicro insulation providing radius ratios of the outer insulation versus the disk electrode (RG values) suitable for SECM experiments. Characterization of PFE-insulated AFM-SECM probes will be presented along with combined AFM-SECM approach curves and imaging.

  15. In situ observation of the impact of surface oxidation on the crystallization mechanism of GeTe phase-change thin films by scanning transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Berthier, R.; Bernier, N.; Cooper, D.; Sabbione, C.; Hippert, F.; Noé, P.

    2017-09-01

    The crystallization mechanisms of prototypical GeTe phase-change material thin films have been investigated by in situ scanning transmission electron microscopy annealing experiments. A novel sample preparation method has been developed to improve sample quality and stability during in situ annealing, enabling quantitative analysis and live recording of phase change events. Results show that for an uncapped 100 nm thick GeTe layer, exposure to air after fabrication leads to composition changes which promote heterogeneous nucleation at the oxidized surface. We also demonstrate that protecting the GeTe layer with a 10 nm SiN capping layer prevents nucleation at the surface and allows volume nucleation at a temperature 50 °C higher than the onset of crystallization in the oxidized sample. Our results have important implications regarding the integration of these materials in confined memory cells.

  16. Fluorescence lifetime imaging microscopy for the monitoring of green fluorescent protein-tagged androgen receptors in living cells.

    PubMed

    Miyake, Rina; Uchimura, Tomohiro; Li, Xu; Imasaka, Totaro

    2013-01-01

    Fluorescence lifetime imaging microscopy (FLIM) was used to monitor the interaction between androgen receptor (AR) tagging of a green fluorescent protein (GFP) and the ligands in living cells. The fluorescence lifetime of the AR-GFP without ligands was ca. 3.1 ns, which was reduced to ca. 2.5 ns after treatment with agonist 5α-dihydrotestosterone. On the other hand, the fluorescence lifetime of AR-GFP was not changed after treatment with antagonist hydroxyflutamide. The reaction kinetics was simulated in the present study, and the obtained results indicated the possibility of the presence of an intermediate complex during the reaction. FLIM can be used to record the ratio of the AR as it reacts with an agonist, and, therefore, it is useful for acquiring information concerning the interaction between AR and ligands in living cells.

  17. 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.

  18. Localized electroporation and molecular delivery into single living cells by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Nawarathna, D.; Unal, K.; Wickramasinghe, H. Kumar

    2008-10-01

    We present an efficient and fast method for selective and localized electroporation of a single living cell from a population of millions to tens of cells using the modified tip of an atomic force microscope. Electroporation was observed in real time using an inverted microscope. This technique is proposed as a tool for efficient and controlled delivery of biomolecules, proteins, drugs, and genes.

  19. Transient absorption microscopy of gold nanorods as spectrally orthogonal labels in live cells

    NASA Astrophysics Data System (ADS)

    Chen, Tao; Chen, Shouhui; Zhou, Jihan; Liang, Dehai; Chen, Xiaoyuan; Huang, Yanyi

    2014-08-01

    Gold nanorods (AuNRs) have shown great potential as bio-compatible imaging probes in various biological applications. Probing nanomaterials in live cells is essential to reveal the interaction between them. In this study, we used a transient absorption microscope to selectively image AuNRs in live cells. The transient absorption signals were monitored through lock-in amplification. This provides a new way of observing AuNRs with no interference from background autofluorescence.Gold nanorods (AuNRs) have shown great potential as bio-compatible imaging probes in various biological applications. Probing nanomaterials in live cells is essential to reveal the interaction between them. In this study, we used a transient absorption microscope to selectively image AuNRs in live cells. The transient absorption signals were monitored through lock-in amplification. This provides a new way of observing AuNRs with no interference from background autofluorescence. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr03413a

  20. 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

  1. 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.

  2. 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.

  3. Atomic force microscopy studies of functional and dysfunctional pulmonary surfactant films, II: albumin-inhibited pulmonary surfactant films and the effect of SP-A.

    PubMed

    Zuo, Yi Y; Tadayyon, Seyed M; Keating, Eleonora; Zhao, Lin; Veldhuizen, Ruud A W; Petersen, Nils O; Amrein, Matthias W; Possmayer, Fred

    2008-09-15

    Pulmonary surfactant (PS) dysfunction because of the leakage of serum proteins into the alveolar space could be an operative pathogenesis in acute respiratory distress syndrome. Albumin-inhibited PS is a commonly used in vitro model for studying surfactant abnormality in acute respiratory distress syndrome. However, the mechanism by which PS is inhibited by albumin remains controversial. This study investigated the film organization of albumin-inhibited bovine lipid extract surfactant (BLES) with and without surfactant protein A (SP-A), using atomic force microscopy. The BLES and albumin (1:4 w/w) were cospread at an air-water interface from aqueous media. Cospreading minimized the adsorption barrier for phospholipid vesicles imposed by preadsorbed albumin molecules, i.e., inhibition because of competitive adsorption. Atomic force microscopy revealed distinct variations in film organization, persisting up to 40 mN/m, compared with pure BLES monolayers. Fluorescence confocal microscopy confirmed that albumin remained within the liquid-expanded phase of the monolayer at surface pressures higher than the equilibrium surface pressure of albumin. The remaining albumin mixed with the BLES monolayer so as to increase film compressibility. Such an inhibitory effect could not be relieved by repeated compression-expansion cycles or by adding surfactant protein A. These experimental data indicate a new mechanism of surfactant inhibition by serum proteins, complementing the traditional competitive adsorption mechanism.

  4. 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.

  5. 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.

  6. Multi-photon excitation fluorescence and third-harmonic generation microscopy measurements combined with confocal Raman microscopy for the analysis of layered samples of varnished oil films

    NASA Astrophysics Data System (ADS)

    Nevin, A.; Comelli, D.; Osticioli, I.; Filippidis, G.; Melessanaki, K.; Valentini, G.; Cubeddu, R.; Fotakis, C.

    2010-09-01

    The non-destructive determination of layer structures in works of art remains a significant challenge. Non-linear microscopy and confocal Raman microscopy (CRM) were employed for characterisation of varnish-media layers in model samples, providing important information regarding the thickness of materials and the identification of different media in depth. Commonly found triterpenoid varnishes mastic and dammar were applied over a single layer of films of linseed oil. Non-linear microscopy of samples was carried out using a 1028-nm femtosecond laser source; both third-harmonic generation signals (THG) and three-photon fluorescence signals (3PEF) of samples were collected in an effort to measure the thickness of mono- and bi-layers; in parallel scans of larger areas were undertaken to assess heterogeneities in samples with spatial resolution of ˜2 μm. Complementary spectroscopic information from CRM collected with both a 514.5-nm argon-ion and a 785-nm diode lasers coupled with a 100X objective and a motorised stage was carried out. Comparison of C-H stretching regions of Raman spectra allowed the differentiation between different molecular materials and the fingerprint region was employed for the depth profiling of the samples.

  7. High-resolution electron microscopy study of Ni 81Fe 19 film with Co 33Cr 67 buffer layer

    NASA Astrophysics Data System (ADS)

    Xu, Q. Y.; Wang, Z. M.; Shen, F.; Du, Y. W.; Zhang, Z.

    2003-04-01

    The anisotropic magnetoresistance (AMR) in permalloy Ni 81Fe 19 film deposited on a 1.2 nm Co 33Cr 67 buffer layer was significantly enhanced. The high-resolution electron microscopy was used to study the microstructure of Ni 81Fe 19 film with and without Co 33Cr 67 buffer layer. It was found that Co 33Cr 67 buffer layer can induce good (1 1 1) texture, while without Co 33Cr 67 buffer layer, Ni 81Fe 19 film show randomly oriented grain structure. The Δ ρ/ ρ enhancement is attributed to the decrease in the resistivity ρ of the Ni 81Fe 19 film due to the formation of the large (1 1 1) textured grains in Ni 81Fe 19 film with Co 33Cr 67 buffer layer. However, the surface roughness of substrate may limit the (1 1 1) textured grain size and induce additional grain boundaries in Ni 81Fe 19 film with Co 33Cr 67 buffer layer, limit the enhancement of the AMR effect.

  8. 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.

  9. 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.

  10. 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.

  11. 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

  12. Detecting cells in time varying intensity images in confocal microscopy for gene expression studies in living cells

    NASA Astrophysics Data System (ADS)

    Mitra, Debasis; Boutchko, Rostyslav; Ray, Judhajeet; Nilsen-Hamilton, Marit

    2015-03-01

    In this work we present a time-lapsed confocal microscopy image analysis technique for an automated gene expression study of multiple single living cells. Fluorescence Resonance Energy Transfer (FRET) is a technology by which molecule-to-molecule interactions are visualized. We analyzed a dynamic series of ~102 images obtained using confocal microscopy of fluorescence in yeast cells containing RNA reporters that give a FRET signal when the gene promoter is activated. For each time frame, separate images are available for three spectral channels and the integrated intensity snapshot of the system. A large number of time-lapsed frames must be analyzed to identify each cell individually across time and space, as it is moving in and out of the focal plane of the microscope. This makes it a difficult image processing problem. We have proposed an algorithm here, based on scale-space technique, which solves the problem satisfactorily. The algorithm has multiple directions for even further improvement. The ability to rapidly measure changes in gene expression simultaneously in many cells in a population will open the opportunity for real-time studies of the heterogeneity of genetic response in a living cell population and the interactions between cells that occur in a mixed population, such as the ones found in the organs and tissues of multicellular organisms.

  13. Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy

    PubMed Central

    Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

    2013-01-01

    Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens. PMID:23267183

  14. Reversible optical control of cyanine fluorescence in fixed and living cells: optical lock-in detection immunofluorescence imaging microscopy.

    PubMed

    Yan, Yuling; Petchprayoon, Chutima; Mao, Shu; Marriott, Gerard

    2013-02-05

    Optical switch probes undergo rapid and reversible transitions between two distinct states, one of which may fluoresce. This class of probe is used in various super-resolution imaging techniques and in the high-contrast imaging technique of optical lock-in detection (OLID) microscopy. Here, we introduce optimized optical switches for studies in living cells under standard conditions of cell culture. In particular, a highly fluorescent cyanine probe (Cy or Cy3) is directly or indirectly linked to naphthoxazine (NISO), a highly efficient optical switch that undergoes robust, 405/532 nm-driven transitions between a colourless spiro (SP) state and a colourful merocyanine (MC) state. The intensity of Cy fluorescence in these Cy/Cy3-NISO probes is reversibly modulated between a low and high value in SP and MC states, respectively, as a result of Förster resonance energy transfer. Cy/Cy3-NISO probes are targeted to specific proteins in living cells where defined waveforms of Cy3 fluorescence are generated by optical switching of the SP and MC states. Finally, we introduce a new imaging technique (called OLID-immunofluorescence microscopy) that combines optical modulation of Cy3 fluorescence from Cy3/NISO co-labelled antibodies within fixed cells and OLID analysis to significantly improve image contrast in samples having high background or rare antigens.

  15. 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.

  16. Atomic oxygen effects on thin film space coatings studied by spectroscopic ellipsometry, atomic force microscopy, and laser light scattering

    NASA Technical Reports Server (NTRS)

    Synowicki, R. A.; Hale, Jeffrey S.; Woollam, John A.

    1992-01-01

    The University of Nebraska is currently evaluating Low Earth Orbit (LEO) simulation techniques as well as a variety of thin film protective coatings to withstand atomic oxygen (AO) degradation. Both oxygen plasma ashers and an electron cyclotron resonance (ECR) source are being used for LEO simulation. Thin film coatings are characterized by optical techniques including Variable Angle Spectroscopic Ellipsometry, Optical spectrophotometry, and laser light scatterometry. Atomic Force Microscopy (AFM) is also used to characterize surface morphology. Results on diamondlike carbon (DLC) films show that DLC degrades with simulated AO exposure at a rate comparable to Kapton polyimide. Since DLC is not as susceptible to environmental factors such as moisture absorption, it could potentially provide more accurate measurements of AO fluence on short space flights.

  17. 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.

  18. 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.

  19. 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.

  20. 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.

  1. 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.

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

    PubMed

    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 ? m 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.

  3. Coherent Brightfield Microscopy Provides the Spatiotemporal Resolution To Study Early Stage Viral Infection in Live Cells.

    PubMed

    Huang, Yi-Fan; Zhuo, Guan-Yu; Chou, Chun-Yu; Lin, Cheng-Hao; Chang, Wen; Hsieh, Chia-Lung

    2017-03-28

    Viral infection starts with a virus particle landing on a cell surface followed by penetration of the plasma membrane. Due to the difficulty of measuring the rapid motion of small-sized virus particles on the membrane, little is known about how a virus particle reaches an endocytic site after landing at a random location. Here, we use coherent brightfield (COBRI) microscopy to investigate early stage viral infection with ultrahigh spatiotemporal resolution. By detecting intrinsic scattered light via imaging-based interferometry, COBRI microscopy allows us to track the motion of a single vaccinia virus particle with nanometer spatial precision (<3 nm) in 3D and microsecond temporal resolution (up to 100,000 frames per second). We explore the possibility of differentiating the virus signal from cell background based on their distinct spatial and temporal behaviors via digital image processing. Through image postprocessing, relatively stationary background scattering of cellular structures is effectively removed, generating a background-free image of the diffusive virus particle for precise localization. Using our method, we unveil single virus particles exploring cell plasma membranes after attachment. We found that immediately after attaching to the membrane (within a second), the virus particle is locally confined within hundreds of nanometers where the virus particle diffuses laterally with a very high diffusion coefficient (∼1 μm(2)/s) at microsecond time scales. Ultrahigh-speed scattering-based optical imaging may provide opportunities for resolving rapid virus-receptor interactions with nanometer clarity.

  4. 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.

  5. Metastable structures in Al thin films before the onset of corrosion pitting as observed using liquid cell transmission electron microscopy.

    PubMed

    Chee, See Wee; Duquette, David J; Ross, Frances M; Hull, Robert

    2014-04-01

    One of the fundamental challenges in understanding the early stages of corrosion pitting in metals protected with an oxide film is that there are relatively few techniques that can probe microstructure with sufficient resolution while maintaining a wet environment. Here, we demonstrate that microstructural changes in Al thin films caused by aqueous NaCl solutions of varying chloride concentrations can be directly observed using a liquid flow cell enclosed within a transmission electron microscope (TEM) holder. In the absence of chloride, Al thin films did not exhibit significant corrosion when immersed in de-ionized water for 2 days. However, introducing 0.01 M NaCl solutions led to extensive random formation of blisters over the sample surface, while 0.1 M NaCl solutions formed anomalous structures that were larger than the typical grain size. Immersion in 1.0 M NaCl solutions led to fractal corrosion consistent with previously reported studies of Al thin films using optical microscopy. These results show the potential of in situ liquid cell electron microscopy for probing the processes that take place before the onset of pitting and for correlating pit locations with the underlying microstructure of the material.

  6. 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.

  7. 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

  8. 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.

  9. High-resolution live imaging of plant growth in near physiological bright conditions using light sheet fluorescence microscopy.

    PubMed

    Maizel, Alexis; von Wangenheim, Daniel; Federici, Fernán; Haseloff, Jim; Stelzer, Ernst H K

    2011-10-01

    Most plant growth occurs post-embryonically and is characterized by the constant and iterative formation of new organs. Non-invasive time-resolved imaging of intact, fully functional organisms allows studies of the dynamics involved in shaping complex organisms. Conventional and confocal fluorescence microscopy suffer from limitations when whole living organisms are imaged at single-cell resolution. We applied light sheet-based fluorescence microscopy to overcome these limitations and study the dynamics of plant growth. We designed a special imaging chamber in which the plant is maintained vertically under controlled illumination with its leaves in the air and its root in the medium. We show that minimally invasive, multi-color, three-dimensional imaging of live Arabidopsis thaliana samples can be achieved at organ, cellular and subcellular scales over periods of time ranging from seconds to days with minimal damage to the sample. We illustrate the capabilities of the method by recording the growth of primary root tips and lateral root primordia over several hours. This allowed us to quantify the contribution of cell elongation to the early morphogenesis of lateral root primordia and uncover the diurnal growth rhythm of lateral roots. We demonstrate the applicability of our approach at varying spatial and temporal scales by following the division of plant cells as well as the movement of single endosomes in live growing root samples. This multi-dimensional approach will have an important impact on plant developmental and cell biology and paves the way to a truly quantitative description of growth processes at several scales. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

  10. 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.

  11. 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.

  12. 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.

  13. 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

  14. [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.

  15. 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.

  16. 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

  17. 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.

  18. 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.

  19. 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

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

    PubMed Central

    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

    2008-01-01

    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. PMID:19004775

  1. 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

  2. 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.

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

    PubMed

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

    2016-11-22

    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.

  4. 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

  5. Microfluidic approaches to synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy of living biosystems

    DOE PAGES

    Loutherback, Kevin; Birarda, Giovanni; Chen, Liang; ...

    2016-02-15

    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 watermore » 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.« less

  6. Microfluidic approaches to synchrotron radiation-based Fourier transform infrared (SR-FTIR) spectral microscopy of living biosystems

    SciTech Connect

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

    2016-02-15

    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.

  7. Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated.

    PubMed

    Hill, D B; Macosko, J C; Holzwarth, G M

    2008-09-01

    Video-enhanced differential interference contrast microscopy with background subtraction has made visible many structures and processes in living cells. In video-enhanced differential interference contrast, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved video-enhanced differential interference contrast by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced differential interference contrast. By carrying out background subtraction with 12-bit images, the number of grey levels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background and motion-enhanced differential interference contrast images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron.

  8. Motion-enhanced, differential interference contrast (MEDIC) microscopy of moving vesicles in live cells: VE-DIC updated

    PubMed Central

    Hill, David B.; Macosko, Jed C.; Holzwarth, George M.

    2009-01-01

    Summary Video-enhanced DIC microscopy (VE-DIC) with background subtraction has made visible many structures and processes in living cells. In VE-DIC, the background image is stored manually by defocusing the microscope before images are acquired. We have updated and improved VE-DIC by adding automatic generation of the background image as a rolling average of the incoming image stream. Subtraction of this continuously updated 12-bit background image from the incoming 12-bit image stream provides a flat background which allows the contrast of moving objects, such as vesicles, to be strongly enhanced while suppressing stationary features such as the overall cell shape. We call our method MEDIC, for motion-enhanced DIC. By carrying out background subtraction with 12-bit images, the number of graylevels in the moving vesicles can be maximized and a single look-up table can be applied to the entire image, enhancing the contrast of all vesicles simultaneously. Contrast is increased by as much as a factor of 13. The method is illustrated with raw, background, and MEDIC images of moving vesicles within a neurite of a live PC12 cell and a live chick motorneuron. PMID:18754997

  9. 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.

  10. 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.

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

    NASA Astrophysics Data System (ADS)

    Min, Junwei; Yao, Baoli; Ketelhut, Steffi; Kemper, Björn

    2017-02-01

    The modular combination of optical microscopes with digital holographic microscopy (DHM) has been proven to be a powerful tool for quantitative live cell imaging. The introduction of condenser and different microscope objectives (MO) simplifies the usage of the technique and makes it easier to measure different kinds of specimens with different magnifications. However, the high flexibility of illumination and imaging also causes variable phase aberrations that need to be eliminated for high resolution quantitative phase imaging. The existent phase aberrations compensation methods either require add additional elements into the reference arm or need specimen free reference areas or separate reference holograms to build up suitable digital phase masks. These inherent requirements make them unpractical for usage with highly variable illumination and imaging systems and prevent on-line monitoring of living cells. In this paper, we present a simple numerical method for phase aberration compensation based on the analysis of holograms in spatial frequency domain with capabilities for on-line quantitative phase imaging. From a single shot off-axis hologram, the whole phase aberration can be eliminated automatically without numerical fitting or pre-knowledge of the setup. The capabilities and robustness for quantitative phase imaging of living cancer cells are demonstrated.

  12. 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

  13. 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

  14. 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.

  15. 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.

  16. 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.

  17. 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.

  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. Detection of a live cell in a microfluidic system by scanning capacitance microscopy

    NASA Astrophysics Data System (ADS)

    Sung, S. Y.; Yi, I. J.; Choi, Y. J.; Kim, J. Y.; Kim, Y. S.; Kang, C. J.

    2007-03-01

    In recent years, many studies on the biosensors using a microfluidic system have been performed. The system fabricated with polydimethylsiloxane (PDMS) has many advantages such that it is portable, disposable, cost effective, and automatable. Scanning capacitance microscope (SCM) that has a good capacitance pickup sensor attached to an atomic force microscope (AFM) is capable of measuring the capacitance variation with a resolution of better than 10-18F/V between a conducting tip and the sample. In this work, we present possibility of SCM as a biosensor by measuring a live cell which flows in the microchannel. By measuring the consecutive capacitance line profiles of a cell, which represent the charge distribution of a cell surface resulting from the ion channel or cell activity, we can get more information on the cell analysis and provide one solution for the realization of a lab-on-a-chip.

  20. Investigation of Membrane Receptors' Oligomers Using Fluorescence Resonance Energy Transfer and Multiphoton Microscopy in Living Cells

    NASA Astrophysics Data System (ADS)

    Mishra, Ashish K.

    Investigating quaternary structure (oligomerization) of macromolecules (such as proteins and nucleic acids) in living systems (in vivo) has been a great challenge in biophysics, due to molecular diffusion, fluctuations in several biochemical parameters such as pH, quenching of fluorescence by oxygen (when fluorescence methods are used), etc. We studied oligomerization of membrane receptors in living cells by means of Fluorescence (Forster) Resonance Energy Transfer (FRET) using fluorescent markers and two photon excitation fluorescence micro-spectroscopy. Using suitable FRET models, we determined the stoichiometry and quaternary structure of various macromolecular complexes. The proteins of interest for this work are : (1) sigma-1 receptor and (2) rhodopsin, are described as below. (1) Sigma-1 receptors are molecular chaperone proteins, which also regulate ion channels. S1R seems to be involved in substance abuse, as well as several diseases such as Alzheimer's. We studied S1R in the presence and absence of its ligands haloperidol (an antagonist) and pentazocine +/- (an agonist), and found that at low concentration they reside as a mixture of monomers and dimers and that they may form higher order oligomers at higher concentrations. (2) Rhodopsin is a prototypical G protein coupled receptor (GPCR) and is directly involved in vision. GPCRs form a large family of receptors that participate in cell signaling by responding to external stimuli such as drugs, thus being a major drug target (more than 40% drugs target GPCRs). Their oligomerization has been largely controversial. Understanding this may help to understand the functional role of GPCRs oligomerization, and may lead to the discovery of more drugs targeting GPCR oligomers. It may also contribute toward finding a cure for Retinitis Pigmentosa, which is caused by a mutation (G188R) in rhodopsin, a disease which causes blindness and has no cure so far. Comparing healthy rhodopsin's oligomeric structure with that

  1. 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…

  2. 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.

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

    PubMed

    Huebinger, Jan; Han, Hong-Mei; Grabenbauer, Markus

    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.

  4. Dissection of mechanical force in living cells by super-resolved traction force microscopy.

    PubMed

    Colin-York, Huw; Eggeling, Christian; Fritzsche, Marco

    2017-04-01

    Cells continuously exert or respond to mechanical force. Measurement of these nanoscale forces is a major challenge in cell biology; yet such measurement is essential to the understanding of cell regulation and function. Current methods for examining mechanical force generation either necessitate dedicated equipment or limit themselves to coarse-grained force measurements on the micron scale. In this protocol, we describe stimulated emission depletion traction force microscopy-STED-TFM (STFM), which allows higher sampling of the forces generated by the cell than conventional TFM, leading to a twofold increase in spatial resolution (of up to 500 nm). The procedure involves the preparation of functionalized polyacrylamide gels loaded with fluorescent beads, as well as the acquisition of STED images and their analysis. We illustrate the approach using the example of HeLa cells expressing paxillin-EGFP to visualize focal adhesions. Our protocol uses widely available laser-scanning confocal microscopes equipped with a conventional STED laser, open-source software and common molecular biology techniques. The entire STFM experiment preparation, data acquisition and analysis require 2-3 d and could be completed by someone with minimal experience in molecular biology or biophysics.

  5. 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.

  6. 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

  7. Magnetic-field-modulated written bits in TbFeCo thin films: Transmission electron microscopy Lorentz and scanning electron microscopy with polarization analysis studies

    NASA Astrophysics Data System (ADS)

    Aeschlimann, M.; Scheinfein, M.; Unguris, J.; Greidanus, F. J. A. M.; Klahn, S.

    1990-11-01

    Domains written thermomagnetically in TbFeCo thin films are studied with Lorentz transmission electron microscopy (TEM) and scanning electron microscopy with polarization analysis (SEMPA). Four different rare-earth/transition-metal compositions TbxFeyCo1-x-y are examined. The domain structures observed with both techniques are similar even though TEM Lorentz only detects the transverse component of the net magnetic field along the electron's trajectory through the sample, while SEMPA detects the surface electron-spin polarization (magnetization). We find that the magnetic contrast in the SEMPA measurements is proportional to the magnetization of the transition-metal (TM) subnetwork which is antiferromagnetically coupled to the rare-earth (RE) subnetwork. This allows high-contrast SEMPA images to be acquired even when the system is magnetically compensated (Ms=‖MRE-MTM‖=0). The surface magnetization can be explained by assuming that the surface of the TbFeCo alloy consists of an outermost thin oxide layer followed by an Fe-rich subsurface layer. The importance of the demagnetizing field on the switching and domain nucleation process for thermomagnetically written bits is examined.

  8. Correlative live-cell and superresolution microscopy reveals cargo transport dynamics at microtubule intersections

    PubMed Central

    Bálint, Štefan; Verdeny Vilanova, Ione; Sandoval Álvarez, Ángel; Lakadamyali, Melike

    2013-01-01

    Intracellular transport plays an essential role in maintaining the organization of polarized cells. Motor proteins tether and move cargos along microtubules during long-range transport to deliver them to their proper location of function. To reach their destination, cargo-bound motors must overcome barriers to their forward motion such as intersection points between microtubules. The ability to visualize how motors navigate these barriers can give important information about the mechanisms that lead to efficient transport. Here, we first develop an all-optical correlative imaging method based on single-particle tracking and superresolution microscopy to map the transport trajectories of cargos to individual microtubules with high spatiotemporal resolution. We then use this method to study the behavior of lysosomes at microtubule–microtubule intersections. Our results show that the intersection poses a significant hindrance that leads to long pauses in transport only when the separation distance of the intersecting microtubules is smaller than ∼100 nm. However, the obstructions are typically overcome by the motors with high fidelity by either switching to the intersecting microtubule or eventually passing through the intersection. Interestingly, there is a large tendency to maintain the polarity of motion (anterograde or retrograde) after the intersection, suggesting a high degree of regulation of motor activity to maintain transport in a given direction. These results give insights into the effect of the cytoskeletal geometry on cargo transport and have important implications for the mechanisms that cargo-bound motors use to maneuver through the obstructions set up by the complex cytoskeletal network. PMID:23401534

  9. 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.

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. Extended two-photon microscopy in live samples with Bessel beams: steadier focus, faster volume scans, and simpler stereoscopic imaging

    PubMed Central

    Thériault, Gabrielle; Cottet, Martin; Castonguay, Annie; McCarthy, Nathalie; De Koninck, Yves

    2014-01-01

    Two-photon microscopy has revolutionized functional cellular imaging in tissue, but although the highly confined depth of field (DOF) of standard set-ups yields great optical sectioning, it also limits imaging speed in volume samples and ease of use. For this reason, we recently presented a simple and retrofittable modification to the two-photon laser-scanning microscope which extends the DOF through the use of an axicon (conical lens). Here we demonstrate three significant benefits of this technique using biological samples commonly employed in the field of neuroscience. First, we use a sample of neurons grown in culture and move it along the z-axis, showing that a more stable focus is achieved without compromise on transverse resolution. Second, we monitor 3D population dynamics in an acute slice of live mouse cortex, demonstrating that faster volumetric scans can be conducted. Third, we acquire a stereoscopic image of neurons and their dendrites in a fixed sample of mouse cortex, using only two scans instead of the complete stack and calculations required by standard systems. Taken together, these advantages, combined with the ease of integration into pre-existing systems, make the extended depth-of-field imaging based on Bessel beams a strong asset for the field of microscopy and life sciences in general. PMID:24904284

  15. Contact X-ray microscopy of living cells by using LiF crystal as imaging detector.

    PubMed

    Reale, L; Bonfigli, F; Lai, A; Flora, F; Albertano, P; DI Giorgio, M L; Mezi, L; Montereali, R M; Faenov, A; Pikuz, T; Almaviva, S; Francucci, M; Gaudio, P; Martellucci, S; Richetta, M; Poma, A

    2015-05-01

    In this paper, the use of lithium fluoride (LiF) as imaging radiation detector to analyse living cells by single-shot soft X-ray contact microscopy is presented. High resolved X-ray images on LiF of cyanobacterium Leptolyngbya VRUC135, two unicellular microalgae of the genus Chlamydomonas and mouse macrophage cells (line RAW 264.7) have been obtained utilizing X-ray radiation in the water window energy range from a laser plasma source. The used method is based on loading of the samples, the cell suspension, in a special holder where they are in close contact with a LiF crystal solid-state X-ray imaging detector. After exposure and sample removal, the images stored in LiF by the soft X-ray contact microscopy technique are read by an optical microscope in fluorescence mode. The clear image of the mucilaginous sheath the structure of the filamentous Leptolyngbya and the visible nucleolus in the macrophage cells image, are noteworthiness results. The peculiarities of the used X-ray radiation and of the LiF imaging detector allow obtaining images in absorption contrast revealing the internal structures of the investigated samples at high spatial resolution. Moreover, the wide dynamic range of the LiF imaging detector contributes to obtain high-quality images. In particular, we demonstrate that this peculiar characteristic of LiF detector allows enhancing the contrast and reveal details even when they were obscured by a nonuniform stray light. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  16. 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.

  17. Evaluation of fluorophores to label SNAP-tag fused proteins for multicolor single-molecule tracking microscopy in live cells.

    PubMed

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

    2014-08-19

    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. Copyright © 2014 Biophysical Society. Published by Elsevier

  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. Scanning electrochemical microscopy of Belousov-Zhabotinsky reaction: how confined oscillations reveal short lived radicals and auto-catalytic species.

    PubMed

    Stockmann, T Jane; Noël, Jean-Marc; Ristori, Sandra; Combellas, Catherine; Abou-Hassan, Ali; Rossi, Federico; Kanoufi, Frédéric

    2015-10-06

    Oscillating chemical reactions, encapsulated within artificial vesicles have been demonstrated as a powerful analogy of living cells for the investigation of chemical communication and morphogenesis. However, little is understood with regards to the influence of confinement on the reactivity of such systems. Herein, the effect of confinement on the Belousov-Zhabotinsky (BZ) oscillating reaction in bulk solution, (employing ferroin as a catalyst and malonic acid as the organic substrate) is investigated using scanning electrochemical microscopy (SECM) toward different insulating surfaces such as glass, silanized glass, or PTFE. An unexpected increase in the amplitude of the BZ reaction at a tip-substrate distance of ∼12-15 μm is observed. By simulating different reaction mechanisms, from simple EC' catalysis to more sophisticated Oregonator or even an 11-reaction scheme, it is shown that such behavior reveals the intervention of redox catalysis processes and particularly the short-lived highly reactive radical intermediate BrO2(•) indirectly detected at micromolar concentrations. The reinspection of the EC' mechanism shows that the homogeneous catalysis route is confirmed and kinetically characterized from SECM toward an insulating substrate, with promising potentiality in many systems. More specifically to the complex chemical case of BZ reactions, the mechanism is understood from the envelope curves of the oscillations, which are assessed in the absence of the oscillation (absence of organic substrate).

  20. 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

  1. 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

  2. Closed-loop ARS mode for scanning ion conductance microscopy with improved speed and stability for live cell imaging applications

    NASA Astrophysics Data System (ADS)

    Jung, Goo-Eun; Noh, Hanaul; Shin, Yong Kyun; Kahng, Se-Jong; Baik, Ku Youn; Kim, Hong-Bae; Cho, Nam-Joon; Cho, Sang-Joon

    2015-06-01

    Scanning ion conductance microscopy (SICM) is an increasingly useful nanotechnology tool for non-contact, high resolution imaging of live biological specimens such as cellular membranes. In particular, approach-retract-scanning (ARS) mode enables fast probing of delicate biological structures by rapid and repeated approach/retraction of a nano-pipette tip. For optimal performance, accurate control of the tip position is a critical issue. Herein, we present a novel closed-loop control strategy for the ARS mode that achieves higher operating speeds with increased stability. The algorithm differs from that of most conventional (i.e., constant velocity) approach schemes as it includes a deceleration phase near the sample surface, which is intended to minimize the possibility of contact with the surface. Analysis of the ion current and tip position demonstrates that the new mode is able to operate at approach speeds of up to 250 μm s-1. As a result of the improved stability, SICM imaging with the new approach scheme enables significantly improved, high resolution imaging of subtle features of fixed and live cells (e.g., filamentous structures & membrane edges). Taken together, the results suggest that optimization of the tip approach speed can substantially improve SICM imaging performance, further enabling SICM to become widely adopted as a general and versatile research tool for biological studies at the nanoscale level.

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

  8. Retrieving spin textures on curved magnetic thin films with full-field soft X-ray microscopies.

    PubMed

    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. 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.

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

    SciTech Connect

    Sutter, P. Sutter, E.

    2014-09-01

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

  10. 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.

  11. 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

  12. Nanoscale multilevel switching in Ge2Sb2Te5 thin film with conductive atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Fei; Xu, Ling; Chen, Jing; Xu, Jun; Yu, Yao; Ma, Zhongyuan; Chen, Kunji

    2016-01-01

    We demonstrate three-level data storage in amorphous Ge2Sb2Te5 (GST) thin film by conductive atomic force microscopy (C-AFM). Due to the high resolution and current sensitivity of AFM, the electrical properties of GST are investigated in the nanoscale. By applying an electric field between an AFM probe tip and the GST surface, well-resolved threshold switching and memory switching are obtained successively in a current-voltage sweeping. Correspondingly, three states with high, intermediate and low resistances, which are assigned data values ‘0’, ‘1’ and ‘2’ respectively, are observed in an IV-spectrum. The electrical resistance of GST thin film decreases by over two orders of magnitude in both switching processes, which provides a clear contrast to distinguish the three logical states. We also discuss the threshold electrical field of threshold switching in the amorphous GST thin film. Nanoscale conductive marks in the amorphous ON state and crystalline state are successfully fabricated by applying IV-spectra with different voltage ranges on the GST thin films.

  13. Direct Visualization of Nucleation and Growth Processes of Solid Electrolyte Interphase Film Using in Situ Atomic Force Microscopy.

    PubMed

    Shi, Yang; Yan, Hui-Juan; Wen, Rui; Wan, Li-Jun

    2017-07-05

    An understanding of the formation mechanism of solid electrolyte interphase (SEI) film at the nanoscale is paramount because it is one of the key issues at interfaces in lithium-ion batteries (LIBs). Herein, we explored the nucleation, growth, and formation of SEI film on highly oriented pyrolytic graphite (HOPG) substrate in ionic liquid-based electrolytes 1-butyl-1-methyl-pyrrolidinium bis(fluorosulfonyl)imide ([BMP](+)[FSI](-)) and 1-butyl-1-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide ([BMP](+)[TFSI](-)) by in situ atomic force microscopy (AFM) and found that the types of anions have significant influence on the structure of the formed SEI. In [BMP](+)[FSI](-) containing LiFSI, a compact and thin SEI film prefers to grow in the plane of HOPG substrate, while a rough and loose film tends to form in [BMP](+)[TFSI](-) containing LiTFSI. On the basis of in situ AFM observations, the relationship between the SEI structure and the electrochemical performance was clarified.

  14. 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

  15. Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: An atomic force microscopy study

    NASA Astrophysics Data System (ADS)

    Fernandes, Anwesha N.; Chen, Xinyong; Scotchford, Colin A.; Walker, James; Wells, Darren M.; Roberts, Clive J.; Everitt, Nicola M.

    2012-02-01

    The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of “Index of Plasticity” (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus ((H)/(E*)).

  16. 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.

  17. Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: an atomic force microscopy study.

    PubMed

    Fernandes, Anwesha N; Chen, Xinyong; Scotchford, Colin A; Walker, James; Wells, Darren M; Roberts, Clive J; Everitt, Nicola M

    2012-02-01

    The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of "Index of Plasticity" (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus (H/E(*)). © 2012 American Physical Society

  18. 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.

  19. 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.

  20. Determination of sound velocity and acoustic impedance of thin chitosan films by phase-sensitive acoustic microscopy

    NASA Astrophysics Data System (ADS)

    Kamanyi, Albert E.; Ahmed Mohamed, Esam T.; Ngwa, Wilfred; Grill, Wolfgang

    2010-03-01

    The biomaterial chitosan is used in the paper manufacturing industry, as a wound healing agent and in filtration amongst others. In this paper the longitudinal sound velocity and acoustic impedance of thin films of chitosan of varying thicknesses are determined by vector-contrast acoustic microscopy. The exploitation of the relative reflectivity information from the maximum amplitude images and a comparison of the experimentally obtained V(z) curves with simulations using appropriate models are applied for the evaluation of the sound velocity. These results were compared to those previously obtained results with the same instrument.

  1. X-ray absorption spectroscopy and atomic force microscopy study of bias-enhanced nucleation of diamond films

    SciTech Connect

    Garcia, M.M.; Jimenez, I.; Vazquez, L.; Gomez-Aleixandre, C.; Albella, J.M.; Sanchez, O.; Terminello, L.J.; Himpsel, F.J.

    1998-04-01

    The bias-enhanced nucleation of diamond on Si(100) has been studied by x-ray absorption near-edge spectroscopy (XANES) and atomic force microscopy, two techniques well suited to characterize nanometric crystallites. Diamond nuclei of {approximately}15nm are formed after 5 min of bias-enhanced treatment. The number of nuclei and its size increases with the time of application of the bias voltage. A nanocrystalline diamond film is attained after 20 min of bias-enhanced nucleation. At the initial nucleation stages, the Si substrate appears covered with diamond crystallites and graphite, without SiC being detected by XANES. {copyright} {ital 1998 American Institute of Physics.}

  2. 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.

  3. 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.

  4. 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.

  5. 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.

  6. Resolving rotational motions of nano-objects in engineered environments and live cells with gold nanorods and differential interference contrast microscopy.

    PubMed

    Wang, Gufeng; Sun, Wei; Luo, Yong; Fang, Ning

    2010-11-24

    Gold nanorods are excellent orientation probes due to their anisotropic optical properties. Their dynamic rotational motion in the 3D space can be disclosed with Nomarski-type differential interference contrast (DIC) microscopy. We demonstrate that by using the combination of gold nanorod probes and DIC microscopy, we are able to resolve rotational motions of nano-cargos transported by motor proteins at video rate not only on engineered surfaces but also on cytoskeleton tracks in live cells.

  7. 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

  8. Nanoscale modification of electrical and magnetic properties of Fe{sub 3}O{sub 4} thin film by atomic force microscopy lithography

    SciTech Connect

    Hirooka, Motoyuki; Tanaka, Hidekazu; Li, Runwei; Kawai, Tomoji

    2004-09-06

    We present a report on the nanopatterning of an epitaxial ultrathin film of Fe{sub 3}O{sub 4} with room-temperature (ferri)magnetism using atomic force microscopy (AFM). Fe{sub 3}O{sub 4} thin films with atomically flat surfaces were grown using laser molecular-beam epitaxy on a MgAl{sub 2}O{sub 4}(111) single-crystal substrate. (Nanowire) were constructed on Fe{sub 3}O{sub 4} thin film by applying an electric field between an AFM conductive tip and the surface of the film. The minimum width and height in the resulting nanowire are 48 nm and 2 nm, respectively. The patterned region of the Fe{sub 3}O{sub 4} film surface possesses a resistance which is 10{sup 5} times higher than the unpatterned region. Furthermore, magnetic force microscopy measurements also revealed that magnetization of the patterned region is strongly suppressed.

  9. 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.

  10. Top electrode size effects in the piezoresponse force microscopy of piezoelectric thin films attached to a rigid substrate

    NASA Astrophysics Data System (ADS)

    Wang, J. H.

    2017-10-01

    In order to avoid the highly concentrated electric field induced beneath the sharp tip, the technique using a top coating electrode in the piezoresponse force microscopy (PFM) has been developed to detect the piezoelectric coefficients. Reliable theory should be erected to explain the broadly reported top electrode size effects and relate the responses with material constants. In this paper, the surface displacement, electric potential inside the film, electric charge and effective piezoelectric coefficient are expressed as a set of integral equations. Analytical solutions are obtained for two limiting cases, i.e., half space (HS) and infinitely thin film (IT). The effective piezoelectric coefficient of the HS case is proved to be the same as that from the PFM of a piezoelectric half plane without a top coating. For the IT case, it is identical to the well-known piezoelectric coefficient result of piezoelectric thin film clamped between flat rigid electrodes subject to homogeneous external electric field. For PZT4 thin layer, numerical results reveal that the surface displacement obviously decreases and the electric potential distributions inside the film become more and more homogeneous as the electrode radius to film thickness ratio (a/t) enlarges. The electric charge dramatically increases while the effective piezoelectric coefficient evidently decreases and they both transfer from the HS solutions to the IT results when a/t varies from 0.001 to 20. The transition occurs at about a/t = 1 in agreement with the experimental observations. A critical top electrode size, i.e., a/t > 10, is obtained and applicable to other piezoelectric materials. Under such circumstances, one can readily gain the piezoelectric coefficients e 33, d 33 and the dielectric coefficient {\\in }33 if other mechanical coefficients and one piezoelectric constant are known a prior.

  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 live cells at the nanometer-scale with single-molecule microscopy: obstacles and achievements in experiment optimization for microbiology.

    PubMed

    Haas, Beth L; Matson, Jyl S; DiRita, Victor J; Biteen, Julie S

    2014-08-13

    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.

  13. Imaging the uptake of gold nanoshells in live cells using plasmon resonance enhanced four wave mixing microscopy

    PubMed Central

    Garrett, Natalie; Whiteman, Matt; Moger, Julian

    2014-01-01

    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. PMID:21935123

  14. 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.

  15. 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

  16. Measurement of the tear film and anterior chamber by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Buttenschön, Kim K.; Girkin, John M.; Wilson, Clive G.; Daly, Daniel J.

    2010-02-01

    We report on the development of a novel, low cost instrument that is capable of accurately measuring small, short and long term changes in the thickness of the cornea and tear film at high speed. The performance of the instrument was tested by measuring the influence of Allergan's OPTIVETM lubricating eye drops on the thickness of the cornea and tear film. Comparative measurements to quantify the performance were taken using Haag-Streit's LenStar. It was found that the newly developed instrument accurately measured a change in thickness of around 9 μm with an accuracy comparable to the LenStar, and with a standard deviation of less than 1 micrometer. Since the new instrument was not configured to resolve the tear film from the cornea, we are not yet able to distinguish the cause of the thickening.

  17. Optical detection of transverse spin-Seebeck effect in permalloy film using Sagnac interferometer microscopy

    NASA Astrophysics Data System (ADS)

    McLaughlin, Ryan; Sun, Dali; Zhang, Chuang; Groesbeck, Matthew; Vardeny, Z. Valy

    2017-05-01

    Macroscopic spatial spin distribution caused by the application of an in-plane thermal gradient in a conducting ferromagnetic film, known as transverse spin-Seebeck effect (TSSE), is in many cases overshadowed by thermoelectric and magnetothermoelectric effects when using the conventional electrical detection via the inverse spin Hall effect. Here we report an optical method for the detection and characterization of TSSE response in permalloy films using magneto-optical Kerr effect with an ultrasensitive fiber-optic Sagnac interferometer microscope that is free of magnetothermoelectric artifacts, which also allows measurements with field direction parallel and perpendicular to the film surface. We found a substantial anisotropy in the permalloy TSSE coefficient, where the "in-plane magnetization" coefficient is much larger than that in the "out-of-plane magnetization."

  18. 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.

  19. 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.