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Sample records for 3d electron microscopy

  1. Atomic resolution 3D electron diffraction microscopy

    SciTech Connect

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

    2002-03-01

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

  2. Navigating 3D electron microscopy maps with EM-SURFER.

    PubMed

    Esquivel-Rodríguez, Juan; Xiong, Yi; Han, Xusi; Guang, Shuomeng; Christoffer, Charles; Kihara, Daisuke

    2015-05-30

    The Electron Microscopy DataBank (EMDB) is growing rapidly, accumulating biological structural data obtained mainly by electron microscopy and tomography, which are emerging techniques for determining large biomolecular complex and subcellular structures. Together with the Protein Data Bank (PDB), EMDB is becoming a fundamental resource of the tertiary structures of biological macromolecules. To take full advantage of this indispensable resource, the ability to search the database by structural similarity is essential. However, unlike high-resolution structures stored in PDB, methods for comparing low-resolution electron microscopy (EM) density maps in EMDB are not well established. We developed a computational method for efficiently searching low-resolution EM maps. The method uses a compact fingerprint representation of EM maps based on the 3D Zernike descriptor, which is derived from a mathematical series expansion for EM maps that are considered as 3D functions. The method is implemented in a web server named EM-SURFER, which allows users to search against the entire EMDB in real-time. EM-SURFER compares the global shapes of EM maps. Examples of search results from different types of query structures are discussed. We developed EM-SURFER, which retrieves structurally relevant matches for query EM maps from EMDB within seconds. The unique capability of EM-SURFER to detect 3D shape similarity of low-resolution EM maps should prove invaluable in structural biology.

  3. 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

    PubMed Central

    Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

    2017-01-01

    ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

  4. 3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy.

    PubMed

    Russell, Matthew R G; Lerner, Thomas R; Burden, Jemima J; Nkwe, David O; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L; Peddie, Christopher J; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G; Collinson, Lucy M

    2017-01-01

    The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. © 2017. Published by The Company of Biologists Ltd.

  5. 3D image reconstruction algorithms for cryo-electron-microscopy images of virus particles

    NASA Astrophysics Data System (ADS)

    Doerschuk, Peter C.; Johnson, John E.

    2000-11-01

    A statistical model for the object and the complete image formation process in cryo electron microscopy of viruses is presented. Using this model, maximum likelihood reconstructions of the 3D structure of viruses are computed using the expectation maximization algorithm and an example based on Cowpea mosaic virus is provided.

  6. 3D structure of individual nanocrystals in solution by electron microscopy

    SciTech Connect

    Park, Jungwok; Elmlund, Hans; Ercius, Peter; Yuk, Jong Min; Limmer, David T.; Chen, Qian; Kim, Kwanpyo; Han, Sang Hoon; Weitz, David A.; Zettl, A.; Alivisatos, A. P.

    2015-07-17

    Here, knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unordered nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale.

  7. 3D structure of individual nanocrystals in solution by electron microscopy

    DOE PAGES

    Park, Jungwok; Elmlund, Hans; Ercius, Peter; ...

    2015-07-17

    Here, knowledge about the synthesis, growth mechanisms, and physical properties of colloidal nanoparticles has been limited by technical impediments. We introduce a method for determining three-dimensional (3D) structures of individual nanoparticles in solution. We combine a graphene liquid cell, high-resolution transmission electron microscopy, a direct electron detector, and an algorithm for single-particle 3D reconstruction originally developed for analysis of biological molecules. This method yielded two 3D structures of individual platinum nanocrystals at near-atomic resolution. Because our method derives the 3D structure from images of individual nanoparticles rotating freely in solution, it enables the analysis of heterogeneous populations of potentially unorderedmore » nanoparticles that are synthesized in solution, thereby providing a means to understand the structure and stability of defects at the nanoscale.« less

  8. Novel scanning electron microscopy methods for analyzing the 3D structure of the Golgi apparatus.

    PubMed

    Koga, Daisuke; Ushiki, Tatsuo; Watanabe, Tsuyoshi

    2017-01-01

    The structure of the Golgi apparatus has been extensively examined by light and electron microscopy, but details of its three-dimensional (3D) structure have remained unclear because of the technical limitations of conventional microscopy techniques. To overcome this problem, we have developed several novel scanning electron microscopy (SEM) methods for observing the 3D structure of subcellular organelles including the Golgi apparatus: (1) an osmium maceration method that facilitates SEM observation of membranous organelles, including the Golgi apparatus, by selectively removing soluble cytoplasmic proteins, (2) an osmium impregnation/maceration method that combines an osmium impregnation method with the osmium maceration method to determine the polarity of the Golgi apparatus by SEM, (3) a correlative light and SEM method that combines a cryosectioning technique with the osmium maceration method to enable correlation of the immunocytochemical distribution of molecules with the 3D ultrastructure of the Golgi apparatus, and (4) array tomography based on the systematic collection and integration of SEM images of serial ultrathin sections on glass slides for revealing the 3D ultrastructure of the entire Golgi apparatus. Together, the novel SEM techniques listed above can reveal the complete 3D structure of the Golgi apparatus in different cell types.

  9. Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy.

    PubMed

    Jaros, Josef; Petrov, Michal; Tesarova, Marketa; Hampl, Ales

    2017-01-01

    Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.

  10. Automatic 3D reconstruction of quasi-planar stereo Scanning Electron Microscopy (SEM) images.

    PubMed

    Roy, S; Meunier, J; Marian, A M; Vidal, F; Brunette, I; Costantino, S

    2012-01-01

    Scanning Electron Microscopy (SEM) is widely used in science to characterize the surface roughness of materials. Three-dimensional information can be obtained with SEM based on stereovision techniques. A stereo pair is typically obtained by tilting the sample by a few degrees. In this paper we present a fully automated method for 3D reconstruction from a SEM stereo pair without any particular constraint. Results are presented for corneal stromal surfaces.

  11. 3D imaging by serial block face scanning electron microscopy for materials science using ultramicrotomy.

    PubMed

    Hashimoto, Teruo; Thompson, George E; Zhou, Xiaorong; Withers, Philip J

    2016-04-01

    Mechanical serial block face scanning electron microscopy (SBFSEM) has emerged as a means of obtaining three dimensional (3D) electron images over volumes much larger than possible by focused ion beam (FIB) serial sectioning and at higher spatial resolution than achievable with conventional X-ray computed tomography (CT). Such high resolution 3D electron images can be employed for precisely determining the shape, volume fraction, distribution and connectivity of important microstructural features. While soft (fixed or frozen) biological samples are particularly well suited for nanoscale sectioning using an ultramicrotome, the technique can also produce excellent 3D images at electron microscope resolution in a time and resource-efficient manner for engineering materials. Currently, a lack of appreciation of the capabilities of ultramicrotomy and the operational challenges associated with minimising artefacts for different materials is limiting its wider application to engineering materials. Consequently, this paper outlines the current state of the art for SBFSEM examining in detail how damage is introduced during slicing and highlighting strategies for minimising such damage. A particular focus of the study is the acquisition of 3D images for a variety of metallic and coated systems. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  12. Staining and embedding of human chromosomes for 3-d serial block-face scanning electron microscopy.

    PubMed

    Yusuf, Mohammed; Chen, Bo; Hashimoto, Teruo; Estandarte, Ana Katrina; Thompson, George; Robinson, Ian

    2014-12-01

    The high-order structure of human chromosomes is an important biological question that is still under investigation. Studies have been done on imaging human mitotic chromosomes using mostly 2-D microscopy methods. To image micron-sized human chromosomes in 3-D, we developed a procedure for preparing samples for serial block-face scanning electron microscopy (SBFSEM). Polyamine chromosomes are first separated using a simple filtration method and then stained with heavy metal. We show that the DNA-specific platinum blue provides higher contrast than osmium tetroxide. A two-step procedure for embedding chromosomes in resin is then used to concentrate the chromosome samples. After stacking the SBFSEM images, a familiar X-shaped chromosome was observed in 3-D.

  13. Correlated fluorescence and 3D electron microscopy with high sensitivity and spatial precision

    PubMed Central

    Kukulski, Wanda; Schorb, Martin; Welsch, Sonja; Picco, Andrea

    2011-01-01

    Correlative electron and fluorescence microscopy has the potential to elucidate the ultrastructural details of dynamic and rare cellular events, but has been limited by low precision and sensitivity. Here we present a method for direct mapping of signals originating from ∼20 fluorescent protein molecules to 3D electron tomograms with a precision of less than 100 nm. We demonstrate that this method can be used to identify individual HIV particles bound to mammalian cell surfaces. We also apply the method to image microtubule end structures bound to mal3p in fission yeast, and demonstrate that growing microtubule plus-ends are flared in vivo. We localize Rvs167 to endocytic sites in budding yeast, and show that scission takes place halfway through a 10-s time period during which amphiphysins are bound to the vesicle neck. This new technique opens the door for direct correlation of fluorescence and electron microscopy to visualize cellular processes at the ultrastructural scale. PMID:21200030

  14. Cellulose Nanocrystals as Chiral Inducers: Enantioselective Catalysis and Transmission Electron Microscopy 3D Characterization.

    PubMed

    Kaushik, Madhu; Basu, Kaustuv; Benoit, Charles; Cirtiu, Ciprian M; Vali, Hojatollah; Moores, Audrey

    2015-05-20

    Cellulose nanocrystals (CNCs), derived from cellulose, provide us with an opportunity to devise more sustainable solutions to current technological challenges. Enantioselective catalysis, especially heterogeneous, is the preferred method for the synthesis of pure chiral molecules in the fine chemical industries. Cellulose has been long sought as a chiral inducer in enantioselective catalysis. We report herein an unprecedentedly high enantiomeric excess (ee) for Pd patches deposited onto CNCs used as catalysts for the hydrogenation of prochiral ketones in water at room temperature and 4 bar H2. Our system, where CNCs acted as support and sole chiral source, achieved an ee of 65% with 100% conversions. Cryo-electron microscopy, high-resolution transmission electron microscopy, and tomography were used for the first time to study the 3D structure of a metal functionalized CNC hybrid. It established the presence of sub-nanometer-thick Pd patches at the surface of CNCs and provided insight into the chiral induction mechanism.

  15. Computational methods for constructing protein structure models from 3D electron microscopy maps.

    PubMed

    Esquivel-Rodríguez, Juan; Kihara, Daisuke

    2013-10-01

    Protein structure determination by cryo-electron microscopy (EM) has made significant progress in the past decades. Resolutions of EM maps have been improving as evidenced by recently reported structures that are solved at high resolutions close to 3Å. Computational methods play a key role in interpreting EM data. Among many computational procedures applied to an EM map to obtain protein structure information, in this article we focus on reviewing computational methods that model protein three-dimensional (3D) structures from a 3D EM density map that is constructed from two-dimensional (2D) maps. The computational methods we discuss range from de novo methods, which identify structural elements in an EM map, to structure fitting methods, where known high resolution structures are fit into a low-resolution EM map. A list of available computational tools is also provided.

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

    PubMed Central

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

    2014-01-01

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

  17. Electron Microscopy: From 2D to 3D Images with Special Reference to Muscle

    PubMed Central

    2015-01-01

    This is a brief and necessarily very sketchy presentation of the evolution in electron microscopy (EM) imaging that was driven by the necessity of extracting 3-D views from the essentially 2-D images produced by the electron beam. The lens design of standard transmission electron microscope has not been greatly altered since its inception. However, technical advances in specimen preparation, image collection and analysis gradually induced an astounding progression over a period of about 50 years. From the early images that redefined tissues, cell and cell organelles at the sub-micron level, to the current nano-resolution reconstructions of organelles and proteins the step is very large. The review is written by an investigator who has followed the field for many years, but often from the sidelines, and with great wonder. Her interest in muscle ultrastructure colors the writing. More specific detailed reviews are presented in this issue. PMID:26913146

  18. 3D Imaging of Diatoms with Ion-abrasion Scanning Electron Microscopy

    PubMed Central

    Hildebrand, Mark; Kim, Sang; Shi, Dan; Scott, Keana; Subramaniam, Sriram

    2009-01-01

    Ion-abrasion scanning electron microscopy (IASEM) takes advantage of focused ion beams to abrade thin sections from the surface of bulk specimens, coupled with SEM to image the surface of each section, enabling 3D reconstructions of subcellular architecture at ~ 30 nm resolution. Here, we report the first application of IASEM for imaging a biomineralizing organism, the marine diatom Thalassiosira pseudonana. Diatoms have highly patterned silica-based cell wall structures that are unique models for the study and application of directed nanomaterials synthesis by biological systems. Our study provides new insights into the architecture and assembly principles of both the “hard” (siliceous) and “soft” (organic) components of the cell. From 3D reconstructions of developmentally synchronized diatoms captured at different stages, we show that both micro- and nanoscale siliceous structures can be visualized at specific stages in their formation. We show that not only are structures visualized in a whole-cell context, but demonstrate that fragile, early-stage structures are visible, and that this can be combined with elemental mapping in the exposed slice. We demonstrate that the 3D architectures of silica structures, and the cellular components that mediate their creation and positioning can be visualized simultaneously, providing new opportunities to study and manipulate mineral nanostructures in a genetically tractable system. PMID:19269330

  19. Transmission electron microscopy studies and modeling of 3D reciprocal space of ω forming alloy.

    PubMed

    Alphy George; Sharma, Vinayak; Divakar, R; Sabeena, M; Mohandas, E

    2017-09-12

    Initial stage of ω phase formation and associated anomalous features that appear in diffraction patterns of a metastable β transition metal alloy have been investigated in this study with the aid of transmission electron microscopy, simulation and modeling. The paper explores discrete features that emerge in selected area diffraction patterns of quenched Ti-15wt%Mo alloy and analyzes the correlation between ω reflections and diffuse arcs by considering all variants of ω phase as per the formation kinetics of ω phase in β matrix while quenching. Superimposed simulated diffraction patterns have been compared with experimental counterparts and it is deduced that there is lack of congruence between ω reflections and diffuse arcs even after considering trigonal ω with varying degrees of displacement. Direct lattice imaging of trigonal ω in β matrix has been demonstrated by phase contrast microscopy coupled with Fourier filtering techniques. By investigating the nature of ω reflections and diffuse arcs with the aid of electron diffraction pattern calculations and phase contrast microscopy, it is shown that, existing model of three-dimensional (3D) reciprocal space of ω forming alloy at quenched stage is not complete. A new model incorporating a patterned intensity distribution is fitted at the octahedral sites of an fcc reciprocal lattice whose planar intersections with Ewald's sphere show a better fit with the observed experimental diffraction patterns. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

    PubMed

    Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

    2017-04-03

    The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. Immuno- and correlative light microscopy-electron tomography methods for 3D protein localization in yeast.

    PubMed

    Mari, Muriel; Geerts, Willie J C; Reggiori, Fulvio

    2014-10-01

    Compartmentalization of eukaryotic cells is created and maintained through membrane rearrangements that include membrane transport and organelle biogenesis. Three-dimensional reconstructions with nanoscale resolution in combination with protein localization are essential for an accurate molecular dissection of these processes. The yeast Saccharomyces cerevisiae is a key model system for identifying genes and characterizing pathways essential for the organization of cellular ultrastructures. Electron microscopy studies of yeast, however, have been hampered by the presence of a cell wall that obstructs penetration of resins and cryoprotectants, and by the protein dense cytoplasm, which obscures the membrane details. Here we present an immuno-electron tomography (IET) method, which allows the determination of protein distribution patterns on reconstructed organelles from yeast. In addition, we extend this IET approach into a correlative light microscopy-electron tomography procedure where structures positive for a specific protein localized through a fluorescent signal are resolved in 3D. These new investigative tools for yeast will help to advance our understanding of the endomembrane system organization in eukaryotic cells.

  2. Optimizing the 3D-reconstruction technique for serial block-face scanning electron microscopy.

    PubMed

    Wernitznig, Stefan; Sele, Mariella; Urschler, Martin; Zankel, Armin; Pölt, Peter; Rind, F Claire; Leitinger, Gerd

    2016-05-01

    Elucidating the anatomy of neuronal circuits and localizing the synaptic connections between neurons, can give us important insights in how the neuronal circuits work. We are using serial block-face scanning electron microscopy (SBEM) to investigate the anatomy of a collision detection circuit including the Lobula Giant Movement Detector (LGMD) neuron in the locust, Locusta migratoria. For this, thousands of serial electron micrographs are produced that allow us to trace the neuronal branching pattern. The reconstruction of neurons was previously done manually by drawing cell outlines of each cell in each image separately. This approach was very time consuming and troublesome. To make the process more efficient a new interactive software was developed. It uses the contrast between the neuron under investigation and its surrounding for semi-automatic segmentation. For segmentation the user sets starting regions manually and the algorithm automatically selects a volume within the neuron until the edges corresponding to the neuronal outline are reached. Internally the algorithm optimizes a 3D active contour segmentation model formulated as a cost function taking the SEM image edges into account. This reduced the reconstruction time, while staying close to the manual reference segmentation result. Our algorithm is easy to use for a fast segmentation process, unlike previous methods it does not require image training nor an extended computing capacity. Our semi-automatic segmentation algorithm led to a dramatic reduction in processing time for the 3D-reconstruction of identified neurons. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. A toolbox for ab initio 3-D reconstructions in single-particle electron microscopy.

    PubMed

    Voss, Neil R; Lyumkis, Dmitry; Cheng, Anchi; Lau, Pick-Wei; Mulder, Anke; Lander, Gabriel C; Brignole, Edward J; Fellmann, Denis; Irving, Christopher; Jacovetty, Erica L; Leung, Albert; Pulokas, James; Quispe, Joel D; Winkler, Hanspeter; Yoshioka, Craig; Carragher, Bridget; Potter, Clinton S

    2010-03-01

    Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a "toolbox" of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map.

  4. A Toolbox for Ab Initio 3-D Reconstructions in Single-particle Electron Microscopy

    PubMed Central

    Voss, Neil R; Lyumkis, Dmitry; Cheng, Anchi; Lau, Pick-Wei; Mulder, Anke; Lander, Gabriel C; Brignole, Edward J; Fellmann, Denis; Irving, Christopher; Jacovetty, Erica L; Leung, Albert; Pulokas, James; Quispe, Joel D; Winkler, Hanspeter; Yoshioka, Craig; Carragher, Bridget; Potter, Clinton S

    2010-01-01

    Structure determination of a novel macromolecular complex via single-particle electron microscopy depends upon overcoming the challenge of establishing a reliable 3-D reconstruction using only 2-D images. There are a variety of strategies that deal with this issue, but not all of them are readily accessible and straightforward to use. We have developed a “toolbox” of ab initio reconstruction techniques that provide several options for calculating 3-D volumes in an easily managed and tightly controlled work-flow that adheres to standard conventions and formats. This toolbox is designed to streamline the reconstruction process by removing the necessity for bookkeeping, while facilitating transparent data transfer between different software packages. It currently includes procedures for calculating ab initio reconstructions via random or orthogonal tilt geometry, tomograms, and common lines, all of which have been tested using the 50S ribosomal subunit. Our goal is that the accessibility of multiple independent reconstruction algorithms via this toolbox will improve the ease with which models can be generated, and provide a means of evaluating the confidence and reliability of the final reconstructed map. PMID:20018246

  5. Single Particle Cryo-electron Microscopy and 3-D Reconstruction of Viruses

    PubMed Central

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3–4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced. PMID:24357374

  6. Single particle cryo-electron microscopy and 3-D reconstruction of viruses.

    PubMed

    Guo, Fei; Jiang, Wen

    2014-01-01

    With fast progresses in instrumentation, image processing algorithms, and computational resources, single particle electron cryo-microscopy (cryo-EM) 3-D reconstruction of icosahedral viruses has now reached near-atomic resolutions (3-4 Å). With comparable resolutions and more predictable outcomes, cryo-EM is now considered a preferred method over X-ray crystallography for determination of atomic structure of icosahedral viruses. At near-atomic resolutions, all-atom models or backbone models can be reliably built that allow residue level understanding of viral assembly and conformational changes among different stages of viral life cycle. With the developments of asymmetric reconstruction, it is now possible to visualize the complete structure of a complex virus with not only its icosahedral shell but also its multiple non-icosahedral structural features. In this chapter, we will describe single particle cryo-EM experimental and computational procedures for both near-atomic resolution reconstruction of icosahedral viruses and asymmetric reconstruction of viruses with both icosahedral and non-icosahedral structure components. Procedures for rigorous validation of the reconstructions and resolution evaluations using truly independent de novo initial models and refinements are also introduced.

  7. A resource from 3D electron microscopy of hippocampal neuropil for user training and tool development

    PubMed Central

    Harris, Kristen M.; Spacek, Josef; Bell, Maria Elizabeth; Parker, Patrick H.; Lindsey, Laurence F.; Baden, Alexander D.; Vogelstein, Joshua T.; Burns, Randal

    2015-01-01

    Resurgent interest in synaptic circuitry and plasticity has emphasized the importance of 3D reconstruction from serial section electron microscopy (3DEM). Three volumes of hippocampal CA1 neuropil from adult rat were imaged at X-Y resolution of ~2 nm on serial sections of ~50–60 nm thickness. These are the first densely reconstructed hippocampal volumes. All axons, dendrites, glia, and synapses were reconstructed in a cube (~10 μm3) surrounding a large dendritic spine, a cylinder (~43 μm3) surrounding an oblique dendritic segment (3.4 μm long), and a parallelepiped (~178 μm3) surrounding an apical dendritic segment (4.9 μm long). The data provide standards for identifying ultrastructural objects in 3DEM, realistic reconstructions for modeling biophysical properties of synaptic transmission, and a test bed for enhancing reconstruction tools. Representative synapses are quantified from varying section planes, and microtubules, polyribosomes, smooth endoplasmic reticulum, and endosomes are identified and reconstructed in a subset of dendrites. The original images, traces, and Reconstruct software and files are freely available and visualized at the Open Connectome Project (Data Citation 1). PMID:26347348

  8. Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

    PubMed

    Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

    2015-08-01

    In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  9. 3D scanning electron microscopy applied to surface characterization of fluorosed dental enamel.

    PubMed

    Limandri, Silvina; Galván Josa, Víctor; Valentinuzzi, María Cecilia; Chena, María Emilia; Castellano, Gustavo

    2016-05-01

    The enamel surfaces of fluorotic teeth were studied by scanning electron stereomicroscopy. Different whitening treatments were applied to 25 pieces to remove stains caused by fluorosis and their surfaces were characterized by stereomicroscopy in order to obtain functional and amplitude parameters. The topographic features resulting for each treatment were determined through these parameters. The results obtained show that the 3D reconstruction achieved from the SEM stereo pairs is a valuable potential alternative for the surface characterization of this kind of samples.

  10. Correlative 3D superresolution fluorescence and electron microscopy reveal the relationship of mitochondrial nucleoids to membranes

    PubMed Central

    Kopek, Benjamin G.; Shtengel, Gleb; Xu, C. Shan; Clayton, David A.; Hess, Harald F.

    2012-01-01

    Microscopic images of specific proteins in their cellular context yield important insights into biological processes and cellular architecture. The advent of superresolution optical microscopy techniques provides the possibility to augment EM with nanometer-resolution fluorescence microscopy to access the precise location of proteins in the context of cellular ultrastructure. Unfortunately, efforts to combine superresolution fluorescence and EM have been stymied by the divergent and incompatible sample preparation protocols of the two methods. Here, we describe a protocol that preserves both the delicate photoactivatable fluorescent protein labels essential for superresolution microscopy and the fine ultrastructural context of EM. This preparation enables direct 3D imaging in 500- to 750-nm sections with interferometric photoactivatable localization microscopy followed by scanning EM images generated by focused ion beam ablation. We use this process to “colorize” detailed EM images of the mitochondrion with the position of labeled proteins. The approach presented here has provided a new level of definition of the in vivo nature of organization of mitochondrial nucleoids, and we expect this straightforward method to be applicable to many other biological questions that can be answered by direct imaging. PMID:22474357

  11. 3D multiplexed immunoplasmonics microscopy

    NASA Astrophysics Data System (ADS)

    Bergeron, Éric; Patskovsky, Sergiy; Rioux, David; Meunier, Michel

    2016-07-01

    -scanning by RLM with three spectral filters corresponding to the plasmon peak wavelengths of the immunoplasmonics labels in the cellular environment (500 nm for 80 nm AgNSs, 580 nm for 100 nm AuNSs and 700 nm for 40 nm × 92 nm AuNRs). Third, the developed technology is simple and compatible with standard epi-fluorescence microscopes used in biological and clinical laboratories. Thus, 3D multiplexed immunoplasmonics microscopy is ready for clinical applications as a cost-efficient alternative to immunofluorescence. Electronic supplementary information (ESI) available: Characterization of functionalized nanoparticles by UV-visible-NIR spectroscopy, standard dark field microscopy and reflected light microscopy. Immunofluorescence of cells. See DOI: 10.1039/c6nr01257d

  12. Isolation, Electron Microscopy and 3D Reconstruction of Invertebrate Muscle Myofilaments

    PubMed Central

    Craig, Roger

    2011-01-01

    Understanding the molecular mechanism of muscle contraction and its regulation has been greatly influenced and aided by studies of myofilament structure in invertebrate muscles. Invertebrates are easily obtained and cover a broad spectrum of species and functional specializations. The thick (myosin-containing) filaments from some invertebrates are especially stable and simple in structure and thus much more amenable to structural analysis than those of vertebrates. Comparative studies of invertebrate filaments by electron microscopy and image processing have provided important generalizations of muscle molecular structure and function. This article reviews methods for preparing thick and thin filaments from invertebrate muscle, for imaging filaments by electron microscopy, and for determining their three dimensional structure by image processing. It also highlights some of the key insights into filament function that have come from these studies. PMID:22155190

  13. Ultra-high voltage electron microscopy of primitive algae illuminates 3D ultrastructures of the first photosynthetic eukaryote

    PubMed Central

    Takahashi, Toshiyuki; Nishida, Tomoki; Saito, Chieko; Yasuda, Hidehiro; Nozaki, Hisayoshi

    2015-01-01

    A heterotrophic organism 1–2 billion years ago enslaved a cyanobacterium to become the first photosynthetic eukaryote, and has diverged globally. The primary phototrophs, glaucophytes, are thought to retain ancestral features of the first photosynthetic eukaryote, but examining the protoplast ultrastructure has previously been problematic in the coccoid glaucophyte Glaucocystis due to its thick cell wall. Here, we examined the three-dimensional (3D) ultrastructure in two divergent species of Glaucocystis using ultra-high voltage electron microscopy. Three-dimensional modelling of Glaucocystis cells using electron tomography clearly showed that numerous, leaflet-like flattened vesicles are distributed throughout the protoplast periphery just underneath a single-layered plasma membrane. This 3D feature is essentially identical to that of another glaucophyte genus Cyanophora, as well as the secondary phototrophs in Alveolata. Thus, the common ancestor of glaucophytes and/or the first photosynthetic eukaryote may have shown similar 3D structures. PMID:26439276

  14. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB.

    PubMed

    Lagerstedt, Ingvar; Moore, William J; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R; Kleywegt, Gerard J

    2013-11-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed.

  15. Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB☆

    PubMed Central

    Lagerstedt, Ingvar; Moore, William J.; Patwardhan, Ardan; Sanz-García, Eduardo; Best, Christoph; Swedlow, Jason R.; Kleywegt, Gerard J.

    2013-01-01

    The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed. PMID:24113529

  16. Visualization of Mesenchymal Stromal Cells in 2Dand 3D-Cultures by Scanning Electron Microscopy with Lanthanide Contrasting.

    PubMed

    Novikov, I A; Vakhrushev, I V; Antonov, E N; Yarygin, K N; Subbot, A M

    2017-02-01

    Mesenchymal stromal cells from deciduous teeth in 2D- and 3D-cultures on culture plastic, silicate glass, porous polystyrene, and experimental polylactoglycolide matrices were visualized by scanning electron microscopy with lanthanide contrasting. Supravital staining of cell cultures with a lanthanide-based dye (neodymium chloride) preserved normal cell morphology and allowed assessment of the matrix properties of the carriers. The developed approach can be used for the development of biomaterials for tissue engineering.

  17. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy

    SciTech Connect

    Barad, Benjamin A.; Echols, Nathaniel; Wang, Ray Yu-Ruei; Cheng, Yifan; DiMaio, Frank; Adams, Paul D.; Fraser, James S.

    2015-08-17

    Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report that EMRinger is a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.

  18. EMRinger: side chain–directed model and map validation for 3D cryo-electron microscopy

    DOE PAGES

    Barad, Benjamin A.; Echols, Nathaniel; Wang, Ray Yu-Ruei; ...

    2015-08-17

    Advances in high-resolution cryo-electron microscopy (cryo-EM) require the development of validation metrics to independently assess map quality and model geometry. We report that EMRinger is a tool that assesses the precise fitting of an atomic model into the map during refinement and shows how radiation damage alters scattering from negatively charged amino acids. EMRinger (https://github.com/fraser-lab/EMRinger) will be useful for monitoring progress in resolving and modeling high-resolution features in cryo-EM.

  19. 3D structured illumination microscopy

    NASA Astrophysics Data System (ADS)

    Dougherty, William M.; Goodwin, Paul C.

    2011-03-01

    Three-dimensional structured illumination microscopy achieves double the lateral and axial resolution of wide-field microscopy, using conventional fluorescent dyes, proteins and sample preparation techniques. A three-dimensional interference-fringe pattern excites the fluorescence, filling in the "missing cone" of the wide field optical transfer function, thereby enabling axial (z) discrimination. The pattern acts as a spatial carrier frequency that mixes with the higher spatial frequency components of the image, which usually succumb to the diffraction limit. The fluorescence image encodes the high frequency content as a down-mixed, moiré-like pattern. A series of images is required, wherein the 3D pattern is shifted and rotated, providing down-mixed data for a system of linear equations. Super-resolution is obtained by solving these equations. The speed with which the image series can be obtained can be a problem for the microscopy of living cells. Challenges include pattern-switching speeds, optical efficiency, wavefront quality and fringe contrast, fringe pitch optimization, and polarization issues. We will review some recent developments in 3D-SIM hardware with the goal of super-resolved z-stacks of motile cells.

  20. 3D structure of Syk kinase determined by single particle electron microscopy

    PubMed Central

    Arias-Palomo, Ernesto; Recuero-Checa, María A.; Bustelo, Xosé R.; Llorca, Oscar

    2008-01-01

    The cytoplasmic Syk kinase plays key roles in immune responses and comprises two N-terminal regulatory Src homology 2 (SH2) domains followed by a catalytic region. Atomic structures of these domains have only been solved in isolation. To gain insights into the three-dimensional structure of full-length Syk, we have used single-particle electron microscopy. Syk acquires a closed conformation resembling the inhibited structure of Zap-70, another member of the Syk family. Such configuration suggests an inhibition of the N-terminal domains on its catalytic activity. The phosphotyrosine binding pockets of both SH2 domains are not occluded and they could interact with other phosphoproteins. PMID:18021750

  1. From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

    PubMed Central

    Tsai, Wen-Ting; Hassan, Ahmed; Sarkar, Purbasha; Correa, Joaquin; Metlagel, Zoltan; Jorgens, Danielle M.; Auer, Manfred

    2014-01-01

    Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation. The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data

  2. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells.

    PubMed

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-12-03

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications.

  3. Viral Infection at High Magnification: 3D Electron Microscopy Methods to Analyze the Architecture of Infected Cells

    PubMed Central

    Romero-Brey, Inés; Bartenschlager, Ralf

    2015-01-01

    As obligate intracellular parasites, viruses need to hijack their cellular hosts and reprogram their machineries in order to replicate their genomes and produce new virions. For the direct visualization of the different steps of a viral life cycle (attachment, entry, replication, assembly and egress) electron microscopy (EM) methods are extremely helpful. While conventional EM has given important information about virus-host cell interactions, the development of three-dimensional EM (3D-EM) approaches provides unprecedented insights into how viruses remodel the intracellular architecture of the host cell. During the last years several 3D-EM methods have been developed. Here we will provide a description of the main approaches and examples of innovative applications. PMID:26633469

  4. PF2 fit: Polar Fast Fourier Matched Alignment of Atomistic Structures with 3D Electron Microscopy Maps

    PubMed Central

    Bettadapura, Radhakrishna; Rasheed, Muhibur; Vollrath, Antje; Bajaj, Chandrajit

    2015-01-01

    There continue to be increasing occurrences of both atomistic structure models in the PDB (possibly reconstructed from X-ray diffraction or NMR data), and 3D reconstructed cryo-electron microscopy (3D EM) maps (albeit at coarser resolution) of the same or homologous molecule or molecular assembly, deposited in the EMDB. To obtain the best possible structural model of the molecule at the best achievable resolution, and without any missing gaps, one typically aligns (match and fits) the atomistic structure model with the 3D EM map. We discuss a new algorithm and generalized framework, named PF2 fit (Polar Fast Fourier Fitting) for the best possible structural alignment of atomistic structures with 3D EM. While PF2 fit enables only a rigid, six dimensional (6D) alignment method, it augments prior work on 6D X-ray structure and 3D EM alignment in multiple ways: Scoring. PF2 fit includes a new scoring scheme that, in addition to rewarding overlaps between the volumes occupied by the atomistic structure and 3D EM map, rewards overlaps between the volumes complementary to them. We quantitatively demonstrate how this new complementary scoring scheme improves upon existing approaches. PF2 fit also includes two scoring functions, the non-uniform exterior penalty and the skeleton-secondary structure score, and implements the scattering potential score as an alternative to traditional Gaussian blurring. Search. PF2 fit utilizes a fast polar Fourier search scheme, whose main advantage is the ability to search over uniformly and adaptively sampled subsets of the space of rigid-body motions. PF2 fit also implements a new reranking search and scoring methodology that considerably improves alignment metrics in results obtained from the initial search. PMID:26469938

  5. Genetically targeted 3D visualisation of Drosophila neurons under Electron Microscopy and X-Ray Microscopy using miniSOG

    PubMed Central

    Ng, Julian; Browning, Alyssa; Lechner, Lorenz; Terada, Masako; Howard, Gillian; Jefferis, Gregory S. X. E.

    2016-01-01

    Large dimension, high-resolution imaging is important for neural circuit visualisation as neurons have both long- and short-range patterns: from axons and dendrites to the numerous synapses at terminal endings. Electron Microscopy (EM) is the favoured approach for synaptic resolution imaging but how such structures can be segmented from high-density images within large volume datasets remains challenging. Fluorescent probes are widely used to localise synapses, identify cell-types and in tracing studies. The equivalent EM approach would benefit visualising such labelled structures from within sub-cellular, cellular, tissue and neuroanatomical contexts. Here we developed genetically-encoded, electron-dense markers using miniSOG. We demonstrate their ability in 1) labelling cellular sub-compartments of genetically-targeted neurons, 2) generating contrast under different EM modalities, and 3) segmenting labelled structures from EM volumes using computer-assisted strategies. We also tested non-destructive X-ray imaging on whole Drosophila brains to evaluate contrast staining. This enabled us to target specific regions for EM volume acquisition. PMID:27958322

  6. Web-based volume slicer for 3D electron-microscopy data from EMDB.

    PubMed

    Salavert-Torres, José; Iudin, Andrii; Lagerstedt, Ingvar; Sanz-García, Eduardo; Kleywegt, Gerard J; Patwardhan, Ardan

    2016-05-01

    We describe the functionality and design of the Volume slicer - a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale.

  7. Uncertainty studies of topographical measurements on steel surface corrosion by 3D scanning electron microscopy.

    PubMed

    Kang, K W; Pereda, M D; Canafoglia, M E; Bilmes, P; Llorente, C; Bonetto, R

    2012-02-01

    Pitting corrosion is a damage mechanism quite serious and dangerous in both carbon steel boiler tubes for power plants which are vital to most industries and stainless steels for orthopedic human implants whose demand, due to the increase of life expectation and rate of traffic accidents, has sharply increased. Reliable methods to characterize this kind of damage are becoming increasingly necessary, when trying to evaluate the advance of damage and to establish the best procedures for component inspection in order to determine remaining lives and failure mitigation. A study about the uncertainties on the topographies of corrosion pits from 3D SEM images, obtained at low magnifications (where errors are greater) and different stage tilt angles were carried out using an in-house software previously developed. Additionally, measurements of pit depths on biomaterial surfaces, subjected to two different surface treatments on stainless steels, were carried out. The different depth distributions observed were in agreement with electrochemical measurements.

  8. Measuring surface topography with scanning electron microscopy. I. EZEImage: a program to obtain 3D surface data.

    PubMed

    Ponz, Ezequiel; Ladaga, Juan Luis; Bonetto, Rita Dominga

    2006-04-01

    Scanning electron microscopy (SEM) is widely used in the science of materials and different parameters were developed to characterize the surface roughness. In a previous work, we studied the surface topography with fractal dimension at low scale and two parameters at high scale by using the variogram, that is, variance vs. step log-log graph, of a SEM image. Those studies were carried out with the FERImage program, previously developed by us. To verify the previously accepted hypothesis by working with only an image, it is indispensable to have reliable three-dimensional (3D) surface data. In this work, a new program (EZEImage) to characterize 3D surface topography in SEM has been developed. It uses fast cross correlation and dynamic programming to obtain reliable dense height maps in a few seconds which can be displayed as an image where each gray level represents a height value. This image can be used for the FERImage program or any other software to obtain surface topography characteristics. EZEImage also generates anaglyph images as well as characterizes 3D surface topography by means of a parameter set to describe amplitude properties and three functional indices for characterizing bearing and fluid properties.

  9. Web-based volume slicer for 3D electron-microscopy data from EMDB

    PubMed Central

    Salavert-Torres, José; Iudin, Andrii; Lagerstedt, Ingvar; Sanz-García, Eduardo; Kleywegt, Gerard J.; Patwardhan, Ardan

    2016-01-01

    We describe the functionality and design of the Volume slicer – a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale. PMID:26876163

  10. Efficient Semi-Automatic 3D Segmentation for Neuron Tracing in Electron Microscopy Images

    PubMed Central

    Jones, Cory; Liu, Ting; Cohan, Nathaniel Wood; Ellisman, Mark; Tasdizen, Tolga

    2015-01-01

    0.1. Background In the area of connectomics, there is a significant gap between the time required for data acquisition and dense reconstruction of the neural processes contained in the same dataset. Automatic methods are able to eliminate this timing gap, but the state-of-the-art accuracy so far is insufficient for use without user corrections. If completed naively, this process of correction can be tedious and time consuming. 0.2. New Method We present a new semi-automatic method that can be used to perform 3D segmentation of neurites in EM image stacks. It utilizes an automatic method that creates a hierarchical structure for recommended merges of superpixels. The user is then guided through each predicted region to quickly identify errors and establish correct links. 0.3. Results We tested our method on three datasets with both novice and expert users. Accuracy and timing were compared with published automatic, semi-automatic, and manual results. 0.4. Comparison with Existing Methods Post-automatic correction methods have also been used in [1] and [2]. These methods do not provide navigation or suggestions in the manner we present. Other semi-automatic methods require user input prior to the automatic segmentation such as [3] and [4] and are inherently different than our method. 0.5. Conclusion Using this method on the three datasets, novice users achieved accuracy exceeding state-of-the-art automatic results, and expert users achieved accuracy on par with full manual labeling but with a 70% time improvement when compared with other examples in publication. PMID:25769273

  11. Efficient semi-automatic 3D segmentation for neuron tracing in electron microscopy images.

    PubMed

    Jones, Cory; Liu, Ting; Cohan, Nathaniel Wood; Ellisman, Mark; Tasdizen, Tolga

    2015-05-15

    In the area of connectomics, there is a significant gap between the time required for data acquisition and dense reconstruction of the neural processes contained in the same dataset. Automatic methods are able to eliminate this timing gap, but the state-of-the-art accuracy so far is insufficient for use without user corrections. If completed naively, this process of correction can be tedious and time consuming. We present a new semi-automatic method that can be used to perform 3D segmentation of neurites in EM image stacks. It utilizes an automatic method that creates a hierarchical structure for recommended merges of superpixels. The user is then guided through each predicted region to quickly identify errors and establish correct links. We tested our method on three datasets with both novice and expert users. Accuracy and timing were compared with published automatic, semi-automatic, and manual results. Post-automatic correction methods have also been used in Mishchenko et al. (2010) and Haehn et al. (2014). These methods do not provide navigation or suggestions in the manner we present. Other semi-automatic methods require user input prior to the automatic segmentation such as Jeong et al. (2009) and Cardona et al. (2010) and are inherently different than our method. Using this method on the three datasets, novice users achieved accuracy exceeding state-of-the-art automatic results, and expert users achieved accuracy on par with full manual labeling but with a 70% time improvement when compared with other examples in publication. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. A correlative approach for combining microCT, light and transmission electron microscopy in a single 3D scenario

    PubMed Central

    2013-01-01

    Background In biomedical research, a huge variety of different techniques is currently available for the structural examination of small specimens, including conventional light microscopy (LM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), microscopic X-ray computed tomography (microCT), and many others. Since every imaging method is physically limited by certain parameters, a correlative use of complementary methods often yields a significant broader range of information. Here we demonstrate the advantages of the correlative use of microCT, light microscopy, and transmission electron microscopy for the analysis of small biological samples. Results We used a small juvenile bivalve mollusc (Mytilus galloprovincialis, approximately 0.8 mm length) to demonstrate the workflow of a correlative examination by microCT, LM serial section analysis, and TEM-re-sectioning. Initially these three datasets were analyzed separately, and subsequently they were fused in one 3D scene. This workflow is very straightforward. The specimen was processed as usual for transmission electron microscopy including post-fixation in osmium tetroxide and embedding in epoxy resin. Subsequently it was imaged with microCT. Post-fixation in osmium tetroxide yielded sufficient X-ray contrast for microCT imaging, since the X-ray absorption of epoxy resin is low. Thereafter, the same specimen was serially sectioned for LM investigation. The serial section images were aligned and specific organ systems were reconstructed based on manual segmentation and surface rendering. According to the region of interest (ROI), specific LM sections were detached from the slides, re-mounted on resin blocks and re-sectioned (ultrathin) for TEM. For analysis, image data from the three different modalities was co-registered into a single 3D scene using the software AMIRA®. We were able to register both the LM section series volume and TEM slices neatly to the microCT dataset, with

  13. Structure-Function Studies of Blood and Air Capillaries in Chicken Lung Using 3D Electron Microscopy

    PubMed Central

    West, John B.; Fu, Zhenxing; Deerinck, Thomas J.; Mackey, Mason R.; Obayashi, James T.; Ellisman, Mark H.

    2010-01-01

    Avian pulmonary capillaries differ from those of mammals in three important ways. The blood-gas barrier is much thinner, it is more uniform in thickness, and the capillaries are far more rigid when their transmural pressure is altered. The thinness of the barrier is surprising because it predisposes the capillaries to stress failure. A possible mechanism for these differences is that avian pulmonary capillaries, unlike mammalian, are supported from the outside by air capillaries, but the details of the support are poorly understood. To clarify this we studied the blood and air capillaries in chicken lung using transmission electron microscopy (EM) and two relatively new techniques that allow 3D visualization: electron tomography and serial block-face scanning EM. These studies show that the pulmonary capillaries are flanked by epithelial bridges composed of two extremely thin epithelial cells with large surface areas. The junctions of the bridges with the capillary walls show thickening of the epithelial cells and an accumulation of extracellular matrix. Collapse of the pulmonary capillaries when the pressure outside them is increased is apparently prevented by the guy wire-like action of the epithelial bridges. The enlarged junctions between the bridges and the walls could provide a mechanism that limits the hoop stress in the capillary walls when the pressure inside them is increased. The support of the pulmonary capillaries may also be explained by an interdependence mechanism whereby the capillaries are linked to a rigid assemblage of air capillaries. These EM studies show the supporting structures in greater detail than has previously been possible, particularly in 3D, and they allow a more complete analysis of the mechanical forces affecting avian pulmonary capillaries. PMID:20038456

  14. Multi-scale 3D investigations of a commercial 18650 Li-ion battery with correlative electron- and X-ray microscopy

    NASA Astrophysics Data System (ADS)

    Gelb, Jeff; Finegan, Donal P.; Brett, Dan J. L.; Shearing, Paul R.

    2017-07-01

    In the present study, a commercial 18650 Li-ion cylindrical cell is investigated with non-destructive 3D X-ray microscopy across a range of length scales, beginning with a survey of the entire cell and non-destructively enlarging a smaller section. Active materials are extracted from a disassembled cell and imaging performed using a combination of sub-micron X-ray microscopy and 2D scanning-electron microscopy, which point toward the need for multi-scale analysis in order to accurately characterize the cell. Furthermore, a small section is physically isolated for 3D nano-scale X-ray microscopy, which provides a measurement of porosity and enables the effective diffusivity and 3-dimensional tortuosities to be calculated via computer simulation. Finally, the 3D X-ray microscopy data is loaded into a correlative microscopy environment, where a representative sub-surface region is identified and, subsequently, analyzed using electron microscopy and energy-dispersive X-ray spectroscopy. The results of this study elucidate the microstructural characteristics and potential degradation mechanisms of a commercial NCA battery and, further, establish a technique for extracting the Bruggeman exponent for a real-world microstructure using correlative microscopy.

  15. 3D reconstruction of VZV infected cell nuclei and PML nuclear cages by serial section array scanning electron microscopy and electron tomography.

    PubMed

    Reichelt, Mike; Joubert, Lydia; Perrino, John; Koh, Ai Leen; Phanwar, Ibanri; Arvin, Ann M

    2012-01-01

    Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell

  16. 3D Reconstruction of VZV Infected Cell Nuclei and PML Nuclear Cages by Serial Section Array Scanning Electron Microscopy and Electron Tomography

    PubMed Central

    Reichelt, Mike; Joubert, Lydia; Perrino, John; Koh, Ai Leen; Phanwar, Ibanri; Arvin, Ann M.

    2012-01-01

    Varicella-zoster virus (VZV) is a human alphaherpesvirus that causes varicella (chickenpox) and herpes zoster (shingles). Like all herpesviruses, the VZV DNA genome is replicated in the nucleus and packaged into nucleocapsids that must egress across the nuclear membrane for incorporation into virus particles in the cytoplasm. Our recent work showed that VZV nucleocapsids are sequestered in nuclear cages formed from promyelocytic leukemia protein (PML) in vitro and in human dorsal root ganglia and skin xenografts in vivo. We sought a method to determine the three-dimensional (3D) distribution of nucleocapsids in the nuclei of herpesvirus-infected cells as well as the 3D shape, volume and ultrastructure of these unique PML subnuclear domains. Here we report the development of a novel 3D imaging and reconstruction strategy that we term Serial Section Array-Scanning Electron Microscopy (SSA-SEM) and its application to the analysis of VZV-infected cells and these nuclear PML cages. We show that SSA-SEM permits large volume imaging and 3D reconstruction at a resolution sufficient to localize, count and distinguish different types of VZV nucleocapsids and to visualize complete PML cages. This method allowed a quantitative determination of how many nucleocapsids can be sequestered within individual PML cages (sequestration capacity), what proportion of nucleocapsids are entrapped in single nuclei (sequestration efficiency) and revealed the ultrastructural detail of the PML cages. More than 98% of all nucleocapsids in reconstructed nuclear volumes were contained in PML cages and single PML cages sequestered up to 2,780 nucleocapsids, which were shown by electron tomography to be embedded and cross-linked by an filamentous electron-dense meshwork within these unique subnuclear domains. This SSA-SEM analysis extends our recent characterization of PML cages and provides a proof of concept for this new strategy to investigate events during virion assembly at the single cell

  17. Visualizing the 3D Architecture of Multiple Erythrocytes Infected with Plasmodium at Nanoscale by Focused Ion Beam-Scanning Electron Microscopy

    PubMed Central

    Soares Medeiros, Lia Carolina; De Souza, Wanderley; Jiao, Chengge; Barrabin, Hector; Miranda, Kildare

    2012-01-01

    Different methods for three-dimensional visualization of biological structures have been developed and extensively applied by different research groups. In the field of electron microscopy, a new technique that has emerged is the use of a focused ion beam and scanning electron microscopy for 3D reconstruction at nanoscale resolution. The higher extent of volume that can be reconstructed with this instrument represent one of the main benefits of this technique, which can provide statistically relevant 3D morphometrical data. As the life cycle of Plasmodium species is a process that involves several structurally complex developmental stages that are responsible for a series of modifications in the erythrocyte surface and cytoplasm, a high number of features within the parasites and the host cells has to be sampled for the correct interpretation of their 3D organization. Here, we used FIB-SEM to visualize the 3D architecture of multiple erythrocytes infected with Plasmodium chabaudi and analyzed their morphometrical parameters in a 3D space. We analyzed and quantified alterations on the host cells, such as the variety of shapes and sizes of their membrane profiles and parasite internal structures such as a polymorphic organization of hemoglobin-filled tubules. The results show the complex 3D organization of Plasmodium and infected erythrocyte, and demonstrate the contribution of FIB-SEM for the obtainment of statistical data for an accurate interpretation of complex biological structures. PMID:22432024

  18. Correlative two-photon and serial block face scanning electron microscopy in neuronal tissue using 3D near-infrared branding maps.

    PubMed

    Lees, Robert M; Peddie, Christopher J; Collinson, Lucy M; Ashby, Michael C; Verkade, Paul

    2017-01-01

    Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM). This provides a snapshot of high resolution structural information pertaining to the shape, organization, and localization of multiple subcellular structures at the same time. The pairing of these two imaging modalities in the same specimen provides key information to relate cellular dynamics to the ultrastructural environment. Until recently, approaches to relocate a region of interest (ROI) in tissue from 2P microscopy for SBF-SEM have been inefficient or unreliable. However, near-infrared branding (NIRB) overcomes this by using the laser from a multiphoton microscope to create fiducial markers for accurate correlation of 2P and electron microscopy (EM) imaging volumes. The process is quick and can be user defined for each sample. Here, to increase the efficiency of ROI relocation, multiple NIRB marks are used in 3D to target ultramicrotomy. A workflow is described and discussed to obtain a data set for 3D correlated light and electron microscopy, using three different preparations of brain tissue as examples. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. 3D Viscoelastic traction force microscopy.

    PubMed

    Toyjanova, Jennet; Hannen, Erin; Bar-Kochba, Eyal; Darling, Eric M; Henann, David L; Franck, Christian

    2014-10-28

    Native cell-material interactions occur on materials differing in their structural composition, chemistry, and physical compliance. While the last two decades have shown the importance of traction forces during cell-material interactions, they have been almost exclusively presented on purely elastic in vitro materials. Yet, most bodily tissue materials exhibit some level of viscoelasticity, which could play an important role in how cells sense and transduce tractions. To expand the realm of cell traction measurements and to encompass all materials from elastic to viscoelastic, this paper presents a general, and comprehensive approach for quantifying 3D cell tractions in viscoelastic materials. This methodology includes the experimental characterization of the time-dependent material properties for any viscoelastic material with the subsequent mathematical implementation of the determined material model into a 3D traction force microscopy (3D TFM) framework. Utilizing this new 3D viscoelastic TFM (3D VTFM) approach, we quantify the influence of viscosity on the overall material traction calculations and quantify the error associated with omitting time-dependent material effects, as is the case for all other TFM formulations. We anticipate that the 3D VTFM technique will open up new avenues of cell-material investigations on even more physiologically relevant time-dependent materials including collagen and fibrin gels.

  20. How precise can atoms of a nanocluster be located in 3D using a tilt series of scanning transmission electron microscopy images?

    PubMed

    Alania, M; De Backer, A; Lobato, I; Krause, F F; Van Dyck, D; Rosenauer, A; Van Aert, S

    2017-10-01

    In this paper, we investigate how precise atoms of a small nanocluster can ultimately be located in three dimensions (3D) from a tilt series of images acquired using annular dark field (ADF) scanning transmission electron microscopy (STEM). Therefore, we derive an expression for the statistical precision with which the 3D atomic position coordinates can be estimated in a quantitative analysis. Evaluating this statistical precision as a function of the microscope settings also allows us to derive the optimal experimental design. In this manner, the optimal angular tilt range, required electron dose, optimal detector angles, and number of projection images can be determined. Copyright © 2016 Elsevier B.V. All rights reserved.

  1. 3D imaging of cells and tissues by focused ion beam/scanning electron microscopy (FIB/SEM).

    PubMed

    Drobne, Damjana

    2013-01-01

    Integration of a scanning electron microscope (SEM) and focused ion beam (FIB) technology into a single FIB/SEM system permits use of the FIB as a nano-scalpel to reveal site-specific subsurface microstructures which can be examined in great detail by SEM. The FIB/SEM technology is widely used in the semiconductor industry and material sciences, and recently its use in the life sciences has been initiated. Samples for FIB/SEM investigation can be either embedded in a plastic matrix, the traditional means of preparation of transmission electron microscopy (TEM) specimens, or simply dried as in samples prepared for SEM imaging. Currently, FIB/SEM is used in the life sciences for (a) preparation by the lift-out technique of lamella for TEM analysis, (b) tomography of samples embedded in a matrix, and (c) in situ site-specific FIB milling and SEM imaging using a wide range of magnifications. Site-specific milling and imaging has attracted wide interest as a technique in structural research of single eukaryotic and prokaryotic cells, small animals, and different animal tissue, but it still remains to be explored more thoroughly. In the past, preparation of samples for site-specific milling and imaging by FIB/SEM has typically adopted the embedding techniques used for TEM samples, and which have been very well described in the literature. Sample preparation protocols for the use of dried samples in FIB/SEM have been less well investigated. The aim of this chapter is to encourage application of FIB/SEM on dried biological samples. A detailed description of conventional dried sample preparation and FIB/SEM investigation of dried biological samples is presented. The important steps are described and illustrated, and direct comparison between embedded and dried samples of same tissues is provided. The ability to discover links between gross morphology of the tissue or organ, surface characteristics of any selected region, and intracellular structural details on the nanometer

  2. 3D microscopy - new powerful tools in geomaterials characterization

    NASA Astrophysics Data System (ADS)

    Mauko Pranjić, Alenka; Mladenovič, Ana; Turk, Janez; Šajna, Aljoša; Čretnik, Janko

    2016-04-01

    Microtomography (microCT) is becoming more and more widely recognized in geological sciences as a powerful tool for the spatial characterization of rock and other geological materials. Together with 3D image analysis and other complementary techniques, it has the characteristics of an innovative and non-destructive 3D microscopical technique. On the other hand its main disadvantages are low availability (only a few geological laboratories are equipped with high resolution tomographs), the relatively high prices of testing connected with the use of an xray source, technical limitations connected to the resolution and imaging of certain materials, as well as timeconsuming and complex 3D image analysis, necessary for quantification of 3D tomographic data sets. In this work three examples are presented of optimal 3D microscopy analysis of geomaterials in construction such as porosity characterization of impregnated sandstone, aerated concrete and marble prone to bowing. Studies include processes of microCT imaging, 3D data analysis and fitting of data with complementary analysis, such as confocal microscopy, mercury porosimetry, gas sorption, optical/fluorescent microscopy and scanning electron microscopy. Present work has been done in the frame of national research project 3D and 4D microscopy development of new powerful tools in geosciences (ARRS J1-7148) funded by Slovenian Research Agency.

  3. Patterns of organelle ontogeny through a cell cycle revealed by whole-cell reconstructions using 3D electron microscopy.

    PubMed

    Hughes, Louise; Borrett, Samantha; Towers, Katie; Starborg, Tobias; Vaughan, Sue

    2017-02-01

    The major mammalian bloodstream form of the African sleeping sickness parasite Trypanosoma brucei multiplies rapidly, and it is important to understand how these cells divide. Organelle inheritance involves complex spatiotemporal re-arrangements to ensure correct distribution to daughter cells. Here, serial block face scanning electron microscopy (SBF-SEM) was used to reconstruct whole individual cells at different stages of the cell cycle to give an unprecedented temporal, spatial and quantitative view of organelle division, inheritance and abscission in a eukaryotic cell. Extensive mitochondrial branching occurred only along the ventral surface of the parasite, but the mitochondria returned to a tubular form during cytokinesis. Fission of the mitochondrion occurred within the cytoplasmic bridge during the final stage of cell division, correlating with cell abscission. The nuclei were located underneath each flagellum at mitosis and the mitotic spindle was located along the ventral surface, further demonstrating the asymmetric arrangement of cell cleavage in trypanosomes. Finally, measurements demonstrated that multiple Golgi bodies were accurately positioned along the flagellum attachment zone, suggesting a mechanism for determining the location of Golgi bodies along each flagellum during the cell cycle. © 2017. Published by The Company of Biologists Ltd.

  4. From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

    PubMed Central

    Spiegelhalter, Coralie; Tosch, Valérie; Hentsch, Didier; Koch, Marc; Kessler, Pascal; Schwab, Yannick; Laporte, Jocelyn

    2010-01-01

    Background In cell biology, the study of proteins and organelles requires the combination of different imaging approaches, from live recordings with light microscopy (LM) to electron microscopy (EM). Methodology To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization. We provide examples of the dynamic oligomerization of GFP-tagged myotubularin (MTM1) phosphoinositides phosphatase induced by osmotic stress, and of the ultrastructure of membrane tubules dependent on amphiphysin 2 (BIN1) expression. Conclusion Accessible and versatile, we show that this approach is efficient to routinely correlate functional and dynamic LM with high resolution morphology by EM, with immuno-EM labeling, with 3D reconstruction using serial immuno-EM or tomography, and with scanning-EM. PMID:20140253

  5. Synthesis of solid textures based on a 2D example: application to the synthesis of 3D carbon structures observed by transmission electronic microscopy

    NASA Astrophysics Data System (ADS)

    Da Costa, Jean-Pierre; Germain, Christian

    2010-01-01

    We propose a novel parametric approach which aims at the synthesis of anisotropic solid textures from the analysis of a single 2D exemplar. This approach is an extension of the pyramidal scheme of Portilla and Simoncelli. It proceeds in three main steps: first, a 2D analysis of the example is performed which produces a set of reference statistics. Then, 3D reference statistics are inferred from the 2D ones thanks to specific anisotropy assumptions. The final step aims at the synthesis itself: the 3D target statistics are imposed on a random 3D block according to a specific multi resolution pyramidal scheme. The approach is applied to the synthesis of solid textures representative of the structure of dense pre-graphitic carbons. The samples are lattice fringe images obtained by high resolution transmission electronic microscopy (HRTEM). HRTEM samples with increasing structural order are used for the experimental evaluation. The produced solid textures exhibit anisotropy properties similar to those observed in the HRTEM samples. Such an approach can easily be extended to any 3D anisotropic structures showing stacks of layers such as wood grain images, seismic data, etc.

  6. Electron Microscopy.

    ERIC Educational Resources Information Center

    Beer, Michael

    1980-01-01

    Reviews technical aspects of structure determination in biological electron microscopy (EM). Discusses low dose EM, low temperature microscopy, electron energy loss spectra, determination of mass or molecular weight, and EM of labeled systems. Cites 34 references. (CS)

  7. 3D analysis of synaptic vesicle density and distribution after acute foot-shock stress by using serial section transmission electron microscopy.

    PubMed

    Khanmohammadi, M; Darkner, S; Nava, N; Nyengaard, J R; Wegener, G; Popoli, M; Sporring, J

    2017-01-01

    Behavioural stress has shown to strongly affect neurotransmission within the neocortex. In this study, we analysed the effect of an acute stress model on density and distribution of neurotransmitter-containing vesicles within medial prefrontal cortex. Serial section transmission electron microscopy was employed to compare two groups of male rats: (1) rats subjected to foot-shock stress and (2) rats with sham stress as control group. Two-dimensional (2D) density measures are common in microscopic images and are estimated by following a 2D path in-section. However, this method ignores the slant of the active zone and thickness of the section. In fact, the active zone is a surface in three-dimension (3D) and the 2D measures do not accurately reflect the geometric configuration unless the active zone is perpendicular to the sectioning angle. We investigated synaptic vesicle density as a function of distance from the active zone in 3D. We reconstructed a 3D dataset by estimating the thickness of all sections and by registering all the image sections into a common coordinate system. Finally, we estimated the density as the average number of vesicles per area and volume and modelled the synaptic vesicle distribution by fitting a one-dimensional parametrized distribution that took into account the location uncertainty due to section thickness. Our results showed a clear structural difference in synaptic vesicle density and distribution between stressed and control group with improved separation by 3D measures in comparison to the 2D measures. Our results showed that acute foot-shock stress exposure significantly affected both the spatial distribution and density of the synaptic vesicles within the presynaptic terminal.

  8. Multi-class maximum likelihood symmetry determination and motif reconstruction of 3-D helical objects from projection images for electron microscopy

    PubMed Central

    Lee, Seunghee; Johnson, John E.

    2011-01-01

    Many micro- to nano-scale 3-D biological objects have a helical symmetry. Cryo electron microscopy provides 2-D projection images where, however, the images have low SNR and unknown projection directions. The object is described as a helical array of identical motifs, where both the parameters of the helical symmetry and the motif are unknown. Using a detailed image formation model, a maximum likelihood estimator for the parameters of the symmetry and the 3-D motif based on images of many objects and algorithms for computing the estimate are described. The possibility that the objects are not identical but rather come from a small set of homogeneous classes is included. The first example is based on 316 128×100 pixel experimental images of Tobacco Mosaic Virus, has one class, and achieves 12.40Å spatial resolution in the reconstruction. The second example is based on 400 128 × 128 pixel synthetic images of helical objects constructed from NaK ion channel pore macromolecular complexes, has two classes differing in helical symmetry, and achieves 7.84Å and 7.90Å spatial resolution in the reconstructions for the two classes. PMID:21335314

  9. 3D differential phase contrast microscopy

    NASA Astrophysics Data System (ADS)

    Chen, Michael; Tian, Lei; Waller, Laura

    2016-03-01

    We demonstrate three-dimensional (3D) optical phase and amplitude reconstruction based on coded source illumination using a programmable LED array. Multiple stacks of images along the optical axis are computed from recorded intensities captured by multiple images under off-axis illumination. Based on the first Born approximation, a linear differential phase contrast (DPC) model is built between 3D complex index of refraction and the intensity stacks. Therefore, 3D volume reconstruction can be achieved via a fast inversion method, without the intermediate 2D phase retrieval step. Our system employs spatially partially coherent illumination, so the transverse resolution achieves twice the NA of coherent systems, while axial resolution is also improved 2× as compared to holographic imaging.

  10. Simple buffers for 3D STORM microscopy.

    PubMed

    Olivier, Nicolas; Keller, Debora; Rajan, Vinoth Sundar; Gönczy, Pierre; Manley, Suliana

    2013-06-01

    3D STORM is one of the leading methods for super-resolution imaging, with resolution down to 10 nm in the lateral direction, and 30-50 nm in the axial direction. However, there is one important requirement to perform this type of imaging: making dye molecules blink. This usually relies on the utilization of complex buffers, containing different chemicals and sensitive enzymatic systems, limiting the reproducibility of the method. We report here that the commercial mounting medium Vectashield can be used for STORM of Alexa-647, and yields images comparable or superior to those obtained with more complex buffers, especially for 3D imaging. We expect that this advance will promote the versatile utilization of 3D STORM by removing one of its entry barriers, as well as provide a more reproducible way to compare optical setups and data processing algorithms.

  11. X-ray fluorescence (conventional and 3D) and scanning electron microscopy for the investigation of Portuguese polychrome glazed ceramics: Advances in the knowledge of the manufacturing techniques

    NASA Astrophysics Data System (ADS)

    Guilherme, A.; Coroado, J.; dos Santos, J. M. F.; Lühl, L.; Wolff, T.; Kanngießer, B.; Carvalho, M. L.

    2011-05-01

    This work shows the first analytical results obtained by X-Ray Fluorescence (XRF) (conventional and 3D) and Scanning Electron Microscopy with Energy Dispersive System (SEM-EDS) on original Portuguese ceramic pieces produced between the 16th and 18th centuries in Coimbra and Lisbon. Experts distinguished these productions based only on the color, texture and brightness, which originates mislabeling in some cases. Thanks to lateral and spatial resolution in the micrometer regime, the results obtained with μ-XRF were essential in determining the glaze and pigment thicknesses by monitoring the profile of the most abundant element in each "layer". Furthermore, the dissemination of these elements throughout the glaze is different depending on the glaze composition, firing temperature and on the pigment itself. Hence, the crucial point of this investigation was to analyze and understand the interfaces color/glaze and glaze/ceramic support. Together with the XRF results, images captured by SEM and the corresponding semi-quantitative EDS data revealed different manufacturing processes used by the two production centers. Different capture modes were suitable to distinguish different crystals from the minerals that confer the color of the pigments used and to enhance the fact that some of them are very well spread through the glassy matrix, sustaining the theory of an evolved and careful procedure in the manufacturing process of the glaze.

  12. 3D differential phase contrast microscopy

    PubMed Central

    Chen, Michael; Tian, Lei; Waller, Laura

    2016-01-01

    We demonstrate 3D phase and absorption recovery from partially coherent intensity images captured with a programmable LED array source. Images are captured through-focus with four different illumination patterns. Using first Born and weak object approximations (WOA), a linear 3D differential phase contrast (DPC) model is derived. The partially coherent transfer functions relate the sample’s complex refractive index distribution to intensity measurements at varying defocus. Volumetric reconstruction is achieved by a global FFT-based method, without an intermediate 2D phase retrieval step. Because the illumination is spatially partially coherent, the transverse resolution of the reconstructed field achieves twice the NA of coherent systems and improved axial resolution. PMID:27867705

  13. Resolution improvement by 3D particle averaging in localization microscopy

    NASA Astrophysics Data System (ADS)

    Broeken, Jordi; Johnson, Hannah; Lidke, Diane S.; Liu, Sheng; Nieuwenhuizen, Robert P. J.; Stallinga, Sjoerd; Lidke, Keith A.; Rieger, Bernd

    2015-03-01

    Inspired by recent developments in localization microscopy that applied averaging of identical particles in 2D for increasing the resolution even further, we discuss considerations for alignment (registration) methods for particles in general and for 3D in particular. We detail that traditional techniques for particle registration from cryo electron microscopy based on cross-correlation are not suitable, as the underlying image formation process is fundamentally different. We argue that only localizations, i.e. a set of coordinates with associated uncertainties, are recorded and not a continuous intensity distribution. We present a method that owes to this fact and that is inspired by the field of statistical pattern recognition. In particular we suggest to use an adapted version of the Bhattacharyya distance as a merit function for registration. We evaluate the method in simulations and demonstrate it on 3D super-resolution data of Alexa 647 labelled to the Nup133 protein in the nuclear pore complex of Hela cells. From the simulations we find suggestions that for successful registration the localization uncertainty must be smaller than the distance between labeling sites on a particle. These suggestions are supported by theoretical considerations concerning the attainable resolution in localization microscopy and its scaling behavior as a function of labeling density and localization precision.

  14. Frames-Based Denoising in 3D Confocal Microscopy Imaging.

    PubMed

    Konstantinidis, Ioannis; Santamaria-Pang, Alberto; Kakadiaris, Ioannis

    2005-01-01

    In this paper, we propose a novel denoising method for 3D confocal microscopy data based on robust edge detection. Our approach relies on the construction of a non-separable frame system in 3D that incorporates the Sobel operator in dual spatial directions. This multidirectional set of digital filters is capable of robustly detecting edge information by ensemble thresholding of the filtered data. We demonstrate the application of our method to both synthetic and real confocal microscopy data by comparing it to denoising methods based on separable 3D wavelets and 3D median filtering, and report very encouraging results.

  15. Towards Single Cell Traction Microscopy within 3D Collagen Matrices

    PubMed Central

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-01-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cells migration within collagen gels. PMID:23806281

  16. Toward single cell traction microscopy within 3D collagen matrices.

    PubMed

    Hall, Matthew S; Long, Rong; Feng, Xinzeng; Huang, Yuling; Hui, Chung-Yuen; Wu, Mingming

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. © 2013 Elsevier Inc. All rights reserved.

  17. Microscopy in 3D: a biologist’s toolbox

    PubMed Central

    Fischer, Robert S.; Wu, Yicong; Kanchanawong, Pakorn; Shroff, Hari; Waterman, Clare M.

    2012-01-01

    The power of fluorescence microscopy to study cellular structures and macromolecular complexes spans a wide range of size scales, from studies of cell behavior and function in physiological, three-dimensional (3D) environments, to understanding the molecular architecture of organelles. At each length scale, the challenge in 3D imaging is to extract the most spatial and temporal resolution possible while limiting photodamage/bleaching to living cells. A number of advancements in 3D fluorescence microscopy now offer higher resolution, improved speed, and reduced photobleaching relative to traditional point-scanning microscopy methods. Here, we discuss a few specific microscopy modalities that we believe will be particularly advantageous in imaging cells and subcellular structures in physiologically relevant 3D environments. PMID:22047760

  18. Adaptive optics enables 3D STED microscopy in aberrating specimens

    PubMed Central

    Gould, Travis J.; Burke, Daniel; Bewersdorf, Joerg; Booth, Martin J.

    2012-01-01

    Stimulated emission depletion (STED) microscopy allows fluorescence far-field imaging with diffraction-unlimited resolution. Unfortunately, extending this technique to three-dimensional (3D) imaging of thick specimens has been inhibited by sample-induced aberrations. Here we present the first implementation of adaptive optics in STED microscopy to allow 3D super-resolution imaging in strongly aberrated imaging conditions, such as those introduced by thick biological tissue. PMID:23037223

  19. Ultra-thin resin embedding method for scanning electron microscopy of individual cells on high and low aspect ratio 3D nanostructures.

    PubMed

    Belu, A; Schnitker, J; Bertazzo, S; Neumann, E; Mayer, D; Offenhäusser, A; Santoro, F

    2016-07-01

    The preparation of biological cells for either scanning or transmission electron microscopy requires a complex process of fixation, dehydration and drying. Critical point drying is commonly used for samples investigated with a scanning electron beam, whereas resin-infiltration is typically used for transmission electron microscopy. Critical point drying may cause cracks at the cellular surface and a sponge-like morphology of nondistinguishable intracellular compartments. Resin-infiltrated biological samples result in a solid block of resin, which can be further processed by mechanical sectioning, however that does not allow a top view examination of small cell-cell and cell-surface contacts. Here, we propose a method for removing resin excess on biological samples before effective polymerization. In this way the cells result to be embedded in an ultra-thin layer of epoxy resin. This novel method highlights in contrast to standard methods the imaging of individual cells not only on nanostructured planar surfaces but also on topologically challenging substrates with high aspect ratio three-dimensional features by scanning electron microscopy. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

  20. Toward single cell traction microscopy within 3D collagen matrices

    SciTech Connect

    Hall, Matthew S.; Long, Rong; Feng, Xinzeng; Huang, YuLing; Hui, Chung-Yuen; Wu, Mingming

    2013-10-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion, and migration. Cells require the three-dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, current understanding of cell–ECM and cell–cell mechanical interactions is largely derived from 2D cell traction force microscopy, in which cells are cultured on a flat substrate. 3D cell traction microscopy is emerging for mapping traction fields of single animal cells embedded in either synthetic or natively derived fibrous gels. We discuss here the development of 3D cell traction microscopy, its current limitations, and perspectives on the future of this technology. Emphasis is placed on strategies for applying 3D cell traction microscopy to individual tumor cell migration within collagen gels. - Highlights: • Review of the current state of the art in 3D cell traction force microscopy. • Bulk and micro-characterization of remodelable fibrous collagen gels. • Strategies for performing 3D cell traction microscopy within collagen gels.

  1. A novel 2D and 3D method for automated insulin granule measurement and its application in assessing accepted preparation methods for electron microscopy

    NASA Astrophysics Data System (ADS)

    Mantell, J.; Nam, D.; Bull, D.; Achim, A.; Verkade, P.

    2014-06-01

    Transmission electron microscopy images of insulin-producing beta cells in the islets of Langerhans contain many complex structures, making it difficult to accurately segment insulin granules. Furthermore the appearance of the granules and surrounding halo and limiting membrane can vary enormously depending on the methods used for sample preparation. An automated method has been developed using active contours to segment the insulin core initially and then expand to segment the halos [1]. The method has been validated against manual measurements and also yields higher accuracy than other automated methods [2]. It has then been extended to three dimensions to analyse a tomographic reconstruction from a thick section of the same material. The final step has been to produce a GUI and use the automated process to compare a number of different electron microscopy preparation protocols including chemical fixation (where many of halos are often distended) and to explore the many subtleties of high pressure freezing (where the halos are often minimal, [3]).

  2. Find your way with X-Ray: Using microCT to correlate in vivo imaging with 3D electron microscopy.

    PubMed

    Karreman, Matthia A; Ruthensteiner, Bernhard; Mercier, Luc; Schieber, Nicole L; Solecki, Gergely; Winkler, Frank; Goetz, Jacky G; Schwab, Yannick

    2017-01-01

    Combining in vivo imaging with electron microscopy (EM) uniquely allows monitoring rare and critical events in living tissue, followed by their high-resolution visualization in their native context. A major hurdle, however, is to keep track of the region of interest (ROI) when moving from intravital microscopy (IVM) to EM. Here, we present a workflow that relies on correlating IVM and microscopic X-ray computed tomography to predict the position of the ROI inside the EM-processed sample. The ROI can then be accurately and quickly targeted using ultramicrotomy and imaged using EM. We outline how this procedure is used to retrieve and image tumor cells arrested in the vasculature of the mouse brain. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Hybrid 3D Printing of Soft Electronics.

    PubMed

    Valentine, Alexander D; Busbee, Travis A; Boley, John William; Raney, Jordan R; Chortos, Alex; Kotikian, Arda; Berrigan, John Daniel; Durstock, Michael F; Lewis, Jennifer A

    2017-09-06

    Hybrid 3D printing is a new method for producing soft electronics that combines direct ink writing of conductive and dielectric elastomeric materials with automated pick-and-place of surface mount electronic components within an integrated additive manufacturing platform. Using this approach, insulating matrix and conductive electrode inks are directly printed in specific layouts. Passive and active electrical components are then integrated to produce the desired electronic circuitry by using an empty nozzle (in vacuum-on mode) to pick up individual components, place them onto the substrate, and then deposit them (in vacuum-off mode) in the desired location. The components are then interconnected via printed conductive traces to yield soft electronic devices that may find potential application in wearable electronics, soft robotics, and biomedical devices. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. 3D Micro-topography of Transferred Laboratory and Natural Ice Crystal Surfaces Imaged by Cryo and Environmental Scanning Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Magee, N. B.; Boaggio, K.; Bancroft, L.; Bandamede, M.

    2015-12-01

    Recent work has highlighted micro-scale roughness on the surfaces of ice crystals grown and imaged in-situ within the chambers of environmental scanning electron microscopes (ESEM). These observations appear to align with theoretical and satellite observations that suggest a prevalence of rough ice in cirrus clouds. However, the atmospheric application of the lab observations are indeterminate because the observations have been based only on crystals grown on substrates and in pure-water vapor environments. In this work, we present details and results from the development of a transfer technique which allows natural and lab-grown ice and snow crystals to be captured, preserved, and transferred into the ESEM for 3D imaging. Ice crystals were gathered from 1) natural snow, 2) a balloon-borne cirrus particle capture device, and 3) lab-grown ice crystals from a diffusion chamber. Ice crystals were captured in a pre-conditioned small-volume (~1 cm3) cryo-containment cell. The cell was then sealed closed and transferred to a specially-designed cryogenic dewer (filled with liquid nitrogen or crushed dry ice) for transport to a new Hitachi Field Emission, Variable Pressure SEM (SU-5000). The cryo-cell was then removed from the dewer and quickly placed onto the pre-conditioned cryo transfer stage attached to the ESEM (Quorum 3010T). Quantitative 3D topographical digital elevation models of ice surfaces are reported from SEM for the first time, including a variety of objective measures of statistical surface roughness. The surfaces of the transported crystals clearly exhibit signatures of mesoscopic roughening that are similar to examples of roughness seen in ESEM-grown crystals. For most transported crystals, the habits and crystal edges are more intricate that those observed for ice grown directly on substrates within the ESEM chamber. Portions of some crystals do appear smooth even at magnification greater than 1000x, a rare observation in our ESEM-grown crystals. The

  5. Single Cell Traction Microscopy within 3D Collagen Matrices

    NASA Astrophysics Data System (ADS)

    Wu, Mingming

    2014-03-01

    Mechanical interaction between the cell and its extracellular matrix (ECM) regulates cellular behaviors, including proliferation, differentiation, adhesion and migration. Cells require the three dimensional (3D) architectural support of the ECM to perform physiologically realistic functions. However, our current understanding of cell-ECM and cell-cell mechanical interactions is largely derived from 2D traction force microscopy, in which cells are cultured on a flat substrate. It is now clear that what we learn about cellular behavior on a 2D substrate does not always apply to cells embedded within a 3D biomatrix. 3D traction microscopy is emerging for mapping traction fields of single cells embedded in 3D gel, but current methods cannot account for the fibrous and nonlinear properties of collagen gel. In this talk, I will present a forward computation algorithm that we have developed for 3D cell traction measurements within collagen gels. The application of this technology to understanding cancer migration and invasion will be discussed. This work is supported by the National Center for Research Resources (5R21RR025801-03, NIH) and the National Institute of General Medical Sciences (8 R21 GM103388-03,NIH), and the Cornell Center on the Microenvironment & Metastasis.

  6. 3D super-resolution microscopy of bacterial division machinery

    NASA Astrophysics Data System (ADS)

    Vedyaykin, A. D.; Sabantsev, A. V.; Vishnyakov, I. E.; Morozova, N. E.; Polinovskaya, V. S.; Khodorkovskii, M. A.

    2016-08-01

    Super-resolution microscopy is a promising tool for the field of microbiology, as bacteria sizes are comparable to the resolution limit of light microscopy. Bacterial division machinery and FtsZ protein in particular attract much attention of scientists who use different super-resolution microscopy techniques, but most of the available data on FtsZ structures was obtained using two-dimensional (2D) super-resolution microscopy. Using 3D single-molecule localization microscopy (SMLM, namely dSTORM) to visualize FtsZ, we demonstrate that this approach allows more accurate interpretation of super-resolution images and provides new opportunities for the study of complex structures like bacterial divisome.

  7. Single molecule microscopy in 3D cell cultures and tissues.

    PubMed

    Lauer, Florian M; Kaemmerer, Elke; Meckel, Tobias

    2014-12-15

    From the onset of the first microscopic visualization of single fluorescent molecules in living cells at the beginning of this century, to the present, almost routine application of single molecule microscopy, the method has well-proven its ability to contribute unmatched detailed insight into the heterogeneous and dynamic molecular world life is composed of. Except for investigations on bacteria and yeast, almost the entire story of success is based on studies on adherent mammalian 2D cell cultures. However, despite this continuous progress, the technique was not able to keep pace with the move of the cell biology community to adapt 3D cell culture models for basic research, regenerative medicine, or drug development and screening. In this review, we will summarize the progress, which only recently allowed for the application of single molecule microscopy to 3D cell systems and give an overview of the technical advances that led to it. While initially posing a challenge, we finally conclude that relevant 3D cell models will become an integral part of the on-going success of single molecule microscopy. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Quantitative 3D structured illumination microscopy of nuclear structures.

    PubMed

    Kraus, Felix; Miron, Ezequiel; Demmerle, Justin; Chitiashvili, Tsotne; Budco, Alexei; Alle, Quentin; Matsuda, Atsushi; Leonhardt, Heinrich; Schermelleh, Lothar; Markaki, Yolanda

    2017-05-01

    3D structured illumination microscopy (3D-SIM) is the super-resolution technique of choice for multicolor volumetric imaging. Here we provide a validated sample preparation protocol for labeling nuclei of cultured mammalian cells, image acquisition and registration practices, and downstream image analysis of nuclear structures and epigenetic marks. Using immunostaining and replication labeling combined with image segmentation, centroid mapping and nearest-neighbor analyses in open-source environments, 3D maps of nuclear structures are analyzed in individual cells and normalized to fluorescence standards on the nanometer scale. This protocol fills an unmet need for the application of 3D-SIM to the technically challenging nuclear environment, and subsequent quantitative analysis of 3D nuclear structures and epigenetic modifications. In addition, it establishes practical guidelines and open-source solutions using ImageJ/Fiji and the TANGO plugin for high-quality and routinely comparable data generation in immunostaining experiments that apply across model systems. From sample preparation through image analysis, the protocol can be executed within one week.

  9. Resolution in 3D in multifocal plane microscopy

    NASA Astrophysics Data System (ADS)

    Chao, Jerry; Ram, Sripad; Abraham, Anish V.; Ward, E. Sally; Ober, Raimund J.

    2008-02-01

    Using single molecule microscopy, biological interactions can be imaged and studied at the level of individual biomolecules. When characterizing an imaged biological interaction, the distance separating the two participating biomolecules can provide valuable information. Therefore, the resolvability of an imaging setup is of practical significance in the analysis of the acquired image data. Importantly, the resolvability of the imaging setup needs evaluation in the 3D context, since in general biomolecules reside in 3D space within the cellular environment. We recently introduced an information-theoretic 2D resolution measure which shows that the resolution limit due to Rayleigh's criterion can be overcome. This new result predicts that the resolution of optical microscopes is not limited, but rather can be improved with increased photon counts detected from the single molecules. The 2D result was subsequently extended to the 3D context, and the proposed information-theoretic 3D resolution measure can readily be used to determine the resolvability of a conventional single focal plane imaging setup. Here, we consider the 3D resolution measure for a multifocal plane microscope setup, an imaging system which allows the concurrent imaging of multiple focal planes within a specimen. The technique is useful in applications such as the tracking of subcellular objects in 3D. By comparing their 3D resolution measures, we find a two-plane setup to outperform a comparable conventional single-plane setup in resolvability over a range of axial locations for the single molecule pair. Moreover, we investigate and compare the impact of noise on the resolvability of the two setups.

  10. Mesoscale morphology at nanoscale resolution: serial block-face scanning electron microscopy reveals fine 3D detail of a novel silk spinneret system in a tube-building tanaid crustacean.

    PubMed

    Kaji, Tomonari; Kakui, Keiichi; Miyazaki, Naoyuki; Murata, Kazuyoshi; Palmer, A Richard

    2016-01-01

    The study of morphology is experiencing a renaissance due to rapid improvements in technologies for 3D visualization of complex internal and external structures. But 3D visualization of the internal structure of mesoscale objects - those in the 10-1000 μm range - remains problematic. They are too small for microCT, many lack suitable specific fluorescent markers for confocal microscopy, or they require labor-intensive stacking and smoothing of individual TEM images. Here we illustrate the first comprehensive morphological description of a complete mesoscale biological system at nanoscopic resolution using ultra-modern technology for 3D visualization - serial block-face scanning electron microscopy (SBF-SEM). The SBF-SEM machine combines an in-chamber ultramicrotome, which creates a serial array of exposed surfaces, with an SEM that images each surface as it is exposed. The serial images are then stacked automatically by 3D reconstruction software. We used SBF-SEM to study the spinneret (thread-producing) system of a small, tube-dwelling crustacean that weaves tubes of silk. Thread-producing ability is critical for the survival of many small-bodied animals but the basic morphology of these systems remains mysterious due to the limits of traditional microscopy. SBF-SEM allowed us to describe - in full 3D - well-resolved components (glands, ducts, pores, and associated nerves and muscles) of the spinneret system in the thoracic legs and body segments of Sinelobus sp. (Crustacea, Peracarida, Tanaidacea), a tube-building tanaid only 2 mm in body length. The 3D reconstruction by SBF-SEM revealed at nanoscale resolution a unique structure to the gland and duct systems: In each of three thread-producing thoracic segments, two separate ducts, derived from two separate glands located in the body, run through the entire leg and merge at the leg tip just before the spinneret pore opening. We also resolved nerves connecting to individual setae, spines and pores on the

  11. Unsupervised noise removal algorithms for 3-D confocal fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Roysam, Badrinath; Bhattacharjya, Anoop K.; Srinivas, Chukka; Szarowski, Donald H.; Turner, James N.

    1992-06-01

    Fast algorithms are presented for effective removal of the noise artifact in 3-D confocal fluorescence microscopy images of extended spatial objects such as neurons. The algorithms are unsupervised in the sense that they automatically estimate and adapt to the spatially and temporally varying noise level in the microscopy data. An important feature of the algorithms is the fact that a 3-D segmentation of the field emerges jointly with the intensity estimate. The role of the segmentation is to limit any smoothing to the interiors of regions and hence avoid the blurring that is associated with conventional noise removal algorithms. Fast computation is achieved by parallel computation methods, rather than by algorithmic or modelling compromises. The noise-removal proceeds iteratively, starting from a set of approximate user- supplied, or default initial guesses of the underlying random process parameters. An expectation maximization algorithm is used to obtain a more precise characterization of these parameters, that are then input to a hierarchical estimation algorithm. This algorithm computes a joint solution of the related problems corresponding to intensity estimation, segmentation, and boundary-surface estimation subject to a combination of stochastic priors and syntactic pattern constraints. Three-dimensional stereoscopic renderings of processed 3-D images of murine hippocampal neurons are presented to demonstrate the effectiveness of the method. The processed images exhibit increased contrast and significant smoothing and reduction of the background intensity while avoiding any blurring of the neuronal structures.

  12. Validation of image processing tools for 3-D fluorescence microscopy.

    PubMed

    Dieterlen, Alain; Xu, Chengqi; Gramain, Marie-Pierre; Haeberlé, Olivier; Colicchio, Bruno; Cudel, Christophe; Jacquey, Serge; Ginglinger, Emanuelle; Jung, Georges; Jeandidier, Eric

    2002-04-01

    3-D optical fluorescent microscopy becomes nowadays an efficient tool for volumic investigation of living biological samples. Using optical sectioning technique, a stack of 2-D images is obtained. However, due to the nature of the system optical transfer function and non-optimal experimental conditions, acquired raw data usually suffer from some distortions. In order to carry out biological analysis, raw data have to be restored by deconvolution. The system identification by the point-spread function is useful to obtain the knowledge of the actual system and experimental parameters, which is necessary to restore raw data. It is furthermore helpful to precise the experimental protocol. In order to facilitate the use of image processing techniques, a multi-platform-compatible software package called VIEW3D has been developed. It integrates a set of tools for the analysis of fluorescence images from 3-D wide-field or confocal microscopy. A number of regularisation parameters for data restoration are determined automatically. Common geometrical measurements and morphological descriptors of fluorescent sites are also implemented to facilitate the characterisation of biological samples. An example of this method concerning cytogenetics is presented.

  13. High Resolution, Large Deformation 3D Traction Force Microscopy

    PubMed Central

    López-Fagundo, Cristina; Reichner, Jonathan; Hoffman-Kim, Diane; Franck, Christian

    2014-01-01

    Traction Force Microscopy (TFM) is a powerful approach for quantifying cell-material interactions that over the last two decades has contributed significantly to our understanding of cellular mechanosensing and mechanotransduction. In addition, recent advances in three-dimensional (3D) imaging and traction force analysis (3D TFM) have highlighted the significance of the third dimension in influencing various cellular processes. Yet irrespective of dimensionality, almost all TFM approaches have relied on a linear elastic theory framework to calculate cell surface tractions. Here we present a new high resolution 3D TFM algorithm which utilizes a large deformation formulation to quantify cellular displacement fields with unprecedented resolution. The results feature some of the first experimental evidence that cells are indeed capable of exerting large material deformations, which require the formulation of a new theoretical TFM framework to accurately calculate the traction forces. Based on our previous 3D TFM technique, we reformulate our approach to accurately account for large material deformation and quantitatively contrast and compare both linear and large deformation frameworks as a function of the applied cell deformation. Particular attention is paid in estimating the accuracy penalty associated with utilizing a traditional linear elastic approach in the presence of large deformation gradients. PMID:24740435

  14. High resolution, large deformation 3D traction force microscopy.

    PubMed

    Toyjanova, Jennet; Bar-Kochba, Eyal; López-Fagundo, Cristina; Reichner, Jonathan; Hoffman-Kim, Diane; Franck, Christian

    2014-01-01

    Traction Force Microscopy (TFM) is a powerful approach for quantifying cell-material interactions that over the last two decades has contributed significantly to our understanding of cellular mechanosensing and mechanotransduction. In addition, recent advances in three-dimensional (3D) imaging and traction force analysis (3D TFM) have highlighted the significance of the third dimension in influencing various cellular processes. Yet irrespective of dimensionality, almost all TFM approaches have relied on a linear elastic theory framework to calculate cell surface tractions. Here we present a new high resolution 3D TFM algorithm which utilizes a large deformation formulation to quantify cellular displacement fields with unprecedented resolution. The results feature some of the first experimental evidence that cells are indeed capable of exerting large material deformations, which require the formulation of a new theoretical TFM framework to accurately calculate the traction forces. Based on our previous 3D TFM technique, we reformulate our approach to accurately account for large material deformation and quantitatively contrast and compare both linear and large deformation frameworks as a function of the applied cell deformation. Particular attention is paid in estimating the accuracy penalty associated with utilizing a traditional linear elastic approach in the presence of large deformation gradients.

  15. Applied 3D printing for microscopy in health science research

    NASA Astrophysics Data System (ADS)

    Brideau, Craig; Zareinia, Kourosh; Stys, Peter

    2015-03-01

    The rapid prototyping capability offered by 3D printing is considered advantageous for commercial applications. However, the ability to quickly produce precision custom devices is highly beneficial in the research laboratory setting as well. Biological laboratories require the manipulation and analysis of delicate living samples, thus the ability to create custom holders, support equipment, and adapters allow the extension of existing laboratory machines. Applications include camera adapters and stage sample holders for microscopes, surgical guides for tissue preparation, and small precision tools customized to unique specifications. Where high precision is needed, especially the reproduction of fine features, a printer with a high resolution is needed. However, the introduction of cheaper, lower resolution commercial printers have been shown to be more than adequate for less demanding projects. For direct manipulation of delicate samples, biocompatible raw materials are often required, complicating the printing process. This paper will examine some examples of 3D-printed objects for laboratory use, and provide an overview of the requirements for 3D printing for this application. Materials, printing resolution, production, and ease of use will all be reviewed with an eye to producing better printers and techniques for laboratory applications. Specific case studies will highlight applications for 3D-printed devices in live animal imaging for both microscopy and Magnetic Resonance Imaging.

  16. Phase mask optimization for 3D parallax EDF microscopy

    NASA Astrophysics Data System (ADS)

    Beckers, Ingeborg E.; Gierlack, Michael; Höppel, Robert; Landskron, Jürgen

    2014-03-01

    Extended depth-of-field (EDF) microscopy is a well-investigated and very simple method to obtain projection images with an extended depth of focus. Despite its advantages of being a real-time method applicable to any microscopic mode with high lateral resolution that can be simply realized by extending a commercial microscope, the lack of z-correlation is still a problem. In this work we present a combined technique of EDF and stereomicroscopy. By cross-correlation depth information is obtained. Finally, 3D images are reconstructed for best phase masks and simulation results are evaluated experimentally.

  17. 3D scanning Hall probe microscopy with 700 nm resolution

    NASA Astrophysics Data System (ADS)

    Dede, M.; Akram, R.; Oral, A.

    2016-10-01

    In this report, we present a three dimensional (3D) imaging of magnetic field vector B → (x,y,z) emanating from the magnetic material surfaces using a scanning Hall probe microscopy (3D-SHPM) down to a 700 nm spatial resolution. The Hall probe is used to measure Bz(x,y) on the specimen surface at different heights with the step size of Δz = 250 nm, as we move away from the surface in z direction, until the field decays to zero. These set of images are then used to get ∂Bz(x,y)/∂x and ∂Bz(x,y)/∂y at different z by numerical differentiation. Using the Maxwell's equations in the source free region, Bx(x,y) and By(x,y) can be calculated by integrating ∂Bz(x,y)/∂x and ∂Bz(x,y)/∂y in the z direction. Alternatively, the gradients can also be measured in the Hall gradiometer configuration directly. The operation of the 3D-SHPM is demonstrated by imaging Bx(x,y), By(x,y) and Bz(x,y) on a hard disk specimen at a 700 nm resolution, using both of these methods at 77 K. The system is capable of operating from 300 K down to 4 K range.

  18. 3D imaging of neutron tracks using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Gillmore, Gavin; Wertheim, David; Flowers, Alan

    2016-04-01

    Neutron detection and neutron flux assessment are important aspects in monitoring nuclear energy production. Neutron flux measurements can also provide information on potential biological damage from exposure. In addition to the applications for neutron measurement in nuclear energy, neutron detection has been proposed as a method of enhancing neutrino detectors and cosmic ray flux has also been assessed using ground-level neutron detectors. Solid State Nuclear Track Detectors (or SSNTDs) have been used extensively to examine cosmic rays, long-lived radioactive elements, radon concentrations in buildings and the age of geological samples. Passive SSNTDs consisting of a CR-39 plastic are commonly used to measure radon because they respond to incident charged particles such as alpha particles from radon gas in air. They have a large dynamic range and a linear flux response. We have previously applied confocal microscopy to obtain 3D images of alpha particle tracks in SSNTDs from radon track monitoring (1). As a charged particle traverses through the polymer it creates an ionisation trail along its path. The trail or track is normally enhanced by chemical etching to better expose radiation damage, as the damaged area is more sensitive to the etchant than the bulk material. Particle tracks in CR-39 are usually assessed using 2D optical microscopy. In this study 6 detectors were examined using an Olympus OLS4100 LEXT 3D laser scanning confocal microscope (Olympus Corporation, Japan). The detectors had been etched for 2 hours 50 minutes at 85 °C in 6.25M NaOH. Post etch the plastics had been treated with a 10 minute immersion in a 2% acetic acid stop bath, followed by rinsing in deionised water. The detectors examined had been irradiated with a 2mSv neutron dose from an Am(Be) neutron source (producing roughly 20 tracks per mm2). We were able to successfully acquire 3D images of neutron tracks in the detectors studied. The range of track diameter observed was between 4

  19. Sample drift correction in 3D fluorescence photoactivation localization microscopy

    NASA Astrophysics Data System (ADS)

    Mlodzianoski, Michael J.; Schreiner, John M.; Callahan, Steven P.; Smolková, Katarina; Dlasková, Andrea; Šantorová, Jitka; Ježek, Petr; Bewersdorf, Joerg

    2011-08-01

    The recent development of diffraction-unlimited far-field fluorescence microscopy has overcome the classical resolution limit of ~250 nm of conventional light microscopy by about a factor of ten. The improved resolution, however, reveals not only biological structures at an unprecedented resolution, but is also susceptible to sample drift on a much finer scale than previously relevant. Without correction, sample drift leads to smeared images with decreased resolution, and in the worst case to misinterpretation of the imaged structures. This poses a problem especially for techniques such as Fluorescence Photoactivation Localization Microscopy (FPALM/PALM) or Stochastic Optical Reconstruction Microscopy (STORM), which often require minutes recording time. Here we discuss an approach that corrects for three-dimensional (3D) drift in images of fixed samples without the requirement for fiduciary markers or instrument modifications. Drift is determined by calculating the spatial cross-correlation function between subsets of localized particles imaged at different times. Correction down to ~5 nm precision is achieved despite the fact that different molecules are imaged in each frame. We demonstrate the performance of our drift correction algorithm with different simulated structures and analyze its dependence on particle density and localization precision. By imaging mitochondria with Biplane FPALM we show our algorithm's feasibility in a practical application.

  20. Sub-nanometer surface chemistry and orbital hybridization in lanthanum-doped ceria nano-catalysts revealed by 3D electron microscopy.

    PubMed

    Collins, Sean M; Fernandez-Garcia, Susana; Calvino, José J; Midgley, Paul A

    2017-07-14

    Surface chemical composition, electronic structure, and bonding characteristics determine catalytic activity but are not resolved for individual catalyst particles by conventional spectroscopy. In particular, the nano-scale three-dimensional distribution of aliovalent lanthanide dopants in ceria catalysts and their effect on the surface electronic structure remains unclear. Here, we reveal the surface segregation of dopant cations and oxygen vacancies and observe bonding changes in lanthanum-doped ceria catalyst particle aggregates with sub-nanometer precision using a new model-based spectroscopic tomography approach. These findings refine our understanding of the spatially varying electronic structure and bonding in ceria-based nanoparticle aggregates with aliovalent cation concentrations and identify new strategies for advancing high efficiency doped ceria nano-catalysts.

  1. 3D high resolution pure optical photoacoustic microscopy

    NASA Astrophysics Data System (ADS)

    Xie, Zhixing; Chen, Sung-Liang; Ling, Tao; Guo, L. Jay; Carson, Paul L.; Wang, Xueding

    2012-02-01

    The concept of pure optical photoacoustic microscopy(POPAM) was proposed based on optical rastering of a focused excitation beam and optically sensing the photoacoustic signal using a microring resonator fabricated by a nanoimprinting technique. After some refinedment of in the resonator structure and mold fabrication, an ultrahigh Q factor of 3.0×105 was achieved which provided high sensitivity with a noise equivalent detectable pressure(NEDP) value of 29Pa. This NEDP is much lower than the hundreds of Pascals achieved with existing optical resonant structures such as etalons, fiber gratings and dielectric multilayer interference filters available for acoustic measurement. The featured high sensitivity allowed the microring resonator to detect the weak photoacoustic signals from micro- or submicroscale objects. The inherent superbroad bandwidth of the optical microring resonator combined with an optically focused scanning beam provided POPAM of high resolution in the axial as well as both lateral directions while the axial resolution of conventional photoacoustic microscopy (PAM) suffers from the limited bandwidth of PZT detectors. Furthermore, the broadband microring resonator showed similar sensitivity to that of our most sensitive PZT detector. The current POPAM system provides a lateral resolution of 5μm and an axial resolution of 8μm, comparable to that achieved by optical microscopy while presenting the unique contrast of optical absorption and functional information complementing other optical modalities. The 3D structure of microvasculature, including capillary networks, and even individual red blood cells have been discerned successfully in the proof-of-concept experiments on mouse bladders ex vivo and mouse ears in vivo. The potential of approximately GHz bandwidth of the microring resonator also might allow much higher resolution than shown here in microscopy of optical absorption and acoustic propagation properties at depths in unfrozen tissue

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

    PubMed

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

    2013-05-03

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

  3. 3D Image Analysis of Geomaterials using Confocal Microscopy

    NASA Astrophysics Data System (ADS)

    Mulukutla, G.; Proussevitch, A.; Sahagian, D.

    2009-05-01

    Confocal microscopy is one of the most significant advances in optical microscopy of the last century. It is widely used in biological sciences but its application to geomaterials lingers due to a number of technical problems. Potentially the technique can perform non-invasive testing on a laser illuminated sample that fluoresces using a unique optical sectioning capability that rejects out-of-focus light reaching the confocal aperture. Fluorescence in geomaterials is commonly induced using epoxy doped with a fluorochrome that is impregnated into the sample to enable discrimination of various features such as void space or material boundaries. However, for many geomaterials, this method cannot be used because they do not naturally fluoresce and because epoxy cannot be impregnated into inaccessible parts of the sample due to lack of permeability. As a result, the confocal images of most geomaterials that have not been pre-processed with extensive sample preparation techniques are of poor quality and lack the necessary image and edge contrast necessary to apply any commonly used segmentation techniques to conduct any quantitative study of its features such as vesicularity, internal structure, etc. In our present work, we are developing a methodology to conduct a quantitative 3D analysis of images of geomaterials collected using a confocal microscope with minimal amount of prior sample preparation and no addition of fluorescence. Two sample geomaterials, a volcanic melt sample and a crystal chip containing fluid inclusions are used to assess the feasibility of the method. A step-by-step process of image analysis includes application of image filtration to enhance the edges or material interfaces and is based on two segmentation techniques: geodesic active contours and region competition. Both techniques have been applied extensively to the analysis of medical MRI images to segment anatomical structures. Preliminary analysis suggests that there is distortion in the

  4. 3D geometry-based quantification of colocalizations in multichannel 3D microscopy images of human soft tissue tumors.

    PubMed

    Wörz, Stefan; Sander, Petra; Pfannmöller, Martin; Rieker, Ralf J; Joos, Stefan; Mechtersheimer, Gunhild; Boukamp, Petra; Lichter, Peter; Rohr, Karl

    2010-08-01

    We introduce a new model-based approach for automatic quantification of colocalizations in multichannel 3D microscopy images. The approach uses different 3D parametric intensity models in conjunction with a model fitting scheme to localize and quantify subcellular structures with high accuracy. The central idea is to determine colocalizations between different channels based on the estimated geometry of the subcellular structures as well as to differentiate between different types of colocalizations. A statistical analysis was performed to assess the significance of the determined colocalizations. This approach was used to successfully analyze about 500 three-channel 3D microscopy images of human soft tissue tumors and controls.

  5. Angular reconstitution-based 3D reconstructions of nanomolecular structures from superresolution light-microscopy images.

    PubMed

    Salas, Desirée; Le Gall, Antoine; Fiche, Jean-Bernard; Valeri, Alessandro; Ke, Yonggang; Bron, Patrick; Bellot, Gaetan; Nollmann, Marcelo

    2017-08-29

    Superresolution light microscopy allows the imaging of labeled supramolecular assemblies at a resolution surpassing the classical diffraction limit. A serious limitation of the superresolution approach is sample heterogeneity and the stochastic character of the labeling procedure. To increase the reproducibility and the resolution of the superresolution results, we apply multivariate statistical analysis methods and 3D reconstruction approaches originally developed for cryogenic electron microscopy of single particles. These methods allow for the reference-free 3D reconstruction of nanomolecular structures from two-dimensional superresolution projection images. Since these 2D projection images all show the structure in high-resolution directions of the optical microscope, the resulting 3D reconstructions have the best possible isotropic resolution in all directions.

  6. Holographic microscopy for 3D tracking of bacteria

    NASA Astrophysics Data System (ADS)

    Nadeau, Jay; Cho, Yong Bin; El-Kholy, Marwan; Bedrossian, Manuel; Rider, Stephanie; Lindensmith, Christian; Wallace, J. Kent

    2016-03-01

    Understanding when, how, and if bacteria swim is key to understanding critical ecological and biological processes, from carbon cycling to infection. Imaging motility by traditional light microscopy is limited by focus depth, requiring cells to be constrained in z. Holographic microscopy offers an instantaneous 3D snapshot of a large sample volume, and is therefore ideal in principle for quantifying unconstrained bacterial motility. However, resolving and tracking individual cells is difficult due to the low amplitude and phase contrast of the cells; the index of refraction of typical bacteria differs from that of water only at the second decimal place. In this work we present a combination of optical and sample-handling approaches to facilitating bacterial tracking by holographic phase imaging. The first is the design of the microscope, which is an off-axis design with the optics along a common path, which minimizes alignment issues while providing all of the advantages of off-axis holography. Second, we use anti-reflective coated etalon glass in the design of sample chambers, which reduce internal reflections. Improvement seen with the antireflective coating is seen primarily in phase imaging, and its quantification is presented here. Finally, dyes may be used to increase phase contrast according to the Kramers-Kronig relations. Results using three test strains are presented, illustrating the different types of bacterial motility characterized by an enteric organism (Escherichia coli), an environmental organism (Bacillus subtilis), and a marine organism (Vibrio alginolyticus). Data processing steps to increase the quality of the phase images and facilitate tracking are also discussed.

  7. 3D super-resolution imaging by localization microscopy.

    PubMed

    Magenau, Astrid; Gaus, Katharina

    2015-01-01

    Fluorescence microscopy is an important tool in all fields of biology to visualize structures and monitor dynamic processes and distributions. Contrary to conventional microscopy techniques such as confocal microscopy, which are limited by their spatial resolution, super-resolution techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have made it possible to observe and quantify structure and processes on the single molecule level. Here, we describe a method to image and quantify the molecular distribution of membrane-associated proteins in two and three dimensions with nanometer resolution.

  8. Hybrid additive manufacturing of 3D electronic systems

    NASA Astrophysics Data System (ADS)

    Li, J.; Wasley, T.; Nguyen, T. T.; Ta, V. D.; Shephard, J. D.; Stringer, J.; Smith, P.; Esenturk, E.; Connaughton, C.; Kay, R.

    2016-10-01

    A novel hybrid additive manufacturing (AM) technology combining digital light projection (DLP) stereolithography (SL) with 3D micro-dispensing alongside conventional surface mount packaging is presented in this work. This technology overcomes the inherent limitations of individual AM processes and integrates seamlessly with conventional packaging processes to enable the deposition of multiple materials. This facilitates the creation of bespoke end-use products with complex 3D geometry and multi-layer embedded electronic systems. Through a combination of four-point probe measurement and non-contact focus variation microscopy, it was identified that there was no obvious adverse effect of DLP SL embedding process on the electrical conductivity of printed conductors. The resistivity maintained to be less than 4  ×  10-4 Ω · cm before and after DLP SL embedding when cured at 100 °C for 1 h. The mechanical strength of SL specimens with thick polymerized layers was also identified through tensile testing. It was found that the polymerization thickness should be minimised (less than 2 mm) to maximise the bonding strength. As a demonstrator a polymer pyramid with embedded triple-layer 555 LED blinking circuitry was successfully fabricated to prove the technical viability.

  9. Advanced 3D Optical Microscopy in ENS Research.

    PubMed

    Vanden Berghe, Pieter

    2016-01-01

    Microscopic techniques are among the few approaches that have survived the test of time. Being invented half way the seventeenth century by Antonie van Leeuwenhoek and Robert Hooke, this technology is still essential in modern biomedical labs. Many microscopy techniques have been used in ENS research to guide researchers in their dissections and later to enable electrode recordings. Apart from this, microscopy has been instrumental in the identification of subpopulations of cells in the ENS, using a variety of staining methods. A significant step forward in the use of microscopy was the introduction of fluorescence approaches. Due to the fact that intense excitation light is now filtered away from the longer wavelength emission light, the contrast can be improved drastically, which helped to identify subpopulations of enteric neurons in a variety of species. Later functionalized fluorescent probes were used to measure and film activity in muscle and neuronal cells. Another important impetus to the use of microscopy was the discovery and isolation of the green fluorescent protein (GFP), as it gave rise to the development of many different color variants and functionalized constructs. Recent advances in microscopy are the result of a continuous search to enhance contrast between the item of interest and its background but also to improve resolving power to tell two small objects apart. In this chapter three different microscopy approaches will be discussed that can aid to improve our understanding of ENS function within the gut wall.

  10. Four-dimensional electron microscopy.

    PubMed

    Zewail, Ahmed H

    2010-04-09

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

  11. Precision 3-D microscopy with intensity modulated fibre optic scanners

    NASA Astrophysics Data System (ADS)

    Olmos, P.

    2016-01-01

    Optical 3-D imagers constitute a family of precision and useful instruments, easily available on the market in a wide variety of configurations and performances. However, besides their cost they usually provide an image of the object (i.e. a more or less faithful representation of the reality) instead of a truly object's reconstruction. Depending on the detailed working principles of the equipment, this reconstruction may become a challenging task. Here a very simple yet reliable device is described; it is able to form images of opaque objects by illuminating them with an optical fibre and collecting the reflected light with another fibre. Its 3-D capability comes from the spatial filtering imposed by the fibres together with their movement (scanning) along the three directions: transversal (surface) and vertical. This unsophisticated approach allows one to model accurately the entire optical process and to perform the desired reconstruction, finding that information about the surface which is of interest: its profile and its reflectance, ultimately related to the type of material.

  12. Two-Photon Microscopy Analysis of Gold Nanoparticle Uptake in 3D Cell Spheroids

    PubMed Central

    Rane, Tushar D.; Armani, Andrea M.

    2016-01-01

    Nanomaterials can be synthesized from a wide range of material systems in numerous morphologies, creating an extremely diverse portfolio. As result of this tunability, these materials are emerging as a new class of nanotherapeutics and imaging agents. One particularly interesting nanomaterial is the gold nanoparticle. Due to its inherent biocompatibility and tunable photothermal behavior, it has made a rapid transition from the lab setting to in vivo testing. In most nanotherapeutic applications, the efficacy of the agent is directly related to the target of interest. However, the optimization of the AuNP size and shape for efficacy in vitro, prior to testing in in vivo models of a disease, has been largely limited to two dimensional monolayers of cells. Two dimensional cell cultures are unable to reproduce conditions experienced by AuNP in the body. In this article, we systematically investigate the effect of different properties of AuNP on the penetration depth into 3D cell spheroids using two-photon microscopy. The 3D spheroids are formed from the HCT116 cell line, a colorectal carcinoma cell line. In addition to studying different sizes and shapes of AuNPs, we also study the effect of an oligo surface chemistry. There is a significant difference between AuNP uptake profiles in the 2D monolayers of cells as compared to the 3D cell spheroids. Additionally, the range of sizes and shapes studied here also exhibit marked differences in uptake penetration depth and efficacy. Finally, our results demonstrate that two-photon microscopy enables quantitative AuNP localization and concentration data to be obtained at the single spheroid level without fluorescent labeling of the AuNP, thus, providing a viable technique for large scale screening of AuNP properties in 3D cell spheroids as compared to tedious and time consuming techniques like electron microscopy. PMID:27936027

  13. 3D fluorescence anisotropy imaging using selective plane illumination microscopy

    PubMed Central

    Hedde, Per Niklas; Ranjit, Suman; Gratton, Enrico

    2015-01-01

    Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein. PMID:26368202

  14. Cytology 3D structure formation based on optical microscopy images

    NASA Astrophysics Data System (ADS)

    Pronichev, A. N.; Polyakov, E. V.; Shabalova, I. P.; Djangirova, T. V.; Zaitsev, S. M.

    2017-01-01

    The article the article is devoted to optimization of the parameters of imaging of biological preparations in optical microscopy using a multispectral camera in visible range of electromagnetic radiation. A model for the image forming of virtual preparations was proposed. The optimum number of layers was determined for the object scan in depth and holistic perception of its switching according to the results of the experiment.

  15. 3D Printing of Plant Golgi Stacks from Their Electron Tomographic Models.

    PubMed

    Mai, Keith Ka Ki; Kang, Madison J; Kang, Byung-Ho

    2017-01-01

    Three-dimensional (3D) printing is an effective tool for preparing tangible 3D models from computer visualizations to assist in scientific research and education. With the recent popularization of 3D printing processes, it is now possible for individual laboratories to convert their scientific data into a physical form suitable for presentation or teaching purposes. Electron tomography is an electron microscopy method by which 3D structures of subcellular organelles or macromolecular complexes are determined at nanometer-level resolutions. Electron tomography analyses have revealed the convoluted membrane architectures of Golgi stacks, chloroplasts, and mitochondria. But the intricacy of their 3D organizations is difficult to grasp from tomographic models illustrated on computer screens. Despite the rapid development of 3D printing technologies, production of organelle models based on experimental data with 3D printing has rarely been documented. In this chapter, we present a simple guide to creating 3D prints of electron tomographic models of plant Golgi stacks using the two most accessible 3D printing technologies.

  16. Scanning ultrafast electron microscopy.

    PubMed

    Yang, Ding-Shyue; Mohammed, Omar F; Zewail, Ahmed H

    2010-08-24

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability.

  17. Scanning ultrafast electron microscopy

    PubMed Central

    Yang, Ding-Shyue; Mohammed, Omar F.; Zewail, Ahmed H.

    2010-01-01

    Progress has been made in the development of four-dimensional ultrafast electron microscopy, which enables space-time imaging of structural dynamics in the condensed phase. In ultrafast electron microscopy, the electrons are accelerated, typically to 200 keV, and the microscope operates in the transmission mode. Here, we report the development of scanning ultrafast electron microscopy using a field-emission-source configuration. Scanning of pulses is made in the single-electron mode, for which the pulse contains at most one or a few electrons, thus achieving imaging without the space-charge effect between electrons, and still in ten(s) of seconds. For imaging, the secondary electrons from surface structures are detected, as demonstrated here for material surfaces and biological specimens. By recording backscattered electrons, diffraction patterns from single crystals were also obtained. Scanning pulsed-electron microscopy with the acquired spatiotemporal resolutions, and its efficient heat-dissipation feature, is now poised to provide in situ 4D imaging and with environmental capability. PMID:20696933

  18. Registration and 3D visualization of large microscopy images

    NASA Astrophysics Data System (ADS)

    Mosaliganti, Kishore; Pan, Tony; Sharp, Richard; Ridgway, Randall; Iyengar, Srivathsan; Gulacy, Alexandra; Wenzel, Pamela; de Bruin, Alain; Machiraju, Raghu; Huang, Kun; Leone, Gustavo; Saltz, Joel

    2006-03-01

    Inactivation of the retinoblastoma gene in mouse embryos causes tissue infiltrations into critical sections of the placenta, which has been shown to affect fetal survivability. Our collaborators in cancer genetics are extremely interested in examining the three dimensional nature of these infiltrations given a stack of two dimensional light microscopy images. Three sets of wildtype and mutant placentas was sectioned serially and digitized using a commercial light microscopy scanner. Each individual placenta dataset consisted of approximately 1000 images totaling 700 GB in size, which were registered into a volumetric dataset using National Library of Medicine's (NIH/NLM) Insight Segmentation and Registration Toolkit (ITK). This paper describes our method for image registration to aid in volume visualization of tissue level intermixing for both wildtype and Rb - specimens. The registration process faces many challenges arising from the large image sizes, damages during sectioning, staining gradients both within and across sections, and background noise. These issues limit the direct application of standard registration techniques due to frequent convergence to local solutions. In this work, we develop a mixture of automated and semi-automated enhancements with ground-truth validation for the mutual information-based registration algorithm. Our final volume renderings clearly show tissue intermixing differences between both wildtype and Rb - specimens which are not obvious prior to registration.

  19. High performance 3D printed electronics using electroless plated copper

    NASA Astrophysics Data System (ADS)

    Jian, Jin Rong; Kim, Taeil; Park, Jae Sung; Wang, Jiacheng; Kim, Woo Soo

    2017-03-01

    This paper presents design and performance validation of 3D printed electronic components, 3D toroidal air-core inductors, fabricated by multi-material based Fused Deposition Modelling (FDM) 3D printing technology and electroless copper plating. Designs of toroidal inductor is investigated with different core shapes and winding numbers; circular and half-circular cores with 10 and 13 turns of windings. Electroless plated copper thin film ensures 3D printed toroidal plastic structures to possess inductive behaviors. The inductance is demonstrated reliably with an applied source frequency from 100 kHz to 2 MHz as designs vary. An RL circuit is utilized to test the fabricated inductors' phase-leading characteristics with corresponding phase angle changes.

  20. Two-Layer Elastographic 3-D Traction Force Microscopy

    PubMed Central

    Álvarez-González, Begoña; Zhang, Shun; Gómez-González, Manuel; Meili, Ruedi; Firtel, Richard A.; Lasheras, Juan C.; del Álamo, Juan C.

    2017-01-01

    Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum’s Poisson’s ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson’s ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson’s ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions. PMID:28074837

  1. Two-Layer Elastographic 3-D Traction Force Microscopy

    NASA Astrophysics Data System (ADS)

    Álvarez-González, Begoña; Zhang, Shun; Gómez-González, Manuel; Meili, Ruedi; Firtel, Richard A.; Lasheras, Juan C.; Del Álamo, Juan C.

    2017-01-01

    Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum’s Poisson’s ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson’s ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson’s ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.

  2. Two-Layer Elastographic 3-D Traction Force Microscopy.

    PubMed

    Álvarez-González, Begoña; Zhang, Shun; Gómez-González, Manuel; Meili, Ruedi; Firtel, Richard A; Lasheras, Juan C; Del Álamo, Juan C

    2017-01-11

    Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.

  3. Astigmatic multifocus microscopy enables deep 3D super-resolved imaging

    PubMed Central

    Oudjedi, Laura; Fiche, Jean-Bernard; Abrahamsson, Sara; Mazenq, Laurent; Lecestre, Aurélie; Calmon, Pierre-François; Cerf, Aline; Nöllmann, Marcelo

    2016-01-01

    We have developed a 3D super-resolution microscopy method that enables deep imaging in cells. This technique relies on the effective combination of multifocus microscopy and astigmatic 3D single-molecule localization microscopy. We describe the optical system and the fabrication process of its key element, the multifocus grating. Then, two strategies for localizing emitters with our imaging method are presented and compared with a previously described deep 3D localization algorithm. Finally, we demonstrate the performance of the method by imaging the nuclear envelope of eukaryotic cells reaching a depth of field of ~4µm. PMID:27375935

  4. Experimental characterization of 3D localization techniques for particle-tracking and super-resolution microscopy.

    PubMed

    Mlodzianoski, Michael J; Juette, Manuel F; Beane, Glen L; Bewersdorf, Joerg

    2009-05-11

    Three-dimensional (3D) particle localization at the nanometer scale plays a central role in 3D particle tracking and 3D localization-based super-resolution microscopy. Here we introduce a localization algorithm that is independent of theoretical models and therefore generally applicable to a large number of experimental realizations. Applying this algorithm and a convertible experimental setup we compare the performance of the two major 3D techniques based on astigmatic distortions and on multiplane detection. In both methods we obtain experimental 3D localization accuracies in agreement with theoretical predictions and characterize the depth dependence of the localization accuracy in detail.

  5. Conventional transmission electron microscopy

    PubMed Central

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

    2014-01-01

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

  6. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy.

    PubMed

    Gualda, Emilio J; Simão, Daniel; Pinto, Catarina; Alves, Paula M; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment.

  7. Imaging of human differentiated 3D neural aggregates using light sheet fluorescence microscopy

    PubMed Central

    Gualda, Emilio J.; Simão, Daniel; Pinto, Catarina; Alves, Paula M.; Brito, Catarina

    2014-01-01

    The development of three dimensional (3D) cell cultures represents a big step for the better understanding of cell behavior and disease in a more natural like environment, providing not only single but multiple cell type interactions in a complex 3D matrix, highly resembling physiological conditions. Light sheet fluorescence microscopy (LSFM) is becoming an excellent tool for fast imaging of such 3D biological structures. We demonstrate the potential of this technique for the imaging of human differentiated 3D neural aggregates in fixed and live samples, namely calcium imaging and cell death processes, showing the power of imaging modality compared with traditional microscopy. The combination of light sheet microscopy and 3D neural cultures will open the door to more challenging experiments involving drug testing at large scale as well as a better understanding of relevant biological processes in a more realistic environment. PMID:25161607

  8. 3D Observation of GEMS by Electron Tomography

    NASA Technical Reports Server (NTRS)

    Matsuno, Junya; Miyake, Akira; Tsuchiyama, Akira; Nakamura-Messenger, Keiko; Messenger, Scott

    2014-01-01

    Amorphous silicates in chondritic porous interplanetary dust particles (CP-IDPs) coming from comets are dominated by glass with embedded metal and sulfides (GEMS). GEMS grains are submicron-sized rounded objects (typically 100-500) nm in diameter) with anaometer-sized (10-50 nm) Fe-Ni metal and sulfide grains embedded in an amorphous silicate matrix. Several formation processes for GEMS grains have been proposed so far, but these models are still being debated [2-5]. Bradley et al. proposed that GEMS grains are interstellar silicate dust that survived various metamorphism or alteration processes in the protoplanetary disk and that they are amorphiation products of crystalline silicates in the interstellar medium by sputter-deposition of cosmic ray irradiation, similar to space weathering [2,4]. This consideration is based on the observation of nano-sized crystals (approximately 10 nm) called relict grains in GEMS grains and their shapes are pseudomorphs to the host GEMS grains. On the other hand, Keller and Messenger proposed that most GEMS formed in the protoplanetary disk as condensates from high temperature gas [3,5]. This model is based on the fact that most GEMS grains have solar isotopic compositions and have extremely heterogeneous and non-solar elemental compositions. Keller and Messenger (2011) also reported that amorphous silicates in GEMS grains are surrounded by sulfide grains, which formed as sulfidization of metallic iron grains located on the GEMS surface. The previous studies were performed with 2D observation by using transmission electron microscopy (TEM) or scanning TEM (STEM). In order to understand the structure of GEMS grains described above more clearly, we observed 3D structure of GEMS grains by electron tomography using a TEM/STEM (JEM-2100F, JEOL) at Kyoto University. Electron tomography gives not only 3D structures but also gives higher spatial resolution (approximately a few nm) than that in conventional 2D image, which is restricted by

  9. Fluorescence in situ hybridization applications for super-resolution 3D structured illumination microscopy.

    PubMed

    Markaki, Yolanda; Smeets, Daniel; Cremer, Marion; Schermelleh, Lothar

    2013-01-01

    Fluorescence in situ hybridization on three-dimensionally preserved cells (3D-FISH) is an efficient tool to analyze the subcellular localization and spatial arrangement of targeted DNA sequences and RNA transcripts at the single cell level. 3D reconstructions from serial optical sections obtained by confocal laser scanning microscopy (CLSM) have long been considered the gold standard for 3D-FISH analyses. Recent super-resolution techniques circumvent the diffraction-limit of optical resolution and have defined a new state-of-the-art in bioimaging. Three-dimensional structured illumination microscopy (3D-SIM) represents one of these technologies. Notably, 3D-SIM renders an eightfold improved volumetric resolution over conventional imaging, and allows the simultaneous visualization of differently labeled target structures. These features make this approach highly attractive for the analysis of spatial relations and substructures of nuclear targets that escape detection by conventional light microscopy. Here, we focus on the application of 3D-SIM for the visualization of subnuclear 3D-FISH preparations. In comparison with conventional fluorescence microscopy, the quality of 3D-SIM data is dependent to a much greater extent on the optimal sample preparation, labeling and acquisition conditions. We describe typical problems encountered with super-resolution imaging of in situ hybridizations in mammalian tissue culture cells and provide optimized DNA-/(RNA)-FISH protocols including combinations with immunofluorescence staining (Immuno-FISH) and DNA replication labeling using click chemistry.

  10. Using 3-D Super-Resolution Microscopy to Probe Breast Cancer Stem Cells and Their Microenvironment

    DTIC Science & Technology

    2013-03-01

    microscope, which now has the capabilities to perform both 2D and 3D multi-color Stochastic Optical Reconstruction Microscopy (STORM) and Photoactivated...this cellular phenotype. To realize this goal, we had proposed to use 3D super-resolution microscopy to visualize how individual breast CaSCs and...techniques. Using AutoCAD , we create a mask and use soft lithography to create PDMS (Sylgard 184) microposts with a given aspect ratio and

  11. Laser embedding electronics on 3D printed objects

    NASA Astrophysics Data System (ADS)

    Kirleis, Matthew A.; Simonson, Duane; Charipar, Nicholas A.; Kim, Heungsoo; Charipar, Kristin M.; Auyeung, Ray C. Y.; Mathews, Scott A.; Piqué, Alberto

    2014-03-01

    Additive manufacturing techniques such as 3D printing are able to generate reproductions of a part in free space without the use of molds; however, the objects produced lack electrical functionality from an applications perspective. At the same time, techniques such as inkjet and laser direct-write (LDW) can be used to print electronic components and connections onto already existing objects, but are not capable of generating a full object on their own. The approach missing to date is the combination of 3D printing processes with direct-write of electronic circuits. Among the numerous direct write techniques available, LDW offers unique advantages and capabilities given its compatibility with a wide range of materials, surface chemistries and surface morphologies. The Naval Research Laboratory (NRL) has developed various LDW processes ranging from the non-phase transformative direct printing of complex suspensions or inks to lase-and-place for embedding entire semiconductor devices. These processes have been demonstrated in digital manufacturing of a wide variety of microelectronic elements ranging from circuit components such as electrical interconnects and passives to antennas, sensors, actuators and power sources. At NRL we are investigating the combination of LDW with 3D printing to demonstrate the digital fabrication of functional parts, such as 3D circuits. Merging these techniques will make possible the development of a new generation of structures capable of detecting, processing, communicating and interacting with their surroundings in ways never imagined before. This paper shows the latest results achieved at NRL in this area, describing the various approaches developed for generating 3D printed electronics with LDW.

  12. How to use 3D shadows for simple microscopy and vibrometry

    NASA Astrophysics Data System (ADS)

    Parikesit, Gea O. F.; Kusumaningtyas, Indraswari

    2017-07-01

    In 2014, we reported that shadows can be displayed in 3D using a stereoscopic setup. We now report that the 3D shadows can also be used to perform simple measurements, which are suitable for physics education in schools and colleges. Two different types of measurements are demonstrated, i.e. microscopy and vibrometry. Both types of measurements take advantage of the geometrical optics of the 3D shadows, where the 3D position of an object can be estimated using the coordinates of the colored light sources and the coordinates of the colored shadow images. We also include several student activities that can raise the students’ curiosity and capability.

  13. Dual-Color 3D Superresolution Microscopy by Combined Spectral-Demixing and Biplane Imaging

    PubMed Central

    Winterflood, Christian M.; Platonova, Evgenia; Albrecht, David; Ewers, Helge

    2015-01-01

    Multicolor three-dimensional (3D) superresolution techniques allow important insight into the relative organization of cellular structures. While a number of innovative solutions have emerged, multicolor 3D techniques still face significant technical challenges. In this Letter we provide a straightforward approach to single-molecule localization microscopy imaging in three dimensions and two colors. We combine biplane imaging and spectral-demixing, which eliminates a number of problems, including color cross-talk, chromatic aberration effects, and problems with color registration. We present 3D dual-color images of nanoscopic structures in hippocampal neurons with a 3D compound resolution routinely achieved only in a single color. PMID:26153696

  14. Electronic detectors for electron microscopy.

    PubMed

    Faruqi, A R; McMullan, G

    2011-08-01

    Electron microscopy (EM) is an important tool for high-resolution structure determination in applications ranging from condensed matter to biology. Electronic detectors are now used in most applications in EM as they offer convenience and immediate feedback that is not possible with film or image plates. The earliest forms of electronic detector used routinely in transmission electron microscopy (TEM) were charge coupled devices (CCDs) and for many applications these remain perfectly adequate. There are however applications, such as the study of radiation-sensitive biological samples, where film is still used and improved detectors would be of great value. The emphasis in this review is therefore on detectors for use in such applications. Two of the most promising candidates for improved detection are: monolithic active pixel sensors (MAPS) and hybrid pixel detectors (of which Medipix2 was chosen for this study). From the studies described in this review, a back-thinned MAPS detector appears well suited to replace film in for the study of radiation-sensitive samples at 300 keV, while Medipix2 is suited to use at lower energies and especially in situations with very low count rates. The performance of a detector depends on the energy of electrons to be recorded, which in turn is dependent on the application it is being used for; results are described for a wide range of electron energies ranging from 40 to 300 keV. The basic properties of detectors are discussed in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE) as a function of spatial frequency.

  15. 3D-Printed Self-Folding Electronics.

    PubMed

    Sundaram, Subramanian; Kim, David S; Baldo, Marc A; Hayward, Ryan C; Matusik, Wojciech

    2017-09-20

    Self-transforming structures are gaining prominence due to their general ability to adopt programmed shapes each tailored for specific functions. Composites that self-fold have so far relied on using the stimuli-responsive mechanisms focusing on reversible shape change. Integrating additional functions within these composites can rapidly enhance their practical applicability; however, this remains a challenging problem. Here, we demonstrate a method for spontaneous folding of three-dimensional (3D)-printed composites with embedded electronics at room temperature. The composite is printed using a multimaterial 3D-printing process with no external processing steps. Upon peeling from the print platform, the composite self-shapes itself using the residual forces resulting from polymer swelling during the layer-by-layer fabrication process. As a specific example, electrochromic elements are printed within the composite and can be electrically controlled through its folded legs. Our shape-transformation scheme provides a route to transform planar electronics into nonplanar geometries containing the overhangs. Integrating electronics within complex 3D shapes can enable new applications in sensing and robotics.

  16. Hollow Cone Electron Imaging for Single Particle 3D Reconstruction of Proteins

    PubMed Central

    Tsai, Chun-Ying; Chang, Yuan-Chih; Lobato, Ivan; Van Dyck, Dirk; Chen, Fu-Rong

    2016-01-01

    The main bottlenecks for high-resolution biological imaging in electron microscopy are radiation sensitivity and low contrast. The phase contrast at low spatial frequencies can be enhanced by using a large defocus but this strongly reduces the resolution. Recently, phase plates have been developed to enhance the contrast at small defocus but electrical charging remains a problem. Single particle cryo-electron microscopy is mostly used to minimize the radiation damage and to enhance the resolution of the 3D reconstructions but it requires averaging images of a massive number of individual particles. Here we present a new route to achieve the same goals by hollow cone dark field imaging using thermal diffuse scattered electrons giving about a 4 times contrast increase as compared to bright field imaging. We demonstrate the 3D reconstruction of a stained GroEL particle can yield about 13.5 Å resolution but using a strongly reduced number of images. PMID:27292544

  17. Hollow Cone Electron Imaging for Single Particle 3D Reconstruction of Proteins.

    PubMed

    Tsai, Chun-Ying; Chang, Yuan-Chih; Lobato, Ivan; Van Dyck, Dirk; Chen, Fu-Rong

    2016-06-13

    The main bottlenecks for high-resolution biological imaging in electron microscopy are radiation sensitivity and low contrast. The phase contrast at low spatial frequencies can be enhanced by using a large defocus but this strongly reduces the resolution. Recently, phase plates have been developed to enhance the contrast at small defocus but electrical charging remains a problem. Single particle cryo-electron microscopy is mostly used to minimize the radiation damage and to enhance the resolution of the 3D reconstructions but it requires averaging images of a massive number of individual particles. Here we present a new route to achieve the same goals by hollow cone dark field imaging using thermal diffuse scattered electrons giving about a 4 times contrast increase as compared to bright field imaging. We demonstrate the 3D reconstruction of a stained GroEL particle can yield about 13.5 Å resolution but using a strongly reduced number of images.

  18. Hollow Cone Electron Imaging for Single Particle 3D Reconstruction of Proteins

    NASA Astrophysics Data System (ADS)

    Tsai, Chun-Ying; Chang, Yuan-Chih; Lobato, Ivan; van Dyck, Dirk; Chen, Fu-Rong

    2016-06-01

    The main bottlenecks for high-resolution biological imaging in electron microscopy are radiation sensitivity and low contrast. The phase contrast at low spatial frequencies can be enhanced by using a large defocus but this strongly reduces the resolution. Recently, phase plates have been developed to enhance the contrast at small defocus but electrical charging remains a problem. Single particle cryo-electron microscopy is mostly used to minimize the radiation damage and to enhance the resolution of the 3D reconstructions but it requires averaging images of a massive number of individual particles. Here we present a new route to achieve the same goals by hollow cone dark field imaging using thermal diffuse scattered electrons giving about a 4 times contrast increase as compared to bright field imaging. We demonstrate the 3D reconstruction of a stained GroEL particle can yield about 13.5 Å resolution but using a strongly reduced number of images.

  19. FIJI Macro 3D ART VeSElecT: 3D Automated Reconstruction Tool for Vesicle Structures of Electron Tomograms

    PubMed Central

    Kaltdorf, Kristin Verena; Schulze, Katja; Helmprobst, Frederik; Kollmannsberger, Philip; Stigloher, Christian

    2017-01-01

    Automatic image reconstruction is critical to cope with steadily increasing data from advanced microscopy. We describe here the Fiji macro 3D ART VeSElecT which we developed to study synaptic vesicles in electron tomograms. We apply this tool to quantify vesicle properties (i) in embryonic Danio rerio 4 and 8 days past fertilization (dpf) and (ii) to compare Caenorhabditis elegans N2 neuromuscular junctions (NMJ) wild-type and its septin mutant (unc-59(e261)). We demonstrate development-specific and mutant-specific changes in synaptic vesicle pools in both models. We confirm the functionality of our macro by applying our 3D ART VeSElecT on zebrafish NMJ showing smaller vesicles in 8 dpf embryos then 4 dpf, which was validated by manual reconstruction of the vesicle pool. Furthermore, we analyze the impact of C. elegans septin mutant unc-59(e261) on vesicle pool formation and vesicle size. Automated vesicle registration and characterization was implemented in Fiji as two macros (registration and measurement). This flexible arrangement allows in particular reducing false positives by an optional manual revision step. Preprocessing and contrast enhancement work on image-stacks of 1nm/pixel in x and y direction. Semi-automated cell selection was integrated. 3D ART VeSElecT removes interfering components, detects vesicles by 3D segmentation and calculates vesicle volume and diameter (spherical approximation, inner/outer diameter). Results are collected in color using the RoiManager plugin including the possibility of manual removal of non-matching confounder vesicles. Detailed evaluation considered performance (detected vesicles) and specificity (true vesicles) as well as precision and recall. We furthermore show gain in segmentation and morphological filtering compared to learning based methods and a large time gain compared to manual segmentation. 3D ART VeSElecT shows small error rates and its speed gain can be up to 68 times faster in comparison to manual annotation

  20. Brain morphology imaging by 3D microscopy and fluorescent Nissl staining.

    PubMed

    Lazutkin, A A; Komissarova, N V; Toptunov, D M; Anokhin, K V

    2013-07-01

    Modern optical methods (multiphoton and light-sheet fluorescent microscopy) allow 3D imaging of large specimens of the brain with cell resolution. It is therefore essential to refer the resultant 3D pictures of expression of transgene, protein, and other markers in the brain to the corresponding structures in the atlas. This implies counterstaining of specimens with morphological dyes. However, there are no methods for contrasting large samples of the brain without their preliminary slicing. We have developed a method for fluorescent Nissl staining of whole brain samples. 3D reconstructions of specimens of the hippocampus, olfactory bulbs, and cortex were created. The method can be used for morphological control and evaluation of the effects of various factors on the brain using 3D microscopy technique.

  1. Hybrid wide-field and scanning microscopy for high-speed 3D imaging.

    PubMed

    Duan, Yubo; Chen, Nanguang

    2015-11-15

    Wide-field optical microscopy is efficient and robust in biological imaging, but it lacks depth sectioning. In contrast, scanning microscopic techniques, such as confocal microscopy and multiphoton microscopy, have been successfully used for three-dimensional (3D) imaging with optical sectioning capability. However, these microscopic techniques are not very suitable for dynamic real-time imaging because they usually take a long time for temporal and spatial scanning. Here, a hybrid imaging technique combining wide-field microscopy and scanning microscopy is proposed to accelerate the image acquisition process while maintaining the 3D optical sectioning capability. The performance was demonstrated by proof-of-concept imaging experiments with fluorescent beads and zebrafish liver.

  2. Digital holographic microscopy for imaging growth and treatment response in 3D tumor models

    NASA Astrophysics Data System (ADS)

    Li, Yuyu; Petrovic, Ljubica; Celli, Jonathan P.; Yelleswarapu, Chandra S.

    2014-03-01

    While three-dimensional tumor models have emerged as valuable tools in cancer research, the ability to longitudinally visualize the 3D tumor architecture restored by these systems is limited with microscopy techniques that provide only qualitative insight into sample depth, or which require terminal fixation for depth-resolved 3D imaging. Here we report the use of digital holographic microscopy (DHM) as a viable microscopy approach for quantitative, non-destructive longitudinal imaging of in vitro 3D tumor models. Following established methods we prepared 3D cultures of pancreatic cancer cells in overlay geometry on extracellular matrix beds and obtained digital holograms at multiple timepoints throughout the duration of growth. The holograms were digitally processed and the unwrapped phase images were obtained to quantify nodule thickness over time under normal growth, and in cultures subject to chemotherapy treatment. In this manner total nodule volumes are rapidly estimated and demonstrated here to show contrasting time dependent changes during growth and in response to treatment. This work suggests the utility of DHM to quantify changes in 3D structure over time and suggests the further development of this approach for time-lapse monitoring of 3D morphological changes during growth and in response to treatment that would otherwise be impractical to visualize.

  3. Recent progress in printed 2/3D electronic devices

    NASA Astrophysics Data System (ADS)

    Klug, Andreas; Patter, Paul; Popovic, Karl; Blümel, Alexander; Sax, Stefan; Lenz, Martin; Glushko, Oleksandr; Cordill, Megan J.; List-Kratochvil, Emil J. W.

    2015-09-01

    New, energy-saving, efficient and cost-effective processing technologies such as 2D and 3D inkjet printing (IJP) for the production and integration of intelligent components will be opening up very interesting possibilities for industrial applications of molecular materials in the near future. Beyond the use of home and office based printers, "inkjet printing technology" allows for the additive structured deposition of photonic and electronic materials on a wide variety of substrates such as textiles, plastics, wood, stone, tiles or cardboard. Great interest also exists in applying IJP in industrial manufacturing such as the manufacturing of PCBs, of solar cells, printed organic electronics and medical products. In all these cases inkjet printing is a flexible (digital), additive, selective and cost-efficient material deposition method. Due to these advantages, there is the prospect that currently used standard patterning processes can be replaced through this innovative material deposition technique. A main issue in this research area is the formulation of novel functional inks or the adaptation of commercially available inks for specific industrial applications and/or processes. In this contribution we report on the design, realization and characterization of novel active and passive inkjet printed electronic devices including circuitry and sensors based on metal nanoparticle ink formulations and the heterogeneous integration into 2/3D printed demonstrators. The main emphasis of this paper will be on how to convert scientific inkjet knowledge into industrially relevant processes and applications.

  4. Bridging fluorescence microscopy and electron microscopy

    PubMed Central

    2008-01-01

    Development of new fluorescent probes and fluorescence microscopes has led to new ways to study cell biology. With the emergence of specialized microscopy units at most universities and research centers, the use of these techniques is well within reach for a broad research community. A major breakthrough in fluorescence microscopy in biology is the ability to follow specific targets on or in living cells, revealing dynamic localization and/or function of target molecules. One of the inherent limitations of fluorescence microscopy is the resolution. Several efforts are undertaken to overcome this limit. The traditional and most well-known way to achieve higher resolution imaging is by electron microscopy. Moreover, electron microscopy reveals organelles, membranes, macromolecules, and thus aids in the understanding of cellular complexity and localization of molecules of interest in relation to other structures. With the new probe development, a solid bridge between fluorescence microscopy and electron microscopy is being built, even leading to correlative imaging. This connection provides several benefits, both scientifically as well as practically. Here, I summarize recent developments in bridging microscopy. PMID:18575880

  5. Optimum conditions for high-quality 3D reconstruction in confocal scanning microscopy

    NASA Astrophysics Data System (ADS)

    Kim, Taehoon; Kim, Taejoong; Lee, SeungWoo; Gweon, Dae-Gab; Seo, Jungwoo

    2006-02-01

    Confocal Scanning Microscopy (CSM) is very useful to reconstruct 3D image of Bio-cells and the objects that have specification shape in higher axial and lateral resolution and widely used as measurement instrument. A 3D reconstruction is used to visualize confocal images and consists of following processes. The First process is to get 3D data by collecting a series of images at regular focus intervals (Optical Sectioning). The Second process is to fit a curve to a series of 3D data points each pixel. The Third process is to search height information that has maximum value from curve-fitting. However, because of various systematic errors (NOISE) occurred when collecting the information of images through Optical Sectioning and large peak deviation occurred from curve-fitting error, high quality 3D reconstruction is not expected. Also, it takes much time to 3d Reconstruction by using many 3D data in order to acquire high quality and much cost to improve signal-to-noise (SNR) using a higher power laser. So, we are going to define SNR, peak deviation and the order of curve-fitting as important factors and simulate the relation between the factors in order to find a optimum condition for high quality 3D reconstruction in Confoal Scanning Microscopy. If we use optimum condition obtained by this simulation, using a suitable SNR and the suitable number of data and the suitable n-th order curve-fitting, small peak deviation is expected and then, 3D reconstruction of little better quality is expected. Also, it is expected to save.

  6. Ligand Electron Density Shape Recognition Using 3D Zernike Descriptors

    NASA Astrophysics Data System (ADS)

    Gunasekaran, Prasad; Grandison, Scott; Cowtan, Kevin; Mak, Lora; Lawson, David M.; Morris, Richard J.

    We present a novel approach to crystallographic ligand density interpretation based on Zernike shape descriptors. Electron density for a bound ligand is expanded in an orthogonal polynomial series (3D Zernike polynomials) and the coefficients from this expansion are employed to construct rotation-invariant descriptors. These descriptors can be compared highly efficiently against large databases of descriptors computed from other molecules. In this manuscript we describe this process and show initial results from an electron density interpretation study on a dataset containing over a hundred OMIT maps. We could identify the correct ligand as the first hit in about 30 % of the cases, within the top five in a further 30 % of the cases, and giving rise to an 80 % probability of getting the correct ligand within the top ten matches. In all but a few examples, the top hit was highly similar to the correct ligand in both shape and chemistry. Further extensions and intrinsic limitations of the method are discussed.

  7. Quantitative IR microscopy and spectromics open the way to 3D digital pathology.

    PubMed

    Bobroff, Vladimir; Chen, Hsiang-Hsin; Delugin, Maylis; Javerzat, Sophie; Petibois, Cyril

    2016-06-01

    Currently, only mass-spectrometry (MS) microscopy brings a quantitative analysis of chemical contents of tissue samples in 3D. Here, the reconstruction of a 3D quantitative chemical images of a biological tissue by FTIR spectro-microscopy is reported. An automated curve-fitting method is developed to extract all intense absorption bands constituting IR spectra. This innovation benefits from three critical features: (1) the correction of raw IR spectra to make them quantitatively comparable; (2) the automated and iterative data treatment allowing to transfer the IR-absorption spectrum into a IR-band spectrum; (3) the reconstruction of an 3D IR-band matrix (x, y, z for voxel position and a 4(th) dimension with all IR-band parameters). Spectromics, which is a new method for exploiting spectral data for tissue metadata reconstruction, is proposed to further translate the related chemical information in 3D, as biochemical and anatomical tissue parameters. An example is given with oxidative stress distribution and the reconstruction of blood vessels in tissues. The requirements of IR microscopy instrumentation to propose 3D digital histology as a clinical routine technology is briefly discussed.

  8. Electron microscopy and forensic practice

    NASA Astrophysics Data System (ADS)

    Kotrlý, Marek; Turková, Ivana

    2013-05-01

    Electron microanalysis in forensic practice ranks among basic applications used in investigation of traces (latents, stains, etc.) from crime scenes. Applying electron microscope allows for rapid screening and receiving initial information for a wide range of traces. SEM with EDS/WDS makes it possible to observe topography surface and morphology samples and examination of chemical components. Physical laboratory of the Institute of Criminalistics Prague use SEM especially for examination of inorganic samples, rarely for biology and other material. Recently, possibilities of electron microscopy have been extended considerably using dual systems with focused ion beam. These systems are applied mainly in study of inner micro and nanoparticles , thin layers (intersecting lines in graphical forensic examinations, analysis of layers of functional glass, etc.), study of alloys microdefects, creating 3D particles and aggregates models, etc. Automated mineralogical analyses are a great asset to analysis of mineral phases, particularly soils, similarly it holds for cathode luminescence, predominantly colour one and precise quantitative measurement of their spectral characteristics. Among latest innovations that are becoming to appear also at ordinary laboratories are TOF - SIMS systems and micro Raman spectroscopy with a resolution comparable to EDS/WDS analysis (capable of achieving similar level as through EDS/WDS analysis).

  9. Silver stain for electron microscopy

    NASA Technical Reports Server (NTRS)

    Corbett, R. L.

    1972-01-01

    Ammoniacal silver stain used for light microscopy was adapted advantageously for use with very thin biological sections required for electron microscopy. Silver stain can be performed in short time, has more contrast, and is especially useful for low power electron microscopy.

  10. 3D reconstruction of the glycocalyx structure in mammalian capillaries using electron tomography.

    PubMed

    Arkill, Kenton P; Neal, Chris R; Mantell, Judith M; Michel, Charles C; Qvortrup, Klaus; Rostgaard, Jørgen; Bates, Dave O; Knupp, Carlo; Squire, John M

    2012-05-01

    Visualising the molecular strands making up the glycocalyx in the lumen of small blood vessels has proved to be difficult using conventional transmission electron microscopy techniques. Images obtained from tissue stained in a variety of ways have revealed a regularity in the organisation of the proteoglycan components of the glycocalyx layer (fundamental spacing about 20 nm), but require a large sample number. Attempts to visualise the glycocalyx face-on (i.e. in a direction perpendicular to the endothelial cell layer in the lumen and directly applicable for permeability modelling) has had limited success (e.g. freeze fracture). A new approach is therefore needed. Here we demonstrate the effectiveness of using the relatively novel electron microscopy technique of 3D electron tomography on two differently stained glycocalyx preparations. A tannic acid staining method and a novel staining technique using Lanthanum Dysprosium Glycosamino Glycan adhesion (the LaDy GAGa method). 3D electron tomography reveals details of the architecture of the glycocalyx just above the endothelial cell layer. The LaDy GAGa method visually appears to show more complete coverage and more depth than the Tannic Acid staining method. The tomographic reconstructions show a potentially significant improvement in determining glycocalyx structure over standard transmission electron microscopy. © 2012 John Wiley & Sons Ltd.

  11. Large scale superres 3D imaging: light-sheet single-molecule localization microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Lu, Chieh Han; Chen, Peilin; Chen, Bi-Chang

    2017-02-01

    Optical imaging techniques provide much important information in understanding life science especially cellular structure and morphology because "seeing is believing". However, the resolution of optical imaging is limited by the diffraction limit, which is discovered by Ernst Abbe, i.e. λ/2(NA) (NA is the numerical aperture of the objective lens). Fluorescence super-resolution microscopic techniques such as Stimulated emission depletion microscopy (STED), Photoactivated localization microscopy (PALM), and Stochastic optical reconstruction microscopy (STORM) are invented to have the capability of seeing biological entities down to molecular level that are smaller than the diffraction limit (around 200-nm in lateral resolution). These techniques do not physically violate the Abbe limit of resolution but exploit the photoluminescence properties and labelling specificity of fluorescence molecules to achieve super-resolution imaging. However, these super-resolution techniques limit most of their applications to the 2D imaging of fixed or dead samples due to the high laser power needed or slow speed for the localization process. Extended from 2D imaging, light sheet microscopy has been proven to have a lot of applications on 3D imaging at much better spatiotemporal resolutions due to its intrinsic optical sectioning and high imaging speed. Herein, we combine the advantage of localization microscopy and light-sheet microscopy to have super-resolved cellular imaging in 3D across large field of view. With high-density labeled spontaneous blinking fluorophore and wide-field detection of light-sheet microscopy, these allow us to construct 3D super-resolution multi-cellular imaging at high speed ( minutes) by light-sheet single-molecule localization microscopy.

  12. 3D motion picture recording by parallel phase-shifting digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Tahara, Tatsuki; Xia, Peng; Kakue, Takashi; Awatsuji, Yasuhiro; Nishio, Kenzo; Ura, Shogo; Kubota, Toshihiro; Matoba, Osamu

    2013-12-01

    Three-dimensional (3-D) motion-picture recording by parallel phase-shifting digital holographic microscopy that has the ability of instantaneous 3-D recording of dynamic phenomena in the microscopic field of view is presented. Parallel phase-shifting digital holography is a scheme to record multiple phase-shifted holograms with a single-shot exposure, and to achieve 3-D motion-picture recording of objects with high accuracy and wide 3-D area, based on space-division multiplexing of phase-shifted holograms. Parallel phase-shifting digital holographic microscopy is implemented by an optical interferometer and an image sensor on which polarization-detection function is introduced pixel by pixel. This time, we constructed a parallel phase-shifting digital holographic microscope for recording high-speed dynamic phenomena, and then motions of biological objects in water were recorded at more than 10,000 frames per second, which is the fastest among the previous reports on 3-D imaging of biological objects.

  13. Coherent Microscopy for 3-D Movement Monitoring and Super-Resolved Imaging

    NASA Astrophysics Data System (ADS)

    Beiderman, Yevgeny; Amsel, Avigail; Tzadka, Yaniv; Fixler, Dror; Teicher, Mina; Micó, Vicente; Garcí, Javier; Javidi, Bahram; DaneshPanah, Mehdi; Moon, Inkyu; Zalevsky, Zeev

    In this chapter we present three types of microscopy-related configurations while the first one is used for 3-D movement monitoring of the inspected samples, the second one is used for super-resolved 3-D imaging, and the last one presents an overview digital holographic microscopy applications. The first configuration is based on temporal tracking of secondary reflected speckles when imaged by properly defocused optics. We validate the proposed scheme by using it to monitor 3-D spontaneous contraction of rat's cardiac muscle cells while allowing nanometric tracking accuracy without interferometric recording. The second configuration includes projection of temporally varying speckle patterns on top of the sample and by proper decoding exceeding the diffraction as well as the geometrical-related lateral resolution limitation. In the final part of the chapter, we overview applications of digital holographic microscopy (DHM) for real-time non-invasive 3-D sensing, tracking, and recognition of living microorganisms such as single- or multiple-cell organisms and bacteria.

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

    PubMed Central

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

    2015-01-01

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

  15. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    PubMed Central

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-01-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions. PMID:27488312

  16. Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions

    NASA Astrophysics Data System (ADS)

    Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D. C.; Kelly, Kevin F.; Landes, Christy F.

    2016-08-01

    Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.

  17. Phase retrieval and 3D imaging in gold nanoparticles based fluorescence microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ilovitsh, Tali; Ilovitsh, Asaf; Weiss, Aryeh M.; Meir, Rinat; Zalevsky, Zeev

    2017-02-01

    Optical sectioning microscopy can provide highly detailed three dimensional (3D) images of biological samples. However, it requires acquisition of many images per volume, and is therefore time consuming, and may not be suitable for live cell 3D imaging. We propose the use of the modified Gerchberg-Saxton phase retrieval algorithm to enable full 3D imaging of gold nanoparticles tagged sample using only two images. The reconstructed field is free space propagated to all other focus planes using post processing, and the 2D z-stack is merged to create a 3D image of the sample with high fidelity. Because we propose to apply the phase retrieving on nano particles, the regular ambiguities typical to the Gerchberg-Saxton algorithm, are eliminated. The proposed concept is then further enhanced also for tracking of single fluorescent particles within a three dimensional (3D) cellular environment based on image processing algorithms that can significantly increases localization accuracy of the 3D point spread function in respect to regular Gaussian fitting. All proposed concepts are validated both on simulated data as well as experimentally.

  18. Deep Learning Segmentation of Optical Microscopy Images Improves 3D Neuron Reconstruction.

    PubMed

    Li, Rongjian; Zeng, Tao; Peng, Hanchuan; Ji, Shuiwang

    2017-03-08

    Digital reconstruction, or tracing, of 3-dimensional (3D) neuron structure from microscopy images is a critical step toward reversing engineering the wiring and anatomy of a brain. Despite a number of prior attempts, this task remains very challenging, especially when images are contaminated by noises or have discontinued segments of neurite patterns. An approach for addressing such problems is to identify the locations of neuronal voxels using image segmentation methods prior to applying tracing or reconstruction techniques. This preprocessing step is expected to remove noises in the data, thereby leading to improved reconstruction results. In this work, we proposed to use 3D Convolutional neural networks (CNNs) for segmenting the neuronal microscopy images. Specifically, we designed a novel CNN architecture that takes volumetric images as the inputs and their voxel-wise segmentation maps as the outputs. The developed architecture allows us to train and predict using large microscopy images in an end-to-end manner. We evaluated the performance of our model on a variety of challenging 3D microscopy images from different organisms. Results showed that the proposed methods improved the tracing performance significantly when combined with different reconstruction algorithms.

  19. Video lensfree microscopy of 2D and 3D culture of cells

    NASA Astrophysics Data System (ADS)

    Allier, C. P.; Vinjimore Kesavan, S.; Coutard, J.-G.; Cioni, O.; Momey, F.; Navarro, F.; Menneteau, M.; Chalmond, B.; Obeid, P.; Haguet, V.; David-Watine, B.; Dubrulle, N.; Shorte, S.; van der Sanden, B.; Di Natale, C.; Hamard, L.; Wion, D.; Dolega, M. E.; Picollet-D'hahan, N.; Gidrol, X.; Dinten, J.-M.

    2014-03-01

    Innovative imaging methods are continuously developed to investigate the function of biological systems at the microscopic scale. As an alternative to advanced cell microscopy techniques, we are developing lensfree video microscopy that opens new ranges of capabilities, in particular at the mesoscopic level. Lensfree video microscopy allows the observation of a cell culture in an incubator over a very large field of view (24 mm2) for extended periods of time. As a result, a large set of comprehensive data can be gathered with strong statistics, both in space and time. Video lensfree microscopy can capture images of cells cultured in various physical environments. We emphasize on two different case studies: the quantitative analysis of the spontaneous network formation of HUVEC endothelial cells, and by coupling lensfree microscopy with 3D cell culture in the study of epithelial tissue morphogenesis. In summary, we demonstrate that lensfree video microscopy is a powerful tool to conduct cell assays in 2D and 3D culture experiments. The applications are in the realms of fundamental biology, tissue regeneration, drug development and toxicology studies.

  20. Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures.

    PubMed

    Chen, Yong; Cai, Jiye; Zhao, Tao; Wang, Chenxi; Dong, Shuo; Luo, Shuqian; Chen, Zheng W

    2005-06-01

    The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale.

  1. Advanced prior modeling for 3D bright field electron tomography

    NASA Astrophysics Data System (ADS)

    Sreehari, Suhas; Venkatakrishnan, S. V.; Drummy, Lawrence F.; Simmons, Jeffrey P.; Bouman, Charles A.

    2015-03-01

    Many important imaging problems in material science involve reconstruction of images containing repetitive non-local structures. Model-based iterative reconstruction (MBIR) could in principle exploit such redundancies through the selection of a log prior probability term. However, in practice, determining such a log prior term that accounts for the similarity between distant structures in the image is quite challenging. Much progress has been made in the development of denoising algorithms like non-local means and BM3D, and these are known to successfully capture non-local redundancies in images. But the fact that these denoising operations are not explicitly formulated as cost functions makes it unclear as to how to incorporate them in the MBIR framework. In this paper, we formulate a solution to bright field electron tomography by augmenting the existing bright field MBIR method to incorporate any non-local denoising operator as a prior model. We accomplish this using a framework we call plug-and-play priors that decouples the log likelihood and the log prior probability terms in the MBIR cost function. We specifically use 3D non-local means (NLM) as the prior model in the plug-and-play framework, and showcase high quality tomographic reconstructions of a simulated aluminum spheres dataset, and two real datasets of aluminum spheres and ferritin structures. We observe that streak and smear artifacts are visibly suppressed, and that edges are preserved. Also, we report lower RMSE values compared to the conventional MBIR reconstruction using qGGMRF as the prior model.

  2. Automated Identification and Localization of Hematopoietic Stem Cells in 3D Intravital Microscopy Data

    PubMed Central

    Khorshed, Reema A.; Hawkins, Edwin D.; Duarte, Delfim; Scott, Mark K.; Akinduro, Olufolake A.; Rashidi, Narges M.; Spitaler, Martin; Lo Celso, Cristina

    2015-01-01

    Summary Measuring three-dimensional (3D) localization of hematopoietic stem cells (HSCs) within the bone marrow microenvironment using intravital microscopy is a rapidly expanding research theme. This approach holds the key to understanding the detail of HSC-niche interactions, which are critical for appropriate stem cell function. Due to the complex tissue architecture of the bone marrow and to the progressive introduction of scattering and signal loss at increasing imaging depths, there is no ready-made software to handle efficient segmentation and unbiased analysis of the data. To address this, we developed an automated image analysis tool that simplifies and standardizes the biological interpretation of 3D HSC microenvironment images. The algorithm identifies HSCs and measures their localization relative to surrounding osteoblast cells and bone collagen. We demonstrate here the effectiveness, consistency, and accuracy of the proposed approach compared to current manual analysis and its wider applicability to analyze other 3D bone marrow components. PMID:26120058

  3. Investigation on 3D morphological changes of in vitro cells through digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Memmolo, Pasquale; Miccio, Lisa; Merola, Francesco; Netti, Paolo A.; Coppola, Giuseppe; Ferraro, Pietro

    2013-04-01

    We report the investigation of the identification and measurement of region of interest (ROI) in quantitative phase-contrast maps (QPMs) of biological cells by digital holographic microscopy (DHM), with the aim to analyze the 3D positions and 3D morphology together. We consider as test case for our tool the in vitro bull sperm head morphometry analysis. Extraction and measurement of various morphological parameters are performed by using two methods: the anisotropic diffusion filter, that is based on the Gaussian diffusivity function which allows more accuracy of the edge position, and the simple thresholding filter. In particular we consider the calculation of area, ellipticity, perimeter, major axis, minor axis and shape factor as a morphological parameter, instead, for the estimation of 3D position, we compute the centroid, the weighted centroid and the maximum phase values. A statistical analysis on a data set composed by N = 14 holograms relative to bovine spermatozoa and its reference holograms is reported.

  4. Correlative light and volume electron microscopy: using focused ion beam scanning electron microscopy to image transient events in model organisms.

    PubMed

    Bushby, Andrew J; Mariggi, Giovanni; Armer, Hannah E J; Collinson, Lucy M

    2012-01-01

    The study of a biological event within a live model organism has become routine through the use of fluorescent labeling of specific proteins in conjunction with laser confocal imaging. These methods allow 3D visualization of temporal events that can elucidate biological function but cannot resolve the tissue organization, extracellular and subcellular details of the tissues. Here, we present a method for correlating electron microscopy image data with the light microscopy data from the same sample volume to reveal the 3D structural information: "correlative light and volume electron microscopy." The methods for live video confocal microscopy, fixation and embedding of the tissue for electron microscopy, the focused ion beam scanning electron microscopy method for sequentially slicing and imaging the volume of interest, and the treatment of the resulting 3D dataset are presented. The method is illustrated with data collected during the angiogenesis of blood vessels in a transgenic zebrafish embryo. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. New data-driven method from 3D confocal microscopy for calculating phytoplankton cell biovolume.

    PubMed

    Roselli, L; Paparella, F; Stanca, E; Basset, A

    2015-06-01

    Confocal laser scanner microscopy coupled with an image analysis system was used to directly determine the shape and calculate the biovolume of phytoplankton organisms by constructing 3D models of cells. The study was performed on Biceratium furca (Ehrenberg) Vanhoeffen, which is one of the most complex-shaped phytoplankton. Traditionally, biovolume is obtained from a standardized set of geometric models based on linear dimensions measured by light microscopy. However, especially in the case of complex-shaped cells, biovolume is affected by very large errors associated with the numerous manual measurements that this entails. We evaluate the accuracy of these traditional methods by comparing the results obtained using geometric models with direct biovolume measurement by image analysis. Our results show cell biovolume measurement based on decomposition into simple geometrical shapes can be highly inaccurate. Although we assume that the most accurate cell shape is obtained by 3D direct biovolume measurement, which is based on voxel counting, the intrinsic uncertainty of this method is explored and assessed. Finally, we implement a data-driven formula-based approach to the calculation of biovolume of this complex-shaped organism. On one hand, the model is obtained from 3D direct calculation. On the other hand, it is based on just two linear dimensions which can easily be measured by hand. This approach has already been used for investigating the complexities of morphology and for determining the 3D structure of cells. It could also represent a novel way to generalize scaling laws for biovolume calculation.

  6. Infrared differential interference contrast microscopy for 3D interconnect overlay metrology.

    PubMed

    Ku, Yi-sha; Shyu, Deh-Ming; Lin, Yeou-Sung; Cho, Chia-Hung

    2013-08-12

    One of the main challenges for 3D interconnect metrology of bonded wafers is measuring through opaque silicon wafers using conventional optical microscopy. We demonstrate here the use infrared microscopy, enhanced by implementing the differential interference contrast (DIC) technique, to measure the wafer bonding overlay. A pair of two dimensional symmetric overlay marks were processed at both the front and back sides of thinned wafers to evaluate the bonding overlay. A self-developed analysis algorithm and theoretical fitting model was used to map the overlay error between the bonded wafers and the interconnect structures. The measurement accuracy was found to be better than 1.0 micron.

  7. Imaging of Bacterial Chromosome Organization by 3D Super-Resolution Microscopy.

    PubMed

    Le Gall, Antoine; Cattoni, Diego I; Nollmann, Marcelo

    2017-01-01

    The bacterial nucleoid is highly organized, yet it is dynamically remodeled by cellular processes such as transcription, replication, or segregation. Many principles of nucleoid organization have remained obscure due to the inability of conventional microscopy methods to retrieve structural information beyond the diffraction limit of light. Structured illumination microscopy has recently been shown to provide new levels of spatial details on bacterial chromosome organization by surpassing the diffraction limit. Its ease of use and fast 3D multicolor capabilities make it a method of choice for imaging fluorescently labeled specimens at the nanoscale. We describe a simple high-throughput method for imaging bacterial chromosomes using this technique.

  8. Chondrocytes provide a model for in-situ confocal microscopy and 3D reconstructions

    NASA Astrophysics Data System (ADS)

    Hirsch, Michelle S.; Svoboda, Kathy K. H.

    1994-04-01

    Hyaline cartilage is composed of chondrocytes that reside in lacunae surrounded by extracellular matrix molecules. Microscopic and histochemical features of cartilage have been studied with many techniques. Many of these techniques can be time consuming and may alter natural cartilage characteristics. In addition, the orientation and order of sectioned tissue must be maintained to create 3D reconstructions. We show that confocal laser scanning microscopy may replace traditional methods for studying cartilage.

  9. Photometry unlocks 3D information from 2D localization microscopy data.

    PubMed

    Franke, Christian; Sauer, Markus; van de Linde, Sebastian

    2017-01-01

    We developed a straightforward photometric method, temporal, radial-aperture-based intensity estimation (TRABI), that allows users to extract 3D information from existing 2D localization microscopy data. TRABI uses the accurate determination of photon numbers in different regions of the emission pattern of single emitters to generate a z-dependent photometric parameter. This method can determine fluorophore positions up to 600 nm from the focal plane and can be combined with biplane detection to further improve axial localization.

  10. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    NASA Astrophysics Data System (ADS)

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-02-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

  11. Geometrical characterization of fluorescently labelled surfaces from noisy 3D microscopy data.

    PubMed

    Shelton, Elijah; Serwane, Friedhelm; Campàs, Otger

    2017-09-01

    Modern fluorescence microscopy enables fast 3D imaging of biological and inert systems alike. In many studies, it is important to detect the surface of objects and quantitatively characterize its local geometry, including its mean curvature. We present a fully automated algorithm to determine the location and curvatures of an object from 3D fluorescence images, such as those obtained using confocal or light-sheet microscopy. The algorithm aims at reconstructing surface labelled objects with spherical topology and mild deformations from the spherical geometry with high accuracy, rather than reconstructing arbitrarily deformed objects with lower fidelity. Using both synthetic data with known geometrical characteristics and experimental data of spherical objects, we characterize the algorithm's accuracy over the range of conditions and parameters typically encountered in 3D fluorescence imaging. We show that the algorithm can detect the location of the surface and obtain a map of local mean curvatures with relative errors typically below 2% and 20%, respectively, even in the presence of substantial levels of noise. Finally, we apply this algorithm to analyse the shape and curvature map of fluorescently labelled oil droplets embedded within multicellular aggregates and deformed by cellular forces. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  12. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide.

    PubMed

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-02-10

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab.

  13. All-in-one 3D printed microscopy chamber for multidimensional imaging, the UniverSlide

    PubMed Central

    Alessandri, Kevin; Andrique, Laetitia; Feyeux, Maxime; Bikfalvi, Andreas; Nassoy, Pierre; Recher, Gaëlle

    2017-01-01

    While live 3D high resolution microscopy techniques are developing rapidly, their use for biological applications is partially hampered by practical difficulties such as the lack of a versatile sample chamber. Here, we propose the design of a multi-usage observation chamber adapted for live 3D bio-imaging. We show the usefulness and practicality of this chamber, which we named the UniverSlide, for live imaging of two case examples, namely multicellular systems encapsulated in sub-millimeter hydrogel shells and zebrafish larvae. We also demonstrate its versatility and compatibility with all microscopy devices by using upright or inverted microscope configurations after loading the UniverSlide with fixed or living samples. Further, the device is applicable for medium/high throughput screening and automatized multi-position image acquisition, providing a constraint-free but stable and parallelized immobilization of the samples. The frame of the UniverSlide is fabricated using a stereolithography 3D printer, has the size of a microscopy slide, is autoclavable and sealed with a removable lid, which makes it suitable for use in a controlled culture environment. We describe in details how to build this chamber and we provide all the files necessary to print the different pieces in the lab. PMID:28186188

  14. Ultrasound array photoacoustic microscopy for dynamic in vivo 3D imaging

    NASA Astrophysics Data System (ADS)

    Song, Liang; Maslov, Konstantin; Shung, K. Kirk; Wang, Lihong V.

    2010-02-01

    Using realtime ultrasound array photoacoustic microscopy (UA-PAM), we demonstrated the feasibility of noninvasive in vivo imaging of human pulsatile dynamics, as well as 3-D dynamic imaging of sentinel lymph nodes (SLNs) in a murine model. The system, capable of realtime B-scan imaging at 50 Hz and high-speed 3-D imaging, was validated by imaging the subcutaneous microvasculature in rats and humans. After the validation, a human superficial palmar was imaged, and its pulsatile dynamics monitored, with 20-ms B-scan imaging temporal resolution. In addition, noninvasive photoacoustic sentinel lymph node (SLN) mapping with high spatial resolution has the potential to reduce the false negative rate and eliminate the use of radioactive tracers. Upon intra-dermal injection of Evans blue, the system maps SLNs accurately in mice and rats. Furthermore, the ~6 s 3-D imaging temporal resolution offers the capability to quantitatively and noninvasively monitor the dye dynamics in SLNs in vivo through sequential 3-D imaging. The demonstrated capability suggests that high-speed 3-D photoacoustic imaging should facilitate the understanding of the dynamics of various dyes in SLNs, and potentially help identify SLNs with high accuracy. With the results shown in this study, we believe that UA-PAM can potentially enable many new possibilities for studying functional and physiological dynamics in both preclinical and clinical imaging settings.

  15. Quantitative analyses of the 3D nuclear landscape recorded with super-resolved fluorescence microscopy.

    PubMed

    Schmid, Volker J; Cremer, Marion; Cremer, Thomas

    2017-03-18

    Recent advancements of super-resolved fluorescence microscopy have revolutionized microscopic studies of cells, including the exceedingly complex structural organization of cell nuclei in space and time. In this paper we describe and discuss tools for (semi-) automated, quantitative 3D analyses of the spatial nuclear organization. These tools allow the quantitative assessment of highly resolved different chromatin compaction levels in individual cell nuclei, which reflect functionally different regions or sub-compartments of the 3D nuclear landscape, and measurements of absolute distances between sites of different chromatin compaction. In addition, these tools allow 3D mapping of specific DNA/RNA sequences and nuclear proteins relative to the 3D chromatin compaction maps and comparisons of multiple cell nuclei. The tools are available in the free and open source R packages nucim and bioimagetools. We discuss the use of masks for the segmentation of nuclei and the use of DNA stains, such as DAPI, as a proxy for local differences in chromatin compaction. We further discuss the limitations of 3D maps of the nuclear landscape as well as problems of the biological interpretation of such data.

  16. Towards non-invasive 3D hepatotoxicity assays with optical coherence phase microscopy

    NASA Astrophysics Data System (ADS)

    Nelson, Leonard J.; Koulovasilopoulos, Andreas; Treskes, Philipp; Hayes, Peter C.; Plevris, John N.; Bagnaninchi, Pierre O.

    2015-03-01

    Three-dimensional tissue-engineered models are increasingly recognised as more physiologically-relevant than standard 2D cell culture for pre-clinical drug toxicity testing. However, many types of conventional toxicity assays are incompatible with dense 3D tissues. This study investigated the use of optical coherence phase microscopy (OCPM) as a novel approach to assess cell death in 3D tissue culture. For 3D micro-spheroid formation Human hepatic C3A cells were encapsulated in hyaluronic acid gels and cultured in 100μl MEME/10%FBS in 96-well plates. After spheroid formation the 3D liver constructs were exposed to acetaminophen on culture day 8. Acetaminophen hepatotoxicity in 3D cultures was evaluated using standard biochemical assays. An inverted OCPM in common path configuration was developed with a Callisto OCT engine (Thorlabs), centred at 930nm and a custom scanning head. Intensity data were used to perform in-depth microstructural imaging. In addition, phase fluctuations were measured by collecting several successive B scans at the same location, and statistics on the first time derivative of the phase, i.e. time fluctuations, were analysed over the acquisition time interval to retrieve overall cell viability. OCPM intensity (cell cluster size) and phase fluctuation statistics were directly compared with biochemical assays. In this study, we investigated optical coherence phase tomography to assess cell death in a 3d liver model after exposure to a prototypical hepatotoxin, acetaminophen. We showed that OCPM has the potential to assess noninvasively and label-free drug toxicity in 3D tissue models.

  17. A new 3D tracking method exploiting the capabilities of digital holography in microscopy

    NASA Astrophysics Data System (ADS)

    Miccio, L.; Memmolo, P.; Merola, F.; Fusco, S.; Embrione, V.; Netti, P. A.; Ferraro, P.

    2013-04-01

    A method for 3D tracking has been developed exploiting Digital Holographic Microscopy (DHM) features. In the framework of self-consistent platform for manipulation and measurement of biological specimen we use DHM for quantitative and completely label free analysis of specimen with low amplitude contrast. Tracking capability extend the potentiality of DHM allowing to monitor the motion of appropriate probes and correlate it with sample properties. Complete 3D tracking has been obtained for the probes avoiding the issue of amplitude refocusing in traditional tracking processing. Our technique belongs to the video tracking methods that, conversely from Quadrant Photo-Diode method, opens the possibility to track multiples probes. All the common used video tracking algorithms are based on the numerical analysis of amplitude images in the focus plane and the shift of the maxima in the image plane are measured after the application of an appropriate threshold. Our approach for video tracking uses different theoretical basis. A set of interferograms is recorded and the complex wavefields are managed numerically to obtain three dimensional displacements of the probes. The procedure works properly on an higher number of probes and independently from their size. This method overcomes the traditional video tracking issues as the inability to measure the axial movement and the choice of suitable threshold mask. The novel configuration allows 3D tracking of micro-particles and simultaneously can furnish Quantitative Phase-contrast maps of tracked micro-objects by interference microscopy, without changing the configuration. In this paper, we show a new concept for a compact interferometric microscope that can ensure the multifunctionality, accomplishing accurate 3D tracking and quantitative phase-contrast analysis. Experimental results are presented and discussed for in vitro cells. Through a very simple and compact optical arrangement we show how two different functionalities

  18. In vivo multiphoton microscopy associated to 3D image processing for human skin characterization

    NASA Astrophysics Data System (ADS)

    Baldeweck, T.; Tancrède, E.; Dokladal, P.; Koudoro, S.; Morard, V.; Meyer, F.; Decencière, E.; Pena, A.-M.

    2012-03-01

    Multiphoton microscopy has emerged in the past decade as a promising non-invasive skin imaging technique. The aim of this study was to assess whether multiphoton microscopy coupled to specific 3D image processing tools could provide new insights into the organization of different skin components and their age-related changes. For that purpose, we performed a clinical trial on 15 young and 15 aged human female volunteers on the ventral and dorsal side of the forearm using the DermaInspectR medical imaging device. We visualized the skin by taking advantage of intrinsic multiphoton signals from cells, elastic and collagen fibers. We also developed 3D image processing algorithms adapted to in vivo multiphoton images of human skin in order to extract quantitative parameters in each layer of the skin (epidermis and superficial dermis). The results show that in vivo multiphoton microscopy is able to evidence several skin alterations due to skin aging: morphological changes in the epidermis and modifications in the quantity and organization of the collagen and elastic fibers network. In conclusion, the association of multiphoton microscopy with specific image processing allows the three-dimensional organization of skin components to be visualized and quantified thus providing a powerful tool for cosmetic and dermatological investigations.

  19. 3D Printing: 3D Printing of Shape Memory Polymers for Flexible Electronic Devices (Adv. Mater. 22/2016).

    PubMed

    Zarek, Matt; Layani, Michael; Cooperstein, Ido; Sachyani, Ela; Cohn, Daniel; Magdassi, Shlomo

    2016-06-01

    On page 4449, D. Cohn, S. Magdassi, and co-workers describe a general and facile method based on 3D printing of methacrylated macromonomers to fabricate shape-memory objects that can be used in flexible and responsive electrical circuits. Such responsive objects can be used in the fabrication of soft robotics, minimal invasive medical devices, sensors, and wearable electronics. The use of 3D printing overcomes the poor processing characteristics of thermosets and enables complex geometries that are not easily accessible by other techniques.

  20. Point scanning confocal microscopy facilitates 3D human hair follicle imaging in tissue sections.

    PubMed

    Kloepper, Jennifer E; Bíró, Tamás; Paus, Ralf; Cseresnyés, Zoltán

    2010-07-01

    Efficiency is a key factor in determining whether a scientific method becomes widely accepted in practical applications. In dermatology, morphological characterisation of intact hair follicles by traditional methods can be rather inefficient. Samples are embedded, sliced, imaged and digitally reconstructed, which can be time-consuming. Confocal microscopy, on the other hand, is more efficient and readily applicable to study intact hair follicles. Modern confocal microscopes deliver and collect light very efficiently and thus allow high spatial resolution imaging of relatively thick samples. In this letter, we report that we successfully imaged entire intact human hair follicles using point scanning confocal microscopy. Light delivery and light-collection were further improved by preparing the samples in 2,2'-Thiodiethanol (TDE), thus reducing refractive index gradients. The relatively short total scan times and the high quality of the acquired 3D images make confocal microscopy a desirable method for studying intact hair follicles under normal and pathological conditions.

  1. Probing the Morphology and Evolving Dynamics of 3D Printed Nanostructures Using High-Speed Atomic Force Microscopy.

    PubMed

    Yang, Chen; Winkler, Robert; Dukic, Maja; Zhao, Jie; Plank, Harald; Fantner, Georg E

    2017-07-26

    Focused electron beam induced deposition (FEBID) has been demonstrated as a promising solution for synthesizing truly three-dimensional (3D) nanostructures. However, the lack of morphological feedback during growth complicates further development toward higher spatial fabrication precision. Here, we show that by combining in situ high speed atomic force microscopy (HS-AFM) with FEBID, morphologies in multistep fabrication process can be accessed. More importantly, the proposed method enables simultaneous imaging and fabrication operation, which opens new possibilities to investigate evolving mechanical properties of the deposit. The experiments indicate an exponential increase law of the mechanical resistance, meaning that a mechanically stable state establishes around 4 min after deposition.

  2. 3D reconstruction of cortical microtubules using multi-angle total internal reflection fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Jin, Luhong; Xiu, Peng; Zhou, Xiaoxu; Fan, Jiannan; Kuang, Cuifang; Liu, Xu; Xu, Yingke

    2017-01-01

    Total internal reflection fluorescence microscopy (TIRFM) has been widely used in biomedical research to visualize cellular processes near the cell surface. In this study, a novel multi-angle ring-illuminated TIRFM system, equipped with two galvo mirrors that are on conjugate plan of a 4f optical system was developed. Multi-angle TIRFM generates images with different penetration depths through the controlled variation of the incident angle of illuminating laser. We presented a method to perform three-dimensional (3-D) reconstruction of microtubules from multi-angle TIRFM images. The performance of our method was validated in simulated microtubules with variable signal-to-noise ratios (SNR) and the axial resolution and accuracy of reconstruction were evaluated in selecting different numbers of illumination angles or in different SNR conditions. In U373 cells, we reconstructed the 3-D localization of microtubules near the cell surface with high resolution using over a hundred different illumination angles. Theoretically, the presented TIRFM setup and 3-D reconstruction method can achieve 40 nm axial resolution in experimental conditions where SNR is as low as 2, with 35 different illumination angles. Moreover, our system and reconstruction method have the potential to be used in live cells to track membrane dynamics in 3-D.

  3. Imaging the behavior of molecules in biological systems: breaking the 3D speed barrier with 3D multi-resolution microscopy.

    PubMed

    Welsher, Kevin; Yang, Haw

    2015-01-01

    The overwhelming effort in the development of new microscopy methods has been focused on increasing the spatial and temporal resolution in all three dimensions to enable the measurement of the molecular scale phenomena at the heart of biological processes. However, there exists a significant speed barrier to existing 3D imaging methods, which is associated with the overhead required to image large volumes. This overhead can be overcome to provide nearly unlimited temporal precision by simply focusing on a single molecule or particle via real-time 3D single-particle tracking and the newly developed 3D Multi-resolution Microscopy (3D-MM). Here, we investigate the optical and mechanical limits of real-time 3D single-particle tracking in the context of other methods. In particular, we investigate the use of an optical cantilever for position sensitive detection, finding that this method yields system magnifications of over 3000×. We also investigate the ideal PID control parameters and their effect on the power spectrum of simulated trajectories. Taken together, these data suggest that the speed limit in real-time 3D single particle-tracking is a result of slow piezoelectric stage response as opposed to optical sensitivity or PID control.

  4. Investigation of osteoblast cells behavior in polymeric 3D micropatterned scaffolds using digital holographic microscopy.

    PubMed

    Mihailescu, M; Popescu, R C; Matei, A; Acasandrei, A; Paun, I A; Dinescu, M

    2014-08-01

    The effect of micropatterned polymeric scaffolds on the features of the cultured cells at different time intervals after seeding was investigated by digital holographic microscopy. Both parallel and perpendicular walls, with different heights, were fabricated using two-photon lithography on photopolymers. The walls were subsequently coated with polypyrrole-based thin films using the matrix assisted pulsed laser evaporation technique. Osteoblast-like cells, MG-63 line, were cultured on these polymeric 3D micropatterned scaffolds. To analyze these scaffolds with/without cultured cells, an inverted digital holographic microscope, which provides 3D images, was used. Information about the samples' refractive indices and heights was obtained from the phase shift introduced in the optical path. Characteristics of cell adhesion, alignment, orientation, and morphology as a function of the wall heights and time from seeding were highlighted.

  5. 3D map distribution of metallic nanoparticles in whole cells using MeV ion microscopy.

    PubMed

    Vasco, M S; Alves, L C; Corregidor, V; Correia, D; Godinho, C P; Sá-Correia, I; Bettiol, A; Watt, F; Pinheiro, T

    2017-08-01

    In this work, a new tool was developed, the MORIA program that readily translates Rutherford backscattering spectrometry (RBS) output data into visual information, creating a display of the distribution of elements in a true three-dimensional (3D) environment. The program methodology is illustrated with the analysis of yeast Saccharomyces cerevisiae cells, exposed to copper oxide nanoparticles (CuO-NP) and HeLa cells in the presence of gold nanoparticles (Au-NP), using different beam species, energies and nuclear microscopy systems. Results demonstrate that for both cell types, the NP internalization can be clearly perceived. The 3D models of the distribution of CuO-NP in S. cerevisiae cells indicate the nonuniform distribution of NP in the cellular environment and a relevant confinement of CuO-NP to the cell wall. This suggests the impenetrability of certain cellular organelles or compartments for NP. By contrast, using a high-resolution ion beam system, discretized agglomerates of Au-NP were visualized inside the HeLa cell. This is consistent with the mechanism of entry of these NPs in the cellular space by endocytosis enclosed in endosomal vesicles. This approach shows RBS to be a powerful imaging technique assigning to nuclear microscopy unparalleled potential to assess nanoparticle distribution inside the cellular volume. © 2017 The Authors Journal of Microscopy © 2017 Royal Microscopical Society.

  6. Note: An improved 3D imaging system for electron-electron coincidence measurements

    SciTech Connect

    Lin, Yun Fei; Lee, Suk Kyoung; Adhikari, Pradip; Herath, Thushani; Lingenfelter, Steven; Winney, Alexander H.; Li, Wen

    2015-09-15

    We demonstrate an improved imaging system that can achieve highly efficient 3D detection of two electrons in coincidence. The imaging system is based on a fast frame complementary metal-oxide semiconductor camera and a high-speed waveform digitizer. We have shown previously that this detection system is capable of 3D detection of ions and electrons with good temporal and spatial resolution. Here, we show that with a new timing analysis algorithm, this system can achieve an unprecedented dead-time (<0.7 ns) and dead-space (<1 mm) when detecting two electrons. A true zero dead-time detection is also demonstrated.

  7. 3D structure tensor analysis of light microscopy data for validating diffusion MRI.

    PubMed

    Khan, Ahmad Raza; Cornea, Anda; Leigland, Lindsey A; Kohama, Steven G; Jespersen, Sune Nørhøj; Kroenke, Christopher D

    2015-05-01

    Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image "stacks" acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations that

  8. 3D structure tensor analysis of light microscopy data for validating diffusion MRI

    PubMed Central

    Khan, Ahmad Raza; Cornea, Anda; Leigland, Lindsey A.; Kohama, Steven G.; Jespersen, Sune Nørhøj; Kroenke, Christopher D.

    2015-01-01

    Diffusion magnetic resonance imaging (d-MRI) is a powerful non-invasive and non-destructive technique for characterizing brain tissue on the microscopic scale. However, the lack of validation of d-MRI by independent experimental means poses an obstacle to accurate interpretation of data acquired using this method. Recently, structure tensor analysis has been applied to light microscopy images, and this technique holds promise to be a powerful validation strategy for d-MRI. Advantages of this approach include its similarity to d-MRI in terms of averaging the effects of a large number of cellular structures, and its simplicity, which enables it to be implemented in a high-throughput manner. However, a drawback of previous implementations of this technique arises from it being restricted to 2D. As a result, structure tensor analyses have been limited to tissue sectioned in a direction orthogonal to the direction of interest. Here we describe the analytical framework for extending structure tensor analysis to 3D, and utilize the results to analyze serial image “stacks” acquired with confocal microscopy of rhesus macaque hippocampal tissue. Implementation of 3D structure tensor procedures requires removal of sources of anisotropy introduced in tissue preparation and confocal imaging. This is accomplished with image processing steps to mitigate the effects of anisotropic tissue shrinkage, and the effects of anisotropy in the point spread function (PSF). In order to address the latter confound, we describe procedures for measuring the dependence of PSF anisotropy on distance from the microscope objective within tissue. Prior to microscopy, ex vivo d-MRI measurements performed on the hippocampal tissue revealed three regions of tissue with mutually orthogonal directions of least restricted diffusion that correspond to CA1, alveus and inferior longitudinal fasciculus. We demonstrate the ability of 3D structure tensor analysis to identify structure tensor orientations

  9. Computational-optical microscopy for 3D biological imaging beyond the diffraction limit

    NASA Astrophysics Data System (ADS)

    Grover, Ginni

    In recent years, super-resolution imaging has become an important fluorescent microscopy tool. It has enabled imaging of structures smaller than the optical diffraction limit with resolution less than 50 nm. Extension to high-resolution volume imaging has been achieved by integration with various optical techniques. In this thesis, development of a fluorescent microscope to enable high resolution, extended depth, three dimensional (3D) imaging is discussed; which is achieved by integration of computational methods with optical systems. In the first part of the thesis, point spread function (PSF) engineering for volume imaging is discussed. A class of PSFs, referred to as double-helix (DH) PSFs, is generated. The PSFs exhibit two focused spots in the image plane which rotate about the optical axis, encoding depth in rotation of the image. These PSFs extend the depth-of-field up to a factor of ˜5. Precision performance of the DH-PSFs, based on an information theoretical analysis, is compared with other 3D methods with conclusion that the DH-PSFs provide the best precision and the longest depth-of-field. Out of various possible DH-PSFs, a suitable PSF is obtained for super-resolution microscopy. The DH-PSFs are implemented in imaging systems, such as a microscope, with a special phase modulation at the pupil plane. Surface-relief elements which are polarization-insensitive and ˜90% light efficient are developed for phase modulation. The photon-efficient DH-PSF microscopes thus developed are used, along with optimal position estimation algorithms, for tracking and super-resolution imaging in 3D. Imaging at depths-of-field of up to 2.5 microm is achieved without focus scanning. Microtubules were imaged with 3D resolution of (6, 9, 39) nm, which is in close agreement with the theoretical limit. A quantitative study of co-localization of two proteins in volume was conducted in live bacteria. In the last part of the thesis practical aspects of the DH-PSF microscope are

  10. Soil microstructure and electron microscopy

    NASA Technical Reports Server (NTRS)

    Smart, P.; Fryer, J. R.

    1988-01-01

    As part of the process of comparing Martian soils with terrestial soils, high resolution electron microscopy and associated techniques should be used to examine the finer soil particles, and various techniques of electron and optical microscopy should be used to examine the undisturbed structure of Martian soils. To examine the structure of fine grained portions of the soil, transmission electron microscopy may be required. A striking feature of many Martian soils is their red color. Although the present-day Martian climate appears to be cold, this color is reminiscent of terrestial tropical red clays. Their chemical contents are broadly similar.

  11. Methods For Electronic 3-D Moving Pictures Without Glasses

    NASA Astrophysics Data System (ADS)

    Collender, Robert B.

    1987-06-01

    This paper describes implementation approaches in image acquisition and playback for 3-D computer graphics, 3-D television and 3-D theatre movies without special glasses. Projection lamps, spatial light modulators, CRT's and dynamic scanning are all eliminated by the application of an active image array, all static components and a semi-specular screen. The resulting picture shows horizontal parallax with a wide horizontal view field (up to 360 de-grees) giving a holographic appearance in full color with smooth continuous viewing without speckle. Static component systems are compared with dynamic component systems using both linear and circular arrays. Implementation of computer graphic systems are shown that allow complex shaded color images to extend from the viewer's eyes to infinity. Large screen systems visible by hundreds of people are feasible by the use of low f-stops and high gain screens in projection. Screen geometries and special screen properties are shown. Viewing characteristics offer no restrictions in view-position over the entire view-field and have a "look-around" feature for all the categories of computer graphics, television and movies. Standard video cassettes and optical discs can also interface the system to generate a 3-D window viewable without glasses. A prognosis is given for technology application to 3-D pictures without glasses that replicate the daily viewing experience. Super-position of computer graphics on real-world pictures is shown feasible.

  12. Utilization of 3D printing for an intravital microscopy platform to study the intestinal microcirculation.

    PubMed

    Burkovskiy, I; Lehmann, C; Jiang, C; Zhou, J

    2016-11-01

    Intravital microscopy of the intestine is a sophisticated technique that allows qualitative and quantitative in vivo observation of dynamic cellular interactions and blood flow at a high resolution. Physiological conditions of the animal and in particular of the observed organ, such as temperature and moisture are crucial for intravital imaging. Often, the microscopy stage with the animal or the organ of interest imposes limitations on how well the animal can be maintained. In addition, the access for additional oxygen supply or drug administration during the procedure is rather restricted. To address these limitations, we developed a novel intravital microscopy platform, allowing us to have improved access to the animal during the intravital microscopy procedure, as well as improved microenvironmental maintenance. The production process of this prototype platform is based on 3D printing of device parts in a single-step process. The simplicity of production and the advantages of this versatile and customizable design are shown and discussed in this paper. Our design potentially represents a major step forward in facilitating intestinal intravital imaging using fluorescent microscopy.

  13. Applications of Digital Holography: From Microscopy to 3D-Television

    NASA Astrophysics Data System (ADS)

    Kreis, T.

    2012-03-01

    The paper gives an overview of the applications of digital holography based on the one hand on CCD-recording, computer storage, and numerical reconstruction of the wave fields, and on the other hand on numerical calculation of computer generated holograms (CGH) and the transfer of these CGHs to spatial light modulators (SLM) for optical reconstruction of the wave fields. The first mentioned type of digital holography finds applications in digital holographic microscopy, particle analysis, and interferometric form and deformation measurement, while the second type constitutes the basis for holographic 3D TV. The space-bandwidth-problem occuring in this context is addressed and first partial solutions are presented.

  14. X-ray Laue Diffraction Microscopy in 3D at the Advanced Photon Source

    SciTech Connect

    Liu, W.; Zschack, P.; Tischler, Jonathan Zachary; Ice, Gene E; Larson, Ben C

    2011-01-01

    Studies of materials on mesoscopic length-scales require a penetrating structural probe with submicron point-to-point spatial resolution. The principle research activities at beamline 34-ID-E of the Advanced Photon Source (APS) involve development of exciting new micro-/nano-diffraction techniques for characterization and microscopy in support of both applied engineering and fundamental materials research. Taking advantage of the high brightness of the source, advanced focusing mirrors, a novel depth profiling technique, and high-speed area detectors, three-dimensional scanning Laue diffraction microscopy provides detailed local structural information of crystalline materials, such as crystallographic orientation, orientation gradients, and strain tensors. It is general and applicable to single-crystal, polycrystalline, composite, deformed, and functionally graded materials. Applications include 3D diffraction investigations for a diverse and growing user community with interests in materials deformation, electro-migration, recrystallization, fatigue, solid-solution precipitation, high-pressure environments, and condensed matter physics.

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

    PubMed

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

    2015-02-28

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

  16. Electronic Blending in Virtual Microscopy

    ERIC Educational Resources Information Center

    Maybury, Terrence S.; Farah, Camile S.

    2010-01-01

    Virtual microscopy (VM) is a relatively new technology that transforms the computer into a microscope. In essence, VM allows for the scanning and transfer of glass slides from light microscopy technology to the digital environment of the computer. This transition is also a function of the change from print knowledge to electronic knowledge, or as…

  17. A one-piece 3D printed flexure translation stage for open-source microscopy

    NASA Astrophysics Data System (ADS)

    Sharkey, James P.; Foo, Darryl C. W.; Kabla, Alexandre; Baumberg, Jeremy J.; Bowman, Richard W.

    2016-02-01

    Open source hardware has the potential to revolutionise the way we build scientific instruments; with the advent of readily available 3D printers, mechanical designs can now be shared, improved, and replicated faster and more easily than ever before. However, printed parts are typically plastic and often perform poorly compared to traditionally machined mechanisms. We have overcome many of the limitations of 3D printed mechanisms by exploiting the compliance of the plastic to produce a monolithic 3D printed flexure translation stage, capable of sub-micron-scale motion over a range of 8 × 8 × 4 mm. This requires minimal post-print clean-up and can be automated with readily available stepper motors. The resulting plastic composite structure is very stiff and exhibits remarkably low drift, moving less than 20 μm over the course of a week, without temperature stabilisation. This enables us to construct a miniature microscope with excellent mechanical stability, perfect for time-lapse measurements in situ in an incubator or fume hood. The ease of manufacture lends itself to use in containment facilities where disposability is advantageous and to experiments requiring many microscopes in parallel. High performance mechanisms based on printed flexures need not be limited to microscopy, and we anticipate their use in other devices both within the laboratory and beyond.

  18. Automated Identification and Localization of Hematopoietic Stem Cells in 3D Intravital Microscopy Data.

    PubMed

    Khorshed, Reema A; Hawkins, Edwin D; Duarte, Delfim; Scott, Mark K; Akinduro, Olufolake A; Rashidi, Narges M; Spitaler, Martin; Lo Celso, Cristina

    2015-07-14

    Measuring three-dimensional (3D) localization of hematopoietic stem cells (HSCs) within the bone marrow microenvironment using intravital microscopy is a rapidly expanding research theme. This approach holds the key to understanding the detail of HSC-niche interactions, which are critical for appropriate stem cell function. Due to the complex tissue architecture of the bone marrow and to the progressive introduction of scattering and signal loss at increasing imaging depths, there is no ready-made software to handle efficient segmentation and unbiased analysis of the data. To address this, we developed an automated image analysis tool that simplifies and standardizes the biological interpretation of 3D HSC microenvironment images. The algorithm identifies HSCs and measures their localization relative to surrounding osteoblast cells and bone collagen. We demonstrate here the effectiveness, consistency, and accuracy of the proposed approach compared to current manual analysis and its wider applicability to analyze other 3D bone marrow components. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  19. X-ray microscopy for in situ characterization of 3D nanostructural evolution in the laboratory

    NASA Astrophysics Data System (ADS)

    Hornberger, Benjamin; Bale, Hrishikesh; Merkle, Arno; Feser, Michael; Harris, William; Etchin, Sergey; Leibowitz, Marty; Qiu, Wei; Tkachuk, Andrei; Gu, Allen; Bradley, Robert S.; Lu, Xuekun; Withers, Philip J.; Clarke, Amy; Henderson, Kevin; Cordes, Nikolaus; Patterson, Brian M.

    2015-09-01

    X-ray microscopy (XRM) has emerged as a powerful technique that reveals 3D images and quantitative information of interior structures. XRM executed both in the laboratory and at the synchrotron have demonstrated critical analysis and materials characterization on meso-, micro-, and nanoscales, with spatial resolution down to 50 nm in laboratory systems. The non-destructive nature of X-rays has made the technique widely appealing, with potential for "4D" characterization, delivering 3D micro- and nanostructural information on the same sample as a function of sequential processing or experimental conditions. Understanding volumetric and nanostructural changes, such as solid deformation, pore evolution, and crack propagation are fundamental to understanding how materials form, deform, and perform. We will present recent instrumentation developments in laboratory based XRM including a novel in situ nanomechanical testing stage. These developments bridge the gap between existing in situ stages for micro scale XRM, and SEM/TEM techniques that offer nanometer resolution but are limited to analysis of surfaces or extremely thin samples whose behavior is strongly influenced by surface effects. Several applications will be presented including 3D-characterization and in situ mechanical testing of polymers, metal alloys, composites and biomaterials. They span multiple length scales from the micro- to the nanoscale and different mechanical testing modes such as compression, indentation and tension.

  20. Single-Particle Cryo-EM and 3D Reconstruction of Hybrid Nanoparticles with Electron-Dense Components.

    PubMed

    Yu, Guimei; Yan, Rui; Zhang, Chuan; Mao, Chengde; Jiang, Wen

    2015-10-01

    Single-particle cryo-electron microscopy (cryo-EM), accompanied with 3D reconstruction, is a broadly applicable tool for the structural characterization of macromolecules and nanoparticles. Recently, the cryo-EM field has pushed the limits of this technique to higher resolutions and samples of smaller molecular mass, however, some samples still present hurdles to this technique. Hybrid particles with electron-dense components, which have been studied using single-particle cryo-EM yet with limited success in 3D reconstruction due to the interference caused by electron-dense elements, constitute one group of such challenging samples. To process such hybrid particles, a masking method is developed in this work to adaptively remove pixels arising from electron-dense portions in individual projection images while maintaining maximal biomass signals for subsequent 2D alignment, 3D reconstruction, and iterative refinements. As demonstrated by the success in 3D reconstruction of an octahedron DNA/gold hybrid particle, which has been previously published without a 3D reconstruction, the devised strategy that combines adaptive masking and standard single-particle 3D reconstruction approach has overcome the hurdle of electron-dense elements interference, and is generally applicable to cryo-EM structural characterization of most, if not all, hybrid nanomaterials with electron-dense components.

  1. 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.

  2. Imaging brain vasculature with BOLD 3D microscopy: MR detection limits determined by in vivo two-photon microscopy

    PubMed Central

    Park, Sung-Hong; Masamoto, Kazuto; Hendrich, Kristy; Kanno, Iwao; Kim, Seong-Gi

    2008-01-01

    Rat brain vasculature was imaged at 9.4 T with blood oxygenation level dependent (BOLD) microscopy. Data were acquired without exogenous contrast agent in < 35 min using 3D gradient-echo imaging with 78-μm isotropic resolution (N = 6). Detailed vascular patterns including intracortical veins and some branches were observed in simple magnitude-contrast data acquired at an experimentally-optimized echo time. The venous origin of the dark patterns was confirmed by oxygenation-dependent studies, and when the systemic arterial oxygen saturation level was < 80%, BOLD microscopy revealed additional intracortical vessels presumed to be of arterial orgin. Quantification shows a decrease of intracortical venous density with depth. The full width at half-minimum intensity was 90–190 μm for most intracortical venous vessels identifiable by BOLD venography. Since actual diameters are not directly quantifiable by BOLD, we also measured diameter-dependent intracortical venous density in vivo by two-photon excitation fluorescent microscopy (N = 5). Density comparisons between the two modalities, along with computer simulations, show that venous vessels as small as ~16–30 μm diameter are detectable with 9.4-T BOLD microscopy under our experimental conditions. PMID:18383285

  3. 3D X-ray ultra-microscopy of bone tissue.

    PubMed

    Langer, M; Peyrin, F

    2016-02-01

    We review the current X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. We further review the different ultra-structural features that have so far been resolved: the lacuno-canalicular network, collagen orientation, nano-scale mineralization and their use as basis for mechanical simulations. X-ray computed tomography at the micro-metric scale is increasingly considered as the reference technique in imaging of bone micro-structure. The trend has been to push towards increasingly higher resolution. Due to the difficulty of realizing optics in the hard X-ray regime, the magnification has mainly been due to the use of visible light optics and indirect detection of the X-rays, which limits the attainable resolution with respect to the wavelength of the visible light used in detection. Recent developments in X-ray optics and instrumentation have allowed to implement several types of methods that achieve imaging that is limited in resolution by the X-ray wavelength, thus enabling computed tomography at the nano-scale. We review here the X-ray techniques with 3D imaging capability at the nano-scale: transmission X-ray microscopy, ptychography and in-line phase nano-tomography. Further, we review the different ultra-structural features that have so far been resolved and the applications that have been reported: imaging of the lacuno-canalicular network, direct analysis of collagen orientation, analysis of mineralization on the nano-scale and use of 3D images at the nano-scale to drive mechanical simulations. Finally, we discuss the issue of going beyond qualitative description to quantification of ultra-structural features.

  4. Rapid reconstruction of 3D neuronal morphology from light microscopy images with augmented rayburst sampling.

    PubMed

    Ming, Xing; Li, Anan; Wu, Jingpeng; Yan, Cheng; Ding, Wenxiang; Gong, Hui; Zeng, Shaoqun; Liu, Qian

    2013-01-01

    Digital reconstruction of three-dimensional (3D) neuronal morphology from light microscopy images provides a powerful technique for analysis of neural circuits. It is time-consuming to manually perform this process. Thus, efficient computer-assisted approaches are preferable. In this paper, we present an innovative method for the tracing and reconstruction of 3D neuronal morphology from light microscopy images. The method uses a prediction and refinement strategy that is based on exploration of local neuron structural features. We extended the rayburst sampling algorithm to a marching fashion, which starts from a single or a few seed points and marches recursively forward along neurite branches to trace and reconstruct the whole tree-like structure. A local radius-related but size-independent hemispherical sampling was used to predict the neurite centerline and detect branches. Iterative rayburst sampling was performed in the orthogonal plane, to refine the centerline location and to estimate the local radius. We implemented the method in a cooperative 3D interactive visualization-assisted system named flNeuronTool. The source code in C++ and the binaries are freely available at http://sourceforge.net/projects/flneurontool/. We validated and evaluated the proposed method using synthetic data and real datasets from the Digital Reconstruction of Axonal and Dendritic Morphology (DIADEM) challenge. Then, flNeuronTool was applied to mouse brain images acquired with the Micro-Optical Sectioning Tomography (MOST) system, to reconstruct single neurons and local neural circuits. The results showed that the system achieves a reasonable balance between fast speed and acceptable accuracy, which is promising for interactive applications in neuronal image analysis.

  5. Comparison of 3D Orientation Distribution Functions Measured with Confocal Microscopy and Diffusion MRI

    PubMed Central

    Schilling, Kurt; Janve, Vaibhav; Gao, Yurui; Stepniewska, Iwona; Landman, Bennett A; Anderson, Adam W

    2016-01-01

    The ability of diffusion MRI (dMRI) fiber tractography to non-invasively map three-dimensional (3D) anatomical networks in the human brain has made it a valuable tool in both clinical and research settings. However, there are many assumptions inherent to any tractography algorithm that can limit the accuracy of the reconstructed fiber tracts. Among them is the assumption that the diffusion-weighted images accurately reflect the underlying fiber orientation distribution (FOD) in the MRI voxel. Consequently, validating dMRI’s ability to assess the underlying fiber orientation in each voxel is critical for its use as a biomedical tool. Here, using post-mortem histology and confocal microscopy, we present a method to perform histological validation of orientation functions in 3D, which has previously been limited to two-dimensional analysis of tissue sections. We demonstrate the ability to extract the 3D FOD from confocal z-stacks, and quantify the agreement between the MRI estimates of orientation information obtained using constrained spherical deconvolution (CSD) and the true geometry of the fibers. We find an orientation error of approximately 6° in voxels containing nearly parallel fibers, and 10-11° in crossing fiber regions, and note that CSD was unable to resolve fibers crossing at angles below 60° in our dataset. This is the first time the 3D white matter orientation distribution is calculated from histology and compared to dMRI. Thus, this technique serves as a gold standard for dMRI validation studies - providing the ability to determine the extent to which the dMRI signal is consistent with the histological FOD, and to establish how well different dMRI models can predict the ground truth FOD. PMID:26804781

  6. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy

    PubMed Central

    Arnold, Jan; Mahamid, Julia; Lucic, Vladan; de Marco, Alex; Fernandez, Jose-Jesus; Laugks, Tim; Mayer, Tobias; Hyman, Anthony A.; Baumeister, Wolfgang; Plitzko, Jürgen M.

    2016-01-01

    The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy. PMID:26769364

  7. Site-Specific Cryo-focused Ion Beam Sample Preparation Guided by 3D Correlative Microscopy.

    PubMed

    Arnold, Jan; Mahamid, Julia; Lucic, Vladan; de Marco, Alex; Fernandez, Jose-Jesus; Laugks, Tim; Mayer, Tobias; Hyman, Anthony A; Baumeister, Wolfgang; Plitzko, Jürgen M

    2016-02-23

    The development of cryo-focused ion beam (cryo-FIB) for the thinning of frozen-hydrated biological specimens enabled cryo-electron tomography (cryo-ET) analysis in unperturbed cells and tissues. However, the volume represented within a typical FIB lamella constitutes a small fraction of the biological specimen. Retaining low-abundance and dynamic subcellular structures or macromolecular assemblies within such limited volumes requires precise targeting of the FIB milling process. In this study, we present the development of a cryo-stage allowing for spinning-disk confocal light microscopy at cryogenic temperatures and describe the incorporation of the new hardware into existing workflows for cellular sample preparation by cryo-FIB. Introduction of fiducial markers and subsequent computation of three-dimensional coordinate transformations provide correlation between light microscopy and scanning electron microscopy/FIB. The correlative approach is employed to guide the FIB milling process of vitrified cellular samples and to capture specific structures, namely fluorescently labeled lipid droplets, in lamellas that are 300 nm thick. The correlation procedure is then applied to localize the fluorescently labeled structures in the transmission electron microscopy image of the lamella. This approach can be employed to navigate the acquisition of cryo-ET data within FIB-lamellas at specific locations, unambiguously identified by fluorescence microscopy. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  8. Materials science through electron microscopy

    NASA Astrophysics Data System (ADS)

    Fujita, Hiroshi

    1992-03-01

    Electron microscopy has greatly contributed as a powerful tool in both the characterization and identification of materials in the atomic scale. In these contributions, the most important advantage is it's ability for dynamic study of phenomena, i.e., in situ experiments. This research has been carried out using high voltage electron microscopes, but some results have been obtained with high resolution electron microscopes under critical conditions. Electron microscopy has been improved further to become an indispensable ?Micro-Laboratory? in which formation of various advance materials can also be carried out precisely in the atomic scale. Electron beam science and engineering is a typical example in this research field, and detailed processes of crystalline-amorphous transition and electron irradiation induced foreign atom implantation have been clarified by this method. Recently, new applications to the research fields of non-linear material behavior, such as the behavior of atom clusters and the role of electric dipoles on diffusion, have been carried out.

  9. Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography

    PubMed Central

    Beckwith, Marianne Sandvold; Beckwith, Kai Sandvold; Sikorski, Pawel; Skogaker, Nan Tostrup

    2015-01-01

    Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D. PMID:26406896

  10. Seeing a Mycobacterium-Infected Cell in Nanoscale 3D: Correlative Imaging by Light Microscopy and FIB/SEM Tomography.

    PubMed

    Beckwith, Marianne Sandvold; Beckwith, Kai Sandvold; Sikorski, Pawel; Skogaker, Nan Tostrup; Flo, Trude Helen; Halaas, Øyvind

    2015-01-01

    Mycobacteria pose a threat to the world health today, with pathogenic and opportunistic bacteria causing tuberculosis and non-tuberculous disease in large parts of the population. Much is still unknown about the interplay between bacteria and host during infection and disease, and more research is needed to meet the challenge of drug resistance and inefficient vaccines. This work establishes a reliable and reproducible method for performing correlative imaging of human macrophages infected with mycobacteria at an ultra-high resolution and in 3D. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) tomography is applied, together with confocal fluorescence microscopy for localization of appropriately infected cells. The method is based on an Aclar poly(chloro-tri-fluoro)ethylene substrate, micropatterned into an advantageous geometry by a simple thermomoulding process. The platform increases the throughput and quality of FIB/SEM tomography analyses, and was successfully applied to detail the intracellular environment of a whole mycobacterium-infected macrophage in 3D.

  11. The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

    PubMed

    Dubrovin, E V; Presnova, G V; Rubtsova, M Yu; Egorov, A M; Grigorenko, V G; Yaminsky, I V

    2015-01-01

    Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

  12. 3D elemental sensitive imaging using transmission X-ray microscopy.

    PubMed

    Liu, Yijin; Meirer, Florian; Wang, Junyue; Requena, Guillermo; Williams, Phillip; Nelson, Johanna; Mehta, Apurva; Andrews, Joy C; Pianetta, Piero

    2012-09-01

    Determination of the heterogeneous distribution of metals in alloy/battery/catalyst and biological materials is critical to fully characterize and/or evaluate the functionality of the materials. Using synchrotron-based transmission x-ray microscopy (TXM), it is now feasible to perform nanoscale-resolution imaging over a wide X-ray energy range covering the absorption edges of many elements; combining elemental sensitive imaging with determination of sample morphology. We present an efficient and reliable methodology to perform 3D elemental sensitive imaging with excellent sample penetration (tens of microns) using hard X-ray TXM. A sample of an Al-Si piston alloy is used to demonstrate the capability of the proposed method.

  13. Automated 3D dendritic spine detection and analysis from two-photon microscopy

    NASA Astrophysics Data System (ADS)

    Koh, Ingrid Y. Y.; Lindquist, W. Brent

    2001-04-01

    The functional significance of dendritic spines and their plasticity to a wide spectrum of developmental and pathological conditions has led to extensive studies based on spine morphology. The advances in image acquisition techniques and the associated generation of large 3D data sets of optical micrographs have not been accompanied by comparable advances in data analysis techniques. We present an automated 3D spine detection and quantification procedure suitable for images obtained by laser scanning microscopy. The image is first processed by deconvolution and the dendritic phase consisting of the neuronal cytoplasm is extracted by segmentation. Spines are detected as geometrical protrusions relative to the dendritic backbone. As very thin necks may not be imaged, some spine `heads' may be detached from the dendrite and are detected as detached components. These detected heads are merged with spine `bases' where appropriate. Morphological characterizations on spine length, volume, density and shape classifications are obtained. For time-lapse data, images are registered and individual spines are traced through the image sequence. Successful comparison results on spine lengths and densities with manual analysis are obtained. This method is highly automatic and allows detailed and objective quantification of the structure and dynamics of dendritic spines, which can be important predictors for the function of neural networks.

  14. 3D tracking the Brownian motion of colloidal particles using digital holographic microscopy and joint reconstruction.

    PubMed

    Verrier, Nicolas; Fournier, Corinne; Fournel, Thierry

    2015-06-01

    In-line digital holography is a valuable tool for sizing, locating, and tracking micro- or nano-objects in a volume. When a parametric imaging model is available, inverse problem approaches provide a straightforward estimate of the object parameters by fitting data with the model, thereby allowing accurate reconstruction. As recently proposed and demonstrated, combining pixel super-resolution techniques with inverse problem approaches improves the estimation of particle size and 3D position. Here, we demonstrate the accurate tracking of colloidal particles in Brownian motion. Particle size and 3D position are jointly optimized from video holograms acquired with a digital holographic microscopy setup based on a low-end microscope objective (×20, NA 0.5). Exploiting information redundancy makes it possible to characterize particles with a standard deviation of 15 nm in size and a theoretical resolution of 2×2×5  nm3 for position under additive white Gaussian noise assumption.

  15. Quantitative analysis of 3D hydrodynamic focusing of microparticles by digital holographic microscopy

    NASA Astrophysics Data System (ADS)

    Perfetti, C.; Iorio, C. S.; El Mallahi, A.; Dubois, F.

    2014-02-01

    In the field of life sciences, the monitoring of biological samples has become a great concern to control the ecosystem evolution. However, their characterization is often time-consuming because the typical size of the organisms/particles of interest is several orders of magnitude smaller than the size of the sample under observation. Optical visualization systems require, then, high magnifications that severely limit the depth of focus and consequently decrease the sampling rate. To tackle this issue, the most straightforward technique consists in focusing the samples to fit the observation field of view by means of so-called "sheath flows". This expedient allows for increasing the overall flow rate, inversely related to the sampling time. In this article, a cost-effective 3D hydro-focusing device is presented. Several flow rates have been tested for both sample and sheath flows, and a thorough investigation of the shape of the focused streamlines conducted in order to validate the prototype design. The 3D position of the sampled micro-objects has been located by digital holographic microscopy and their distribution in cross-sections downstream the injection nozzle compared to numerical simulations. A maximum constriction—ratio between the part of the cross-sections where particles are present with and without focusing sheath flow—of 4.4 % has been observed confirming the potentiality of the technique. Also, a successful match between experiment and numerical simulation has been noted.

  16. 3D Analysis of Porosity in a Ceramic Coating Using X-ray Microscopy

    NASA Astrophysics Data System (ADS)

    Klement, Uta; Ekberg, Johanna; Kelly, Stephen T.

    2017-02-01

    Suspension plasma spraying (SPS) is a new, innovative plasma spray technique using a feedstock consisting of fine powder particles suspended in a liquid. Using SPS, ceramic coatings with columnar microstructures have been produced which are used as topcoats in thermal barrier coatings. The microstructure contains a wide pore size range consisting of inter-columnar spacings, micro-pores and nano-pores. Hence, determination of total porosity and pore size distribution is a challenge. Here, x-ray microscopy (XRM) has been applied for describing the complex pore space of the coatings because of its capability to image the (local) porosity within the coating in 3D at a resolution down to 50 nm. The possibility to quantitatively segment the analyzed volume allows analysis of both open and closed porosity. For an yttria-stabilized zirconia coating with feathery microstructure, both open and closed porosity were determined and it could be revealed that 11% of the pore volumes (1.4% of the total volume) are closed pores. The analyzed volume was reconstructed to illustrate the distribution of open and closed pores in 3D. Moreover, pore widths and pore volumes were determined. The results on the complex pore space obtained by XRM are discussed in connection with other porosimetry techniques.

  17. Automated Atom-By-Atom Three-Dimensional (3D) Reconstruction of Field Ion Microscopy Data.

    PubMed

    Dagan, Michal; Gault, Baptiste; Smith, George D W; Bagot, Paul A J; Moody, Michael P

    2017-03-20

    An automated procedure has been developed for the reconstruction of field ion microscopy (FIM) data that maintains its atomistic nature. FIM characterizes individual atoms on the specimen's surface, evolving subject to field evaporation, in a series of two-dimensional (2D) images. Its unique spatial resolution enables direct imaging of crystal defects as small as single vacancies. To fully exploit FIM's potential, automated analysis tools are required. The reconstruction algorithm developed here relies on minimal assumptions and is sensitive to atomic coordinates of all imaged atoms. It tracks the atoms across a sequence of images, allocating each to its respective crystallographic plane. The result is a highly accurate 3D lattice-resolved reconstruction. The procedure is applied to over 2000 tungsten atoms, including ion-implanted planes. The approach is further adapted to analyze carbides in a steel matrix, demonstrating its applicability to a range of materials. A vast amount of information is collected during the experiment that can underpin advanced analyses such as automated detection of "out of sequence" events, subangstrom surface displacements and defects effects on neighboring atoms. These analyses have the potential to reveal new insights into the field evaporation process and contribute to improving accuracy and scope of 3D FIM and atom probe characterization.

  18. Automatic segmentation and analysis of fibrin networks in 3D confocal microscopy images

    NASA Astrophysics Data System (ADS)

    Liu, Xiaomin; Mu, Jian; Machlus, Kellie R.; Wolberg, Alisa S.; Rosen, Elliot D.; Xu, Zhiliang; Alber, Mark S.; Chen, Danny Z.

    2012-02-01

    Fibrin networks are a major component of blood clots that provides structural support to the formation of growing clots. Abnormal fibrin networks that are too rigid or too unstable can promote cardiovascular problems and/or bleeding. However, current biological studies of fibrin networks rarely perform quantitative analysis of their structural properties (e.g., the density of branch points) due to the massive branching structures of the networks. In this paper, we present a new approach for segmenting and analyzing fibrin networks in 3D confocal microscopy images. We first identify the target fibrin network by applying the 3D region growing method with global thresholding. We then produce a one-voxel wide centerline for each fiber segment along which the branch points and other structural information of the network can be obtained. Branch points are identified by a novel approach based on the outer medial axis. Cells within the fibrin network are segmented by a new algorithm that combines cluster detection and surface reconstruction based on the α-shape approach. Our algorithm has been evaluated on computer phantom images of fibrin networks for identifying branch points. Experiments on z-stack images of different types of fibrin networks yielded results that are consistent with biological observations.

  19. Fluorescence fluctuation microscopy to reveal 3D architecture and function in the cell nucleus.

    PubMed

    Lenser, Thorsten; Weisshart, Klaus; Ulbricht, Tobias; Klement, Karolin; Hemmerich, Peter

    2010-01-01

    The three-dimensional (3D) architecture of the cell nucleus is determined not only by the presence of subnuclear domains, such as the nuclear envelope, chromosome territories, and nuclear bodies, but also by smaller domains which form in response to specific functions, such as RNA transcription, DNA replication, and DNA repair. Since both stable and dynamic structures contribute to nuclear morphology, it is important to study the biophysical principles of the formation of macromolecular assemblies within the nucleus. For this purpose, a variety of fluorescence fluctuation microscopy techniques can be applied. Here, we summarize our current knowledge on the 3D architecture of the mammalian cell nucleus and describe in detail how the assembly of functional nuclear protein complexes can be analyzed in living cells using fluorescence bleaching techniques, fluorescence correlation spectroscopy, raster image correlation spectroscopy, and mathematical modeling. In conclusion, the application of all these techniques in combination is a powerful tool to assess the full spectrum of nuclear protein dynamics and to understand the biophysical principles underlying nuclear structure and function.

  20. Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking.

    PubMed

    Dettmer, Simon L; Keyser, Ulrich F; Pagliara, Stefano

    2014-02-01

    In this article we present methods for measuring hindered Brownian motion in the confinement of complex 3D geometries using digital video microscopy. Here we discuss essential features of automated 3D particle tracking as well as diffusion data analysis. By introducing local mean squared displacement-vs-time curves, we are able to simultaneously measure the spatial dependence of diffusion coefficients, tracking accuracies and drift velocities. Such local measurements allow a more detailed and appropriate description of strongly heterogeneous systems as opposed to global measurements. Finite size effects of the tracking region on measuring mean squared displacements are also discussed. The use of these methods was crucial for the measurement of the diffusive behavior of spherical polystyrene particles (505 nm diameter) in a microfluidic chip. The particles explored an array of parallel channels with different cross sections as well as the bulk reservoirs. For this experiment we present the measurement of local tracking accuracies in all three axial directions as well as the diffusivity parallel to the channel axis while we observed no significant flow but purely Brownian motion. Finally, the presented algorithm is suitable also for tracking of fluorescently labeled particles and particles driven by an external force, e.g., electrokinetic or dielectrophoretic forces.

  1. Local characterization of hindered Brownian motion by using digital video microscopy and 3D particle tracking

    SciTech Connect

    Dettmer, Simon L.; Keyser, Ulrich F.; Pagliara, Stefano

    2014-02-15

    In this article we present methods for measuring hindered Brownian motion in the confinement of complex 3D geometries using digital video microscopy. Here we discuss essential features of automated 3D particle tracking as well as diffusion data analysis. By introducing local mean squared displacement-vs-time curves, we are able to simultaneously measure the spatial dependence of diffusion coefficients, tracking accuracies and drift velocities. Such local measurements allow a more detailed and appropriate description of strongly heterogeneous systems as opposed to global measurements. Finite size effects of the tracking region on measuring mean squared displacements are also discussed. The use of these methods was crucial for the measurement of the diffusive behavior of spherical polystyrene particles (505 nm diameter) in a microfluidic chip. The particles explored an array of parallel channels with different cross sections as well as the bulk reservoirs. For this experiment we present the measurement of local tracking accuracies in all three axial directions as well as the diffusivity parallel to the channel axis while we observed no significant flow but purely Brownian motion. Finally, the presented algorithm is suitable also for tracking of fluorescently labeled particles and particles driven by an external force, e.g., electrokinetic or dielectrophoretic forces.

  2. Electric fields in Scanning Electron Microscopy simulations

    NASA Astrophysics Data System (ADS)

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

    2016-03-01

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

  3. Electron microscopy of electromagnetic waveforms

    NASA Astrophysics Data System (ADS)

    Ryabov, A.; Baum, P.

    2016-07-01

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

  4. Analysis of incomplete excisions of basal-cell carcinomas after breadloaf microscopy compared with 3D-microscopy: a prospective randomized and blinded study.

    PubMed

    Boehringer, Alexandra; Adam, Patrick; Schnabl, Saskia; Häfner, Hans-Martin; Breuninger, Helmut

    2015-08-01

    Basal-cell carcinomas may show irregular, asymmetric subclinical growth. This study analyzed the efficacy of 'breadloaf' microscopy (serial sectioning) and three-dimensional (3D) microscopy in detecting positive tumor margins. Two hundred eighty-three (283) tumors (51.2%) were put into the breadloaf microscopy group; 270 tumors (48.8%) into the 3D microscopy group. The position of any detected tumor outgrowths was identified in clock face fashion. The time required for cutting and embedding the specimens and the examination of the microscopic slides was measured. Patient/tumor characteristics and surgical margins did not differ significantly. Tumor outgrowths at the excision margin were found in 62 of 283 cases (21.9%) in the breadloaf microscopy group and in 115 of 270 cases (42.6%) in the 3D microscopy group, constituting a highly significant difference (p < 0.001). This difference held true with incomplete excision of fibrosing (infiltrative/sclerosing/morpheaform) tumors [32.9% in the breadloaf microscopy group and 57.5% in the 3D microscopy group (p = 0.003)] and also with solid (nodular) tumors [16.1 and 34.2%, respectively (p < 0.001)]. The mean overall examination time required showed no important difference. In summary, for detection of tumor outgrowths, 3D microscopy has almost twice the sensitivity of breadloaf microscopy, particularly in the situation of aggressive/infiltrative carcinomas.

  5. Super-resolution imaging of the cytokinetic Z ring in live bacteria using fast 3D-structured illumination microscopy (f3D-SIM).

    PubMed

    Turnbull, Lynne; Strauss, Michael P; Liew, Andrew T F; Monahan, Leigh G; Whitchurch, Cynthia B; Harry, Elizabeth J

    2014-09-29

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques - stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

  6. Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

    PubMed Central

    Liew, Andrew T. F.; Monahan, Leigh G.; Whitchurch, Cynthia B.; Harry, Elizabeth J.

    2014-01-01

    Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques – stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 µm from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide. PMID:25286090

  7. Fast Segmentation of Stained Nuclei in Terabyte-Scale, Time Resolved 3D Microscopy Image Stacks

    PubMed Central

    Stegmaier, Johannes; Otte, Jens C.; Kobitski, Andrei; Bartschat, Andreas; Garcia, Ariel; Nienhaus, G. Ulrich; Strähle, Uwe; Mikut, Ralf

    2014-01-01

    Automated analysis of multi-dimensional microscopy images has become an integral part of modern research in life science. Most available algorithms that provide sufficient segmentation quality, however, are infeasible for a large amount of data due to their high complexity. In this contribution we present a fast parallelized segmentation method that is especially suited for the extraction of stained nuclei from microscopy images, e.g., of developing zebrafish embryos. The idea is to transform the input image based on gradient and normal directions in the proximity of detected seed points such that it can be handled by straightforward global thresholding like Otsu’s method. We evaluate the quality of the obtained segmentation results on a set of real and simulated benchmark images in 2D and 3D and show the algorithm’s superior performance compared to other state-of-the-art algorithms. We achieve an up to ten-fold decrease in processing times, allowing us to process large data sets while still providing reasonable segmentation results. PMID:24587204

  8. High-resolution nonlinear ellipse rotation measurements for 3D microscopy

    NASA Astrophysics Data System (ADS)

    Miguez, M. L.; Barbano, E. C.; Coura, J. A.; Zilio, S. C.; Misoguti, L.

    2015-03-01

    Nonlinear optical effects have been widely explored for microscopy due to the possibility of three-dimension (3D) image acquisition. Harmonic generation and nonlinear absorption, for instance, were used for this purpose. Each nonlinear effect has its own characteristic, complexity, type of contrast, advantage and disadvantage, etc. Recently, we developed a new simple and sensitive method for measuring nonlinear ellipse rotation (NER) using a dual-phase lock-in amplifier, which could be successfully applied for measuring local nonlinearity distribution on a sample and, consequently, the image acquisition. The NER is a particular refractive nonlinear effect which appears when strong elliptical polarized laser beam propagates along one nonlinear material. It is type of refractive Kerr nonlinearity similar to self-focalization responsible for the signal in the Z-scan technique. The self-focalization is one of the most important refractive effects, but it cannot be used for image acquisition. On the other hand, NER does. Furthermore, such refractive nonlinearities signal can be very strong and serves as a new contrast for nonlinear microscopy.

  9. Gradient light interference microscopy for 3D imaging of unlabeled specimens.

    PubMed

    Nguyen, Tan H; Kandel, Mikhail E; Rubessa, Marcello; Wheeler, Matthew B; Popescu, Gabriel

    2017-08-08

    Multiple scattering limits the contrast in optical imaging of thick specimens. Here, we present gradient light interference microscopy (GLIM) to extract three-dimensional information from both thin and thick unlabeled specimens. GLIM exploits a special case of low-coherence interferometry to extract phase information from the specimen, which in turn can be used to measure cell mass, volume, surface area, and their evolutions in time. Because it combines multiple intensity images that correspond to controlled phase shifts between two interfering waves, gradient light interference microscopy is capable of suppressing the incoherent background due to multiple scattering. GLIM can potentially become a valuable tool for in vitro fertilization, where contrast agents and fluorophores may impact the viability of the embryo. Since GLIM is implemented as an add-on module to an existing inverted microscope, we anticipate that it will be adopted rapidly by the biological community.Challenges in biological imaging include labeling, photobleaching and phototoxicity, as well as light scattering. Here, Nguyen et al. develop a quantitative phase method that uses low-coherence interferometry for label-free 3D imaging in scattering tissue.

  10. Dynamic imaging with electron microscopy

    ScienceCinema

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2016-07-12

    Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

  11. Dynamic imaging with electron microscopy

    SciTech Connect

    Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

    2014-02-20

    Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

  12. The 3d International Workshop on Computational Electronics

    NASA Astrophysics Data System (ADS)

    Goodnick, Stephen M.

    1994-09-01

    The Third International Workshop on Computational Electronics (IWCE) was held at the Benson Hotel in downtown Portland, Oregon, on May 18, 19, and 20, 1994. The workshop was devoted to a broad range of topics in computational electronics related to the simulation of electronic transport in semiconductors and semiconductor devices, particularly those which use large computational resources. The workshop was supported by the National Science Foundation (NSF), the Office of Naval Research and the Army Research Office, as well as local support from the Oregon Joint Graduate Schools of Engineering and the Oregon Center for Advanced Technology Education. There were over 100 participants in the Portland workshop, of which more than one quarter represented research groups outside of the United States from Austria, Canada, France, Germany, Italy, Japan, Switzerland, and the United Kingdom. There were a total 81 papers presented at the workshop, 9 invited talks, 26 oral presentations and 46 poster presentations. The emphasis of the contributions reflected the interdisciplinary nature of computational electronics with researchers from the Chemistry, Computer Science, Mathematics, Engineering, and Physics communities participating in the workshop.

  13. Non-destructive mapping of grain orientations in 3D by laboratory X-ray microscopy

    PubMed Central

    McDonald, S. A.; Reischig, P.; Holzner, C.; Lauridsen, E. M.; Withers, P. J.; Merkle, A. P.; Feser, M.

    2015-01-01

    The ability to characterise crystallographic microstructure, non-destructively and in three-dimensions, is a powerful tool for understanding many aspects related to damage and deformation mechanisms in polycrystalline materials. To this end, the technique of X-ray diffraction contrast tomography (DCT) using monochromatic synchrotron and polychromatic laboratory X-ray sources has been shown to be capable of mapping crystal grains and their orientations non-destructively in 3D. Here we describe a novel laboratory-based X-ray DCT modality (LabDCT), enabling the wider accessibility of the DCT technique for routine use and in-depth studies of, for example, temporal changes in crystallographic grain structure non-destructively over time through ‘4D’ in situ time-lapse studies. The capability of the technique is demonstrated by studying a titanium alloy (Ti-β21S) sample. In the current implementation the smallest grains that can be reliably detected are around 40 μm. The individual grain locations and orientations are reconstructed using the LabDCT method and the results are validated against independent measurements from phase contrast tomography and electron backscatter diffraction respectively. Application of the technique promises to provide important insights related to the roles of recrystallization and grain growth on materials properties as well as supporting 3D polycrystalline modelling of materials performance. PMID:26494523

  14. Tilting and moving-object lens for a 3D electron microscope.

    PubMed

    Ura, Katsumi

    2016-10-01

    I investigated the tilting and movement of the objective lens of a 3D electron microscope electrically as an extension of the moving-objective lens concept. The electric or magnetic potential along the tilted optical axis is analytically expressed by a multipole potential expansion about the fixed central axis. The field distributions for axially symmetric dipole and quadrupole components are numerically shown, where the optical axis of a bell-shaped magnetic lens is tilted around the lens center by up to 60°. The hexapole and octapole components are also shown at a tilt angle of 45°. © 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.

  15. Confocal laser scanning microscopy and 3-D reconstructions of neuronal structures in human brain cortex.

    PubMed

    Belichenko, P V; Dahlström, A

    1995-09-01

    Human brain material was studied with Lucifer yellow (LY) microinjections, indirect Texas red immunofluorescence, and confocal laser scanning microscopy (CLSM). The scanned images were transferred to a Silicon Graphics (SG) IRIS computer equipped with software for reconstructing the 3-D architecture of cells. By employing dual channel CLSM (Bio-Rad MRC 600), LY-injected cells and Texas red immunofluorescence could be studied simultaneously. Autopsy material with 2- to 48-h postmortem delays (6 control and 2 Rett's syndrome cases) as well as biopsy material (14 cases with therapy-resistant partial epilepsy--TRPE--undergoing neurosurgery) were used. In each specimen, 100-200 pyramidal and nonpyramidal neurons were visualized by LY microinjection. Single neurons were imaged and 2-D reconstructions of each neuron were made using z-projections of serial optical images; 3-D reconstructions and rotations were computed using the SG workstation, with VoxelView software from Vital Images (UK), and stored in a "neuronal library" on laser or magnetic optical disks. In Ret's syndrome cases and in patients with TRPE various abnormalities in the dendritic geometry of pyramidal and nonpyramidal cells have been found. The combination of LY injections with immunofluorescence allows the investigation of transmitter-related substances around the LY-injected cells. Using antibodies to synaptic vesicle proteins, presynaptic elements docking onto individual spines have been demonstrated. This approach may contribute to the understanding of different neurological and psychiatric disorders and may be useful in the Mapping of the Human Brain project. It may also be integrated with functional imaging by PET scan and with the human genome project.

  16. Electronic structure of 3d metals at finite temperatures

    SciTech Connect

    Delgadillo, I.; Gollisch, H.; Feder, R.

    1996-07-01

    A theoretical approach to the electronic structure of crystalline solids at finite temperature has been developed on the basis of the adiabatic approximation. For any given temperature, correlated ion core displacement configurations on large clusters with periodic boundary conditions are determined such that they are consistent with experimental phonon dispersion relations. Total and {ital k}{searrow}-resolved densities of states are obtained by a tight-binding recursion method for each configuration followed by a configurational average. In the case of ferromagnetic crystals, the above treatment is augmented by including the influence of spin fluctuations. The local magnetic moments associated with the atomic sites are assumed to fluctuate subject to an average magnetization and a short-range order specific for the given temperature. The spin-resolved electronic structure for temperatures up to the Curie temperature and beyond can thus be obtained. Numerical calculations are performed on Cu and Ni and the results compared to experimental photoemission data. {copyright} {ital 1996 American Institute of Physics.}

  17. Dynamics of electronically inelastic collisions from 3D Doppler measurements

    SciTech Connect

    Suits, A.G.; de Pujo, P.; Sublemontier, O.; Visticot, J.; Berlande, J.; Cuvellier, J.; Gustavsson, T.; Mestdagh, J.; Meynadier, P. ); Lee, Y.T. )

    1991-11-25

    Flux-velocity contour maps were obtained for the inelastic collision process Ba({sup 1}{ital P}{sub 1})+O{sub 2}N{sub 2}{r arrow}Ba({sup 3}{ital P}{sub 2})+O{sub 2}N{sub 2} from Doppler scans of scattered Ba({sup 3}{ital P}{sub 2}) taken over a range of probe laser directions in a crossed-beam experiment. Collision with O{sub 2} resulted in sharply forward scattered Ba({sup 3}{ital P}{sub 2}), with efficient conversion of inital electronic energy into O{sub 2} internal energy and little momentum transfer. Collision with N{sub 2} was dominated by wide-angle scattering with most of the available electronic energy appearing in product translation. The results suggest the importance of large-impact-parameter collisions and a near-resonant energy transfer in the case of O{sub 2}, while for N{sub 2} close collisions dominate despite the presence of an analogous near-resonant channel. The results represent the first direct experimental demonstration of a near-resonant quenching process.

  18. 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.

  19. Clean localization super-resolution microscopy for 3D biological imaging

    NASA Astrophysics Data System (ADS)

    Mondal, Partha P.; Curthoys, Nikki M.; Hess, Samuel T.

    2016-01-01

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  20. Real Time Gabor-Domain Optical Coherence Microscopy for 3D Imaging.

    PubMed

    Rolland, Jannick P; Canavesi, Cristina; Tankam, Patrice; Cogliati, Andrea; Lanis, Mara; Santhanam, Anand P

    2016-01-01

    Fast, robust, nondestructive 3D imaging is needed for the characterization of microscopic tissue structures across various clinical applications. A custom microelectromechanical system (MEMS)-based 2D scanner was developed to achieve, together with a multi-level GPU architecture, 55 kHz fast-axis A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) custom instrument. GD-OCM yields high-definition micrometer-class volumetric images. A dynamic depth of focusing capability through a bio-inspired liquid lens-based microscope design, as in whales' eyes, was developed to enable the high definition instrument throughout a large field of view of 1 mm3 volume of imaging. Developing this technology is prime to enable integration within the workflow of clinical environments. Imaging at an invariant resolution of 2 μm has been achieved throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. Volumetric scans of human skin in vivo and an excised human cornea are presented.

  1. Clean localization super-resolution microscopy for 3D biological imaging

    SciTech Connect

    Mondal, Partha P.; Curthoys, Nikki M.; Hess, Samuel T.

    2016-01-15

    We propose clean localization microscopy (a variant of fPALM) using a molecule filtering technique. Localization imaging involves acquiring a large number of images containing single molecule signatures followed by one-to-one mapping to render a super-resolution image. In principle, this process can be repeated for other z-planes to construct a 3D image. But, single molecules observed from off-focal planes result in false representation of their presence in the focal plane, resulting in incorrect quantification and analysis. We overcome this with a single molecule filtering technique that imposes constraints on the diffraction limited spot size of single molecules in the image plane. Calibration with sub-diffraction size beads puts a natural cutoff on the actual diffraction-limited size of single molecules in the focal plane. This helps in distinguishing beads present in the focal plane from those in the off-focal planes thereby providing an estimate of the single molecules in the focal plane. We study the distribution of actin (labeled with a photoactivatable CAGE 552 dye) in NIH 3T3 mouse fibroblast cells.

  2. Jamming of a soft granular system of hollow elastic shells in 3D using confocal microscopy

    NASA Astrophysics Data System (ADS)

    Jose, Jissy; van Blaaderen, Alfons; Imhof, Arnout

    2014-03-01

    We introduce a new system for jammed matter research consisting of monodisperse, fluorescent, hollow deformable shells, dispersed in an index matched solvent. The interesting fact about these elastic shells is that they undergo buckling: in each contact one of the shells receives an indentation from its neighbor under compressive stress. This kind of deformation is different from the soft granular systems experimentally studied so far like photo elastic disks, emulsions and foams, where the particles are flattened in the region of contact and conserve their volume. Using confocal microscopy and image analysis routines (ImageJ software) we identified the 3D position of the particles with sub pixel resolution. The force law to find the contact forces between pairs of particle is derived from the theory of elasticity of thin shells, where force is proportional to the square root of indentation depth. The distribution of normalized contact forces showed a similar trend like other jammed systems with a peak around the mean and a tail that decayed faster than exponential away from jamming threshold. Further, we also investigated the structure of the jammed packings and contact number distribution with distance to jamming.

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

  4. 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.

  5. Model-based segmentation and quantification of subcellular structures in 2D and 3D fluorescent microscopy images

    NASA Astrophysics Data System (ADS)

    Wörz, Stefan; Heinzer, Stephan; Weiss, Matthias; Rohr, Karl

    2008-03-01

    We introduce a model-based approach for segmenting and quantifying GFP-tagged subcellular structures of the Golgi apparatus in 2D and 3D microscopy images. The approach is based on 2D and 3D intensity models, which are directly fitted to an image within 2D circular or 3D spherical regions-of-interest (ROIs). We also propose automatic approaches for the detection of candidates, for the initialization of the model parameters, and for adapting the size of the ROI used for model fitting. Based on the fitting results, we determine statistical information about the spatial distribution and the total amount of intensity (fluorescence) of the subcellular structures. We demonstrate the applicability of our new approach based on 2D and 3D microscopy images.

  6. Electron Microscopy of Intracellular Protozoa.

    DTIC Science & Technology

    1982-08-01

    the erythrocytes infected with P. falciparum. Scannning electron microscopy demonstrated numerous cone-shaped knobs evenly distributed over the entire...Mystromys albicaudatus and are being used as an excellent model of American cutaneous leishmaniasis In anti-leishmanial drug screen tests at WRAIR1 s...available liquid media for rapid cultivation. J Parasitol 1978, 76:309- .316. - 16 - I- ~. . . ... .. . . " " :" "". . REFERENCES (cont’d) 17. Cohn ZA

  7. Tracking 3D Picometer-Scale Motions of Single Nanoparticles with High-Energy Electron Probes

    PubMed Central

    Ogawa, Naoki; Hoshisashi, Kentaro; Sekiguchi, Hiroshi; Ichiyanagi, Kouhei; Matsushita, Yufuku; Hirohata, Yasuhisa; Suzuki, Seiichi; Ishikawa, Akira; Sasaki, Yuji C.

    2013-01-01

    We observed the high-speed anisotropic motion of an individual gold nanoparticle in 3D at the picometer scale using a high-energy electron probe. Diffracted electron tracking (DET) using the electron back-scattered diffraction (EBSD) patterns of labeled nanoparticles under wet-SEM allowed us to super-accurately measure the time-resolved 3D motion of individual nanoparticles in aqueous conditions. The highly precise DET data corresponded to the 3D anisotropic log-normal Gaussian distributions over time at the millisecond scale. PMID:23868465

  8. Volumetric label-free imaging and 3D reconstruction of mammalian cochlea based on two-photon excitation fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Zhang, Xianzeng; Geng, Yang; Ye, Qing; Zhan, Zhenlin; Xie, Shusen

    2013-11-01

    The visualization of the delicate structure and spatial relationship of intracochlear sensory cells has relied on the laborious procedures of tissue excision, fixation, sectioning and staining for light and electron microscopy. Confocal microscopy is advantageous for its high resolution and deep penetration depth, yet disadvantageous due to the necessity of exogenous labeling. In this study, we present the volumetric imaging of rat cochlea without exogenous dyes using a near-infrared femtosecond laser as the excitation mechanism and endogenous two-photon excitation fluorescence (TPEF) as the contrast mechanism. We find that TPEF exhibits strong contrast, allowing cellular and even subcellular resolution imaging of the cochlea, differentiating cell types, visualizing delicate structures and the radial nerve fiber. Our results further demonstrate that 3D reconstruction rendered with z-stacks of optical sections enables better revealment of fine structures and spatial relationships, and easily performed morphometric analysis. The TPEF-based optical biopsy technique provides great potential for new and sensitive diagnostic tools for hearing loss or hearing disorders, especially when combined with fiber-based microendoscopy.

  9. A 3D High Frequency Array Based 16 Channel Photoacoustic Microscopy System for In Vivo Micro-vascular Imaging

    PubMed Central

    Zemp, Roger; Yen, Jesse; Wang, L.V.; Shung, K. Kirk

    2009-01-01

    This paper discusses the design of a novel photoacoustic microscopy imaging system with promise for studying the structure of tissue microvasculature for applications in visualizing angiogenesis. A new sixteen channel analog and digital high frequency array based photoacoustic microscopy system (PAM) was developed using an Nd:YLF pumped tunable dye laser, a 30MHz piezo composite linear array transducer and a custom multi-channel receiver electronics system. Using offline delay and sum beamforming and beamsteering, phantom images were obtained from a 6µm carbon fiber in water at a depth of 8mm. The measured -6dB lateral and axial spatial resolution of the system was 100±5µm and 45±5µm, respectively. The dynamic focusing capability of the system was demonstrated by imaging a composite carbon fiber matrix through a 12.5mm imaging depth. Next, 2-D in vivo images were formed of vessels around 100µm in diameter in the human hand. 3-D in vivo images were also formed of micro-vessels 3mm below the surface of the skin in two Sprague Dawley rats. PMID:19131292

  10. 3D Printing of Shape Memory Polymers for Flexible Electronic Devices.

    PubMed

    Zarek, Matt; Layani, Michael; Cooperstein, Ido; Sachyani, Ela; Cohn, Daniel; Magdassi, Shlomo

    2016-06-01

    The formation of 3D objects composed of shape memory polymers for flexible electronics is described. Layer-by-layer photopolymerization of methacrylated semicrystalline molten macromonomers by a 3D digital light processing printer enables rapid fabrication of complex objects and imparts shape memory functionality for electrical circuits.

  11. Preparation of cryofixed cells for improved 3D ultrastructure with scanning transmission electron tomography.

    PubMed

    Höhn, Katharina; Sailer, Michaela; Wang, Li; Lorenz, Myriam; Schneider, Marion E; Walther, Paul

    2011-01-01

    Scanning transmission electron tomography offers enhanced contrast compared to regular transmission electron microscopy, and thicker samples, up to 1 μm or more, can be analyzed, since the depth of focus and inelastic scattering are not limitations. In this study, we combine this novel imaging approach with state of the art specimen preparation by using novel light transparent sapphire specimen carrier for high-pressure freezing and a freeze substitution protocol for better contrast of membranes. This combination allows for imaging membranes and other subcellular structures with unsurpassed quality. This is demonstrated with mitochondria, where the inner and outer mitochondrial membranes as well as the membranes in the cristae appear in very close apposition with a minimal intermembrane space. These findings correspond well with old observations using freeze fracturing. In 880-nm thick sections of hemophagocytes, the three-dimensional structure of membrane sheets could be observed in the virtual sections of the tomogram. Microtubules, actin and intermediate filaments could be visualized within one sample. Intermediate filaments, however, could even be better observed in 3D using surface scanning electron tomography.

  12. Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

    PubMed Central

    Song, Changyong; Takagi, Masatoshi; Park, Jaehyun; Xu, Rui; Gallagher-Jones, Marcus; Imamoto, Naoko; Ishikawa, Tetsuya

    2014-01-01

    Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens. PMID:25185543

  13. Spectroscopic imaging in electron microscopy

    SciTech Connect

    Pennycook, Stephen J; Colliex, C.

    2012-01-01

    In the scanning transmission electron microscope, multiple signals can be simultaneously collected, including the transmitted and scattered electron signals (bright field and annular dark field or Z-contrast images), along with spectroscopic signals such as inelastically scattered electrons and emitted photons. In the last few years, the successful development of aberration correctors for the electron microscope has transformed the field of electron microscopy, opening up new possibilities for correlating structure to functionality. Aberration correction not only allows for enhanced structural resolution with incident probes into the sub-angstrom range, but can also provide greater probe currents to facilitate mapping of intrinsically weak spectroscopic signals at the nanoscale or even the atomic level. In this issue of MRS Bulletin, we illustrate the power of the new generation of electron microscopes with a combination of imaging and spectroscopy. We show the mapping of elemental distributions at atomic resolution and also the mapping of electronic and optical properties at unprecedented spatial resolution, with applications ranging from graphene to plasmonic nanostructures, and oxide interfaces to biology.

  14. Immunogold Labeling for Scanning Electron Microscopy.

    PubMed

    Goldberg, Martin W; Fišerová, Jindřiška

    2016-01-01

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

  15. Immunogold labelling for scanning electron microscopy.

    PubMed

    Goldberg, Martin W; Fiserova, Jindriska

    2010-01-01

    Scanning electron microscopes are useful biological tools that can be used to image the surface of whole organisms, tissues, cells, cellular components and macromolecules. Processes and structures that exist at surfaces can be imaged in pseudo or real 3D at magnifications of anything from about x10 to x1,000,000. Therefore a whole multicellular organism, such as a fly, or a single protein embedded in one of its cell membranes can be visualised. In order to identify that protein at high resolution, or to see and quantify its distribution at lower magnifications, samples can be labelled with antibodies. Any surface that can be exposed can potentially be studied in this way. Presented here is a generic method for immunogold labelling for scanning electron microscopy, using two examples of specimens: isolated nuclear envelopes and the cytoskeleton of mammalian culture cells. Various parameters for sample preparation, fixation, immunogold labelling, drying, metal coating and imaging are discussed so that the best immunogold scanning electron microscopy results can be obtained from different types of specimens.

  16. Electronic structure and properties of rare earth and 3d transition metal compounds

    SciTech Connect

    Dagys, R.; Babonas, G.J. )

    1994-03-01

    Excitation energies of various electronic configurations in rare earth and 3d transition metal compounds are considered and related to the peculiarities of the observed electrical and optical properties. Intraionic excitations of 4f, 3d electrons to less localized nl states are shown to be equally important as interionic d-d or charge transfer transitions usually considered, and to be even more significant in compounds containing low valence metals.

  17. Contiguous 3 d and 4 f Magnetism: Strongly Correlated 3 d Electrons in YbFe2Al10

    NASA Astrophysics Data System (ADS)

    Khuntia, P.; Peratheepan, P.; Strydom, A. M.; Utsumi, Y.; Ko, K.-T.; Tsuei, K.-D.; Tjeng, L. H.; Steglich, F.; Baenitz, M.

    2014-11-01

    We present magnetization, specific heat, and Al 27 NMR investigations on YbFe2Al10 over a wide range in temperature and magnetic field. The magnetic susceptibility at low temperatures is strongly enhanced at weak magnetic fields, accompanied by a ln (T0/T ) divergence of the low-T specific heat coefficient in zero field, which indicates a ground state of correlated electrons. From our hard-x-ray photoemission spectroscopy study, the Yb valence at 50 K is evaluated to be 2.38. The system displays valence fluctuating behavior in the low to intermediate temperature range, whereas above 400 K, Yb3 + carries a full and stable moment, and Fe carries a moment of about 3.1 μB. The enhanced value of the Sommerfeld-Wilson ratio and the dynamic scaling of the spin-lattice relaxation rate divided by T [(1 /T1T ) 27 ] with static susceptibility suggests admixed ferromagnetic correlations. (1 /T1T ) 27 simultaneously tracks the valence fluctuations from the 4 f Yb ions in the high temperature range and field dependent antiferromagnetic correlations among partially Kondo screened Fe 3 d moments at low temperature; the latter evolve out of an Yb 4 f admixed conduction band.

  18. Infrared differential interference contrast microscopy for overlay metrology on 3D-interconnect bonded wafers

    NASA Astrophysics Data System (ADS)

    Ku, Yi-sha; Shyu, Deh-Ming; Lin, Yeou-Sung; Cho, Chia-Hung

    2013-04-01

    Overlay metrology for stacked layers will be playing a key role in bringing 3D IC devices into manufacturing. However, such bonded wafer pairs present a metrology challenge for optical microscopy tools by the opaque nature of silicon. Using infrared microscopy, silicon wafers become transparent to the near-infrared (NIR) wavelengths of the electromagnetic spectrum, enabling metrology at the interface of bonded wafer pairs. Wafers can be bonded face to face (F2F) or face to back (F2B) which the stacking direction is dictated by how the stacks are carried in the process and functionality required. For example, Memory stacks tend to use F2B stacking enables a better managed design. Current commercial tools use single image technique for F2F bonding overlay measurement because depth of focus is sufficient to include both surfaces; and use multiple image techniques for F2B overlay measurement application for the depth of focus is no longer sufficient to include both stacked wafer surfaces. There is a need to specify the Z coordinate or stacking wafer number through the silicon when visiting measurement wafer sites. Two shown images are of the same (X, Y) but separate Z location acquired at focus position of each wafer surface containing overlay marks. Usually the top surface image is bright and clear; however, the bottom surface image is somewhat darker and noisier as an adhesive layer is used in between to bond the silicon wafers. Thus the top and bottom surface images are further processed to achieve similar brightness and noise level before merged for overlay measurement. This paper presents a special overlay measurement technique, using the infrared differential interference contrast (DIC) microscopy technique to measure the F2B wafer bonding overlay by a single shot image. A pair of thinned wafers at 50 and 150 μm thickness is bonded on top of a carrier wafer to evaluate the bonding overlay. It works on the principle of interferometry to gain information about the

  19. Examination of heterogeneous crossing sequences between toner and rollerball pen strokes by digital microscopy and 3-D laser profilometry.

    PubMed

    Montani, Isabelle; Mazzella, Williams; Guichard, Marion; Marquis, Raymond

    2012-07-01

    The determination of line crossing sequences between rollerball pens and laser printers presents difficulties that may not be overcome using traditional techniques. This research aimed to study the potential of digital microscopy and 3-D laser profilometry to determine line crossing sequences between a toner and an aqueous ink line. Different paper types, rollerball pens, and writing pressure were tested. Correct opinions of the sequence were given for all case scenarios, using both techniques. When the toner was printed before the ink, a light reflection was observed in all crossing specimens, while this was never observed in the other sequence types. The 3-D laser profilometry, more time-consuming, presented the main advantage of providing quantitative results. The findings confirm the potential of the 3-D laser profilometry and demonstrate the efficiency of digital microscopy as a new technique for determining the sequence of line crossings involving rollerball pen ink and toner.

  20. Synthetic incoherence for electron microscopy.

    PubMed

    Levine, Zachary H; Dunstan, Robyn M

    2007-08-01

    Tomographic studies of submicrometer samples in materials science using electron microscopy have been inhibited by diffraction effects. In the present work, we describe a practical method for ameliorating these effects. First, we find an analytic expression for the mutual coherence function for hollow-cone illumination. Then, we use this analytic expression to propose a Gaussian weighting of hollow-cone illumination, which we name tapered solid-cone illumination, and present an analytic expression for its mutual coherence function. Finally, we investigate numerically an n-ring approximation to tapered solid-cone illumination. The results suggest a method for removing diffraction effects and hence enabling tomography.

  1. Liquid Cell Transmission Electron Microscopy.

    PubMed

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-27

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

  2. Liquid Cell Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Liao, Hong-Gang; Zheng, Haimei

    2016-05-01

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

  3. Angular distribution of Auger electrons due to 3d-shell impact ionization of krypton

    NASA Technical Reports Server (NTRS)

    Omidvar, K.

    1977-01-01

    Cross sections for electron impact ionization of krypton due to ejection of a 3d-shell electron have been calculated using screened hydrogenic and Hartree-Slater wavefunctions for the target atom. While the total ionization cross sections in the two approximations are within 10% of each other, the Auger electron angular distribution, related to cross sections for specific magnetic quantum numbers of the 3d electrons, are widely different in the two approximations. The angular distribution due to the Hartree-Slater approximation is in excellent agreement with measurement. The physical reason for the discrepancies in the two approximations is explained.

  4. Epoxy Resins in Electron Microscopy

    PubMed Central

    Finck, Henry

    1960-01-01

    A method of embedding biological specimens in araldite 502 (Ciba) has been developed for materials available in the United States. Araldite-embedded tissues are suitable for electron microscopy, but the cutting qualities of the resin necessitates more than routine attention during microtomy. The rather high viscosity of araldite 502 also seems to be an unnecessary handicap. The less viscous epoxy epon 812 (Shell) produces specimens with improved cutting qualities, and has several features—low shrinkage and absence of specimen damage during cure, minimal compression of sections, relative absence of electron beam-induced section damage, etc.—which recommends it as a routine embedding material. The hardness of the cured resin can be easily adjusted by several methods to suit the materials embedded in it. Several problems and advantages of working with sections of epoxy resins are also discussed. PMID:13822825

  5. 3D Quantitative Confocal Laser Microscopy of Ilmenite Volume Distribution in Alpe Arami Olivine

    NASA Astrophysics Data System (ADS)

    Bozhilov, K. N.

    2001-12-01

    The deep origin of the Alpe Arami garnet lherzolite massif in the Swiss Alps proposed by Dobrzhinetskaya et al. (Science, 1996) has been a focus of heated debate. One of the lines of evidence supporting an exhumation from more than 200 km depth includes the abundance, distribution, and orientation of magnesian ilmenite rods in the oldest generation of olivine. This argument has been disputed in terms of the abundance of ilmenite and consequently the maximum TiO2 content in the discussed olivine. In order to address this issue, we have directly measured the volume fraction of ilmenite of the oldest generation of olivine by applying confocal laser scanning microscopy (CLSM). CLSM is a method which allows for three-dimensional imaging and quantitative volume determination by optical sectioning of the objects. The images for 3D reconstruction and measurements were acquired from petrographic thin sections in reflected laser light with 488 nm wavelength. Measurements of more than 80 olivine grains in six thin sections of our material yielded an average volume fraction of 0.31% ilmenite in the oldest generation of olivine from Alpe Arami. This translates into 0.23 wt.% TiO2 in olivine with error in determination of ±0.097 wt.%, a value significantly different from that of 0.02 to 0.03 wt.% TiO2 determined by Hacker et al. (Science, 1997) by a broad-beam microanalysis technique. During the complex geological history of the Alpe Arami massif, several events of metamorphism are recorded which all could have caused increased mobility of the mineral components. Evidence for loss of TiO2 from olivine is the tendency for high densities of ilmenite to be restricted to cores of old grains, the complete absence of ilmenite inclusions from the younger, recrystallized, generation of olivine, and reduction in ilmenite size and abundance in more serpentinized specimens. These observations suggest that only olivine grains with the highest concentrations of ilmenite are close to the

  6. Liquid crystal lens array for 3D microscopy and endoscope application

    NASA Astrophysics Data System (ADS)

    Huang, Yi-Pai; Hsieh, Po-Yuan; Hassanfiroozi, Amir; Chu, Chao-Yu; Hsuan, Yun; Martinez, Manuel; Javidi, Bahram

    2016-06-01

    In this paper, we demonstrate two liquid crystal (LC) lens array devices for 3D microscope and 3D endoscope applications respectively. Compared with the previous 3D biomedical system, the proposed LC lens arrays are not only switchable between 2D and 3D modes, but also are able to adjust focus in both modes. The multi-function liquid crystal lens (MFLC-lens) array with dual layer electrode has diameter 1.42 mm, which is much smaller than the conventional 3D endoscope with double fixed lenses. The hexagonal liquid crystal micro-lens array (HLC-MLA) instead of fixed micro-lens array in 3D light field microscope can extend the effective depth of field from 60 um to 780 um. To achieve the LC lens arrays, a high-resistance layer needs to be coated on the electrodes to generate an ideal gradient electric-field distribution, which can induce a lens-like form of LC molecules. The parameters and characteristics of high-resistance layer are investigated and discussed with an aim to optimize the performance of liquid crystal lens arrays.

  7. 3D objects enlargement technique using an optical system and multiple SLMs for electronic holography.

    PubMed

    Yamamoto, Kenji; Ichihashi, Yasuyuki; Senoh, Takanori; Oi, Ryutaro; Kurita, Taiichiro

    2012-09-10

    One problem in electronic holography, which is caused by the display performance of spatial light modulators (SLM), is that the size of reconstructed 3D objects is small. Although methods for increasing the size using multiple SLMs have been considered, they typically had the problem that some parts of 3D objects were missing as a result of the gap between adjacent SLMs or 3D objects lost the vertical parallax. This paper proposes a method of resolving this problem by locating an optical system containing a lens array and other components in front of multiple SLMs. We used an optical system and 9 SLMs to construct a device equivalent to an SLM with approximately 74,600,000 pixels and used this to reconstruct 3D objects in both the horizontal and vertical parallax with an image size of 63 mm without losing any part of 3D objects.

  8. Effect of 3d doping on the electronic structure of BaFe2As2

    SciTech Connect

    McLeod, John A.; Buling, A.; Green, R.J.; Boyko, T.D.; Skorikov, N.A.; Kurmaev, E.Z.; Neumann, M.; Finkelstein, L.D.; Ni, Ni; Thaler, Alexander; Budko, Serguei L.; Canfield, Paul; Moewes, A.

    2012-04-25

    The electronic structure of BaFe2As2 doped with Co, Ni and Cu has been studied by a variety of experimental and theoretical methods, but a clear picture of the dopant 3d states has not yet emerged. Herein we provide experimental evidence of the distribution of Co, Ni and Cu 3d states in the valence band. We conclude that the Co and Ni 3d states provide additional free carriers to the Fermi level, while the Cu 3d states are found at the bottom of the valence band in a localized 3d10 shell. These findings help shed light on why superconductivity can occur in BaFe2As2 doped with Co and Ni but not Cu.

  9. Measurement of dynamic cell-induced 3D displacement fields in vitro for traction force optical coherence microscopy.

    PubMed

    Mulligan, Jeffrey A; Bordeleau, François; Reinhart-King, Cynthia A; Adie, Steven G

    2017-02-01

    Traction force microscopy (TFM) is a method used to study the forces exerted by cells as they sense and interact with their environment. Cell forces play a role in processes that take place over a wide range of spatiotemporal scales, and so it is desirable that TFM makes use of imaging modalities that can effectively capture the dynamics associated with these processes. To date, confocal microscopy has been the imaging modality of choice to perform TFM in 3D settings, although multiple factors limit its spatiotemporal coverage. We propose traction force optical coherence microscopy (TF-OCM) as a novel technique that may offer enhanced spatial coverage and temporal sampling compared to current methods used for volumetric TFM studies. Reconstructed volumetric OCM data sets were used to compute time-lapse extracellular matrix deformations resulting from cell forces in 3D culture. These matrix deformations revealed clear differences that can be attributed to the dynamic forces exerted by normal versus contractility-inhibited NIH-3T3 fibroblasts embedded within 3D Matrigel matrices. Our results are the first step toward the realization of 3D TF-OCM, and they highlight the potential use of OCM as a platform for advancing cell mechanics research.

  10. Measurement of dynamic cell-induced 3D displacement fields in vitro for traction force optical coherence microscopy

    PubMed Central

    Mulligan, Jeffrey A.; Bordeleau, François; Reinhart-King, Cynthia A.; Adie, Steven G.

    2017-01-01

    Traction force microscopy (TFM) is a method used to study the forces exerted by cells as they sense and interact with their environment. Cell forces play a role in processes that take place over a wide range of spatiotemporal scales, and so it is desirable that TFM makes use of imaging modalities that can effectively capture the dynamics associated with these processes. To date, confocal microscopy has been the imaging modality of choice to perform TFM in 3D settings, although multiple factors limit its spatiotemporal coverage. We propose traction force optical coherence microscopy (TF-OCM) as a novel technique that may offer enhanced spatial coverage and temporal sampling compared to current methods used for volumetric TFM studies. Reconstructed volumetric OCM data sets were used to compute time-lapse extracellular matrix deformations resulting from cell forces in 3D culture. These matrix deformations revealed clear differences that can be attributed to the dynamic forces exerted by normal versus contractility-inhibited NIH-3T3 fibroblasts embedded within 3D Matrigel matrices. Our results are the first step toward the realization of 3D TF-OCM, and they highlight the potential use of OCM as a platform for advancing cell mechanics research. PMID:28271010

  11. 3D Modeling Activity for Novel High Power Electron Guns at SLAC

    SciTech Connect

    Krasnykh, Anatoly

    2003-07-29

    The next generation of powerful electronic devices requires new approaches to overcome the known limitations of existing tube technology. Multi-beam and sheet beam approaches are novel concepts for the high power microwave devices. Direct and indirect modeling methods are being developed at SLAC to meet the new requirements in the 3D modeling. The direct method of solving of Poisson's equations for the multi-beam and sheet beam guns is employed in the TOPAZ 3D tool. The combination of TOPAZ 2D and EGUN (in the beginning) with MAFIA 3D and MAGIC 3D (at the end) is used in an indirect method to model the high power electron guns. Both methods complement each other to get reliable representation of the beam trajectories. Several gun ideas are under consideration at the present time. The collected results of these simulations are discussed.

  12. 3-D laser confocal microscopy study of the oxidation of NdFeB magnets in atmospheric conditions

    NASA Astrophysics Data System (ADS)

    Meakin, J. P.; Speight, J. D.; Sheridan, R. S.; Bradshaw, A.; Harris, I. R.; Williams, A. J.; Walton, A.

    2016-08-01

    Neodymium iron boron (NdFeB) magnets are used in a number of important applications, such as generators in gearless wind turbines, motors in electric vehicles and electronic goods (e.g.- computer hard disk drives, HDD). Hydrogen can be used as a processing gas to separate and recycle scrap sintered Nd-Fe-B magnets from end-of-life products to form a powder suitable for recycling. However, the magnets are likely to have been exposed to atmospheric conditions prior to processing, and any oxidation could lead to activation problems for the hydrogen decrepitation reaction. Many previous studies on the oxidation of NdFeB magnets have been performed at elevated temperatures; however, few studies have been formed under atmospheric conditions. In this paper a combination of 3-D laser confocal microscopy and Raman spectroscopy have been used to assess the composition, morphology and rate of oxidation/corrosion on scrap sintered NdFeB magnets. Confocal microscopy has been employed to measure the growth of surface reaction products at room temperature, immediately after exposure to air. The results showed that there was a significant height increase at the triple junctions of the Nd-rich grain boundaries. Using Raman spectroscopy, the product was shown to consist of Nd2O3 and formed only on the Nd-rich triple junctions. The diffusion coefficient of the triple junction reaction product growth at 20 °C was determined to be approximately 4 × 10-13 cm2/sec. This value is several orders of magnitude larger than values derived from the diffusion controlled oxide growth observations at elevated temperatures in the literature. This indicates that the growth of the room temperature oxidation products are likely defect enhanced processes at the NdFeB triple junctions.

  13. Analysis of the 3D distribution of stacked self-assembled quantum dots by electron tomography

    PubMed Central

    2012-01-01

    The 3D distribution of self-assembled stacked quantum dots (QDs) is a key parameter to obtain the highest performance in a variety of optoelectronic devices. In this work, we have measured this distribution in 3D using a combined procedure of needle-shaped specimen preparation and electron tomography. We show that conventional 2D measurements of the distribution of QDs are not reliable, and only 3D analysis allows an accurate correlation between the growth design and the structural characteristics. PMID:23249477

  14. Adaptive Geometric Tessellation for 3D Reconstruction of Anisotropically Developing Cells in Multilayer Tissues from Sparse Volumetric Microscopy Images

    PubMed Central

    Chakraborty, Anirban; Perales, Mariano M.; Reddy, G. Venugopala; Roy-Chowdhury, Amit K.

    2013-01-01

    The need for quantification of cell growth patterns in a multilayer, multi-cellular tissue necessitates the development of a 3D reconstruction technique that can estimate 3D shapes and sizes of individual cells from Confocal Microscopy (CLSM) image slices. However, the current methods of 3D reconstruction using CLSM imaging require large number of image slices per cell. But, in case of Live Cell Imaging of an actively developing tissue, large depth resolution is not feasible in order to avoid damage to cells from prolonged exposure to laser radiation. In the present work, we have proposed an anisotropic Voronoi tessellation based 3D reconstruction framework for a tightly packed multilayer tissue with extreme z-sparsity (2–4 slices/cell) and wide range of cell shapes and sizes. The proposed method, named as the ‘Adaptive Quadratic Voronoi Tessellation’ (AQVT), is capable of handling both the sparsity problem and the non-uniformity in cell shapes by estimating the tessellation parameters for each cell from the sparse data-points on its boundaries. We have tested the proposed 3D reconstruction method on time-lapse CLSM image stacks of the Arabidopsis Shoot Apical Meristem (SAM) and have shown that the AQVT based reconstruction method can correctly estimate the 3D shapes of a large number of SAM cells. PMID:23940509

  15. A virtually imaged defocused array (VIDA) for high-speed 3D microscopy.

    PubMed

    Schonbrun, Ethan; Di Caprio, Giuseppe

    2016-10-01

    We report a method to capture a multifocus image stack based on recording multiple reflections generated by imaging through a custom etalon. The focus stack is collected in a single camera exposure and consequently the information needed for 3D reconstruction is recorded in the camera integration time, which is only 100 µs. We have used the VIDA microscope to temporally resolve the multi-lobed 3D morphology of neutrophil nuclei as they rotate and deform through a microfluidic constriction. In addition, we have constructed a 3D imaging flow cytometer and quantified the nuclear morphology of nearly a thousand white blood cells flowing at a velocity of 3 mm per second. The VIDA microscope is compact and simple to construct, intrinsically achromatic, and the field-of-view and stack number can be easily reconfigured without redesigning diffraction gratings and prisms.

  16. Picosecond Fresnel transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Schliep, Karl B.; Quarterman, P.; Wang, Jian-Ping; Flannigan, David J.

    2017-05-01

    We report the demonstration of picosecond Fresnel imaging with an ultrafast transmission electron microscope (UEM). By operating with a low instrument repetition rate (5 kHz) and without objective-lens excitation, the picosecond demagnetization of an FePt film, via in situ, femtosecond laser excitation, is directly imaged. The dynamics are quantified and monitored as a time-dependent change in the degree of electron coherence within the magnetic domain walls. The relative coherence of conventional (thermionic) Fresnel transmission electron microscopy is also directly compared to that of Fresnel UEM through the domain-wall size. Further, the robustness and reversibility of the domain-wall dynamics are illustrated by repeating the picosecond image scans at defocus values having the same magnitude but different signs (e.g., +25 mm vs. -25 mm). Control experiments and approaches to identifying and isolating systematic errors and sources of artifacts are also described. This work, and continued future developments also described here, opens the way to direct correlation of transient structure, morphology, and magnetic dynamics in magnetic thin films and spintronic devices.

  17. A simple, low-cost conductive composite material for 3D printing of electronic sensors.

    PubMed

    Leigh, Simon J; Bradley, Robert J; Purssell, Christopher P; Billson, Duncan R; Hutchins, David A

    2012-01-01

    3D printing technology can produce complex objects directly from computer aided digital designs. The technology has traditionally been used by large companies to produce fit and form concept prototypes ('rapid prototyping') before production. In recent years however there has been a move to adopt the technology as full-scale manufacturing solution. The advent of low-cost, desktop 3D printers such as the RepRap and Fab@Home has meant a wider user base are now able to have access to desktop manufacturing platforms enabling them to produce highly customised products for personal use and sale. This uptake in usage has been coupled with a demand for printing technology and materials able to print functional elements such as electronic sensors. Here we present formulation of a simple conductive thermoplastic composite we term 'carbomorph' and demonstrate how it can be used in an unmodified low-cost 3D printer to print electronic sensors able to sense mechanical flexing and capacitance changes. We show how this capability can be used to produce custom sensing devices and user interface devices along with printed objects with embedded sensing capability. This advance in low-cost 3D printing with offer a new paradigm in the 3D printing field with printed sensors and electronics embedded inside 3D printed objects in a single build process without requiring complex or expensive materials incorporating additives such as carbon nanotubes.

  18. A Simple, Low-Cost Conductive Composite Material for 3D Printing of Electronic Sensors

    PubMed Central

    Leigh, Simon J.; Bradley, Robert J.; Purssell, Christopher P.; Billson, Duncan R.; Hutchins, David A.

    2012-01-01

    3D printing technology can produce complex objects directly from computer aided digital designs. The technology has traditionally been used by large companies to produce fit and form concept prototypes (‘rapid prototyping’) before production. In recent years however there has been a move to adopt the technology as full-scale manufacturing solution. The advent of low-cost, desktop 3D printers such as the RepRap and Fab@Home has meant a wider user base are now able to have access to desktop manufacturing platforms enabling them to produce highly customised products for personal use and sale. This uptake in usage has been coupled with a demand for printing technology and materials able to print functional elements such as electronic sensors. Here we present formulation of a simple conductive thermoplastic composite we term ‘carbomorph’ and demonstrate how it can be used in an unmodified low-cost 3D printer to print electronic sensors able to sense mechanical flexing and capacitance changes. We show how this capability can be used to produce custom sensing devices and user interface devices along with printed objects with embedded sensing capability. This advance in low-cost 3D printing with offer a new paradigm in the 3D printing field with printed sensors and electronics embedded inside 3D printed objects in a single build process without requiring complex or expensive materials incorporating additives such as carbon nanotubes. PMID:23185319

  19. Polyhedral 3D structure of human plasma very low density lipoproteins by individual particle cryo-electron tomography

    SciTech Connect

    Yu, Yadong; Kuang, Yu-Lin; Lei, Dongsheng; Zhai, Xiaobo; Zhang, Meng; Krauss, Ronald M.; Ren, Gang

    2016-08-18

    Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures. The 3D reconstructions of VLDL and VLDL bound to antibodies revealed an unexpected polyhedral shape, in contrast to the generally accepted model of a spherical emulsion-like particle. The smaller curvature of surface lipids compared with HDL may also reduce surface hydrophobicity, resulting in lower binding affinity to the hydrophobic distal end of the N-terminal β-barrel domain of cholesteryl ester transfer protein (CETP) compared with HDL. The directional binding of CETP to HDL and VLDL may explain the function of CETP in transferring TGs and cholesteryl esters between these particles. This first visualization of the 3D structure of VLDL could improve our understanding of the role of VLDL in atherogenesis.

  20. Polyhedral 3D structure of human plasma very low density lipoproteins by individual particle cryo-electron tomography

    DOE PAGES

    Yu, Yadong; Kuang, Yu-Lin; Lei, Dongsheng; ...

    2016-08-18

    Human VLDLs assembled in the liver and secreted into the circulation supply energy to peripheral tissues. VLDL lipolysis yields atherogenic LDLs and VLDL remnants that strongly correlate with CVD. Although the composition of VLDL particles has been well-characterized, their 3D structure is elusive because of their variations in size, heterogeneity in composition, structural flexibility, and mobility in solution. Here, we employed cryo-electron microscopy and individual-particle electron tomography to study the 3D structure of individual VLDL particles (without averaging) at both below and above their lipid phase transition temperatures. The 3D reconstructions of VLDL and VLDL bound to antibodies revealed anmore » unexpected polyhedral shape, in contrast to the generally accepted model of a spherical emulsion-like particle. The smaller curvature of surface lipids compared with HDL may also reduce surface hydrophobicity, resulting in lower binding affinity to the hydrophobic distal end of the N-terminal β-barrel domain of cholesteryl ester transfer protein (CETP) compared with HDL. The directional binding of CETP to HDL and VLDL may explain the function of CETP in transferring TGs and cholesteryl esters between these particles. This first visualization of the 3D structure of VLDL could improve our understanding of the role of VLDL in atherogenesis.« less

  1. Bulk crystal growth and electronic characterization of the 3D Dirac semimetal Na3Bi

    NASA Astrophysics Data System (ADS)

    Kushwaha, Satya K.; Krizan, Jason W.; Feldman, Benjamin E.; Gyenis, András; Randeria, Mallika T.; Xiong, Jun; Xu, Su-Yang; Alidoust, Nasser; Belopolski, Ilya; Liang, Tian; Zahid Hasan, M.; Ong, N. P.; Yazdani, A.; Cava, R. J.

    2015-04-01

    High quality hexagon plate-like Na3Bi crystals with large (001) plane surfaces were grown from a molten Na flux. The freshly cleaved crystals were analyzed by low temperature scanning tunneling microscopy and angle-resolved photoemission spectroscopy, allowing for the characterization of the three-dimensional (3D) Dirac semimetal (TDS) behavior and the observation of the topological surface states. Landau levels were observed, and the energy-momentum relations exhibited a linear dispersion relationship, characteristic of the 3D TDS nature of Na3Bi. In transport measurements on Na3Bi crystals, the linear magnetoresistance and Shubnikov-de Haas quantum oscillations are observed for the first time.

  2. Quantification of Cardiomyocyte Alignment from Three-Dimensional (3D) Confocal Microscopy of Engineered Tissue.

    PubMed

    Kowalski, William J; Yuan, Fangping; Nakane, Takeichiro; Masumoto, Hidetoshi; Dwenger, Marc; Ye, Fei; Tinney, Joseph P; Keller, Bradley B

    2017-08-01

    Biological tissues have complex, three-dimensional (3D) organizations of cells and matrix factors that provide the architecture necessary to meet morphogenic and functional demands. Disordered cell alignment is associated with congenital heart disease, cardiomyopathy, and neurodegenerative diseases and repairing or replacing these tissues using engineered constructs may improve regenerative capacity. However, optimizing cell alignment within engineered tissues requires quantitative 3D data on cell orientations and both efficient and validated processing algorithms. We developed an automated method to measure local 3D orientations based on structure tensor analysis and incorporated an adaptive subregion size to account for multiple scales. Our method calculates the statistical concentration parameter, κ, to quantify alignment, as well as the traditional orientational order parameter. We validated our method using synthetic images and accurately measured principal axis and concentration. We then applied our method to confocal stacks of cleared, whole-mount engineered cardiac tissues generated from human-induced pluripotent stem cells or embryonic chick cardiac cells and quantified cardiomyocyte alignment. We found significant differences in alignment based on cellular composition and tissue geometry. These results from our synthetic images and confocal data demonstrate the efficiency and accuracy of our method to measure alignment in 3D tissues.

  3. Fluorescent stereo microscopy for 3D surface profilometry and deformation mapping.

    PubMed

    Hu, Zhenxing; Luo, Huiyang; Du, Yingjie; Lu, Hongbing

    2013-05-20

    Recently, mechanobiology has received increased attention. For investigation of biofilm and cellular tissue, measurements of the surface topography and deformation in real-time are a pre-requisite for understanding the growth mechanisms. In this paper, a novel three-dimensional (3D) fluorescent microscopic method for surface profilometry and deformation measurements is developed. In this technique a pair of cameras are connected to a binocular fluorescent microscope to acquire micrographs from two different viewing angles of a sample surface doped or sprayed with fluorescent microparticles. Digital image correlation technique is used to search for matching points in the pairing fluorescence micrographs. After calibration of the system, the 3D surface topography is reconstructed from the pair of planar images. When the deformed surface topography is compared with undeformed topography using fluorescent microparticles for movement tracking of individual material points, the full field deformation of the surface is determined. The technique is demonstrated on topography measurement of a biofilm, and also on surface deformation measurement of the biofilm during growth. The use of 3D imaging of the fluorescent microparticles eliminates the formation of bright parts in an image caused by specular reflections. The technique is appropriate for non-contact, full-field and real-time 3D surface profilometry and deformation measurements of materials and structures at the microscale.

  4. High-purity 3D nano-objects grown by focused-electron-beam induced deposition

    NASA Astrophysics Data System (ADS)

    Córdoba, Rosa; Sharma, Nidhi; Kölling, Sebastian; Koenraad, Paul M.; Koopmans, Bert

    2016-09-01

    To increase the efficiency of current electronics, a specific challenge for the next generation of memory, sensing and logic devices is to find suitable strategies to move from two- to three-dimensional (3D) architectures. However, the creation of real 3D nano-objects is not trivial. Emerging non-conventional nanofabrication tools are required for this purpose. One attractive method is focused-electron-beam induced deposition (FEBID), a direct-write process of 3D nano-objects. Here, we grow 3D iron and cobalt nanopillars by FEBID using diiron nonacarbonyl Fe2(CO)9, and dicobalt octacarbonyl Co2(CO)8, respectively, as starting materials. In addition, we systematically study the composition of these nanopillars at the sub-nanometer scale by atom probe tomography, explicitly mapping the homogeneity of the radial and longitudinal composition distributions. We show a way of fabricating high-purity 3D vertical nanostructures of ˜50 nm in diameter and a few micrometers in length. Our results suggest that the purity of such 3D nanoelements (above 90 at% Fe and above 95 at% Co) is directly linked to their growth regime, in which the selected deposition conditions are crucial for the final quality of the nanostructure. Moreover, we demonstrate that FEBID and the proposed characterization technique not only allow for growth and chemical analysis of single-element structures, but also offers a new way to directly study 3D core-shell architectures. This straightforward concept could establish a promising route to the design of 3D elements for future nano-electronic devices.

  5. Imaging bacterial 3D motion using digital in-line holographic microscopy and correlation-based de-noising algorithm

    PubMed Central

    Molaei, Mehdi; Sheng, Jian

    2014-01-01

    Abstract: Better understanding of bacteria environment interactions in the context of biofilm formation requires accurate 3-dimentional measurements of bacteria motility. Digital Holographic Microscopy (DHM) has demonstrated its capability in resolving 3D distribution and mobility of particulates in a dense suspension. Due to their low scattering efficiency, bacteria are substantially difficult to be imaged by DHM. In this paper, we introduce a novel correlation-based de-noising algorithm to remove the background noise and enhance the quality of the hologram. Implemented in conjunction with DHM, we demonstrate that the method allows DHM to resolve 3-D E. coli bacteria locations of a dense suspension (>107 cells/ml) with submicron resolutions (<0.5 µm) over substantial depth and to obtain thousands of 3D cell trajectories. PMID:25607177

  6. Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging.

    PubMed

    Boulanger, Jérôme; Gueudry, Charles; Münch, Daniel; Cinquin, Bertrand; Paul-Gilloteaux, Perrine; Bardin, Sabine; Guérin, Christophe; Senger, Fabrice; Blanchoin, Laurent; Salamero, Jean

    2014-12-02

    Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second.

  7. Fast high-resolution 3D total internal reflection fluorescence microscopy by incidence angle scanning and azimuthal averaging

    PubMed Central

    Boulanger, Jérôme; Gueudry, Charles; Münch, Daniel; Cinquin, Bertrand; Paul-Gilloteaux, Perrine; Bardin, Sabine; Guérin, Christophe; Senger, Fabrice; Blanchoin, Laurent; Salamero, Jean

    2014-01-01

    Total internal reflection fluorescence microscopy (TIRFM) is the method of choice to visualize a variety of cellular processes in particular events localized near the plasma membrane of live adherent cells. This imaging technique not relying on particular fluorescent probes provides a high sectioning capability. It is, however, restricted to a single plane. We present here a method based on a versatile design enabling fast multiwavelength azimuthal averaging and incidence angles scanning to computationally reconstruct 3D images sequences. We achieve unprecedented 50-nm axial resolution over a range of 800 nm above the coverslip. We apply this imaging modality to obtain structural and dynamical information about 3D actin architectures. We also temporally decipher distinct Rab11a-dependent exocytosis events in 3D at a rate of seven stacks per second. PMID:25404337

  8. Alterations of filopodia by near infrared photoimmunotherapy: evaluation with 3D low-coherent quantitative phase microscopy

    PubMed Central

    Nakamura, Yuko; Nagaya, Tadanobu; Sato, Kazuhide; Harada, Toshiko; Okuyama, Shuhei; Choyke, Peter L.; Yamauchi, Toyohiko; Kobayashi, Hisataka

    2016-01-01

    Filopodia are highly organized cellular membrane structures that facilitate intercellular communication. Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that causes necrotic cell death. Three-dimensional low-coherent quantitative phase microscopy (3D LC-QPM) is based on a newly established low-coherent interference microscope designed to obtain serial topographic images of the cellular membrane. Herein, we report rapid involution of filopodia after NIR-PIT using 3D LC-QPM. For 3T3/HER2 cells, the number of filopodia decreased immediately after treatment with significant differences. Volume and relative height of 3T3/HER2 cells increased immediately after NIR light exposure, but significant differences were not observed. Thus, disappearance of filopodia, evaluated by 3D LC-QPM, is an early indicator of cell membrane damage after NIR-PIT. PMID:27446702

  9. 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

  10. Dual array 3D electron cyclotron emission imaging at ASDEX Upgrade

    SciTech Connect

    Classen, I. G. J. Bogomolov, A. V.; Domier, C. W.; Luhmann, N. C.; Suttrop, W.; Boom, J. E.; Tobias, B. J.; Donné, A. J. H.

    2014-11-15

    In a major upgrade, the (2D) electron cyclotron emission imaging diagnostic (ECEI) at ASDEX Upgrade has been equipped with a second detector array, observing a different toroidal position in the plasma, to enable quasi-3D measurements of the electron temperature. The new system will measure a total of 288 channels, in two 2D arrays, toroidally separated by 40 cm. The two detector arrays observe the plasma through the same vacuum window, both under a slight toroidal angle. The majority of the field lines are observed by both arrays simultaneously, thereby enabling a direct measurement of the 3D properties of plasma instabilities like edge localized mode filaments.

  11. Twin-beams digital holography for 3D tracking and quantitative phase-contrast microscopy in microfluidics.

    PubMed

    Memmolo, Pasquale; Finizio, Andrea; Paturzo, Melania; Miccio, Lisa; Ferraro, Pietro

    2011-12-05

    We report on a compact twin-beam interferometer that can be adopted as a flexible diagnostic tool in microfluidic platforms with twofold functionality. The novel configuration allows 3D tracking of micro-particles and, at same time, can simultaneously furnish Quantitative Phase-contrast maps of tracked micro-objects by interference microscopy, without changing the configuration. Experimental demonstration is given on for in vitro cells in a microfluidic environment.

  12. Integrated profiling of three dimensional cell culture models and 3D microscopy

    PubMed Central

    Bilgin, Cemal Cagatay; Kim, Sun; Leung, Elle; Chang, Hang; Parvin, Bahram

    2013-01-01

    Motivation: Our goal is to develop a screening platform for quantitative profiling of colony organizations in 3D cell culture models. The 3D cell culture models, which are also imaged in 3D, are functional assays that mimic the in vivo characteristics of the tissue architecture more faithfully than the 2D cultures. However, they also introduce significant computational challenges, with the main barriers being the effects of growth conditions, fixations and inherent complexities in segmentation that need to be resolved in the 3D volume. Results: A segmentation strategy has been developed to delineate each nucleus in a colony that overcomes (i) the effects of growth conditions, (ii) variations in chromatin distribution and (iii) ambiguities formed by perceptual boundaries from adjacent nuclei. The strategy uses a cascade of geometric filters that are insensitive to spatial non-uniformity and partitions a clump of nuclei based on the grouping of points of maximum curvature at the interface of two neighboring nuclei. These points of maximum curvature are clustered together based on their coplanarity and proximity to define dissecting planes that separate the touching nuclei. The proposed curvature-based partitioning method is validated with both synthetic and real data, and is shown to have a superior performance against previous techniques. Validation and sensitivity analysis are coupled with the experimental design that includes a non-transformed cell line and three tumorigenic cell lines, which covers a wide range of phenotypic diversity in breast cancer. Colony profiling, derived from nuclear segmentation, reveals distinct indices for the morphogenesis of each cell line. Availability: All software are developed in ITK/VTK and are available at https://vision.lbl.gov/Software/3DMorphometry. Contact: b_parvin@lbl.gov or hchang@lbl.gov Supplementary information: Supplementary data are available at Bioinformatics online. PMID:24045773

  13. Integrated profiling of three dimensional cell culture models and 3D microscopy.

    PubMed

    Bilgin, Cemal Cagatay; Kim, Sun; Leung, Elle; Chang, Hang; Parvin, Bahram

    2013-12-01

    Our goal is to develop a screening platform for quantitative profiling of colony organizations in 3D cell culture models. The 3D cell culture models, which are also imaged in 3D, are functional assays that mimic the in vivo characteristics of the tissue architecture more faithfully than the 2D cultures. However, they also introduce significant computational challenges, with the main barriers being the effects of growth conditions, fixations and inherent complexities in segmentation that need to be resolved in the 3D volume. A segmentation strategy has been developed to delineate each nucleus in a colony that overcomes (i) the effects of growth conditions, (ii) variations in chromatin distribution and (iii) ambiguities formed by perceptual boundaries from adjacent nuclei. The strategy uses a cascade of geometric filters that are insensitive to spatial non-uniformity and partitions a clump of nuclei based on the grouping of points of maximum curvature at the interface of two neighboring nuclei. These points of maximum curvature are clustered together based on their coplanarity and proximity to define dissecting planes that separate the touching nuclei. The proposed curvature-based partitioning method is validated with both synthetic and real data, and is shown to have a superior performance against previous techniques. Validation and sensitivity analysis are coupled with the experimental design that includes a non-transformed cell line and three tumorigenic cell lines, which covers a wide range of phenotypic diversity in breast cancer. Colony profiling, derived from nuclear segmentation, reveals distinct indices for the morphogenesis of each cell line.

  14. 3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy

    PubMed Central

    Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A.; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

    2016-01-01

    Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis. PMID:27869673

  15. 3D Visualization of Developmental Toxicity of 2,4,6-Trinitrotoluene in Zebrafish Embryogenesis Using Light-Sheet Microscopy.

    PubMed

    Eum, Juneyong; Kwak, Jina; Kim, Hee Joung; Ki, Seoyoung; Lee, Kooyeon; Raslan, Ahmed A; Park, Ok Kyu; Chowdhury, Md Ashraf Uddin; Her, Song; Kee, Yun; Kwon, Seung-Hae; Hwang, Byung Joon

    2016-11-17

    Environmental contamination by trinitrotoluene is of global concern due to its widespread use in military ordnance and commercial explosives. Despite known long-term persistence in groundwater and soil, the toxicological profile of trinitrotoluene and other explosive wastes have not been systematically measured using in vivo biological assays. Zebrafish embryos are ideal model vertebrates for high-throughput toxicity screening and live in vivo imaging due to their small size and transparency during embryogenesis. Here, we used Single Plane Illumination Microscopy (SPIM)/light sheet microscopy to assess the developmental toxicity of explosive-contaminated water in zebrafish embryos and report 2,4,6-trinitrotoluene-associated developmental abnormalities, including defects in heart formation and circulation, in 3D. Levels of apoptotic cell death were higher in the actively developing tissues of trinitrotoluene-treated embryos than controls. Live 3D imaging of heart tube development at cellular resolution by light-sheet microscopy revealed trinitrotoluene-associated cardiac toxicity, including hypoplastic heart chamber formation and cardiac looping defects, while the real time PCR (polymerase chain reaction) quantitatively measured the molecular changes in the heart and blood development supporting the developmental defects at the molecular level. Identification of cellular toxicity in zebrafish using the state-of-the-art 3D imaging system could form the basis of a sensitive biosensor for environmental contaminants and be further valued by combining it with molecular analysis.

  16. Micron-Resolution X-ray Structural Microscopy Studies of 3-D Grain Growth in Polycrystalline Aluminum

    NASA Astrophysics Data System (ADS)

    Budai, J. D.; Yang, W.; Tischler, J. Z.; Liu, W.; Larson, B. C.; Ice, G. E.

    2004-03-01

    We describe a new polychromatic x-ray microdiffraction technique providing 3D measurements of lattice structure, orientation and strain with submicron point-to-point spatial resolution. The instrument is located on the UNI-CAT II undulator beamline at the Advanced Photon Source and uses Kirkpatrick-Baez focusing mirrors, differential aperture CCD measurements and automated analysis of spatially-resolved Laue patterns. 3D x-ray structural microscopy is applicable to a wide range of materials investigations and here we describe 3D thermal grain growth studies in polycrystalline aluminum ( ˜1% Fe,Si) from Alcoa. The morphology and orientations of the grains in a hot-rolled aluminum sample were initially mapped. The sample was then annealed to induce grain growth, cooled to room temperature, and the same volume region was re-mapped to determine the thermal migration of all grain boundaries. Significant grain growth was observed after annealing above ˜350^oC where both low-angle and high-angle boundaries were mobile. These measurements will provide the detailed 3D experimental input needed for testing theories and computer models of 3D grain growth in bulk materials.

  17. A 3D Image Filter for Parameter-Free Segmentation of Macromolecular Structures from Electron Tomograms

    PubMed Central

    Ali, Rubbiya A.; Landsberg, Michael J.; Knauth, Emily; Morgan, Garry P.; Marsh, Brad J.; Hankamer, Ben

    2012-01-01

    3D image reconstruction of large cellular volumes by electron tomography (ET) at high (≤5 nm) resolution can now routinely resolve organellar and compartmental membrane structures, protein coats, cytoskeletal filaments, and macromolecules. However, current image analysis methods for identifying in situ macromolecular structures within the crowded 3D ultrastructural landscape of a cell remain labor-intensive, time-consuming, and prone to user-bias and/or error. This paper demonstrates the development and application of a parameter-free, 3D implementation of the bilateral edge-detection (BLE) algorithm for the rapid and accurate segmentation of cellular tomograms. The performance of the 3D BLE filter has been tested on a range of synthetic and real biological data sets and validated against current leading filters—the pseudo 3D recursive and Canny filters. The performance of the 3D BLE filter was found to be comparable to or better than that of both the 3D recursive and Canny filters while offering the significant advantage that it requires no parameter input or optimisation. Edge widths as little as 2 pixels are reproducibly detected with signal intensity and grey scale values as low as 0.72% above the mean of the background noise. The 3D BLE thus provides an efficient method for the automated segmentation of complex cellular structures across multiple scales for further downstream processing, such as cellular annotation and sub-tomogram averaging, and provides a valuable tool for the accurate and high-throughput identification and annotation of 3D structural complexity at the subcellular level, as well as for mapping the spatial and temporal rearrangement of macromolecular assemblies in situ within cellular tomograms. PMID:22479430

  18. Sample holder for axial rotation of specimens in 3D microscopy.

    PubMed

    Bruns, T; Schickinger, S; Schneckenburger, H

    2015-10-01

    In common light microscopy, observation of samples is only possible from one perspective. However, especially for larger three-dimensional specimens observation from different views is desirable. Therefore, we are presenting a sample holder permitting rotation of the specimen around an axis perpendicular to the light path of the microscope. Thus, images can be put into a defined multidimensional context, enabling reliable three-dimensional reconstructions. The device can be easily adapted to a great variety of common light microscopes and is suitable for various applications in science, education and industry, where the observation of three-dimensional specimens is essential. Fluorescence z-projection images of copepods and ixodidae ticks at different rotation angles obtained by confocal laser scanning microscopy and light sheet fluorescence microscopy are reported as representative results. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

  19. 3-D Cellular Ultrastructure Can Be Resolved by X-ray Microscopy | Center for Cancer Research

    Cancer.gov

    X-ray microscopy (XRM) is more rapid than cryoelectron tomography or super-resolution fluorescence microscopy and could fill an important gap in current technologies used to investigate in situ three-dimensional structure of cells. New XRM methods developed by first author Gerd Schneider, Ph.D., working with James McNally. Ph.D., and a team of colleagues, is capable of revealing full cellular ultrastructure without requiring fixation, staining, or sectioning.

  20. Band like Electronic Structures in Square Hollow Quantum Dots by 3D-MHFKS Calculation

    NASA Astrophysics Data System (ADS)

    Takizawa, Tokihiro; Okada, Hoshihito; Matsuse, Takehiro

    To find novel aspects of the electronic structures in quantum dots (QD) from a view point of spatial broken symmetry, 3-dimensional-mesh Hartree-Fock-Kohn-Sham (3D-MHFKS) calculations1 are applied to the interacting electron system of electron number N in a symmetry broken hollow QD. For the case of a square hollow quantum dot confined in square hard wall (HW) potential (SSHQD), the magnetic (B) field dependence of the obtained single particle energy levels and chemical potentials in B-N diagram are shown to have a band like electronic structures over the wide B-field range up to 20T. To clarify the origin of the band like electronic structures in SSHQD, 3D-MHFKS calculations are also applied for the mixed symmetry QD's with a circular hollow in square HW potential (SCHQD) and with a square hollow in circular HW potential (CSHQD).

  1. 3D structure of eukaryotic flagella/cilia by cryo-electron tomography.

    PubMed

    Ishikawa, Takashi

    2013-01-01

    Flagella/cilia are motile organelles with more than 400 proteins. To understand the mechanism of such complex systems, we need methods to describe molecular arrange-ments and conformations three-dimensionally in vivo. Cryo-electron tomography enabled us such a 3D structural analysis. Our group has been working on 3D structure of flagella/cilia using this method and revealed highly ordered and beautifully organized molecular arrangement. 3D structure gave us insights into the mechanism to gener-ate bending motion with well defined waveforms. In this review, I summarize our recent structural studies on fla-gella/cilia by cryo-electron tomography, mainly focusing on dynein microtubule-based ATPase motor proteins and the radial spoke, a regulatory protein complex.

  2. Building Proteins in a Day: Efficient 3D Molecular Structure Estimation with Electron Cryomicroscopy.

    PubMed

    Punjani, Ali; Brubaker, Marcus A; Fleet, David J

    2017-04-01

    Discovering the 3D atomic-resolution structure of molecules such as proteins and viruses is one of the foremost research problems in biology and medicine. Electron Cryomicroscopy (cryo-EM) is a promising vision-based technique for structure estimation which attempts to reconstruct 3D atomic structures from a large set of 2D transmission electron microscope images. This paper presents a new Bayesian framework for cryo-EM structure estimation that builds on modern stochastic optimization techniques to allow one to scale to very large datasets. We also introduce a novel Monte-Carlo technique that reduces the cost of evaluating the objective function during optimization by over five orders of magnitude. The net result is an approach capable of estimating 3D molecular structure from large-scale datasets in about a day on a single CPU workstation.

  3. A new method for imaging and 3D reconstruction of mammalian cochlea by fluorescent confocal microscopy.

    PubMed

    Hardie, Natalie A; MacDonald, Glen; Rubel, Edwin W

    2004-03-12

    Traditional methods for anatomical and morphometric studies of cochlear tissues have relied upon either microdissection of the organ of Corti or the generation of serial sections of the cochlea. Such methods are time-consuming, disruptive to three-dimensional relationships and often restrict sampling to very limited numbers of cells. We have found that cells and tissue components of the cochlear duct may be labelled by fluorescent markers within intact cochleae, which are then embedded in epoxy resin for subsequent viewing by fluorescent microscopy methods. This approach allows imaging through thick optical volumes with preservation of three-dimensional relationships. Unlike sectioned tissue, alignment of the sample relative to the focal axis may be easily corrected by re-orientation of the optical volume with common image processing software. Fluorescently labelled cochleae embedded in epoxy can be viewed by most fluorescent microscopy methods including laser scanning confocal microscopy, multi-photon confocal microscopy and widefield epi-fluorescence microscopy with deconvolution. Furthermore, semi-thin sections made from these preparations are compatible with traditional histological stains, as well as allowing brightly labelled epi-fluorescent images.

  4. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy.

    PubMed

    Park, HyunJoo; Hong, Sung-Hee; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, Sang-Eun; Park, YongKeun

    2015-06-03

    Babesia microti causes "emergency" human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability.

  5. Simultaneous whole-animal 3D-imaging of neuronal activity using light-field microscopy

    PubMed Central

    Hoffmann, Maximilian; Pak, Nikita; Wetzstein, Gordon; Kato, Saul; Schrödel, Tina; Raskar, Ramesh; Zimmer, Manuel; Boyden, Edward S.; Vaziri, Alipasha

    2014-01-01

    High-speed large-scale 3D imaging of neuronal activity poses a major challenge in neuroscience. Here, we demonstrate intrinsically simultaneous functional imaging of neuronal activity at single neuron resolution for an entire Caenorhabditis elegans as well as for the whole-brain of larval zebrafish. Our technique captures dynamics of spiking neurons in volumes of ~700 μm x 700 μm x 200 μm at 20 Hz and its simplicity makes it an attractive tool for high-speed volumetric calcium imaging. PMID:24836920

  6. Characterizations of individual mouse red blood cells parasitized by Babesia microti using 3-D holographic microscopy

    PubMed Central

    Park, HyunJoo; Hong, Sung-Hee; Kim, Kyoohyun; Cho, Shin-Hyeong; Lee, Won-Ja; Kim, Youngchan; Lee, Sang-Eun; Park, YongKeun

    2015-01-01

    Babesia microti causes “emergency” human babesiosis. However, little is known about the alterations in B. microti invaded red blood cells (Bm-RBCs) at the individual cell level. Through quantitative phase imaging techniques based on laser interferometry, we present the simultaneous measurements of structural, chemical, and mechanical modifications in individual mouse Bm-RBCs. 3-D refractive index maps of individual RBCs and in situ parasite vacuoles are imaged, from which total contents and concentration of dry mass are also precisely quantified. In addition, we examine the dynamic membrane fluctuation of Bm-RBCs, which provide information on cell membrane deformability. PMID:26039793

  7. Multi-modal digital holographic microscopy for wide-field fluorescence and 3D phase imaging

    NASA Astrophysics Data System (ADS)

    Quan, Xiangyu; Xia, Peng; Matoba, Osamu; Nitta, Koichi; Awatsuji, Yasuhiro

    2016-03-01

    Multi-modal digital holographic microscopy is a combination of epifluorescence microscopy and digital holographic microscopy, the main function of which is to obtain images from fluorescence intensity and quantified phase contrasts, simultaneously. The proposed system is mostly beneficial to biological studies, with the reason that often the studies are depending on fluorescent labeling techniques to detect certain intracellular molecules, while phase information reflecting properties of unstained transparent elements. This paper is presenting our latest researches on applications such as randomly moving micro-fluorescent beads and living cells of Physcomitrella patens. The experiments are succeeded on obtaining a succession of wide-field fluorescent images and holograms from micro-beads, and different depths focusing is realized via numerical reconstruction. Living cells of Physcomitrella patens are recorded in the static manner, the reconstruction distance indicates thickness of cellular structure. These results are implementing practical applications toward many biomedical science researches.

  8. Determining optimum red filter slide distance on creating 3D electron microscope images using anaglyph method

    NASA Astrophysics Data System (ADS)

    Tresna, W. P.; Isnaeni

    2017-04-01

    Scanning Electron Microscope (SEM) is a proven instrument for analyzing material in which a 2D image of an object is produced. However, the optimization of a 3D image in the SEM system is usually difficult and costly. There is a simple method to produce a 3D image by using two light sources with a red and a blue filter combined in a certain angle. In this experiment, the authors conducted a simulation of the 3D image formation using anaglyph method by finding the optimum point of shifting the red and blue filters in an SEM image. The method used in this experiment was an image processing that employed a digital manipulation on a certain deviation distance of the central point of the main object. The simulation result of an SEM image with a magnification of 5000 times showed an optimal 3D effect that was achieved when the red filter was shifted by 1 μm to the right and the blue filter was shifted by 1 µm to the left from the central position. The result of this simulation can be used to understand better the viewing angle and the optimal position of the two light sources, i.e. red and blue filter pairs. The produced 3D image can be clearly seen using 3D glasses.

  9. Electrochemical fields within 3D reconstructed microstructures of mixed ionic and electronic conducting devices

    NASA Astrophysics Data System (ADS)

    Zhang, Yanxiang; Chen, Yu; Lin, Ye; Yan, Mufu; Harris, William M.; Chiu, Wilson K. S.; Ni, Meng; Chen, Fanglin

    2016-11-01

    The performance and stability of the mixed ionic and electronic conducting (MIEC) membrane devices, such as solid oxide cells (SOCs) and oxygen separation membranes (OSMs) interplay tightly with the transport properties and the three-dimensional (3D) microstructure of the membrane. However, development of the MIEC devices is hindered by the limited knowledge about the distribution of electrochemical fields within the 3D local microstructures, especially at surface and interface. In this work, a generic model conforming to local thermodynamic equilibrium is developed to calculate the electrochemical fields, such as electric potential and oxygen chemical potential, within the 3D microstructure of the MIEC membrane. Stability of the MIEC membrane is evaluated by the distribution of oxygen partial pressure. The cell-level performance such as polarization resistance and voltage vs. current curve can be further calculated. Case studies are performed to demonstrate the capability of the framework by using X-ray computed tomography reconstructed 3D microstructures of a SOC and an OSM. The calculation method demonstrates high computational efficiency for large size 3D tomographic microstructures, and permits parallel calculation. The framework can serve as a powerful tool for correlating the transport properties and the 3D microstructure to the performance and the stability of MIEC devices.

  10. Combined Scanning Transmission Electron Microscopy Tilt- and Focal Series

    SciTech Connect

    Dahmen, Tim; Baudoin, Jean-Pierre G; Lupini, Andrew R; Kubel, Christian; Slusallek, Phillip; De Jonge, Niels

    2014-01-01

    In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller missing wedge artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.

  11. Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

    PubMed

    Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

    2014-03-01

    One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

  12. Shape‐Controlled, Self‐Wrapped Carbon Nanotube 3D Electronics

    PubMed Central

    Wang, Huiliang; Wang, Yanming; Tee, Benjamin C.‐K.; Kim, Kwanpyo; Lopez, Jeffrey; Cai, Wei

    2015-01-01

    The mechanical flexibility and structural softness of ultrathin devices based on organic thin films and low‐dimensional nanomaterials have enabled a wide range of applications including flexible display, artificial skin, and health monitoring devices. However, both living systems and inanimate systems that are encountered in daily lives are all 3D. It is therefore desirable to either create freestanding electronics in a 3D form or to incorporate electronics onto 3D objects. Here, a technique is reported to utilize shape‐memory polymers together with carbon nanotube flexible electronics to achieve this goal. Temperature‐assisted shape control of these freestanding electronics in a programmable manner is demonstrated, with theoretical analysis for understanding the shape evolution. The shape control process can be executed with prepatterned heaters, desirable for 3D shape formation in an enclosed environment. The incorporation of carbon nanotube transistors, gas sensors, temperature sensors, and memory devices that are capable of self‐wrapping onto any irregular shaped‐objects without degradations in device performance is demonstrated. PMID:27980972

  13. Shape-Controlled, Self-Wrapped Carbon Nanotube 3D Electronics.

    PubMed

    Wang, Huiliang; Wang, Yanming; Tee, Benjamin C-K; Kim, Kwanpyo; Lopez, Jeffrey; Cai, Wei; Bao, Zhenan

    2015-09-01

    The mechanical flexibility and structural softness of ultrathin devices based on organic thin films and low-dimensional nanomaterials have enabled a wide range of applications including flexible display, artificial skin, and health monitoring devices. However, both living systems and inanimate systems that are encountered in daily lives are all 3D. It is therefore desirable to either create freestanding electronics in a 3D form or to incorporate electronics onto 3D objects. Here, a technique is reported to utilize shape-memory polymers together with carbon nanotube flexible electronics to achieve this goal. Temperature-assisted shape control of these freestanding electronics in a programmable manner is demonstrated, with theoretical analysis for understanding the shape evolution. The shape control process can be executed with prepatterned heaters, desirable for 3D shape formation in an enclosed environment. The incorporation of carbon nanotube transistors, gas sensors, temperature sensors, and memory devices that are capable of self-wrapping onto any irregular shaped-objects without degradations in device performance is demonstrated.

  14. A Review on Energy Harvesting Using 3D Printed Fabrics for Wearable Electronics

    NASA Astrophysics Data System (ADS)

    Gowthaman, Swaminathan; Chidambaram, Gowri Shankar; Rao, Dilli Babu Govardhana; Subramya, Hemakumar Vyudhayagiri; Chandrasekhar, Udhayagiri

    2016-06-01

    Embedding of energy harvesting systems into wearable health and environment monitoring systems, like integration of smart piezoelectric fibers into soldier fabric structures opens up avenues in generating electricity from natural mechanical movements for self-powering of wearable electronics. Emergence of multitudinous of materials and manufacturing technologies has enabled realization of various energy harvesting systems from mechanical movements. The materials and manufacturing related to 3D printing of energy harvesting fabrics are reviewed in this paper. State-of-the-art energy harvesting sources are briefly described following which an in-depth analysis on the materials and 3D printing techniques for energy harvesting fabrics are presented. While tremendous motivation and opportunity exists for wider-scale adoption of 3D printing for this niche area, the success depends on efficient design of three critical factors namely materials, process and structure. The present review discusses on the complex issues of materials selection, modelling and processing of 3D printed fabrics. The paper culminates by presenting a discussion on how future advancements in 3D printing technology might be useful for development of wearable electronics.

  15. Advances in automated 3-D image analyses of cell populations imaged by confocal microscopy.

    PubMed

    Ancin, H; Roysam, B; Dufresne, T E; Chestnut, M M; Ridder, G M; Szarowski, D H; Turner, J N

    1996-11-01

    Automated three-dimensional (3-D) image analysis methods are presented for rapid and effective analysis of populations of fluorescently labeled cells or nuclei in thick tissue sections that have been imaged three dimensionally using a confocal microscope. The methods presented here greatly improve upon our earlier work (Roysam et al.:J Microsc 173: 115-126, 1994). The principal advances reported are: algorithms for efficient data pre-processing and adaptive segmentation, effective handling of image anisotrophy, and fast 3-D morphological algorithms for separating overlapping or connected clusters utilizing image gradient information whenever available. A particular feature of this method is its ability to separate densely packed and connected clusters of cell nuclei. Some of the challenges overcome in this work include the efficient and effective handling of imaging noise, anisotrophy, and large variations in image parameters such as intensity, object size, and shape. The method is able to handle significant inter-cell, intra-cell, inter-image, and intra-image variations. Studies indicate that this method is rapid, robust, and adaptable. Examples were presented to illustrate the applicability of this approach to analyzing images of nuclei from densely packed regions in thick sections of rat liver, and brain that were labeled with a fluorescent Schiff reagent.

  16. Phenotypic feature quantification of patient derived 3D cancer spheroids in fluorescence microscopy image

    NASA Astrophysics Data System (ADS)

    Kang, Mi-Sun; Rhee, Seon-Min; Seo, Ji-Hyun; Kim, Myoung-Hee

    2017-03-01

    Patients' responses to a drug differ at the cellular level. Here, we present an image-based cell phenotypic feature quantification method for predicting the responses of patient-derived glioblastoma cells to a particular drug. We used high-content imaging to understand the features of patient-derived cancer cells. A 3D spheroid culture formation resembles the in vivo environment more closely than 2D adherent cultures do, and it allows for the observation of cellular aggregate characteristics. However, cell analysis at the individual level is more challenging. In this paper, we demonstrate image-based phenotypic screening of the nuclei of patient-derived cancer cells. We first stitched the images of each well of the 384-well plate with the same state. We then used intensity information to detect the colonies. The nuclear intensity and morphological characteristics were used for the segmentation of individual nuclei. Next, we calculated the position of each nucleus that is appeal of the spatial pattern of cells in the well environment. Finally, we compared the results obtained using 3D spheroid culture cells with those obtained using 2D adherent culture cells from the same patient being treated with the same drugs. This technique could be applied for image-based phenotypic screening of cells to determine the patient's response to the drug.

  17. Structured illumination multimodal 3D-resolved quantitative phase and fluorescence sub-diffraction microscopy

    PubMed Central

    Chowdhury, Shwetadwip; Eldridge, Will J.; Wax, Adam; Izatt, Joseph A.

    2017-01-01

    Sub-diffraction resolution imaging has played a pivotal role in biological research by visualizing key, but previously unresolvable, sub-cellular structures. Unfortunately, applications of far-field sub-diffraction resolution are currently divided between fluorescent and coherent-diffraction regimes, and a multimodal sub-diffraction technique that bridges this gap has not yet been demonstrated. Here we report that structured illumination (SI) allows multimodal sub-diffraction imaging of both coherent quantitative-phase (QP) and fluorescence. Due to SI’s conventionally fluorescent applications, we first demonstrate the principle of SI-enabled three-dimensional (3D) QP sub-diffraction imaging with calibration microspheres. Image analysis confirmed enhanced lateral and axial resolutions over diffraction-limited QP imaging, and established striking parallels between coherent SI and conventional optical diffraction tomography. We next introduce an optical system utilizing SI to achieve 3D sub-diffraction, multimodal QP/fluorescent visualization of A549 biological cells fluorescently tagged for F-actin. Our results suggest that SI has a unique utility in studying biological phenomena with significant molecular, biophysical, and biochemical components. PMID:28663887

  18. Pico-projector-based optical sectioning microscopy for 3D chlorophyll fluorescence imaging of mesophyll cells

    NASA Astrophysics Data System (ADS)

    Chen, Szu-Yu; Hsu, Yu John; Yeh, Chia-Hua; Chen, S.-Wei; Chung, Chien-Han

    2015-03-01

    A pico-projector-based optical sectioning microscope (POSM) was constructed using a pico-projector to generate structured illumination patterns. A net rate of 5.8 × 106 pixel/s and sub-micron spatial resolution in three-dimensions (3D) were achieved. Based on the pico-projector’s flexibility in pattern generation, the characteristics of POSM with different modulation periods and at different imaging depths were measured and discussed. With the application of different modulation periods, 3D chlorophyll fluorescence imaging of mesophyll cells was carried out in freshly plucked leaves of four species without sectioning or staining. For each leaf, an average penetration depth of 120 μm was achieved. Increasing the modulation period along with the increment of imaging depth, optical sectioning images can be obtained with a compromise between the axial resolution and signal-to-noise ratio. After ∼30 min imaging on the same area, photodamage was hardly observed. Taking the advantages of high speed and low damages of POSM, the investigation of the dynamic fluorescence responses to temperature changes was performed under three different treatment temperatures. The three embedded blue, green and red light-emitting diode light sources were applied to observe the responses of the leaves with different wavelength excitation.

  19. Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  20. Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  1. Confocal Microscopy of thick tissue sections: 3D Visualization of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  2. Confocal microscopy of thick tissue sections: 3D visualizaiton of rat kidney glomeruli

    EPA Science Inventory

    Confocal laser scanning microscopy (CLSM) as a technique capable of generating serial sections of whole-mount tissue and then reassembling the computer-acquired images as a virtual 3-dimentional structure. In many ways CLSM offers an alternative to traditional sectioning approac...

  3. Prediction of spin-dependent electronic structure in 3d-transition-metal doped antimonene

    NASA Astrophysics Data System (ADS)

    Yang, L. F.; Song, Y.; Mi, W. B.; Wang, X. C.

    2016-07-01

    We investigate the geometric structure and electronic and magnetic properties of 3d-transition-metal atom doped antimonene using spin-polarized first-principles calculations. Strong orbital hybridization exhibits between 3d-transition-metal and Sb atoms, where covalent bonds form in antimonene. A spin-polarized semiconducting state appears in Cr-doped antimonene, while half-metallic states appear by doping Ti, V, and Mn. These findings indicate that once combined with doping states, the bands of antimonene systems offer a variety of features. Specific dopants lead to half-metallic characters with high spin polarization that has potential application in spintronics.

  4. Carbon nanotube based 3-D matrix for enabling three-dimensional nano-magneto-electronics [corrected].

    PubMed

    Hong, Jeongmin; Stefanescu, Eugenia; Liang, Ping; Joshi, Nikhil; Xue, Song; Litvinov, Dmitri; Khizroev, Sakhrat

    2012-01-01

    This letter describes the use of vertically aligned carbon nanotubes (CNT)-based arrays with estimated 2-nm thick cobalt (Co) nanoparticles deposited inside individual tubes to unravel the possibility of using the unique templates for ultra-high-density low-energy 3-D nano-magneto-electronic devices. The presence of oriented 2-nm thick Co layers within individual nanotubes in the CNT-based 3-D matrix is confirmed through VSM measurements as well as an energy-dispersive X-ray spectroscopy (EDS).

  5. Prediction of spin-dependent electronic structure in 3d-transition-metal doped antimonene

    SciTech Connect

    Yang, L. F.; Song, Y.; Mi, W. B.; Wang, X. C.

    2016-07-11

    We investigate the geometric structure and electronic and magnetic properties of 3d-transition-metal atom doped antimonene using spin-polarized first-principles calculations. Strong orbital hybridization exhibits between 3d-transition-metal and Sb atoms, where covalent bonds form in antimonene. A spin-polarized semiconducting state appears in Cr-doped antimonene, while half-metallic states appear by doping Ti, V, and Mn. These findings indicate that once combined with doping states, the bands of antimonene systems offer a variety of features. Specific dopants lead to half-metallic characters with high spin polarization that has potential application in spintronics.

  6. High-resolution 3D reconstruction of microtubule structures by quantitative multi-angle total internal reflection fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Jin, Luhong; Wu, Jian; Xiu, Peng; Fan, Jiannan; Hu, Miao; Kuang, Cuifang; Xu, Yingke; Zheng, Xiaoxiang; Liu, Xu

    2017-07-01

    Total internal reflection fluorescence microscopy (TIRFM) has been widely used in biomedical research to visualize cellular processes near the cell surface. In this study, a novel multi-angle ring-illuminated TIRFM system, equipped with two galvo mirrors that are on conjugate plan of a 4f optical system was developed. Multi-angle TIRFM generates images with different penetration depths through the controlled variation of the incident angle of illuminating laser. We presented a method to perform three-dimensional (3-D) reconstruction of microtubules from multi-angle TIRFM images. The performance of our method was validated in simulated microtubules with variable signal-to-noise ratios (SNR) and the axial resolution and accuracy of reconstruction were evaluated in selecting different numbers of illumination angles or in different SNR conditions. In U373 cells, we reconstructed the 3-D localization of microtubules near the cell surface with high resolution using over a hundred different angles. Theoretically, the presented TIRFM setup and 3-D reconstruction method can achieve 40 nm axial resolution in experimental conditions where SNR is as low as 2, with 35 different illumination angles. Moreover, our system and reconstruction method have the potential to be used in live cells to track membrane dynamics in 3-D.

  7. Analysis of thin baked-on silicone layers by FTIR and 3D-Laser Scanning Microscopy.

    PubMed

    Funke, Stefanie; Matilainen, Julia; Nalenz, Heiko; Bechtold-Peters, Karoline; Mahler, Hanns-Christian; Friess, Wolfgang

    2015-10-01

    Pre-filled syringes (PFS) and auto-injection devices with cartridges are increasingly used for parenteral administration. To assure functionality, silicone oil is applied to the inner surface of the glass barrel. Silicone oil migration into the product can be minimized by applying a thin but sufficient layer of silicone oil emulsion followed by thermal bake-on versus spraying-on silicone oil. Silicone layers thicker than 100nm resulting from regular spray-on siliconization can be characterized using interferometric profilometers. However, the analysis of thin silicone layers generated by bake-on siliconization is more challenging. In this paper, we have evaluated Fourier transform infrared (FTIR) spectroscopy after solvent extraction and a new 3D-Laser Scanning Microscopy (3D-LSM) to overcome this challenge. A multi-step solvent extraction and subsequent FTIR spectroscopy enabled to quantify baked-on silicone levels as low as 21-325μg per 5mL cartridge. 3D-LSM was successfully established to visualize and measure baked-on silicone layers as thin as 10nm. 3D-LSM was additionally used to analyze the silicone oil distribution within cartridges at such low levels. Both methods provided new, highly valuable insights to characterize the siliconization after processing, in order to achieve functionality. Copyright © 2015 Elsevier B.V. All rights reserved.

  8. Fast 3-D temporal focusing microscopy using an electrically tunable lens.

    PubMed

    Jiang, Jun; Zhang, Dapeng; Walker, Steven; Gu, Chenglin; Ke, Ya; Yung, Wing Ho; Chen, Shih-chi

    2015-09-21

    In this paper, we present a 3-D temporal focusing microscope based on an electrically tunable lens (ETL) and a femtosecond regenerative laser amplifier. The focus-tunable lens provides a fast and compact way to perform non-mechanical z-scanning and resolves the blurry image issue compared with GVD-based z-scanning methods. The optical performance of the temporal focusing system, including z-scanning characteristics, the associated the magnification variation, and the lateral and axial resolution, has been studied and characterized using calibrated Rhodamine-6G thin film sample, fluorescent beads, and pollen samples. Lastly, we demonstrate the optical cross-sectioning and z-scanning capability with an in vivo experiment, where Ca(2+) imaging of neurons in GaCamp6 labeled zebrafish was performed.

  9. Time lapse microscopy of temperature control during self-assembly of 3D DNA crystals

    NASA Astrophysics Data System (ADS)

    Conn, Fiona W.; Jong, Michael Alexander; Tan, Andre; Tseng, Robert; Park, Eunice; Ohayon, Yoel P.; Sha, Ruojie; Mao, Chengde; Seeman, Nadrian C.

    2017-10-01

    DNA nanostructures are created by exploiting the high fidelity base-pairing interactions of double-stranded branched DNA molecules. These structures present a convenient medium for the self-assembly of macroscopic 3D crystals. In some self-assemblies in this system, crystals can be formed by lowering the temperature, and they can be dissolved by raising it. The ability to monitor the formation and melting of these crystals yields information that can be used to monitor crystal formation and growth. Here, we describe the development of an inexpensive tool that enables direct observation of the crystal growth process as a function of both time and temperature. Using the hanging-drop crystallization of the well-characterized 2-turn DNA tensegrity triangle motif for our model system, its response to temperature has been characterized visually.

  10. 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.

  11. Fast 3D visualization of endogenous brain signals with high-sensitivity laser scanning photothermal microscopy

    PubMed Central

    Miyazaki, Jun; Iida, Tadatsune; Tanaka, Shinji; Hayashi-Takagi, Akiko; Kasai, Haruo; Okabe, Shigeo; Kobayashi, Takayoshi

    2016-01-01

    A fast, high-sensitivity photothermal microscope was developed by implementing a spatially segmented balanced detection scheme into a laser scanning microscope. We confirmed a 4.9 times improvement in signal-to-noise ratio in the spatially segmented balanced detection compared with that of conventional detection. The system demonstrated simultaneous bi-modal photothermal and confocal fluorescence imaging of transgenic mouse brain tissue with a pixel dwell time of 20 μs. The fluorescence image visualized neurons expressing yellow fluorescence proteins, while the photothermal signal detected endogenous chromophores in the mouse brain, allowing 3D visualization of the distribution of various features such as blood cells and fine structures probably due to lipids. This imaging modality was constructed using compact and cost-effective laser diodes, and will thus be widely useful in the life and medical sciences. PMID:27231615

  12. 3D-confocal microscopy for surface analysis of microstructured materials

    NASA Astrophysics Data System (ADS)

    Kagerer, Bernd; Brodmann, Rainer; Valentin, Juergen; Filzek, Jan; Popp, Uwe

    2002-06-01

    The surface of technical materials is playing an ever more important part in modern production processes. However, standard roughness values, which are obtained from a profile, frequently no longer provide sufficient descriptions. What are desired are three-dimensional measurements of surfaces over a macroscopic range with a high degree of vertical and lateral resolution. This has become necessary to be able to describe both deterministic and non-deterministic structures in the same fashion. Due to increased requirements for data and the measuring speed demanded by industry, only optical systems are a possibility. Using the example of tribology, the capability of this technology is shown in this article on the basis of the commercial confocal 3D white light microscope, the NanoFocusTMμSurfTM. On the one hand, the technology and data preparation used are discussed, and on the other, a comparison is drawn with other standard optical measuring methods.

  13. Dynamics of electron emission in double photoionization processes near the krypton 3d threshold

    NASA Astrophysics Data System (ADS)

    Penent, F.; Sheinerman, S.; Andric, L.; Lablanquie, P.; Palaudoux, J.; Becker, U.; Braune, M.; Viefhaus, J.; Eland, J. H. D.

    2008-02-01

    Two-electron emission following photoabsorption near the Kr 3d threshold is investigated both experimentally and theoretically. On the experimental side, electron/electron coincidences using a magnetic bottle time-of-flight spectrometer allow us to observe the complete double photo ionization (DPI) continua of selected Kr2+ final states, and to see how these continua are affected by resonant processes in the vicinity of the Kr 3d threshold. The analysis is based on a quantum mechanical approach that takes into account the contribution of three different processes: (A) Auger decay of the inner 3d vacancy with the associated post-collision interaction (PCI) effects, (B) capture of slow photoelectrons into discrete states followed by valence multiplet decay (VMD) of the excited ionic states and (C) valence shell DPI. The dominant process for each Kr2+(4p-2) final state is the photoionization of the inner shell followed by Auger decay of the 3d vacancies. Moreover, for the 4p-2(3P) and 4p-2(1D) final ionic states an important contribution comes from the processes of slow photoelectron capture followed by VMD as well as from double ionization of the outer shell involving also VMD.

  14. Multicolor 3D super-resolution imaging by quantum dot stochastic optical reconstruction microscopy.

    PubMed

    Xu, Jianquan; Tehrani, Kayvan F; Kner, Peter

    2015-03-24

    We demonstrate multicolor three-dimensional super-resolution imaging with quantum dots (QSTORM). By combining quantum dot asynchronous spectral blueing with stochastic optical reconstruction microscopy and adaptive optics, we achieve three-dimensional imaging with 24 nm lateral and 37 nm axial resolution. By pairing two short-pass filters with two appropriate quantum dots, we are able to image single blueing quantum dots on two channels simultaneously, enabling multicolor imaging with high photon counts.

  15. Resonant structure of the 3d electron`s angular distribution in a free Mn{sup +}Ion

    SciTech Connect

    Amusia, M.Y.; Dolmatov, V.K.

    1995-08-01

    The 3d-electron angular anisotropy parameter of the free Mn{sup +} ion is calculated using the {open_quotes}spin-polarized{close_quotes} random-phase approximation with exchange. Strong resonance structure is discovered, which is due to interference with the powerful 3p {yields} 3d discrete excitation. The effect of the 3p {yields} 4s transition is also noticeable. The ordering of these respective resonances with phonon energy increase proved to be opposite in angular anisotropy parameter to that in 3d-photoionization cross section. A paper describing these results was published.

  16. High-Resolution Solid Modeling of Biological Samples Imaged with 3D Fluorescence Microscopy

    PubMed Central

    Ferko, Michael C.; Patterson, Brian W.; Butler, Peter J.

    2011-01-01

    Optical-sectioning, digital fluorescence microscopy provides images representing temporally- and spatially-resolved molecular-scale details of the substructures of living cells. To render such images into solid models for further computational analyses, we have developed an integrated system of image acquisition, processing, and rendering, which includes a new empirical technique to correct for axial distortions inherent in fluorescence microscopy due to refractive index mismatches between microscope objective immersion medium, coverslip glass, and water. This system takes advantage of the capabilities of ultra-high numerical aperture objectives (e.g. total internal reflection fluorescence microscopy) and enables faithful three-dimensional rendering of living cells into solid models amenable to further computational analysis. An example of solid modeling of bovine aortic endothelial cells and their nuclei is presented. Since many cellular level events are temporally and spatially confined, such integrated image acquisition, processing, rendering, and computational analysis, will enable, in silico, the generation of new computational models for cell mechanics and signaling. PMID:16758474

  17. Ultra-fast 3D scanning and holographic illumination in non-linear microscopy using acousto-optic deflectors

    NASA Astrophysics Data System (ADS)

    Akemann, Walther; Ventalon, Cathie; Léger, Jean-François; Mathieu, Benjamin; Dieudonné, Stéphane; Blochet, Baptiste; Gigan, Sylvain; Bourdieu, Laurent

    2017-04-01

    Decoding of information in the brain requires the imaging of large neuronal networks using e.g. two-photon microscopy (TPM). Fast control of the focus in 3D can be achieved with phase shaping of the light beam using acoustooptic deflectors (AODs). However, beam shaping using AODs is not straightforward because of non-stationary of acousto-optic diffraction. Here, we demonstrated a new stable AOD-based phase modulator, which operates at a rate of up to about hundred kHz. It provides opportunity for 3D scanning in TPM with the possibility to correct aberrations independently for every focus position or to achieve refocusing of scattered photons in rapidly decorrelating tissues.

  18. Rapid, High-Throughput Tracking of Bacterial Motility in 3D via Phase-Contrast Holographic Video Microscopy

    PubMed Central

    Cheong, Fook Chiong; Wong, Chui Ching; Gao, YunFeng; Nai, Mui Hoon; Cui, Yidan; Park, Sungsu; Kenney, Linda J.; Lim, Chwee Teck

    2015-01-01

    Tracking fast-swimming bacteria in three dimensions can be extremely challenging with current optical techniques and a microscopic approach that can rapidly acquire volumetric information is required. Here, we introduce phase-contrast holographic video microscopy as a solution for the simultaneous tracking of multiple fast moving cells in three dimensions. This technique uses interference patterns formed between the scattered and the incident field to infer the three-dimensional (3D) position and size of bacteria. Using this optical approach, motility dynamics of multiple bacteria in three dimensions, such as speed and turn angles, can be obtained within minutes. We demonstrated the feasibility of this method by effectively tracking multiple bacteria species, including Escherichia coli, Agrobacterium tumefaciens, and Pseudomonas aeruginosa. In addition, we combined our fast 3D imaging technique with a microfluidic device to present an example of a drug/chemical assay to study effects on bacterial motility. PMID:25762336

  19. Quasi-3D electron cyclotron emission imaging on J-TEXT

    NASA Astrophysics Data System (ADS)

    Zhao, Zhenling; Zhu, Yilun; Tong, Li; Xie, Jinlin; Liu, Wandong; Yu, Changxuan; Yang, Zhoujun; Zhuang, Ge; Luhmann, N. C., Jr.; Domier, C. W.

    2017-09-01

    Electron cyclotron emission imaging (ECEI) can provide measurements of 2D electron temperature fluctuation with high temporal and spatial resolution in magnetic fusion plasma devices. Two ECEI systems located in different toroidal ports with 67.5 degree separation have been implemented on J-TEXT to study the 3D structure of magnetohydrodynamic (MHD) instabilities. Each system consists of 12 (vertical) × 16 (horizontal) = 192 channels and the image of the 2nd harmonic X-mode electron cyclotron emission can be captured continuously in the core plasma region. The field curvature adjustment lens concept is developed to control the imaging plane for receiving optics of the ECEI systems. Field curvature of the image can be controlled to match the emission layer. Consequently, a quasi-3D image of the MHD instability in the core of the plasma has been achieved.

  20. Injectable 3-D Fabrication of Medical Electronics at the Target Biological Tissues

    PubMed Central

    Jin, Chao; Zhang, Jie; Li, Xiaokang; Yang, Xueyao; Li, Jingjing; Liu, Jing

    2013-01-01

    Conventional transplantable biomedical devices generally request sophisticated surgery which however often causes big trauma and serious pain to the patients. Here, we show an alternative way of directly making three-dimensional (3-D) medical electronics inside the biological body through sequential injections of biocompatible packaging material and liquid metal ink. As the most typical electronics, a variety of medical electrodes with different embedded structures were demonstrated to be easily formed at the target tissues. Conceptual in vitro experiments provide strong evidences for the excellent performances of the injectable electrodes. Further in vivo animal experiments disclosed that the formed electrode could serve as both highly efficient ECG (Electrocardiograph) electrode and stimulator electrode. These findings clarified the unique features and practicability of the liquid metal based injectable 3-D fabrication of medical electronics. The present strategy opens the way for directly manufacturing electrophysiological sensors or therapeutic devices in situ via a truly minimally invasive approach. PMID:24309385

  1. Injectable 3-D Fabrication of Medical Electronics at the Target Biological Tissues

    NASA Astrophysics Data System (ADS)

    Jin, Chao; Zhang, Jie; Li, Xiaokang; Yang, Xueyao; Li, Jingjing; Liu, Jing

    2013-12-01

    Conventional transplantable biomedical devices generally request sophisticated surgery which however often causes big trauma and serious pain to the patients. Here, we show an alternative way of directly making three-dimensional (3-D) medical electronics inside the biological body through sequential injections of biocompatible packaging material and liquid metal ink. As the most typical electronics, a variety of medical electrodes with different embedded structures were demonstrated to be easily formed at the target tissues. Conceptual in vitro experiments provide strong evidences for the excellent performances of the injectable electrodes. Further in vivo animal experiments disclosed that the formed electrode could serve as both highly efficient ECG (Electrocardiograph) electrode and stimulator electrode. These findings clarified the unique features and practicability of the liquid metal based injectable 3-D fabrication of medical electronics. The present strategy opens the way for directly manufacturing electrophysiological sensors or therapeutic devices in situ via a truly minimally invasive approach.

  2. Injectable 3-D fabrication of medical electronics at the target biological tissues.

    PubMed

    Jin, Chao; Zhang, Jie; Li, Xiaokang; Yang, Xueyao; Li, Jingjing; Liu, Jing

    2013-12-06

    Conventional transplantable biomedical devices generally request sophisticated surgery which however often causes big trauma and serious pain to the patients. Here, we show an alternative way of directly making three-dimensional (3-D) medical electronics inside the biological body through sequential injections of biocompatible packaging material and liquid metal ink. As the most typical electronics, a variety of medical electrodes with different embedded structures were demonstrated to be easily formed at the target tissues. Conceptual in vitro experiments provide strong evidences for the excellent performances of the injectable electrodes. Further in vivo animal experiments disclosed that the formed electrode could serve as both highly efficient ECG (Electrocardiograph) electrode and stimulator electrode. These findings clarified the unique features and practicability of the liquid metal based injectable 3-D fabrication of medical electronics. The present strategy opens the way for directly manufacturing electrophysiological sensors or therapeutic devices in situ via a truly minimally invasive approach.

  3. Tuning the electronic and magnetic properties of borophene by 3d transition-metal atom adsorption

    NASA Astrophysics Data System (ADS)

    Li, J. Y.; Lv, H. Y.; Lu, W. J.; Shao, D. F.; Xiao, R. C.; Sun, Y. P.

    2016-12-01

    The electronic and magnetic properties of borophene functionalized by 3d transition metal (TM) atom adsorption are investigated by using first-principles calculations. The results show that the 3d TM atoms can be adsorbed on borophene with high binding energies ranging between 5.9 and 8.3 eV. Interestingly, the originally nonmagnetic borophene tends to be ferromagnetic when Ti, V, Cr, Mn, and Fe atoms are adsorbed, and the magnetic moments are dominated by the TM atoms. The origin of the ferromagnetism is discussed based on the Stoner criterion. Our results indicate that the magnetic properties of borophene can be effectively tuned through the adsorption of 3d TM atoms, which could have promising applications in spintronics and nanoelectronics.

  4. A high-throughput comparative characterization of laser-induced soft tissue damage using 3D digital microscopy.

    PubMed

    Das, Debobrato; Reed, Stephanie; Klokkevold, Perry R; Wu, Benjamin M

    2013-02-01

    3D digital microscopy was used to develop a rapid alternative approach to quantify the effects of specific laser parameters on soft tissue ablation and charring in vitro without the use of conventional tissue processing techniques. Two diode lasers operating at 810 and 980 nm wavelengths were used to ablate three tissue types (bovine liver, turkey breast, and bovine muscle) at varying laser power (0.3, 1.0, and 2.0 W) and velocities (1-50 mm/s). Spectrophotometric analyses were performed on each tissue to determine tissue-specific absorption coefficients and were considered in creating wavelength-dependent energy attenuation models to evaluate minimum heat of tissue ablations. 3D surface contour profiles characterizing tissue damage revealed that ablation depth and tissue charring increased with laser power and decreased with lateral velocity independent of wavelength and tissue type. While bovine liver ablation and charring were statistically higher at 810 than 980 nm (p < 0.05), turkey breast and bovine muscle ablated and charred more at 980 than 810 nm (p < 0.05). Spectrophotometric analysis revealed that bovine liver tissue had a greater tissue-specific absorption coefficient at 810 than 980 nm, while turkey breast and bovine muscle had a larger absorption coefficient at 980 nm (p < 0.05). This rapid 3D microscopic analysis of robot-driven laser ablation yielded highly reproducible data that supported well-defined trends related to laser-tissue interactions and enabled high throughput characterization of many laser-tissue permutations. Since 3D microscopy quantifies entire lesions without altering the tissue specimens, conventional and immunohistologic techniques can be used, if desired, to further interrogate specific sections of the digitized lesions.

  5. Simulation-Guided 3D Nanomanufacturing via Focused Electron Beam Induced Deposition

    DOE PAGES

    Fowlkes, Jason D.; Winkler, Robert; Lewis, Brett B.; ...

    2016-06-10

    Focused electron beam induced deposition (FEBID) is one of the few techniques that enables direct-write synthesis of free-standing 3D nanostructures. While the fabrication of simple architectures such as vertical or curving nanowires has been achieved by simple trial and error, processing complex 3D structures is not tractable with this approach. This is due, inpart, to the dynamic interplay between electron–solid interactions and the transient spatial distribution of absorbed precursor molecules on the solid surface. Here, we demonstrate the ability to controllably deposit 3D lattice structures at the micro/nanoscale, which have received recent interest owing to superior mechanical and optical properties.more » Moreover, a hybrid Monte Carlo–continuum simulation is briefly overviewed, and subsequently FEBID experiments and simulations are directly compared. Finally, a 3D computer-aided design (CAD) program is introduced, which generates the beam parameters necessary for FEBID by both simulation and experiment. In using this approach, we demonstrate the fabrication of various 3D lattice structures using Pt-, Au-, and W-based precursors.« less

  6. 3D Magnetic Induction Maps of Nanoscale Materials Revealed by Electron Holographic Tomography

    PubMed Central

    2015-01-01

    The investigation of three-dimensional (3D) ferromagnetic nanoscale materials constitutes one of the key research areas of the current magnetism roadmap and carries great potential to impact areas such as data storage, sensing, and biomagnetism. The properties of such nanostructures are closely connected with their 3D magnetic nanostructure, making their determination highly valuable. Up to now, quantitative 3D maps providing both the internal magnetic and electric configuration of the same specimen with high spatial resolution are missing. Here, we demonstrate the quantitative 3D reconstruction of the dominant axial component of the magnetic induction and electrostatic potential within a cobalt nanowire (NW) of 100 nm in diameter with spatial resolution below 10 nm by applying electron holographic tomography. The tomogram was obtained using a dedicated TEM sample holder for acquisition, in combination with advanced alignment and tomographic reconstruction routines. The powerful approach presented here is widely applicable to a broad range of 3D magnetic nanostructures and may trigger the progress of novel spintronic nonplanar nanodevices. PMID:27182110

  7. Simulation-Guided 3D Nanomanufacturing via Focused Electron Beam Induced Deposition

    SciTech Connect

    Fowlkes, Jason D.; Winkler, Robert; Lewis, Brett B.; Stanford, Michael G.; Plank, Harald; Rack, Philip D.

    2016-06-10

    Focused electron beam induced deposition (FEBID) is one of the few techniques that enables direct-write synthesis of free-standing 3D nanostructures. While the fabrication of simple architectures such as vertical or curving nanowires has been achieved by simple trial and error, processing complex 3D structures is not tractable with this approach. This is due, inpart, to the dynamic interplay between electron–solid interactions and the transient spatial distribution of absorbed precursor molecules on the solid surface. Here, we demonstrate the ability to controllably deposit 3D lattice structures at the micro/nanoscale, which have received recent interest owing to superior mechanical and optical properties. Moreover, a hybrid Monte Carlo–continuum simulation is briefly overviewed, and subsequently FEBID experiments and simulations are directly compared. Finally, a 3D computer-aided design (CAD) program is introduced, which generates the beam parameters necessary for FEBID by both simulation and experiment. In using this approach, we demonstrate the fabrication of various 3D lattice structures using Pt-, Au-, and W-based precursors.

  8. 3D Magnetic Induction Maps of Nanoscale Materials Revealed by Electron Holographic Tomography.

    PubMed

    Wolf, Daniel; Rodriguez, Luis A; Béché, Armand; Javon, Elsa; Serrano, Luis; Magen, Cesar; Gatel, Christophe; Lubk, Axel; Lichte, Hannes; Bals, Sara; Van Tendeloo, Gustaaf; Fernández-Pacheco, Amalio; De Teresa, José M; Snoeck, Etienne

    2015-10-13

    The investigation of three-dimensional (3D) ferromagnetic nanoscale materials constitutes one of the key research areas of the current magnetism roadmap and carries great potential to impact areas such as data storage, sensing, and biomagnetism. The properties of such nanostructures are closely connected with their 3D magnetic nanostructure, making their determination highly valuable. Up to now, quantitative 3D maps providing both the internal magnetic and electric configuration of the same specimen with high spatial resolution are missing. Here, we demonstrate the quantitative 3D reconstruction of the dominant axial component of the magnetic induction and electrostatic potential within a cobalt nanowire (NW) of 100 nm in diameter with spatial resolution below 10 nm by applying electron holographic tomography. The tomogram was obtained using a dedicated TEM sample holder for acquisition, in combination with advanced alignment and tomographic reconstruction routines. The powerful approach presented here is widely applicable to a broad range of 3D magnetic nanostructures and may trigger the progress of novel spintronic nonplanar nanodevices.

  9. Simulation-Guided 3D Nanomanufacturing via Focused Electron Beam Induced Deposition

    SciTech Connect

    Fowlkes, Jason D.; Winkler, Robert; Lewis, Brett B.; Stanford, Michael G.; Plank, Harald; Rack, Philip D.

    2016-06-10

    Focused electron beam induced deposition (FEBID) is one of the few techniques that enables direct-write synthesis of free-standing 3D nanostructures. While the fabrication of simple architectures such as vertical or curving nanowires has been achieved by simple trial and error, processing complex 3D structures is not tractable with this approach. This is due, inpart, to the dynamic interplay between electron–solid interactions and the transient spatial distribution of absorbed precursor molecules on the solid surface. Here, we demonstrate the ability to controllably deposit 3D lattice structures at the micro/nanoscale, which have received recent interest owing to superior mechanical and optical properties. Moreover, a hybrid Monte Carlo–continuum simulation is briefly overviewed, and subsequently FEBID experiments and simulations are directly compared. Finally, a 3D computer-aided design (CAD) program is introduced, which generates the beam parameters necessary for FEBID by both simulation and experiment. In using this approach, we demonstrate the fabrication of various 3D lattice structures using Pt-, Au-, and W-based precursors.

  10. Analytical transmission electron microscopy in materials science

    SciTech Connect

    Fraser, H.L.

    1980-01-01

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

  11. 3-D localization of flaws in structural ceramics by use of scanning laser acoustic microscopy

    SciTech Connect

    Wey, A.C.; Kessler, L.W.

    1989-08-01

    Scanning laser acoustic microscopy (SLAM), which operates in a transmission mode, accomplishes not merely the real-time nondestructive inspection of samples but high-resolution, two-dimensional shadowgraphic imaging of the detailed structure of small objects. Due to diffraction, however, image ambiguities can arise; a digital image-processing technique has accordingly been developed which uses acoustical holography principles in conjunction with wave-field propagation calculations to yield three-dimensional SLAM. Attention is given to the modifications required so that conventional SLAM acquires the amplitude and phase data needed for image reconstruction. 7 refs.

  12. Articular cartilage zonal differentiation via 3D Second-Harmonic Generation imaging microscopy.

    PubMed

    Chaudhary, Rajeev; Campbell, Kirby R; Tilbury, Karissa B; Vanderby, Ray; Block, Walter F; Kijowski, Richard; Campagnola, Paul J

    2015-04-01

    The collagen structure throughout the patella has not been thoroughly investigated by 3D imaging, where the majority of the existing data come from histological cross sections. It is important to have a better understanding of the architecture in normal tissues, where this could then be applied to imaging of diseased states. To address this shortcoming, we investigated the combined use of collagen-specific Second-Harmonic Generation (SHG) imaging and measurement of bulk optical properties to characterize collagen fiber orientations of the histologically defined zones of bovine articular cartilage. Forward and backward SHG intensities of sections from superficial, middle and deep zones were collected as a function of depth and analyzed by Monte Carlo simulations to extract the SHG creation direction, which is related to the fibrillar assembly. Our results revealed differences in SHG forward-backward response between the three zones, where these are consistent with a previously developed model of SHG emission. Some of the findings are consistent with that from other modalities; however, SHG analysis showed the middle zone had the most organized fibril assembly. While not distinct, we also report bulk optical property values for these different zones within the patella. Collectively, these results provide quantitative measurements of structural changes at both the fiber and fibril assembly of the different cartilage zones and reveals structural information not possible by other microscope modalities. This can provide quantitative insight to the collagen fiber network in normal cartilage, which may ultimately be developed as a biomarker for osteoarthritis.

  13. 3D reconstruction and characterization of laser induced craters by in situ optical microscopy

    NASA Astrophysics Data System (ADS)

    Casal, A.; Cerrato, R.; Mateo, M. P.; Nicolas, G.

    2016-06-01

    A low-cost optical microscope was developed and coupled to an irradiation system in order to study the induced effects on material during a multipulse regime by an in situ visual inspection of the surface, in particular of the spot generated at different pulses. In the case of laser ablation, a reconstruction of the crater in 3D was made from the images of the sample surface taken during the irradiation process, and the subsequent profiles of ablated material were extracted. The implementation of this homemade optical device gives an added value to the irradiation system, providing information about morphology evolution of irradiated area when successive pulses are applied. In particular, the determination of ablation rates in real time can be especially useful for a better understanding and controlling of the ablation process in applications where removal of material is involved, such as laser cleaning and in-depth characterization of multilayered samples and diffusion processes. The validation of the developed microscope was made by a comparison with a commercial confocal microscope configured for the characterization of materials where similar results of crater depth and diameter were obtained for both systems.

  14. Image reconstruction for 3D light microscopy with a regularized linear method incorporating a smoothness prior

    NASA Astrophysics Data System (ADS)

    Preza, Chrysanthe; Miller, Michael I.; Conchello, Jose-Angel

    1993-07-01

    We have shown that the linear least-squares (LLS) estimate of the intensities of a 3-D object obtained from a set of optical sections is unstable due to the inversion of small and zero-valued eigenvalues of the point-spread function (PSF) operator. The LLS solution was regularized by constraining it to lie in a subspace spanned by the eigenvectors corresponding to a selected number of the largest eigenvalues. In this paper we extend the regularized LLS solution to a maximum a posteriori (MAP) solution induced by a prior formed from a 'Good's like' smoothness penalty. This approach also yields a regularized linear estimator which reduces noise as well as edge artifacts in the reconstruction. The advantage of the linear MAP (LMAP) estimate over the current regularized LLS (RLLS) is its ability to regularize the inverse problem by smoothly penalizing components in the image associated with small eigenvalues. Computer simulations were performed using a theoretical PSF and a simple phantom to compare the two regularization techniques. It is shown that the reconstructions using the smoothness prior, give superior variance and bias results compared to the RLLS reconstructions. Encouraging reconstructions obtained with the LMAP method from real microscopical images of a 10 micrometers fluorescent bead, and a four-cell Volvox embryo are shown.

  15. 3D PIC modeling of laser acceleration of electrons from two-dimensional inhomogeneous plasma corona

    NASA Astrophysics Data System (ADS)

    Pugachev, L. P.; Levashov, P. R.; Andreev, N. E.

    2015-11-01

    This paper presents the results of three-dimensional (3D3V) particle-in-cell modeling of the interaction of a femtosecond laser pulse with a two-dimensional inhomogeneous plasma corona of subcritical density. It was shown that in the presence of sufficiently steep temporal pulse edge the excitation of plasma waves, electron trapping and generation of collimated beams of accelerated electrons with energy of about 0.2-0.5 MeV may occur. The simulation results are compared with experiments on the generation of collimated beams of accelerated electrons from metal targets irradiated by intense femtosecond laser radiation.

  16. Vibration insensitive 3D-profilometry: a new type of white light interferometric microscopy

    NASA Astrophysics Data System (ADS)

    Cohen-Sabban, J.; Reolon, D.

    2008-08-01

    The measurement accuracy in non contact profilometric techniques is generally limited by mechanical vibrations and by geometrical defaults of the micro-scanning table. In order to free the measurement from these environnemental perturbations, we describe a novel type of interferometric microscopy based on the well-known Spectroscopic Analysis of White Light Interferograms (SAWLI). The originality of the presented set-up lies in the fixation of the reference plate on the inspected object. As reference plate and sample are fixed together, the mechanical vibrations do not affect the measurements. As a result the potential nanometric accuracy of interferometric microscopy is effective. This method consists in measuring the air gap thickness between the reference plate and the sample. At the output of the spectral interferometric microscope a channelled spectrum is observed. From this signal, the spectral phase is calculated using a numerical seven points phase shifting algorithm allowing the measurement of the local height of the analyzed surface. These preliminary results demonstrate the ability of this method as a point sensor. Then this technique will be implemented in a high frequency scanning STIL technology named MPLS 180.

  17. Fabrication of 3D polymer microstructures using electron beam lithography and nanoimprinting technologies

    NASA Astrophysics Data System (ADS)

    Chen, Kuo-Shen; Lin, I.-Kuan; Ko, Fu-Hsang

    2005-10-01

    Recently, with the advancement in bio-MEMS and micro optoelectromechanical systems (MOEMS), 3D microstructures have become increasingly important and efficient fabrication processes are currently being sought. In this paper, a novel 3D fabrication process has been proposed by utilizing the proximity effect of electron beam lithography (EBL) to create 3D microstructures on negative photoresists as the primary molds, which are subsequently transferred to their corresponding negative molds using nanoimprinting lithography (NIL), and to form the final replicas by either electroforming or polymer spin casting to reduce cost. The effect of electron backscattering on the 3D topography is firstly investigated and the relationship among the spatial distribution of electron beam irradiation, the spot size and the dosage level of irradiation is experimentally characterized in SU-8 to establish a dosage kernel distribution function. A mathematical procedure based on linear operation of this kernel function is then proposed to mimic the EBL fabrication process. The subsequent experiments indicate that the predicted surface profiles agree with the experimental results to large extent and the proposed mathematical operations are valid for the purpose of designing the fabrication process. Finally, the SU-8 primary molds are transferred to NEB to form secondary molds via the nanoimprinting process. It shows that the nanoimprinting process can essentially reproduce the shape and geometry of the primary molds. However, due to the nature of polymer-to-polymer contact printing, the elastic restitution of materials induces a slight deviation of the final device size and a further study should be made in the future to minimize such types of error. Although the above problems are reported, nevertheless, the primary experimental results indicate that this proposed fabrication process is capable of creating 3D shape microstructure in the order of 1 µm and should be useful for related

  18. 3D structural fluctuation of IgG1 antibody revealed by individual particle electron tomography

    DOE PAGES

    Zhang, Xing; Zhang, Lei; Tong, Huimin; ...

    2015-05-05

    Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, wemore » derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.« less

  19. 3D Structural Fluctuation of IgG1 Antibody Revealed by Individual Particle Electron Tomography

    PubMed Central

    Zhang, Xing; Zhang, Lei; Tong, Huimin; Peng, Bo; Rames, Matthew J.; Zhang, Shengli; Ren, Gang

    2015-01-01

    Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions. PMID:25940394

  20. 3D structural fluctuation of IgG1 antibody revealed by individual particle electron tomography

    SciTech Connect

    Zhang, Xing; Zhang, Lei; Tong, Huimin; Peng, Bo; Rames, Matthew J.; Zhang, Shengli; Ren, Gang

    2015-05-05

    Commonly used methods for determining protein structure, including X-ray crystallography and single-particle reconstruction, often provide a single and unique three-dimensional (3D) structure. However, in these methods, the protein dynamics and flexibility/fluctuation remain mostly unknown. Here, we utilized advances in electron tomography (ET) to study the antibody flexibility and fluctuation through structural determination of individual antibody particles rather than averaging multiple antibody particles together. Through individual-particle electron tomography (IPET) 3D reconstruction from negatively-stained ET images, we obtained 120 ab-initio 3D density maps at an intermediate resolution (~1–3 nm) from 120 individual IgG1 antibody particles. Using these maps as a constraint, we derived 120 conformations of the antibody via structural flexible docking of the crystal structure to these maps by targeted molecular dynamics simulations. Statistical analysis of the various conformations disclosed the antibody 3D conformational flexibility through the distribution of its domain distances and orientations. This blueprint approach, if extended to other flexible proteins, may serve as a useful methodology towards understanding protein dynamics and functions.

  1. Extensions of 1d Bgk Electron Solitary Wave Solutions To 3d Magnetized and Unmagnetized Plasmas

    NASA Astrophysics Data System (ADS)

    Chen, Li-Jen; Parks, George K.

    This paper will compare the key results for BGK electron solitary waves in 3D mag- netized and unmagnetized plasmas. For 3D magnetized plasmas with highly magnetic field-aligned electrons, our results predict that the parallel widths of the solitary waves can be smaller than one Debye length, the solitary waves can be large scale features of the magnetosphere, and the parallel width-amplitude relation has a dependence on the perpendicular size. We can thus obtain an estimate on the typical perpendicular size of the observed solitary waves assuming a series of consecutive solitary waves are in the same flux tude with a particular perpendicular span. In 3D unmagnetized plasma systems such as the neutral sheet and magnetic reconnection sites, our theory indi- cates that although mathematical solutions can be constructed as the time-stationary solutions for the nonlinear Vlasov-Poisson equations, there does not exist a param- eter range for the solutions to be physical. We conclude that single-humped solitary potential pulses cannot be self-consistently supported by charged particles in 3D un- magnetized plasmas.

  2. Real time 3-D electron density reconstruction over Europe by using TaD profiler

    NASA Astrophysics Data System (ADS)

    Kutiev, I.; Marinov, P.; Belehaki, A.

    2016-07-01

    The TaD (Topside Sounder Model (TSM)-assisted Digisonde) profiler, developed on the basis of the Topside Sounder Model (TSM), provides vertical electron density profiles (EDP) over Digisondes from the bottomside ionosphere up to Global Navigation Satellite Systems (GNSS) orbit heights. TaD EDP uses the Digisonde bottomside profile and extends it above the F2 layer peak, representing O+ distribution by α-Chapman formula and H+ distribution by a single exponent. Topside scale height HT and transition height hT are taken from TSM, while the plasmasphere scale height Hp is defined as a function of HT. All profile parameters are adjusted to the current conditions comparing the profile integral with the GNSS vertical total electron content (TEC) retrieved from the European Reference Frame (EUREF) maps. To expand to three dimensions (3-D), European maps of foF2 and hmF2 are produced, based on Digisonde data, with spatial resolution 1°×1° in latitude and longitude, and TaD profiles are calculated at each grid node. Electron density (ED) at any point of the 3-D space is obtained by linear interpolation of TaD parameters between neighbor nodes. Samples of two dimensional (2-D) electron density distribution (EDD) at different cross sections of the 3-D space between 200 km and 1150 km over the mapping area are presented, along with distributions of the electron density along various raypaths of GNSS signals. The modeled 3-D EDD is compared with vertical (vTEC) and slant (sTEC) TEC parameters calculated from individual GNSS receivers. The model error (relative deviation of model from the data), based on 6780 data values, is 10% for sTEC and 6% for vTEC.

  3. Potential of 3D printing technologies for fabrication of electron bolus and proton compensators.

    PubMed

    Zou, Wei; Fisher, Ted; Zhang, Miao; Kim, Leonard; Chen, Ting; Narra, Venkat; Swann, Beth; Singh, Rachana; Siderit, Richard; Yin, Lingshu; Teo, Boon-Keng Kevin; McKenna, Michael; McDonough, James; Ning, Yue J

    2015-05-08

    In electron and proton radiotherapy, applications of patient-specific electron bolus or proton compensators during radiation treatments are often necessary to accommodate patient body surface irregularities, tissue inhomogeneity, and variations in PTV depths to achieve desired dose distributions. Emerging 3D printing technologies provide alternative fabrication methods for these bolus and compensators. This study investigated the potential of utilizing 3D printing technologies for the fabrication of the electron bolus and proton compensators. Two printing technologies, fused deposition modeling (FDM) and selective laser sintering (SLS), and two printing materials, PLA and polyamide, were investigated. Samples were printed and characterized with CT scan and under electron and proton beams. In addition, a software package was developed to convert electron bolus and proton compensator designs to printable Standard Tessellation Language file format. A phantom scalp electron bolus was printed with FDM technology with PLA material. The HU of the printed electron bolus was 106.5 ± 15.2. A prostate patient proton compensator was printed with SLS technology and polyamide material with -70.1 ± 8.1 HU. The profiles of the electron bolus and proton compensator were compared with the original designs. The average over all the CT slices of the largest Euclidean distance between the design and the fabricated bolus on each CT slice was found to be 0.84 ± 0.45 mm and for the compensator to be 0.40 ± 0.42 mm. It is recommended that the properties of specific 3D printed objects are understood before being applied to radiotherapy treatments.

  4. Fast electron microscopy via compressive sensing

    SciTech Connect

    Larson, Kurt W; Anderson, Hyrum S; Wheeler, Jason W

    2014-12-09

    Various technologies described herein pertain to compressive sensing electron microscopy. A compressive sensing electron microscope includes a multi-beam generator and a detector. The multi-beam generator emits a sequence of electron patterns over time. Each of the electron patterns can include a plurality of electron beams, where the plurality of electron beams is configured to impart a spatially varying electron density on a sample. Further, the spatially varying electron density varies between each of the electron patterns in the sequence. Moreover, the detector collects signals respectively corresponding to interactions between the sample and each of the electron patterns in the sequence.

  5. PreMosa: extracting 2D surfaces from 3D microscopy mosaics.

    PubMed

    Blasse, Corinna; Saalfeld, Stephan; Etournay, Raphaël; Sagner, Andreas; Eaton, Suzanne; Myers, Eugene W

    2017-08-15

    A significant focus of biological research is to understand the development, organization and function of tissues. A particularly productive area of study is on single layer epithelial tissues in which the adherence junctions of cells form a 2D manifold that is fluorescently labeled. Given the size of the tissue, a microscope must collect a mosaic of overlapping 3D stacks encompassing the stained surface. Downstream interpretation is greatly simplified by preprocessing such a dataset as follows: (i) extracting and mapping the stained manifold in each stack into a single 2D projection plane, (ii) correcting uneven illumination artifacts, (iii) stitching the mosaic planes into a single, large 2D image and (iv) adjusting the contrast. We have developed PreMosa, an efficient, fully automatic pipeline to perform the four preprocessing tasks above resulting in a single 2D image of the stained manifold across which contrast is optimized and illumination is even. Notable features are as follows. First, the 2D projection step employs a specially developed algorithm that actually finds the manifold in the stack based on maximizing contrast, intensity and smoothness. Second, the projection step comes first, implying all subsequent tasks are more rapidly solved in 2D. And last, the mosaic melding employs an algorithm that globally adjusts contrasts amongst the 2D tiles so as to produce a seamless, high-contrast image. We conclude with an evaluation using ground-truth datasets and present results on datasets from Drosophila melanogaster wings and Schmidtae mediterranea ciliary components. PreMosa is available under https://cblasse.github.io/premosa. blasse@mpi-cbg.de or myers@mpi-cbg.de. Supplementary data are available at Bioinformatics online.

  6. Visual Understanding of Light Absorption and Waveguiding in Standing Nanowires with 3D Fluorescence Confocal Microscopy.

    PubMed

    Frederiksen, Rune; Tutuncuoglu, Gozde; Matteini, Federico; Martinez, Karen L; Fontcuberta I Morral, Anna; Alarcon-Llado, Esther

    2017-09-20

    Semiconductor nanowires are promising building blocks for next-generation photonics. Indirect proofs of large absorption cross sections have been reported in nanostructures with subwavelength diameters, an effect that is even more prominent in vertically standing nanowires. In this work we provide a three-dimensional map of the light around vertical GaAs nanowires standing on a substrate by using fluorescence confocal microscopy, where the strong long-range disruption of the light path along the nanowire is illustrated. We find that the actual long-distance perturbation is much larger in size than calculated extinction cross sections. While the size of the perturbation remains similar, the intensity of the interaction changes dramatically over the visible spectrum. Numerical simulations allow us to distinguish the effects of scattering and absorption in the nanowire leading to these phenomena. This work provides a visual understanding of light absorption in semiconductor nanowire structures, which is of high interest for solar energy conversion applications.

  7. Visual Understanding of Light Absorption and Waveguiding in Standing Nanowires with 3D Fluorescence Confocal Microscopy

    PubMed Central

    2017-01-01

    Semiconductor nanowires are promising building blocks for next-generation photonics. Indirect proofs of large absorption cross sections have been reported in nanostructures with subwavelength diameters, an effect that is even more prominent in vertically standing nanowires. In this work we provide a three-dimensional map of the light around vertical GaAs nanowires standing on a substrate by using fluorescence confocal microscopy, where the strong long-range disruption of the light path along the nanowire is illustrated. We find that the actual long-distance perturbation is much larger in size than calculated extinction cross sections. While the size of the perturbation remains similar, the intensity of the interaction changes dramatically over the visible spectrum. Numerical simulations allow us to distinguish the effects of scattering and absorption in the nanowire leading to these phenomena. This work provides a visual understanding of light absorption in semiconductor nanowire structures, which is of high interest for solar energy conversion applications. PMID:28966933

  8. A surface-based 3-D dendritic spine detection approach from confocal microscopy images.

    PubMed

    Li, Qing; Deng, Zhigang

    2012-03-01

    Determining the relationship between the dendritic spine morphology and its functional properties is a fundamental challenge in neurobiology research. In particular, how to accurately and automatically analyse meaningful structural information from a large microscopy image data set is far away from being resolved. As pointed out in existing literature, one remaining challenge in spine detection and segmentation is how to automatically separate touching spines. In this paper, based on various global and local geometric features of the dendrite structure, we propose a novel approach to detect and segment neuronal spines, in particular, a breaking-down and stitching-up algorithm to accurately separate touching spines. Extensive performance comparisons show that our approach is more accurate and robust than two state-of-the-art spine detection and segmentation algorithms.

  9. Parallel deconvolution of large 3D images obtained by confocal laser scanning microscopy.

    PubMed

    Pawliczek, Piotr; Romanowska-Pawliczek, Anna; Soltys, Zbigniew

    2010-03-01

    Various deconvolution algorithms are often used for restoration of digital images. Image deconvolution is especially needed for the correction of three-dimensional images obtained by confocal laser scanning microscopy. Such images suffer from distortions, particularly in the Z dimension. As a result, reliable automatic segmentation of these images may be difficult or even impossible. Effective deconvolution algorithms are memory-intensive and time-consuming. In this work, we propose a parallel version of the well-known Richardson-Lucy deconvolution algorithm developed for a system with distributed memory and implemented with the use of Message Passing Interface (MPI). It enables significantly more rapid deconvolution of two-dimensional and three-dimensional images by efficiently splitting the computation across multiple computers. The implementation of this algorithm can be used on professional clusters provided by computing centers as well as on simple networks of ordinary PC machines.

  10. Ultrafast Science Opportunities with Electron Microscopy

    SciTech Connect

    Durr, Hermann

    2016-04-28

    X-rays and electrons are two of the most fundamental probes of matter. When the Linac Coherent Light Source (LCLS), the world’s first x-ray free electron laser, began operation in 2009, it transformed ultrafast science with the ability to generate laser-like x-ray pulses from the manipulation of relativistic electron beams. This document describes a similar future transformation. In Transmission Electron Microscopy, ultrafast relativistic (MeV energy) electron pulses can achieve unsurpassed spatial and temporal resolution. Ultrafast temporal resolution will be the next frontier in electron microscopy and can ideally complement ultrafast x-ray science done with free electron lasers. This document describes the Grand Challenge science opportunities in chemistry, material science, physics and biology that arise from an MeV ultrafast electron diffraction & microscopy facility, especially when coupled with linac-based intense THz and X-ray pump capabilities.

  11. Real-time monitoring of quorum sensing in 3D-printed bacterial aggregates using scanning electrochemical microscopy.

    PubMed

    Connell, Jodi L; Kim, Jiyeon; Shear, Jason B; Bard, Allen J; Whiteley, Marvin

    2014-12-23

    Microbes frequently live in nature as small, densely packed aggregates containing ∼10(1)-10(5) cells. These aggregates not only display distinct phenotypes, including resistance to antibiotics, but also, serve as building blocks for larger biofilm communities. Aggregates within these larger communities display nonrandom spatial organization, and recent evidence indicates that this spatial organization is critical for fitness. Studying single aggregates as well as spatially organized aggregates remains challenging because of the technical difficulties associated with manipulating small populations. Micro-3D printing is a lithographic technique capable of creating aggregates in situ by printing protein-based walls around individual cells or small populations. This 3D-printing strategy can organize bacteria in complex arrangements to investigate how spatial and environmental parameters influence social behaviors. Here, we combined micro-3D printing and scanning electrochemical microscopy (SECM) to probe quorum sensing (QS)-mediated communication in the bacterium Pseudomonas aeruginosa. Our results reveal that QS-dependent behaviors are observed within aggregates as small as 500 cells; however, aggregates larger than 2,000 bacteria are required to stimulate QS in neighboring aggregates positioned 8 μm away. These studies provide a powerful system to analyze the impact of spatial organization and aggregate size on microbial behaviors.

  12. GPU-based rapid reconstruction of cellular 3D refractive index maps from tomographic phase microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Dardikman, Gili; Shaked, Natan T.

    2016-03-01

    We present highly parallel and efficient algorithms for real-time reconstruction of the quantitative three-dimensional (3-D) refractive-index maps of biological cells without labeling, as obtained from the interferometric projections acquired by tomographic phase microscopy (TPM). The new algorithms are implemented on the graphic processing unit (GPU) of the computer using CUDA programming environment. The reconstruction process includes two main parts. First, we used parallel complex wave-front reconstruction of the TPM-based interferometric projections acquired at various angles. The complex wave front reconstructions are done on the GPU in parallel, while minimizing the calculation time of the Fourier transforms and phase unwrapping needed. Next, we implemented on the GPU in parallel the 3-D refractive index map retrieval using the TPM filtered-back projection algorithm. The incorporation of algorithms that are inherently parallel with a programming environment such as Nvidia's CUDA makes it possible to obtain real-time processing rate, and enables high-throughput platform for label-free, 3-D cell visualization and diagnosis.

  13. 3D restoration microscopy improves quantification of enzyme-labeled fluorescence-based single-cell phosphatase activity in plankton.

    PubMed

    Diaz-de-Quijano, Daniel; Palacios, Pilar; Horňák, Karel; Felip, Marisol

    2014-10-01

    The ELF or fluorescence-labeled enzyme activity (FLEA) technique is a culture-independent single-cell tool for assessing plankton enzyme activity in close-to-in situ conditions. We demonstrate that single-cell FLEA quantifications based on two-dimensional (2D) image analysis were biased by up to one order of magnitude relative to deconvolved 3D. This was basically attributed to out-of-focus light, and partially to object size. Nevertheless, if sufficient cells were measured (25-40 cells), biases in individual 2D cell measurements were partially compensated, providing useful and comparable results to deconvolved 3D. We also discuss how much caution should be used when comparing the single-cell enzyme activities of different sized bacterio- and/or phytoplankton populations measured on 2D images. Finally, a novel method based on deconvolved 3D images (wide field restoration microscopy; WFR) was devised to improve the discrimination of similar single-cell enzyme activities, the comparison of enzyme activities between different size cells, the measurement of low fluorescence intensities, the quantification of less numerous species, and the combination of the FLEA technique with other single-cell methods. These improvements in cell enzyme activity measurements will provide a more precise picture of individual species' behavior in nature, which is essential to understand their functional role and evolutionary history.

  14. 3-D Raman Imagery and Atomic Force Microscopy of Ancient Microscopic Fossils

    NASA Astrophysics Data System (ADS)

    Schopf, J.

    2003-12-01

    Investigations of the Precambrian (~540- to ~3,500-Ma-old) fossil record depend critically on identification of authentic microbial fossils. Combined with standard paleontologic studies (e.g., of paleoecologic setting, population structure, cellular morphology, preservational variants), two techniques recently introduced to such studies -- Raman imagery and atomic force microscopy -- can help meet this need. Laser-Raman imagery is a non-intrusive, non-destructive technique that can be used to demonstrate a micron-scale one-to-one correlation between optically discernable morphology and the organic (kerogenous) composition of individual microbial fossils(1,2), a prime indicator of biogencity. Such analyses can be used to characterize the molecular-structural makeup of organic-walled microscopic fossils both in acid-resistant residues and in petrographic thin sections, and whether the fossils analyzed are exposed at the upper surface of, or are embedded within (to depths >65 microns), the section studied. By providing means to map chemically, in three dimensions, whole fossils or parts of such fossils(3), Raman imagery can also show the presence of cell lumina, interior cellular cavities, another prime indicator of biogenicity. Atomic force microscopy (AFM) has been used to visualize the nanometer-scale structure of the kerogenous components of single Precambrian microscopic fossils(4). Capable of analyzing minute fragments of ancient organic matter exposed at the upper surface of thin sections (or of kerogen particles deposited on flat surfaces), such analyses hold promise not only for discriminating between biotic and abiotic micro-objects but for elucidation of the domain size -- and, thus, the degree of graphitization -- of the graphene subunits of the carbonaceous matter analyzed. These techniques -- both new to paleobiology -- can provide useful insight into the biogenicity and geochemical maturity of ancient organic matter. References: (1) Kudryavtsev, A.B. et

  15. The future of electron microscopy

    SciTech Connect

    Zhu, Yimei; Durr, Hermann

    2015-04-01

    Seeing is believing. So goes the old adage and seen evidence is undoubtedly satisfying because it can be interpreted easily, though not always correctly. For centuries, humans have developed such instruments as telescopes that observe the heavens and microscopes that reveal bacteria and viruses. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their foundational work on superresolution fluorescence microscopy in which they overcame the Abbe diffraction limit for the resolving power of conventional light microscopes. (See Physics Today, December 2014, page 18.) That breakthrough enabled discoveries in biological research and testifies to the importance of modern microscopy.

  16. The future of electron microscopy

    DOE PAGES

    Zhu, Yimei; Durr, Hermann

    2015-04-01

    Seeing is believing. So goes the old adage and seen evidence is undoubtedly satisfying because it can be interpreted easily, though not always correctly. For centuries, humans have developed such instruments as telescopes that observe the heavens and microscopes that reveal bacteria and viruses. The 2014 Nobel Prize in Chemistry was awarded to Eric Betzig, Stefan Hell, and William Moerner for their foundational work on superresolution fluorescence microscopy in which they overcame the Abbe diffraction limit for the resolving power of conventional light microscopes. (See Physics Today, December 2014, page 18.) That breakthrough enabled discoveries in biological research and testifiesmore » to the importance of modern microscopy.« less

  17. SU-C-213-06: Dosimetric Verification of 3D Printed Electron Bolus

    SciTech Connect

    Rasmussen, K; Corbett, M; Pelletier, C; Huang, Z; Feng, Y; Jung, J

    2015-06-15

    Purpose: To determine the dosimetric effect of 3D printed bolus in an anthropomorphic phantom. Methods: Conformable bolus material was generated for an anthropomorphic phantom from a DICOM volume. The bolus generated was a uniform expansion of 5mm applied to the nose region of the phantom, as this is a difficult area to uniformly apply bolus clinically. A Printrbot metal 3D Printer using PLA plastic generated the bolus. A 9MeV anterior beam with a 5cm cone was used to deliver dose to the nose of the phantom. TLD measurements were compared to predicted values at the phantom surface. Film planes were analyzed for the printed bolus, a standard 5mm bolus sheet placed on the phantom, and the phantom with no bolus applied to determine depth and dose distributions. Results: TLDs measured within 2.5% of predicted value for the 3D bolus. Film demonstrated a more uniform dose distribution in the nostril region for the 3d printed bolus than the standard bolus. This difference is caused by the air gap created around the nostrils by the standard bolus, creating a secondary build-up region. Both demonstrated a 50% central axis dose shift of 5mm relative to the no bolus film. HU for the bolus calculated the PLA electron density to be ∼1.1g/cc. Physical density was measured to be 1.3g/cc overall. Conclusion: 3D printed PLA bolus demonstrates improved dosimetric performance to standard bolus for electron beams with complex phantom geometry.

  18. Tensor decomposition in electronic structure calculations on 3D Cartesian grids

    SciTech Connect

    Khoromskij, B.N. Khoromskaia, V.; Chinnamsetty, S.R.; Flad, H.-J.

    2009-09-01

    In this paper, we investigate a novel approach based on the combination of Tucker-type and canonical tensor decomposition techniques for the efficient numerical approximation of functions and operators in electronic structure calculations. In particular, we study applicability of tensor approximations for the numerical solution of Hartree-Fock and Kohn-Sham equations on 3D Cartesian grids. We show that the orthogonal Tucker-type tensor approximation of electron density and Hartree potential of simple molecules leads to low tensor rank representations. This enables an efficient tensor-product convolution scheme for the computation of the Hartree potential using a collocation-type approximation via piecewise constant basis functions on a uniform nxnxn grid. Combined with the Richardson extrapolation, our approach exhibits O(h{sup 3}) convergence in the grid-size h=O(n{sup -1}). Moreover, this requires O(3rn+r{sup 3}) storage, where r denotes the Tucker rank of the electron density with r=O(logn), almost uniformly in n. For example, calculations of the Coulomb matrix and the Hartree-Fock energy for the CH{sub 4} molecule, with a pseudopotential on the C atom, achieved accuracies of the order of 10{sup -6} hartree with a grid-size n of several hundreds. Since the tensor-product convolution in 3D is performed via 1D convolution transforms, our scheme markedly outperforms the 3D-FFT in both the computing time and storage requirements.

  19. Quantitative comparison of 3D third harmonic generation and fluorescence microscopy images.

    PubMed

    Zhang, Zhiqing; Kuzmin, Nikolay V; Groot, Marie Louise; de Munck, Jan C

    2017-05-02

    Third harmonic generation (THG) microscopy is a label-free imaging technique that shows great potential for rapid pathology of brain tissue during brain tumor surgery. However, the interpretation of THG brain images should be quantitatively linked to images of more standard imaging techniques, which so far has been done qualitatively only. We establish here such a quantitative link between THG images of mouse brain tissue and all-nuclei-highlighted fluorescence images, acquired simultaneously from the same tissue area. For quantitative comparison of a substantial pair of images, we present here a segmentation workflow that is applicable for both THG and fluorescence images, with a precision of 91.3 % and 95.8 % achieved respectively. We find that the correspondence between the main features of the two imaging modalities amounts to 88.9 %, providing quantitative evidence of the interpretation of dark holes as brain cells. Moreover, 80 % bright objects in THG images overlap with nuclei highlighted in the fluorescence images, and they are 2 times smaller than the dark holes, showing that cells of different morphologies can be recognized in THG images. We expect that the described quantitative comparison is applicable to other types of brain tissue and with more specific staining experiments for cell type identification. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Spatial light modulator phase mask implementation of wavefront encoded 3D computational-optical microscopy.

    PubMed

    King, Sharon V; Doblas, Ana; Patwary, Nurmohammed; Saavedra, Genaro; Martínez-Corral, Manuel; Preza, Chrysanthe

    2015-10-10

    Spatial light modulator (SLM) implementation of wavefront encoding enables various types of engineered point-spread functions (PSFs), including the generalized-cubic and squared-cubic phase mask wavefront encoded (WFE) PSFs, shown to reduce the impact of sample-induced spherical aberration in fluorescence microscopy. This investigation validates dynamic experimental parameter variation of these WFE-PSFs. We find that particular design parameter bounds exist, within which the divergence of computed and experimental WFE-PSFs is of the same order of magnitude as that of computed and experimental conventional PSFs, such that model-based approaches for solving the inverse imaging problem can be applied to a wide range of SLM-WFE systems. Interferometric measurements were obtained to evaluate the SLM implementation of the numeric mask. Agreement between experiment and theory in terms of a wrapped phase, 0-2π, validates the phase mask implementation and allows characterization of the SLM response. These measurements substantiate experimental practice of computational-optical microscope imaging with an SLM-engineered PSF.

  1. Step-by-step guide to building an inexpensive 3D printed motorized positioning stage for automated high-content screening microscopy.

    PubMed

    Schneidereit, Dominik; Kraus, Larissa; Meier, Jochen C; Friedrich, Oliver; Gilbert, Daniel F

    2017-06-15

    High-content screening microscopy relies on automation infrastructure that is typically proprietary, non-customizable, costly and requires a high level of skill to use and maintain. The increasing availability of rapid prototyping technology makes it possible to quickly engineer alternatives to conventional automation infrastructure that are low-cost and user-friendly. Here, we describe a 3D printed inexpensive open source and scalable motorized positioning stage for automated high-content screening microscopy and provide detailed step-by-step instructions to re-building the device, including a comprehensive parts list, 3D design files in STEP (Standard for the Exchange of Product model data) and STL (Standard Tessellation Language) format, electronic circuits and wiring diagrams as well as software code. System assembly including 3D printing requires approx. 30h. The fully assembled device is light-weight (1.1kg), small (33×20×8cm) and extremely low-cost (approx. EUR 250). We describe positioning characteristics of the stage, including spatial resolution, accuracy and repeatability, compare imaging data generated with our device to data obtained using a commercially available microplate reader, demonstrate its suitability to high-content microscopy in 96-well high-throughput screening format and validate its applicability to automated functional Cl(-)- and Ca(2+)-imaging with recombinant HEK293 cells as a model system. A time-lapse video of the stage during operation and as part of a custom assembled screening robot can be found at https://vimeo.com/158813199. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  2. 3D imaging of optically cleared tissue using a simplified CLARITY method and on-chip microscopy

    PubMed Central

    Zhang, Yibo; Shin, Yoonjung; Sung, Kevin; Yang, Sam; Chen, Harrison; Wang, Hongda; Teng, Da; Rivenson, Yair; Kulkarni, Rajan P.; Ozcan, Aydogan

    2017-01-01

    High-throughput sectioning and optical imaging of tissue samples using traditional immunohistochemical techniques can be costly and inaccessible in resource-limited areas. We demonstrate three-dimensional (3D) imaging and phenotyping in optically transparent tissue using lens-free holographic on-chip microscopy as a low-cost, simple, and high-throughput alternative to conventional approaches. The tissue sample is passively cleared using a simplified CLARITY method and stained using 3,3′-diaminobenzidine to target cells of interest, enabling bright-field optical imaging and 3D sectioning of thick samples. The lens-free computational microscope uses pixel super-resolution and multi-height phase recovery algorithms to digitally refocus throughout the cleared tissue and obtain a 3D stack of complex-valued images of the sample, containing both phase and amplitude information. We optimized the tissue-clearing and imaging system by finding the optimal illumination wavelength, tissue thickness, sample preparation parameters, and the number of heights of the lens-free image acquisition and implemented a sparsity-based denoising algorithm to maximize the imaging volume and minimize the amount of the acquired data while also preserving the contrast-to-noise ratio of the reconstructed images. As a proof of concept, we achieved 3D imaging of neurons in a 200-μm-thick cleared mouse brain tissue over a wide field of view of 20.5 mm2. The lens-free microscope also achieved more than an order-of-magnitude reduction in raw data compared to a conventional scanning optical microscope imaging the same sample volume. Being low cost, simple, high-throughput, and data-efficient, we believe that this CLARITY-enabled computational tissue imaging technique could find numerous applications in biomedical diagnosis and research in low-resource settings. PMID:28819645

  3. Conductive Cellulose Composites with Low Percolation Threshold for 3D Printed Electronics.

    PubMed

    Park, Jae Sung; Kim, Taeil; Kim, Woo Soo

    2017-06-12

    We are reporting a 3D printable composite paste having strong thixotropic rheology. The composite has been designed and investigated with highly conductive silver nanowires. The optimized electrical percolation threshold from both simulation and experiment is shown from 0.7 vol. % of silver nanowires which is significantly lower than other composites using conductive nano-materials. Reliable conductivity of 1.19 × 10(2) S/cm has been achieved from the demonstrated 3D printable composite with 1.9 vol. % loading of silver nanowires. Utilizing the high conductivity of the printable composites, 3D printing of designed battery electrode pastes is demonstrated. Rheology study shows superior printability of the electrode pastes aided by the cellulose's strong thixotropic rheology. The designed anode, electrolyte, and cathode pastes are sequentially printed to form a three-layered lithium battery for the demonstration of a charging profile. This study opens opportunities of 3D printable conductive materials to create printed electronics with the next generation additive manufacturing process.

  4. Magnetic and Electronic Phase Diagram of Sr2VFeAsO3-d

    NASA Astrophysics Data System (ADS)

    Tojo, Yujiro; Shibuya, Taizo; Nakamura, Tetsuro; Shoji, Koichiro; Matoba, Masanori; Yasui, Shintaro; Itoh, Mitsuru; Kitao, Shinji; Seto, Makoto; Kamihara, Yoichi; Research Grants From Keio Univ. Collaboration; Msl Collaborative Research Project Collaboration; Joint Usage in Krri Collaboration

    2014-03-01

    Ae2MFePnO3 (so-called 21113 systems) with perovskite-type layers such as Ae2MO3, where Ae denotes alkaline earth metals, M does Sc, Ti, Cr, V and other transition metal atoms and Pn does As and P shows superconductivity at T <46 K. Sr2VFeAsO3-d is a representative compound in 21113 systems. [Zhu et al, Phys. Rev. B (2009); Ogino et al, Supercond. Sci. Technol. (2009); Ogino et al, Supercond. Sci. Technol. (2009)] Although the oxygen deficiency (d) as a function of Tc is still controversial in Sr2VFeAsO3-d, many samples have been reported as superconductors with Tc = 24-37 K. In this study, a polycrystalline Sr2VFeAsO3-d (d = ~ 0.1- 0.6) were prepared by a solid state reaction using an alumina tube in a sealed silica tube. DC electrical resistivity was measured by a four-probe technique. Magnetization measurements were performed on a superconducting quantum interference device. 57Fe Mossbauer spectra were obtained using conventional equipment. The electronic and magnetic phase diagram of Sr2VFeAsO3-d is elucidated.

  5. 3D elemental mapping with nanometer scale depth resolution via electron optical sectioning

    DOE PAGES

    Pennycook, Timothy J.; Yang, Hao; Jones, Lewys; ...

    2016-12-05

    Electron energy loss spectroscopy in the scanning transmission electron microscope has long been used to perform elemental mapping but has not previously exhibited depth sensitivity. The key to depth resolution with optical sectioning is the transfer of sufficiently high lateral spatial frequencies. By performing spectrum imaging with atomic resolution we achieve in this paper nanometer scale depth resolution, enabling us to optically section an oxide heterostructure spectroscopically. Finally, such 3D elemental mapping is sensitive to atomic scale changes in structure and composition and is more interpretable than Z-contrast imaging alone.

  6. Suppression of electron-hole correlations in 3D polymer materials.

    PubMed

    Puschnig, Peter; Ambrosch-Draxl, Claudia

    2002-07-29

    We present an ab initio study of the optical absorption spectra of isolated as well as crystalline trans-polyacetylene. We include excitonic effects by solving the Bethe-Salpeter equation for the electron-hole two-particle correlation function. We observe that the strength of the electron-hole interactions drastically reduces when going from an isolated polymer chain to a crystalline arrangement. This is not only a result of enhanced screening in the 3D material, but also of the increased spatial extent of the exciton perpendicular to the polymer chains. We point out that these findings apply to crystalline phases of conjugated polymers and molecular crystals in general.

  7. Techniques in electron microscopy of animal tissue.

    PubMed

    Cheville, N F; Stasko, J

    2014-01-01

    Technical improvements in electron microscopy, both instrumental and preparative, permit increasingly accurate analyses. Digital images for transmission electron microscopy (TEM) can be processed by software programs that automate tasks and create custom tools that allow for image enhancement for brightness, contrast and coloration; for creation of rectangular, ellipsoidal or irregular area selections; and for measurement of mean area and standard deviation. Sample preparation remains a source of error since organelles and spatial arrangements of macromolecules rapidly change after anoxia. Guidelines for maintaining consistency in preparation, examination and interpretation are presented for different electron microscopy (EM) modalities.

  8. Probing the Proton with Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Friedman, Jerome I.

    2014-01-01

    This article is written as a tribute and memorial to Dr. Akira Tonomura who was an outstanding experimental physicist and a friend. Early in his career, he opened a new era in electron microscopy by demonstrating in 1968 that electron holography, proposed by Gabor in 1949, was possible; and later he developed Lorentz "phase" microscopy, which allows one to generate real-space, real-time images. All through his career, he perfected these designs into superb instruments that he employed to investigate fundamental questions in physics. Dr. Tonomura set world standards for electron microscopy.

  9. 3D image reconstruction using optical sectioning in confocal scanning microscopy

    NASA Astrophysics Data System (ADS)

    Seo, Jungwoo; Kang, Dong Kyun; Park, Sunglim; Gweon, Dae gab

    2001-10-01

    Confocal scanning microscopy (CSM) has been used in biological application, materials science, semiconductor quality measurement and other non-destructive microscopic application. Small spot of light illuminates a sample, and a small detector that is ideally a point detector collects the reflected or transmitted light having the information of specimen. An image distribution can be reconstructed by a correlation analysis of spots with the high bandwidth. The mechanism for two-dimensional beam scanning and optical sectioning has an important role in CSM as the three-dimensional profiler. The parasitic motion of focus on the detector gives rise to the fatal distortion of an image profile named the extinction effect while using acousto-optical (AO) deflector. The intensity profile for the open loop scanning should be matched with its response for the standard. The non-linearity can be minimized with the optical sectioning or the optical probe of the closed loop control. This paper shows the mathematical expression of the light such as the extinction curve in the optical fields of system using AO deflector, the axial/lateral response experimentally when the error sources change, and the methods of optical sectioning. We propose the progressive methods for the high quality image as the following. At first, for having the corrected image, small spot and long scan range, this paper shows that the optimal design having the multi-objects can be used by choosing the unitary lens device in CSM. At second, in order to compensate for the intensity cancellation at the end profile that may be the cause of waviness for the optical image, this paper shows that it is efficient to schedule the frequency of scan. According to characteristics of the extinction curve and axial/lateral response having the error property, we can define the frequency and sensitivity of as their robustness. Finally, the axial response gives an important motive for the optical section, and the limit of

  10. Front-end receiver electronics for a matrix transducer for 3-D transesophageal echocardiography.

    PubMed

    Yu, Zili; Blaak, Sandra; Chang, Zu-yao; Yao, Jiajian; Bosch, Johan G; Prins, Christian; Lancée, Charles T; de Jong, Nico; Pertijs, Michiel A P; Meijer, Gerard C M

    2012-07-01

    There is a clear clinical need for creating 3-D images of the heart. One promising technique is the use of transesophageal echocardiography (TEE). To enable 3-D TEE, we are developing a miniature ultrasound probe containing a matrix piezoelectric transducer with more than 2000 elements. Because a gastroscopic tube cannot accommodate the cables needed to connect all transducer elements directly to an imaging system, a major challenge is to locally reduce the number of channels, while maintaining a sufficient signal-to-noise ratio. This can be achieved by using front-end receiver electronics bonded to the transducers to provide appropriate signal conditioning in the tip of the probe. This paper presents the design of such electronics, realizing time-gain compensation (TGC) and micro-beamforming using simple, low-power circuits. Prototypes of TGC amplifiers and micro-beamforming cells have been fabricated in 0.35-μm CMOS technology. These prototype chips have been combined on a printed circuit board (PCB) to form an ultrasound-receiver system capable of reading and combining the signals of three transducer elements. Experimental results show that this design is a suitable candidate for 3-D TEE.

  11. A 3D technique for simulation of irregular electron treatment fields using a digital camera

    SciTech Connect

    Bassalow, Roustem; Sidhu, Narinder P

    2003-09-30

    Cerrobend inserts, which define electron field apertures, are manufactured at our institution using perspex templates. Contours are reproduced manually on these templates at the simulator from the field outlines drawn on the skin or mask of a patient. A previously reported technique for simulation of electron treatment fields uses a digital camera to eliminate the need for such templates. However, avoidance of the image distortions introduced by non-flat surfaces on which the electron field outlines were drawn could only be achieved by limiting the application of this technique to surfaces which were flat or near flat. We present a technique that employs a digital camera and allows simulation of electron treatment fields contoured on an anatomical surface of an arbitrary three-dimensional (3D) shape, such as that of the neck, extremities, face, or breast. The procedure is fast, accurate, and easy to perform.

  12. Effect of Single-Electron Interface Trapping in Decanano MOSFETs: A 3D Atomistic Simulation Study

    NASA Technical Reports Server (NTRS)

    Asenov, Asen; Balasubramaniam, R.; Brown, A. R.; Davies, J. H.

    2000-01-01

    We study the effect of trapping/detrapping of a single-electron in interface states in the channel of n-type MOSFETs with decanano dimensions using 3D atomistic simulation techniques. In order to highlight the basic dependencies, the simulations are carried out initially assuming continuous doping charge, and discrete localized charge only for the trapped electron. The dependence of the random telegraph signal (RTS) amplitudes on the device dimensions and on the position of the trapped charge in the channel are studied in detail. Later, in full-scale, atomistic simulations assuming discrete charge for both randomly placed dopants and the trapped electron, we highlight the importance of current percolation and of traps with strategic position where the trapped electron blocks a dominant current path.

  13. Strain measurement of a mouse bone by 3D-electronic speckle pattern interferometry (3D-ESPI)

    NASA Astrophysics Data System (ADS)

    Samala, Praveen R.; Su, Min; Liu, Sheng; Jiang, Hui H.; Yokota, Hiroki; Yang, Lianxiang

    2005-08-01

    Bone is a mechanosensitive tissue that adapts its mass, architecture and mechanical properties to mechanical loading. Appropriate mechanical loads provide an effective means to stimulate bone remodeling and prevent from bone loss. It is controversial whether in situ strain in bone is a critical determinant in enhancement of bone formation, and it is therefore important to evaluate load-driven strain in bone. Using electronic speckle pattern interferometry, we determined high-resolution three-dimensional strains on the mouse femur in response to two loading modalities: an axial loading modality (ALM) and a knee loading modality (KLM). We demonstrated that these two loading modalities induced a different pattern of strain distributions. ALM generated strain in the midshaft of cortical bone, while strains with KLM were concentrated on the distal epiphysis of the mouse femur. Since KLM is capable of enhancing bone formation in cortical bone distant from the knee, the current results indicate that in situ strain is not always necessary for load-driven bone formation.

  14. Synthesizing a 3D auditory scene for use in an electronic travel aid for the blind

    NASA Astrophysics Data System (ADS)

    Bujacz, Michał; Strumiłło, Paweł

    2008-01-01

    A system for auditory presentation of 3D scenes to the blind is presented, with the focus of the paper on the synthesis of sound codes suitable to carry important scene information. First, a short review of existing electronic travel aids for the blind (ETAs) is provided. Second, the project of the wearable ETA device, currently under development at the Technical University of Lodz, is outlined, along with the system modules: 3D scene reconstruction, object (obstacle) selection, synthesis of the sound code and the application of head related transfer functions (HRTFs) for generating spatialized sound. The importance of psychoacoustics, especially Bregman's theory of sound streams, is analyzed and proposed methods of sound code synthesis are presented, along with the software used for their verification.

  15. Correlative super-resolution fluorescence and metal replica transmission electron microscopy

    PubMed Central

    Sochacki, Kem A.; Shtengel, Gleb; van Engelenburg, Schuyler B.; Hess, Harald F.; Taraska, Justin W.

    2014-01-01

    Super-resolution localization microscopy is combined with a complementary imaging technique, transmission electron microscopy of metal replicas, to locate proteins on the landscape of the cellular plasma membrane at the nanoscale. Robust correlation on the scale of 20 nm is validated by imaging endogenous clathrin (with 2D and 3D PALM/TEM) and the method is further used to find the previously unknown 3D position of epsin on clathrin coated structures. PMID:24464288

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

    PubMed

    Sims, Paul A; Hardin, Jeff D

    2007-01-01

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

  17. Computing the 3-D structure of viruses from unoriented cryo electron microscope images: a fast algorithm for a statistical approach.

    PubMed

    Lee, Junghoon; Zheng, Yili; Doerschuk, Peter C

    2006-01-01

    In a cryo electron microscopy experiment, the data is noisy 2-D projection images of the 3-D electron scattering intensity where the orientation of the projections is not known. In previous work we have developed a solution for this problem based on a maximum likelihood estimator that is computed by an expectation maximization algorithm. In the expectation maximization algorithm the expensive step is the expectation which requires numerical evaluation of 3- or 5-dimensional integrations of a square matrix of dimension equal to the number of Fourier series coefficients used to describe the 3-D reconstruction. By taking advantage of the rotational properties of spherical harmonics, we can reduce the integrations of a matrix to integrations of a scalar. The key property is that a rotated spherical harmonic can be expressed as a linear combination of the other harmonics of the same order and the weights in the linear combination factor so that each of the three factors is a function of only one of the Euler angles describing the orientation of the projection. Numerical example of the reconstructions is provided based on Nudaurelia Omega Capensis virus.

  18. Transmission electron microscopy: Imaging of materials

    SciTech Connect

    Thomas, G.

    1988-10-01

    This report was an invited paper for a symposium and only covers general aspects of transmission electron microscopy. A history, and examples of work done on ceramics and alloys are covered. 6 refs., 44 figs. (JL)

  19. Characterization of mesoporosity in ceria particles using electron microscopy.

    PubMed

    Shih, Shao-Ju; Herrero, Pilar Rodrigo; Li, Guoqiang; Chen, Chin-Yi; Lozano-Perez, Sergio

    2011-02-01

    The geometry and three-dimensional (3D) morphology of the ceria particles synthesized by spray pyrolysis (SP) from two different precursors--cerium acetate hydrate and cerium nitrate hydrate (CeA and CeN ceria particles)--were characterized by transmission electron microscopy and electron tomography. Results were compared with surface area measurements, confirming that the surface area of CeA ceria particles is twice as large as that of CeN ceria particles. This result was supported by 3D microstructural observations, which have revealed that CeA ceria particles contain open pores (connected to surfaces) and closed pores (embedded in particles), while CeN ceria particles only contained closed pores. This experimental result suggests that the type of porosity is controlled by the precursors and could be related to their melting temperature during the heating process in SP.

  20. A nanofiber based artificial electronic skin with high pressure sensitivity and 3D conformability.

    PubMed

    Zhong, Weibin; Liu, Qiongzhen; Wu, Yongzhi; Wang, Yuedan; Qing, Xing; Li, Mufang; Liu, Ke; Wang, Wenwen; Wang, Dong

    2016-06-16

    Pressure sensors with 3D conformability are highly desirable components for artificial electronic skin or e-textiles that can mimic natural skin, especially for application in real-time monitoring of human physiological signals. Here, a nanofiber based electronic skin with ultra-high pressure sensitivity and 3D conformability is designed and built by interlocking two elastic patterned nanofibrous membranes. The patterned membrane is facilely prepared by casting conductive nanofiber ink into a silicon mould to form an array of semi-spheroid-like protuberances. The protuberances composed of intertwined elastic POE nanofibers and PPy@PVA-co-PE nanofibers afford a tunable effective elastic modulus that is capable of capturing varied strains and stresses, thereby contributing to a high sensitivity for pressure sensing. This electronic skin-like sensor demonstrates an ultra-high sensitivity (1.24 kPa(-1)) below 150 Pa with a detection limit as low as about 1.3 Pa. The pixelated sensor array and a RGB-LED light are then assembled into a circuit and show a feasibility for visual detection of spatial pressure. Furthermore, a nanofiber based proof-of-concept wireless pressure sensor with a bluetooth module as a signal transmitter is proposed and has demonstrated great promise for wireless monitoring of human physiological signals, indicating a potential for large scale wearable electronic devices or e-skin.

  1. Advanced electron microscopy characterization of multimetallic nanoparticles

    NASA Astrophysics Data System (ADS)

    Khanal, Subarna Raj

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

  2. Three-Dimensional Electron Microscopy Simulation with the CASINO Monte Carlo Software

    PubMed Central

    Demers, Hendrix; Poirier-Demers, Nicolas; Couture, Alexandre Réal; Joly, Dany; Guilmain, Marc; de Jonge, Niels; Drouin, Dominique

    2011-01-01

    Monte Carlo softwares are widely used to understand the capabilities of electron microscopes. To study more realistic applications with complex samples, 3D Monte Carlo softwares are needed. In this paper, the development of the 3D version of CASINO is presented. The software feature a graphical user interface, an efficient (in relation to simulation time and memory use) 3D simulation model, accurate physic models for electron microscopy applications, and it is available freely to the scientific community at this website: www.gel.usherbrooke.ca/casino/index.html. It can be used to model backscattered, secondary, and transmitted electron signals as well as absorbed energy. The software features like scan points and shot noise allow the simulation and study of realistic experimental conditions. This software has an improved energy range for scanning electron microscopy and scanning transmission electron microscopy applications. PMID:21769885

  3. New highlights on phytolith structure and occluded carbon location: 3-D X-ray microscopy and NanoSIMS results

    NASA Astrophysics Data System (ADS)

    Alexandre, A.; Basile-Doelsch, I.; Delhaye, T.; Borshneck, D.; Mazur, J. C.; Reyerson, P.; Santos, G. M.

    2014-10-01

    Phytoliths contain occluded organic compounds called phytC. Recently, phytC content, nature, origin, paleoenvironmental meaning and impact in the global C cycle has been the subject of increasing debate. Inconsistencies were fed by the scarcity of in-situ characterization of phytC in phytoliths. Here we reconstructed at high spatial resolution the 3-dimensional (3-D) structure of harvested grass short cell (GSC) phytoliths using 3-D X-ray microscopy. While this technic has been widely used for 3-D reconstruction of biological systems it has never been applied in high resolution mode to silica particles. Simultaneously, we investigated the location of phytC using Nano-scale Secondary Ion Mass Spectrometry (NanoSIMS). Our data evidenced that the silica structure contains micrometric internal cavities. These internal cavities were sometimes observed isolated from the outside. Their opening may be an original feature or may result from a beginning of dissolution of silica during the chemical extraction procedure, mimicking the progressive dissolution process that can happen in natural environments. The phytC that may originally occupy the cavities is thus susceptible to rapid oxidation. It was not detected by the nanoSIMS technique. To the contrary another pool of phytC, continuously distributed in and protected by the silica structure was evidenced. Its N/C ratio (0.27) is in agreement with the presence of amino acids. These findings allowed to discuss discrepancies in phytC quantification, evaluate phytC accessibility to oxidation, and reassess the paleo-environmental meaning of opaque features observed in phytoliths by natural light (NL) microcopy. They also should help to reappraise the significance of phytC in the global C cycle.

  4. New highlights of phytolith structure and occluded carbon location: 3-D X-ray microscopy and NanoSIMS results

    NASA Astrophysics Data System (ADS)

    Alexandre, A.; Basile-Doelsch, I.; Delhaye, T.; Borshneck, D.; Mazur, J. C.; Reyerson, P.; Santos, G. M.

    2015-02-01

    Phytoliths contain occluded organic compounds called phytC. Recently, phytC content, nature, origin, paleoenvironmental meaning and impact in the global C cycle have been the subject of increasing debate. Inconsistencies were fed by the scarcity of in situ characterizations of phytC in phytoliths. Here we reconstructed at high spatial resolution the 3-D structure of harvested grass short cell (GSC) phytoliths using 3-D X-ray microscopy. While this technique has been widely used for 3-D reconstruction of biological systems it has never been applied in high-resolution mode to silica particles. Simultaneously, we investigated the location of phytC using nanoscale secondary ion mass spectrometry (NanoSIMS). Our data evidenced that the silica structure contains micrometric internal cavities. These internal cavities were sometimes observed isolated from the outside. Their opening may be an original feature or may result from a beginning of dissolution of silica during the chemical extraction procedure, mimicking the progressive dissolution process that can happen in natural environments. The phytC that may originally occupy the cavities is thus susceptible to rapid oxidation. It was not detected by the NanoSIMS technique. However, another pool of phytC, continuously distributed in and protected by the silica structure, was observed. Its N/C ratio (0.27) is in agreement with the presence of amino acids. These findings constitute a basis to further characterize the origin, occlusion process, nature and accessibility of phytC, as a prerequisite for assessing its significance in the global C cycle.

  5. Statistical characterization of ensembles of symmetric virus particles: 3-D stochastic signal reconstruction from electron microscope images.

    PubMed

    Nan Xu; Doerschuk, Peter C

    2016-08-01

    Stochastic models of nano-biomachines have been studied by 3-D reconstruction from cryo electron microscopy images in recent years. The image data is the projection of many heterogeneous instances of the object under study (e.g., a virus). Initial reconstruction algorithms require different instances of the object, while still heterogeneous, to have the same symmetry. This paper presents a maximum likelihood reconstruction approach which allows each object to lack symmetry while constraining the statistics of the ensemble of objects to have symmetry. This algorithm is demonstrated on bacteriophage HK97 and is contrasted with the former algorithm. Reconstruction results show that the proposed algorithm provides estimates that make more biological sense.

  6. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution.

    PubMed

    Meddens, Marjolein B M; Liu, Sheng; Finnegan, Patrick S; Edwards, Thayne L; James, Conrad D; Lidke, Keith A

    2016-06-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

  7. Direct Determination of 3D Distribution of Elemental Composition in Single Semiconductor Nanoislands by Scanning Auger Microscopy

    NASA Astrophysics Data System (ADS)

    Ponomaryov, Semyon S.; Yukhymchuk, Volodymyr O.; Lytvyn, Peter M.; Valakh, Mykhailo Ya

    2016-02-01

    An application of scanning Auger microscopy with ion etching technique and effective compensation of thermal drift of the surface analyzed area is proposed for direct local study of composition distribution in the bulk of single nanoislands. For GexSi1 - x-nanoislands obtained by MBE of Ge on Si-substrate gigantic interdiffusion mixing takes place both in the open and capped nanostructures. Lateral distributions of the elemental composition as well as concentration-depth profiles were recorded. 3D distribution of the elemental composition in the d-cluster bulk was obtained using the interpolation approach by lateral composition distributions in its several cross sections and concentration-depth profile. It was shown that there is a germanium core in the nanoislands of both nanostructure types, which even penetrates the substrate. In studied nanostructures maximal Ge content in the nanoislands may reach about 40 at.%.

  8. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    SciTech Connect

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

    2016-01-01

    Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.

  9. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    DOE PAGES

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; ...

    2016-01-01

    Here, we have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single moleculemore » super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet.« less

  10. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    PubMed Central

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

    2016-01-01

    We have developed a method for performing light-sheet microscopy with a single high numerical aperture lens by integrating reflective side walls into a microfluidic chip. These 45° side walls generate light-sheet illumination by reflecting a vertical light-sheet into the focal plane of the objective. Light-sheet illumination of cells loaded in the channels increases image quality in diffraction limited imaging via reduction of out-of-focus background light. Single molecule super-resolution is also improved by the decreased background resulting in better localization precision and decreased photo-bleaching, leading to more accepted localizations overall and higher quality images. Moreover, 2D and 3D single molecule super-resolution data can be acquired faster by taking advantage of the increased illumination intensities as compared to wide field, in the focused light-sheet. PMID:27375939

  11. High throughput 3D super-resolution microscopy reveals Caulobacter crescentus in vivo Z-ring organization

    PubMed Central

    Holden, Seamus J.; Pengo, Thomas; Meibom, Karin L.; Fernandez Fernandez, Carmen; Collier, Justine; Manley, Suliana

    2014-01-01

    We created a high-throughput modality of photoactivated localization microscopy (PALM) that enables automated 3D PALM imaging of hundreds of synchronized bacteria during all stages of the cell cycle. We used high-throughput PALM to investigate the nanoscale organization of the bacterial cell division protein FtsZ in live Caulobacter crescentus. We observed that FtsZ predominantly localizes as a patchy midcell band, and only rarely as a continuous ring, supporting a model of “Z-ring” organization whereby FtsZ protofilaments are randomly distributed within the band and interact only weakly. We found evidence for a previously unidentified period of rapid ring contraction in the final stages of the cell cycle. We also found that DNA damage resulted in production of high-density continuous Z-rings, which may obstruct cytokinesis. Our results provide a detailed quantitative picture of in vivo Z-ring organization. PMID:24616530

  12. Self-Consistent 3D Modeling of Electron Cloud Dynamics and Beam Response

    SciTech Connect

    Furman, Miguel; Furman, M.A.; Celata, C.M.; Kireeff-Covo, M.; Sonnad, K.G.; Vay, J.-L.; Venturini, M.; Cohen, R.; Friedman, A.; Grote, D.; Molvik, A.; Stoltz, P.

    2007-04-02

    We present recent advances in the modeling of beam electron-cloud dynamics, including surface effects such as secondary electron emission, gas desorption, etc, and volumetric effects such as ionization of residual gas and charge-exchange reactions. Simulations for the HCX facility with the code WARP/POSINST will be described and their validity demonstrated by benchmarks against measurements. The code models a wide range of physical processes and uses a number of novel techniques, including a large-timestep electron mover that smoothly interpolates between direct orbit calculation and guiding-center drift equations, and a new computational technique, based on a Lorentz transformation to a moving frame, that allows the cost of a fully 3D simulation to be reduced to that of a quasi-static approximation.

  13. 3-D readout-electronics packaging for high-bandwidth massively paralleled imager

    DOEpatents

    Kwiatkowski, Kris; Lyke, James

    2007-12-18

    Dense, massively parallel signal processing electronics are co-packaged behind associated sensor pixels. Microchips containing a linear or bilinear arrangement of photo-sensors, together with associated complex electronics, are integrated into a simple 3-D structure (a "mirror cube"). An array of photo-sensitive cells are disposed on a stacked CMOS chip's surface at a 45.degree. angle from light reflecting mirror surfaces formed on a neighboring CMOS chip surface. Image processing electronics are held within the stacked CMOS chip layers. Electrical connections couple each of said stacked CMOS chip layers and a distribution grid, the connections for distributing power and signals to components associated with each stacked CSMO chip layer.

  14. In vivo 3D measurement of moxifloxacin and gatifloxacin distributions in the mouse cornea using multiphoton microscopy

    NASA Astrophysics Data System (ADS)

    Lee, Seunghun; Lee, Jun Ho; Park, Jin Hyoung; Yoon, Yeoreum; Chung, Wan Kyun; Tchah, Hungwon; Kim, Myoung Joon; Kim, Ki Hean

    2016-05-01

    Moxifloxacin and gatifloxacin are fourth-generation fluoroquinolone antibiotics used in the clinic to prevent or treat ocular infections. Their pharmacokinetics in the cornea is usually measured from extracted ocular fluids or tissues, and in vivo direct measurement is difficult. In this study multiphoton microscopy (MPM), which is a 3D optical microscopic technique based on multiphoton fluorescence, was applied to the measurement of moxifloxacin and gatifloxacin distribution in the cornea. Intrinsic multiphoton fluorescence properties of moxifloxacin and gatifloxacin were characterized, and their distributions in mouse cornea in vivo were measured by 3D MPM imaging. Both moxifloxacin and gatifloxacin had similar multiphoton spectra, while moxifloxacin had stronger fluorescence than gatifloxacin. MPM imaging of mouse cornea in vivo showed (1) moxifloxacin had good penetration through the superficial corneal epithelium, while gatifloxacin had relatively poor penetration, (2) both ophthalmic solutions had high intracellular distribution. In vivo MPM results were consistent with previous studies. This study demonstrates the feasibility of MPM as a method for in vivo direct measurement of moxifloxacin and gatifloxacin in the cornea.

  15. Holographic microscopy and microfluidics platform for measuring wall stress and 3D flow over surfaces textured by micro-pillars.

    PubMed

    Bocanegra Evans, Humberto; Gorumlu, Serdar; Aksak, Burak; Castillo, Luciano; Sheng, Jian

    2016-06-29

    Understanding how fluid flow interacts with micro-textured surfaces is crucial for a broad range of key biological processes and engineering applications including particle dispersion, pathogenic infections, and drag manipulation by surface topology. We use high-speed digital holographic microscopy (DHM) in combination with a correlation based de-noising algorithm to overcome the optical interference generated by surface roughness and to capture a large number of 3D particle trajectories in a microfluidic channel with one surface patterned with micropillars. It allows us to obtain a 3D ensembled velocity field with an uncertainty of 0.06% and 2D wall shear stress distribution at the resolution of ~65 μPa. Contrary to laminar flow in most microfluidics, we find that the flow is three-dimensional and complex for the textured microchannel. While the micropillars affect the velocity flow field locally, their presence is felt globally in terms of wall shear stresses at the channel walls. These findings imply that micro-scale mixing and wall stress sensing/manipulation can be achieved through hydro-dynamically smooth but topologically rough micropillars.

  16. Holographic microscopy and microfluidics platform for measuring wall stress and 3D flow over surfaces textured by micro-pillars

    PubMed Central

    Bocanegra Evans, Humberto; Gorumlu, Serdar; Aksak, Burak; Castillo, Luciano; Sheng, Jian

    2016-01-01

    Understanding how fluid flow interacts with micro-textured surfaces is crucial for a broad range of key biological processes and engineering applications including particle dispersion, pathogenic infections, and drag manipulation by surface topology. We use high-speed digital holographic microscopy (DHM) in combination with a correlation based de-noising algorithm to overcome the optical interference generated by surface roughness and to capture a large number of 3D particle trajectories in a microfluidic channel with one surface patterned with micropillars. It allows us to obtain a 3D ensembled velocity field with an uncertainty of 0.06% and 2D wall shear stress distribution at the resolution of ~65 μPa. Contrary to laminar flow in most microfluidics, we find that the flow is three-dimensional and complex for the textured microchannel. While the micropillars affect the velocity flow field locally, their presence is felt globally in terms of wall shear stresses at the channel walls. These findings imply that micro-scale mixing and wall stress sensing/manipulation can be achieved through hydro-dynamically smooth but topologically rough micropillars. PMID:27353632

  17. Full 3D opto-electronic simulation tool for nanotextured solar cells (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Michallon, Jérôme; Collin, Stéphane

    2017-04-01

    Increasing efforts on the photovoltaics research have recently been devoted to material savings, leading to the emergence of new designs based on nanotextured and nanowire-based solar cells. The use of small absorber volumes, light-trapping nanostructures and unconventional carrier collection schemes (radial nanowire junctions, point contacts in planar structures,…) increases the impact of surfaces recombination and induces homogeneity in the photogenerated carrier concentrations. The investigation of their impacts on the device performances need to be addressed using full 3D coupled opto-electrical modeling. In this context, we have developed a new tool for full 3D opto-electrical simulation using the most advanced optical and electrical simulation techniques. We will present an overview of its simulation capabilities and the key issues that have been solved to make it fully operational and reliable. We will provide various examples of opto-electronic simulation of (i) nanostructured solar cells with localized contacts and (ii) nanowire solar cells. We will also show how opto-electronic simulation can be used to simulate light- and electron-beam induced current (LBIC/EBIC) experiments, targeting quantitative analysis of the passivation properties of surfaces.

  18. Low voltage transmission electron microscopy of graphene.

    PubMed

    Bachmatiuk, Alicja; Zhao, Jiong; Gorantla, Sandeep Madhukar; Martinez, Ignacio Guillermo Gonzalez; Wiedermann, Jerzy; Lee, Changgu; Eckert, Juergen; Rummeli, Mark Hermann

    2015-02-04

    The initial isolation of graphene in 2004 spawned massive interest in this two-dimensional pure sp(2) carbon structure due to its incredible electrical, optical, mechanical, and thermal effects. This in turn led to the rapid development of various characterization tools for graphene. Examples include Raman spectroscopy and scanning tunneling microscopy. However, the one tool with the greatest prowess for characterizing and studying graphene is the transmission electron microscope. State-of-the-art (scanning) transmission electron microscopes enable one to image graphene with atomic resolution, and also to conduct various other characterizations simultaneously. The advent of aberration correctors was timely in that it allowed transmission electron microscopes to operate with reduced acceleration voltages, so that damage to graphene is avoided while still providing atomic resolution. In this comprehensive review, a brief introduction is provided to the technical aspects of transmission electron microscopes relevant to graphene. The reader is then introduced to different specimen preparation techniques for graphene. The different characterization approaches in both transmission electron microscopy and scanning transmission electron microscopy are then discussed, along with the different aspects of electron diffraction and electron energy loss spectroscopy. The use of graphene for other electron microscopy approaches such as in-situ investigations is also presented.

  19. 3D image analysis of plants using electron tomography and micro-CT.

    PubMed

    Mineyuki, Yoshinobu

    2014-11-01

    help to promote MT bundling. Cell plate attachment to the parental wall leads to the fusion of the newly formed middle lamellae in the cell plate to the middle lamella of parental cell wall, and a three-way junction is created. Air space develops from the three-way junction. To determine 3D arrangement of cells and air spaces, we used X-ray micro-CT at the SPring-8 synchrotron radiation facility. Using micro-CT available in BL20XU (8 keV, 0.2 µm/pixel), we were able to elucidate ∼90% of the cortical cell outlines in the hypocotyl-radicle axis of arabidopsis seeds [4] and to analyze cell geometrical properties. As the strength of the system X-ray is too strong for seed survival, we used another beam line BL20B2 (10-15 keV, 2.4-2.7 µm/pixel) to examine air space development during seed imbibition [4,5]. Using this system, we were able to detect air space development at the early imbibition stages of seeds without causing damage during seed germination. AcknowledgmentThe author would like to thank Dr. Ichirou Karahara (Univ. Toyama), Dr. L. Andrew Staehelin (Univ. Colorado), Ms. Naoko Kajimura, Dr. Akio Takaoka (Osaka Univ.), Dr. Kazuyo Misaki, Dr. Shigenobu Yonemura (RIKEN CDB), Dr. Kazuyoshi Murata (NIP), Dr. Kentaro Uesugi, Dr. Akihisa Takeuchi, Dr. Yoshio Suzuki (JASRI), Dr. Miyuki Takeuchi, Dr. Daisuke Tamaoki, Dr. Daisuke Yamauchi, and Ms. Aki Fukuda (Univ. Hyogo) for their collaborations in the work presented here. © 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.

  20. 3D Distribution of the Coronal Electron Density and its Evolution with Solar Cycle

    NASA Astrophysics Data System (ADS)

    Wang, Tongjiang; Reginald, Nelson Leslie; Davila, Joseph M.; St. Cyr, Orville Chris

    2016-05-01

    The variability of the solar white-light corona and its connection to the solar activity has been studied for more than a half century. It is widely accepted that the temporal variation of the total radiance of the K-corona follows the solar cycle pattern (e.g., correlated with sunspot number). However, the origin of this variation and its relationships with regard to coronal mass ejections and the solar wind are yet to be clearly understood. We know that the COR1-A and -B instruments onboard the STEREO spacecraft have continued to perform high-cadence (5 min) polarized brightness measurements from two different vantage points over a long period of time that encompasses the solar minimum of Solar Cycle 23 to the solar maximum of Solar Cycle 24. This extended period of polarized brightness measurements can now be used to reconstruct 3D electron density distributions of the corona between the heliocentric heights of 1.5-4.0 solar radii. In this study we have constructed the 3D coronal density models for 100 Carrington rotations (CRs) from 2007 to 2014 using the spherically symmetric inversion (SSI) method. The validity of these 3D density models is verified by comparing with similar 3D density models created by other means such as tomography, MHD modeling, and 2D density distributions inverted from the polarized brightness images from LASCO/C2 instrument onboard the SOHO spacecraft. When examining the causes for the temporal variation of the global electron content we find that its increase from the solar minimum to maximum depends on changes to both the total area and mean density of coronal streamers. We also find that the global and hemispheric electron contents show quasi-periodic variations with a period of 8-9 CRs during the ascending and maximum phases of Solar Cycle 24 through wavelet analysis. In addition, we also explore any obvious relationships between temporal variation of the global electron content with the photospheric magnetic flux, total mass of

  1. 3-D analysis of bacterial cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches.

    PubMed

    Schmid, G; Zeitvogel, F; Hao, L; Ingino, P; Floetenmeyer, M; Stierhof, Y-D; Schroeppel, B; Burkhardt, C J; Kappler, A; Obst, M

    2014-07-01

    The formation of cell-(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2-D microscopy techniques, the 3-D and internal structure remain obscure. In this study, we examined the 3-D structure of cell-(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate-reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3-D microscopy techniques. We obtained 3-D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4-200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike- or platelet-shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell-(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell-(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3-D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X-ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a

  2. Transmission electron microscopy of composites

    NASA Technical Reports Server (NTRS)

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

    1988-01-01

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

  3. Multiphotonic Confocal Microscopy 3D imaging: Application to mantle sulfides in sub-arc environment (Avacha Volcano, Kamchatka)

    NASA Astrophysics Data System (ADS)

    Antoine, Bénard; Luc-Serge, Doucet; Sabine, Palle; Dmitri A., Ionov

    2010-05-01

    Petrogenetic relations in igneous rocks are usually studied in natural samples using classical optical microscopy and subsequent geochemical data acquisition. Multiphotonic Laser Scanning Confocal Microscopy (MLSCM) can be a powerful tool to section geological materials optically with sub-micrometric resolution and then generate a three-dimensional (3D) reconstruction (ca. 106 μm3 stack). MLSCM is used here to investigate textural relations of Monosulfide Solid Solution (MSS) with silicate phases in fresh spinel harzburgite xenoliths from the andesitic Avacha volcano (Kamchatka, Russia). The xenoliths contain MSS disseminated in olivine and orthopyroxene (opx) neoblasts as well as MSS-rich quenched magmatic opx veins [1]. First, Reflection Mode (RM) was tested on vein sulfides in resin-impregnated thick (120 μm) polished rock sections. Then we used a combination of Differential Interference Contrast (DIC) with a transmitted light detector, two photons-excited fluorescence (2PEF) and Second Harmonic Generation (SHG). Sequential imaging feature of the Leica TCS-SP2 software was applied. The excitation laser used for 2PEF was a COHERENT MIRA 900 with a 76Hz repetition rate and 800nm wavelength. Image stacks were analysed using ImageJ software [2]. The aim of the tests was to try to discriminate sulfides in silicate matrix as a tool for a better assessment of equilibrium conditions between the two phases. Preliminary results show that Fe-Ni rich MSS from vein and host rock have a strong auto-fluorescence in the Near UV-VIS domain (392-715 nm) whereas silicate matrix is only revealed through DIC. SHG is obtained only from dense nanocentrosymmetrical structures such as embedded medium (organic matter like glue and resin). The three images were recorded sequentially enabling efficient discrimination between the different components of the rock slices. RM permits reconstruction of the complete 3D structure of the rock slice. High resolution (ca. 0.2 μm along X-Y axis vs

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

    NASA Astrophysics Data System (ADS)

    Yankovich, Andrew B.

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

  5. Electron Microscopy of Natural and Epitaxial Diamond

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  6. Electron Microscopy of Natural and Epitaxial Diamond

    NASA Technical Reports Server (NTRS)

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

    1993-01-01

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

  7. Analysis of cytokinesis by electron microscopy.

    PubMed

    König, J; Borrego-Pinto, J; Streichert, D; Munzig, M; Lenart, P; Müller-Reichert, T

    2017-01-01

    Following up on a chapter on the Correlative Light and Electron Microscopy of Early Caenorhabditis elegans Embryos in Mitosis (MCB 79, 101-119), we present an adaptation of our established protocol for the ultrastructural analysis of either permeabilized or injected embryonic systems. We prepared both drug-treated early C. elegans embryos and fluorescently labeled sea urchin embryos of Lytechinus pictus for ultrastructural studies on animal cytokinesis. Here we focus on the initial preparation steps of postmitotic embryos for high-pressure freezing and subsequent electron microscopy with an emphasis on electron tomography. The advantages and limitations of our extended protocol will be discussed. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. A nanofiber based artificial electronic skin with high pressure sensitivity and 3D conformability

    NASA Astrophysics Data System (ADS)

    Zhong, Weibin; Liu, Qiongzhen; Wu, Yongzhi; Wang, Yuedan; Qing, Xing; Li, Mufang; Liu, Ke; Wang, Wenwen; Wang, Dong

    2016-06-01

    Pressure sensors with 3D conformability are highly desirable components for artificial electronic skin or e-textiles that can mimic natural skin, especially for application in real-time monitoring of human physiological signals. Here, a nanofiber based electronic skin with ultra-high pressure sensitivity and 3D conformability is designed and built by interlocking two elastic patterned nanofibrous membranes. The patterned membrane is facilely prepared by casting conductive nanofiber ink into a silicon mould to form an array of semi-spheroid-like protuberances. The protuberances composed of intertwined elastic POE nanofibers and PPy@PVA-co-PE nanofibers afford a tunable effective elastic modulus that is capable of capturing varied strains and stresses, thereby contributing to a high sensitivity for pressure sensing. This electronic skin-like sensor demonstrates an ultra-high sensitivity (1.24 kPa-1) below 150 Pa with a detection limit as low as about 1.3 Pa. The pixelated sensor array and a RGB-LED light are then assembled into a circuit and show a feasibility for visual detection of spatial pressure. Furthermore, a nanofiber based proof-of-concept wireless pressure sensor with a bluetooth module as a signal transmitter is proposed and has demonstrated great promise for wireless monitoring of human physiological signals, indicating a potential for large scale wearable electronic devices or e-skin.Pressure sensors with 3D conformability are highly desirable components for artificial electronic skin or e-textiles that can mimic natural skin, especially for application in real-time monitoring of human physiological signals. Here, a nanofiber based electronic skin with ultra-high pressure sensitivity and 3D conformability is designed and built by interlocking two elastic patterned nanofibrous membranes. The patterned membrane is facilely prepared by casting conductive nanofiber ink into a silicon mould to form an array of semi-spheroid-like protuberances. The

  9. Environmental scanning electron microscopy in cell biology.

    PubMed

    McGregor, J E; Staniewicz, L T L; Guthrie Neé Kirk, S E; Donald, A M

    2013-01-01

    Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

  10. Perspectives on in situ electron microscopy

    DOE PAGES

    Zheng, Haimei; Zhu, Yimei

    2017-03-29

    In situ transmission electron microscopy (TEM) with the ability to reveal materials dynamic processes with high spatial and temporal resolution has attracted significant interest. The recent advances in in situ methods, including liquid and gas sample environment, pump-probe ultrafast microscopy, nanomechanics and ferroelectric domain switching the aberration corrected electron optics as well as fast electron detector has opened new opportunities to extend the impact of in situ TEM in broad areas of research ranging from materials science to chemistry, physics and biology. Here in this paper, we highlight the development of liquid environment electron microscopy and its applications in themore » study of colloidal nanoparticle growth, electrochemical processes and others; in situ study of topological vortices in ferroelectric and ferromagnetic materials. At the end, perspectives of future in situ TEM are provided.« less

  11. Multiphoton microscopy of engineered dermal substitutes: assessment of 3D collagen matrix remodeling induced by fibroblasts contraction

    NASA Astrophysics Data System (ADS)

    Pena, A.-M.; Olive, C.; Michelet, J.-F.; Galey, J.-B.; Fagot, D.; Leroy, F.; Martin, J.-L.; Colonna, A.; Schanne-Klein, M.-C.

    2010-02-01

    One of the main functions of dermal fibroblasts is the generation of mechanical forces within their surrounding extracellular matrix. Investigating molecules that could modulate fibroblast contraction and act as potent anti aging ingredients requires the development of three-dimensional in situ imaging methodologies for dermal substitute analysis. Here we use multiphoton microscopy in order to investigate the fibroblast-induced collagen matrix reorganization in engineered dermal tissue and to evaluate the effect of Y27632, a RhoA kinase inhibitor on dermal substitutes contraction. We observe that collagen fibrils rearrange around fibroblast with increasing density in control samples, whereas collagen fibrils show no remodeling in the samples containing the RhoA kinase inhibitor. Moreover, when the culture medium containing the inhibitor was replaced with a control medium, the dermal substitutes presented the same 3D reorganization as the control samples, which indicates that the inhibitory effects are reversible. In conclusion, our study demonstrates the relevance of multiphoton microscopy to visualize three-dimensional remodeling of the matrix induced by fibroblast contraction.

  12. Scanning Electron Microscopy Sample Preparation and Imaging.

    PubMed

    Nguyen, Jenny Ngoc Tran; Harbison, Amanda M

    2017-01-01

    Scanning electron microscopes allow us to reach magnifications of 20-130,000× and resolve compositional and topographical images with intense detail. These images are created by bombarding a sample with electrons in a focused manner to generate a black and white image from the electrons that bounce off of the sample. The electrons are detected using positively charged detectors. Scanning electron microscopy permits three-dimensional imaging of desiccated specimens or wet cells and tissues by using variable pressure chambers. SEM ultrastructural analysis and intracellular imaging supplement light microscopy for molecular profiling of prokaryotes, plants, and mammals. This chapter demonstrates how to prepare and image samples that are (a) desiccated and conductive, (b) desiccated and nonconductive but coated with an electron conductive film using a gold sputter coater, and (c) wet and maintained in a hydrated state using a Deben Coolstage.

  13. 3D mapping of nanoscale electric potentials in semiconductor structures using electron-holographic tomography

    NASA Astrophysics Data System (ADS)

    Wolf, Daniel; Lubk, Axel; Prete, Paola; Lovergine, Nico; Lichte, Hannes

    2016-09-01

    Off-axis electron holography (EH) is a powerful method for mapping projected electric potentials, such as built-in potentials in semiconductor devices, in two dimensions (2D) at nanometer resolution. However, not well-defined thickness profiles, surface effects, and composition changes of the sample under investigation complicate the interpretation of the projected potentials. Here, we demonstrate how these problems can be overcome by combining EH with tomographic techniques, that is, electron holographic tomography (EHT), reconstructing electric potentials in 3D. We present EHT reconstructions of an n-type MOSFET including its dopant-related built-in potentials inside the device, as well as of a GaAs/AlGaAs core-multishell nanowire containing a 5 nm thick quantum well tube.

  14. Electronic Properties of COPPER-3d Transition-Metal Pairs in Silicon

    NASA Astrophysics Data System (ADS)

    Justo, João F.; Assali, Lucy V. C.

    We report a theoretical investigation of the chemical trends in the electronic properties of the substitutional Cu-interstitial 3d-transition-metal (Cr, Mn, Fe) trigonal pairs in silicon. The calculations were carried out in the framework of the multiple-scattering Xα molecular cluster model. The electronic structures show that the stability of these pairs is mostly the result of a covalent interaction between the molecular orbitals coming not only from the Cu and TM atoms but also from the neighboring Si atoms. These results are in contrast to an ionic model which has been generally invoked to explain the stability of those pairs, but in agreement with some recent experimental findings. The Fermi contact terms for all the stable pairs in different charge states were computed and compared to available experimental data. We speculate on the existence of a different microscopic structure to explain these Cu-related complex pairs.

  15. 3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography

    PubMed Central

    Nicastro, Daniela; McIntosh, J. Richard; Baumeister, Wolfgang

    2005-01-01

    We have used cryo-electron tomography to investigate the 3D structure and macromolecular organization of intact, frozen-hydrated sea urchin sperm flagella in a quiescent state. The tomographic reconstructions provide information at a resolution better than 6 nm about the in situ arrangements of macromolecules that are key for flagellar motility. We have visualized the heptameric rings of the motor domains in the outer dynein arm complex and determined that they lie parallel to the plane that contains the axes of neighboring flagellar microtubules. Both the material associated with the central pair of microtubules and the radial spokes display a plane of symmetry that helps to explain the planar beat pattern of these flagella. Cryo-electron tomography has proven to be a powerful technique for helping us understand the relationships between flagellar structure and function and the design of macromolecular machines in situ. PMID:16246999

  16. Three-dimensional cryo-electron microscopy on intermediate filaments.

    PubMed

    Kirmse, Robert; Bouchet-Marquis, Cédric; Page, Cynthia; Hoenger, Andreas

    2010-01-01

    Together with microtubules and actin filaments (F-actin), intermediate filaments (IFs) form the cytoskeleton of metazoan cells. However, unlike the other two entities that are extremely conserved, IFs are much more diverse and are grouped into five different families. In contrast to microtubules and F-actin, IFs do not exhibit a polarity, which may be the reason that no molecular motors travel along them. The molecular structure of IFs is less well resolved than that of the other cytoskeletal systems. This is partially due to their functional variability, tissue-specific expression, and their intrinsic structural properties. IFs are composed mostly of relatively smooth protofibrils formed by antiparallel arranged α-helical coiled-coil bundles flanked by small globular domains at either end. These features make them difficult to study by various electron microscopy methods or atomic force microscopy (AFM). Furthermore, the elongated shape of monomeric or dimeric IF units interferes with the formation of highly ordered three-dimensional (3-D) crystals suitable for atomic resolution crystallography. So far, most of the data we currently have on IF macromolecular structures come from electron microscopy of negatively stained samples, and fragmented α-helical coiled-coil units solved by X-ray diffraction. In addition, AFM allows the observation of the dynamic states of IFs in solution and delivers a new view into the assembly properties of IFs. Here, we discuss the applicability of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for the field. Both methods are strongly related and have only recently been applied to IFs. However, cryo-EM revealed distinct new features within IFs that have not been seen before, and cryo-ET adds a 3-D view of IFs revealing the path and number of protofilaments within the various IF assemblies.

  17. Electron Microscopy of the Cell

    PubMed Central

    Leeson, T. S.

    1965-01-01

    The use of the electron microscope has added much to our knowledge of the cell. The fine structure of the component parts of the nucleus and the cytoplasm is described, and their functions are indicated. The nature and structural modifications of the plasma membrane are illustrated with particular reference to function. To illustrate the interrelationships of the nucleus and cytoplasm, the theory of protein secretion is discussed, the secretion of a particular protein or polypeptide being determined by a particular nucleotide sequence in the desoxyribonucleic acid of a chromosome, that is, by a gene. This information is transferred from nucleus to cytoplasm. It is in the cytoplasm that the majority of the work is performed while the nucleus directs the work of the cell. ImagesFig. 2Fig. 3Fig. 4Fig. 5Fig. 6Fig. 7Fig. 8Fig. 9Fig. 10Fig. 11Fig. 12Fig. 13Fig. 14Fig. 15Fig. 16Fig. 17Fig. 18Fig. 19Fig. 20Fig. 21Fig. 22Fig. 23Fig. 24Fig. 25Fig. 26 PMID:5829410

  18. Electron crystallography of ultrathin 3D protein crystals: atomic model with charges.

    PubMed

    Yonekura, Koji; Kato, Kazuyuki; Ogasawara, Mitsuo; Tomita, Masahiro; Toyoshima, Chikashi

    2015-03-17

    Membrane proteins and macromolecular complexes often yield crystals too small or too thin for even the modern synchrotron X-ray beam. Electron crystallography could provide a powerful means for structure determination with such undersized crystals, as protein atoms diffract electrons four to five orders of magnitude more strongly than they do X-rays. Furthermore, as electron crystallography yields Coulomb potential maps rather than electron density maps, it could provide a unique method to visualize the charged states of amino acid residues and metals. Here we describe an attempt to develop a methodology for electron crystallography of ultrathin (only a few layers thick) 3D protein crystals and present the Coulomb potential maps at 3.4-Å and 3.2-Å resolution, respectively, obtained from Ca(2+)-ATPase and catalase crystals. These maps demonstrate that it is indeed possible to build atomic models from such crystals and even to determine the charged states of amino acid residues in the Ca(2+)-binding sites of Ca(2+)-ATPase and that of the iron atom in the heme in catalase.

  19. Predicting the Electronic Properties of 3D, Million-atom Semiconductor nanostructure Architectures

    SciTech Connect

    Jack Dongarra; Stanimire Tomov

    2012-03-15

    This final report describes the work done by Jack Dongarra (University Distinguished Professor) and Stanimire Tomov (Research Scientist) related to the DOE project entitled Predicting the Electronic Properties of 3D, Million-Atom Semiconductor Nanostructure Architectures. In this project we addressed the mathematical methodology required to calculate the electronic and transport properties of large nanostructures with comparable accuracy and reliability to that of current ab initio methods. This capability is critical for further developing the field, yet it is missing in all the existing computational methods. Additionally, quantitative comparisons with experiments are often needed for a qualitative understanding of the physics, and for guiding the design of new nanostructures. We focused on the mathematical challenges of the project, in particular on solvers and preconditioners for large scale eigenvalue problems that occur in the computation of electronic states of large nanosystems. Usually, the states of interest lie in the interior of the spectrum and their computation poses great difficulties for existing algorithms. The electronic properties of a semiconductor nanostructure architecture can be predicted/determined by computing its band structure. Of particular importance are the 'band edge states' (electronic states near the energy gap) which can be computed from a properly defined interior eigenvalue problem. Our primary mathematics and computational challenge here has been to develop an efficient solution methodology for finding these interior states for very large systems. Our work has produced excellent results in terms of developing both new and extending current state-of-the-art techniques.

  20. Advanced Electron Microscopy in Materials Physics

    SciTech Connect

    Zhu, Y.; Jarausch, K.

    2009-06-01

    Aberration correction has opened a new frontier in electron microscopy by overcoming the limitations of conventional round lenses, providing sub-angstrom-sized probes and extending information limits. The imaging and analytical performance of these corrector-equipped microscopes affords an unprecedented opportunity to study structure-property relationships of matter at the atomic scale. This new generation of microscopes is able to retrieve high-quality structural information comparable to neutron and synchrotron x-ray experiments, but with local atomic resolution. These advances in instrumentation are accelerating the research and development of various functional materials ranging from those for energy generation, conversion, transportation and storage to those for catalysis and nano-device applications. The dramatic improvements in electron-beam illumination and detection also present a host of new challenges for the interpretation and optimization of experiments. During 7-9 November 2007, a workshop, entitled 'Aberration Corrected Electron Microscopy in Material Physics', was convened at the Center for Functional Nanomaterials, Brookhaven National Laboratories (BNL) to address these opportunities and challenges. The workshop was co-sponsored by Hitachi High Technologies, a leader in electron microscopy instrumentation, and BNL's Institute of Advanced Electron Microscopy, a leader in materials physics research using electron microscopy. The workshop featured presentations by internationally prominent scientists working at the frontiers of electron microscopy, both on developing instrumentation and applying it in materials physics. The meeting, structured to stimulate scientific exchanges and explore new capabilities, brought together {approx}100 people from over 10 countries. This special issue complies many of the advances in instrument performance and materials physics reported by the invited speakers and attendees at the workshop.

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

    PubMed Central

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

    2010-01-01

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

  2. Three-dimensional scanning transmission electron microscopy of biological specimens

    SciTech Connect

    De Jonge, Niels; Sougrat, Rachid; Northan, Brian; Pennycook, Stephen J

    2010-01-01

    A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2 - 3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original data set. The precision of the height determination was 0.2 nm. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy (TEM). However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved data set.

  3. Three-dimensional scanning transmission electron microscopy of biological specimens.

    PubMed

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

    2010-02-01

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

  4. A 3D tomographic reconstruction method to analyze Jupiter's electron-belt emission observations

    NASA Astrophysics Data System (ADS)

    Santos-Costa, Daniel; Girard, Julien; Tasse, Cyril; Zarka, Philippe; Kita, Hajime; Tsuchiya, Fuminori; Misawa, Hiroaki; Clark, George; Bagenal, Fran; Imai, Masafumi; Becker, Heidi N.; Janssen, Michael A.; Bolton, Scott J.; Levin, Steve M.; Connerney, John E. P.

    2017-04-01

    Multi-dimensional reconstruction techniques of Jupiter's synchrotron radiation from radio-interferometric observations were first developed by Sault et al. [Astron. Astrophys., 324, 1190-1196, 1997]. The tomographic-like technique introduced 20 years ago had permitted the first 3-dimensional mapping of the brightness distribution around the planet. This technique has demonstrated the advantage to be weakly dependent on planetary field models. It also does not require any knowledge on the energy and spatial distributions of the radiating electrons. On the downside, it is assumed that the volume emissivity of any punctual point source around the planet is isotropic. This assumption becomes incorrect when mapping the brightness distribution for non-equatorial point sources or any point sources from Juno's perspective. In this paper, we present our modeling effort to bypass the isotropy issue. Our approach is to use radio-interferometric observations and determine the 3-D brightness distribution in a cylindrical coordinate system. For each set (z, r), we constrain the longitudinal distribution with a Fourier series and the anisotropy is addressed with a simple periodic function when possible. We develop this new method over a wide range of frequencies using past VLA and LOFAR observations of Jupiter. We plan to test this reconstruction method with observations of Jupiter that are currently being carried out with LOFAR and GMRT in support to the Juno mission. We describe how this new 3D tomographic reconstruction method provides new model constraints on the energy and spatial distributions of Jupiter's ultra-relativistic electrons close to the planet and be used to interpret Juno MWR observations of Jupiter's electron-belt emission and assist in evaluating the background noise from the radiation environment in the atmospheric measurements.

  5. BioMEA: a versatile high-density 3D microelectrode array system using integrated electronics.

    PubMed

    Charvet, Guillaume; Rousseau, Lionel; Billoint, Olivier; Gharbi, Sadok; Rostaing, Jean-Pierre; Joucla, Sébastien; Trevisiol, Michel; Bourgerette, Alain; Chauvet, Philippe; Moulin, Céline; Goy, François; Mercier, Bruno; Colin, Mikael; Spirkovitch, Serge; Fanet, Hervé; Meyrand, Pierre; Guillemaud, Régis; Yvert, Blaise

    2010-04-15

    Microelectrode arrays (MEAs) offer a powerful tool to both record activity and deliver electrical microstimulations to neural networks either in vitro or in vivo. Microelectronics microfabrication technologies now allow building high-density MEAs containing several hundreds of microelectrodes. However, dense arrays of 3D micro-needle electrodes, providing closer contact with the neural tissue than planar electrodes, are not achievable using conventional isotropic etching processes. Moreover, increasing the number of electrodes using conventional electronics is difficult to achieve into compact devices addressing all channels independently for simultaneous recording and stimulation. Here, we present a full modular and versatile 256-channel MEA system based on integrated electronics. First, transparent high-density arrays of 3D-shaped microelectrodes were realized by deep reactive ion etching techniques of a silicon substrate reported on glass. This approach allowed achieving high electrode aspect ratios, and different shapes of tip electrodes. Next, we developed a dedicated analog 64-channel Application Specific Integrated Circuit (ASIC) including one amplification stage and one current generator per channel, and analog output multiplexing. A full modular system, called BIOMEA, has been designed, allowing connecting different types of MEAs (64, 128, or 256 electrodes) to different numbers of ASICs for simultaneous recording and/or stimulation on all channels. Finally, this system has been validated experimentally by recording and electrically eliciting low-amplitude spontaneous rhythmic activity (both LFPs and spikes) in the developing mouse CNS. The availability of high-density MEA systems with integrated electronics will offer new possibilities for both in vitro and in vivo studies of large neural networks.

  6. The 3d Rydberg (3A2) electronic state observed by Herzberg and Shoosmith for methylene

    NASA Astrophysics Data System (ADS)

    Yamaguchi, Yukio; Schaefer, Henry F., III

    1997-06-01

    In 1959 and 1961 Herzberg and Shoosmith reported the vacuum ultraviolet spectrum of the triplet state of CH2. The present study focuses on a characterization of the upper state, the 3d Rydberg (3A2) state, observed at 1415 Å. The theoretical interpretation of these experiments is greatly complicated by the presence of a lower-lying 3A2 valence state with a very small equilibrium bond angle. Ab initio electronic structure methods involving self-consistent-field (SCF), configuration interaction with single and double excitations (CISD), complete active space (CAS) SCF, state-averaged (SA) CASSCF, coupled cluster with single and double excitations (CCSD), CCSD with perturbative triple excitations [CCSD(T)], CASSCF second-order (SO) CI, and SACASSCF-SOCI have been employed with six distinct basis sets. With the largest basis set, triple zeta plus triple polarization with two sets of higher angular momentum functions and three sets of diffuse functions TZ3P(2 f,2d)+3diff, the CISD level of theory predicts the equilibrium geometry of the 3d Rydberg (3A2) state to be re=1.093 Å and θe=141.3 deg. With the same basis set the energy (Te value) of the 3d Rydberg state relative to the ground (X˜ 3B1) state has been determined to be 201.6 kcal mol-1 (70 500 cm-1) at the CCSD (T) level, 200.92kcal mol-1 (70 270 cm-1) at the CASSCF-SOCI level, and 200.89kcal mol-1 (70 260 cm-1) at the SACASSCF-SOCI level of theory. These predictions are in excellent agreement with the experimental T0 value of 201.95 kcalmol-1 (70 634 cm-1) reported by Herzberg.

  7. Active Pixel Sensors for electron microscopy

    NASA Astrophysics Data System (ADS)

    Denes, P.; Bussat, J.-M.; Lee, Z.; Radmillovic, V.

    2007-09-01

    The technology used for monolithic CMOS imagers, popular for cell phone cameras and other photographic applications, has been explored for charged particle tracking by the high-energy physics community for several years. This technology also lends itself to certain imaging detector applications in electron microscopy. We have been developing such detectors for several years at Lawrence Berkeley National Laboratory, and we and others have shown that this technology can offer excellent point-spread function, direct detection and high readout speed. In this paper, we describe some of the design constraints peculiar to electron microscopy and summarize where such detectors could play a useful role.

  8. Three-Dimensional (3D) Nanometrology Based on Scanning Electron Microscope (SEM) Stereophotogrammetry.

    PubMed

    Tondare, Vipin N; Villarrubia, John S; Vlada R, András E

    2017-09-18

    Three-dimensional (3D) reconstruction of a sample surface from scanning electron microscope (SEM) images taken at two perspectives has been known for decades. Nowadays, there exist several commercially available stereophotogrammetry software packages. For testing these software packages, in this study we used Monte Carlo simulated SEM images of virtual samples. A virtual sample is a model in a computer, and its true dimensions are known exactly, which is impossible for real SEM samples due to measurement uncertainty. The simulated SEM images can be used for algorithm testing, development, and validation. We tested two stereophotogrammetry software packages and compared their reconstructed 3D models with the known geometry of the virtual samples used to create the simulated SEM images. Both packages performed relatively well with simulated SEM images of a sample with a rough surface. However, in a sample containing nearly uniform and therefore low-contrast zones, the height reconstruction error was ≈46%. The present stereophotogrammetry software packages need further improvement before they can be used reliably with SEM images with uniform zones.

  9. Optical microscopy versus scanning electron microscopy in urolithiasis.

    PubMed

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

    2009-10-01

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

  10. Three-dimensional scanning near field optical microscopy (3D-SNOM) imaging of random arrays of copper nanoparticles: implications for plasmonic solar cell enhancement

    NASA Astrophysics Data System (ADS)

    Ezugwu, Sabastine; Ye, Hanyang; Fanchini, Giovanni

    2014-11-01

    In order to investigate the suitability of random arrays of nanoparticles for plasmonic enhancement in the visible-near infrared range, we introduced three-dimensional scanning near-field optical microscopy (3D-SNOM) imaging as a useful technique to probe the intensity of near-field radiation scattered by random systems of nanoparticles at heights up to several hundred nm from their surface. We demonstrated our technique using random arrays of copper nanoparticles (Cu-NPs) at different particle diameter and concentration. Bright regions in the 3D-SNOM images, corresponding to constructive interference of forward-scattered plasmonic waves, were obtained at heights Δz >= 220 nm from the surface for random arrays of Cu-NPs of ~60-100 nm in diameter. These heights are too large to use Cu-NPs in contact of the active layer for light harvesting in thin organic solar cells, which are typically no thicker than 200 nm. Using a 200 nm transparent spacer between the system of Cu-NPs and the solar cell active layer, we demonstrate that forward-scattered light can be conveyed in 200 nm thin film solar cells. This architecture increases the solar cell photoconversion efficiency by a factor of 3. Our 3D-SNOM technique is general enough to be suitable for a large number of other applications in nanoplasmonics.In order to investigate the suitability of random arrays of nanoparticles for plasmonic enhancement in the visible-near infrared range, we introduced three-dimensional scanning near-field optical microscopy (3D-SNOM) imaging as a useful technique to probe the intensity of near-field radiation scattered by random systems of nanoparticles at heights up to several hundred nm from their surface. We demonstrated our technique using random arrays of copper nanoparticles (Cu-NPs) at different particle diameter and concentration. Bright regions in the 3D-SNOM images, corresponding to constructive interference of forward-scattered plasmonic waves, were obtained at heights Δz >= 220

  11. 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

  12. 3D hybrid simulations with gyrokinetic particle ions and fluid electrons

    SciTech Connect

    Belova, E.V.; Park, W.; Fu, G.Y.; Strauss, H.R.; Sugiyama, L.E.

    1998-12-31

    The previous hybrid MHD/particle model (MH3D-K code) represented energetic ions as gyrokinetic (or drift-kinetic) particles coupled to MHD equations using the pressure or current coupling scheme. A small energetic to bulk ion density ratio was assumed, n{sub h}/n{sub b} {much_lt} 1, allowing the neglect of the energetic ion perpendicular inertia in the momentum equation and the use of MHD Ohm`s law E = {minus}v{sub b} {times} B. A generalization of this model in which all ions are treated as gyrokinetic/drift-kinetic particles and fluid description is used for the electron dynamics is considered in this paper.

  13. Lateral error reduction in the 3D characterization of deep MOEMS devices using white light interference microscopy

    NASA Astrophysics Data System (ADS)

    Montgomery, Paul C.; Montaner, Denis; Manzardo, Omar; Herzig, Hans-Peter

    2004-08-01

    White light scanning interference microscopy, with its high axial resolution, is particularly useful for the rapid 3D characterization of MOEMS micro-systems. Although this technique can be used for submicron critical dimension measurement on micron high microelectronic structures, recent tests using a standard system have revealed errors of up to 3 μm in the measurement of lateral position of deep square steps. Thus the 2 μm wide, 75 μm deep teeth of an electrostatic comb structure in a FT MOEMS spectrometer were measured to be nearly 7 μm wide using a Mirau interference objective with the aperture diaphragm of the illumination system fully open in white light. Tests under different conditions show that the error is greatest for the Mirau objective, with the aperture diaphragm fully open at longer wavelengths. In addition, the location of the centre of such structures can vary by up to 1 μm depending on the degree of reference mirror tilt. Investigations of the XZ images of square steps have revealed the presence of "ghost" fringes resulting from diffraction and the conical illumination used. The errors in edge position can be reduced using a Linnik type objective with the aperture diaphragm closed down using shorter wavelength light.

  14. Neuronal nuclei localization in 3D using level set and watershed segmentation from laser scanning microscopy images

    NASA Astrophysics Data System (ADS)

    Zhu, Yingxuan; Olson, Eric; Subramanian, Arun; Feiglin, David; Varshney, Pramod K.; Krol, Andrzej

    2008-03-01

    Abnormalities of the number and location of cells are hallmarks of both developmental and degenerative neurological diseases. However, standard stereological methods are impractical for assigning each cell's nucleus position within a large volume of brain tissue. We propose an automated approach for segmentation and localization of the brain cell nuclei in laser scanning microscopy (LSM) embryonic mouse brain images. The nuclei in these images are first segmented by using the level set (LS) and watershed methods in each optical plane. The segmentation results are further refined by application of information from adjacent optical planes and prior knowledge of nuclear shape. Segmentation is then followed with an algorithm for 3D localization of the centroid of nucleus (CN). Each volume of tissue is thus represented by a collection of centroids leading to an approximate 10,000-fold reduction in the data set size, as compared to the original image series. Our method has been tested on LSM images obtained from an embryonic mouse brain, and compared to the segmentation and CN localization performed by an expert. The average Euclidian distance between locations of CNs obtained using our method and those obtained by an expert is 1.58+/-1.24 µm, a value well within the ~5 µm average radius of each nucleus. We conclude that our approach accurately segments and localizes CNs within cell dense embryonic tissue.

  15. A workflow to process 3D+time microscopy images of developing organisms and reconstruct their cell lineage

    PubMed Central

    Faure, Emmanuel; Savy, Thierry; Rizzi, Barbara; Melani, Camilo; Stašová, Olga; Fabrèges, Dimitri; Špir, Róbert; Hammons, Mark; Čúnderlík, Róbert; Recher, Gaëlle; Lombardot, Benoît; Duloquin, Louise; Colin, Ingrid; Kollár, Jozef; Desnoulez, Sophie; Affaticati, Pierre; Maury, Benoît; Boyreau, Adeline; Nief, Jean-Yves; Calvat, Pascal; Vernier, Philippe; Frain, Monique; Lutfalla, Georges; Kergosien, Yannick; Suret, Pierre; Remešíková, Mariana; Doursat, René; Sarti, Alessandro; Mikula, Karol; Peyriéras, Nadine; Bourgine, Paul

    2016-01-01

    The quantitative and systematic analysis of embryonic cell dynamics from in vivo 3D+time image data sets is a major challenge at the forefront of developmental biology. Despite recent breakthroughs in the microscopy imaging of living systems, producing an accurate cell lineage tree for any developing organism remains a difficult task. We present here the BioEmergences workflow integrating all reconstruction steps from image acquisition and processing to the interactive visualization of reconstructed data. Original mathematical methods and algorithms underlie image filtering, nucleus centre detection, nucleus and membrane segmentation, and cell tracking. They are demonstrated on zebrafish, ascidian and sea urchin embryos with stained nuclei and membranes. Subsequent validation and annotations are carried out using Mov-IT, a custom-made graphical interface. Compared with eight other software tools, our workflow achieved the best lineage score. Delivered in standalone or web service mode, BioEmergences and Mov-IT offer a unique set of tools for in silico experimental embryology. PMID:26912388

  16. Correction of Depth-Dependent Aberrations in 3D Single Molecule Localization and Super-resolution Microscopy

    PubMed Central

    McGorty, Ryan; Schnitzbauer, Joerg; Zhang, Wei; Huang, Bo

    2014-01-01

    Single molecule switching based super-resolution microscopy techniques have been extended into three dimensions through various 3D single molecule localization methods. However, the localization accuracy in z can be severely degraded by the presence of aberrations, particularly the spherical aberration introduced by the refractive-index-mismatch when imaging into an aqueous sample with an oil immersion objective. This aberration confines the imaging depth in most experiments to regions close to the coverslip. Here, we show a method to obtain accurate, depth dependent z calibrations by measuring the point spread function (PSF) at the coverslip surface, calculating the microscope pupil function through phase retrieval, and then computing the depth dependent PSF with the addition of spherical aberrations. We demonstrate experimentally that this method can maintain z localization accuracy over a large range of imaging depths. Our super-resolution images of a mammalian cell nucleus acquired between 0 and 2.5 μm past the coverslip show that this method produces accurate z localizations even in the deepest focal plane. PMID:24562125

  17. Multiphoton microscopy observations of 3D elastin and collagen fiber microstructure changes during pressurization in aortic media.

    PubMed

    Sugita, Shukei; Matsumoto, Takeo

    2017-06-01

    Elastin and collagen fibers play important roles in the mechanical properties of aortic media. Because knowledge of local fiber structures is required for detailed analysis of blood vessel wall mechanics, we investigated 3D microstructures of elastin and collagen fibers in thoracic aortas and monitored changes during pressurization. Using multiphoton microscopy, autofluorescence images from elastin and second harmonic generation signals from collagen were acquired in media from rabbit thoracic aortas that were stretched biaxially to restore physiological dimensions. Both elastin and collagen fibers were observed in all longitudinal-circumferential plane images, whereas alternate bright and dark layers were observed along the radial direction and were recognized as elastic laminas (ELs) and smooth muscle-rich layers (SMLs), respectively. Elastin and collagen fibers are mainly oriented in the circumferential direction, and waviness of collagen fibers was significantly higher than that of elastin fibers. Collagen fibers were more undulated in longitudinal than in radial direction, whereas undulation of elastin fibers was equibiaxial. Changes in waviness of collagen fibers during pressurization were then evaluated using 2-dimensional fast Fourier transform in mouse aortas, and indices of waviness of collagen fibers decreased with increases in intraluminal pressure. These indices also showed that collagen fibers in SMLs became straight at lower intraluminal pressures than those in EL, indicating that SMLs stretched more than ELs. These results indicate that deformation of the aorta due to pressurization is complicated because of the heterogeneity of tissue layers and differences in elastic properties of ELs, SMLs, and surrounding collagen and elastin.

  18. Introduction to high-resolution cryo-electron microscopy.

    PubMed

    Czarnocki-Cieciura, Mariusz; Nowotny, Marcin

    2016-01-01

    For many years two techniques have dominated structural biology - X-ray crystallography and NMR spectroscopy. Traditional cryo-electron microscopy of biological macromolecules produced macromolecular reconstructions at resolution limited to 6-10 Å. Recent development of transmission electron microscopes, in particular the development of direct electron detectors, and continuous improvements in the available software, have led to the "resolution revolution" in cryo-EM. It is now possible to routinely obtain near-atomic-resolution 3D maps of intact biological macromolecules as small as ~100 kDa. Thus, cryo-EM is now becoming the method of choice for structural analysis of many complex assemblies that are unsuitable for structure determination by other methods.

  19. Tunable electronic behavior in 3d transition metal doped 2H-WSe2

    NASA Astrophysics Data System (ADS)

    Liu, Shuai; Huang, Songlei; Li, Hongping; Zhang, Quan; Li, Changsheng; Liu, Xiaojuan; Meng, Jian; Tian, Yi

    2017-03-01

    Structural and electronic properties of 3d transition metal Sc, Ti, Cr and Mn incorporated 2H-WSe2 have been systematically investigated by first-principles calculations based on density functional theory. The calculated formation energies reveal that all the doped systems are thermodynamically more favorable under Se-rich condition than W-rich condition. The geometry structures almost hold that of the pristine 2H-WSe2 albeit with slight lattice distortion. More importantly, the electronic properties have been significantly tuned by the dopants, i.e., metal and semimetal behavior has been found in Sc, Ti and Mn-doped 2H-WSe2, respectively, semiconducting nature with narrowed band gap is expected in Cr-doped case, just as that of the pristine 2H-WSe2. In particular, magnetic character is realized by incorporation of Mn impurity with a total magnetic moment of 0.96 μB. Our results suggest chemical doping is an effective way to precisely tailor the electronic structure of layered transition metal dichalcogenide 2H-WSe2 for target technological applications.

  20. Interaction of 3d transition metal atoms with charged ion projectiles from Electron Nuclear Dynamics computation

    NASA Astrophysics Data System (ADS)

    Hagelberg, Frank

    2003-03-01

    Computational results on atomic scattering between charged projectiles and transition metal target atoms are presented. This work aims at obtaining detailed information about charge, spin and energy transfer processes that occur between the interacting particles. An in-depth understanding of these phenomena is expected to provide a theoretical basis for the interpretation of various types of ion beam experiments, ranging from gas phase chromatography to spectroscopic observations of fast ions in ferromagnetic media. This contribution focuses on the scattering of light projectiles ranging from He to O, that are prepared in various initial charge states, by 3d transition metal atoms. The presented computations are performed in the framework of Electron Nuclear Dynamics (END)^1 theory which incorporates the coupling between electronic and nuclear degrees of freedom without reliance on the computationally cumbersome and frequently intractable determination of potential energy surfaces. In the present application of END theory to ion - transition metal atom scattering, a supermolecule approach is utilized in conjunction with a spin-unrestricted single determinantal wave function describing the electronic system. Integral scattering, charge and spin exchange cross sections are discussed as functions of the elementary parameters of the problem, such as projectile and target atomic numbers as well as projectile charge and initial kinetic energy. ^1 E.Deumens, A.Diz, R.Longo, Y.Oehrn, Rev.Mod.Phys. 66, 917 (1994)

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

    PubMed

    Dahmen, Tim; Baudoin, Jean-Pierre; Lupini, Andrew R; Kübel, Christian; Slusallek, Philipp; de Jonge, Niels

    2014-04-01

    In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt-focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller "missing wedge" artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.

  2. Scanning electron microscopy study of Trichomonas gallinae.

    PubMed

    Tasca, Tiana; De Carli, Geraldo A

    2003-12-01

    A scanning electron microscopy (SEM) study of Trichomonas gallinae (Rivolta, 1878), provided more information about the morphology of this flagellated protozoan. SEM showed the morphological features of the trophozoites; the emergence of the anterior flagella, the structure of the undulating membrane, the position and shape of the pelta, axostyle and posterior flagellum. Of special interest were the pseudocyst forms.

  3. Photon-induced near field electron microscopy

    NASA Astrophysics Data System (ADS)

    Park, Sang Tae; Zewail, Ahmed H.

    2013-09-01

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

  4. Application of Electron Diffraction to Biological Electron Microscopy

    PubMed Central

    Glaeser, Robert M.; Thomas, Gareth

    1969-01-01

    Three methods by which electron diffraction may be applied to problems in electron microscopy are discussed from a fundamental point of view, and experimental applications with biological specimens are demonstrated for each case. It is shown that wide-angle electron diffraction provides valuable information for evaluating specimen damage that can occur either during specimen preparation or while in the electron beam. Dark-field electron microscopy can be used both to enhance the image contrast and to provide highly restricted and therefore highly specific information about the object. Low-angle electron diffraction provides quantitative information about the object structure in the range from 20 A to ∼ 1000 A. Lowangle electron diffraction also demonstrates the important role of Fourier contrast with biological specimens, which are usually characterized by structural features with dimensions of 20 A or larger. ImagesFigure 1Figure 2Figure 5Figure 6Figure 7Figure 8Figure 9Figure 10Figure 11Figure 13 PMID:4896898

  5. Mudrocks examined by backscattered electron microscopy

    NASA Technical Reports Server (NTRS)

    Pye, K.; Krinsley, D.

    1983-01-01

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

  6. Mudrocks examined by backscattered electron microscopy

    NASA Technical Reports Server (NTRS)

    Pye, K.; Krinsley, D.

    1983-01-01

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

  7. 3-D structures of crack-tip dislocations and their shielding effect revealed by electron tomography.

    PubMed

    Tanaka, Masaki; Honda, Masaki; Sadamatsu, Sunao; Higashida, Kenji

    2010-08-01

    Three-dimensional structures of crack-tip dislocations in silicon crystals have been examined by combining scanning transmission electron microscopy and computed tomography. Cracks were introduced by a Vickers hardness tester at room temperature, and the sample was heated at 823 K for 1 h in order to introduce dislocations around the crack tips. Dislocation segments cut out from loops were observed around the crack tip, the three-dimensional structure of which was characterized by using by electron tomography. Their Burgers vectors including the sings were also determined by oscillating contrasts along dislocations. In order to investigate the effect of the dislocations on fracture behaviours, local stress intensity factor due to one dislocation was calculated, which indicates the dislocations observed were shielding type to increase fracture toughness.

  8. Cellular uptake mechanisms of functionalised multi-walled carbon nanotubes by 3D electron tomography imaging

    NASA Astrophysics Data System (ADS)

    Al-Jamal, Khuloud T.; Nerl, Hannah; Müller, Karin H.; Ali-Boucetta, Hanene; Li, Shouping; Haynes, Peter D.; Jinschek, Joerg R.; Prato, Maurizio; Bianco, Alberto; Kostarelos, Kostas; Porter, Alexandra E.

    2011-06-01

    Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed that MWNT-NH3+ were internalised in both phagocytic and non-phagocytic cells by any one of three mechanisms: (a) individually via membrane wrapping; (b) individually by direct membrane translocation; and (c) in clusters within vesicular compartments. At early time points following intracellular translocation, we noticed accumulation of nanotube material within various intracellular compartments, while a long-term (14-day) study using primary human macrophages revealed that MWNT-NH3+ were able to escape vesicular (phagosome) entrapment by translocating directly into the cytoplasm.Carbon nanotubes (CNTs) are being investigated for a variety of biomedical applications. Despite numerous studies, the pathways by which carbon nanotubes enter cells and their subsequent intracellular trafficking and distribution remain poorly determined. Here, we use 3-D electron tomography techniques that offer optimum enhancement of contrast between carbon nanotubes and the plasma membrane to investigate the mechanisms involved in the cellular uptake of shortened, functionalised multi-walled carbon nanotubes (MWNT-NH3+). Both human lung epithelial (A549) cells, that are almost incapable of phagocytosis and primary macrophages, capable of extremely efficient phagocytosis, were used. We observed

  9. Photoemission electron microscopy and scanning electron microscopy of Magnetospirillum magnetotacticum's magnetosome chains.

    PubMed

    Keutner, Christoph; von Bohlen, Alex; Berges, Ulf; Espeter, Philipp; Schneider, Claus M; Westphal, Carsten

    2014-10-07

    Magnetotactic bacteria are of great interdisciplinary interest, since a vast field of applications from magnetic recording media to medical nanorobots is conceivable. A key feature for a further understanding is the detailed knowledge about the magnetosome chain within the bacteria. We report on two preparation procedures suitable for UHV experiments in reflective geometry. Further, we present the results of scanning electron microscopy, as well as the first photoemission electron microscopy experiments, both accessing the magnetosomes within intact magnetotactic bacteria and compare these to scanning electron microscopy data from the literature. From the images, we can clearly identify individual magnetosomes within their chains.

  10. Electronic structure and local magnetism of 3d-5d impurity substituted CeFe2

    NASA Astrophysics Data System (ADS)

    Das, Rakesh; Das, G. P.; Srivastava, S. K.

    2016-04-01

    We present here a systematic first-principles study of electronic structure and local magnetic properties of Ce[Fe0.75M0.25]2 compounds, where M is a 3d, 4d or 5d transition or post-transition element, using the generalized gradient approximation of the density functional theory. The d-f band hybridizations existing in CeFe2 get modified by the impurity M in an orderly manner across a period for each impurity series: the hybridization is strongest for the Mn group impurity in the period and gets diminished on either side of it. The weakening of the d-f hybridization strength is also associated with a relative localization of the Ce 4f states with respect to the delocalized 4f states in CeFe2. The above effects are most prominent for 3d impurity series, while for 4d and 5d impurities, the hybridizations and relocalizations are relatively weak due primarily to the relatively extended nature of 4d and 5d wavefunctions. The Ce local moment is found to decrease from the CeFe2 value in proportion to the strength of relocalization, thus following almost the same orderly trend as obeyed by the d-f hybridization. Further, depending on the way the spin-up and spin-down densities of states of an impurity shift relative to the Fermi energy, the impurity local moments are highest for Mn or Fe group, reduce on either side, become zero for Ni to Ga, and are small but negative for V and Ti. The Ce hyperfine field is found to follow the M local moment in a linear fashion, and vice-versa.

  11. DNA base identification by electron microscopy.

    PubMed

    Bell, David C; Thomas, W Kelley; Murtagh, Katelyn M; Dionne, Cheryl A; Graham, Adam C; Anderson, Jobriah E; Glover, William R

    2012-10-01

    Advances in DNA sequencing, based on fluorescent microscopy, have transformed many areas of biological research. However, only relatively short molecules can be sequenced by these technologies. Dramatic improvements in genomic research will require accurate sequencing of long (>10,000 base-pairs), intact DNA molecules. Our approach directly visualizes the sequence of DNA molecules using electron microscopy. This report represents the first identification of DNA base pairs within intact DNA molecules by electron microscopy. By enzymatically incorporating modified bases, which contain atoms of increased atomic number, direct visualization and identification of individually labeled bases within a synthetic 3,272 base-pair DNA molecule and a 7,249 base-pair viral genome have been accomplished. This proof of principle is made possible by the use of a dUTP nucleotide, substituted with a single mercury atom attached to the nitrogenous base. One of these contrast-enhanced, heavy-atom-labeled bases is paired with each adenosine base in the template molecule and then built into a double-stranded DNA molecule by a template-directed DNA polymerase enzyme. This modification is small enough to allow very long molecules with labels at each A-U position. Image contrast is further enhanced by using annular dark-field scanning transmission electron microscopy (ADF-STEM). Further refinements to identify additional base types and more precisely determine the location of identified bases would allow full sequencing of long, intact DNA molecules, significantly improving the pace of complex genomic discoveries.

  12. Aberration corrected Lorentz scanning transmission electron microscopy.

    PubMed

    McVitie, S; McGrouther, D; McFadzean, S; MacLaren, D A; O'Shea, K J; Benitez, M J

    2015-05-01

    We present results from an aberration corrected scanning transmission electron microscope which has been customised for high resolution quantitative Lorentz microscopy with the sample located in a magnetic field free or low field environment. We discuss the innovations in microscope instrumentation and additional hardware that underpin the imaging improvements in resolution and detection with a focus on developments in differential phase contrast microscopy. Examples from materials possessing nanometre scale variations in magnetisation illustrate the potential for aberration corrected Lorentz imaging as a tool to further our understanding of magnetism on this lengthscale.

  13. Detection of latent fingerprints using high-resolution 3D confocal microscopy in non-planar acquisition scenarios

    NASA Astrophysics Data System (ADS)

    Kirst, Stefan; Vielhauer, Claus

    2015-03-01

    In digitized forensics the support of investigators in any manner is one of the main goals. Using conservative lifting methods, the detection of traces is done manually. For non-destructive contactless methods, the necessity for detecting traces is obvious for further biometric analysis. High resolutional 3D confocal laser scanning microscopy (CLSM) grants the possibility for a detection by segmentation approach with improved detection results. Optimal scan results with CLSM are achieved on surfaces orthogonal to the sensor, which is not always possible due to environmental circumstances or the surface's shape. This introduces additional noise, outliers and a lack of contrast, making a detection of traces even harder. Prior work showed the possibility of determining angle-independent classification models for the detection of latent fingerprints (LFP). Enhancing this approach, we introduce a larger feature space containing a variety of statistical-, roughness-, color-, edge-directivity-, histogram-, Gabor-, gradient- and Tamura features based on raw data and gray-level co-occurrence matrices (GLCM) using high resolutional data. Our test set consists of eight different surfaces for the detection of LFP in four different acquisition angles with a total of 1920 single scans. For each surface and angles in steps of 10, we capture samples from five donors to introduce variance by a variety of sweat compositions and application influences such as pressure or differences in ridge thickness. By analyzing the present test set with our approach, we intend to determine angle- and substrate-dependent classification models to determine optimal surface specific acquisition setups and also classification models for a general detection purpose for both, angles and substrates. The results on overall models with classification rates up to 75.15% (kappa 0.50) already show a positive tendency regarding the usability of the proposed methods for LFP detection on varying surfaces in non

  14. Gabor-domain optical coherence microscopy with integrated dual-axis MEMS scanner for fast 3D imaging and metrology

    NASA Astrophysics Data System (ADS)

    Canavesi, Cristina; Cogliati, Andrea; Hayes, Adam; Santhanam, Anand P.; Tankam, Patrice; Rolland, Jannick P.

    2015-10-01

    Fast, robust, nondestructive 3D imaging is needed for characterization of microscopic structures in industrial and clinical applications. A custom micro-electromechanical system (MEMS)-based 2D scanner system was developed to achieve 55 kHz A-scan acquisition in a Gabor-domain optical coherence microscopy (GD-OCM) instrument with a novel multilevel GPU architecture for high-speed imaging. GD-OCM yields high-definition volumetric imaging with dynamic depth of focusing through a bio-inspired liquid lens-based microscope design, which has no moving parts and is suitable for use in a manufacturing setting or in a medical environment. A dual-axis MEMS mirror was chosen to replace two single-axis galvanometer mirrors; as a result, the astigmatism caused by the mismatch between the optical pupil and the scanning location was eliminated and a 12x reduction in volume of the scanning system was achieved. Imaging at an invariant resolution of 2 μm was demonstrated throughout a volume of 1 × 1 × 0.6 mm3, acquired in less than 2 minutes. The MEMS-based scanner resulted in improved image quality, increased robustness and lighter weight of the system - all factors that are critical for on-field deployment. A custom integrated feedback system consisting of a laser diode and a position-sensing detector was developed to investigate the impact of the resonant frequency of the MEMS and the driving signal of the scanner on the movement of the mirror. Results on the metrology of manufactured materials and characterization of tissue samples with GD-OCM are presented.

  15. Multi-pass transmission electron microscopy

    DOE PAGES

    Juffmann, Thomas; Koppell, Stewart A.; Klopfer, Brannon B.; ...

    2017-05-10

    Feynman once asked physicists to build better electron microscopes to be able to watch biology at work. While electron microscopes can now provide atomic resolution, electron beam induced specimen damage precludes high resolution imaging of sensitive materials, such as single proteins or polymers. Here, we use simulations to show that an electron m