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Sample records for advanced light microscopy

  1. Advances in Light Microscopy for Neuroscience

    PubMed Central

    Wilt, Brian A.; Burns, Laurie D.; Ho, Eric Tatt Wei; Ghosh, Kunal K.; Mukamel, Eran A.

    2010-01-01

    Since the work of Golgi and Cajal, light microscopy has remained a key tool for neuroscientists to observe cellular properties. Ongoing advances have enabled new experimental capabilities using light to inspect the nervous system across multiple spatial scales, including ultrastructural scales finer than the optical diffraction limit. Other progress permits functional imaging at faster speeds, at greater depths in brain tissue, and over larger tissue volumes than previously possible. Portable, miniaturized fluorescence microscopes now allow brain imaging in freely behaving mice. Complementary progress on animal preparations has enabled imaging in head-restrained behaving animals, as well as time-lapse microscopy studies in the brains of live subjects. Mouse genetic approaches permit mosaic and inducible fluorescence-labeling strategies, whereas intrinsic contrast mechanisms allow in vivo imaging of animals and humans without use of exogenous markers. This review surveys such advances and highlights emerging capabilities of particular interest to neuroscientists. PMID:19555292

  2. Invited Review Article: Advanced light microscopy for biological space research

    NASA Astrophysics Data System (ADS)

    De Vos, Winnok H.; Beghuin, Didier; Schwarz, Christian J.; Jones, David B.; van Loon, Jack J. W. A.; Bereiter-Hahn, Juergen; Stelzer, Ernst H. K.

    2014-10-01

    As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

  3. Invited review article: Advanced light microscopy for biological space research.

    PubMed

    De Vos, Winnok H; Beghuin, Didier; Schwarz, Christian J; Jones, David B; van Loon, Jack J W A; Bereiter-Hahn, Juergen; Stelzer, Ernst H K

    2014-10-01

    As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy. PMID:25362364

  4. Invited Review Article: Advanced light microscopy for biological space research

    SciTech Connect

    De Vos, Winnok H.; Beghuin, Didier; Schwarz, Christian J.; Jones, David B.; Loon, Jack J. W. A. van

    2014-10-15

    As commercial space flights have become feasible and long-term extraterrestrial missions are planned, it is imperative that the impact of space travel and the space environment on human physiology be thoroughly characterized. Scrutinizing the effects of potentially detrimental factors such as ionizing radiation and microgravity at the cellular and tissue level demands adequate visualization technology. Advanced light microscopy (ALM) is the leading tool for non-destructive structural and functional investigation of static as well as dynamic biological systems. In recent years, technological developments and advances in photochemistry and genetic engineering have boosted all aspects of resolution, readout and throughput, rendering ALM ideally suited for biological space research. While various microscopy-based studies have addressed cellular response to space-related environmental stressors, biological endpoints have typically been determined only after the mission, leaving an experimental gap that is prone to bias results. An on-board, real-time microscopical monitoring device can bridge this gap. Breadboards and even fully operational microscope setups have been conceived, but they need to be rendered more compact and versatile. Most importantly, they must allow addressing the impact of gravity, or the lack thereof, on physiologically relevant biological systems in space and in ground-based simulations. In order to delineate the essential functionalities for such a system, we have reviewed the pending questions in space science, the relevant biological model systems, and the state-of-the art in ALM. Based on a rigorous trade-off, in which we recognize the relevance of multi-cellular systems and the cellular microenvironment, we propose a compact, but flexible concept for space-related cell biological research that is based on light sheet microscopy.

  5. Advanced light microscopy core facilities: Balancing service, science and career.

    PubMed

    Ferrando-May, Elisa; Hartmann, Hella; Reymann, Jürgen; Ansari, Nariman; Utz, Nadine; Fried, Hans-Ulrich; Kukat, Christian; Peychl, Jan; Liebig, Christian; Terjung, Stefan; Laketa, Vibor; Sporbert, Anje; Weidtkamp-Peters, Stefanie; Schauss, Astrid; Zuschratter, Werner; Avilov, Sergiy

    2016-06-01

    Core Facilities (CF) for advanced light microscopy (ALM) have become indispensable support units for research in the life sciences. Their organizational structure and technical characteristics are quite diverse, although the tasks they pursue and the services they offer are similar. Therefore, throughout Europe, scientists from ALM-CFs are forming networks to promote interactions and discuss best practice models. Here, we present recommendations for ALM-CF operations elaborated by the workgroups of the German network of ALM-CFs, German Bio-Imaging (GerBI). We address technical aspects of CF planning and instrument maintainance, give advice on the organization and management of an ALM-CF, propose a scheme for the training of CF users, and provide an overview of current resources for image processing and analysis. Further, we elaborate on the new challenges and opportunities for professional development and careers created by CFs. While some information specifically refers to the German academic system, most of the content of this article is of general interest for CFs in the life sciences. Microsc. Res. Tech. 79:463-479, 2016. © 2016 THE AUTHORS MICROSCOPY RESEARCH AND TECHNIQUE PUBLISHED BY WILEY PERIODICALS, INC. PMID:27040755

  6. X-ray Microscopy Resource Center at the Advanced Light Source

    SciTech Connect

    Meyer-Ilse, W.; Attwood, D.; Koike, M.

    1992-08-01

    The high spectral brightness of undulator radiation from the Advanced Light Source (ALS) offers a great scientific opportunity for biological x-ray microscopy. X-ray microscopy extends visible light microscopy to higher resolution and makes use of unique contrast mechanisms. It does not compete with techniques such as electron microscopy in terms of resolution, but rather offers unique advantages, including the opportunity to take images of samples in an aqueous environment. For a considerable range of resolution and sample thickness the radiation dose in x-ray microscopy is lower than in electron microscopy under the same imaging conditions. To exploit this opportunity a Biological X-ray Microscopy Resource Center will be built at the ALS. An x-ray microscope (XM) and a scanning x-ray microscope (SXM) are to be built. These two microscopes serve complementary needs. The XM gives high quality images at comparably short exposure times, while the SXM is optimized for low radiation dose. High resolution is accomplished in both microscopes with Fresnel zone plate lenses. The SXM produces a diffraction-limited focus point, which is scanned across the sample; therefore the SXM can use only the spatially coherent portion of the radiation. The SXM is best operated on an undulator source with its small phase space. An XM can use the full brightness, including the incoherent fraction of the source. It can be operated with either a bending magnet or an undulator source. The XM can be installed initially at a bending magnet, which can be available at an earlier time, and thus permits the development of diverse biological community at an earlier time. Later this XM can be moved to the undulator, or left at the bending magnet for developmental and less demanding experiments.

  7. Soft X-Ray Microscopy and Spectroscopy at the Molecular Environmental Science Beamline at the Advanced Light Source

    SciTech Connect

    Bluhm, Hendrik; Andersson, Klas J.; Araki, Tohru; Benzerara, Karim; Brown, Gordon E.; Dynes, Jay J.; Ghosal, Sutapa; Gilles, Mary K.; Hansen, Hans C.; Hemminger, J. C.; Hitchcock, Adam P.; Ketteler, Guido; Kilcoyne, Arthur L.; Kneedler, Eric M.; Lawrence, John R.; Leppard, Gary G.; Majzlam, Juraj; Mun, B. S.; Myneni, Satish C.; Nilsson, Anders R.; Ogasawara, Hirohito; Ogletree, D. F.; Pecher, Klaus H.; Salmeron, Miquel B.; Shuh, David K.; Tonner, Brian; Tyliszczak, Tolek; Warwick, Tony; Yoon, T. H.

    2006-02-01

    We present examples of the application of synchrotron-based spectroscopies and microscopies to environmentally-relevant samples. The experiments were performed at the Molecular Environmental Science beamline (11.0.2) at the Advanced Light Source, Lawrence Berkeley National Laboratory. Examples range from the study of water monolayers on Pt(111) single crystal surfaces using X-ray emission spectroscopy and the examination of alkali halide solution/water vapor interfaces using ambient pressure photoemission spectroscopy, to the investigation of actinides, river-water biofilms, Al-containing colloids and mineral-bacteria suspensions using scanning transmission X-ray spectromicroscopy. The results of our experiments show that spectroscopy and microscopy in the soft X-ray energy range are excellent tools for the investigation of environmentally relevant samples under realistic conditions, i.e. with water or water vapor present at ambient temperature.

  8. Advances in Urine Microscopy.

    PubMed

    Becker, Gavin J; Garigali, Giuseppe; Fogazzi, Giovanni B

    2016-06-01

    Urine microscopy is an important tool for the diagnosis and management of several conditions affecting the kidneys and urinary tract. In this review, we describe the automated instruments, based either on flow cytometry or digitized microscopy, that are currently in use in large clinical laboratories. These tools allow the examination of large numbers of samples in short periods. We also discuss manual urinary microscopy commonly performed by nephrologists, which we encourage. After discussing the advantages of phase contrast microscopy over bright field microscopy, we describe the advancements of urine microscopy in various clinical conditions. These include persistent isolated microscopic hematuria (which can be classified as glomerular or nonglomerular on the basis of urinary erythrocyte morphology), drug- and toxin-related cystalluria (which can be a clue for the diagnosis of acute kidney injury associated with intrarenal crystal precipitation), and some inherited conditions (eg, adenine phosphoribosyltransferase deficiency, which is associated with 2,8-dihydroxyadenine crystalluria, and Fabry disease, which is characterized by unique urinary lamellated fatty particles). Finally, we describe the utility of identifying "decoy cells" and atypical malignant cells, which can be easily done with phase contrast microscopy in unfixed samples. PMID:26806004

  9. Quantitative analysis with advanced compensated polarized light microscopy on wavelength dependence of linear birefringence of single crystals causing arthritis

    NASA Astrophysics Data System (ADS)

    Takanabe, Akifumi; Tanaka, Masahito; Taniguchi, Atsuo; Yamanaka, Hisashi; Asahi, Toru

    2014-07-01

    To improve our ability to identify single crystals causing arthritis, we have developed a practical measurement system of polarized light microscopy called advanced compensated polarized light microscopy (A-CPLM). The A-CPLM system is constructed by employing a conventional phase retardation plate, an optical fibre and a charge-coupled device spectrometer in a polarized light microscope. We applied the A-CPLM system to measure linear birefringence (LB) in the visible region, which is an optical anisotropic property, for tiny single crystals causing arthritis, i.e. monosodium urate monohydrate (MSUM) and calcium pyrophosphate dihydrate (CPPD). The A-CPLM system performance was evaluated by comparing the obtained experimental data using the A-CPLM system with (i) literature data for a standard sample, MgF2, and (ii) experimental data obtained using an established optical method, high-accuracy universal polarimeter, for the MSUM. The A-CPLM system was found to be applicable for measuring the LB spectra of the single crystals of MSUM and CPPD, which cause arthritis, in the visible regions. We quantitatively reveal the large difference in LB between MSUM and CPPD crystals. These results demonstrate the usefulness of the A-CPLM system for distinguishing the crystals causing arthritis.

  10. A new bend magnet beam line for scanning transmission x-ray microscopy at the Advanced Light Source

    SciTech Connect

    Warwick, Tony; Ade, Harald; Kilcoyne, A.L. David; Kritscher, Michael; Tylisczcak, Tolek; Fakra, Sirine; Hitchcock, Adam P.; Hitchcock, Peter; Padmore, Howard A.

    2001-12-12

    The high brightness of the bend magnets at the Advanced Light Source has been exploited to illuminate a Scanning Transmission X-ray Microscope (STXM). This is the first diffraction-limited scanning x-ray microscope to operate with useful count rate on a synchrotron bend magnet source. A simple, dedicated beam line has been built covering the range of photon energy from 250 eV to 600 eV. Ease of use and operational availability are radically improved compared to previous installations using undulator beams. This facility provides radiation for C 1s, N 1s and O 1s near edge x-ray absorption spectro-microscopy with a spectral resolution up to about 1:5000 and with STXM count rates in excess of 1 MHz.

  11. Techniques of advanced light microscopy and their applications to morphological analysis of human extra-embryonic membranes.

    PubMed

    Ockleford, C D; Mongan, L C; Hubbard, A R

    The science of light microscopy has advanced dramatically in recent years through the introduction of new technology. A brief description of scanning light microscopes, laser illumination, the confocal principle, digital imaging, and image processing reveals a number of theoretical advantages which are particularly useful in improving epifluorescence microscope images. Examples of results from several studies of human extra-embryonic membranes conducted in our laboratory show how the application of these techniques has been used to describe structures such as microtrabeculae and rivets for the first time, to map the microscopic distribution of a wide range of proteins, and to observe the activity of placental villi at the microscopic level in an environmentally controlled microscope stage. High-sensitivity detectors have permitted the "super-resolution" detection of structures smaller than the theoretically calculated limits of light microscope resolution. Rendering images in false colour is demonstrably useful in detecting subtle variations in fluorescence intensity at different intracellular sites and at different sites within tissues of fetal membranes. Processing stacks of digital images using appropriate software allows the 3-D reconstruction of suitably sized extra-embryonic membrane components. These digital images created from optical sections through the tissue are obtained non-destructively, and the relationships in space of the components are well preserved. PMID:9260846

  12. Hyperspectral light sheet microscopy.

    PubMed

    Jahr, Wiebke; Schmid, Benjamin; Schmied, Christopher; Fahrbach, Florian O; Huisken, Jan

    2015-01-01

    To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos. PMID:26329685

  13. Hyperspectral light sheet microscopy

    NASA Astrophysics Data System (ADS)

    Jahr, Wiebke; Schmid, Benjamin; Schmied, Christopher; Fahrbach, Florian O.; Huisken, Jan

    2015-09-01

    To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos.

  14. Hyperspectral light sheet microscopy

    PubMed Central

    Jahr, Wiebke; Schmid, Benjamin; Schmied, Christopher; Fahrbach, Florian O.; Huisken, Jan

    2015-01-01

    To study the development and interactions of cells and tissues, multiple fluorescent markers need to be imaged efficiently in a single living organism. Instead of acquiring individual colours sequentially with filters, we created a platform based on line-scanning light sheet microscopy to record the entire spectrum for each pixel in a three-dimensional volume. We evaluated data sets with varying spectral sampling and determined the optimal channel width to be around 5 nm. With the help of these data sets, we show that our setup outperforms filter-based approaches with regard to image quality and discrimination of fluorophores. By spectral unmixing we resolved overlapping fluorophores with up to nanometre resolution and removed autofluorescence in zebrafish and fruit fly embryos. PMID:26329685

  15. Further development of soft X-ray scanning microscopy with anelliptical undulator at the Advanced Light Source

    SciTech Connect

    Warwick, Tony; Ade, Harald; Fakra, Sirine; Gilles, Mary; Hitchcock, Adam; Kilcoyne, David; Shuh, David; Tyliszczak, Tolek

    2003-04-02

    Soft x-ray scanning microscopy (1) is under continuing development at the Advanced Light Source. Significant progress has been made implementing new scan control systems in both operational microscopes (2) and they now operate at beam lines 5.3.2 and 11.0.2 with interferometer servo scanning and stabilization. The interferometer servo loop registers the images on a universal x/y coordinate system and locks the x-ray spot on selected features for spectro-microscopic studies. At the present time zone plates are in use with 35nm outer zone width and the imaging spatial resolution is at the diffraction limit of these lenses. Current research programs are underway in areas of polymer chemistry, environmental chemistry and materials science. A dedicated polymer STXM is in operation on a bend magnet beam line (4) and is the subject of a separate article (3) in this issue. Here we focus on the capabilities of STXM at a new beam line that employs an elliptical undulator (5) to give control of the polarization of the x-ray beam. This facility is in the process of commissioning and some results are available, other capabilities will be developed during the first half of 2003.

  16. Virtual reflected-light microscopy.

    PubMed

    Harrison, A P; Wong, C M; Joseph, D

    2011-12-01

    Research on better methods to digitally represent microscopic specimens has increased over recent decades. Opaque specimens, such as microfossils and metallurgic specimens, are often viewed using reflected light microscopy. Existing 3D surface estimation techniques for reflected light microscopy do not model reflectance, restricting the representation to only one illumination condition and making them an imperfect recreation of the experience of using an actual microscope. This paper introduces a virtual reflected-light microscopy (VRLM) system that estimates both shape and reflectance from a set of specimen images. When coupled with anaglyph creation, the system can depict both depth information and illumination cues under any desired lighting configuration. Digital representations are compact and easily viewed in an online setting. A prototype used to construct VRLM representations is comprised only of a microscope, a digital camera, a motorized stage and software. Such a system automatically acquires VRLM representations of large batches of specimens. VRLM representations are then disseminated in an interactive online environment, which allows users to change the virtual light source direction and type. Experiments demonstrate high quality VRLM representations of 500 microfossils. PMID:21919903

  17. The DIADEM data sets: representative light microscopy images of neuronal morphology to advance automation of digital reconstructions.

    PubMed

    Brown, Kerry M; Barrionuevo, Germán; Canty, Alison J; De Paola, Vincenzo; Hirsch, Judith A; Jefferis, Gregory S X E; Lu, Ju; Snippe, Marjolein; Sugihara, Izumi; Ascoli, Giorgio A

    2011-09-01

    The comprehensive characterization of neuronal morphology requires tracing extensive axonal and dendritic arbors imaged with light microscopy into digital reconstructions. Considerable effort is ongoing to automate this greatly labor-intensive and currently rate-determining process. Experimental data in the form of manually traced digital reconstructions and corresponding image stacks play a vital role in developing increasingly more powerful reconstruction algorithms. The DIADEM challenge (short for DIgital reconstruction of Axonal and DEndritic Morphology) successfully stimulated progress in this area by utilizing six data set collections from different animal species, brain regions, neuron types, and visualization methods. The original research projects that provided these data are representative of the diverse scientific questions addressed in this field. At the same time, these data provide a benchmark for the types of demands automated software must meet to achieve the quality of manual reconstructions while minimizing human involvement. The DIADEM data underwent extensive curation, including quality control, metadata annotation, and format standardization, to focus the challenge on the most substantial technical obstacles. This data set package is now freely released ( http://diademchallenge.org ) to train, test, and aid development of automated reconstruction algorithms. PMID:21249531

  18. Light Sheet Fluorescence Microscopy (LSFM)

    PubMed Central

    Adams, Michael W.; Loftus, Andrew F.; Dunn, Sarah E.; Joens, Matthew S.; Fitzpatrick, James A.J.

    2015-01-01

    The development of confocal microscopy techniques introduced the ability to optically section fluorescent samples in the axial dimension, perpendicular to the image plane. These approaches, via the placement of a pinhole in the conjugate image plane, provided superior resolution in the axial (z) dimension resulting in nearly isotropic optical sections. However, increased axial resolution, via pinhole optics, comes at the cost of both speed and excitation efficiency. Light Sheet Fluorescent Microscopy (LSFM), a century old idea (Siedentopf and Zsigmondy, 1902) made possible with modern developments in both excitation and detection optics, provides sub-cellular resolution and optical sectioning capabilities without compromising speed or excitation efficiency. Over the past decade, several variations of LSFM have been implemented each with its own benefits and deficiencies. Here we discuss LSFM fundamentals and outline the basic principles of several major light sheet based imaging modalities (SPIM, inverted SPIM, multi-view SPIM, Bessel beam SPIM, and stimulated emission depletion SPIM while considering their biological relevance in terms of intrusiveness, temporal resolution, and sample requirements. PMID:25559221

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

    PubMed

    Begemann, Isabell; Galic, Milos

    2016-01-01

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

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

    PubMed Central

    Begemann, Isabell; Galic, Milos

    2016-01-01

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

  1. Correlated light and electron microscopy: ultrastructure lights up!

    PubMed

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

    2015-06-01

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

  2. Advances in multiphoton microscopy technology

    PubMed Central

    Hoover, Erich E.; Squier, Jeff A.

    2013-01-01

    Multiphoton microscopy has enabled unprecedented dynamic exploration in living organisms. A significant challenge in biological research is the dynamic imaging of features deep within living organisms, which permits the real-time analysis of cellular structure and function. To make progress in our understanding of biological machinery, optical microscopes must be capable of rapid, targeted access deep within samples at high resolution. In this Review, we discuss the basic architecture of a multiphoton microscope capable of such analysis and summarize the state-of-the-art technologies for the quantitative imaging of biological phenomena. PMID:24307915

  3. Value of Reflected Light Microscopy in Teaching.

    ERIC Educational Resources Information Center

    Pasteris, Jill Dill

    1983-01-01

    Briefly reviews some optical and other physical properties of minerals that can be determined in reflected/incident light. Topics include optical properties of minerals, reflectance, internal reflections, color, bireflectance and reflection pleochroism, anisotropism, zonation, and reflected light microscopy as a teaching tool in undergraduate…

  4. Advances in fiber lasers for nonlinear microscopy

    NASA Astrophysics Data System (ADS)

    Wise, F. W.; Ouzounov, D.; Kieu, K.; Renninger, W.; Chong, A.; Liu, H.

    2008-02-01

    In the past 30 years major advances in medical imaging have been made in areas such as magnetic resonance imaging, computed tomography, and ultrasound. These techniques have become quite effective for structural imaging at the organ or tissue level, but do not address the clear need for imaging technologies that exploit existing knowledge of the genetic and molecular bases of disease. Techniques that can provide similar information on the cellular and molecular scale would be very powerful, and ultimately the extension of such techniques to in vivo measurements will be desired. The availability of these imaging capabilities would allow monitoring of the early stages of disease or therapy, for example. Optical techniques provide excellent imaging capabilities, with sub-micron spatial resolution, and are noninvasive. An overall goal of biomedical imaging is to obtain diagnostic or functional information about biological structures. The difficulty of acquiring high-resolution images of structures deep in tissue presents a major challenge, however, owing to strong scattering of light. As a consequence, optical imaging has been limited to thin (typically ~0.5 mm) samples or superficial tissue. In contrast, techniques such as ultrasound and magnetic resonance provide images of structures centimeters deep in tissue, with ~100-micron resolution. It is desirable to develop techniques that offer the resolution of optics with the depth-penetration of other techniques. Since 1990, a variety of nonlinear microscopies have been demonstrated. These include 2- and 3-photon fluorescence microscopy, and 2nd- and 3rd-harmonic generation microscopies. These typically employ femtosecond-pulse excitation, for maximum peak power (and thus nonlinear excitation) for a given pulse energy. A relative newcomer to the group is CARS microscopy [1], which exploits resonant vibrational excitation of molecules or bonds. The CARS signal contrast arises from intrinsic elements of cells, and thus

  5. Lighting up microscopy with random Raman lasing

    NASA Astrophysics Data System (ADS)

    Hokr, Brett H.; Nodurft, Dawson T.; Thompson, Jonathan V.; Bixler, Joel N.; Noojin, Gary D.; Redding, Brandon; Thomas, Robert J.; Cao, Hui; Rockwell, Benjamin A.; Scully, Marlan O.; Yakovlev, Vladislav V.

    2016-03-01

    Wide-field microscopy, where full images are obtained simultaneously, is limited by the power available from speckle-free light sources. Currently, the vast majority of wide-field microscopes use either mercury arc lamps, or LEDs as the illumination source. The power available from these sources limits wide-field fluorescent microscopy to tens of microseconds temporal resolution. Lasers, while capable of producing high power and short pulses, have high spatial coherence. This leads to the formation of laser speckle that makes such sources unsuitable for wide-field imaging applications. Random Raman lasers offer the best of both worlds by producing laser-like intensities, short, nanosecond-scale, pulses, and low spatial coherence, speckle-free, output. These qualities combine to make random Raman lasers 4 orders of magnitude brighter than traditional wide-field microscopy light sources. Furthermore, the unique properties of random Raman lasers make possible the entirely new possibilities of wide-field fluorescence lifetime imaging or wide-field Raman microscopy. We will introduce the relevant physics that give rise to the unique properties of random Raman lasing, and demonstrate early proof of principle results demonstrating random Raman lasing emission being used as an imaging light source. Finally, we will discuss future directions and elucidate the benefits of using random Raman lasers as a wide-field microscopy light source.

  6. Polarized light microscopy: principles and practice.

    PubMed

    Oldenbourg, Rudolf

    2013-11-01

    Polarized light microscopy provides unique opportunities for analyzing the molecular order in heterogeneous systems, such as living cells and tissues, without using exogenous dyes or labels. This article briefly discusses the theory of polarized light microscopy and elaborates on its practice using a traditional polarized light microscope and more specialized polarization microscopes such as the LC-PolScope, Oosight, or Abrio. The microscope components specific to analyzing the polarization of light, such as polarizer and compensator, are introduced, and quantitative techniques for measuring the birefringence of the specimen point by point using a traditional polarizing microscope are discussed. The new LC-PolScope greatly improves the analytic power of the technique, providing quantitative birefringence data simultaneously for every image point, thereby revealing molecular order with unprecedented sensitivity and at the highest resolution of the light microscope. Practical aspects discussed include the choice of optics, sample preparation, and combining polarized light with differential interference contrast and fluorescence microscopy. A glossary of polarization optical terms is also included to facilitate the discussion of observations made with a polarized light microscope. PMID:24184765

  7. Light Microscopy Module (LMM)-Emulator

    NASA Technical Reports Server (NTRS)

    Levine, Howard G.; Smith, Trent M.; Richards, Stephanie E.

    2016-01-01

    The Light Microscopy Module (LMM) is a microscope facility developed at Glenn Research Center (GRC) that provides researchers with powerful imaging capability onboard the International Space Station (ISS). LMM has the ability to have its hardware recongured on-orbit to accommodate a wide variety of investigations, with the capability of remotely acquiring and downloading digital images across multiple levels of magnication.

  8. Confocal multiview light-sheet microscopy

    PubMed Central

    Medeiros, Gustavo de; Norlin, Nils; Gunther, Stefan; Albert, Marvin; Panavaite, Laura; Fiuza, Ulla-Maj; Peri, Francesca; Hiiragi, Takashi; Krzic, Uros; Hufnagel, Lars

    2015-01-01

    Selective-plane illumination microscopy has proven to be a powerful imaging technique due to its unsurpassed acquisition speed and gentle optical sectioning. However, even in the case of multiview imaging techniques that illuminate and image the sample from multiple directions, light scattering inside tissues often severely impairs image contrast. Here we combine multiview light-sheet imaging with electronic confocal slit detection implemented on modern camera sensors. In addition to improved imaging quality, the electronic confocal slit detection doubles the acquisition speed in multiview setups with two opposing illumination directions allowing simultaneous dual-sided illumination. Confocal multiview light-sheet microscopy eliminates the need for specimen-specific data fusion algorithms, streamlines image post-processing, easing data handling and storage. PMID:26602977

  9. Camera array based light field microscopy.

    PubMed

    Lin, Xing; Wu, Jiamin; Zheng, Guoan; Dai, Qionghai

    2015-09-01

    This paper proposes a novel approach for high-resolution light field microscopy imaging by using a camera array. In this approach, we apply a two-stage relay system for expanding the aperture plane of the microscope into the size of an imaging lens array, and utilize a sensor array for acquiring different sub-apertures images formed by corresponding imaging lenses. By combining the rectified and synchronized images from 5 × 5 viewpoints with our prototype system, we successfully recovered color light field videos for various fast-moving microscopic specimens with a spatial resolution of 0.79 megapixels at 30 frames per second, corresponding to an unprecedented data throughput of 562.5 MB/s for light field microscopy. We also demonstrated the use of the reported platform for different applications, including post-capture refocusing, phase reconstruction, 3D imaging, and optical metrology. PMID:26417490

  10. Camera array based light field microscopy

    PubMed Central

    Lin, Xing; Wu, Jiamin; Zheng, Guoan; Dai, Qionghai

    2015-01-01

    This paper proposes a novel approach for high-resolution light field microscopy imaging by using a camera array. In this approach, we apply a two-stage relay system for expanding the aperture plane of the microscope into the size of an imaging lens array, and utilize a sensor array for acquiring different sub-apertures images formed by corresponding imaging lenses. By combining the rectified and synchronized images from 5 × 5 viewpoints with our prototype system, we successfully recovered color light field videos for various fast-moving microscopic specimens with a spatial resolution of 0.79 megapixels at 30 frames per second, corresponding to an unprecedented data throughput of 562.5 MB/s for light field microscopy. We also demonstrated the use of the reported platform for different applications, including post-capture refocusing, phase reconstruction, 3D imaging, and optical metrology. PMID:26417490

  11. 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. PMID:27379646

  12. Light microscopy of whole plant organs.

    PubMed

    Timmers, Antonius C J

    2016-08-01

    Plants are ideal organisms for light microscopical studies of cellular mechanisms controlling cell organisation and cell functioning. However, most plant organs are not transparent to light which prevents high resolution imaging deep within plant tissues. Classically, access into plant organs is achieved by sectioning or whole-mount tissue clearing. Until recently, the protocols for clearing destroyed the signal from fluorescent markers which prevented the imaging of the distribution of fluorescent proteins and the three-dimensional reconstruction from optical slices of whole plant organs. From 2011, a number of protocols have been developed for whole brain and whole organism imaging for animal studies. Now, these protocols have been adapted for in-depth imaging of whole plant organs. Here, I present an overview of clearing techniques of plant organs and highlight the latest developments of plant tissue clearing in combination with high resolution fluorescence microscopy. PMID:27027806

  13. Recent advances in magnetic force microscopy.

    PubMed

    Koblischka, M R; Hartmann, U

    2003-01-01

    During the past ten years magnetic force microscopy (MFM) has become probably the most powerful general-purpose method for magnetic imaging. MFM can be applied under various environmental conditions and requires only little sample preparation. Basic research on magnetic materials as well as the mentioned industrial applications create an increasing demand for high-resolution magnetic imaging methods. This contribution will review some new concepts which have been realized in the field of advanced probe preparation, based on electron beam methods in order to improve the spatial resolution beyond 100nm. It is shown that the advanced probes allow high-resolution imaging of magnetic fine structures within thin film permalloy elements exhibiting a complicated cooperative magnetization reversal process. These investigations are of importance for various concepts underlying modern magnetic data storage developments. Furthermore, we present some developments of MFM to suit the needs of the magnetic recording industry. PMID:12801662

  14. Light Microscopy Module Imaging Tested and Demonstrated

    NASA Technical Reports Server (NTRS)

    Gati, Frank

    2004-01-01

    The Fluids Integrated Rack (FIR), a facility-class payload, and the Light Microscopy Module (LMM), a subrack payload, are integrated research facilities that will fly in the U.S. Laboratory module, Destiny, aboard the International Space Station. Both facilities are being engineered, designed, and developed at the NASA Glenn Research Center by Northrop Grumman Information Technology. The FIR is a modular, multiuser scientific research facility that is one of two racks that make up the Fluids and Combustion Facility (the other being the Combustion Integrated Rack). The FIR has a large volume dedicated for experimental hardware; easily reconfigurable diagnostics, power, and data systems that allow for unique experiment configurations; and customizable software. The FIR will also provide imagers, light sources, power management and control, command and data handling for facility and experiment hardware, and data processing and storage. The first payload in the FIR will be the LMM. The LMM integrated with the FIR is a remotely controllable, automated, on-orbit microscope subrack facility, with key diagnostic capabilities for meeting science requirements--including video microscopy to observe microscopic phenonema and dynamic interactions, interferometry to make thin-film measurements with nanometer resolution, laser tweezers to manipulate micrometer-sized particles, confocal microscopy to provide enhanced three-dimensional visualization of structures, and spectrophotometry to measure the photonic properties of materials. Vibration disturbances were identified early in the LMM development phase as a high risk for contaminating the science microgravity environment. An integrated FIR-LMM test was conducted in Glenn's Acoustics Test Laboratory to assess mechanical sources of vibration and their impact to microscopic imaging. The primary purpose of the test was to characterize the LMM response at the sample location, the x-y stage within the microscope, to vibration

  15. Advanced electron microscopy characterization of multimetallic nanoparticles

    NASA Astrophysics Data System (ADS)

    Khanal, Subarna Raj

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

  16. Immunogold silver staining for light microscopy.

    PubMed

    Lackie, P M

    1996-07-01

    The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly deposit metallic silver only at the site of heavy metals, including gold and silver, because of their catalytic activity. The developing solution contains the silver ions and reducing agent necessary for this reaction. Using different silver salts as ion donors and by selecting an appropriate temperature and pH, visible amounts of silver can be deposited in a few minutes at the site of colloidal gold labelling while little non-specific background deposition occurs. Inclusion of protective colloids in the solution can also be used to control the reaction. Although studies of the chemical basis of silver deposition around unlabelled colloidal gold date back to 1939, immunogold enhancement by silver was established in 1983. The IGSS method evolved from the combination of disparate photographic, histochemical and immunogold techniques which have been effectively combined and optimised over the last 10 years to provide a visualisation system which is well suited to many immunohistochemical studies. PMID:8858363

  17. A quick guide to light microscopy in cell biology

    PubMed Central

    Thorn, Kurt

    2016-01-01

    Light microscopy is a key tool in modern cell biology. Light microscopy has several features that make it ideally suited for imaging biology in living cells: the resolution is well-matched to the sizes of subcellular structures, a diverse range of available fluorescent probes makes it possible to mark proteins, organelles, and other structures for imaging, and the relatively nonperturbing nature of light means that living cells can be imaged for long periods of time to follow their dynamics. Here I provide a brief introduction to using light microscopy in cell biology, with particular emphasis on factors to be considered when starting microscopy experiments. PMID:26768859

  18. Restoration of uneven illumination in light sheet microscopy images.

    PubMed

    Uddin, Mohammad Shorif; Lee, Hwee Kuan; Preibisch, Stephan; Tomancak, Pavel

    2011-08-01

    Light microscopy images suffer from poor contrast due to light absorption and scattering by the media. The resulting decay in contrast varies exponentially across the image along the incident light path. Classical space invariant deconvolution approaches, while very effective in deblurring, are not designed for the restoration of uneven illumination in microscopy images. In this article, we present a modified radiative transfer theory approach to solve the contrast degradation problem of light sheet microscopy (LSM) images. We confirmed the effectiveness of our approach through simulation as well as real LSM images. PMID:21682937

  19. Light-sheet optimization for microscopy

    NASA Astrophysics Data System (ADS)

    Wilding, Dean; Pozzi, Paolo; Soloviev, Oleg; Vdovin, Gleb; Verhaegen, Michel

    2016-03-01

    Aberrations, scattering and absorption degrade the performance light-sheet fluorescence microscopes (LSFM). An adaptive optics system to correct for these artefacts and to optimize the light-sheet illumination is presented. This system allows a higher axial resolution to be recovered over the field-of-view of the detection objective. It is standard selective plane illumination microscope (SPIM) configuration modified with the addition of a spatial light modulator (SLM) and a third objective for the detection of transmitted light. Optimization protocols use this transmission light allowing the extension the depth-of-field and correction of aberrations whilst retaining a thin optical section.

  20. Light microscopy: an ongoing contemporary revolution

    NASA Astrophysics Data System (ADS)

    Weisenburger, Siegfried; Sandoghdar, Vahid

    2015-04-01

    The optical microscope is one of the oldest scientific instruments that is still used in forefront research. Ernst Abbe's nineteenth century formulation of the resolution limit in microscopy let generations of scientists believe that optical studies of individual molecules and resolving subwavelength structures were not feasible. The Nobel Prize in 2014 for super-resolution fluorescence microscopy marks a clear recognition that the old beliefs have to be revisited. In this article, we present a critical overview of various recent developments in optical microscopy. In addition to the popular super-resolution fluorescence methods, we discuss the prospects of various other techniques and imaging contrasts and consider some of the fundamental and practical challenges that lie ahead.

  1. Modular platform for low-light microscopy

    PubMed Central

    Kim, Tae Jin; Tuerkcan, Silvan; Ceballos, Andrew; Pratx, Guillem

    2015-01-01

    Cell imaging using low-light techniques such as bioluminescence, radioluminescence, and low-excitation fluorescence has received increased attention, particularly due to broad commercialization of highly sensitive detectors. However, the dim signals are still regarded as difficult to image using conventional microscopes, where the only low-light microscope in the market is primarily optimized for bioluminescence imaging. Here, we developed a novel modular microscope that is cost-effective and suitable for imaging different low-light luminescence modes. Results show that this microscope system features excellent aberration correction capabilities and enhanced image resolution, where bioluminescence, radioluminescence and epifluorescence images were captured and compared with the commercial bioluminescence microscope. PMID:26601020

  2. Advanced Light Source elliptical wiggler

    SciTech Connect

    Hoyer, E.; Akre, J.; Humphries, D.; Marks, S.; Minamihara, Y.; Pipersky, P.

    1994-07-01

    A 3.5m long elliptical wiggler, optimized to produce elliptically polarized light in the 50 eV to 10 keV range, is currently under design and construction at the Advanced Light Source (ALS) at Lawrence Berkeley Laboratory. Calculations of spectral performance show that the flux of circularly polarized photons exceeds 10{sup 13} photons/sec over the 50 eV to 10 keV operating range for current of 0.4 amps and 1.5 GeV electron energy. This device features vertical and horizontal magnetic structures of 14 and 14{1/2} periods respectively. The period length is 20.0 cm. The vertical structure is a hybrid permanent magnet design with tapered pole tips that produce a peak field of 2.0 T. The horizontal structure is an iron core electromagnetic design, shifted longitudinally {1/4} period, that is tucked between the upper and lower vertical magnetic structure sections. A maximum peak oscillating field of 0.095 T at a frequency up to 1 Hz will be achieved by excitation of the horizontal poles with a trapezoidal current waveform. The vacuum chamber is an unconventional design that is removable from the magnetic structure, after magnetic measurements, for UHV processing. The chamber is fabricated from non-magnetic stainless steel to minimize the effects of eddy currents. Device design is presented.

  3. Fixation-resistant photoactivatable fluorescent proteins for correlative light and electron microscopy

    PubMed Central

    Paez Segala, Maria G.; Sun, Mei G.; Shtengel, Gleb; Viswanathan, Sarada; Baird, Michelle A.; Macklin, John J.; Patel, Ronak; Allen, John R.; Howe, Elizabeth S.; Piszczek, Grzegorz; Hess, Harald F.; Davidson, Michael W.; Wang, Yalin; Looger, Loren L.

    2014-01-01

    Fluorescent proteins facilitate a variety of imaging paradigms in live and fixed samples. However, they cease to function following heavy fixation, hindering advanced applications such as correlative light and electron microscopy. Here we report engineered variants of the photoconvertible Eos fluorescent protein that function normally in heavily fixed (0.5–1% OsO4), plastic resin-embedded samples, enabling correlative super-resolution fluorescence imaging and high-quality electron microscopy. PMID:25581799

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

    PubMed

    Killingsworth, Murray C; Bobryshev, Yuri V

    2016-01-01

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

  5. Hyperspectral microscopy to identify foodborne bacteria with optimum lighting source

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Hyperspectral microscopy is an emerging technology for rapid detection of foodborne pathogenic bacteria. Since scattering spectral signatures from hyperspectral microscopic images (HMI) vary with lighting sources, it is important to select optimal lights. The objective of this study is to compare t...

  6. Stroboscopic white-light interference microscopy.

    PubMed

    de Groot, Peter

    2006-08-10

    The principle of stroboscopic motion freezing of oscillating objects extends directly to interference microscopes that use coherence as part of the measurement principle. Analysis shows, however, that the fringe contrast loss for out-of-plane motion in stroboscopic interferometry is a wavelength-dependent phenomenon, which can alter the apparent nominal center wavelength of the white-light source. As in monochromatic systems, the key adjustable parameter is the duty cycle, equal to the product of the vibrational frequency and the pulse width. This theoretical study provides detailed graphs of expected errors as a function of the duty cycle, including fringe contrast loss, apparent wavelength shift, and measurement error. PMID:16926870

  7. Stroboscopic white-light interference microscopy

    SciTech Connect

    Groot, Peter de

    2006-08-10

    The principle of stroboscopic motion freezing of oscillating objects extends directly to interference microscopes that use coherence as part of the measurement principle. Analysis shows, however, that the fringe contrast loss for out-of-plan emotion in stroboscopic interferometry is a wavelength-dependent phenomenon,which can alter the apparent nominal center wavelength of the white-light source.As in monochromatic systems, the key adjustable parameter is the duty cycle,equal to the product of the vibrational frequency and the pulse width. This theoretical study provides detailed graphs of expected errors as a function of the duty cycle, including fringe contrast loss, apparent wavelength shift, and measurement error.

  8. A Simplified, Low-Cost Method for Polarized Light Microscopy

    PubMed Central

    Maude, Richard J.; Buapetch, Wanchana; Silamut, Kamolrat

    2009-01-01

    Malaria pigment is an intracellular inclusion body that appears in blood and tissue specimens on microscopic examination and can help in establishing the diagnosis of malaria. In simple light microscopy, it can be difficult to discern from cellular background and artifacts. It has long been known that if polarized light microscopy is used, malaria pigment can be much easier to distinguish. However, this technique is rarely used because of the need for a relatively costly polarization microscope. We describe a simple and economical technique to convert any standard light microscope suitable for examination of malaria films into a polarization microscope. PMID:19861611

  9. Microscopy imaging device with advanced imaging properties

    SciTech Connect

    Ghosh, Kunal; Burns, Laurie; El Gamal, Abbas; Schnitzer, Mark J.; Cocker, Eric; Ho, Tatt Wei

    2015-11-24

    Systems, methods and devices are implemented for microscope imaging solutions. One embodiment of the present disclosure is directed toward an epifluorescence microscope. The microscope includes an image capture circuit including an array of optical sensor. An optical arrangement is configured to direct excitation light of less than about 1 mW to a target object in a field of view of that is at least 0.5 mm.sup.2 and to direct epi-fluorescence emission caused by the excitation light to the array of optical sensors. The optical arrangement and array of optical sensors are each sufficiently close to the target object to provide at least 2.5 .mu.m resolution for an image of the field of view.

  10. Multimodal light-sheet microscopy for fluorescence live imaging

    NASA Astrophysics Data System (ADS)

    Oshima, Y.; Kajiura-Kobayashi, H.; Nonaka, S.

    2012-03-01

    Light-sheet microscopy, it is known as single plane illumination microscope (SPIM), is a fluorescence imaging technique which can avoid phototoxic effects to living cells and gives high contrast and high spatial resolution by optical sectioning with light-sheet illumination in developmental biology. We have been developed a multifunctional light-sheet fluorescence microscopy system with a near infrared femto-second fiber laser, a high sensitive image sensor and a high throughput spectrometer. We performed that multiphoton fluorescence images of a transgenic fish and a mouse embryo were observed on the light-sheet microscope. As the results, two photon images with high contrast and high spatial resolution were successfully obtained in the microscopy system. The system has multimodality, not only mutiphoton fluorescence imaging, but also hyperspectral imaging, which can be applicable to fluorescence unmixing analysis and Raman imaging. It enables to obtain high specific and high throughput molecular imaging in vivo and in vitro.

  11. The LBL advanced light source

    SciTech Connect

    Jackson, A.; Chattopadhyay, S.; Keller, R.; Kim, C.; Nishimura, H.; Selph, F.; Zisman, M.

    1988-06-01

    The LBL Advanced Light Source (ALS) will be a third generation synchrotron radiation facility. It is based on a low emittance 1--2 GeV electron storage ring (natural radial emittance <10 nm-rad), optimized to produce extremely bright beams of electromagnetic radiation (in the energy range from a few eV to around one keV) from insertion devices known as undulators. The storage ring is fed from an injection system consisting of a 50 MeV linac and a 1.5 GeV, 1 Hz, booster synchrotron, which can fill the ring to its normal operating current (400 mA, multibunch, or 7.6 mA, single bunch) in a few minutes. As well as high brightness (which is a consequence of the very small electron beam emittance in the storage ring), the design emphasizes: picosecond timestructure, laserlike coherence properties, narrow bandwidth, and long beam lifetimes. The more familiar continuous synchrotron radiation spectrum will be available from bending magnets and from wiggler magnets. This paper gives a general description of the ALS and discusses some of the significant design issues associated with the low emittance storage ring that is required for this new facility. 7 refs., 6 figs., 2 tabs.

  12. Investigating Bacterial-Animal Symbioses with Light Sheet Microscopy

    PubMed Central

    Taormina, Michael J.; Jemielita, Matthew; Stephens, W. Zac; Burns, Adam R.; Troll, Joshua V.; Parthasarathy, Raghuveer; Guillemin, Karen

    2014-01-01

    SUMMARY Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and dynamics of microbial communities in this complex environment. We report the use of light sheet microscopy to provide high resolution imaging of bacterial colonization of the zebrafish intestine. The methodology allows us to characterize bacterial population dynamics across the entire organ and the behaviors of individual bacterial and host cells throughout the colonization process. The large four-dimensional datasets generated by these imaging approaches require new strategies for image analysis. When integrated with other “omics” datasets, information about the spatial and temporal dynamics of microbial cells within the vertebrate intestine will provide new mechanistic insights into how microbial communities assemble and function within hosts. PMID:22983029

  13. Investigating bacterial-animal symbioses with light sheet microscopy.

    PubMed

    Taormina, Michael J; Jemielita, Matthew; Stephens, W Zac; Burns, Adam R; Troll, Joshua V; Parthasarathy, Raghuveer; Guillemin, Karen

    2012-08-01

    Microbial colonization of the digestive tract is a crucial event in vertebrate development, required for maturation of host immunity and establishment of normal digestive physiology. Advances in genomic, proteomic, and metabolomic technologies are providing a more detailed picture of the constituents of the intestinal habitat, but these approaches lack the spatial and temporal resolution needed to characterize the assembly and dynamics of microbial communities in this complex environment. We report the use of light sheet microscopy to provide high-resolution imaging of bacterial colonization of the intestine of Danio rerio, the zebrafish. The method allows us to characterize bacterial population dynamics across the entire organ and the behaviors of individual bacterial and host cells throughout the colonization process. The large four-dimensional data sets generated by these imaging approaches require new strategies for image analysis. When integrated with other "omics" data sets, information about the spatial and temporal dynamics of microbial cells within the vertebrate intestine will provide new mechanistic insights into how microbial communities assemble and function within hosts. PMID:22983029

  14. eduSPIM: Light Sheet Microscopy in the Museum

    PubMed Central

    Schmid, Benjamin; Weber, Michael; Huisken, Jan

    2016-01-01

    Light Sheet Microscopy in the Museum Light sheet microscopy (or selective plane illumination microscopy) is an important imaging technique in the life sciences. At the same time, this technique is also ideally suited for community outreach projects, because it produces visually appealing, highly dynamic images of living organisms and its working principle can be understood with basic optics knowledge. Still, the underlying concepts are widely unknown to the non-scientific public. On the occasion of the UNESCO International Year of Light, a technical museum in Dresden, Germany, launched a special, interactive exhibition. We built a fully functional, educational selective plane illumination microscope (eduSPIM) to demonstrate how developments in microscopy promote discoveries in biology. Design Principles of an Educational Light Sheet Microscope To maximize educational impact, we radically reduced a standard light sheet microscope to its essential components without compromising functionality and incorporated stringent safety concepts beyond those needed in the lab. Our eduSPIM system features one illumination and one detection path and a sealed sample chamber. We image fixed zebrafish embryos with fluorescent vasculature, because the structure is meaningful to laymen and visualises the optical principles of light sheet microscopy. Via a simplified interface, visitors acquire fluorescence and transmission data simultaneously. The eduSPIM Design Is Tailored Easily to Fit Numerous Applications The universal concepts presented here may also apply to other scientific approaches that are communicated to laymen in interactive settings. The specific eduSPIM design is adapted easily for various outreach and teaching activities. eduSPIM may even prove useful for labs needing a simple SPIM. A detailed parts list and schematics to rebuild eduSPIM are provided. PMID:27560188

  15. Advanced lighting guidelines: 1993. Final report

    SciTech Connect

    Eley, C.; Tolen, T.M.; Benya, J.R.; Rubinstein, F.; Verderber, R.

    1993-12-31

    The 1993 Advanced Lighting Guidelines document consists of twelve guidelines that provide an overview of specific lighting technologies and design application techniques utilizing energy-efficient lighting practice. Lighting Design Practice assesses energy-efficient lighting strategies, discusses lighting issues, and explains how to obtain quality lighting design and consulting services. Luminaires and Lighting Systems surveys luminaire equipment designed to take advantage of advanced technology lamp products and includes performance tables that allow for accurate estimation of luminaire light output and power input. The additional ten guidelines -- Computer-Aided Lighting Design, Energy-Efficient Fluorescent Ballasts, Full-Size Fluorescent Lamps, Compact Fluorescent Lamps, Tungsten-Halogen Lamps, Metal Halide and HPS Lamps, Daylighting and Lumen Maintenance, Occupant Sensors, Time Scheduling Systems, and Retrofit Control Technologies -- each provide a product technology overview, discuss current products on the lighting equipment market, and provide application techniques. This document is intended for use by electric utility personnel involved in lighting programs, lighting designers, electrical engineers, architects, lighting manufacturers` representatives, and other lighting professionals.

  16. Microwave Processing of Crowns from Winter Cereals for Light Microscopy.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Microwave processing of tissue considerably shortens the time it takes to prepare samples for light and electron microscopy. However, plant tissues from different species and different regions of the plant respond differently making it impossible to use a single protocol for all plant tissue. The ...

  17. The CryoCapsule: Simplifying correlative light to electron microscopy

    PubMed Central

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

    2014-01-01

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

  18. Structured light illumination for extended resolution in fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Fedosseev, R.; Belyaev, Y.; Frohn, J.; Stemmer, A.

    2005-03-01

    During the last two decades fluorescence microscopy has become a powerful experimental tool in modern biology. Resolution of optical microscopes is limited by the diffraction nature of light and amounts to approximately 200 nm for point objects imaged with green light and high-NA objectives. Recently, several successful attempts have been made to break the resolution limit of microscopes. One of them is the so-called harmonic excitation light microscopy. 2D structured illumination produced by four interfering laser beams improves the lateral resolution by a factor of 2 to reach 100 nm. Structured illumination extends optical resolution since spatial frequencies beyond the classical cut-off frequency are brought into the passband of the optical microscope by frequency mixing. The extended passband is reconstructed computationally from several images acquired with shifted illumination patterns. Here we discuss an extension towards high resolution imaging of thick specimens by combining 2D structured illumination with deconvolution techniques.

  19. Advanced Light Source Activity Report 2000

    SciTech Connect

    Greiner, A.; Moxon, L.; Robinson, A.; Tamura, L.

    2001-04-01

    This is an annual report, detailing activities at the Advanced Light Source for the year 2000. It includes highlights of scientific research by users of the facility as well as information about the development of the facility itself.

  20. Advanced Light Source Activity Report 2002

    SciTech Connect

    Duque, Theresa; Greiner, Annette; Moxon, Elizabeth; Robinson, Arthur; Tamura, Lori

    2003-06-12

    This annual report of the Advanced Light Source details science highlights and facility improvements during the year. It also offers information on events sponsored by the facility, technical specifications, and staff and publication information.

  1. Multimode fibre: Light-sheet microscopy at the tip of a needle

    NASA Astrophysics Data System (ADS)

    Plöschner, Martin; Kollárová, Věra; Dostál, Zbyněk; Nylk, Jonathan; Barton-Owen, Thomas; Ferrier, David E. K.; Chmelík, Radim; Dholakia, Kishan; Čižmár, Tomáš

    2015-12-01

    Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre - an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki.

  2. Multimode fibre: Light-sheet microscopy at the tip of a needle.

    PubMed

    Plöschner, Martin; Kollárová, Věra; Dostál, Zbyněk; Nylk, Jonathan; Barton-Owen, Thomas; Ferrier, David E K; Chmelík, Radim; Dholakia, Kishan; Čižmár, Tomáš

    2015-01-01

    Light-sheet fluorescence microscopy has emerged as a powerful platform for 3-D volumetric imaging in the life sciences. Here, we introduce an important step towards its use deep inside biological tissue. Our new technique, based on digital holography, enables delivery of the light-sheet through a multimode optical fibre--an optical element with extremely small footprint, yet permitting complex control of light transport processes within. We show that this approach supports some of the most advanced methods in light-sheet microscopy: by taking advantage of the cylindrical symmetry of the fibre, we facilitate the wavefront engineering methods for generation of both Bessel and structured Bessel beam plane illumination. Finally, we assess the quality of imaging on a sample of fluorescent beads fixed in agarose gel and we conclude with a proof-of-principle imaging of a biological sample, namely the regenerating operculum prongs of Spirobranchus lamarcki. PMID:26657400

  3. Confocal microscopy of skin cancers: Translational advances toward clinical utility

    PubMed Central

    Rajadhyaksha, Milind

    2014-01-01

    Recent advances in translational research in and technology for confocal microscopy of skin cancers, toward clinical applications, are described. Advances in translational research are in diagnosis of melanoma in vivo, pre-operative mapping of lentigo maligna melanoma margins to guide surgery and intra-operative imaging of residual basal cell carcinomas to guide shave-biopsy. Advances in technology include mosaicing microscopy for detection of basal cell carcinomas in large areas of excised tissue, toward rapid pathology-at-the-bedside, and development of small, simple and low-cost line-scanning confocal microscopes for worldwide use in diverse primary healthcare settings. Current limitations and future opportunities and challenges for both clinicians and technologists are discussed. PMID:19964286

  4. Advances in light curing adhesives

    NASA Astrophysics Data System (ADS)

    Bachmann, Andy

    2001-11-01

    This paper describes the development of a new family of light curing adhesives containing a new reactive additive previously not used in optical grade light curing adhesives are obtained with the addition of functionalized cellulositics. Outgassing as low as 10-6 grams/gram has been observed based on headspace sampling. Other additives have lowered the shrinkage rates of positioning adhesives from near 1 percent to less than 0.1 percent with fractional, percentage movements over thermal range of -40 degrees C to +200 degrees C.

  5. Condenser-free contrast methods for transmitted-light microscopy

    PubMed Central

    WEBB, K F

    2015-01-01

    Phase contrast microscopy allows the study of highly transparent yet detail-rich specimens by producing intensity contrast from phase objects within the sample. Presented here is a generalized phase contrast illumination schema in which condenser optics are entirely abrogated, yielding a condenser-free yet highly effective method of obtaining phase contrast in transmitted-light microscopy. A ring of light emitting diodes (LEDs) is positioned within the light-path such that observation of the objective back focal plane places the illuminating ring in appropriate conjunction with the phase ring. It is demonstrated that true Zernike phase contrast is obtained, whose geometry can be flexibly manipulated to provide an arbitrary working distance between illuminator and sample. Condenser-free phase contrast is demonstrated across a range of magnifications (4–100×), numerical apertures (0.13–1.65NA) and conventional phase positions. Also demonstrated is condenser-free darkfield microscopy as well as combinatorial contrast including Rheinberg illumination and simultaneous, colour-contrasted, brightfield, darkfield and Zernike phase contrast. By providing enhanced and arbitrary working space above the preparation, a range of concurrent imaging and electrophysiological techniques will be technically facilitated. Condenser-free phase contrast is demonstrated in conjunction with scanning ion conductance microscopy (SICM), using a notched ring to admit the scanned probe. The compact, versatile LED illumination schema will further lend itself to novel next-generation transmitted-light microscopy designs. The condenser-free illumination method, using rings of independent or radially-scanned emitters, may be exploited in future in other electromagnetic wavebands, including X-rays or the infrared. PMID:25226859

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

    SciTech Connect

    Timmermans, F. J.; Otto, C.

    2015-01-15

    New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

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

    NASA Astrophysics Data System (ADS)

    Timmermans, F. J.; Otto, C.

    2015-01-01

    New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

  8. High-energy diffraction microscopy at the advanced photon source

    SciTech Connect

    Lienert, U.; Li, S.; Hefferan, C.; Lind, J.; Suter, R.; Bernier, J.; Barton, N.; Brandes, M.; Mills, M.; Miller, M.; Jakobsen, B.; Pantleon, W.

    2012-02-28

    The status of the High Energy Diffraction Microscopy (HEDM) program at the 1-ID beam line of the Advanced Photon Source is reported. HEDM applies high energy synchrotron radiation for the grain and sub-grain scale structural and mechanical characterization of polycrystalline bulk materials in situ during thermomechanical loading. Case studies demonstrate the mapping of grain boundary topology, the evaluation of stress tensors of individual grains during tensile deformation and comparison to a finite element modeling simulation, and the characterization of evolving dislocation structure. Complementary information is obtained by post mortem electron microscopy on the same sample volume previously investigated by HEDM.

  9. High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.

    PubMed

    Koga, Daisuke; Kusumi, Satoshi; Shodo, Ryusuke; Dan, Yukari; Ushiki, Tatsuo

    2015-12-01

    In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. PMID:26206941

  10. Correlative light-ion microscopy for biological applications

    NASA Astrophysics Data System (ADS)

    Bertazzo, Sergio; von Erlach, Thomas; Goldoni, Silvia; Çandarlıoğlu, Pelin L.; Stevens, Molly M.

    2012-04-01

    Here we report a new technique, Correlative Light-Ion Microscopy (CLIM), to correlate SEM-like micrographs with fluorescence images. This technique presents significant advantages over conventional methods in enabling topographical and biochemical information to be correlated with nanoscale resolution without destroying the fluorescence signal. We demonstrate the utility of CLIM for a variety of investigations of cell substrate interactions validating its potential to become a routine procedure in biomedical research.Here we report a new technique, Correlative Light-Ion Microscopy (CLIM), to correlate SEM-like micrographs with fluorescence images. This technique presents significant advantages over conventional methods in enabling topographical and biochemical information to be correlated with nanoscale resolution without destroying the fluorescence signal. We demonstrate the utility of CLIM for a variety of investigations of cell substrate interactions validating its potential to become a routine procedure in biomedical research. Electronic supplementary information (ESI) available. See DOI: 10.1039/c2nr30431g

  11. The Fluids Integrated Rack and Light Microscopy Module Integrated Capabilities

    NASA Technical Reports Server (NTRS)

    Motil, Susan M.; Gati, Frank; Snead, John H.; Hill, Myron E.; Griffin, DeVon W.

    2003-01-01

    The Fluids Integrated Rack (FIR), a facility class payload, and the Light Microscopy Module (LMM), a subrack payload, are scheduled to be launched in 2005. The LMM integrated into the FIR will provide a unique platform for conducting fluids and biological experiments on ISS. The FIR is a modular, multi-user scientific research facility that will fly in the U.S. laboratory module, Destiny, of the International Space Station (ISS). The first payload in the FIR will be the Light Microscopy Module (LMM). The LMM is planned as a remotely controllable, automated, on-orbit microscope subrack facility, allowing flexible scheduling and control of fluids and biology experiments within the FIR. Key diagnostic capabilities for meeting science requirements include video microscopy to observe microscopic phenomena and dynamic interactions, interferometry to make thin film measurements with nanometer resolution, laser tweezers for particle manipulation, confocal microscopy to provide enhanced three-dimensional visualization of structures, and spectrophotometry to measure photonic properties of materials. The LMM also provides experiment sample containment for frangibles and fluids. This paper will provide a description of the current FIR and LMM designs, planned capabilities and key features. In addition a brief description of the initial five experiments planned for LMM/FIR will be provided.

  12. Study of nanoscale structural biology using advanced particle beam microscopy

    NASA Astrophysics Data System (ADS)

    Boseman, Adam J.

    This work investigates developmental and structural biology at the nanoscale using current advancements in particle beam microscopy. Typically the examination of micro- and nanoscale features is performed using scanning electron microscopy (SEM), but in order to decrease surface charging, and increase resolution, an obscuring conductive layer is applied to the sample surface. As magnification increases, this layer begins to limit the ability to identify nanoscale surface structures. A new technology, Helium Ion Microscopy (HIM), is used to examine uncoated surface structures on the cuticle of wild type and mutant fruit flies. Corneal nanostructures observed with HIM are further investigated by FIB/SEM to provide detailed three dimensional information about internal events occurring during early structural development. These techniques are also used to reconstruct a mosquito germarium in order to characterize unknown events in early oogenesis. Findings from these studies, and many more like them, will soon unravel many of the mysteries surrounding the world of developmental biology.

  13. Recent advancements in structured-illumination microscopy toward live-cell imaging.

    PubMed

    Hirano, Yasuhiro; Matsuda, Atsushi; Hiraoka, Yasushi

    2015-08-01

    Fluorescence microscopy allows us to observe fluorescently labeled molecules in diverse biological processes and organelle structures within living cells. However, the diffraction limit restricts its spatial resolution to about half of its wavelength, limiting the capability of biological observation at the molecular level. Structured-illumination microscopy (SIM), a type of super-resolution microscopy, doubles the spatial resolution in all three dimensions by illuminating the sample with a patterned excitation light, followed by computer reconstruction. SIM uses a relatively low illumination power compared with other methods of super-resolution microscopy and is easily available for multicolor imaging. SIM has great potential for meeting the requirements of live-cell imaging. Recent developments in diverse types of SIM have achieved higher spatial (∼50 nm lateral) and temporal (∼100 Hz) resolutions. Here, we review recent advancements in SIM and discuss its application in noninvasive live-cell imaging. PMID:26133185

  14. Three Dimensional Fluorescence Imaging Using Multiple Light-Sheet Microscopy

    PubMed Central

    Mohan, Kavya; Purnapatra, Subhajit B.; Mondal, Partha Pratim

    2014-01-01

    We developed a multiple light-sheet microscopy (MLSM) system capable of 3D fluorescence imaging. Employing spatial filter in the excitation arm of a SPIM system, we successfully generated multiple light-sheets. This improves upon the existing SPIM system and is capable of 3D volume imaging by simultaneously illuminating multiple planes in the sample. Theta detection geometry is employed for data acquisition from multiple specimen layers. This detection scheme inherits many advantages including, background reduction, cross-talk free fluorescence detection and high-resolution at long working distance. Using this technique, we generated equi-intense light-sheets of thickness approximately with an inter-sheet separation of . Moreover, the light-sheets generated by MLSM is found to be 2 times thinner than the state-of-art SPIM system. Imaging of fluorescently coated yeast cells of size (encaged in Agarose gel-matrix) is achieved. Proposed imaging technique may accelerate the field of fluorescence microscopy, cell biology and biophotonics. PMID:24911061

  15. The Advanced Light Source: Technical Design

    SciTech Connect

    Authors, Various

    1984-05-01

    The Advanced Light Source (ALS) is a synchrotron radiation source consisting of a 50-MeV linear accelerator, a 1.3-GeV 'booster' synchrotron, a 1.3-GeV electron storage ring, and a number of photon beam lines, as shown in Figure 1. As an introduction to a detailed description of the Advanced Light Source, this section provides brief discussions on the characteristics of synchrotron radiation and on the theory of storage rings. Appendix A contents: Introduction to Synchrotron-Radiation Sources; Storage Ring; Injection System; Control System; Insertion Devices; Photon Beam Lines; and References.

  16. Light microscopy applications in systems biology: opportunities and challenges

    PubMed Central

    2013-01-01

    Biological systems present multiple scales of complexity, ranging from molecules to entire populations. Light microscopy is one of the least invasive techniques used to access information from various biological scales in living cells. The combination of molecular biology and imaging provides a bottom-up tool for direct insight into how molecular processes work on a cellular scale. However, imaging can also be used as a top-down approach to study the behavior of a system without detailed prior knowledge about its underlying molecular mechanisms. In this review, we highlight the recent developments on microscopy-based systems analyses and discuss the complementary opportunities and different challenges with high-content screening and high-throughput imaging. Furthermore, we provide a comprehensive overview of the available platforms that can be used for image analysis, which enable community-driven efforts in the development of image-based systems biology. PMID:23578051

  17. Light sheet fluorescence microscopy (LSFM): past, present and future.

    PubMed

    Lim, John; Lee, Hwee Kuan; Yu, Weimiao; Ahmed, Sohail

    2014-10-01

    Light sheet fluorescence microscopy (LSFM) has emerged as an important imaging modality to follow biology in live 3D samples over time with reduced phototoxicity and photobleaching. In particular, LSFM has been instrumental in revealing the detail of early embryonic development of Zebrafish, Drosophila, and C. elegans. Open access projects, DIY-SPIM, OpenSPIM, and OpenSPIN, now allow LSFM to be set-up easily and at low cost. The aim of this paper is to facilitate the set-up and use of LSFM by reviewing and comparing open access projects, image processing tools and future challenges. PMID:25118817

  18. Synthetic holography in microscopy: opportunities arising from advanced wavefront shaping

    NASA Astrophysics Data System (ADS)

    Jesacher, Alexander; Ritsch-Marte, Monika

    2016-01-01

    The advent of computer-generated or synthetic holography has created a wealth of possibilities for wavefront shaping in optics. We discuss the impact this has had on optical microscopy. Synthetic Holographic Microscopy utilises wavefront shaping by a computer-generated 'hologram' (CGH) to modify light on the illumination or the detection side, or both. This enables modifications of the general sample appearance concerning contrast, resolution and other aspects. Multiplexing CGHs can perform several tasks at once, for instance splitting the image into sub-images corresponding to different depths in the sample, or displaying differently contrasted images of the sample, e.g. bright field, darkfield or (spiral) phase contrast, in different sub-images. We give an overview of the options and discuss the advantages and disadvantages of using programmable holographic elements inside an optical microscope.

  19. Fast frame scanning camera system for light-sheet microscopy.

    PubMed

    Wu, Di; Zhou, Xing; Yao, Baoli; Li, Runze; Yang, Yanlong; Peng, Tong; Lei, Ming; Dan, Dan; Ye, Tong

    2015-10-10

    In the interest of improving the temporal resolution for light-sheet microscopy, we designed a fast frame scanning camera system that incorporated a galvanometer scanning mirror into the imaging path of a home-built light-sheet microscope. This system transformed a temporal image sequence to a spatial one so that multiple images could be acquired during one exposure period. The improvement factor of the frame rate was dependent on the number of sub-images that could be tiled on the sensor without overlapping each other and was therefore a trade-off with the image size. As a demonstration, we achieved 960 frames/s (fps) on a CCD camera that was originally capable of recording images at only 30 fps (full frame). This allowed us to observe millisecond or sub-millisecond events with ordinary CCD cameras. PMID:26479797

  20. Laser light scattering instrument advanced technology development

    NASA Technical Reports Server (NTRS)

    Wallace, J. F.

    1993-01-01

    The objective of this advanced technology development (ATD) project has been to provide sturdy, miniaturized laser light scattering (LLS) instrumentation for use in microgravity experiments. To do this, we assessed user requirements, explored the capabilities of existing and prospective laser light scattering hardware, and both coordinated and participated in the hardware and software advances needed for a flight hardware instrument. We have successfully breadboarded and evaluated an engineering version of a single-angle glove-box instrument which uses solid state detectors and lasers, along with fiber optics, for beam delivery and detection. Additionally, we have provided the specifications and written verification procedures necessary for procuring a miniature multi-angle LLS instrument which will be used by the flight hardware project which resulted from this work and from this project's interaction with the laser light scattering community.

  1. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.

    PubMed

    Icha, Jaroslav; Schmied, Christopher; Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

  2. Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

    PubMed Central

    Sidhaye, Jaydeep; Tomancak, Pavel; Preibisch, Stephan; Norden, Caren

    2016-01-01

    Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments. PMID:27167079

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

    PubMed

    Beznoussenko, Galina V; Mironov, Alexander A

    2015-01-01

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

  4. Analysis of healing after myocardial infarction using polarized light microscopy.

    PubMed Central

    Whittaker, P.; Boughner, D. R.; Kloner, R. A.

    1989-01-01

    To better understand the healing process after permanent coronary artery occlusion in a canine model, the authors used polarized light microscopy. At 6 weeks after occlusion the scar collagen was mainly type I. Some regions of the scar contained a fiber lattice which appeared to be type III collagen. Collagen orientation was measured using a universal stage; subepicardial collagen was obliquely aligned (-14.0 +/- 3.5 degrees), midmyocardial collagen circumferentially aligned (1.4 +/- 0.4 degrees) and subendocardial collagen obliquely aligned (12.7 +/- 2.1 degrees). The molecular organization of scar collagen increased from 1 to 6 weeks after occlusion. Muscle cell disarray, similar to that in hypertrophic cardiomyopathy, was seen in the viable muscle adjacent to the scar. Such abnormal organization extended as far as 1 cm from the edge of the scar. The ability of polarized light microscopy to assess these different parameters from histologic sections demonstrates that it is a useful adjunct to other methods commonly used to study myocardial healing. Images p[889]-a Figure 7 Figure 1 p883-a Figure 6 PMID:2705508

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

    PubMed

    Shotton, D M

    1995-08-01

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

  6. VIIRS Nighttime Lights: Advances in Satellite Low-Light Imaging

    NASA Astrophysics Data System (ADS)

    Hsu, F.; Baugh, K.; Elvidge, C.; Zhizhin, M. N.

    2013-12-01

    The Soumi National Polar-orbiting Partnership (SNPP) Visible Infrared Imaging Radiometer Suite (VIIRS) Day-Night Band (DNB) represents a major advance in low-light imaging over previous data sources. Building on 18 years of experience compositing nighttime data from the Defense Meteorological Satellite Program (DMSP) Operational Linescan System (OLS), NOAA's NGDC Earth Observation Group created the first global VIIRS nighttime lights composite product by adapting their algorithms to process these new data. Compositing nighttime data involves combining only high quality data components over a period of time to improve sensitivity and coverage. Flag image were compiled to describe image quality. The initial flag categories included: daytime, twilight, stray light, non-zero lunar illuminance, noisy edge of scan data, clouds, and no data. High quality data included in the nighttime lights composite is defined as not having any of these attributes present. After the initial adaptation of heritage OLS algorithms, the authors sought to improve the sharpness of lights in the composite by adding additional flag categories. These include a refined VIIRS cloud mask, a mask based on cloud optical thickness, and a ranking of sharpness of light.. The VIIRS cloud mask, which is a JPSS retained intermediate product cloud mask (IICMO) is refined to reject the misclassification of hot sources like gas flares as cloud. Another JPSS retained intermediate product, cloud optical thickness, also provides valuable information about the clarity of atmosphere. The authors also implemented a sharp light detector to further characterize the quality of light for each pixel. Results of compositing multiple months in 2013 using these new flag categories are presented to demonstrate the improvements in nighttime lights composite quality.

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

    PubMed Central

    Whittaker, Peter; Przyklenk, Karin

    2009-01-01

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

  8. Tracking liquid in drying colloidal fluids with polarized light microscopy

    NASA Astrophysics Data System (ADS)

    Cho, Kun; Park, Jung Soo; Kim, Joon Heon; Weon, Byung Mook

    2014-11-01

    When colloidal fluids dry, tracking liquid surfaces around colloids is difficult with conventional imaging techniques. Here we show that polarized light microscopy (PM) is very useful in tracking liquid surfaces during drying processes of colloidal fluids. In particular, the PM mode is not a new or difficult way but is able to visualize liquid films above colloids in real time. We demonstrate that when liquid films above colloidal particles are broken, the PM patterns appear clearly: this feature is useful to identify the moment of liquid film rupture above colloids in drying colloidal fluids. This result is helpful to improve relevant processes such as inkjet printing, painting, and nanoparticle patterning (K.C. and J.S.P. equally contributed). This work (NRF-2013R1A22A04008115) was supported by Mid-career Researcher Program through NRF grant funded by the MEST.

  9. The Light Microscopy Module Design and Performance Demonstrations

    NASA Technical Reports Server (NTRS)

    Motil, Susan M.; Snead, John H.; Griffin, DeVon W.; Hovenac, Edward A.

    2003-01-01

    The Light Microscopy Module (LMM) is a state-of-the-art space station payload to provide investigations in the fields of fluids, condensed matter physics, and biological sciences. The LMM hardware will reside inside the Fluids Integrated Rack (FIR), a multi-user facility class payload that will provide fundamental services for the LMM and future payloads. LMM and FIR will be launched in 2005 and both will reside in the Destiny module of the International Space Station (ISS). There are five experiments to be performed within the LMM. This paper will provide a description of the initial five experiments: the supporting FIR subsystems; LMM design; capabilities and key features; and a summary of performance demonstrations.

  10. LIGHT SOURCE: Conceptual design of Hefei advanced light source

    NASA Astrophysics Data System (ADS)

    Li, Wei-Min; Wang, Lin; Feng, Guang-Yao; Zhang, Shan-Cai; Wu, Cong-Feng; Xu, Hong-Liang; Liu, Zu-Ping

    2009-06-01

    The conceptual of Hefei Advanced Light Source, which is an advanced VUV and Soft X-ray source, was developed at NSRL of USTC. According to the synchrotron radiation user requirements and the trends of SR source development, some accelerator-based schemes were considered and compared; furthermore storage ring with ultra low emittance was adopted as the baseline scheme of HALS. To achieve ultra low emittance, some focusing structures were studied and optimized in the lattice design. Compromising of emittance, on-momentum and off-momentum dynamic aperture and ring scale, five bend acromat (FBA) was employed. In the preliminary design of HALS, the emittance was reduced to sub nm · rad, thus the radiation up to water window has full lateral coherence. The brilliance of undulator radiation covering several eVs to keVs range is higher than that of HLS by several orders. The HALS should be one of the most advanced synchrotron radiation light sources in the world.

  11. Advanced analytical electron microscopy for alkali-ion batteries

    SciTech Connect

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

    2015-01-01

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

  12. Advanced Motion Compensation Methods for Intravital Optical Microscopy

    PubMed Central

    Vinegoni, Claudio; Lee, Sungon; Feruglio, Paolo Fumene; Weissleder, Ralph

    2013-01-01

    Intravital microscopy has emerged in the recent decade as an indispensible imaging modality for the study of the micro-dynamics of biological processes in live animals. Technical advancements in imaging techniques and hardware components, combined with the development of novel targeted probes and new mice models, have enabled us to address long-standing questions in several biology areas such as oncology, cell biology, immunology and neuroscience. As the instrument resolution has increased, physiological motion activities have become a major obstacle that prevents imaging live animals at resolutions analogue to the ones obtained in vitro. Motion compensation techniques aim at reducing this gap and can effectively increase the in vivo resolution. This paper provides a technical review of some of the latest developments in motion compensation methods, providing organ specific solutions. PMID:24273405

  13. Nanoscale membrane organization: where biochemistry meets advanced microscopy

    PubMed Central

    Cambi, Alessandra; Lidke, Diane S.

    2011-01-01

    Understanding the molecular mechanisms that shape an effective cellular response is a fundamental question in biology. Biochemical measurements have revealed critical information about the order of protein-protein interactions along signaling cascades, but lack the resolution to determine kinetics and localization of interactions on the plasma membrane. Furthermore, the local membrane environment influences membrane receptor distributions and dynamics, which in turn influences signal transduction. To measure dynamic protein interactions and elucidate the consequences of membrane architecture interplay, direct measurements at high spatiotemporal resolution are needed. In this review, we discuss the biochemical principles regulating membrane nanodomain formation and protein function, ranging from the lipid nanoenvironment to the cortical cytoskeleton. We also discuss recent advances in fluorescence microscopy that are making it possible to quantify protein organization and biochemical events at the nanoscale in the living cell membrane. PMID:22004174

  14. Advanced analytical electron microscopy for alkali-ion batteries

    DOE PAGESBeta

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

    2015-01-01

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

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

    PubMed

    Prozorowska, Ewelina; Jackowiak, Hanna

    2015-03-01

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

  16. Total internal reflection and dynamic light scattering microscopy of gels

    NASA Astrophysics Data System (ADS)

    Gregor, Brian F.

    Two different techniques which apply optical microscopy in novel ways to the study of biological systems and materials were built and applied to several samples. The first is a system for adapting the well-known technique of dynamic light scattering (DLS) to an optical microscope. This can detect and scatter light from very small volumes, as compared to standard DLS which studies light scattering from volumes 1000x larger. The small scattering volume also allows for the observation of nonergodic dynamics in appropriate samples. Porcine gastric mucin (PGM) forms a gel at low pH which lines the epithelial cell layer and acts as a protective barrier against the acidic stomach environment. The dynamics and microscopic viscosity of PGM at different pH levels is studied using polystyrene microspheres as tracer particles. The microscopic viscosity and microrheological properties of the commercial basement membrane Matrigel are also studied with this instrument. Matrigel is frequently used to culture cells and its properties remain poorly determined. Well-characterized and purely synthetic Matrigel substitutes will need to have the correct rheological and morphological characteristics. The second instrument designed and built is a microscope which uses an interferometry technique to achieve an improvement in resolution 2.5x better in one dimension than the Abbe diffraction limit. The technique is based upon the interference of the evanescent field generated on the surface of a prism by a laser in a total internal reflection geometry. The enhanced resolution is demonstrated with fluorescent samples. Additionally. Raman imaging microscopy is demonstrated using the evanescent field in resonant and non-resonant samples, although attempts at applying the enhanced resolution technique to the Raman images were ultimately unsuccessful. Applications of this instrument include high resolution imaging of cell membranes and macroscopic structures in gels and proteins. Finally, a third

  17. Wiring patterns in the mouse retina: collecting evidence across the connectome, physiology and light microscopy

    PubMed Central

    Dunn, Felice A; Wong, Rachel O L

    2014-01-01

    The visual system has often been thought of as a parallel processor because distinct regions of the brain process different features of visual information. However, increasing evidence for convergence and divergence of circuit connections, even at the level of the retina where visual information is first processed, chips away at a model of dedicated and distinct pathways for parallel information flow. Instead, our current understanding is that parallel channels may emerge, not from exclusive microcircuits for each channel, but from unique combinations of microcircuits. This review depicts diagrammatically the current knowledge and remaining puzzles about the retinal circuit with a focus on the mouse retina. Advances in techniques for labelling cells and genetic manipulations have popularized the use of transgenic mice. We summarize evidence gained from serial electron microscopy, electrophysiology and light microscopy to illustrate the wiring patterns in mouse retina. We emphasize the need to explore proposed retinal connectivity using multiple methods to verify circuits both structurally and functionally. PMID:25172948

  18. Advanced Light Source beam position monitor

    SciTech Connect

    Hinkson, J.

    1991-10-28

    The Advanced Light Source (ALS) is a synchrotron radiation facility nearing completion at LBL. As a third-generation machine, the ALS is designed to produce intense light from bend magnets, wigglers, and undulators (insertion devices). The facility will include a 50 MeV electron linear accelerator, a 1.5 GeV booster synchrotron, beam transport lines, a 1--2 GeV storage ring, insertion devices, and photon beam lines. Currently, the beam injection systems are being commissioned, and the storage ring is being installed. Electron beam position monitors (BPM) are installed throughout the accelerator and constitute the major part of accelerator beam diagnostics. The design of the BPM instruments is complete, and 50 units have been constructed for use in the injector systems. We are currently fabricating 100 additional instruments for the storage ring. In this paper I discuss engineering fabrication, testing and performance of the beam pickup electrodes and the BPM electronics.

  19. An Upgrade for the Advanced Light Source

    SciTech Connect

    Chemla, Daniel S.; Feinberg, Benedict; Hussain, Zahid; Kirz, Janos; Krebs, Gary F.; Padmore, Howard A.; Robin, David S.; Robinson, Arthur L.; Smith, Neville V.

    2004-09-01

    One of the first third-generation synchrotron light sources, the ALS, has been operating for almost a decade at Berkeley Lab, where experimenters have been exploiting its high brightness for forefront science. However, accelerator and insertion-device technology have significantly changed since the ALS was designed. As a result, the performance of the ALS is in danger of being eclipsed by that of newer, more advanced sources. The ALS upgrade that we are planning includes full-energy, top-off injection with higher storage-ring current and the replacement of five first-generation insertion devices with nine state-of-the art insertion devices and four new application-specific beamlines now being identified in a strategic planning process. The upgrade will help keep the ALS at the forefront of soft x-ray synchrotron light sources for the next two decades.

  20. Operator scheduling at the Advanced Light Source

    SciTech Connect

    Miller, B.

    1998-06-01

    Scheduling Operations staff at the Advanced Light Source (ALS) has evolved from 5 shifts/week for commissioning operations in 1992 to the present 24 hour/day, 21 shift coverage as the ALS went to full operation for users. A number of schedules were developed and implemented in an effort to accommodate changing ALS shift coverage requirements. The present work schedule and the lessons learned, address a number of issues that are useful to any facility that is operating 24 hours/day, 7 days/week.

  1. Magnet costs for the Advanced Light Source

    SciTech Connect

    Tanabe, J.; Krupnick, J.; Hoyer, E.; Paterson, A.

    1993-05-01

    The Advanced Light Source (ALS) accelerator is now completed. The numerous conventional magnets required for the booster ring, the storage ring and the low and high energy transfer lines were installed during the last two years. This paper summarizes the various costs associated with the quantity fabrication of selected magnet families. These costs include the costs of prototypes, tooling, coil and core fabrication, assembly and magnetic measurements. Brief descriptions of the magnets and specialized requirements for magnetic measurements are included in order to associate the costs with the relative complexities of the various magnet systems.

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

    NASA Astrophysics Data System (ADS)

    Pyrz, William D.

    Catalysis will continue to be vitally important to the advancement and sustainability of industrialized societies. Unfortunately, the petroleum-based resources that currently fuel the energy and consumer product needs of an advancing society are becoming increasingly difficult and expensive to extract as supplies diminish and the quality of sources degrade. Therefore, the development of sustainable energy sources and the improvement of the carbon efficiency of existing chemical processes are critical. Further challenges require that these initiatives are accomplished in an environmentally friendly fashion since the effects of carbon-based emissions are proving to be a serious threat to global climate stability. In this dissertation, materials being developed for sustainable energy and process improvement initiatives are studied. Our approach is to use materials characterization, namely advanced electron microscopy, to analyze the targeted systems at the nano- or Angstrom-scale with the goal of developing useful relationships between structure, composition, crystalline order, morphology, and catalytic performance. One area of interest is the complex Mo-V-M-O (M=Te, Sb, Ta, Nb) oxide system currently being developed for the selective oxidation/ammoxidation of propane to acrylic acid or acrylonitrile, respectively. Currently, the production of acrylic acid and acrylonitrile rely on propylene-based processes, yet significant cost savings could be realized if the olefin-based feeds could be replaced by paraffin-based ones. The major challenge preventing this feedstock replacement is the development of a suitable paraffin-activating catalyst. Currently, the best candidate is the Mo-V-Nb-Te-O complex oxide catalyst that is composed of two majority phases that are commonly referred to as M1 and M2. However, there is a limited understanding of the roles of each component with respect to how they contribute to catalyst stability and the reaction mechanism. Aberration

  3. High-performance probes for light and electron microscopy

    PubMed Central

    Viswanathan, Sarada; Williams, Megan E.; Bloss, Erik B.; Stasevich, Timothy J.; Speer, Colenso M.; Nern, Aljoscha; Pfeiffer, Barret D.; Hooks, Bryan M.; Li, Wei-Ping; English, Brian P.; Tian, Teresa; Henry, Gilbert L.; Macklin, John J.; Patel, Ronak; Gerfen, Charles R.; Zhuang, Xiaowei; Wang, Yalin; Rubin, Gerald M.

    2015-01-01

    We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These “spaghetti monster” fluorescent proteins (smFPs) distribute well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localizes weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allow robust, orthogonal multi-color visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers, greatly increase the number of simultaneous imaging channels, and perform well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improve single-molecule image tracking and increase yield for RNA-Seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization. PMID:25915120

  4. High-performance probes for light and electron microscopy.

    PubMed

    Viswanathan, Sarada; Williams, Megan E; Bloss, Erik B; Stasevich, Timothy J; Speer, Colenso M; Nern, Aljoscha; Pfeiffer, Barret D; Hooks, Bryan M; Li, Wei-Ping; English, Brian P; Tian, Teresa; Henry, Gilbert L; Macklin, John J; Patel, Ronak; Gerfen, Charles R; Zhuang, Xiaowei; Wang, Yalin; Rubin, Gerald M; Looger, Loren L

    2015-06-01

    We describe an engineered family of highly antigenic molecules based on GFP-like fluorescent proteins. These molecules contain numerous copies of peptide epitopes and simultaneously bind IgG antibodies at each location. These 'spaghetti monster' fluorescent proteins (smFPs) distributed well in neurons, notably into small dendrites, spines and axons. smFP immunolabeling localized weakly expressed proteins not well resolved with traditional epitope tags. By varying epitope and scaffold, we generated a diverse family of mutually orthogonal antigens. In cultured neurons and mouse and fly brains, smFP probes allowed robust, orthogonal multicolor visualization of proteins, cell populations and neuropil. smFP variants complement existing tracers and greatly increase the number of simultaneous imaging channels, and they performed well in advanced preparations such as array tomography, super-resolution fluorescence imaging and electron microscopy. In living cells, the probes improved single-molecule image tracking and increased yield for RNA-seq. These probes facilitate new experiments in connectomics, transcriptomics and protein localization. PMID:25915120

  5. Light sheet microscopy reveals more gradual light attenuation in light green versus dark green soybean leaves

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This oversaturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light, causing a steep gradient in carbon fixation. Reducing chl content could create a mor...

  6. Breast cancer diagnosis using spatial light interference microscopy

    NASA Astrophysics Data System (ADS)

    Majeed, Hassaan; Kandel, Mikhail E.; Han, Kevin; Luo, Zelun; Macias, Virgilia; Tangella, Krishnarao; Balla, Andre; Popescu, Gabriel

    2015-11-01

    The standard practice in histopathology of breast cancers is to examine a hematoxylin and eosin (H&E) stained tissue biopsy under a microscope to diagnose whether a lesion is benign or malignant. This determination is made based on a manual, qualitative inspection, making it subject to investigator bias and resulting in low throughput. Hence, a quantitative, label-free, and high-throughput diagnosis method is highly desirable. We present here preliminary results showing the potential of quantitative phase imaging for breast cancer screening and help with differential diagnosis. We generated phase maps of unstained breast tissue biopsies using spatial light interference microscopy (SLIM). As a first step toward quantitative diagnosis based on SLIM, we carried out a qualitative evaluation of our label-free images. These images were shown to two pathologists who classified each case as either benign or malignant. This diagnosis was then compared against the diagnosis of the two pathologists on corresponding H&E stained tissue images and the number of agreements were counted. The agreement between SLIM and H&E based diagnosis was 88% for the first pathologist and 87% for the second. Our results demonstrate the potential and promise of SLIM for quantitative, label-free, and high-throughput diagnosis.

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

    PubMed

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

    2013-02-01

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

  8. Quantitative polarized light microscopy of unstained mammalian cochlear sections

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  9. Methods for studying tooth root cementum by light microscopy.

    PubMed

    Foster, Brian L

    2012-09-01

    The tooth root cementum is a thin, mineralized tissue covering the root dentin that is present primarily as acellular cementum on the cervical root and cellular cementum covering the apical root. While cementum shares many properties in common with bone and dentin, it is a unique mineralized tissue and acellular cementum is critical for attachment of the tooth to the surrounding periodontal ligament (PDL). Resources for methodologies for hard tissues often overlook cementum and approaches that may be of value for studying this tissue. To address this issue, this report offers detailed methodology, as well as comparisons of several histological and immunohistochemical stains available for imaging the cementum-PDL complex by light microscopy. Notably, the infrequently used Alcian blue stain with nuclear fast red counterstain provided utility in imaging cementum in mouse, porcine and human teeth. While no truly unique extracellular matrix markers have been identified to differentiate cementum from the other hard tissues, immunohistochemistry for detection of bone sialoprotein (BSP), osteopontin (OPN), and dentin matrix protein 1 (DMP1) is a reliable approach for studying both acellular and cellular cementum and providing insight into developmental biology of these tissues. Histological and immunohistochemical approaches provide insight on developmental biology of cementum. PMID:22996273

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

    PubMed

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

    2016-08-01

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

  11. Scanning magnetoresistive microscopy: An advanced characterization tool for magnetic nanosystems.

    PubMed

    Mitin, D; Grobis, M; Albrecht, M

    2016-02-01

    An advanced scanning magnetoresistive microscopy (SMRM) - a robust magnetic imaging and probing technique - will be presented, which utilizes state-of-the-art recording heads of a hard disk drive as sensors. The spatial resolution of modern tunneling magnetoresistive sensors is nowadays comparable to the more commonly used magnetic force microscopes. Important advantages of SMRM are the ability to detect pure magnetic signals directly proportional to the out-of-plane magnetic stray field, negligible sensor stray fields, and the ability to apply local bipolar magnetic field pulses up to 10 kOe with bandwidths from DC up to 1 GHz. Moreover, the SMRM can be further equipped with a heating stage and external magnetic field units. The performance of this method and corresponding best practices are demonstrated by presenting various examples, including a temperature dependent recording study on hard magnetic L1(0) FeCuPt thin films, imaging of magnetic vortex states in an in-plane magnetic field, and their controlled manipulation by applying local field pulses. PMID:26931856

  12. Scanning magnetoresistive microscopy: An advanced characterization tool for magnetic nanosystems

    NASA Astrophysics Data System (ADS)

    Mitin, D.; Grobis, M.; Albrecht, M.

    2016-02-01

    An advanced scanning magnetoresistive microscopy (SMRM) — a robust magnetic imaging and probing technique — will be presented, which utilizes state-of-the-art recording heads of a hard disk drive as sensors. The spatial resolution of modern tunneling magnetoresistive sensors is nowadays comparable to the more commonly used magnetic force microscopes. Important advantages of SMRM are the ability to detect pure magnetic signals directly proportional to the out-of-plane magnetic stray field, negligible sensor stray fields, and the ability to apply local bipolar magnetic field pulses up to 10 kOe with bandwidths from DC up to 1 GHz. Moreover, the SMRM can be further equipped with a heating stage and external magnetic field units. The performance of this method and corresponding best practices are demonstrated by presenting various examples, including a temperature dependent recording study on hard magnetic L10 FeCuPt thin films, imaging of magnetic vortex states in an in-plane magnetic field, and their controlled manipulation by applying local field pulses.

  13. Light sheet microscopy reveals more gradual light attenuation in light-green versus dark-green soybean leaves

    PubMed Central

    Slattery, Rebecca A.; Grennan, Aleel K.; Sivaguru, Mayandi; Sozzani, Rosangela; Ort, Donald R.

    2016-01-01

    Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This over-saturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light. Reducing chl content could create a more even leaf light distribution and thereby increase leaf light-use efficiency and overall canopy photosynthesis. This was tested on soybean cultivar ‘Clark’ (WT) and a near-isogenic chl b deficient mutant, Y11y11, grown in controlled environment chambers and in the field. Light attenuation was quantified using a novel approach involving light sheet microscopy. Leaf adaxial and abaxial surfaces were illuminated separately with blue, red, and green wavelengths, and chl fluorescence was detected orthogonally to the illumination plane. Relative fluorescence was significantly greater in deeper layers of the Y11y11 mesophyll than in WT, with the greatest differences in blue, then red, and finally green light when illuminated from the adaxial surface. Modeled relative photosynthesis based on chlorophyll profiles and Beer’s Law predicted less steep gradients in mutant relative photosynthesis rates compared to WT. Although photosynthetic light-use efficiency was greater in the field-grown mutant with ~50% lower chl, light-use efficiency was lower in the mutant when grown in chambers where chl was ~80% reduced. This difference is probably due to pleiotropic effects of the mutation that accompany very severe reductions in chlorophyll and may warrant further testing in other low-chl lines. PMID:27329746

  14. Light sheet microscopy reveals more gradual light attenuation in light-green versus dark-green soybean leaves.

    PubMed

    Slattery, Rebecca A; Grennan, Aleel K; Sivaguru, Mayandi; Sozzani, Rosangela; Ort, Donald R

    2016-08-01

    Light wavelengths preferentially absorbed by chlorophyll (chl) often display steep absorption gradients. This over-saturates photosynthesis in upper chloroplasts and deprives lower chloroplasts of blue and red light. Reducing chl content could create a more even leaf light distribution and thereby increase leaf light-use efficiency and overall canopy photosynthesis. This was tested on soybean cultivar 'Clark' (WT) and a near-isogenic chl b deficient mutant, Y11y11, grown in controlled environment chambers and in the field. Light attenuation was quantified using a novel approach involving light sheet microscopy. Leaf adaxial and abaxial surfaces were illuminated separately with blue, red, and green wavelengths, and chl fluorescence was detected orthogonally to the illumination plane. Relative fluorescence was significantly greater in deeper layers of the Y11y11 mesophyll than in WT, with the greatest differences in blue, then red, and finally green light when illuminated from the adaxial surface. Modeled relative photosynthesis based on chlorophyll profiles and Beer's Law predicted less steep gradients in mutant relative photosynthesis rates compared to WT. Although photosynthetic light-use efficiency was greater in the field-grown mutant with ~50% lower chl, light-use efficiency was lower in the mutant when grown in chambers where chl was ~80% reduced. This difference is probably due to pleiotropic effects of the mutation that accompany very severe reductions in chlorophyll and may warrant further testing in other low-chl lines. PMID:27329746

  15. Measuring nanometer, three-dimensional motions with light microscopy

    NASA Astrophysics Data System (ADS)

    Davis, Charles Quentin

    Computational motion analysis of images from a light microscope is used to explore mechanics in the microscopic worlds of cells and man-made micromachines. Images of a moving target are taken using a strobe light and a CCD camera attached to a microscope. Temporal sequences of stop-action images are recorded at multiple planes of focus to characterize three-dimensional motions. We demonstrate that motion resolution is limited by the motion detection algorithm. The largest source of error in gradient-based and matching algorithms is statistical bias. We present a new algorithm with errors that are typically smaller than 0.02 pixels. With this algorithm and our video microscopy system, we can measure motions of submicrometer targets with nanometer accuracy. The system has been used to study both hearing structures and man-made micromachines. Analysis of electrically- induced motions of a microfabricated accelerometer and a microfabricated angular velocity sensor allow visualization and quantification of complex modes of motion that limit the performance of these sensors. Pilot studies demonstrate that the system can also measure sound-induced motions of the human stapes, where it can resolve complex modes of motion thought to be important for loud sounds and for certain middle-ear pathologies. The system has also been used to measure mechanical deformations in the mouse tectorial membrane, a gelatinous inner-ear structure that has previously been a difficult target of study. Our major application has been the study of sound-induced motions of sensory cells and accessory structures in the ear of a lizard. Our results, while still preliminary, are the first direct measurements of sound-induced motions of hair bundles and their overlying tectorial membrane. They indicate that the tectorial membrane does not move as a rigid body but has significant shear throughout its thickness. Therefore, the relative motion between the tectorial membrane and the reticular lamina is

  16. Performance of the Advanced Light Source

    SciTech Connect

    Jackson, A.

    1994-06-01

    The Advanced Light Source (ALS) at the Lawrence Berkeley Laboratory (LBL) is the first of the lower energy (1--2 GeV) third-generation synchrotron radiation facilities to come into operation. Designed with very small electron beam emittances to operate with long insertion devices producing very high brightness beams of synchrotron radiation in the VUV and soft x-ray regions of the spectrum, these facilities are complementary to the higher energy (6--9 GeV) facilities designed for harder x-radiation. The ALS storage ring began operation in October 1993. In this paper, we will review the operational performance of the ALS, including the effects of the 4.5 m long undulators (period 5 cm), and discuss the overall performance of the facility.

  17. Superbend upgrade of the Advanced Light Source

    SciTech Connect

    Robin, D.; Krupnick, J.; Schlueter, R.; Steier, C.; Marks, S.; Wang, B.; Zbasnik, J.; Benjegerdes, R.; Biocca, A.; Bish, P.; Brown, W.; Byrne, W.; Chen, J.; Decking, W.; DeVries, J.; DeMarco, W.R.; Fahmie, M.; Geyer, A.; Harkins, J.; Henderson, T.; Hinkson, J.; Hoyer, E.; Hull, D.; Jacobson, S.; McDonald, J.; Molinari, P.; Mueller, R.; Nadolski, L.; Nishimura, H.; Nishimura, K.; Ottens, F.; Paterson, J.A.; Pipersky, P.; Portmann, G.; Richie, A.; Rossi, S.; Salvant, B.; Scarvie, T.; Schmidt,A.; Spring, J.; Taylor, C.; Thur, W.; Timossi, C.; Wandesforde, A.

    2004-05-26

    The Advanced Light Source (ALS) is a third generation synchrotron light source located at Lawrence Berkeley National Laboratory (LBNL). There was an increasing demand at the ALS for additional high brightness hard x-ray beamlines in the 7 to 40 keV range. In response to that demand, the ALS storage ring was modified in August 2001. Three 1.3 Tesla normal conducting bending magnets were removed and replaced with three 5 Tesla superconducting magnets (Superbends). The radiation produced by these Superbends is an order of magnitude higher in photon brightness and flux at 12 keV than that of the 1.3 Tesla bends, making them excellent sources of hard x-rays for protein crystallography and other hard x-ray applications. At the same time the Superbends did not compromise the performance of the facility in the VUV and soft x-ray regions of the spectrum. The Superbends will eventually feed 12 new beamlines greatly enhancing the facility's capability and capacity in the hard x-ray region. The Superbend project is the biggest upgrade to the ALS storage ring since it was commissioned in 1993. In this paper we present an overview of the Superbend project, its challenges and the resulting impact on the ALS.

  18. Advanced Light Source: Activity report 1993

    SciTech Connect

    Not Available

    1994-11-01

    The Advanced Light Source (ALS) produces the world`s brightest light in the ultraviolet and soft x-ray regions of the spectrum. The first low-energy third-generation synchrotron source in the world, the ALS provides unprecedented opportunities for research in science and technology not possible anywhere else. This year marked the beginning of operations and the start of the user research program at the ALS, which has already produced numerous high quality results. A national user facility located at Lawrence Berkeley Laboratory of the University of California, the ALS is available to researchers from academia, industry, and government laboratories. This report contains the following: (1) director`s message; (2) operations overview; (3) user program; (4) users` executive committee; (5) industrial outreach; (6) accelerator operations; (7) beamline control system; (8) insertion devices; (9) experimental systems; (10) beamline engineering; (11) first results from user beamlines; (12) beamlines for 1994--1995; (13) special events; (14) publications; (15) advisory panels; and (16) ALS staff.

  19. The development of optical microscopy techniques for the advancement of single-particle studies

    NASA Astrophysics Data System (ADS)

    Marchuk, Kyle

    Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called "non-blinking" quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to

  20. The development of optical microscopy techniques for the advancement of single-particle studies

    SciTech Connect

    Marchuk, Kyle

    2013-05-15

    Single particle orientation and rotational tracking (SPORT) has recently become a powerful optical microscopy tool that can expose many molecular motions. Unfortunately, there is not yet a single microscopy technique that can decipher all particle motions in all environmental conditions, thus there are limitations to current technologies. Within, the two powerful microscopy tools of total internal reflection and interferometry are advanced to determine the position, orientation, and optical properties of metallic nanoparticles in a variety of environments. Total internal reflection is an optical phenomenon that has been applied to microscopy to produce either fluorescent or scattered light. The non-invasive far-field imaging technique is coupled with a near-field illumination scheme that allows for better axial resolution than confocal microscopy and epi-fluorescence microscopy. By controlling the incident illumination angle using total internal reflection fluorescence (TIRF) microscopy, a new type of imaging probe called “non-blinking” quantum dots (NBQDs) were super-localized in the axial direction to sub-10-nm precision. These particles were also used to study the rotational motion of microtubules being propelled by the motor protein kinesin across the substrate surface. The same instrument was modified to function under total internal reflection scattering (TIRS) microscopy to study metallic anisotropic nanoparticles and their dynamic interactions with synthetic lipid bilayers. Utilizing two illumination lasers with opposite polarization directions at wavelengths corresponding to the short and long axis surface plasmon resonance (SPR) of the nanoparticles, both the in-plane and out-of-plane movements of many particles could be tracked simultaneously. When combined with Gaussian point spread function (PSF) fitting for particle super-localization, the binding status and rotational movement could be resolved without degeneracy. TIRS microscopy was also used to

  1. Advanced Light Source First-Phase Scientific Program, 1993/1994

    SciTech Connect

    Not Available

    1992-08-01

    This composite document outlines ten different experiments planned for the beamline at the Advanced Light Source. Researchers from various parts of the country have detailed their methods and equipment to be used in experiments in biology and physics. X-ray spectroscopy and microscopy are the common topics to these experiments. (GHH)

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

    PubMed Central

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

    2014-01-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2014-11-01

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

  4. Improved light microscopy counting method for accurately counting Plasmodium parasitemia and reticulocytemia.

    PubMed

    Lim, Caeul; Pereira, Ligia; Shardul, Pritish; Mascarenhas, Anjali; Maki, Jennifer; Rixon, Jordan; Shaw-Saliba, Kathryn; White, John; Silveira, Maria; Gomes, Edwin; Chery, Laura; Rathod, Pradipsinh K; Duraisingh, Manoj T

    2016-08-01

    Even with the advances in molecular or automated methods for detection of red blood cells of interest (such as reticulocytes or parasitized cells), light microscopy continues to be the gold standard especially in laboratories with limited resources. The conventional method for determination of parasitemia and reticulocytemia uses a Miller reticle, a grid with squares of different sizes. However, this method is prone to errors if not used correctly and counts become inaccurate and highly time-consuming at low frequencies of target cells. In this report, we outline the correct guidelines to follow when using a reticle for counting, and present a new counting protocol that is a modified version of the conventional method for increased accuracy in the counting of low parasitemias and reticulocytemias. Am. J. Hematol. 91:852-855, 2016. © 2016 Wiley Periodicals, Inc. PMID:27074559

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

    ERIC Educational Resources Information Center

    Krinsley, David Henry; Manley, Curtis Robert

    1989-01-01

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

  6. Wigglers at the Advanced Light Source

    SciTech Connect

    Hoyer, E.; Akre, J.; Humphries, D.

    1995-04-01

    Two 3.4 m long wigglers are being designed and constructed at Lawrence Berkeley Laboratory`s (LBL) Advanced Light Source (ALS). A 19 period planar wiggler with 16.0 cm period length is designed to provide photons up to 12.4 keV for protein crystallography. This device features a hybrid permanent magnet structure with tapered poles and designed to achieve 2.0 T at a 1.4 cm magnetic gap. An elliptical wiggler is being designed to provide circularly polarized photons in the energy range of 50 eV to 10 keV for magnetic circular dichroism spectroscopy. This device features vertical and horizontal magnetic structures of 14 and 14 {1/2} periods respectively of 20 cm period length. The vertical magnetic structure is a 2.0 T hybrid permanent magnet configuration. The horizontal structure is an iron core electromagnetic design, shifted longitudinally {1/4} period with respect to the vertical magnetic structure. A maximum horizontal peak field of 0.1 T at an oscillating frequency up to 1 Hz will be achieved by excitation of the horizontal poles with a trapezoidal current waveform.

  7. First undulators for the Advanced Light Source

    SciTech Connect

    Hoyer, E.; Akre, J.; Chin, J.

    1993-05-01

    The first three undulators, each 4.6 m in length, for the Advanced Light source (ALS) at Lawrence Berkeley Laboratory (LBL), are near completion and are undergoing qualification tests before installation into the storage ring. Two devices have 5.0-cm period lengths, 89 periods, and achieve an effective field of 0.85 T at the 14 mm minimum magnetic gap. The other device has a period length of 8.0 cm, 55 periods, and an effective field of 1.2 T at the minimum 14 mm gap. Measurements on the first 5 cm period device show the uncorrelated field errors to be 0.23%, which is less than the required 0.25%. Measurements of gap control show reproducibility of {plus_minus}5 microns or better. The first vacuum chamber, 5.0 m long, is flat to within 0.53 mm over the 4.6 m magnetic structure section and a 4 x 10{sup -11} Torr pressure was achieved during vacuum tests. Device description, fabrication, and measurements are presented.

  8. Advances in cryogenic transmission electron microscopy for the characterization of dynamic self-assembling nanostructures

    PubMed Central

    Newcomb, Christina J.; Moyer, Tyson J.; Lee, Sungsoo S.; Stupp, Samuel I.

    2012-01-01

    Elucidating the structural information of nanoscale materials in their solvent-exposed state is crucial, as a result, cryogenic transmission electron microscopy (cryo-TEM) has become an increasingly popular technique in the materials science, chemistry, and biology communities. Cryo-TEM provides a method to directly visualize the specimen structure in a solution-state through a thin film of vitrified solvent. This technique complements X-ray, neutron, and light scattering methods that probe the statistical average of all species present; furthermore, cryo-TEM can be used to observe changes in structure over time. In the area of self-assembly, this tool has been particularly powerful for the characterization of natural and synthetic small molecule assemblies, as well as hybrid organic–inorganic composites. In this review, we discuss recent advances in cryogenic TEM in the context of self-assembling systems with emphasis on characterization of transitions observed in response to external stimuli. PMID:23204913

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

    PubMed

    Kobayashi, Shouhei; Iwamoto, Masaaki; Haraguchi, Tokuko

    2016-08-01

    Fluorescence microscopy (FM) is a powerful tool for observing specific molecular components in living cells, but its spatial resolution is relatively low. In contrast, electron microscopy (EM) provides high-resolution information about cellular structures, but it cannot provide temporal information in living cells. To achieve molecular selectivity in imaging at high resolution, a method combining EM imaging with live-cell fluorescence imaging, known as live correlative light-EM (CLEM), has been developed. In this method, living cells are first observed by FM, fixed in situ during the live observation and then subjected to EM observation. Various fluorescence techniques and tools can be applied for FM, resulting in the generation of various modified methods that are useful for understanding cellular structure in high resolution. Here, we review the methods of CLEM and live-cell imaging associated with CLEM (live CLEM). Such methods can greatly advance the understanding of the function of cellular structures on a molecular level, and thus are useful for medical fields as well as for basic biology. PMID:27385786

  10. Advanced Solid State Lighting for Human Evaluation Project

    NASA Technical Reports Server (NTRS)

    Zeitlin, Nancy; Holbert, Eirik

    2015-01-01

    Lighting intensity and color have a significant impact on human circadian rhythms. Advanced solid state lighting was developed for the Advanced Exploration System (AES) Deep Space Habitat(DSH) concept demonstrator. The latest generation of assemblies using the latest commercially available LED lights were designed for use in the Bigelow Aerospace Environmental Control and Life Support System (ECLSS) simulator and the University of Hawaii's Hawaii Space Exploration Analog and Simulation (Hi-SEAS) habitat. Agreements with both these organizations will allow the government to receive feedback on the lights and lighting algorithms from long term human interaction.

  11. Dynamic Multiphoton Microscopy: Focusing Light on Acute Kidney Injury

    PubMed Central

    Molitoris, Bruce A.

    2014-01-01

    Acute kidney injury (AKI) is a major global health problem; much research has been conducted on AKI, and numerous agents have shown benefit in animal studies, but none have translated into treatments. There is, therefore, a pressing unmet need to increase knowledge of the pathophysiology of AKI. Multiphoton microscopy (MPM) provides a tool to non-invasively visualize dynamic events in real time and at high resolution in rodent kidneys, and in this article we review its application to study novel mechanisms and treatments in different forms of AKI. PMID:25180263

  12. Advances in small animal mesentery models for in vivo flow cytometry, dynamic microscopy, and drug screening

    PubMed Central

    Galanzha, Ekaterina I; Tuchin, Valery V; Zharov, Vladimir P

    2007-01-01

    Using animal mesentery with intravital optical microscopy is a well-established experimental model for studying blood and lymph microcirculation in vivo. Recent advances in cell biology and optical techniques provide the basis for extending this model for new applications, which should generate significantly improved experimental data. This review summarizes the achievements in this specific area, including in vivo label-free blood and lymph photothermal flow cytometry, super-sensitive fluorescence image cytometry, light scattering and speckle flow cytometry, microvessel dynamic microscopy, infrared (IR) angiography, and high-speed imaging of individual cells in fast flow. The capabilities of these techniques, using the rat mesentery model, were demonstrated in various studies; e.g., real-time quantitative detection of circulating and migrating individual blood and cancer cells, studies on vascular dynamics with a focus on lymphatics under normal conditions and under different interventions (e.g. lasers, drugs, nicotine), assessment of lymphatic disturbances from experimental lymphedema, monitoring cell traffic between blood and lymph systems, and high-speed imaging of cell transient deformability in flow. In particular, the obtained results demonstrated that individual cell transportation in living organisms depends on cell type (e.g., normal blood or leukemic cells), the cell’s functional state (e.g., live, apoptotic, or necrotic), and the functional status of the organism. Possible future applications, including in vivo early diagnosis and prevention of disease, monitoring immune response and apoptosis, chemo- and radio-sensitivity tests, and drug screening, are also discussed. PMID:17226898

  13. Transmission Electron Microscopy Advances Reveal Subtle Comet Dust Differences

    NASA Astrophysics Data System (ADS)

    Ishii, H. A.; Bradley, J. P.

    2015-07-01

    TEM advances in multi-SDD-detector EDX mapping applied to Wild 2 dust and likely-cometary CP IDPs demonstrates chondritic fine-grained material at terminal particles is unlike GEMS and consistent with debris generated during the deceleration process.

  14. Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets

    PubMed Central

    Zhao, Teng; Lau, Sze Cheung; Wang, Ying; Su, Yumian; Wang, Hao; Cheng, Aifang; Herrup, Karl; Ip, Nancy Y.; Du, Shengwang; Loy, M. M. T.

    2016-01-01

    We demonstrate a simple and efficient method for producing ultrathin Bessel (‘non-diffracting’) light sheets of any color using a line-shaped beam and an annulus filter. With this robust and cost-effective technology, we obtained two-color, 3D images of biological samples with lateral/axial resolution of 250 nm/400 nm, and high-speed, 4D volume imaging of 20 μm sized live sample at 1 Hz temporal resolution. PMID:27189786

  15. Multifocal multiphoton microscopy based on a spatial light modulator

    PubMed Central

    Shao, Y.; Qin, W.; Liu, H.; Peng, X.; Niu, H.

    2013-01-01

    We present a new multifocal multiphoton microscope that employs a programmable spatial light modulator to generate dynamic multifocus arrays which can be rapidly scanned by changing the incident angle of the laser beam using a pair of galvo scanners. Using this microscope, we can rapidly select the number and the spatial density of focal points in a multifocus array, as well as the locations and shapes of arrays according to the features of the areas of interest in the field of view without any change to the hardware. PMID:23894222

  16. Spectral confocal reflection microscopy using a white light source

    NASA Astrophysics Data System (ADS)

    Booth, M.; Juškaitis, R.; Wilson, T.

    2008-08-01

    We present a reflection confocal microscope incorporating a white light supercontinuum source and spectral detection. The microscope provides images resolved spatially in three-dimensions, in addition to spectral resolution covering the wavelength range 450-650nm. Images and reflection spectra of artificial and natural specimens are presented, showing features that are not normally revealed in conventional microscopes or confocal microscopes using discrete line lasers. The specimens include thin film structures on semiconductor chips, iridescent structures in Papilio blumei butterfly scales, nacre from abalone shells and opal gemstones. Quantitative size and refractive index measurements of transparent beads are derived from spectral interference bands.

  17. Multicolor 4D Fluorescence Microscopy using Ultrathin Bessel Light Sheets.

    PubMed

    Zhao, Teng; Lau, Sze Cheung; Wang, Ying; Su, Yumian; Wang, Hao; Cheng, Aifang; Herrup, Karl; Ip, Nancy Y; Du, Shengwang; Loy, M M T

    2016-01-01

    We demonstrate a simple and efficient method for producing ultrathin Bessel ('non-diffracting') light sheets of any color using a line-shaped beam and an annulus filter. With this robust and cost-effective technology, we obtained two-color, 3D images of biological samples with lateral/axial resolution of 250 nm/400 nm, and high-speed, 4D volume imaging of 20 μm sized live sample at 1 Hz temporal resolution. PMID:27189786

  18. Preparation of Drosophila S2 cells for Light Microscopy

    PubMed Central

    Buster, Daniel W.; Nye, Jonathan; Klebba, Joseph E.; Rogers, Gregory C.

    2010-01-01

    The ideal experimental system would be cheap and easy to maintain, amenable to a variety of techniques, and would be supported by an extensive literature and genome sequence database. Cultured Drosophila S2 cells, the product of disassociated 20-24 hour old embryos1, possess all these properties. Consequently, S2 cells are extremely well-suited for the analysis of cellular processes, including the discovery of the genes encoding the molecular components of the process or mechanism of interest. The features of S2 cells that are most responsible for their utility are the ease with which they are maintained, their exquisite sensitivity to double-stranded (ds)RNA-mediated interference (RNAi), and their tractability to fluorescence microscopy as either live or fixed cells. S2 cells can be grown in a variety of media, including a number of inexpensive, commercially-available, fully-defined, serum-free media2. In addition, they grow optimally and quickly at 21-24°C and can be cultured in a variety of containers. Unlike mammalian cells, S2 cells do not require a regulated atmosphere, but instead do well with normal air and can even be maintained in sealed flasks. Complementing the ease of RNAi in S2 cells is the ability to readily analyze experimentally-induced phenotypes by phase or fluorescence microscopy of fixed or live cells. S2 cells grow in culture as a single monolayer but do not display contact inhibition. Instead, cells tend to grow in colonies in dense cultures. At low density, S2 cultures grown on glass or tissue culture-treated plastic are round and loosely-attached. However, the cytology of S2 cells can be greatly improved by inducing them to flatten extensively by briefly culturing them on a surface coated with the lectin, concanavalin A (ConA)3. S2 cells can also be stably transfected with fluorescently-tagged markers to label structures or organelles of interest in live or fixed cells. Therefore, the usual scenario for the microscopic analysis of cells is

  19. Submicron-resolution photoacoustic microscopy of endogenous light-absorbing biomolecules

    NASA Astrophysics Data System (ADS)

    Zhang, Chi

    Photoacoustic imaging in biomedicine has the unique advantage of probing endogenous light absorbers at various length scales with a 100% relative sensitivity. Among the several modalities of photoacoustic imaging, optical-resolution photoacoustic microscopy (OR-PAM) can achieve high spatial resolution, on the order of optical wavelength, at <1 mm depth in biological tissue (the optical ballistic regime). OR-PAM has been applied successfully to structural and functional imaging of blood vasculature and red blood cells in vivo. Any molecules which absorb sufficient light at certain wavelengths can potentially be imaged by PAM. Compared with pure optical imaging, which typically targets fluorescent markers, label-free PAM avoids the major concerns that the fluorescent labeling probes may disturb the function of biomolecules and may have an insufficient density. This dissertation aims to advance label-free OR-PAM to the subcellular scale. The first part of this dissertation describes the technological advancement of PAM yielding high spatial resolution in 3D. The lateral resolution was improved by using optical objectives with high numerical apertures for optical focusing. The axial resolution was improved by using broadband ultrasonic transducers for ultrasound detection. We achieved 220 nm lateral resolution in transmission mode, 0.43 microm lateral resolution in reflection mode, 7.6 microm axial resolution in normal tissue, and 5.8 microm axial resolution with silicone oil immersion/injection. The achieved lateral resolution and axial resolution were the finest reported at the time. With high-resolution in 3D, PAM was demonstrated to resolve cellular and subcellular structures in vivo, such as red blood cells and melanosomes in melanoma cells. Compared with previous PAM systems, our high-resolution PAM could resolve capillaries in mouse ears more clearly. As an example application, we demonstrated intracellular temperature imaging, assisted by fluorescence signal

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

    PubMed

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

    2013-01-01

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

  1. Serially etched shark enameloid observed by incident light microscopy.

    PubMed

    Risnes, S; Fosse, G

    1979-01-01

    Longitudinal and transverse tooth sections of Isurus oxyrinchus were serially etched in 2.6% nitric acid. The changing optical properties of the etched surfaces were observed during the serial etchings, and the descent of the enameloid surfaces was measured. Shark enameloid seems to be less effectively etched by acid than human enamel; this difference may be due to differences in solubility between fluorapatite and hydroxyapatite. Most of the information regarding the structure of the enameloid was gained during the first five of ten etchings. The reflection of light from the surface was influenced by the orientation of the crystallites, longitudinally sectioned crystallites reflecting the light better than transversely sectioned crystallites. The dentinal extensions were continuous with and of the same structure as the underlying dentine. The radial fibers originated from the dentinal extensions, and they both contained organic material and were accompanied by crystallites. When the specimens were imbibed with water the distinctness of the dentinal extensions and radial fibers was improved. PMID:525241

  2. Status report on the Advanced Light Source control system

    SciTech Connect

    Magyary, S.; Chin, M.; Fahmie, M.; Lancaster, H.; Molinari, P.; Robb, A.; Timossi, C.; Young, J.

    1991-11-11

    This paper is a status report on the ADVANCED LIGHT SOURCE (ALS) control system. The current status, performance data, and future plans will be discussed. Manpower, scheduling, and costs issues are addressed.

  3. Polarized Light Microscopy in Reproductive and Developmental Biology

    PubMed Central

    KOIKE-TANI, MAKI; TANI, TOMOMI; MEHTA, SHALIN B.; VERMA, AMITABH; OLDENBOURG, RUDOLF

    2016-01-01

    SUMMARY The polarized light microscope reveals orientational order in native molecular structures inside living cells, tissues, and whole organisms. It is a powerful tool used to monitor and analyze the early developmental stages of organisms that lend themselves to microscopic observations. In this article, we briefly discuss the components specific to a traditional polarizing microscope and some historically important observations on: chromosome packing in the sperm head, the first zygote division of the sea urchin, and differentiation initiated by the first asymmetric cell division in the sand dollar. We then introduce the LC-PolScope and describe its use for measuring birefringence and polarized fluorescence in living cells and tissues. Applications range from the enucleation of mouse oocytes to analyzing the polarized fluorescence of the water strider acrosome. We end with new results on the birefringence of the developing chick brain, which we analyzed between developmental stages of days 12–20. PMID:23901032

  4. Multiphoton Microscopy and Interaction of Intense Light Pulses with Polymers

    NASA Astrophysics Data System (ADS)

    Guay, Jean-Michel

    2011-07-01

    The nanoscale manipulation of soft-matter, such as biological tissues, in its native environment has promising applications in medicine to correct for defects (eg. eye cataracts) or to destroy malignant regions (eg. cancerous tumours). To achieve this we need the ability to first image and then do precise ablation with sub-micron resolution with the same setup. For this purpose, we designed and built a multiphoton microscope and tested it on goldfish gills and bovine cells. We then studied light-matter interaction on a hard polymer (PMMA) because the nature of ablation of soft-matter in its native environment is complex and not well understood. Ablation and modification thresholds for successive laser shots were obtained. The ablation craters revealed 3D nanostructures and polarization dependent orientation. The interaction also induced localized porosity in PMMA that can be controlled.

  5. Advanced Solid State Lighting for AES Deep Space Hab Project

    NASA Technical Reports Server (NTRS)

    Holbert, Eirik

    2015-01-01

    The advanced Solid State Lighting (SSL) assemblies augmented 2nd generation modules under development for the Advanced Exploration Systems Deep Space Habitat in using color therapy to synchronize crew circadian rhythms. Current RGB LED technology does not produce sufficient brightness to adequately address general lighting in addition to color therapy. The intent is to address both through a mix of white and RGB LEDs designing for fully addressable alertness/relaxation levels as well as more dramatic circadian shifts.

  6. Advanced Indoor Module Light-Soaking Facility

    SciTech Connect

    del Cueto, J. A.; Osterwald, C.; Pruett, J.

    2005-01-01

    An overview of the accelerated, indoor light-soaking test station is presented in this paper, along with data obtained for six modules that underwent exposure. The station comprises a climate-controlled chamber equipped with a solar simulator that allows 1-sun light intensity exposure. Concurrently, we monitor the electrical characteristics of multiple PV modules and exercise active control over their electrical bias using programmable electronic loads, interfaced to a data acquisition system that acquires power-tracking and current-voltage data. This capability allows us to the test different bias conditions and to cyclically alternate between them. Additionally, we can vary the light intensity and module temperatures to garner realistic temperature coefficients of module performance. Data obtained on cadmium telluride (CdTe) and amorphous silicon (a-Si) modules are presented.

  7. Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.

    PubMed

    Faas, F G A; Bárcena, M; Agronskaia, A V; Gerritsen, H C; Moscicka, K B; Diebolder, C A; van Driel, L F; Limpens, R W A L; Bos, E; Ravelli, R B G; Koning, R I; Koster, A J

    2013-03-01

    Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells. PMID:23261400

  8. Studying intracellular transport using high-pressure freezing and Correlative Light Electron Microscopy.

    PubMed

    Brown, Edward; Mantell, Judith; Carter, Debbie; Tilly, Gini; Verkade, Paul

    2009-10-01

    Correlative Light Electron Microscopy (CLEM) aims at combining the best of light and electron microscopy in one experiment. Light microscopy (LM) is especially suited for providing a general overview with data from lots of different cells and by using live cell imaging it can show the history or sequence of events between or inside cells. Electron microscopy (EM) on the other hand can provide a much higher resolution image of a particular event and provide additional spatial information, the so-called reference space. CLEM thus has certain strengths over the application of both LM and EM techniques separately. But combining both modalities however generally also means making compromises in one or both of the techniques. Most often the preservation of ultrastructure for the electron microscopy part is sacrificed. Ideally samples should be visualized in its most native state both in the light microscope as well as the electron microscope. For electron microscopy this currently means that the sample will have to be cryo-fixed instead of the standard chemical fixation. In this paper we will discuss the rationale for using cryofixation for CLEM experiments. In particular we will highlight a CLEM technique using high-pressure freezing in combination with live cell imaging. In addition we examine some of the EM analysis tools that may be useful in combination with CLEM techniques. PMID:19660566

  9. Switching light with light - advanced functional colloidal monolayers

    NASA Astrophysics Data System (ADS)

    Bley, K.; Sinatra, N.; Vogel, N.; Landfester, K.; Weiss, C. K.

    2013-12-01

    Colloidal monolayers comprising of highly ordered two dimensional crystals are of high interest to generate surface patterns for a variety of different applications. Mostly, unfunctionalized polymer or silica colloids are assembled into monolayers. However, the incorporation of functional molecules into such colloids offers a convenient possibility of implementing additional properties to the two-dimensional crystal. Here, we present the formation of novel functional colloidal monolayers with photoswitchable fluorescence. The miniemulsion polymerization technique was used to incorporate an appropriate dye system of a perylene-based fluorophore and a bis-arylethene as a photochrome in polymeric colloids in defined ratios. Upon irradiation with UV or visible light the photochrome reversibly isomerizes from the ring-closed form, which is able to absorb light of the emission wavelength of the fluorescent dye and the ring-open form, which is not. The fluorescence emission of the dye can thus be reversibly switched on and off with light even when embedded in colloids. The colloids were self-assembled at the air-water interface to produce hexagonally ordered functional monolayers and more complex binary crystals. We investigate in detail the influence of the polymeric matrix on the switching properties of the fluorophore/photochrome system and find that the rate constants for the photoswitching, which all lie in the same range, are less influenced by the polymeric environment than expected. We demonstrate the reversible switching of the fluorescence emission in self-assembled colloidal monolayers. The arrangement of broadly distributed functional colloids into ordered monolayers with high addressability was obtained by the formation of binary colloidal monolayers.Colloidal monolayers comprising of highly ordered two dimensional crystals are of high interest to generate surface patterns for a variety of different applications. Mostly, unfunctionalized polymer or silica

  10. Switching light with light - advanced functional colloidal monolayers

    NASA Astrophysics Data System (ADS)

    Bley, K.; Sinatra, N.; Vogel, N.; Landfester, K.; Weiss, C. K.

    2013-12-01

    Colloidal monolayers comprising of highly ordered two dimensional crystals are of high interest to generate surface patterns for a variety of different applications. Mostly, unfunctionalized polymer or silica colloids are assembled into monolayers. However, the incorporation of functional molecules into such colloids offers a convenient possibility of implementing additional properties to the two-dimensional crystal. Here, we present the formation of novel functional colloidal monolayers with photoswitchable fluorescence. The miniemulsion polymerization technique was used to incorporate an appropriate dye system of a perylene-based fluorophore and a bis-arylethene as a photochrome in polymeric colloids in defined ratios. Upon irradiation with UV or visible light the photochrome reversibly isomerizes from the ring-closed form, which is able to absorb light of the emission wavelength of the fluorescent dye and the ring-open form, which is not. The fluorescence emission of the dye can thus be reversibly switched on and off with light even when embedded in colloids. The colloids were self-assembled at the air-water interface to produce hexagonally ordered functional monolayers and more complex binary crystals. We investigate in detail the influence of the polymeric matrix on the switching properties of the fluorophore/photochrome system and find that the rate constants for the photoswitching, which all lie in the same range, are less influenced by the polymeric environment than expected. We demonstrate the reversible switching of the fluorescence emission in self-assembled colloidal monolayers. The arrangement of broadly distributed functional colloids into ordered monolayers with high addressability was obtained by the formation of binary colloidal monolayers.Colloidal monolayers comprising of highly ordered two dimensional crystals are of high interest to generate surface patterns for a variety of different applications. Mostly, unfunctionalized polymer or silica

  11. High-resolution electron microscopy of advanced materials

    SciTech Connect

    Mitchell, T.E.; Kung, H.H.; Sickafus, K.E.; Gray, G.T. III; Field, R.D.; Smith, J.F.

    1997-11-01

    This final report chronicles a three-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The High-Resolution Electron Microscopy Facility has doubled in size and tripled in quality since the beginning of the three-year period. The facility now includes a field-emission scanning electron microscope, a 100 kV field-emission scanning transmission electron microscope (FE-STEM), a 300 kV field-emission high-resolution transmission electron microscope (FE-HRTEM), and a 300 kV analytical transmission electron microscope. A new orientation imaging microscope is being installed. X-ray energy dispersive spectrometers for chemical analysis are available on all four microscopes; parallel electron energy loss spectrometers are operational on the FE-STEM and FE-HRTEM. These systems enable evaluation of local atomic bonding, as well as chemical composition in nanometer-scale regions. The FE-HRTEM has a point-to-point resolution of 1.6 {angstrom}, but the resolution can be pushed to its information limit of 1 {angstrom} by computer reconstruction of a focal series of images. HRTEM has been used to image the atomic structure of defects such as dislocations, grain boundaries, and interfaces in a variety of materials from superconductors and ferroelectrics to structural ceramics and intermetallics.

  12. Thin dielectric film thickness determination by advanced transmission electron microscopy

    SciTech Connect

    Diebold, A.C.; Foran, B.; Kisielowski, C.; Muller, D.; Pennycook, S.; Principe, E.; Stemmer, S.

    2003-09-01

    High Resolution Transmission Electron Microscopy (HR-TEM) has been used as the ultimate method of thickness measurement for thin films. The appearance of phase contrast interference patterns in HR-TEM images has long been confused as the appearance of a crystal lattice by non-specialists. Relatively easy to interpret crystal lattice images are now directly observed with the introduction of annular dark field detectors for scanning TEM (STEM). With the recent development of reliable lattice image processing software that creates crystal structure images from phase contrast data, HR-TEM can also provide crystal lattice images. The resolution of both methods was steadily improved reaching now into the sub Angstrom region. Improvements in electron lens and image analysis software are increasing the spatial resolution of both methods. Optimum resolution for STEM requires that the probe beam be highly localized. In STEM, beam localization is enhanced by selection of the correct aperture. When STEM measurement is done using a highly localized probe beam, HR-TEM and STEM measurement of the thickness of silicon oxynitride films agree within experimental error. In this paper, the optimum conditions for HR-TEM and STEM measurement are discussed along with a method for repeatable film thickness determination. The impact of sample thickness is also discussed. The key result in this paper is the proposal of a reproducible method for film thickness determination.

  13. Advances in high-speed low-latency communications for nanopositioning in advanced microscopy

    NASA Astrophysics Data System (ADS)

    Jordan, Scott C.

    2012-06-01

    We present a comparison of classical and recently developed communications interfacing technologies relevant to scanned imaging. We adopt an applications perspective, with a focus on interfacing techniques as enablers for enhanced resolution, speed, stability, information density or similar benefits. A wealth of such applications have emerged, ranging from nanoscale-stabilized force microscopy yielding 100X resolution improvement thanks to leveraging the latest in interfacing capabilities, to novel approaches in analog interfacing which improve data density and DAC resolution by several orders of magnitude. Our intent is to provide tools to understand, select and implement advanced interfacing to take applications to the next level. We have entered an era in which new interfacing techniques are enablers, in their own right, for novel imaging techniques. For example, clever leveraging of new interfacing technologies has yielded nanoscale stabilization and atomic-force microscopy (AFM) resolution enhancement. To assist in choosing and implementing interfacing strategies that maximize performance and enable new capabilities, we review available interfaces such as USB2, GPIB and Ethernet against the specific needs of positioning for the scanned-imaging community. We spotlight recent developments such as LabVIEW FPGA, which allows non-specialists to quickly devise custom logic and interfaces of unprecedentedly high performance and parallelism. Notable applications are reviewed, including a clever amalgamation of AFM and optical tweezers and a picometer-scaleaccuracy interferometer devised for ultrafine positioning validation. We note the Serial Peripheral Interface (SPI), emerging as a high-speed/low-latency instrumentation interface. The utility of instrument-specific parallel (PIO) and TTL sync/trigger (DIO) interfaces is also discussed. Requirements of tracking and autofocus are reviewed against the time-critical needs of typical applications (to avoid, for example

  14. Correlative light and electron microscopy analysis of the centrosome: A step-by-step protocol.

    PubMed

    Kong, Dong; Loncarek, Jadranka

    2015-01-01

    Correlative light and electron microscopy harnesses the best from each of the two modalities of microscopy it utilizes; while light microscopy provides information about the dynamic properties of the cellular structure or fluorescently labeled protein, electron microscopy provides ultrastructural information in an unsurpassed resolution. However, tracing a particular cell and its rare and small structures such as centrosomes throughout numerous steps of the experiment is not a trivial task. In this chapter, we present the experimental workflow for combining live-cell fluorescence microscopy analysis with classical transmission electron microscopy, adapted for the studies of the centrosomes and basal bodies. We describe, in a step-by-step manner, an approach that can be affordably and successfully employed in any typical cell biology laboratory. The article details all key phases of the analysis starting from cell culture, live-cell microscopy, and sample fixation, through the steps of sample preparation for electron microscopy, to the identification of the target cell on the electron microscope. PMID:26175430

  15. Advanced Lighting Program Development (BG9702800) Final Report

    SciTech Connect

    Rubinstein, Francis; Johnson, Steve

    1998-02-01

    The report presents a long-range plan for a broad-based, coordinated research, development and market transformation program for reducing the lighting energy intensities in commercial and residential buildings in California without compromising lighting quality. An effective program to advance lighting energy efficiency in California must be based on an understanding that lighting is a mature field and the lighting industry has developed many specialized products that meet a wide variety of light needs for different building types. Above all else, the lighting field is diverse and there are applications for a wide range of lighting products, systems, and strategies. Given the range of existing lighting solutions, an effective energy efficient lighting research portfolio must be broad-based and diverse to match the diversity of the lighting market itself. The belief that there is one solution--a magic bullet, such as a better lamp, for example--that will propel lighting efficiency across all uses to new heights is, in the authors' opinion, an illusion. A multi-path program is the only effective means to raising lighting efficiency across all lighting applications in all building types. This report presents a list of 27 lighting technologies and concepts (key activities) that could form the basis of a coordinated research and market transformation plan for significantly reducing lighting energy intensities in California buildings. The total 27 key activities into seven broad classes as follows: Light sources; Ballasts; Luminaires; Lighting Controls; Lighting Systems in Buildings; Human Factors and Education. Each of the above technology classes is discussed in terms of background, key activities, and the energy savings potential for the state. The report concludes that there are many possibilities for targeted research, development, and market transformation activities across all sectors of the building lighting industry. A concerted investment by the state to foster

  16. Advanced light source, User`s Handbook, Revision 1

    SciTech Connect

    1995-07-01

    The Advanced Light Source (ALS) is a national facility for scientific research and development located at the Lawrence Berkeley National Laboratory (LBNL) of the University of California. Its purpose is to generate beams of very bright light in the ultraviolet and soft x-ray regions of the spectrum. The facility is open to researchers from industry, universities, and government laboratories.

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

    PubMed

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

    2015-01-01

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

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

    PubMed Central

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

    2015-01-01

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

  19. Switching light with light--advanced functional colloidal monolayers.

    PubMed

    Bley, K; Sinatra, N; Vogel, N; Landfester, K; Weiss, C K

    2014-01-01

    Colloidal monolayers comprising of highly ordered two dimensional crystals are of high interest to generate surface patterns for a variety of different applications. Mostly, unfunctionalized polymer or silica colloids are assembled into monolayers. However, the incorporation of functional molecules into such colloids offers a convenient possibility of implementing additional properties to the two-dimensional crystal. Here, we present the formation of novel functional colloidal monolayers with photoswitchable fluorescence. The miniemulsion polymerization technique was used to incorporate an appropriate dye system of a perylene-based fluorophore and a bis-arylethene as a photochrome in polymeric colloids in defined ratios. Upon irradiation with UV or visible light the photochrome reversibly isomerizes from the ring-closed form, which is able to absorb light of the emission wavelength of the fluorescent dye and the ring-open form, which is not. The fluorescence emission of the dye can thus be reversibly switched on and off with light even when embedded in colloids. The colloids were self-assembled at the air-water interface to produce hexagonally ordered functional monolayers and more complex binary crystals. We investigate in detail the influence of the polymeric matrix on the switching properties of the fluorophore/photochrome system and find that the rate constants for the photoswitching, which all lie in the same range, are less influenced by the polymeric environment than expected. We demonstrate the reversible switching of the fluorescence emission in self-assembled colloidal monolayers. The arrangement of broadly distributed functional colloids into ordered monolayers with high addressability was obtained by the formation of binary colloidal monolayers. PMID:24227011

  20. Light-emitting diodes in modern microscopy--from David to Goliath?

    PubMed

    Wessels, Johannes T; Pliquett, Uwe; Wouters, Fred S

    2012-03-01

    Proper illumination is essential for light microscopy. Whereas in early years incandescent light was the only illumination, today, more and more specialized light sources, such as lasers or arc lamps are used. Because of the high efficiency and brightness that light-emitting diodes (LED) have reached today, they have become a serious alternative for almost all kinds of illumination in light microscopy. LED have a high durability, do not need expensive electronics, and they can be switched in nanoseconds. Besides this, they are available throughout the UV/Vis/NIR-spectrum with a narrow bandwidth. This makes them ideal light sources for fluorescence microscopy. The white LED, with a color temperature ranging from 2,600 up to 5,000 K is an excellent choice for bright-field illumination with the additional advantage of simple brightness adjustments without changing the spectrum. This review discusses the different LED types, their use in the fluorescence microscope, and discusses LED as specialized illumination sources for Förster resonance energy transfer and fluorescent lifetime imaging microscopy. PMID:22290727

  1. New results in atomic physics at the Advanced Light Source

    SciTech Connect

    Schlachter, A.S.

    1995-01-01

    The Advanced Light Source is the world's first low-energy third-generation synchrotron radiation source. It has been running reliably and exceeding design specifications since it began operation in October 1993. It is available to a wide community of researchers in many scientific fields, including atomic and molecular science and chemistry. Here, new results in atomic physics at the Advanced Light Source demonstrate the opportunities available in atomic and molecular physics at this synchrotron light source. The unprecedented brightness allows experiments with high flux, high spectral resolution, and nearly 100% linear polarization.

  2. Advances and prospects in visible light communications

    NASA Astrophysics Data System (ADS)

    Hongda, Chen; Chunhui, Wu; Honglei, Li; Xiongbin, Chen; Zongyu, Gao; Shigang, Cui; Qin, Wang

    2016-01-01

    Visible light communication (VLC) is an emerging technology in optical wireless communication (OWC) that has attracted worldwide research in recent years. VLC can combine communication and illumination together, which could be applied in many application scenarios such as visible light communication local area networks (VLANs), indoor localization, and intelligent lighting. In recent years, pioneering and significant work have been made in the field of VLC. In this paper, an overview of the recent progress in VLC is presented. We also demonstrate our recent experiment results including bidirectional 100 Mbit/s VLAN or Li-Fi system based on OOK modulation without blue filter. The VLC systems that we proposed are good solutions for high-speed VLC application systems with low-cost and low-complexity. VLC technology shows a bright future due to its inherent advantages, shortage of RF spectra and ever increasing popularity of white LEDs. Project supported by the National High Technology Research and Development Program of China (Nos. 2015AA033303, 2013AA013602, 2013AA013603, 2013AA03A104), the National Natural Science Foundation of China (Nos. 61178051, 61321063, 61335010, 61178048, 61275169), and the National Basic Research Program of China (Nos. 2013CB329205, 2011CBA00608).

  3. The Light Microscopy Module: An On-Orbit Multi-User Microscope Facility

    NASA Technical Reports Server (NTRS)

    Motil, Susan M.; Snead, John H.

    2002-01-01

    The Light Microscopy Module (LMM) is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and operation of fluids and biology experiments within the Fluids and Combustion Facility (FCF) Fluids Integrated Rack (FIR) on the International Space Station (ISS). The LMM will be the first integrated payload with the FIR to conduct four fluid physics experiments. A description of the LMM diagnostic capabilities, including video microscopy, interferometry, laser tweezers, confocal, and spectrophotometry, will be provided.

  4. Limited-view light sheet fluorescence microscopy for three dimensional volume imaging

    NASA Astrophysics Data System (ADS)

    Rasmi, C. K.; Mohan, Kavya; Madhangi, M.; Rajan, K.; Nongthomba, U.; Mondal, Partha P.

    2015-12-01

    We propose and demonstrate a limited-view light sheet microscopy (LV-LSM) for three dimensional (3D) volume imaging. Realizing that longer and frequent image acquisition results in significant photobleaching, we have taken limited angular views (18 views) of the macroscopic specimen and integrated with maximum likelihood (ML) technique for reconstructing high quality 3D volume images. Existing variants of light-sheet microscopy require both rotation and translation with a total of approximately 10-fold more views to render a 3D volume image. Comparatively, LV-LSM technique reduces data acquisition time and consequently minimizes light-exposure by many-folds. Since ML is a post-processing technique and highly parallelizable, this does not cost precious imaging time. Results show noise-free and high contrast volume images when compared to the state-of-the-art selective plane illumination microscopy.

  5. High-resolution computational imaging of leaf hair patterning using polarized light microscopy.

    PubMed

    Pomeranz, Marcelo; Campbell, Jeffrey; Siegal-Gaskins, Dan; Engelmeier, Jacob; Wilson, Tyler; Fernandez, Virginia; Brkljacic, Jelena; Grotewold, Erich

    2013-02-01

    The leaf hairs (trichomes) on the aerial surface of many plant species play important roles in phytochemical production and herbivore protection, and have significant applications in the chemical and agricultural industries. Trichome formation in the model plant Arabidopsis thaliana also presents a tractable experimental system to study cell differentiation and pattern formation in plants and animals. Studies of this developmental process suggest that trichome positioning may be the result of a self-forming pattern, emerging from a lateral inhibition mechanism determined by a network of regulatory factors. Critical to the continued success of these studies is the ability to quantitatively characterize trichome pattern phenotypes in response to mutations in the genes that regulate this process. Advanced protocols for the observation of changes in trichome patterns can be expensive and/or time consuming, and lack user-friendly analysis tools. In order to address some of these challenges, we describe here a strategy based on polarized light microscopy for the quick and accurate measurement of trichome positions, and provide an online tool designed for the quantitative analyses of trichome number, density and patterning. PMID:23163919

  6. Live imaging and quantitative analysis of gastrulation in mouse embryos using light-sheet microscopy and 3D tracking tools.

    PubMed

    Ichikawa, Takehiko; Nakazato, Kenichi; Keller, Philipp J; Kajiura-Kobayashi, Hiroko; Stelzer, Ernst H K; Mochizuki, Atsushi; Nonaka, Shigenori

    2014-03-01

    This protocol describes how to observe gastrulation in living mouse embryos by using light-sheet microscopy and computational tools to analyze the resulting image data at the single-cell level. We describe a series of techniques needed to image the embryos under physiological conditions, including how to hold mouse embryos without agarose embedding, how to transfer embryos without air exposure and how to construct environmental chambers for live imaging by digital scanned light-sheet microscopy (DSLM). Computational tools include manual and semiautomatic tracking programs that are developed for analyzing the large 4D data sets acquired with this system. Note that this protocol does not include details of how to build the light-sheet microscope itself. Time-lapse imaging ends within 12 h, with subsequent tracking analysis requiring 3-6 d. Other than some mouse-handling skills, this protocol requires no advanced skills or knowledge. Light-sheet microscopes are becoming more widely available, and thus the techniques outlined in this paper should be helpful for investigating mouse embryogenesis. PMID:24525751

  7. Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene

    PubMed Central

    Stefaniuk, Marzena; Gualda, Emilio J.; Pawlowska, Monika; Legutko, Diana; Matryba, Paweł; Koza, Paulina; Konopka, Witold; Owczarek, Dorota; Wawrzyniak, Marcin; Loza-Alvarez, Pablo; Kaczmarek, Leszek

    2016-01-01

    Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy. PMID:27312902

  8. Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene.

    PubMed

    Stefaniuk, Marzena; Gualda, Emilio J; Pawlowska, Monika; Legutko, Diana; Matryba, Paweł; Koza, Paulina; Konopka, Witold; Owczarek, Dorota; Wawrzyniak, Marcin; Loza-Alvarez, Pablo; Kaczmarek, Leszek

    2016-01-01

    Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy. PMID:27312902

  9. Structured light optical microscopy for three-dimensional reconstruction of technical surfaces

    NASA Astrophysics Data System (ADS)

    Kettel, Johannes; Reinecke, Holger; Müller, Claas

    2016-04-01

    In microsystems technology quality control of micro structured surfaces with different surface properties is playing an ever more important role. The process of quality control incorporates three-dimensional (3D) reconstruction of specularand diffusive reflecting technical surfaces. Due to the demand on high measurement accuracy and data acquisition rates, structured light optical microscopy has become a valuable solution to solve this problem providing high vertical and lateral resolution. However, 3D reconstruction of specular reflecting technical surfaces still remains a challenge to optical measurement principles. In this paper we present a measurement principle based on structured light optical microscopy which enables 3D reconstruction of specular- and diffusive reflecting technical surfaces. It is realized using two light paths of a stereo microscope equipped with different magnification levels. The right optical path of the stereo microscope is used to project structured light onto the object surface. The left optical path is used to capture the structured illuminated object surface with a camera. Structured light patterns are generated by a Digital Light Processing (DLP) device in combination with a high power Light Emitting Diode (LED). Structured light patterns are realized as a matrix of discrete light spots to illuminate defined areas on the object surface. The introduced measurement principle is based on multiple and parallel processed point measurements. Analysis of the measured Point Spread Function (PSF) by pattern recognition and model fitting algorithms enables the precise calculation of 3D coordinates. Using exemplary technical surfaces we demonstrate the successful application of our measurement principle.

  10. Light Microscopy of the Hair: A Simple Tool to “Untangle” Hair Disorders

    PubMed Central

    Adya, Keshavmurthy A; Inamadar, Arun C; Palit, Aparna; Shivanna, Ragunatha; Deshmukh, Niranjan S

    2011-01-01

    Light microscopy of the hair forms an important bedside clinical tool for the diagnosis of various disorders affecting the hair. Hair abnormalities can be seen in the primary diseases affecting the hair or as a secondary involvement of hair in diseases affecting the scalp. Hair abnormalities also form a part of various genodermatoses and syndromes. In this review, we have briefly highlighted the light microscopic appearance of various infectious and non-infectious conditions affecting the hair. PMID:21769242

  11. Annual meeting of the Advanced Light Source Users` Association

    SciTech Connect

    1995-02-01

    This report contains papers on the following topics: ALS Director`s Report; ALS Operations Update; Recent Results in Machine Physics; Progress in Beamline Commissioning and Overview of New Projects; The ALS Scientific Program; First Results from the SpectroMicroscopy Beamline; Soft X-ray Fluorescence Spectroscopy of Solids; Soft X-Ray Fluorescence Spectroscopy of Molecules; Microstructures and Micromachining at the ALS; High-Resolution Photoemission from Simple Atoms and Molecules; X-Ray Diffraction at the ALS; Utilizing Synchrotron Radiation in Advanced Materials Industries; Polymer Microscopy: About Balls, Rocks and Other ``Stuff``; Infrared Research and Applications; and ALS User Program.

  12. Age related changes and osteochondrosis in swine articular and epiphyseal cartilage: light ane electron microscopy.

    PubMed

    Bhatnagar, R; Christian, R G; Nakano, T; Aherne, F X; Thompson, J R

    1981-04-01

    Age related changes and osteochondrosis in swine were studied using light microscopy and electron microscopy in articular cartilage and light microscopy and epiphyseal cartilage of swine from three days to 30 weeks of age. Thickness, cellularity and vascularity of both the epiphyseal and articular cartilage, decreased as the swine aged. Osteochondrotic changes included formation of "plugs" of cartilage indicating localized failure of ossification and separation and space formation in epiphyseal cartilage. Eosinophilic streaks and space formation in epiphyseal cartilage was observed in relation to epiphyseal separation. Electron microscopy showed a continuous fibrillar layer on the surface of the cartilage corresponding to the lamina splendens of light microscopy. This layer increased in the thickness and showed accumulation of amorphous material between the fibrils with aging. In the matrix, the orientation and distribution of the collagen fibers changed with growth and thicker fibers with clear sub banding were more common in older age groups. Also, necrotic cells, glycogen containing bodies and cellular debris were noticed in the matrix of normal cartilage in old animals. Chondrocytes in the younger cartilage showed accumulation of organelles responsible for protein synthesis; while Golgi bodies, vesicles, lysosomes, well developed foot processes and other inclusions were noticed in older cartilage. Cartilage erosions had a clumped and disrupted lamina splendens on the surface and electron lucent patches in the ground substances of the matrix and chondrocyte cytoplasm. PMID:7260732

  13. Recent advances in conjugated polymers for light emitting devices.

    PubMed

    Alsalhi, Mohamad Saleh; Alam, Javed; Dass, Lawrence Arockiasamy; Raja, Mohan

    2011-01-01

    A recent advance in the field of light emitting polymers has been the discovery of electroluminescent conjugated polymers, that is, kind of fluorescent polymers that emit light when excited by the flow of an electric current. These new generation fluorescent materials may now challenge the domination by inorganic semiconductor materials of the commercial market in light-emitting devices such as light-emitting diodes (LED) and polymer laser devices. This review provides information on unique properties of conjugated polymers and how they have been optimized to generate these properties. The review is organized in three sections focusing on the major advances in light emitting materials, recent literature survey and understanding the desirable properties as well as modern solid state lighting and displays. Recently, developed conjugated polymers are also functioning as roll-up displays for computers and mobile phones, flexible solar panels for power portable equipment as well as organic light emitting diodes in displays, in which television screens, luminous traffic, information signs, and light-emitting wallpaper in homes are also expected to broaden the use of conjugated polymers as light emitting polymers. The purpose of this review paper is to examine conjugated polymers in light emitting diodes (LEDs) in addition to organic solid state laser. Furthermore, since conjugated polymers have been approved as light-emitting organic materials similar to inorganic semiconductors, it is clear to motivate these organic light-emitting devices (OLEDs) and organic lasers for modern lighting in terms of energy saving ability. In addition, future aspects of conjugated polymers in LEDs were also highlighted in this review. PMID:21673938

  14. Recent Advances in Conjugated Polymers for Light Emitting Devices

    PubMed Central

    AlSalhi, Mohamad Saleh; Alam, Javed; Dass, Lawrence Arockiasamy; Raja, Mohan

    2011-01-01

    A recent advance in the field of light emitting polymers has been the discovery of electroluminescent conjugated polymers, that is, kind of fluorescent polymers that emit light when excited by the flow of an electric current. These new generation fluorescent materials may now challenge the domination by inorganic semiconductor materials of the commercial market in light-emitting devices such as light-emitting diodes (LED) and polymer laser devices. This review provides information on unique properties of conjugated polymers and how they have been optimized to generate these properties. The review is organized in three sections focusing on the major advances in light emitting materials, recent literature survey and understanding the desirable properties as well as modern solid state lighting and displays. Recently, developed conjugated polymers are also functioning as roll-up displays for computers and mobile phones, flexible solar panels for power portable equipment as well as organic light emitting diodes in displays, in which television screens, luminous traffic, information signs, and light-emitting wallpaper in homes are also expected to broaden the use of conjugated polymers as light emitting polymers. The purpose of this review paper is to examine conjugated polymers in light emitting diodes (LEDs) in addition to organic solid state laser. Furthermore, since conjugated polymers have been approved as light-emitting organic materials similar to inorganic semiconductors, it is clear to motivate these organic light-emitting devices (OLEDs) and organic lasers for modern lighting in terms of energy saving ability. In addition, future aspects of conjugated polymers in LEDs were also highlighted in this review. PMID:21673938

  15. Optimization of the excitation light sheet in selective plane illumination microscopy.

    PubMed

    Gao, Liang

    2015-03-01

    Selective plane illumination microscopy (SPIM) allows rapid 3D live fluorescence imaging on biological specimens with high 3D spatial resolution, good optical sectioning capability and minimal photobleaching and phototoxic effect. SPIM gains its advantage by confining the excitation light near the detection focal plane, and its performance is determined by the ability to create a thin, large and uniform excitation light sheet. Several methods have been developed to create such an excitation light sheet for SPIM. However, each method has its own strengths and weaknesses, and tradeoffs must be made among different aspects in SPIM imaging. In this work, we present a strategy to select the excitation light sheet among the latest SPIM techniques, and to optimize its geometry based on spatial resolution, field of view, optical sectioning capability, and the sample to be imaged. Besides the light sheets discussed in this work, the proposed strategy is also applicable to estimate the SPIM performance using other excitation light sheets. PMID:25798312

  16. Observation of dendritic cell morphology under light, phase-contrast or confocal laser scanning microscopy.

    PubMed

    Tan, Yuen-Fen; Leong, Chooi-Fun; Cheong, Soon-Keng

    2010-12-01

    Dendritic cells (DCs) are professional antigen presenting cells of the immune system. They can be generated in vitro from peripheral blood monocytes supplemented with GM-CSF, IL-4 and TNF alpha. During induction, DCs will increase in size and acquire multiple cytoplasmic projections when compared to their precursor cells such as monocytes or haematopoietic stem cells which are usually round or spherical. Morphology of DCs can be visualized by conventional light microscopy after staining or phase-contrast inverted microscopy or confocal laser scanning microscopy. In this report, we described the morphological appearances of DCs captured using the above-mentioned techniques. We found that confocal laser scanning microscopy yielded DCs images with greater details but the operating cost for such a technique is high. On the other hand, the images obtained through light microscopy after appropriate staining or phase contrast microscopy were acceptable for identification purpose. Besides, these equipments are readily available in most laboratories and the cost of operation is affordable. Nevertheless, morphological identification is just one of the methods to characterise DCs. Other methods such as phenotypic expression markers and mixed leukocyte reactions are additional tools used in the characterisation of DCs. PMID:21329180

  17. Analyzing cell structure and dynamics with confocal light scattering and absorption spectroscopic microscopy

    NASA Astrophysics Data System (ADS)

    Qiu, Le; Vitkin, Edward; Fang, Hui; Zaman, Munir M.; Andersson, Charlotte; Salahuddin, Saira; Modell, Mark D.; Freedman, Steven D.; Hanlon, Eugene B.; Itzkan, Irving; Perelman, Lev T.

    2007-02-01

    We recently developed a new microscopic optical technique capable of noninvasive analysis of cell structure and cell dynamics on the submicron scale [1]. It combines confocal microscopy, a well-established high-resolution microscopic technique, with light scattering spectroscopy (LSS) and is called confocal light absorption and scattering spectroscopic (CLASS) microscopy. CLASS microscopy requires no exogenous labels and is capable of imaging and continuously monitoring individual viable cells, enabling the observation of cell and organelle functioning at scales on the order of 100 nm. To test the ability of CLASS microscopy to monitor cellular dynamics in vivo we performed experiments with human bronchial epithelial cells treated with DHA and undergoing apoptosis. The treated and untreated cells show not only clear differences in organelle spatial distribution but time sequencing experiments on a single cell show disappearance of certain types of organelles and change of the nuclear shape and density with the progression of apoptosis. In summary, CLASS microscopy provides an insight into metabolic processes within the cell and opens doors for the noninvasive real-time assessment of cellular dynamics. Noninvasive monitoring of cellular dynamics with CLASS microscopy can be used for a real-time dosimetry in a wide variety of medical and environmental applications that have no immediate observable outcome, such as photodynamic therapy, drug screening, and monitoring of toxins.

  18. Correcting spherical aberrations in confocal light sheet microscopy: a theoretical study.

    PubMed

    Silvestri, L; Sacconi, L; Pavone, F S

    2014-07-01

    In the last years, fluorescence light sheet microscopy has attracted an increasing interest among the microscopy community. One of the most promising applications of this technique is the reconstruction of macroscopic biological specimens with microscopic resolution, without physical sectioning. To this aim, light sheet microscopy is combined with clearing protocols based on refractive index matching, which render the tissue transparent. However, these protocols lead to a huge drop in the fluorescence signal, limiting their practical applicability. The reduction of signal to background ratio is commonly ascribed to chemical degradation of the fluorophores by the organic solvents used for clearing. This view however completely neglects another important factor of contrast loss, i.e., optical aberrations. In fact, commercially available objectives suitable for light sheet microscopy are not designed for the refractive index of the clearing solutions, and this mismatch introduces severe spherical aberration. Here we simulated the aberrated point spread function (PSF) of a light sheet microscope with confocal slit detection. We investigated the variation of the PSF as a function of objective numerical aperture (NA) and of imaging depth inside the clearing solution. We also explored the possibility of correcting such spherical aberration by introducing extra optical devices in the detection path. By correcting up to the second order spherical aberration, a quasi-diffraction-limited regime can be recovered, and image quality is restored. PMID:24395714

  19. SPED Light Sheet Microscopy: Fast Mapping of Biological System Structure and Function.

    PubMed

    Tomer, Raju; Lovett-Barron, Matthew; Kauvar, Isaac; Andalman, Aaron; Burns, Vanessa M; Sankaran, Sethuraman; Grosenick, Logan; Broxton, Michael; Yang, Samuel; Deisseroth, Karl

    2015-12-17

    The goal of understanding living nervous systems has driven interest in high-speed and large field-of-view volumetric imaging at cellular resolution. Light sheet microscopy approaches have emerged for cellular-resolution functional brain imaging in small organisms such as larval zebrafish, but remain fundamentally limited in speed. Here, we have developed SPED light sheet microscopy, which combines large volumetric field-of-view via an extended depth of field with the optical sectioning of light sheet microscopy, thereby eliminating the need to physically scan detection objectives for volumetric imaging. SPED enables scanning of thousands of volumes-per-second, limited only by camera acquisition rate, through the harnessing of optical mechanisms that normally result in unwanted spherical aberrations. We demonstrate capabilities of SPED microscopy by performing fast sub-cellular resolution imaging of CLARITY mouse brains and cellular-resolution volumetric Ca(2+) imaging of entire zebrafish nervous systems. Together, SPED light sheet methods enable high-speed cellular-resolution volumetric mapping of biological system structure and function. PMID:26687363

  20. 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

  1. Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: Light and electron microscopy

    USGS Publications Warehouse

    Walker, G.K.; Black, M.G.; Edwards, C.A.

    1996-01-01

    Adult zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondrial "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria.

  2. Developmental Nuclear Localization and Quantification of GFP-Tagged EB1c in Arabidopsis Root Using Light-Sheet Microscopy.

    PubMed

    Novák, Dominik; Kuchařová, Anna; Ovečka, Miroslav; Komis, George; Šamaj, Jozef

    2015-01-01

    The development of the root apex is determined by progress of cells from the meristematic region to the successive post-mitotic developmental zones for transition, cell elongation and final cell differentiation. We addressed root development, tissue architecture and root developmental zonation by means of light-sheet microscopic imaging of Arabidopsis thaliana seedlings expressing END BINDING protein 1c (EB1c) fused to green fluorescent protein (GFP) under control of native EB1c promoter. Unlike the other two members of the EB1 family, plant-specific EB1c shows prominent nuclear localization in non-dividing cells in all developmental zones of the root apex. The nuclear localization of EB1c was previously mentioned solely in meristematic cells, but not further addressed. With the help of advanced light-sheet microscopy, we report quantitative evaluations of developmentally-regulated nuclear levels of the EB1c protein tagged with GFP relatively to the nuclear size in diverse root tissues (epidermis, cortex, and endodermis) and root developmental zones (meristem, transition, and elongation zones). Our results demonstrate a high potential of light-sheet microscopy for 4D live imaging of fluorescently-labeled nuclei in complex samples such as developing roots, showing capacity to quantify parameters at deeper cell layers (e.g., endodermis) with minimal aberrations. The data presented herein further signify the unique role of developmental cell reprogramming in the transition from cell proliferation to cell differentiation in developing root apex. PMID:26779221

  3. Survey, alignment, and beam stability at the Advanced Light Source

    SciTech Connect

    Krebs, G.F.

    1997-10-01

    This paper describes survey and alignment at the Lawrence Berkeley Laboratories Advanced Light Source (ALS) accelerators from 1993 to 1997. The ALS is a third generation light source requiring magnet alignment to within 150 microns. To accomplish this, a network of monuments was established and maintained. Monthly elevation surveys show the movement of the floor over time. Inclinometers have recently been employed to give real time information about magnet, vacuum tank and magnet girder motion in the ALS storage ring.

  4. A Correlative Method for Imaging Identical Regions of Samples by Micro-CT, Light Microscopy, and Electron Microscopy

    PubMed Central

    Sengle, Gerhard; Tufa, Sara F.; Sakai, Lynn Y.; Zulliger, Martin A.

    2013-01-01

    We present a method in which a precise region of interest within an intact organism is spatially mapped in three dimensions by non-invasive micro-computed X-ray tomography (micro-CT), then further evaluated by light microscopy (LM) and transmission electron microscopy (TEM). Tissues are prepared as if for TEM including osmium fixation, which imparts soft tissue contrast in the micro-CT due to its strong X-ray attenuation. This method may therefore be applied to embedded, archived TEM samples. Upon selection of a two-dimensional (2-D) projection from a region of interest (ROI) within the three-dimensional volume, the epoxy-embedded sample is oriented for microtomy so that the sectioning plane is aligned with the micro-CT projection. Registration is verified by overlaying LM images with 2-D micro-CT projections. Structures that are poorly resolved in the micro-CT may be evaluated at TEM resolution by observing the next serial ultrathin section, thereby accessing the same ROI by all three imaging techniques. We compare white adipose tissue within the forelimbs of mice harboring a lipid-altering mutation with their littermate controls. We demonstrate that individual osmium-stained lipid droplets as small as 15 µm and separated by as little as 35 µm may be discerned as separate entities in the micro-CT, validating this to be a high-resolution, non-destructive technique for evaluation of fat content. PMID:23264636

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

    PubMed Central

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

    2014-01-01

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

  6. Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy

    PubMed Central

    Fu, Qinyi; Martin, Benjamin L.; Matus, David Q.; Gao, Liang

    2016-01-01

    Despite the progress made in selective plane illumination microscopy, high-resolution 3D live imaging of multicellular specimens remains challenging. Tiling light-sheet selective plane illumination microscopy (TLS-SPIM) with real-time light-sheet optimization was developed to respond to the challenge. It improves the 3D imaging ability of SPIM in resolving complex structures and optimizes SPIM live imaging performance by using a real-time adjustable tiling light sheet and creating a flexible compromise between spatial and temporal resolution. We demonstrate the 3D live imaging ability of TLS-SPIM by imaging cellular and subcellular behaviours in live C. elegans and zebrafish embryos, and show how TLS-SPIM can facilitate cell biology research in multicellular specimens by studying left-right symmetry breaking behaviour of C. elegans embryos. PMID:27004937

  7. Monitoring of Apoptosis in 3D Cell Cultures by FRET and Light Sheet Fluorescence Microscopy

    PubMed Central

    Weber, Petra; Schickinger, Sarah; Wagner, Michael; Angres, Brigitte; Bruns, Thomas; Schneckenburger, Herbert

    2015-01-01

    Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure. PMID:25761242

  8. How advances in light technology have shaped ENT.

    PubMed

    Mozaffari, M; Fishman, J M; Tolley, N S

    2016-02-01

    The development of light technologies, allowing anatomical visualisation of otherwise hidden structures, led to significant advances in ENT in the nineteenth and twentieth centuries. Natural light from the sun, and from candles, was initially harnessed using mirrors. Later, the invention of limelight and electricity preceded the emergence of the modern-day endoscope, which, in tandem with the discovery of coherent fibre-optics in the 1950s, significantly expanded the surgical repertoire available to otolaryngologists. This study aimed to trace the rich history of ENT through the specialty's use of light. PMID:26669735

  9. Advanced Light Source Activity Report 1997/1998

    SciTech Connect

    Greiner, Annette

    1999-03-01

    This Lawrence Berkeley National Laboratory, Advanced Light Source (ALS) activity report for 1997/98 discusses the following topics: Introduction and Overview; Science Highlights; Facility Report; Special Events; ALS Advisory Panels 1997/98; ALS Staff 1997/98 and Facts and Figures for the year.

  10. Measurement of storage ring motion at the advanced light source

    SciTech Connect

    Krebs, G.F.

    1997-05-01

    The mechanical stability of the Advanced Light Source storage ring is examined over a period of 1.5 years from the point of view of floor motion. The storage ring beam position monitor stability is examined under various operating conditions.

  11. A method of comparing spermatozoa with light and scanning electron microscopy.

    PubMed

    Sauvalle, A M; Prigent, J R; Izard, J Y

    1982-01-01

    A new method of comparing light microscopy and scanning electron microscopy in the study of small cells, such as spermatozoa, that must be examined under oil immersion is described. A grid is etched on the corner of a microscope glass slide, and its inner edges are incised. Its surface area is calculated as a function f the chamber of the critical-point drying apparatus. This method dispenses with the need for any special coverslip and enables the cells to be observed under oil immersion. PMID:6179330

  12. Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development

    PubMed Central

    Bassi, Andrea; Schmid, Benjamin; Huisken, Jan

    2015-01-01

    Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy. Multimodal imaging techniques are now needed to put these distinct structures back into the context of the surrounding tissue. We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity. We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs. PMID:25655702

  13. A novel fibrous duct structure discovered in the brain meninges by using polarized light microscopy

    NASA Astrophysics Data System (ADS)

    Nam, Min-Ho; Jung, Sharon Jiyoon; Soh, Kwang-Sup; Lim, Jaekwan; Seo, Eunseok; Lim, Jun; Baek, Miok; Lee, Sang Joon

    2016-05-01

    We have previously reported the discovery of a novel fibrous structure (NFS) consisting of unidirectionally arranged collagen fibers in the spinal pia mater. Due to its unique structure, it was easily detected using polarized light microscopy. In the current study, we describe the discovery of a similar NFS in the brain meninges of rats by using polarized light microscopy. This NFS is located beneath the superior sagittal sinus. Initially, we systemically analyzed the polarization properties of the NFS. The change in the light intensity of the NFS, with respect to the polarization angle, was eight times greater than that of blood vessels, showing that the collagen fibers are oriented in a particular direction with almost perfect parallelism (0.99). The orientation angle of the polarization ellipse confirmed the orientation of the collagen fibers in the NFS. Histological studies further confirmed that the unidirectionally arranged collagen fibers were responsible for this distinct polarization property. Surprisingly, X-ray microtomography and 3D confocal imaging revealed that the NFS contains within it a duct structure, a putative primo vessel. In conclusion, we report a NFS in the brain meninges, detected by using polarized light microscopy, that provides space for a putative primo vessel, not a blood vessel.

  14. Optimizing and extending light-sculpting microscopy for fast functional imaging in neuroscience

    PubMed Central

    Rupprecht, Peter; Prevedel, Robert; Groessl, Florian; Haubensak, Wulf E.; Vaziri, Alipasha

    2015-01-01

    A number of questions in system biology such as understanding how dynamics of neuronal networks are related to brain function require the ability to capture the functional dynamics of large cellular populations at high speed. Recently, this has driven the development of a number of parallel and high speed imaging techniques such as light-sculpting microscopy, which has been used to capture neuronal dynamics at the whole brain and single cell level in small model organisms. However, the broader applicability of light-sculpting microcopy is limited by the size of volumes for which high speed imaging can be obtained and scattering in brain tissue. Here, we present strategies for optimizing the present tradeoffs in light-sculpting microscopy. Various scanning modalities in light-sculpting microscopy are theoretically and experimentally evaluated, and strategies to maximize the obtainable volume speeds, and depth penetration in brain tissue using different laser systems are provided. Design-choices, important parameters and their trade-offs are experimentally demonstrated by performing calcium-imaging in acute mouse-brain slices. We further show that synchronization of line-scanning techniques with rolling-shutter read-out of the camera can reduce scattering effects and enhance image contrast at depth. PMID:25780729

  15. Use of High Throughput Sequencing and Light Microscopy Show Contrasting Results in a Study of Phytoplankton Occurrence in a Freshwater Environment

    PubMed Central

    Xiao, Xi; Sogge, Hanne; Lagesen, Karin; Tooming-Klunderud, Ave; Jakobsen, Kjetill S.; Rohrlack, Thomas

    2014-01-01

    Assessing phytoplankton diversity is of primary importance for both basic and applied ecological studies. Following the advances in molecular methods, phytoplankton studies are switching from using classical microscopy to high throughput sequencing approaches. However, methodological comparisons of these approaches have rarely been reported. In this study, we compared the two methods, using a unique dataset of multiple water samples taken from a natural freshwater environment. Environmental DNA was extracted from 300 water samples collected weekly during 20 years, followed by high throughput sequencing of amplicons from the 16S and 18S rRNA hypervariable regions. For each water sample, phytoplankton diversity was also estimated using light microscopy. Our study indicates that species compositions detected by light microscopy and 454 high throughput sequencing do not always match. High throughput sequencing detected more rare species and picoplankton than light microscopy, and thus gave a better assessment of phytoplankton diversity. However, when compared to light microscopy, high throughput sequencing of 16S and 18S rRNA amplicons did not adequately identify phytoplankton at the species level. In summary, our study recommends a combined strategy using both morphological and molecular techniques. PMID:25171164

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

    PubMed

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

    2016-01-01

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

  17. Imaging green fluorescent protein-labeled neurons using light and electron microscopy.

    PubMed

    Knott, Graham W

    2013-06-01

    The ability to observe axons and dendrites with transmission electron microscopy (EM) after they have been previously imaged live with laser-scanning microscopy is a useful technique to study their synaptic connectivity. This protocol provides a detailed method by which neurons that were imaged in a live brain or slice culture can be reimaged using EM. First, brain tissue expressing green fluorescent protein (GFP) is chemically fixed. Then, an immunocytochemistry process is used to render the fluorescent protein electron dense so that it can first be located using light microscopy and then serial thin-sectioned for EM so that the ultrastructure of specific parts of neurites can be analyzed in three dimensions. Patterns of blood vessels observed in the live brain are used to locate the previously imaged neurons. The method described here allows for a complete three-dimensional (3D) reconstruction to be made of the imaged structures from serial electron micrographs. PMID:23734023

  18. Age related changes and osteochondrosis in swine articular and epiphyseal cartilage: light ane electron microscopy.

    PubMed Central

    Bhatnagar, R; Christian, R G; Nakano, T; Aherne, F X; Thompson, J R

    1981-01-01

    Age related changes and osteochondrosis in swine were studied using light microscopy and electron microscopy in articular cartilage and light microscopy and epiphyseal cartilage of swine from three days to 30 weeks of age. Thickness, cellularity and vascularity of both the epiphyseal and articular cartilage, decreased as the swine aged. Osteochondrotic changes included formation of "plugs" of cartilage indicating localized failure of ossification and separation and space formation in epiphyseal cartilage. Eosinophilic streaks and space formation in epiphyseal cartilage was observed in relation to epiphyseal separation. Electron microscopy showed a continuous fibrillar layer on the surface of the cartilage corresponding to the lamina splendens of light microscopy. This layer increased in the thickness and showed accumulation of amorphous material between the fibrils with aging. In the matrix, the orientation and distribution of the collagen fibers changed with growth and thicker fibers with clear sub banding were more common in older age groups. Also, necrotic cells, glycogen containing bodies and cellular debris were noticed in the matrix of normal cartilage in old animals. Chondrocytes in the younger cartilage showed accumulation of organelles responsible for protein synthesis; while Golgi bodies, vesicles, lysosomes, well developed foot processes and other inclusions were noticed in older cartilage. Cartilage erosions had a clumped and disrupted lamina splendens on the surface and electron lucent patches in the ground substances of the matrix and chondrocyte cytoplasm. Images Fig. 1. Fig. 2 and 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. Fig. 9. Fig. 10 and 11. Fig. 12. Fig. 13. Fig. 14. Fig. 15. Fig. 16. Fig. 17. Fig. 18. PMID:7260732

  19. Ultrastructural pathogenesis of lesions produced by exposure to oxygen difluoride with correlative light microscopy

    NASA Technical Reports Server (NTRS)

    Harrison, G.; Mackenzie, W.

    1973-01-01

    The lungs of rats exposed to OF2 were examined by light and electron microscopy. The exposures were for 30 to 60 minutes to an average of 4.5 ppm OF2, the minimal lethal dose. Animals were sacrificed after 30 (group 1) and 60 minutes (group 2) exposure and 1 (group 3) and 2 (group 4) hours following 60 minutes exposure. Mild gross changes were observed in groups 3 and 4, but no light microscopic lesions were found. Alterations were noted in all four groups using electron microscopy. These were mostly indicative of fluid change and consisted of blebbing of the endothelial and epithelial layers of the alveolocapillary wall and rarification of the cytoplasm of these cells. The lamellar bodies of the Type II cells showed an increasing and consistent loss of matrix structure and density. These fine structural changes increased in quantity and severity as time of exposure or post-exposure period increased. (Modified author abstract)

  20. Multispectral digital lensless holographic microscopy: from femtosecond laser to white light LED

    NASA Astrophysics Data System (ADS)

    Garcia-Sucerquia, J.

    2015-04-01

    The use of femtosecond laser radiation and super bright white LED in digital lensless holographic microscopy is presented. For the ultrafast laser radiation two different configurations of operation of the microscope are presented and the dissimilar performance of each one analyzed. The microscope operating with a super bright white light LED in combination with optical filters shows very competitive performance as it is compared with more expensive optical sources. The broadband emission of both radiation sources allows the multispectral imaging of biological samples to obtain spectral responses and/or full color images of the microscopic specimens; sections of the head of a Drosophila melanogaster fly are imaged in this contribution. The simple, solid, compact, lightweight, and reliable architecture of digital lensless holographic microscopy operating with broadband light sources to image biological specimens exhibiting micrometer-sized details is evaluated in the present contribution.

  1. Robust atomic resolution imaging of light elements using scanning transmission electron microscopy

    SciTech Connect

    Findlay, S. D.; Shibata, N.; Sawada, H.; Okunishi, E.; Kondo, Y.; Yamamoto, T.; Ikuhara, Y.

    2009-11-09

    We show that an annular detector placed within the bright field cone in scanning transmission electron microscopy allows direct imaging of light elements in crystals. In contrast to common high angle annular dark field imaging, both light and heavy atom columns are visible simultaneously. In contrast to common bright field imaging, the images are directly and robustly interpretable over a large range of thicknesses. We demonstrate this through systematic simulations and present a simple physical model to obtain some insight into the scattering dynamics.

  2. Correlative Light and Scanning X-Ray Scattering Microscopy of Healthy and Pathologic Human Bone Sections

    PubMed Central

    Giannini, C.; Siliqi, D.; Bunk, O.; Beraudi, A.; Ladisa, M.; Altamura, D.; Stea, S.; Baruffaldi, F.

    2012-01-01

    Scanning small and wide angle X-ray scattering (scanning SWAXS) experiments were performed on healthy and pathologic human bone sections. Via crystallographic tools the data were transformed into quantitative images and as such compared with circularly polarized light (CPL) microscopy images. SWAXS and CPL images allowed extracting information of the mineral nanocrystalline phase embedded, with and without preferred orientation, in the collagen fibrils, mapping local changes at sub-osteon resolution. This favorable combination has been applied for the first time to biopsies of dwarfism syndrome and Paget's disease to shed light onto the cortical structure of natural bone in healthy and pathologic sections. PMID:22666538

  3. Plastic embedding and polishing of bone for reflected light and electron microscopy.

    PubMed

    Wighton, A H J; Jones, Chris G; Bell, Lynne S

    2012-01-01

    The successful embedding of bone or any sample for reflected light or electron microscopy is crucial to the success of any analysis that might follow. Different materials present different embedding challenges, and here we discuss bone. Embedding is developed often as an adapted in-house protocol, and will vary from one institution to another, and is barely referenced in any detail in scientific papers. This chapter provides the protocol for bone that has proved successful at the Natural History Museum, both for reflected light and particularly for scanning electron microscopic examination. PMID:22907400

  4. LED Arrays as Cost Effective and Efficient Light Sources for Widefield Microscopy

    PubMed Central

    Sato, Tomokazu F.; Meister, Markus; Murthy, Venkatesh N.

    2008-01-01

    New developments in fluorophores as well as in detection methods have fueled the rapid growth of optical imaging in the life sciences. Commercial widefield microscopes generally use arc lamps, excitation/emission filters and shutters for fluorescence imaging. These components can be expensive, difficult to maintain and preclude stable illumination. Here, we describe methods to construct inexpensive and easy-to-use light sources for optical microscopy using light-emitting diodes (LEDs). We also provide examples of its applicability to biological fluorescence imaging. PMID:18478056

  5. Simultaneous optical coherence tomography and autofluorescence microscopy with a single light source

    NASA Astrophysics Data System (ADS)

    Dai, Cuixia; Liu, Xiaojing; Jiao, Shuliang

    2012-08-01

    We have accomplished simultaneous spectral domain optical coherence tomography (SD-OCT) and autofluorescence (AF) microscopy with a broadband light source centered at 415 nm. The light source was provided by frequency-doubling of an ultra-fast broadband Ti:Sapphire laser. With a bandwidth of 8 nm, the visible SD-OCT achieved a depth resolution of ˜12 μm. Since the two imaging modalities are provided by the same group of photons, their images are intrinsically registered. The dual-modal system is capable of providing OCT imaging and molecular contrasts simultaneously. The imaging system was tested on imaging biological samples ex vivo and in vivo.

  6. Recent advances on Coherent Anti-Strokes Raman scattering (CARS) microscopy

    NASA Astrophysics Data System (ADS)

    Cheng, Ji-xin; Potma, Eric O.; Xie, Sunney

    2002-06-01

    Recent advances in CARS microscopy provided exciting possibilities for imaging living cells with high sensitivity, high 3D spatial resolution and chemical specificity based on vibrational spectroscopy. Epi-detection avoids the forward-going water signal and allows imaging of small features inside cells. Polarization-sensitive detection improves the vibrational contrast in CARS microscopy via suppression of the non-resonant background. High-speed CARS imaging of living cells is realized by raster scanning two collinearly overlapped near infrared picosecond laser beams. Vibrational mapping of lipids and proteins inside living cells is achieved. CARS imaging of unstained cells during mitosis and apoptosis is carried out. Incorporation of CARS microscopy with the photon correlation technique allows probing diffusion dynamics of 50-nm beads with chemical selectivity.

  7. Gastric Tissue Damage Analysis Generated by Ischemia: Bioimpedance, Confocal Endomicroscopy, and Light Microscopy

    PubMed Central

    Beltran, Nohra E.; Garcia, Laura E.; Garcia-Lorenzana, Mario

    2013-01-01

    The gastric mucosa ischemic tissular damage plays an important role in critical care patients' outcome, because it is the first damaged tissue by compensatory mechanism during shock. The aim of the study is to relate bioimpedance changes with tissular damage level generated by ischemia by means of confocal endomicroscopy and light microscopy. Bioimpedance of the gastric mucosa and confocal images were obtained from Wistar male rats during basal and ischemia conditions. They were anesthetized, and stain was applied (fluorescein and/or acriflavine). The impedance spectroscopy catheter was inserted and then confocal endomicroscopy probe. After basal measurements and biopsy, hepatic and gastric arteries clamping induced ischemia. Finally, pyloric antrum tissue was preserved in buffered formaldehyde (10%) for histology processing using light microscopy. Confocal images were equalized, binarized, and boundary defined, and infiltrations were quantified. Impedance and infiltrations increased with ischemia showing significant changes between basal and ischemia conditions (P < 0.01). Light microscopy analysis allows detection of general alterations in cellular and tissular integrity, confirming gastric reactance and confocal images quantification increments obtained during ischemia. PMID:23841094

  8. An advanced image analysis tool for the quantification and characterization of breast cancer in microscopy images.

    PubMed

    Goudas, Theodosios; Maglogiannis, Ilias

    2015-03-01

    The paper presents an advanced image analysis tool for the accurate and fast characterization and quantification of cancer and apoptotic cells in microscopy images. The proposed tool utilizes adaptive thresholding and a Support Vector Machines classifier. The segmentation results are enhanced through a Majority Voting and a Watershed technique, while an object labeling algorithm has been developed for the fast and accurate validation of the recognized cells. Expert pathologists evaluated the tool and the reported results are satisfying and reproducible. PMID:25681102

  9. On the feasibility to investigate point defects by advanced electron microscopy

    SciTech Connect

    Kisielowski, C.; Jinschek, J.R.

    2002-10-02

    Transmission Electron Microscopy evolves rapidly as a primary tool to investigate nano structures on a truly atomic level. Its resolution reaches into the sub Angstrom region by now. Together with a better correction of lens aberrations, sensitivities are drastically enhanced. Utilizing advanced electron microscopes, it is feasible to promote experiments that aim to detect single atoms. This enables local investigations of non-stoichiometry. This paper reviews the current state-of-the-art.

  10. Resonance Raman microscopy in combination with partial dark-field microscopy lights up a new path in malaria diagnostics.

    PubMed

    Wood, Bayden R; Hermelink, Antje; Lasch, Peter; Bambery, Keith R; Webster, Grant T; Khiavi, Mehdi Asghari; Cooke, Brian M; Deed, Samantha; Naumann, Dieter; McNaughton, Don

    2009-06-01

    Our goal is to produce a rapid and accurate diagnostic tool for malaria using resonance Raman spectroscopy to detect small inclusions of haemozoin in Plasmodium falciparum infected red blood cells. In pursuit of this aim we serendipitously discovered a partial dark-field effect generated by our experimental setup, which helps identify in thick blood films potential parasites that are normally difficult to see with conventional bright-field microscopy. The haemozoin deposits 'light up' and these can be selectively targeted with the Raman microscope to confirm the presence or absence of haemozoin by the strong 1569 cm(-1) band, which is a marker for haemozoin. With newly developed imaging Raman microscopes incorporating ultra-sensitive rapid readout CCDs it is possible to obtain spectra with a good signal-to-noise ratio in 1 second. Moreover, images from a smear of potentially infected cells can be recorded and analysed with multivariate methods. The reconstructed images show what appear to be sub-micron-inclusions of haemozoin in some cells indicating that the technique has potential to identify low pigmented forms of the parasite including early trophozoite-stage infected cells. Further work is required to unambiguously confirm the presence of such forms through systematic staining but the results are indeed promising and may lead to the development of a new Raman-based malaria diagnostic. PMID:19475137

  11. Calibration of a wide-field frequency-domain fluorescence lifetime microscopy system using light emitting diodes as light sources.

    PubMed

    Elder, A D; Frank, J H; Swartling, J; Dai, X; Kaminski, C F

    2006-11-01

    High brightness light emitting diodes are an inexpensive and versatile light source for wide-field frequency-domain fluorescence lifetime imaging microscopy. In this paper a full calibration of an LED based fluorescence lifetime imaging microscopy system is presented for the first time. A radio-frequency generator was used for simultaneous modulation of light emitting diode (LED) intensity and the gain of an intensified charge coupled device (CCD) camera. A homodyne detection scheme was employed to measure the demodulation and phase shift of the emitted fluorescence, from which phase and modulation lifetimes were determined at each image pixel. The system was characterized both in terms of its sensitivity to measure short lifetimes (500 ps to 4 ns), and its capability to distinguish image features with small lifetime differences. Calibration measurements were performed in quenched solutions containing Rhodamine 6G dye and the results compared to several independent measurements performed with other measurement methodologies, including time correlated single photon counting, time gated detection, and acousto optical modulator (AOM) based modulation of excitation sources. Results are presented from measurements and simulations. The effects of limited signal-to-noise ratios, baseline drifts and calibration errors are discussed in detail. The implications of limited modulation bandwidth of high brightness, large area LED devices ( approximately 40 MHz for devices used here) are presented. The results show that phase lifetime measurements are robust down to sub ns levels, whereas modulation lifetimes are prone to errors even at large signal-to-noise ratios. Strategies for optimizing measurement fidelity are discussed. Application of the fluorescence lifetime imaging microscopy system is illustrated with examples from studies of molecular mixing in microfluidic devices and targeted drug delivery research. PMID:17204064

  12. Dynamic Aperture Measurements at the Advanced Light Source

    SciTech Connect

    Decking, W.; Robin, D.

    1999-03-12

    A large dynamic aperture for a storage ring is of importance for long lifetimes and a high injection efficiency. Measurements of the dynamic aperture of the third generation synchrotron light source Advanced Light Source (ALS) using beam excitation with kicker magnets are presented. The experiments were done for various accelerator conditions, allowing us to investigate the influence of different working points, chromaticities, insertion devices, etc.. The results are compared both with tracking calculations and a simple model for the dynamic aperture yielding good agreements. This gives us confidence in the predictability of the nonlinear accelerator model. This is especially important for future ALS upgrades as well as new storage ring designs.

  13. Novel interfaces for light directed neuronal stimulation: advances and challenges

    PubMed Central

    Bareket-Keren, Lilach; Hanein, Yael

    2014-01-01

    Light activation of neurons is a growing field with applications ranging from basic investigation of neuronal systems to the development of new therapeutic methods such as artificial retina. Many recent studies currently explore novel methods for optical stimulation with temporal and spatial precision. Novel materials in particular provide an opportunity to enhance contemporary approaches. Here we review recent advances towards light directed interfaces for neuronal stimulation, focusing on state-of-the-art nanoengineered devices. In particular, we highlight challenges and prospects towards improved retinal prostheses. PMID:24872704

  14. The investigation of the light radiation caused polyethylene based materials deterioration by means of atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Sikora, A.; Grabarek, A.; Moroń, L.; Wałecki, M.; Kryla, P.

    2016-02-01

    The impact of the environmental conditions on the materials used in various devices and constructions, in particular in electrotechnical applications, has an critical impact in terms of their reliability and utilization range in specific climatic conditions. Due to increasing utilitarian requirements, technological processes complexity and introducing new materials (for instance nanomaterials), advanced diagnostic techniques are desired. One of such techniques is atomic force microscopy (AFM), which allows to study the changes of the roughness and mechanical properties of the surface at the submicrometer scale, enabling the investigation of the degradation processes. In this work the deterioration of selected group of polyethylene based materials have been measured by means of AFM, as the samples were exposed to the simulated solar light and UV-C radiation. Such an analysis of the environmental conditions impact on the deterioration process using AFM methods for various versions of specific material was not presented before.

  15. Confocal light sheet microscopy: micron-scale neuroanatomy of the entire mouse brain.

    PubMed

    Silvestri, L; Bria, A; Sacconi, L; Iannello, G; Pavone, F S

    2012-08-27

    Elucidating the neural pathways that underlie brain function is one of the greatest challenges in neuroscience. Light sheet based microscopy is a cutting edge method to map cerebral circuitry through optical sectioning of cleared mouse brains. However, the image contrast provided by this method is not sufficient to resolve and reconstruct the entire neuronal network. Here we combined the advantages of light sheet illumination and confocal slit detection to increase the image contrast in real time, with a frame rate of 10 Hz. In fact, in confocal light sheet microscopy (CLSM), the out-of-focus and scattered light is filtered out before detection, without multiple acquisitions or any post-processing of the acquired data. The background rejection capabilities of CLSM were validated in cleared mouse brains by comparison with a structured illumination approach. We show that CLSM allows reconstructing macroscopic brain volumes with sub-cellular resolution. We obtained a comprehensive map of Purkinje cells in the cerebellum of L7-GFP transgenic mice. Further, we were able to trace neuronal projections across brain of thy1-GFP-M transgenic mice. The whole-brain high-resolution fluorescence imaging assured by CLSM may represent a powerful tool to navigate the brain through neuronal pathways. Although this work is focused on brain imaging, the macro-scale high-resolution tomographies affordable with CLSM are ideally suited to explore, at micron-scale resolution, the anatomy of different specimens like murine organs, embryos or flies. PMID:23037106

  16. Environment assisted degradation mechanisms in advanced light metals

    NASA Technical Reports Server (NTRS)

    Gangloff, R. P.; Stoner, G. E.; Swanson, R. E.

    1989-01-01

    A multifaceted research program on the performance of advanced light metallic alloys in aggressive aerospace environments, and associated environmental failure mechanisms was initiated. The general goal is to characterize alloy behavior quantitatively and to develop predictive mechanisms for environmental failure modes. Successes in this regard will provide the basis for metallurgical optimization of alloy performance, for chemical control of aggressive environments, and for engineering life prediction with damage tolerance and long term reliability.

  17. Assessing the imaging performance of light sheet microscopies in highly scattering tissues.

    PubMed

    Glaser, A K; Wang, Y; Liu, J T C

    2016-02-01

    Light sheet microscopy (LSM) has emerged as an optical-imaging method for high spatiotemporal volumetric imaging of relatively transparent samples. While this capability has allowed the technique to be highly impactful in fields such as developmental biology, applications involving highly scattering thick tissues have been largely unexplored. Herein, we employ Monte Carlo simulations to explore the use of LSM for imaging turbid media. In particular, due to its similarity to dual-axis confocal (DAC) microscopy, we compare LSM performance to point-scanned (PS-DAC) and line-scanned (LS-DAC) dual-axis confocal microscopy techniques that have been previously shown to produce high-quality images at round-trip optical lengths of ~9 - 10 and ~3 - 4 respectively. The results of this study indicate that LSM using widefield collection (WF-LSM) provides comparable performance to LS-DAC in thick tissues, due to the fact that they both utilize an illumination beam focused in one dimension (i.e. a line or sheet). On the other hand, LSM using confocal line detection (CL-LSM) is more analogous to PS-DAC microscopy, in which the illumination beam is focused in two dimensions to a point. The imaging depth of LSM is only slightly inferior to DAC (~2 - 3 and ~6 - 7 optical lengths for WF-LSM and CL-LSM respectively) due to the use of a lower numerical aperture (NA) illumination beam for extended imaging along the illumination axis. Therefore, we conclude that the ability to image deeply is dictated most by the confocality of the microscope technique. In addition, we find that imaging resolution is mostly dependent on the collection NA, and is relatively invariant to imaging depth in a homogeneous scattering medium. Our results indicate that superficial imaging of highly scattering tissues using light sheet microscopy is possible. PMID:26977355

  18. Assessing the imaging performance of light sheet microscopies in highly scattering tissues

    PubMed Central

    Glaser, A. K.; Wang, Y.; Liu, J. T.C.

    2016-01-01

    Light sheet microscopy (LSM) has emerged as an optical-imaging method for high spatiotemporal volumetric imaging of relatively transparent samples. While this capability has allowed the technique to be highly impactful in fields such as developmental biology, applications involving highly scattering thick tissues have been largely unexplored. Herein, we employ Monte Carlo simulations to explore the use of LSM for imaging turbid media. In particular, due to its similarity to dual-axis confocal (DAC) microscopy, we compare LSM performance to point-scanned (PS-DAC) and line-scanned (LS-DAC) dual-axis confocal microscopy techniques that have been previously shown to produce high-quality images at round-trip optical lengths of ~9 – 10 and ~3 – 4 respectively. The results of this study indicate that LSM using widefield collection (WF-LSM) provides comparable performance to LS-DAC in thick tissues, due to the fact that they both utilize an illumination beam focused in one dimension (i.e. a line or sheet). On the other hand, LSM using confocal line detection (CL-LSM) is more analogous to PS-DAC microscopy, in which the illumination beam is focused in two dimensions to a point. The imaging depth of LSM is only slightly inferior to DAC (~2 – 3 and ~6 – 7 optical lengths for WF-LSM and CL-LSM respectively) due to the use of a lower numerical aperture (NA) illumination beam for extended imaging along the illumination axis. Therefore, we conclude that the ability to image deeply is dictated most by the confocality of the microscope technique. In addition, we find that imaging resolution is mostly dependent on the collection NA, and is relatively invariant to imaging depth in a homogeneous scattering medium. Our results indicate that superficial imaging of highly scattering tissues using light sheet microscopy is possible. PMID:26977355

  19. High-resolution full-field optical coherence microscopy using a broadband light-emitting diode.

    PubMed

    Ogien, Jonas; Dubois, Arnaud

    2016-05-01

    High-resolution full-field optical coherence microscopy (FF-OCM) is demonstrated using a single broadband light-emitting diode (LED). The characteristics of the LED-illumination FF-OCM system are measured and compared to those obtained using a halogen lamp, the light source of reference in FF-OCM. Both light sources yield identical performance in terms of spatial resolution and detection sensitivity, using the same setup and camera. In particular, an axial resolution of 0.7 μm (in water) is reached. A Xenopus laevis tadpole and ex-vivo human skin have been imaged using both sources, resulting in similar images, showing for the first time that LEDs could favorably replace halogen lamps in high-resolution FF-OCM for biomedical imaging. PMID:27137603

  20. Two-Photon Microscopy with Diffractive Optical Elements and Spatial Light Modulators

    PubMed Central

    Watson, Brendon O.; Nikolenko, Volodymyr; Araya, Roberto; Peterka, Darcy S.; Woodruff, Alan; Yuste, Rafael

    2010-01-01

    Two-photon microscopy is often performed at slow frame rates due to the need to serially scan all points in a field of view with a single laser beam. To overcome this problem, we have developed two optical methods that split and multiplex a laser beam across the sample. In the first method a diffractive optical element (DOE) generates a fixed number of beamlets that are scanned in parallel resulting in a corresponding increase in speed or in signal-to-noise ratio in time-lapse measurements. The second method uses a computer-controlled spatial light modulator (SLM) to generate any arbitrary spatio-temporal light pattern. With an SLM one can image or photostimulate any predefined region of the image such as neurons or dendritic spines. In addition, SLMs can be used to mimic a large number of optical transfer functions including light path corrections as adaptive optics. PMID:20859526

  1. Use of a white light supercontinuum laser for confocal interference-reflection microscopy

    PubMed Central

    Chiu, L-D; Su, L; Reichelt, S; Amos, WB

    2012-01-01

    Shortly after its development, the white light supercontinuum laser was applied to confocal scanning microscopy as a more versatile substitute for the multiple monochromatic lasers normally used for the excitation of fluorescence. This light source is now available coupled to commercial confocal fluorescence microscopes. We have evaluated a supercontinuum laser as a source for a different purpose: confocal interferometric imaging of living cells and artificial models by interference reflection. We used light in the range 460–700 nm where this source provides a reasonably flat spectrum, and obtained images free from fringe artefacts caused by the longer coherence length of conventional lasers. We have also obtained images of cytoskeletal detail that is difficult to see with a monochromatic laser. PMID:22432542

  2. Macro-optical trapping for sample confinement in light sheet microscopy

    PubMed Central

    Yang, Zhengyi; Piksarv, Peeter; Ferrier, David E.K.; Gunn-Moore, Frank J.; Dholakia, Kishan

    2015-01-01

    Light sheet microscopy is a powerful approach to construct three-dimensional images of large specimens with minimal photo-damage and photo-bleaching. To date, the specimens are usually mounted in agents such as agarose, potentially restricting the development of live samples, and also highly mobile specimens need to be anaesthetized before imaging. To overcome these problems, here we demonstrate an integrated light sheet microscope which solely uses optical forces to trap and hold the sample using a counter-propagating laser beam geometry. Specifically, tobacco plant cells and living Spirobranchus lamarcki larvae were successfully trapped and sectional images acquired. This novel approach has the potential to significantly expand the range of applications for light sheet imaging. PMID:26309743

  3. The advanced light source: America`s brightest light for science and industry

    SciTech Connect

    Cross, J.; Lawler, G.

    1994-03-01

    America`s brightest light comes from the Advanced Light Source (ALS), a national facility for scientific research, product development, and manufacturing. Completed in 1993, the ALS produces light in the ultraviolet and x-ray regions of the spectrum. Its extreme brightness provides opportunities for scientific and technical progress not possible anywhere else. Technology is poised on the brink of a major revolution - one in which vital machine components and industrial processes will be drastically miniaturized. Industrialized nations are vying for leadership in this revolution - and the huge economic rewards the leaders will reap.

  4. Issues affecting advanced passive light-water reactor safety analysis

    SciTech Connect

    Beelman, R.J.; Fletcher, C.D.; Modro, S.M.

    1992-08-01

    Next generation commercial reactor designs emphasize enhanced safety through improved safety system reliability and performance by means of system simplification and reliance on immutable natural forces for system operation. Simulating the performance of these safety systems will be central to analytical safety evaluation of advanced passive reactor designs. Yet the characteristically small driving forces of these safety systems pose challenging computational problems to current thermal-hydraulic systems analysis codes. Additionally, the safety systems generally interact closely with one another, requiring accurate, integrated simulation of the nuclear steam supply system, engineered safeguards and containment. Furthermore, numerical safety analysis of these advanced passive reactor designs wig necessitate simulation of long-duration, slowly-developing transients compared with current reactor designs. The composite effects of small computational inaccuracies on induced system interactions and perturbations over long periods may well lead to predicted results which are significantly different than would otherwise be expected or might actually occur. Comparisons between the engineered safety features of competing US advanced light water reactor designs and analogous present day reactor designs are examined relative to the adequacy of existing thermal-hydraulic safety codes in predicting the mechanisms of passive safety. Areas where existing codes might require modification, extension or assessment relative to passive safety designs are identified. Conclusions concerning the applicability of these codes to advanced passive light water reactor safety analysis are presented.

  5. Issues affecting advanced passive light-water reactor safety analysis

    SciTech Connect

    Beelman, R.J.; Fletcher, C.D.; Modro, S.M.

    1992-01-01

    Next generation commercial reactor designs emphasize enhanced safety through improved safety system reliability and performance by means of system simplification and reliance on immutable natural forces for system operation. Simulating the performance of these safety systems will be central to analytical safety evaluation of advanced passive reactor designs. Yet the characteristically small driving forces of these safety systems pose challenging computational problems to current thermal-hydraulic systems analysis codes. Additionally, the safety systems generally interact closely with one another, requiring accurate, integrated simulation of the nuclear steam supply system, engineered safeguards and containment. Furthermore, numerical safety analysis of these advanced passive reactor designs wig necessitate simulation of long-duration, slowly-developing transients compared with current reactor designs. The composite effects of small computational inaccuracies on induced system interactions and perturbations over long periods may well lead to predicted results which are significantly different than would otherwise be expected or might actually occur. Comparisons between the engineered safety features of competing US advanced light water reactor designs and analogous present day reactor designs are examined relative to the adequacy of existing thermal-hydraulic safety codes in predicting the mechanisms of passive safety. Areas where existing codes might require modification, extension or assessment relative to passive safety designs are identified. Conclusions concerning the applicability of these codes to advanced passive light water reactor safety analysis are presented.

  6. Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nehal, Kishwer S.; Rajadhyaksha, Milind

    2016-02-01

    Latest advances in confocal microscopy of skin cancers toward guiding patient care: a Mohs surgeon's review and perspective About 350 publications worldwide have reported the ability of reflectance confocal microscopy (RCM) imaging to detect melanocytic skin lesions in vivo with specificity of 84-88% and sensitivity of 71-92%, and non-melanocytic skin lesions with specificity of 85-97% and sensitivity 100-92%. Lentigo maligna melanoma can be detected with sensitivity of 93% and specificity 82%. While the sensitivity is comparable to that of dermoscopy, the specificity is 2X superior, especially for lightly- and non-pigmented lesions. Dermoscopy combined with RCM imaging is proving to be both highly sensitive and highly specific. Recent studies have reported that the ratio of equivocal (i.e., would have been biopsied) lesions to detected melanomas dropped by ~2X when guided by dermoscopy and RCM imaging, compared to that with dermoscopy alone. Dermoscopy combined with RCM imaging is now being implemented to guide noninvasive diagnosis (to rule out malignancy and biopsy) and to also guide treatment, with promising initial impact: thus far, about 3,000 patients have been saved from biopsies of benign lesions. These are currently under follow-up monitoring. With fluorescence confocal microscopy (FCM) mosaicing, residual basal cell carcinomas can be detected in Mohs surgically excised fresh tissue ex vivo, with sensitivity of 94-97% and specificity 89-94%. FCM mosaicing is now being implemented for guiding Mohs surgery. To date, about 600 Mohs procedures have been performed, guided with mosaicing, and with pathology being performed in parallel to confirm the final outcome. These latest advances demonstrate the promising ability of RCM and FCM to guide patient care.

  7. Spectral Fingerprinting of Individual Cells Visualized by Cavity-Reflection-Enhanced Light-Absorption Microscopy

    PubMed Central

    Arai, Yoshiyuki; Yamamoto, Takayuki; Minamikawa, Takeo; Takamatsu, Tetsuro; Nagai, Takeharu

    2015-01-01

    The absorption spectrum of light is known to be a “molecular fingerprint” that enables analysis of the molecular type and its amount. It would be useful to measure the absorption spectrum in single cell in order to investigate the cellular status. However, cells are too thin for their absorption spectrum to be measured. In this study, we developed an optical-cavity-enhanced absorption spectroscopic microscopy method for two-dimensional absorption imaging. The light absorption is enhanced by an optical cavity system, which allows the detection of the absorption spectrum with samples having an optical path length as small as 10 μm, at a subcellular spatial resolution. Principal component analysis of various types of cultured mammalian cells indicates absorption-based cellular diversity. Interestingly, this diversity is observed among not only different species but also identical cell types. Furthermore, this microscopy technique allows us to observe frozen sections of tissue samples without any staining and is capable of label-free biopsy. Thus, our microscopy method opens the door for imaging the absorption spectra of biological samples and thereby detecting the individuality of cells. PMID:25950513

  8. Light-induced cell damage in live-cell super-resolution microscopy

    NASA Astrophysics Data System (ADS)

    Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus

    2015-10-01

    Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm-2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm-2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities.

  9. Light-induced cell damage in live-cell super-resolution microscopy.

    PubMed

    Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus

    2015-01-01

    Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20-24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm(-2) at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm(-2), emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. PMID:26481189

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

    PubMed

    Li, Yu; Hu, Ying; Cang, Hu

    2013-12-12

    Single particle tracking is a powerful tool to study single molecule dynamics in living biological samples. However, current tracking techniques, which are based mainly on epifluorescence, confocal, or TIRF microscopy, have difficulties in tracking single molecules on the apical surface of a cell. We present here a three-dimensional (3D) single particle tracking technique that is based on prism coupled light-sheet microscopy (PCLSM). This novel design provides a signal-to-noise ratio comparable to confocal microscopy while it has the capability of illuminating at arbitrary depth. We demonstrate tracking of single EGF molcules on the apical surface of live cell membranes from their binding to EGF receptors until they are internalized or photobleached. We found that EGF exhibits multiple diffusion behaviors on live A549 cell membranes. At room temperature, the average diffusion coefficient of EGF on A549 cells was measured to be 0.13 μm(2)/s. Depletion of cellular cholesterol with methyl-β-cyclodextrin leads to a broader distribution of diffusion coefficients and an increase of the average diffusion coefficient at room temperature. This light-sheet based 3D single particle tracking technique solves the technique difficulty of tracking single particles on apical membranes and is able to document the whole "lifetime" of a particle from binding till photobleaching or internalization. PMID:23895420

  11. SLM Microscopy: Scanless Two-Photon Imaging and Photostimulation with Spatial Light Modulators

    PubMed Central

    Nikolenko, Volodymyr; Watson, Brendon O.; Araya, Roberto; Woodruff, Alan; Peterka, Darcy S.; Yuste, Rafael

    2008-01-01

    Laser microscopy has generally poor temporal resolution, caused by the serial scanning of each pixel. This is a significant problem for imaging or optically manipulating neural circuits, since neuronal activity is fast. To help surmount this limitation, we have developed a “scanless” microscope that does not contain mechanically moving parts. This microscope uses a diffractive spatial light modulator (SLM) to shape an incoming two-photon laser beam into any arbitrary light pattern. This allows the simultaneous imaging or photostimulation of different regions of a sample with three-dimensional precision. To demonstrate the usefulness of this microscope, we perform two-photon uncaging of glutamate to activate dendritic spines and cortical neurons in brain slices. We also use it to carry out fast (60 Hz) two-photon calcium imaging of action potentials in neuronal populations. Thus, SLM microscopy appears to be a powerful tool for imaging and optically manipulating neurons and neuronal circuits. Moreover, the use of SLMs expands the flexibility of laser microscopy, as it can substitute traditional simple fixed lenses with any calculated lens function. PMID:19129923

  12. Light-induced cell damage in live-cell super-resolution microscopy

    PubMed Central

    Wäldchen, Sina; Lehmann, Julian; Klein, Teresa; van de Linde, Sebastian; Sauer, Markus

    2015-01-01

    Super-resolution microscopy can unravel previously hidden details of cellular structures but requires high irradiation intensities to use the limited photon budget efficiently. Such high photon densities are likely to induce cellular damage in live-cell experiments. We applied single-molecule localization microscopy conditions and tested the influence of irradiation intensity, illumination-mode, wavelength, light-dose, temperature and fluorescence labeling on the survival probability of different cell lines 20–24 hours after irradiation. In addition, we measured the microtubule growth speed after irradiation. The photo-sensitivity is dramatically increased at lower irradiation wavelength. We observed fixation, plasma membrane permeabilization and cytoskeleton destruction upon irradiation with shorter wavelengths. While cells stand light intensities of ~1 kW cm−2 at 640 nm for several minutes, the maximum dose at 405 nm is only ~50 J cm−2, emphasizing red fluorophores for live-cell localization microscopy. We also present strategies to minimize phototoxic factors and maximize the cells ability to cope with higher irradiation intensities. PMID:26481189

  13. Atomic Force Microscopy and Light Scattering of Small Unilamellar Actin-Containing Liposomes

    PubMed Central

    Palmer, Andre F.; Wingert, Philip; Nickels, Jonathan

    2003-01-01

    Three-dimensional networks of filamentous actin (F-actin) encapsulated inside phosphatidylcholine liposomes are currently being used in an effort to model the cytoskeleton and plasma membrane of eukaryotic cells. In this article, unilamellar lipid vesicles consisting of egg yolk-derived phosphatidylcholine encapsulating monomeric actin (G-actin) were made via extrusion in low ionic strength buffer (G-buffer). Vesicle shape and structure in these dispersions was studied using a combination of fluid-tapping atomic force microscopy, and multiangle static light scattering. After subjecting the liposome dispersion to high ionic strength polymerization buffer (F-buffer) containing K+ ions, atomic force microscopy imaging and light scattering of these liposomes indicated the formation of specialized structures, including an overall liposome structure transformation from spherical to torus, disk-shaped geometries and tubular assemblies. Several atomic force microscopy control measurements were made to ascertain that the specialized structures formed were not due to free G-actin and F-actin self-assembling on the sample surface, plain liposomes exposed to G- and F-buffer, or liposomes encapsulating G-actin. Liposomes encapsulating G-actin assumed mostly thin disk shapes and some large irregularly shaped aggregates. In contrast, liposomes encapsulating polymerized actin assumed mostly torus or disk shapes along with some high aspect ratio tubular structures. PMID:12885667

  14. Shedding new light on lipid functions with CARS and SRS microscopy

    PubMed Central

    Yu, Yong; Ramachandran, Prasanna V.; Wang, Meng C.

    2014-01-01

    Modern optical microscopy has granted biomedical scientists unprecedented access to the inner workings of a cell, and revolutionized our understanding of the molecular mechanisms underlying physiological and disease states. In spite of these advances, however, visualization of certain classes of molecules (e.g. lipids) at the sub-cellular level has remained elusive. Recently developed chemical imaging modalities – Coherent Anti-Stokes Raman Scattering (CARS) microscopy and Stimulated Raman Scattering (SRS) microscopy – have helped bridge this gap. By selectively imaging the vibration of a specific chemical group, these non-invasive techniques allow high-resolution imaging of individual molecules in vivo, and circumvent the need for potentially perturbative extrinsic labels. These tools have already been applied to the study of fat metabolism, helping uncover novel regulators of lipid storage. Here we review the underlying principle of CARS and SRS microscopy, and discuss the advantages and caveats of each technique. We also review recent applications of these tools in the study of lipids as well as other biomolecules, and conclude with a brief guide for interested researchers to build and use CARS/SRS systems for their own research. PMID:24576891

  15. One very rare and one new tracheal tumour found by electron microscopy: glomus tumour and acinic cell tumour resembling carcinoid tumours by light microscopy.

    PubMed Central

    Heard, B E; Dewar, A; Firmin, R K; Lennox, S C

    1982-01-01

    Tracheal tumours were removed surgically from two patients and diagnosed as carcinoid tumours by routine light microscopy. At a later date, electron microscopy was performed on stored tumour tissue and no neurosecretory granules were found in either case. One showed features of a glomus tumour and the other of an acinic cell tumour. Only two glomus tumours appear to have been reported previously in the trachea, and no acinic cell tumours. Electron microscopy is thus sometimes of great assistance in diagnosing accurately unusual tumours of the lower respiratory tract. Images PMID:6281934

  16. Comparison between light and electron microscopy in canine and feline renal pathology: a preliminary study.

    PubMed

    Scaglione, F E; Catalano, D; Bestonso, R; Brovida, C; D'Angelo, A; Zanatta, R; Cornaglia, S; Cornaglia, E; Capucchio, M T

    2008-12-01

    The aim of this study is to compare the accuracy and clinical use of light and transmission electron microscopy in detecting the early stages of renal pathologies in domestic animals. We examined 30 samples of renal tissue from cats and dogs referred to the Veterinary Hospital of the Department of Animal Pathology for different systemic diseases. The progressions of the kidney pathologies were classified using the scheme system proposed by the International Renal Interest Society. All samples were submitted for conventional histology and ultrastructural examination. Our study shows that electron microscopy is necessary to complete the histological examinations, especially to define early stages of kidney diseases (minimal changes disease, epithelial tubular pathologies, tubular basement membrane and glomerular basement membrane changes). Electron microscopy can be more accurate in defining the level of focal lesion, and permits discrimination between different clinical and pathological alterations such as fibrillary deposits. In conclusion, transmission electron microscopy associated with clinical, histological, histochemical and immunological examinations, is an essential method for diagnosis and prognosis of renal disease. PMID:19094015

  17. [Normal light and fluorescence microscopy for authentication of Delphinii Brunoniani Herba of Tibet].

    PubMed

    Wang, Ya-Qiong; Xu, Fu-Chun; Dongzhi, Zhuo-Ma; Liu, E-Hu; Xu, Luo-Shan; Liu, Hui-Juan; Li, Ping

    2012-11-01

    Dried herb of Delphinium brunonianum Royle (Ranunculaceae) has long been used under the herbal name "Xiaguobei" (Delphinii Brunoniani Herba) in traditional Tibetan medicine and prescribed for the treatment of influenza, itchy skin rash and snake bites. In order to find a useful and convenient method for the identification of microscopic features, the technique of fluorescence microscopy was applied to authenticate "Xiaguobei" of Tibet. The transverse sections of stem and leaf, as well as the powder of "Xiaguobei" were observed to seek for typical microscopic features by normal light and fluorescence microscopy. A style-like, single-cell glandular hair containing yellow secretions on the leaf, young stem and sepal of "Xiaguobei" was found. Under the fluorescence microscope, the xylem and pericycle fiber group emitted significant fluorescence. This work indicated that fluorescence microscopy could be an useful additional method for the authentication work. Without the traditional dyeing methods, the main microscopic features could be easily found by fluorescence microscopy. The results provided reliable references for the authentication of "Xiaguobei". PMID:23387092

  18. Developing methods based on light sheet fluorescence microscopy for biophysical investigations of larval zebrafish

    NASA Astrophysics Data System (ADS)

    Taormina, Michael J.

    Adapting the tools of optical microscopy to the large-scale dynamic systems encountered in the development of multicellular organisms provides a path toward understanding the physical processes necessary for complex life to form and function. Obtaining quantitatively meaningful results from such systems has been challenging due to difficulty spanning the spatial and temporal scales representative of the whole, while also observing the many individual members from which complex and collective behavior emerges. A three-dimensional imaging technique known as light sheet fluorescence microscopy provides a number of significant benefits for surmounting these challenges and studying developmental systems. A thin plane of fluorescence excitation light is produced such that it coincides with the focal plane of an imaging system, providing rapid acquisition of optically sectioned images that can be used to construct a three-dimensional rendition of a sample. I discuss the implementation of this technique for use in larva of the model vertebrate Danio rerio (zebrafish). The nature of light sheet imaging makes it especially well suited to the study of large systems while maintaining good spatial resolution and minimizing damage to the specimen from excessive exposure to excitation light. I show the results from a comparative study that demonstrates the ability to image certain developmental processes non-destructively, while in contrast confocal microscopy results in abnormal growth due to phototoxicity. I develop the application of light sheet microscopy to the study of a previously inaccessible system: the bacterial colonization of a host organism. Using the technique, we are able to obtain a survey of the intestinal tract of a larval zebrafish and observe the location of microbes as they grow and establish a stable population in an initially germ free fish. Finally, I describe a new technique to measure the fluid viscosity of this intestinal environment in vivo using

  19. Morphology of the dentin structure of sloths Bradypus tridactylus: a light and scanning electron microscopy investigation.

    PubMed

    Santana, L N S; Barbosa, L V M; Teixeira, F B; Costa, A M P; Fernandes, L M P; Lima, R R

    2013-12-01

    The aim of this study was to describe the dentine morphology of sloths (Bradypus tridactylus). The sloth teeth were removed and prepared for light microscopy (LM) and scanning electron microscopy analyses (SEM). LM revealed two patterns of tubular dentins: an outer with dentinary tubules over the all tooth length and one in the inner part with larger diameter and more spaced tubules, when compared to those present in the outer dentine. These findings were confirmed by SEM, which revealed a tubular pattern in the outer dentine like in humans. The inner dentine displayed pared grouped tubules that were characterized as vascular channels. It can be concluded that this sloth species present two types of dentins: an inner dentin (ortodentin) and an outer dentin characterized as a vascular dentin. This suggests a partial evolutive/adaptive process of this dental tissue, as compared to other mammalian species. PMID:23410180

  20. Lipid vesicle shape analysis from populations using light video microscopy and computer vision.

    PubMed

    Zupanc, Jernej; Drašler, Barbara; Boljte, Sabina; Kralj-Iglič, Veronika; Iglič, Aleš; Erdogmus, Deniz; Drobne, Damjana

    2014-01-01

    We present a method for giant lipid vesicle shape analysis that combines manually guided large-scale video microscopy and computer vision algorithms to enable analyzing vesicle populations. The method retains the benefits of light microscopy and enables non-destructive analysis of vesicles from suspensions containing up to several thousands of lipid vesicles (1-50 µm in diameter). For each sample, image analysis was employed to extract data on vesicle quantity and size distributions of their projected diameters and isoperimetric quotients (measure of contour roundness). This process enables a comparison of samples from the same population over time, or the comparison of a treated population to a control. Although vesicles in suspensions are heterogeneous in sizes and shapes and have distinctively non-homogeneous distribution throughout the suspension, this method allows for the capture and analysis of repeatable vesicle samples that are representative of the population inspected. PMID:25426933

  1. Balamuthia mandrillaris: Further morphological observations of trophozoites by light, scanning and transmission electron microscopy.

    PubMed

    González-Robles, Arturo; Lares-Villa, Fernando; Lares-Jiménez, Luis Fernando; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Martínez-Palomo, Adolfo

    2015-10-01

    Additional morphological features of Balamuthia mandrillaris observed by light and electron microscopy are reported. Trophozoites were extremely pleomorphic: their cell shapes ranged from rounded to elongated and sometimes they appeared exceptionally stretched out and branched. By transmission electron microscopy it was possible to observe two different cytoplasmic areas, the ectoplasm and the endoplasm and often sections of rough endoplasmic reticulum were found in the transition zone. The cytoplasm was very fibrogranular and most of the organelles typically found in eukaryotic cells were observed. A particular finding was the presence of numerous mitochondria with a different structure from those of other free-living amoebae. The observations reported here may reinforce the morphological knowledge of this amoeba and provide a background for further analyses. PMID:26297955

  2. Enhanced light collection in fluorescence microscopy using self-assembled micro-reflectors

    NASA Astrophysics Data System (ADS)

    Göröcs, Zoltán; McLeod, Euan; Ozcan, Aydogan

    2015-06-01

    In fluorescence microscopy, the signal-to-noise ratio (SNR) of the optical system is directly linked to the numerical aperture (NA) of the microscope objective, which creates detection challenges for low-NA, wide-field and high-throughput imaging systems. Here we demonstrate a method to increase the light collection efficiency from micron-scale fluorescent objects using self-assembled vapor-condensed polyethylene glycol droplets, which act as micro-reflectors for fluorescent light. Around each fluorescent particle, a liquid meniscus is formed that increases the excitation efficiency and redirects part of the laterally-emitted fluorescent light towards the detector due to internal reflections at the liquid-air interface of the meniscus. The three-dimensional shape of this micro-reflector can be tuned as a function of time, vapor temperature, and substrate contact angle, providing us optimized SNR performance for fluorescent detection. Based on these self-assembled micro-reflectors, we experimentally demonstrate ~2.5-3 fold enhancement of the fluorescent signal from 2-10 μm sized particles. A theoretical explanation of the formation rate and shapes of these micro-reflectors is presented, along with a ray tracing model of their optical performance. This method can be used as a sample preparation technique for consumer electronics-based microscopy and sensing tools, thus increasing the sensitivity of low-NA systems that image fluorescent micro-objects.

  3. Enhanced light collection in fluorescence microscopy using self-assembled micro-reflectors

    PubMed Central

    Göröcs, Zoltán; McLeod, Euan; Ozcan, Aydogan

    2015-01-01

    In fluorescence microscopy, the signal-to-noise ratio (SNR) of the optical system is directly linked to the numerical aperture (NA) of the microscope objective, which creates detection challenges for low-NA, wide-field and high-throughput imaging systems. Here we demonstrate a method to increase the light collection efficiency from micron-scale fluorescent objects using self-assembled vapor-condensed polyethylene glycol droplets, which act as micro-reflectors for fluorescent light. Around each fluorescent particle, a liquid meniscus is formed that increases the excitation efficiency and redirects part of the laterally-emitted fluorescent light towards the detector due to internal reflections at the liquid-air interface of the meniscus. The three-dimensional shape of this micro-reflector can be tuned as a function of time, vapor temperature, and substrate contact angle, providing us optimized SNR performance for fluorescent detection. Based on these self-assembled micro-reflectors, we experimentally demonstrate ~2.5-3 fold enhancement of the fluorescent signal from 2-10 μm sized particles. A theoretical explanation of the formation rate and shapes of these micro-reflectors is presented, along with a ray tracing model of their optical performance. This method can be used as a sample preparation technique for consumer electronics-based microscopy and sensing tools, thus increasing the sensitivity of low-NA systems that image fluorescent micro-objects. PMID:26083081

  4. Low coherence full field interference microscopy or optical coherence tomography: recent advances, limitations and future trends

    NASA Astrophysics Data System (ADS)

    Abdulhalim, I.

    2013-04-01

    Although low coherence microscopy (LCM) has been known for long time in the context of interference microscopy, coherence radar and white light interferometry, the whole subject has attracted a wide interest in the last two decades particularly accelerated by the entrance of OCT, as a noninvasive powerful technique for biomedical imaging. Today LCM can be classified into two types, both acts as three-dimensional imaging tool. The first is low temporal coherence microscopy; also known as optical coherence tomography (OCT), which is being used for medical diagnostics. The second is full field OCT in various modes and applied to various applications. FF-OCT uses low spatial and temporal coherence similar to the well-known coherence probe microscope (CPM) that have been in use for long time in optical metrology. The CPM has many advantages over conventional microscopy in its ability to discriminate between different transparent layers in a scattering medium thus allowing for precise noninvasive optical probing of dense tissue and other turbid media. In this paper the status of this technology in optical metrology applications will be discussed, on which we have been working to improve its performance, as well as its limitations and future prospective.

  5. Tissue and cellular localization of tannins in Tunisian dates (Phoenix dactylifera L.) by light and transmission electron microscopy.

    PubMed

    Hammouda, Hédi; Alvarado, Camille; Bouchet, Brigitte; Kalthoum-Chérif, Jamila; Trabelsi-Ayadi, Malika; Guyot, Sylvain

    2014-07-16

    A histological approach including light microscopy and transmission electron microscopy (TEM) was used to provide accurate information on the localization of condensed tannins in the edible tissues and in the stone of date fruits (Phoenix dactylifera L.). Light microscopy was carried out on fresh tissues after staining by 4-dimethylaminocinnamaldehyde (DMACA) for a specific detection of condensed tannins. Thus, whether under light microscopy or transmission electron microscopy (TEM), results showed that tannins are not located in the epidermis but more deeply in the mesocarp in the vacuole of very large cells. Regarding the stones, tannins are found in a specific cell layer located at 50 μm from the sclereid cells of the testa. PMID:24987926

  6. Nanoscale Characterization of Mock Explosive Materials Using Advanced Atomic Force Microscopy Methods

    NASA Astrophysics Data System (ADS)

    Xu, Xin; Mares, Jesus; Groven, Lori J.; Son, Steven F.; Reifenberger, Ronald G.; Raman, Arvind

    2015-01-01

    Most explosives are micro- and nanoscale composite material systems consisting of energetic crystals, amorphous particles, binders, and additives whose response to mechanical, thermal, or electromagnetic insults is often controlled by submicrometer-scale heterogeneities and interfaces. Several advanced dynamic atomic force microscopy (AFM) techniques, including phase imaging, force volume mode, and Kelvin probe force microscopy with resonance enhancement for dielectric property mapping, have been used to map the local physical properties of mock explosive materials systems, allowing the identification of submicrometer heterogeneities in electrical and mechanical properties that could lead to the formation of hotspots under electromagnetic or mechanical stimuli. The physical interpretation of the property maps and the methods of image formation are presented. Possible interpretations of the results and future applications to energetic material systems are also discussed.

  7. Multiple correlative immunolabeling for light and electron microscopy using fluorophores and colloidal metal particles.

    PubMed

    Kandela, Irawati K; Bleher, Reiner; Albrecht, Ralph M

    2007-10-01

    Multiple correlative immunolabeling permits colocalization of molecular species for sequential observation of the same sample in light microscopy (LM) and electron microscopy (EM). This technique allows rapid evaluation of labeling via LM, prior to subsequent time-consuming preparation and observation with transmission electric microscopy (TEM). The procedure also yields two different complementary data sets. In LM, different fluorophores are distinguished by their respective excitation and emission wavelengths. In EM, colloidal metal nanoparticles of different elemental composition can be differentiated and mapped by energy-filtering transmission electron microscopy with electron spectroscopic imaging. For the highest level of spatial resolution in TEM, colloidal metal particles were conjugated directly to primary antibodies. For LM, fluorophores were conjugated to secondary antibodies, which did not affect the spatial resolution attainable by fluorescence microscopy but placed the fluorophore at a sufficient distance from the metal particle to limit quenching of the fluorescence signal. It also effectively kept the fluorophore at a sufficient distance from the colloidal metal particles, which resulted in limiting quenching of the fluorescent signal. Two well-defined model systems consisting of myosin and alpha-actinin bands of skeletal muscle tissue and also actin and alpha-actinin of human platelets in ultrathin Epon sections were labeled using both fluorophores (Cy2 and Cy3) as markers for LM and equally sized colloidal gold (cAu) and colloidal palladium (cPd) particles as reporters for TEM. Each sample was labeled by a mixture of conjugates or labels and observed by LM, then further processed for TEM. PMID:17652267

  8. Optimization of the soft x-ray transmission microscopy beamline at the ALBA light source

    NASA Astrophysics Data System (ADS)

    Sorrentino, Andrea; Pereiro, Eva; Valcárcel, Ricardo; Ferrer, Salvador; Nicolas, Josep

    2013-09-01

    Mistral is the soft X-ray full field microscopy beamline at the ALBA light source. The beamline is designed to have large source acceptance and to provide constant magnification at the exit slit for photon energies between 270 and 2600 eV. The monochromator is a variation of the Petersen plane grating monochromator in which a variable line spacing grating is used to maintain the beam focused at the exit slit, independently of the fixed focus constant, and to cancel aberrations. We present the alignment strategy used to compensate errors of the optical elements, and report about the commissioning results.

  9. 3D imaging of the cleared intact murine colon with light sheet microscopy

    NASA Astrophysics Data System (ADS)

    Zufiria, B.; Bocancea, D. I.; Gómez-Gaviro, M. V.; Vaquero, J. J.; Desco, M.; Fresno, M.; Ripoll, J.; Arranz, A.

    2016-03-01

    We here show 3D light sheet microscopy images of fixed and cleared murine colon tissue in-toto, which offer relevant cellular information without the need for physically sectioning the tissue. We have applied the recently developed CUBIC protocol (Susaki et al. Cell 157:726, 2014) for colon tissues and have found that this clearing protocol enables imaging all the way to the central part of the lumen with cellular resolution, thus opening new ways for 3D imaging of colon samples.

  10. Advanced Time-Resolved Fluorescence Microscopy Techniques for the Investigation of Peptide Self-Assembly

    NASA Astrophysics Data System (ADS)

    Anthony, Neil R.

    The ubiquitous cross beta sheet peptide motif is implicated in numerous neurodegenerative diseases while at the same time offers remarkable potential for constructing isomorphic high-performance bionanomaterials. Despite an emerging understanding of the complex folding landscape of cross beta structures in determining disease etiology and final structure, we lack knowledge of the critical initial stages of nucleation and growth. In this dissertation, I advance our understanding of these key stages in the cross-beta nucleation and growth pathways using cutting-edge microscopy techniques. In addition, I present a new combined time-resolved fluorescence analysis technique with the potential to advance our current understanding of subtle molecular level interactions that play a pivotal role in peptide self-assembly. Using the central nucleating core of Alzheimer's Amyloid-beta protein, Abeta(16 22), as a model system, utilizing electron, time-resolved, and non-linear microscopy, I capture the initial and transient nucleation stages of peptide assembly into the cross beta motif. In addition, I have characterized the nucleation pathway, from monomer to paracrystalline nanotubes in terms of morphology and fluorescence lifetime, corroborating the predicted desolvation process that occurs prior to cross-beta nucleation. Concurrently, I have identified unique heterogeneous cross beta domains contained within individual nanotube structures, which have potential bionanomaterials applications. Finally, I describe a combined fluorescence theory and analysis technique that dramatically increases the sensitivity of current time-resolved techniques. Together these studies demonstrate the potential for advanced microscopy techniques in the identification and characterization of the cross-beta folding pathway, which will further our understanding of both amyloidogenesis and bionanomaterials.

  11. Precise measurement of the resolution in light microscopy using Fourier transform.

    PubMed

    Vainrub, Arnold

    2008-04-01

    The resolution power of light microscope has been accurately measured (+/-5%) by Fourier transform of various object images and further evaluation of the highest spatial frequency in Fourier spectrum. Any unknown shape plane object with a shape feature's size smaller than the resolution to be measured was shown to provide a reliable resolution test. This simple method gives a direct measurement of the resolution power as defined by Abbe [Archiv. F. Mikroskopische Anat. 9, 413 (1873)]. The results have been justified by comparison to a standard resolution measurement by using calibrated periodic line patterns. Notably, the approach is applicable in super-resolution light microscopy (transmission, reflection, and fluorescence), where calibrated resolution targets do not occur. It was conveniently implemented by using a compact disk as a test object and free IMAGEJ imaging software. PMID:18447570

  12. Calcium imaging of neural circuits with extended depth-of-field light-sheet microscopy

    PubMed Central

    Quirin, Sean; Vladimirov, Nikita; Yang, Chao-Tsung; Peterka, Darcy S.; Yuste, Rafael; Ahrens, Misha B.

    2016-01-01

    Increasing the volumetric imaging speed of light-sheet microscopy will improve its ability to detect fast changes in neural activity. Here, a system is introduced for brain-wide imaging of neural activity in the larval zebrafish by coupling structured illumination with cubic phase extended depth-of-field (EDoF) pupil encoding. This microscope enables faster light-sheet imaging and facilitates arbitrary plane scanning—removing constraints on acquisition speed, alignment tolerances, and physical motion near the sample. The usefulness of this method is demonstrated by performing multi-plane calcium imaging in the fish brain with a 416 × 832 × 160 µm field of view at 33 Hz. The optomotor response behavior of the zebrafish is monitored at high speeds, and time-locked correlations of neuronal activity are resolved across its brain. PMID:26974063

  13. Precise measurement of the resolution in light microscopy using Fourier transform

    SciTech Connect

    Vainrub, Arnold

    2008-04-15

    The resolution power of light microscope has been accurately measured ({+-}5%) by Fourier transform of various object images and further evaluation of the highest spatial frequency in Fourier spectrum. Any unknown shape plane object with a shape feature's size smaller than the resolution to be measured was shown to provide a reliable resolution test. This simple method gives a direct measurement of the resolution power as defined by Abbe [Archiv. F. Mikroskopische Anat. 9, 413 (1873)]. The results have been justified by comparison to a standard resolution measurement by using calibrated periodic line patterns. Notably, the approach is applicable in super-resolution light microscopy (transmission, reflection, and fluorescence), where calibrated resolution targets do not occur. It was conveniently implemented by using a compact disk as a test object and free IMAGEJ imaging software.

  14. Identification of light elements in silicon nitride by aberration-corrected scanning transmission electron microscopy.

    PubMed

    Idrobo, Juan C; Walkosz, Weronika; Klie, Robert F; Oğüt, Serdar

    2012-12-01

    In silicon nitride structural ceramics, the overall mechanical and thermal properties are controlled by the atomic and electronic structures at the interface between the ceramic grains and the amorphous intergranular films (IGFs) formed by various sintering additives. In the last ten years the atomic arrangements of heavy elements (rare-earths) at the Si(3)N(4)/IGF interfaces have been resolved. However, the atomic position of light elements, without which it is not possible to obtain a complete description of the interfaces, has been lacking. This review article details the authors' efforts to identify the atomic arrangement of light elements such as nitrogen and oxygen at the Si(3)N(4)/SiO(2) interface and in bulk Si(3)N(4) using aberration-corrected scanning transmission electron microscopy. PMID:22726263

  15. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots.

    PubMed

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

  16. Separation of ballistic and diffusive fluorescence photons in confocal Light-Sheet Microscopy of Arabidopsis roots

    PubMed Central

    Meinert, Tobias; Tietz, Olaf; Palme, Klaus J.; Rohrbach, Alexander

    2016-01-01

    Image quality in light-sheet fluorescence microscopy is strongly affected by the shape of the illuminating laser beam inside embryos, plants or tissue. While the phase of Gaussian or Bessel beams propagating through thousands of cells can be partly controlled holographically, the propagation of fluorescence light to the detector is difficult to control. With each scatter process a fluorescence photon loses information necessary for the image generation. Using Arabidopsis root tips we demonstrate that ballistic and diffusive fluorescence photons can be separated by analyzing the image spectra in each plane without a priori knowledge. We introduce a theoretical model allowing to extract typical scattering parameters of the biological material. This allows to attenuate image contributions from diffusive photons and to amplify the relevant image contributions from ballistic photons through a depth dependent deconvolution. In consequence, image contrast and resolution are significantly increased and scattering artefacts are minimized especially for Bessel beams with confocal line detection. PMID:27553506

  17. What advances in microscopy are required for combined MRI and optical functional brain imaging? (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Kleinfeld, David

    2016-03-01

    This overview talk will focus on forward-looking scientific needs and physical limits to images of neuronal processes. The challenge in nervous systems is that the basic unit for "switching" events in the nervous system occurs on the one micrometer scale of synaptic spines, while computations involve communication between individual neurons across the full expanse of cortex, which is ten millimeters for mouse cortex. I will address hoped-for advances in optical microscopy, within the context of existing and proposed contrast mechanisms of neuronal function, that span the four orders of magnitude of length scales for neuronal processing

  18. Gold-copper nanostars as photo-thermal agents: synthesis and advanced electron microscopy characterization.

    PubMed

    Bazán-Díaz, Lourdes; Mendoza-Cruz, Rubén; Velázquez-Salazar, J Jesús; Plascencia-Villa, Germán; Romeu, David; Reyes-Gasga, José; Herrera-Becerra, Raúl; José-Yacamán, Miguel; Guisbiers, Grégory

    2015-12-28

    Nanoalloys have emerged as multi-functional nanoparticles with applications in biomedicine and catalysis. This work reports the efficient production and the advanced transmission electron microscopy characterization of gold-copper pentagonal nanostars. The morphology of the branches is controlled by the adequate choice of the capping agent. When oleylamine is used rounded nanostars are produced, while pointed nanostars are obtained by using hexadecylamine. Both types of nanostars were proved to be thermally stable and could therefore be used as therapeutic agents in photo-thermal therapies as confirmed by the near-infrared absorption spectra. PMID:26602429

  19. Explosive Vessel for Dynamic Experiments at Advanced Light Sources

    NASA Astrophysics Data System (ADS)

    Owens, Charles; Sorensen, Christian; Armstrong, Christopher; Sanchez, Nathaniel; Jensen, Brian

    2015-06-01

    There has been significant effort in coupling dynamic loading platforms to advanced light sources such as the Advanced Photon Source (APS) to take advantage of X-ray diagnostics for examining material physics at extremes. Although the focus of these efforts has been on using gun systems for dynamic compression experiments, there are many experiments that require explosive loading capabilities including studies related to detonator dynamics, small angle X-ray scattering on explosives, and ejecta formation, for example. To this end, an explosive vessel and positioning stage was designed specifically for use at a synchrotron with requirements to confine up to 15 grams of explosives, couple the vessel to the X-ray beam line, and reliably position samples in the X-ray beam remotely with micrometer spatial accuracy. In this work, a description of the system will be provided along with explosive testing results for the robust, reusable positioning system.

  20. Insertion devices for the Advanced Light Source at LBL

    SciTech Connect

    Hassenzahl, W.; Chin, J.; Halbach, K.; Hoyer, E.; Humphries, D.; Kincaid, B.; Savoy, R.

    1989-03-01

    The Advanced Light Source (ALS) at the Lawrence Berkeley Laboratory will be the first of the new generation of dedicated synchrotron light sources to be put into operation. Specially designed insertion devices will be required to realize the high brightness photon beams made possible by the low emittance of the electron beam. The complement of insertion devices on the ALS will include undulators with periods as short as 3.9 cm and one or more high field wigglers. The first device to be designed is a 5 m long, 5 cm period, hybrid undulator. The goal of very high brightness and high harmonic output imposes unusually tight tolerances on the magnetic field quality and thus on the mechanical structure. The design process, using a generic structure for all undulators, is described. 5 refs., 4 figs., 1 tab.

  1. Status report on the Advanced Light Source control system, 1993

    SciTech Connect

    Young, J.; Brown, W. Jr.; Cork, C.

    1993-10-01

    The Advanced Light Source (ALS), under construction for the past seven years, has become operational. The accelerator has been successfully commissioned using a control system based on hundreds of controllers of our own design and high performance personal computers which are the operator interface. The first beamlines are being commissioned using a control system based on VME hardware and the Experimental Physics and Industrial Control System (EPICS) software. The two systems are being integrated, and this paper reports on the current work being done.

  2. Measurement of replication structures at the nanometer scale using super-resolution light microscopy

    PubMed Central

    Baddeley, D.; Chagin, V. O.; Schermelleh, L.; Martin, S.; Pombo, A.; Carlton, P. M.; Gahl, A.; Domaing, P.; Birk, U.; Leonhardt, H.; Cremer, C.; Cardoso, M. C.

    2010-01-01

    DNA replication, similar to other cellular processes, occurs within dynamic macromolecular structures. Any comprehensive understanding ultimately requires quantitative data to establish and test models of genome duplication. We used two different super-resolution light microscopy techniques to directly measure and compare the size and numbers of replication foci in mammalian cells. This analysis showed that replication foci vary in size from 210 nm down to 40 nm. Remarkably, spatially modulated illumination (SMI) and 3D-structured illumination microscopy (3D-SIM) both showed an average size of 125 nm that was conserved throughout S-phase and independent of the labeling method, suggesting a basic unit of genome duplication. Interestingly, the improved optical 3D resolution identified 3- to 5-fold more distinct replication foci than previously reported. These results show that optical nanoscopy techniques enable accurate measurements of cellular structures at a level previously achieved only by electron microscopy and highlight the possibility of high-throughput, multispectral 3D analyses. PMID:19864256

  3. Electron and Light Microscopy Techniques Suitable for Studying Fatigue Damage in a Crystallized Glass Ceramic

    NASA Technical Reports Server (NTRS)

    Harrell, Shelley; Zaretsky, Erwin V.

    1961-01-01

    The crystals of Pyroceram are randomly oriented and highly reflective so that standard microscopy techniques are not satisfactory for studying this material. Standard replicating procedures proved difficult to use. New microscopy techniques and procedures have therefore been developed. A method for locating, orienting, and identifying specific areas to be viewed with an electron microscope is described. This method not require any special equipment. Plastic replicas were found to be unsatisfactory because of their tendency to adhere to Pryoceram. This caused them to tear when released or resulted in artifacts. Preshadowed silicon monoxide replicas were satisfactory but required a releasing agent. A method of depositing the releasing agent is described. To polish specimens without evidence of fire-polishing, it was found necessary to use a vibratory polishing technique. Chrome oxide was used as the abrasive and either water or kerosene as the lubricant. Vibratory polishing is extremely slow, but surfaces so polished show no evidence of fire polishing, even when examined by electron microscopy. The most satisfactory etching process used for Pyroceram 9608 consisted of a primary etch of 5 milliliters of hydrochloric acid (concentrated), 5 milliliters of hydrogen fluoride (45 percent), and 45 milliliters of water, and a secondary etch with methyl alcohol replacing the water. Best results were obtained with total etching times from 25 to 30 seconds. Staining of the Pyroceram surface with a Sanford's marker was found to be an expedient way to reduce the glare of reflected light.

  4. Correlative light and electron microscopy enables viral replication studies at the ultrastructural level.

    PubMed

    Hellström, Kirsi; Vihinen, Helena; Kallio, Katri; Jokitalo, Eija; Ahola, Tero

    2015-11-15

    Electron microscopy (EM) is a powerful tool to study structural changes within cells caused e.g. by ectopic protein expression, gene silencing or virus infection. Correlative light and electron microscopy (CLEM) has proven to be useful in cases when it is problematic to identify a particular cell among a majority of unaffected cells at the EM level. In this technique the cells of interest are first identified by fluorescence microscopy and then further processed for EM. CLEM has become crucial when studying positive-strand RNA virus replication, as it takes place in nanoscale replication sites on specific cellular membranes. Here we have employed CLEM for Semliki Forest virus (SFV) replication studies both by transfecting viral replication components to cells or by infecting different cell types. For the transfection-based system, we developed an RNA template that can be detected in the cells even in the absence of replication and thus allows exploration of lethal mutations in viral proteins. In infected mammalian and mosquito cells, we were able to find replication-positive cells by using a fluorescently labeled viral protein even in the cases of low infection efficiency. The fluorescent region within these cells was shown to correspond to an area rich in modified membranes. These results show that CLEM is a valuable technique for studying virus replication and membrane modifications at the ultrastructural level. PMID:25916619

  5. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

    PubMed

    Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

    2013-12-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. PMID:23242824

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

    NASA Astrophysics Data System (ADS)

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

    2016-05-01

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

  7. Light-sheet-based fluorescence microscopy for three-dimensional imaging of biological samples.

    PubMed

    Swoger, Jim; Pampaloni, Francesco; Stelzer, Ernst H K

    2014-01-01

    In modern biology, most optical imaging technologies are applied to two-dimensional cell culture systems; that is, they are used in a cellular context that is defined by hard and flat surfaces. However, a physiological context is not found in single cells cultivated on coverslips. It requires the complex three-dimensional (3D) relationship of cells cultivated in extracellular matrix (ECM) gels, tissue sections, or in naturally developing organisms. In fact, the number of applications of 3D cell cultures in basic research as well as in drug discovery and toxicity testing has been increasing over the past few years. Unfortunately, the imaging of highly scattering multicellular specimens is still challenging. The main issues are the limited optical penetration depth, the phototoxicity, and the fluorophore bleaching. Light-sheet-based fluorescence microscopy (LSFM) overcomes many drawbacks of conventional fluorescence microscopy by using an orthogonal/azimuthal fluorescence arrangement with independent sets of lenses for illumination and detection. The basic idea is to illuminate the specimen from the side with a thin light sheet that overlaps with the focal plane of a wide-field fluorescence microscope. Optical sectioning and minimal phototoxic damage or photobleaching outside a small volume close to the focal plane are intrinsic properties of LSFM. We discuss the basic principles of LSFM and methods for the preparation, embedding, and imaging of 3D specimens used in the life sciences in an implementation of LSFM known as the single (or selective) plane illumination microscope (SPIM). PMID:24371323

  8. In vivo imaging of cardiac development and function in zebrafish using light sheet microscopy.

    PubMed

    Weber, Michael; Huisken, Jan

    2015-01-01

    Detailed studies of heart development and function are crucial for our understanding of cardiac failures and pave the way for better diagnostics and treatment. However, the constant motion and close incorporation into the cardiovascular system prevent in vivo studies of the living, unperturbed heart. The complementary strengths of the zebrafish model and light sheet microscopy provide a useful platform to fill this gap. High-resolution images of the embryonic vertebrate heart are now recorded from within the living animal: deep inside the unperturbed heart we can follow cardiac contractions and measure action potentials and calcium transients. Three-dimensional reconstructions of the entire beating heart with cellular resolution give new insights into its ever-changing morphology and facilitate studies into how individual cells form the complex cardiac network. In addition, cardiac dynamics and robustness are now examined with targeted optical manipulation. Overall, the combination of zebrafish and light sheet microscopy represents a promising addition for cardiac research and opens the door to a better understanding of heart function and development. PMID:26700795

  9. A Method for Preparing Difficult Plant Tissues for Light and Electron Microscopy.

    PubMed

    Clode, Peta L

    2015-08-01

    Although the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation of Banksia prionotes (Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs. PMID:26189464

  10. Advanced Nuclear Technology: Advanced Light Water Reactors Utility Requirements Document Small Modular Reactors Inclusion Summary

    SciTech Connect

    Loflin, Leonard; McRimmon, Beth

    2014-12-18

    This report summarizes a project by EPRI to include requirements for small modular light water reactors (smLWR) into the EPRI Utility Requirements Document (URD) for Advanced Light Water Reactors. The project was jointly funded by EPRI and the U.S. Department of Energy (DOE). The report covers the scope and content of the URD, the process used to revise the URD to include smLWR requirements, a summary of the major changes to the URD to include smLWR, and how to use the URD as revised to achieve value on new plant projects.

  11. LIGHT SOURCE: Conceptual design of Hefei Advanced Light Source (HALS) injection system

    NASA Astrophysics Data System (ADS)

    Zhang, Shan-Cai; Wang, Lin; Feng, Guang-Yao; Wu, Cong-Feng; Li, Wei-Min; Xu, Hong-Liang; Liu, Zu-Ping

    2009-06-01

    The Hefei Advanced Light Source(HALS) is a super low emittance storage ring and has a very short beam life time. In order to run the ring stablely, top-up injection will be necessary. The injection system will greatly affect the quality of beam. This article first gives a physics design of the injecting system. Then the injecting system is tracked under different errors. The responses of storage beam and injecting beam are given in the article.

  12. Further description of Cruzia tentaculata (Rudolphi, 1819) Travassos, 1917 (Nematoda: Cruzidae) by light and scanning electron microscopy.

    PubMed

    Adnet, F A O; Anjos, D H S; Menezes-Oliveira, A; Lanfredi, R M

    2009-04-01

    Species of Cruzia are parasites of the large intestine of marsupials, reptiles, amphibians, and mammalians. Cruzia tentaculata specimens were collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Colombia (new geographical record) and from Brazil and analyzed by light and scanning electron microscopy. The morphology of males and females by light microscopy corroborated most of the previous description and the ultrastructure by scanning electron microscopy evidence: the topography of the cuticle, deirids, amphids, phasmids in both sexes, a pair of papillae near the vulva opening, and the number and location of male caudal papillae, adding new features for species identification only observed by this technique. PMID:19130086

  13. Gold-copper nanostars as photo-thermal agents: synthesis and advanced electron microscopy characterization

    NASA Astrophysics Data System (ADS)

    Bazán-Díaz, Lourdes; Mendoza-Cruz, Rubén; Velázquez-Salazar, J. Jesús; Plascencia-Villa, Germán; Romeu, David; Reyes-Gasga, José; Herrera-Becerra, Raúl; José-Yacamán, Miguel; Guisbiers, Grégory

    2015-12-01

    Nanoalloys have emerged as multi-functional nanoparticles with applications in biomedicine and catalysis. This work reports the efficient production and the advanced transmission electron microscopy characterization of gold-copper pentagonal nanostars. The morphology of the branches is controlled by the adequate choice of the capping agent. When oleylamine is used rounded nanostars are produced, while pointed nanostars are obtained by using hexadecylamine. Both types of nanostars were proved to be thermally stable and could therefore be used as therapeutic agents in photo-thermal therapies as confirmed by the near-infrared absorption spectra.Nanoalloys have emerged as multi-functional nanoparticles with applications in biomedicine and catalysis. This work reports the efficient production and the advanced transmission electron microscopy characterization of gold-copper pentagonal nanostars. The morphology of the branches is controlled by the adequate choice of the capping agent. When oleylamine is used rounded nanostars are produced, while pointed nanostars are obtained by using hexadecylamine. Both types of nanostars were proved to be thermally stable and could therefore be used as therapeutic agents in photo-thermal therapies as confirmed by the near-infrared absorption spectra. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06491k

  14. CASL: The Consortium for Advanced Simulation of Light Water Reactors

    NASA Astrophysics Data System (ADS)

    Kothe, Douglas B.

    2010-11-01

    Like the fusion community, the nuclear engineering community is embarking on a new computational effort to create integrated, multiphysics simulations. The Consortium for Advanced Simulation of Light Water Reactors (CASL), one of 3 newly-funded DOE Energy Innovation Hubs, brings together an exceptionally capable team that will apply existing modeling and simulation capabilities and develop advanced capabilities to create a usable environment for predictive simulation of light water reactors (LWRs). This environment, designated the Virtual Reactor (VR), will: 1) Enable the use of leadership-class computing for engineering design and analysis to improve reactor capabilities, 2) Promote an enhanced scientific basis and understanding by replacing empirically based design and analysis tools with predictive capabilities, 3) Develop a highly integrated multiphysics environment for engineering analysis through increased fidelity methods, and 4) Incorporate UQ as a basis for developing priorities and supporting, application of the VR tools for predictive simulation. In this presentation, we present the plans for CASL and comment on the similarity and differences with the proposed Fusion Simulation Project (FSP).

  15. Refrigeration options for the Advanced Light Source Superbend Dipole Magnets

    SciTech Connect

    Green, M.A.; Hoyer, E.H.; Schlueter, R.D.; Taylor, C.E.; Zbasnik, J.; Wang, S.T.

    1999-07-09

    The 1.9 GeV Advance Light Source (ALS) at the Lawrence Berkeley National Laboratory (LBNL) produces photons with a critical energy of about 3.1 kev at each of its thirty-six 1.3 T gradient bending magnets. It is proposed that at three locations around the ring the conventional gradient bending magnets be replaced with superconducting bending magnets with a maximum field of 5.6 T. At the point where the photons are extracted, their critical energy will be about 12 keV. In the beam lines where the SuperBend superconducting magnets are installed, the X ray brightness at 20 keV will be increased over two orders of magnitude. This report describes three different refrigeration options for cooling the three SuperBend dipoles. The cooling options include: (1) liquid helium and liquid nitrogen cryogen cooling using stored liquids, (2) a central helium refrigerator (capacity 70 to 100 W) cooling all of the SuperBend magnets, (3) a Gifford McMahon (GM) cryocooler on each of the dipoles. This paper describes the technical and economic reasons for selecting a small GM cryocooler as the method for cooling the SuperBend dipoles on the LBNL Advanced Light Source.

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

    PubMed Central

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

    2016-01-01

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

  17. Individual Particle Analysis of Ambient PM 2.5 Using Advanced Electron Microscopy Techniques

    SciTech Connect

    Gerald J. Keeler; Masako Morishita

    2006-12-31

    The overall goal of this project was to demonstrate a combination of advanced electron microscopy techniques that can be effectively used to identify and characterize individual particles and their sources. Specific techniques to be used include high-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), STEM energy dispersive X-ray spectrometry (EDX), and energy-filtered TEM (EFTEM). A series of ambient PM{sub 2.5} samples were collected in communities in southwestern Detroit, MI (close to multiple combustion sources) and Steubenville, OH (close to several coal fired utility boilers). High-resolution TEM (HRTEM) -imaging showed a series of nano-metal particles including transition metals and elemental composition of individual particles in detail. Submicron and nano-particles with Al, Fe, Ti, Ca, U, V, Cr, Si, Ba, Mn, Ni, K and S were observed and characterized from the samples. Among the identified nano-particles, combinations of Al, Fe, Si, Ca and Ti nano-particles embedded in carbonaceous particles were observed most frequently. These particles showed very similar characteristics of ultrafine coal fly ash particles that were previously reported. By utilizing HAADF-STEM, STEM-EDX, and EF-TEM, this investigation was able to gain information on the size, morphology, structure, and elemental composition of individual nano-particles collected in Detroit and Steubenville. The results showed that the contributions of local combustion sources - including coal fired utilities - to ultrafine particle levels were significant. Although this combination of advanced electron microscopy techniques by itself can not identify source categories, these techniques can be utilized as complementary analytical tools that are capable of providing detailed information on individual particles.

  18. X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

    PubMed Central

    Bushong, Eric A.; Johnson, Donald D.; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T.; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H.

    2015-01-01

    The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging. PMID:25392009

  19. X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.

    PubMed

    Bushong, Eric A; Johnson, Donald D; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H

    2015-02-01

    The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging. PMID:25392009

  20. Highly Automated Module Production Incorporating Advanced Light Management

    SciTech Connect

    Perelli-Minetti, Michael; Roof, Kyle

    2015-08-11

    The objective was to enable a high volume, cost effective solution for increasing the amount of light captured by PV modules through utilization of an advanced Light Re-directing Film and to follow a phased approach to develop and implement this new technology in order to achieve an expected power gain of up to 12 watts per module. Full size PV modules were manufactured using a new Light Redirecting Film (LRF) material applied to two different areas of PV modules in order to increase the amount of light captured by the modules. One configuration involved applying thin strips of LRF film over the tabbing ribbon on the cells in order to redirect the light that is normally absorbed by the tabbing ribbon to the active areas of the cells. A second configuration involved applying thin strips of LRF film over the white spaces between cells within a module in order to capture some of the light that is normally reflected from the white areas back through the front glass of the modules. Significant power increases of 1.4% (3.9 watts) and 1.0% (3.2 watts), respectively, compared to standard PV modules were measured under standard test conditions. The performance of PV modules with LRF applied to the tabbing ribbon was modeled. The results showed that the power increase provided by LRF depended greatly on the angle of incident light with the optimum performance only occurring when the light was within a narrow range of being perpendicular to the solar module. The modeling showed that most of the performance gain would be lost when the angle of incident light was greater than 28 degrees off axis. This effect made the orientation of modules with LRF applied to tabbing ribbons very important as modules mounted in “portrait” mode were predicted to provide little to no power gain from LRF under real world conditions. Based on these results, modules with LRF on tabbing ribbons would have to be mounted in “landscape” mode to realize a performance advantage. In addition

  1. The Consortium for Advanced Simulation of Light Water Reactors

    SciTech Connect

    Ronaldo Szilard; Hongbin Zhang; Doug Kothe; Paul Turinsky

    2011-10-01

    The Consortium for Advanced Simulation of Light Water Reactors (CASL) is a DOE Energy Innovation Hub for modeling and simulation of nuclear reactors. It brings together an exceptionally capable team from national labs, industry and academia that will apply existing modeling and simulation capabilities and develop advanced capabilities to create a usable environment for predictive simulation of light water reactors (LWRs). This environment, designated as the Virtual Environment for Reactor Applications (VERA), will incorporate science-based models, state-of-the-art numerical methods, modern computational science and engineering practices, and uncertainty quantification (UQ) and validation against data from operating pressurized water reactors (PWRs). It will couple state-of-the-art fuel performance, neutronics, thermal-hydraulics (T-H), and structural models with existing tools for systems and safety analysis and will be designed for implementation on both today's leadership-class computers and the advanced architecture platforms now under development by the DOE. CASL focuses on a set of challenge problems such as CRUD induced power shift and localized corrosion, grid-to-rod fretting fuel failures, pellet clad interaction, fuel assembly distortion, etc. that encompass the key phenomena limiting the performance of PWRs. It is expected that much of the capability developed will be applicable to other types of reactors. CASL's mission is to develop and apply modeling and simulation capabilities to address three critical areas of performance for nuclear power plants: (1) reduce capital and operating costs per unit energy by enabling power uprates and plant lifetime extension, (2) reduce nuclear waste volume generated by enabling higher fuel burnup, and (3) enhance nuclear safety by enabling high-fidelity predictive capability for component performance.

  2. Advance in Photosensitizers and Light Delivery for Photodynamic Therapy

    PubMed Central

    Yoon, Il; Li, Jia Zhu

    2013-01-01

    The brief history of photodynamic therapy (PDT) research has been focused on photosensitizers (PSs) and light delivery was introduced recently. The appropriate PSs were developed from the first generation PS Photofrin (QLT) to the second (chlorins or bacteriochlorins derivatives) and third (conjugated PSs on carrier) generations PSs to overcome undesired disadvantages, and to increase selective tumor accumulation and excellent targeting. For the synthesis of new chlorin PSs chlorophyll a is isolated from natural plants or algae, and converted to methyl pheophorbide a (MPa) as an important starting material for further synthesis. MPa has various active functional groups easily modified for the preparation of different kinds of PSs, such as methyl pyropheophorbide a, purpurin-18, purpurinimide, and chlorin e6 derivatives. Combination therapy, such as chemotherapy and photothermal therapy with PDT, is shortly described here. Advanced light delivery system is shown to establish successful clinical applications of PDT. Phtodynamic efficiency of the PSs with light delivery was investigated in vitro and/or in vivo. PMID:23423543

  3. Optical and mechanical detection of near-field light by atomic force microscopy using a piezoelectric cantilever

    NASA Astrophysics Data System (ADS)

    Satoh, Nobuo; Kobayashi, Kei; Watanabe, Shunji; Fujii, Toru; Matsushige, Kazumi; Yamada, Hirofumi

    2016-08-01

    In this study, we developed an atomic force microscopy (AFM) system with scanning near-field optical microscopy (SNOM) using a microfabricated force-sensing cantilever with a lead zirconate titanate (PZT) thin film. Both optical and mechanical detection techniques were adopted in SNOM to detect scattered light induced by the interaction of the PZT cantilever tip apex and evanescent light, and SNOM images were obtained for each detection scheme. The mechanical detection technique did allow for a clear observation of the light scattered from the PZT cantilever without the interference observed by the optical detection technique, which used an objective lens, a pinhole, and a photomultiplier tube.

  4. Cellular imaging of deep organ using two-photon Bessel light-sheet nonlinear structured illumination microscopy

    PubMed Central

    Zhao, Ming; Zhang, Han; Li, Yu; Ashok, Amit; Liang, Rongguang; Zhou, Weibin; Peng, Leilei

    2014-01-01

    In vivo fluorescent cellular imaging of deep internal organs is highly challenging, because the excitation needs to penetrate through strong scattering tissue and the emission signal is degraded significantly by photon diffusion induced by tissue-scattering. We report that by combining two-photon Bessel light-sheet microscopy with nonlinear structured illumination microscopy (SIM), live samples up to 600 microns wide can be imaged by light-sheet microscopy with 500 microns penetration depth, and diffused background in deep tissue light-sheet imaging can be reduced to obtain clear images at cellular resolution in depth beyond 200 microns. We demonstrate in vivo two-color imaging of pronephric glomeruli and vasculature of zebrafish kidney, whose cellular structures located at the center of the fish body are revealed in high clarity by two-color two-photon Bessel light-sheet SIM. PMID:24876996

  5. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques

    PubMed Central

    Jemielita, Matthew; Taormina, Michael J.; DeLaurier, April; Kimmel, Charles B.; Parthasarathy, Raghuveer

    2013-01-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. (A) Schematic: Light sheet microscopy of zebrafish embryos. Opercle-forming osteoblasts following twenty-four hours of (B) light sheet imaging, showing normal growth, and (C) spinning disk confocal imaging, showing aberrant growth. PMID:23242824

  6. Quantitative mapping of collagen fiber alignment in thick tissue samples using transmission polarized-light microscopy

    NASA Astrophysics Data System (ADS)

    Yakovlev, Dmitry D.; Shvachkina, Marina E.; Sherman, Maria M.; Spivak, Andrey V.; Pravdin, Alexander B.; Yakovlev, Dmitry A.

    2016-07-01

    Immersion optical clearing makes it possible to use transmission polarized-light microscopy for characterization of thick (200 to 2000 μm) layers of biological tissues. We discuss polarization properties of thick samples in the context of the problem of characterization of collagen fiber alignment in connective tissues such as sclera and dermis. Optical chirality caused by azimuthal variations of the macroscopic (effective) optic axis of the medium across the sample thickness should be considered in polarization mapping of thick samples of these tissues. We experimentally evaluate to what extent the optical chirality affects the measurement results in typical situations and show under what conditions it can be easily taken into account and does not hinder, but rather helps, in characterization of collagen fiber alignment.

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

    PubMed

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

    2010-03-29

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

  8. Quantitative mapping of collagen fiber alignment in thick tissue samples using transmission polarized-light microscopy.

    PubMed

    Yakovlev, Dmitry D; Shvachkina, Marina E; Sherman, Maria M; Spivak, Andrey V; Pravdin, Alexander B; Yakovlev, Dmitry A

    2016-07-01

    Immersion optical clearing makes it possible to use transmission polarized-light microscopy for characterization of thick (200 to 2000  μm) layers of biological tissues. We discuss polarization properties of thick samples in the context of the problem of characterization of collagen fiber alignment in connective tissues such as sclera and dermis. Optical chirality caused by azimuthal variations of the macroscopic (effective) optic axis of the medium across the sample thickness should be considered in polarization mapping of thick samples of these tissues. We experimentally evaluate to what extent the optical chirality affects the measurement results in typical situations and show under what conditions it can be easily taken into account and does not hinder, but rather helps, in characterization of collagen fiber alignment. PMID:27027930

  9. Light chain crystalline kidney disease: diagnostic urine microscopy as the "liquid kidney biopsy".

    PubMed

    Luciano, Randy L; Castano, Ekaterina; Fogazzi, Giovanni B; Perazella, Mark A

    2014-12-01

    Multiple myeloma (MM) is a plasma cell disorder, which often causes parenchymal kidney disease. Light chain (LC) cast nephropathy represents the most common renal lesion. In some instances, LC crystals precipitate within renal tubular lumens and deposit within proximal tubular cell cytoplasms. Importantly, urine microscopy in such patients can provide insight into the underlying LC-related lesion. Here we present two patients with MM complicated by acute kidney injury (AKI) where LC crystalline casts were observed on urinary sediment analysis. Kidney biopsy revealed acute tubular injury with LC crystal casts within both tubular lumens and renal tubular epithelial cell cytoplasms. These findings suggest that the urinary sediment may be a non-invasive way to diagnose LC crystalline-induced AKI in patients with MM. PMID:25295579

  10. Lattice Light Sheet Microscopy: Imaging Molecules to Embryos at High Spatiotemporal Resolution

    PubMed Central

    Chen, Bi-Chang; Legant, Wesley R.; Wang, Kai; Shao, Lin; Milkie, Daniel E.; Davidson, Michael W.; Janetopoulos, Chris; Wu, Xufeng S.; Hammer, John A.; Liu, Zhe; English, Brian P.; Mimori-Kiyosue, Yuko; Romero, Daniel P.; Ritter, Alex T.; Lippincott-Schwartz, Jennifer; Fritz-Laylin, Lillian; Mullins, R. Dyche; Mitchell, Diana M.; Bembenek, Joshua N.; Reymann, Anne-Cecile; Böhme, Ralph; Grill, Stephan W.; Wang, Jennifer T.; Seydoux, Geraldine; Tulu, U. Serdar; Kiehart, Daniel P.; Betzig, Eric

    2015-01-01

    Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, too small, or occur too rapidly to see clearly with existing tools. We crafted ultra-thin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at sub-second intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and complexity of living systems. PMID:25342811

  11. Automatic segmentation and classification of mycobacterium tuberculosis with conventional light microscopy

    NASA Astrophysics Data System (ADS)

    Xu, Chao; Zhou, Dongxiang; Zhai, Yongping; Liu, Yunhui

    2015-12-01

    This paper realizes the automatic segmentation and classification of Mycobacterium tuberculosis with conventional light microscopy. First, the candidate bacillus objects are segmented by the marker-based watershed transform. The markers are obtained by an adaptive threshold segmentation based on the adaptive scale Gaussian filter. The scale of the Gaussian filter is determined according to the color model of the bacillus objects. Then the candidate objects are extracted integrally after region merging and contaminations elimination. Second, the shape features of the bacillus objects are characterized by the Hu moments, compactness, eccentricity, and roughness, which are used to classify the single, touching and non-bacillus objects. We evaluated the logistic regression, random forest, and intersection kernel support vector machines classifiers in classifying the bacillus objects respectively. Experimental results demonstrate that the proposed method yields to high robustness and accuracy. The logistic regression classifier performs best with an accuracy of 91.68%.

  12. Forensic species identification of elephant (Elephantidae) and giraffe (Giraffidae) tail hair using light microscopy.

    PubMed

    Yates, Bonnie C; Espinoza, Edgard O; Baker, Barry W

    2010-09-01

    Here we present methods for distinguishing tail hairs of African elephants (Loxodonta africana), Asian elephants (Elephas maximus), and giraffes (Giraffa camelopardalis) from forensic contexts. Such hairs are commonly used to manufacture jewelry artifacts that are often sold illegally in the international wildlife trade. Tail hairs from these three species are easily confused macroscopically, and morphological methods for distinguishing African and Asian tail hairs have not been published. We used cross section analysis and light microscopy to analyze the tail hair morphology of 18 individual African elephants, 18 Asian elephants, and 40 giraffes. We found that cross-sectional shape, pigment placement, and pigment density are useful morphological features for distinguishing the three species. These observations provide wildlife forensic scientists with an important analytical tool for enforcing legislation and international treaties regulating the trade in elephant parts. PMID:20549391

  13. Variable multimodal light microscopy with interference contrast and phase contrast; dark or bright field.

    PubMed

    Piper, T; Piper, J

    2014-07-01

    Using the optical methods described, specimens can be observed with modified multimodal light microscopes based on interference contrast combined with phase contrast, dark- or bright-field illumination. Thus, the particular visual information associated with interference and phase contrast, dark- and bright-field illumination is joined in real-time composite images appearing in enhanced clarity and purified from typical artefacts, which are apparent in standard phase contrast and dark-field illumination. In particular, haloing and shade-off are absent or significantly reduced as well as marginal blooming and scattering. The background brightness and thus the range of contrast can be continuously modulated and variable transitions can be achieved between interference contrast and complementary illumination techniques. The methods reported should be of general interest for all disciplines using phase and interference contrast microscopy, especially in biology and medicine, and also in material sciences when implemented in vertical illuminators. PMID:24832212

  14. Soft X-ray Tomography and Cryogenic Light Microscopy: The Cool Combination in Cellular Imaging

    PubMed Central

    McDermott, Gerry; Le Gros, Mark A.; Knoechel, Christian G.; Uchida, Maho; Larabell, Carolyn A.

    2012-01-01

    Soft x-ray tomography (SXT) is ideally suited to imaging sub-cellular architecture and organization, particularly in eukaryotic cells. SXT is similar in concept to the well-established medical diagnostic technique computed axial tomography (CAT), except SXT is capable of imaging with a spatial resolution of 50 nm, or better. In soft x-ray tomography (SXT) cells are imaged using photons from a region of the spectrum known as the ‘water window’. This results in quantitative, high-contrast images of intact, fully hydrated cells without the need to use contrast-enhancing agents. Cells are therefore visualized very close to their native, fully functional state. The utility of SXT has recently been enhanced by the development of high numerical aperture cryogenic light microscopy for correlated imaging. Taking this multi-modal approach now allows labeled molecules to be localized in the context of a high-resolution 3-dimensional tomographic reconstruction of the cell. PMID:19818625

  15. Glycogen in the Nervous System. I; Methods for Light and Electron Microscopy

    NASA Technical Reports Server (NTRS)

    Estable, Rosita F. De; Estable-Puig, J. F.; Miquel, J.

    1964-01-01

    'l'he relative value of different methods for combined light and electron microscopical studies of glycogen in the nervous tissue was investigated. Picroalcoholic fixatives preserve glycogen in a considerable amount but give an inadequate morphological image of glycogen distribution and are unsuitable for ultrastructural studies. Fixation by perfusion, with Dalton's chromeosmic fluid seems adequate for ultrastructural cytochemistry of glycogen. Furthermore it permits routine paraffin embedding of brain slices adjacent to those used for electron microscopy. Dimedone blocking is a necessary step for a selective staining of glycogen with PAS after osmic fixation. Enzymatic removal of glycogen in osmic fixed nervous tissue can be done In paraffin-embedded tissue. It can also be performed in glycolmethacrylate-embedded tissue without removal of the embedding medium. Paraphenylenediamine stains glycogen following periodic acid oxidation.

  16. New Aspidoderidae species parasite of Didelphis aurita (Mammalia: Didelphidae): a light and scanning electron microscopy approach.

    PubMed

    Chagas-Moutinho, V A; Sant'anna, V; Oliveira-Menezes, A; De Souza, W

    2014-02-01

    Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Skrjabin and Schikobalova, 1947, are widely distributed in the Americas. The family Aspidoderidae includes the subfamilies Aspidoderinae Skrjabin and Schikobalova, 1947, and Lauroiinae Skrjabin and Schikobalova, 1951. These two subfamilies are delineated by the presence or absence of cephalic cordons at the anterior region. The nematodes in the subfamily Aspidoderinae, which includes the genus AspidoderaRailliet and Henry, 1912, are represented by nematodes with anterior cephalic cordons at the anterior end. The nematodes of the genus AspidoderaRailliet and Henry, 1912, are found in the cecum and large intestine of mammals of the orders Edentata, Marsupialia and Rodentia. Species within this genus have many morphological similarities. The use of scanning electron microscopy allows the specific characterization of the species within this genus. In the present work, we describe a new species of Aspidodera parasite of the large intestine of Didelphis aurita (Mammalia: Didelphidae) Wied-Neuwied, 1826, collected from Cachoeiras de Macacu, Rio de Janeiro. The combination of light and scanning electron microscopy allowed us a detailed analysis of this nematode. PMID:24129095

  17. Light-sheet photoacoustic microscopy (LIS-PAM) with optical ultrasound detection

    NASA Astrophysics Data System (ADS)

    Nuster, Robert; Slezak, Paul; Paltauf, Guenther

    2016-03-01

    Photoacoustic (or optoacoustic) microscopy has great potential as a diagnostic tool in biomedical research. For in vivo imaging, an important requirement is to keep the measurement time as short as possible. In light-sheet photoacoustic microscopy (LIS-PAM) a cylindrical lens illuminates a thin section perpendicular to the sample surface with a short laser pulse and a projection of the excited acoustic wave pattern leaving the sample is recorded with a camera. From the recorded data, a B-scan photoacoustic image is obtained by applying a two-dimensional reconstruction algorithm, without requiring any mechanical scanning. Hence, LIS-PAM is capable of real-time B-scan imaging with acoustical resolution within the individual B-scans and optical out of plane resolution up to a depth limited by optical diffusion. A 3D image is composed of reconstructed B-scan images recorded while scanning the excitation line along the sample surface. Using a camera with 200 Hz frame rate a C-scan image (5x5 mm2 field of view) can be recorded in less than 5 seconds (without averaging). The achievable sensitivity and resolution of the optical phase contrast detection system were estimated theoretically with 0.34 kPa mm without averaging and 30 μm, respectively. A first experiment on a phantom that mimics tissue properties shows the applicability of this technique for in-vivo imaging.

  18. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis

    PubMed Central

    Park, Ok Kyu; Kwak, Jina; Jung, Yoo Jung; Kim, Young Ho; Hong, Hyun-Seok; Hwang, Byung Joon; Kwon, Seung-Hae; Kee, Yun

    2015-01-01

    Precise 3D spatial mapping of cells and their connections within living tissues is required to fully understand developmental processes and neural activities. Zebrafish embryos are relatively small and optically transparent, making them the vertebrate model of choice for live in vivo imaging. However, embryonic brains cannot be imaged in their entirety by confocal or two-photon microscopy due to limitations in optical range and scanning speed. Here, we use light-sheet fluorescence microscopy to overcome these limitations and image the entire head of live transgenic zebrafish embryos. We simultaneously imaged cranial neurons and blood vessels during embryogenesis, generating comprehensive 3D maps that provide insight into the coordinated morphogenesis of the nervous system and vasculature during early development. In addition, blood cells circulating through the entire head, vagal and cardiac vasculature were also visualized at high resolution in a 3D movie. These data provide the foundation for the construction of a complete 4D atlas of zebrafish embryogenesis and neural activity. PMID:26429501

  19. Mechanisms of dendritic growth investigated by in situ light microscopy during electrodeposition and dissolution of lithium

    NASA Astrophysics Data System (ADS)

    Steiger, Jens; Kramer, Dominik; Mönig, Reiner

    2014-09-01

    Batteries with metallic lithium anodes offer improved volumetric and gravimetric energy densities; therefore, future batteries including the promising lithium-sulfur and lithium-air systems would benefit from them. The electrodeposition of lithium metal - which is an unwanted incident in lithium ion systems - often results in fine filaments or moss, called dendritic lithium, which leads to strong capacity fading and the danger of internal short circuiting. To study the mechanisms of dendritic growth and the behavior during lithium dissolution, lithium deposits have been observed in situ in 1 M LiPF6 in EC:DMC by light microscopy. The high resolution optical microscopy provided information on the growth and electrodissolution of single lithium filaments. The growth areas could be identified in detail: The lithium wires can grow either from the substrate-lithium interface, at kinks or in a region at or close to the tip. Based on these observations, we suggest a growth model for lithium filaments predicated on defect-based insertion of lithium at the aforementioned locations. This type of growth is not compatible with previous models of dendritic growth, for example, it is hardly influenced by electric fields at the tip and does not depend on the direction of the electric field.

  20. Detection of sarcocystosis in goats in Malaysia by light microscopy, histology, and PCR.

    PubMed

    Kutty, Methil Kannan; Latif, Baha; Muslim, Azdayanti; Hussaini, Jamal; Daher, Aqil Mohammad; Heo, Chong Chin; Abdullah, Sulaiman

    2015-04-01

    A number of methods have been used for the detection of the presence of microsarcocysts in animals, but little information exists on the value between the various methods. This study therefore examined for Sarcocystis spp. using three different methods in 105 samples of skeletal muscle collected from goats slaughtered in an abattoir in Selangor, Malaysia from January to February 2014. Three methods were used, direct light microscopy of squashed fresh muscle tissues; histological examination of fixed, sectioned, and hematoxylin and eosin (H&E)-stained samples of muscle; and molecular identification by polymerase chain reaction (PCR). Of the 105 tissue samples, 55 (52.38 %) were positive by light microscopy (LM), 46 (43.8 %) by histology, and 95 (90.48 %) by PCR. Only 29 (27.6 %) and 5 (4.76 %) samples were positive and negative, respectively, by all three methods. The cysts were elongated to a spindle shape with a mean size of 393.30 × 81.6 μm and containing banana-shaped bradyzoites of size 12.32 × 2.08 μm. The wall of the cyst was radially striated with a thickness of 2.83 μm. Samples were tested for the presence of Sarcocystis-specific 18S rRNA and were identified as Sarcocystis capracanis. Of the three methods used, the PCR test appears to be the most useful method for the diagnosis of sarcocystosis especially for species identification. PMID:25740651

  1. Imaging spectroscopic analysis at the Advanced Light Source

    SciTech Connect

    MacDowell, A. A.; Warwick, T.; Anders, S.; Lamble, G.M.; Martin, M.C.; McKinney, W.R.; Padmore, H.A.

    1999-05-12

    One of the major advances at the high brightness third generation synchrotrons is the dramatic improvement of imaging capability. There is a large multi-disciplinary effort underway at the ALS to develop imaging X-ray, UV and Infra-red spectroscopic analysis on a spatial scale from. a few microns to 10nm. These developments make use of light that varies in energy from 6meV to 15KeV. Imaging and spectroscopy are finding applications in surface science, bulk materials analysis, semiconductor structures, particulate contaminants, magnetic thin films, biology and environmental science. This article is an overview and status report from the developers of some of these techniques at the ALS. The following table lists all the currently available microscopes at the. ALS. This article will describe some of the microscopes and some of the early applications.

  2. X-ray Microscopy Studies of Electromigration in Advanced Copper Interconnects

    SciTech Connect

    Schneider, G.; Rudolph, S.; Heim, S.; Rehbein, S.; Guttmann, P.

    2006-02-07

    X-rays have the advantage that they penetrate samples which are several micrometers thick without significant sample damage, and that they provide a chemical image contrast between different dielectric layers of the Cu/low-k on-chip interconnect stack. Therefore, x-ray microscopy is an ideal tool for quantitative 3-D investigations of void dynamics with high spatial resolution of 20 nm. Using the BESSY full-field transmission x-ray microscope (TXM), we performed electromigration studies of advanced backend-of-line (BEoL) stacks of high-performance microprocessors containing copper interconnects and low-k materials. We observed void movement along the top copper/dielectric (SiNx) interface which is found to be the main pathway for electromigration-induced atomic copper transport.

  3. X-ray Microscopy Studies of Electromigration in Advanced Copper Interconnects

    NASA Astrophysics Data System (ADS)

    Schneider, G.; Guttmann, P.; Rudolph, S.; Heim, S.; Rehbein, S.; Meyer, M. A.; Zschech, E.

    2006-02-01

    X-rays have the advantage that they penetrate samples which are several micrometers thick without significant sample damage, and that they provide a chemical image contrast between different dielectric layers of the Cu/low-k on-chip interconnect stack. Therefore, x-ray microscopy is an ideal tool for quantitative 3-D investigations of void dynamics with high spatial resolution of 20 nm. Using the BESSY full-field transmission x-ray microscope (TXM), we performed electromigration studies of advanced backend-of-line (BEoL) stacks of high-performance microprocessors containing copper interconnects and low-k materials. We observed void movement along the top copper/dielectric (SiNx) interface which is found to be the main pathway for electromigration-induced atomic copper transport.

  4. Single virus detection by means of atomic force microscopy in combination with advanced image analysis.

    PubMed

    Bocklitz, Thomas; Kämmer, Evelyn; Stöckel, Stephan; Cialla-May, Dana; Weber, Karina; Zell, Roland; Deckert, Volker; Popp, Jürgen

    2014-10-01

    In the present contribution virions of five different virus species, namely Varicella-zoster virus, Porcine teschovirus, Tobacco mosaic virus, Coliphage M13 and Enterobacteria phage PsP3, are investigated using atomic force microscopy (AFM). From the resulting height images quantitative features like maximal height, area and volume of the viruses could be extracted and compared to reference values. Subsequently, these features were accompanied by image moments, which quantify the morphology of the virions. Both types of features could be utilized for an automatic discrimination of the five virus species. The accuracy of this classification model was 96.8%. Thus, a virus detection on a single-particle level using AFM images is possible. Due to the application of advanced image analysis the morphology could be quantified and used for further analysis. Here, an automatic recognition by means of a classification model could be achieved in a reliable and objective manner. PMID:25196422

  5. Advanced fluorescence microscopy methods for the real-time study of transcription and chromatin dynamics

    PubMed Central

    Annibale, Paolo; Gratton, Enrico

    2014-01-01

    In this contribution we provide an overview of the recent advances allowed by the use of fluorescence microscopy methods in the study of transcriptional processes and their interplay with the chromatin architecture in living cells. Although the use of fluorophores to label nucleic acids dates back at least to about half a century ago,1 two recent breakthroughs have effectively opened the way to use fluorescence routinely for specific and quantitative probing of chromatin organization and transcriptional activity in living cells: namely, the possibility of labeling first the chromatin loci and then the mRNA synthesized from a gene using fluorescent proteins. In this contribution we focus on methods that can probe rapid dynamic processes by analyzing fast fluorescence fluctuations. PMID:25764219

  6. Light Microscopy Module Fan Disturbance Characterized Through Microgravity Emissions Laboratory Testing

    NASA Technical Reports Server (NTRS)

    McNelis, Anne M.; Motil, Susan M.

    2003-01-01

    A Light Microscopy Module (LMM) is being engineered, designed, and developed at the NASA Glenn Research Center. The LMM is planned as a remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and control of physical science and biological science experiments within Glenn s Fluids Integrated Rack on the International Space Station. The LMM concept is a modified commercial research imaging light microscope with powerful laser-diagnostic hardware and interfaces, creating a one-of-a-kind, state-of-the-art microscopic research facility. The microscope will house several different objectives, corresponding to magnifications of 10, 40, 50, 63, and 100. Features of the LMM include high-resolution color video microscopy, brightfield, darkfield, phase contrast, differential interference contrast, spectrophotometry, and confocal microscopy combined in a single configuration. Also, laser tweezers are integrated with the diagnostics as a sample manipulation technique. As part of the development phase of the LMM, it was necessary to quantify the microgravity disturbances generated by the control box fan. Isolating the fan was deemed necessary to reduce the fan speed harmonic amplitudes and to eliminate any broadband disturbances across the 60- to 70-Hz and 160- to 170-Hz frequency ranges. The accelerations generated by a control box fan component of the LMM were measured in the Microgravity Emissions Laboratory (MEL). The MEL is a low-frequency measurement system developed to simulate and verify the on-orbit International Space Station (ISS) microgravity environment. The accelerations generated by various operating components of the ISS, if too large, could hinder the science performed onboard by disturbing the microgravity environment. The MEL facility gives customers a test-verified way of measuring their compliance with ISS limitations on vibratory disturbance levels. The facility is unique in that inertial forces in 6 degrees of freedom can

  7. Advancement of Solidification Processing Technology Through Real Time X-Ray Transmission Microscopy: Sample Preparation

    NASA Technical Reports Server (NTRS)

    Stefanescu, D. M.; Curreri, P. A.

    1996-01-01

    Two types of samples were prepared for the real time X-ray transmission microscopy (XTM) characterization. In the first series directional solidification experiments were carried out to evaluate the critical velocity of engulfment of zirconia particles in the Al and Al-Ni eutectic matrix under ground (l-g) conditions. The particle distribution in the samples was recorded on video before and after the samples were directionally solidified. In the second series samples of the above two type of composites were prepared for directional solidification runs to be carried out on the Advanced Gradient Heating Facility (AGHF) aboard the space shuttle during the LMS mission in June 1996. X-ray microscopy proved to be an invaluable tool for characterizing the particle distribution in the metal matrix samples. This kind of analysis helped in determining accurately the critical velocity of engulfment of ceramic particles by the melt interface in the opaque metal matrix composites. The quality of the cast samples with respect to porosity and instrumented thermocouple sheath breakage or shift could be easily viewed and thus helped in selecting samples for the space shuttle experiments. Summarizing the merits of this technique it can be stated that this technique enabled the use of cast metal matrix composite samples since the particle location was known prior to the experiment.

  8. Soft x-ray spectromicroscopy development for materials science at the Advanced Light Source

    SciTech Connect

    Warwick, T.; Padmore, H.; Ade, H.; Hitchcock, A.P.; Rightor, E.G.; Tonner, B.P.

    1996-08-01

    Several third generation synchrotron radiation facilities are now operational and the high brightness of these photon sources offers new opportunities for x-ray microscopy. Well developed synchrotron radiation spectroscopy techniques are being applied in new instruments capable of imaging the surface of a material with a spatial resolution smaller than one micron. There are two aspects to this. One is to further the field of surface science by exploring the effects of spatial variations across a surface on a scale not previously accessible to x-ray measurements. The other is to open up new analytical techniques in materials science using x-rays, on a spatial scale comparable to that of the processes or devices to be studied. The development of the spectromicroscopy program at the Advanced Light Source will employ a variety of instruments, some are already operational. Their development and use will be discussed, and recent results will be presented to illustrate their capabilities.

  9. Advances in Light-based Imaging of Three-Dimensional Cellular Ultrastructure

    PubMed Central

    Kanchanawong, Pakorn; Waterman, Clare M.

    2012-01-01

    Visualization methods are key to gaining insights into cellular structure and function. Since diffraction has long confined optical microscopes to a resolution no better than hundreds of nanometers, the observation of ultrastructural features has traditionally been the domain of electron microscopes (EM). In the past decade, however, advances in super-resolution fluorescence microscopy have considerably expanded the capability of light-based imaging techniques. Advantages of fluorescent labeling such as high sensitivity, specificity, and multichannel capability, can now be exploited to dissect ultrastructural features of cells. With recent methods capable of imaging specific proteins with a resolution on the order of a few tens of nanometers in 3-dimensions, this has made it possible to elucidate the molecular organization of many complex cellular structures. PMID:22209239

  10. The macromolecular crystallography facility at the advanced light source

    NASA Astrophysics Data System (ADS)

    Earnest, Thomas; Padmore, Howard; Cork, Carl; Behrsing, Rolf; Kim, Sung-Hou

    1996-10-01

    Synchrotron radiation offers several advantages over the use of rotating anode sources for biological crystallography, which allow for the collection of higher-resolution data, substantially more rapid data collection, phasing by multiwavelength anomalous diffraction (MAD) techniques, and time-resolved experiments using polychromatic radiation (Laue diffraction). The use of synchrotron radiation is often necessary to record useful data from crystals which diffract weakly or have very large unit cells. The high brightness and stability characteristics of the advanced light source (ALS) at Lawrence Berkeley National Laboratory, along with the low emittance and long straight sections to accommodate insertion devices present in third generation synchrotrons like the ALS, lead to several advantages in the field of macromolecular crystallography. We are presently constructing a macromolecular crystallography facility at the ALS which is optimized for user-friendliness and high-throughput data collection, with advanced capabilities for MAD and Laue experiments. The X-rays will be directed to three branchlines. A well-equipped support lab will be available for biochemistry, crystal mounting and sample storage, as well as computer hardware and software available, along with staff support, allowing for the complete processing of data on site.

  11. Magnetic design of the advanced light source elliptical wiggler

    SciTech Connect

    Marks, S.; Akre, J.; Hoyer, E.; Humphries, D.; Jackson, T.; Minamihara, Y.; Pipersky, P.; Plate, D.; Schlueter, R.

    1995-06-01

    An elliptical wiggler has been designed for installation in the Advanced Light Source at the Lawrence Berkeley Laboratory. The design has been optimized for the production of circularly polarized light in the 50 eV to 10 KeV energy range. The device will be 3.4 m long consisting of vertical and horizontal periodic structures. The period length for both is 20 cm. The vertical structure is a hybrid permanent magnet design which produces a peak field of 2.0 T. The horizontal magnetic structure is an iron core electromagnetic design shifted longitudinally by one-quarter period relative to the vertical structure; it has a peak field of 0.095 T. The polarity of the horizontal field can be switched at a rate of up to 1 Hz, which results in a modulation of the chirality of the circularly polarized radiation on-axis. This paper discusses the magnetic design and presents the results of radiation spectra calculations used for determining optimal field parameter settings.

  12. Rapid spontaneous Raman light sheet microscopy using cw-lasers and tunable filters

    PubMed Central

    Rocha-Mendoza, Israel; Licea-Rodriguez, Jacob; Marro, Mónica; Olarte, Omar E.; Plata-Sanchez, Marcos; Loza-Alvarez, Pablo

    2015-01-01

    We perform rapid spontaneous Raman 2D imaging in light-sheet microscopy using continuous wave lasers and interferometric tunable filters. By angularly tuning the filter, the cut-on/off edge transitions are scanned along the excited Stokes wavelengths. This allows obtaining cumulative intensity profiles of the scanned vibrational bands, which are recorded on image stacks; resembling a spectral version of the knife-edge technique to measure intensity profiles. A further differentiation of the stack retrieves the Raman spectra at each pixel of the image which inherits the 3D resolution of the host light sheet system. We demonstrate this technique using solvent solutions and composites of polystyrene beads and lipid droplets immersed in agar and by imaging the C–H (2800-3100cm−1) region in a C. elegans worm. The image acquisition time results in 4 orders of magnitude faster than confocal point scanning Raman systems, allowing the possibility of performing fast spontaneous Raman·3D-imaging on biological samples. PMID:26417514

  13. Molecular orientation sensitive second harmonic microscopy by radially and azimuthally polarized light

    PubMed Central

    Ehmke, Tobias; Nitzsche, Tim Heiko; Knebl, Andreas; Heisterkamp, Alexander

    2014-01-01

    We demonstrate the possibility to switch the z-polarization component of the illumination in the vicinity of the focus of high-NA objective lenses by applying radially and azimuthally polarized incident light. The influence of the field distribution on nonlinear effects was first investigated by the means of simulations. These were performed for high-NA objective lenses commonly used in nonlinear microscopy. Special attention is paid to the influence of the polarization of the incoming field. For linearly, circularly and radially polarized light a considerable polarization component in z-direction is generated by high NA focusing. Azimuthal polarization is an exceptional case: even for strong focusing no z-component arises. Furthermore, the influence of the input polarization on the intensity contributing to the nonlinear signal generation was computed. No distinct difference between comparable input polarization states was found for chosen thresholds of nonlinear signal generation. Differences in signal generation for radially and azimuthally polarized vortex beams were experimentally evaluated in native collagen tissue (porcine cornea). The findings are in good agreement with the theoretical predictions and display the possibility to probe the molecular orientation along the optical axis of samples with known nonlinear properties. The combination of simulations regarding the nonlinear response of materials and experiments with different sample orientations and present or non present z-polarization could help to increase the understanding of nonlinear signal formation in yet unstudied materials. PMID:25071961

  14. Fly-by-Light Advanced Systems Hardware (FLASH) program

    NASA Astrophysics Data System (ADS)

    Bedoya, Carlos A.

    1995-05-01

    hundreds of MHz are available. Applications of fiber optic buses would then result in the reduction of wires and connections because of reduction in the number of buses needed for information transfer due to the fact that a large number of different signals can be sent across one fiber by multiplexing each signal. The Advanced Research Projects Agency (ARPA) Technology Reinvestment Project (TRP) Fly-by-Light Advanced Systems Hardware (FLASH) program addresses the development of Fly-by-Light Technology in order to apply the benefits of fiber optics to military and commercial aircraft.

  15. The use of light- and electron microscopy for studies on the cell- and molecular biology of parasites and parasitic diseases.

    PubMed

    Hehl, A B; Hemphill, A

    2006-09-01

    Lightmicroscopical (LM) and electron microscopi cal (EM) techniques, have had a major influence on the development and direction of cell biology, and particularly also on the investigation of complex host-parasite relationships. Earlier, microscopy has been rather descriptive, but new technical and scientific advances have changed the situation. Microscopy has now become analytical, quantitative and three-dimensional, with greater emphasis on analysis of live cells with fluorescent markers. The new or improved techniques that have become available include immunocytochemistry using immunogold labeling techniques or fluorescent probes, cryopreservation and cryosectioning, in situ hybridization, fluorescent reporters for subcellular localization, micro-analytical methods for elemental distribution, confocal laser scanning microscopy, scanning tunneling microscopy and live-imaging. Taken together, these tools are providing both researchers and students with a novel and multidimensional view of the intricate biological processes during parasite development in the host. PMID:17024976

  16. Plasma Membrane Water Permeability of Cultured Cells and Epithelia Measured by Light Microscopy with Spatial Filtering

    PubMed Central

    Farinas, Javier; Kneen, Malea; Moore, Megan; Verkman, A.S.

    1997-01-01

    A method was developed to measure the osmotic water permeability (Pf) of plasma membranes in cell layers and applied to cells and epithelia expressing molecular water channels. It was found that the integrated intensity of monochromatic light in a phase contrast or dark field microscope was dependent on relative cell volume. For cells of different size and shape (Sf9, MDCK, CHO, A549, tracheal epithelia, BHK), increased cell volume was associated with decreased signal intensity; generally the signal decreased 10–20% for a twofold increase in cell volume. A theory relating signal intensity to relative cell volume was developed based on spatial filtering and changes in optical path length associated with cell volume changes. Theory predictions were confirmed by signal measurements of cell layers bathed in solutions of various osmolarities and refractive indices. The excellent signal-to-noise ratio of the transmitted light detection permitted measurement of cell volume changes of <1%. The method was applied to characterize transfected cells and tissues that natively express water channels. Pf in control Chinese hamster ovary cells was low (0.0012 cm/s at 23°C) and increased more than fourfold upon stable transfection with aquaporins 1, 2, 4, or 5. Pf in apical and basolateral membranes in polarized epithelial cells grown on porous supports was measured. Pfbl and Pfap were 0.0011 and 0.0024 cm/s (MDCK cells), and 0.0039 and 0.0052 cm/s (human tracheal cells) at 23°C. In intact toad urinary bladder, basolateral Pf was 0.036 cm/s and apical membrane Pf after vasopressin stimulation was 0.025 cm/s at 23°C. The results establish light microscopy with spatial filtering as a technically simple and quantitative method to measure water permeability in cell layers and provide the first measurement of the apical and basolateral membrane permeabilities of several important epithelial cell types. PMID:9276754

  17. High Resolution Traction Force Microscopy Based on Experimental and Computational Advances

    PubMed Central

    Sabass, Benedikt; Gardel, Margaret L.; Waterman, Clare M.; Schwarz, Ulrich S.

    2008-01-01

    Cell adhesion and migration crucially depend on the transmission of actomyosin-generated forces through sites of focal adhesion to the extracellular matrix. Here we report experimental and computational advances in improving the resolution and reliability of traction force microscopy. First, we introduce the use of two differently colored nanobeads as fiducial markers in polyacrylamide gels and explain how the displacement field can be computationally extracted from the fluorescence data. Second, we present different improvements regarding standard methods for force reconstruction from the displacement field, which are the boundary element method, Fourier-transform traction cytometry, and traction reconstruction with point forces. Using extensive data simulation, we show that the spatial resolution of the boundary element method can be improved considerably by splitting the elastic field into near, intermediate, and far field. Fourier-transform traction cytometry requires considerably less computer time, but can achieve a comparable resolution only when combined with Wiener filtering or appropriate regularization schemes. Both methods tend to underestimate forces, especially at small adhesion sites. Traction reconstruction with point forces does not suffer from this limitation, but is only applicable with stationary and well-developed adhesion sites. Third, we combine these advances and for the first time reconstruct fibroblast traction with a spatial resolution of ∼1 μm. PMID:17827246

  18. Hierarchical super-structure identified by polarized light microscopy, electron microscopy and nanoindentation: Implications for the limits of biological control over the growth mode of abalone sea shells

    PubMed Central

    2012-01-01

    Background Mollusc shells are commonly investigated using high-resolution imaging techniques based on cryo-fixation. Less detailed information is available regarding the light-optical properties. Sea shells of Haliotis pulcherina were embedded for polishing in defined orientations in order to investigate the interface between prismatic calcite and nacreous aragonite by standard materialographic methods. A polished thin section of the interface was prepared with a defined thickness of 60 μm for quantitative birefringence analysis using polarized light and LC-PolScope microscopy. Scanning electron microscopy images were obtained for comparison. In order to study structural-mechanical relationships, nanoindentation experiments were performed. Results Incident light microscopy revealed a super-structure in semi-transparent regions of the polished cross-section under a defined angle. This super-structure is not visible in transmitted birefringence analysis due to the blurred polarization of small nacre platelets and numerous organic interfaces. The relative orientation and homogeneity of calcite prisms was directly identified, some of them with their optical axes exactly normal to the imaging plane. Co-oriented "prism colonies" were identified by polarized light analyses. The nacreous super-structure was also visualized by secondary electron imaging under defined angles. The domains of the super-structure were interpreted to consist of crystallographically aligned platelet stacks. Nanoindentation experiments showed that mechanical properties changed with the same periodicity as the domain size. Conclusions In this study, we have demonstrated that insights into the growth mechanisms of nacre can be obtained by conventional light-optical methods. For example, we observed super-structures formed by co-oriented nacre platelets as previously identified using X-ray Photo-electron Emission Microscopy (X-PEEM) [Gilbert et al., Journal of the American Chemical Society 2008, 130

  19. Light-sheet microscopy with digital Fourier analysis measures transport properties over large field-of-view.

    PubMed

    Wulstein, Devynn M; Regan, Kathryn E; Robertson-Anderson, Rae M; McGorty, Ryan

    2016-09-01

    Using light-sheet microscopy combined with digital Fourier methods we probe the dynamics of colloidal samples and DNA molecules. This combination, referred to as selective-plane illumination differential dynamic microscopy (SPIDDM), has the benefit of optical sectioning to study, with minimal photobleaching, thick samples allowing us to measure the diffusivity of colloidal particles at high volume fractions. Further, SPIDDM exploits the inherent spatially-varying thickness of Gaussian light-sheets. Where the excitation sheet is most focused, we capture high spatial frequency dynamics as the signal-to-background is high. In thicker regions, we capture the slower dynamics as diffusion out of the sheet takes longer. PMID:27607692

  20. New advances in the application of FTIR microscopy and spectroscopy for the characterization of artistic materials.

    PubMed

    Prati, S; Joseph, E; Sciutto, G; Mazzeo, R

    2010-06-15

    Fourier transform infrared (FTIR) spectroscopy is one of the most widely applied techniques for the investigation of cultural heritage materials. FTIR microscopy is well established as an essential tool in the microdestructive analysis of small samples, and the recent introduction of mapping and imaging equipment allows the collection of a large number of FTIR spectra on a surface, providing a distribution map of identified compounds. In this Account, we report recent advances in FTIR spectroscopy and microscopy in our research group. Our laboratory develops, tests, and refines new and less-studied IR spectroscopy and microscopy methods, with the goal of their adoption as routine analytical techniques in conservation laboratories. We discuss (i) the analysis of inorganic materials inactive in the mid-IR region by means of far-IR spectroscopy, (ii) the development of new methods for preparing cross sections, (iii) the characterization and spatial location of thin layers and small particles, and (iv) the evaluation of protective treatments. FTIR spectroscopy and microscopy have been mostly used in the mid-IR region of 4000-600 cm(-1). Some inorganic pigments, however, are inactive in this region, so other spectroscopic techniques have been applied, such as Raman spectroscopy. We suggest an alternative: harnessing the far-IR (600-50 cm(-1)). Our initial results show that far-IR spectroscopy is exceptionally useful with mural paintings or with corrosion products from which larger sample quantities can generally be collected. Moreover, the inorganic composition of a sample can be characterized by the presence of several compounds that are inactive in the mid-IR range (such as sulfides, oxides, and so forth). Stratigraphical analyses by FTIR microscopy can be hindered by the process of cross section preparation, which often involves an embedding organic polymer penetrating the sample's porous structure. Here, the polymer bands may completely cover the bands of organic

  1. Fast, high-contrast imaging of animal development with scanned light sheet-based structured-illumination microscopy.

    PubMed

    Keller, Philipp J; Schmidt, Annette D; Santella, Anthony; Khairy, Khaled; Bao, Zhirong; Wittbrodt, Joachim; Stelzer, Ernst H K

    2010-08-01

    Recording light-microscopy images of large, nontransparent specimens, such as developing multicellular organisms, is complicated by decreased contrast resulting from light scattering. Early zebrafish development can be captured by standard light-sheet microscopy, but new imaging strategies are required to obtain high-quality data of late development or of less transparent organisms. We combined digital scanned laser light-sheet fluorescence microscopy with incoherent structured-illumination microscopy (DSLM-SI) and created structured-illumination patterns with continuously adjustable frequencies. Our method discriminates the specimen-related scattered background from signal fluorescence, thereby removing out-of-focus light and optimizing the contrast of in-focus structures. DSLM-SI provides rapid control of the illumination pattern, exceptional imaging quality and high imaging speeds. We performed long-term imaging of zebrafish development for 58 h and fast multiple-view imaging of early Drosophila melanogaster development. We reconstructed cell positions over time from the Drosophila DSLM-SI data and created a fly digital embryo. PMID:20601950

  2. Light microscopy and molecular identification of Sarcocystis spp. in meat producing animals in Selangor, Malaysia.

    PubMed

    Latif, B; Kannan Kutty, M; Muslim, A; Hussaini, J; Omar, E; Heo, C C; Rossle, N F; Abdullah, S; Kamarudin, M A; Zulkarnain, M A

    2015-09-01

    One thousand and forty-five tissue samples of skeletal muscles, tongue, heart, diaphragm and esophagus were collected from 209 animals (43 sheep, 89 goats and 77 cattle) from an abattoir in Selangor between February and October, 2013. Each sample was divided into three pieces with each piece measuring 2-3 mm3. Each piece was then squeezed between two glass slides and examined microscopically at x 10 magnification for the presence of sarcocystosis. Three positive samples from each animal species were then fixed in 10% formalin for histological processing. Seven positive samples collected from each animal species were preserved at -80°C or 90% ethanol for gene expression studies. Microsarcocysts were detected in 114 (54.5%) animals by light microscopy (LM). The infection rates in sheep, goat and cattle were 86, 61.8 and 28.6% respectively. The highest rate of infection was in the skeletal muscles of sheep (64.9%) and goats (63.6%) and in the heart of cattle (63.6%). The cysts were spindle to oval in shape and two stages were recognized, the peripheral metrocytes and centrally located banana-shaped bradyzoites. 18S rRNA gene expression studies confirmed the isolates from the sheep as S. ovicanis, goats as S. capracanis and cattle as S. bovicanis. This, to the best of our knowledge, is the first molecular identification of an isolate of S. ovicanis and S. capracanis in Malaysia. Further studies with electron microscopy (EM) are required in the future to compare the features of different types of Sarcocysts spp. PMID:26695204

  3. Morphological characterization via light and electron microscopy of Atlantic jackknife clam (Ensis directus) hemocytes.

    PubMed

    Preziosi, Brian M; Bowden, Timothy J

    2016-05-01

    The Atlantic jackknife clam, Ensis directus, is currently being researched as a potential species for aquaculture operations in Maine. The goal of this study was to describe the hemocytes of this species for the first time and provide a morphological classification scheme. We viewed hemocytes under light microscopy (using Hemacolor, neutral red, and Pappenheim's stains) as well as transmission electron microscopy (TEM). The 2 main types of hemocytes found were granulocytes and hyalinocytes (agranular cells). The granulocytes were subdivided into large and small granulocytes while the hyalinocytes were subdivided into large and small hyalinocytes. The large hemocytes had both a larger diameter and smaller nucleus to cell diameter ratio than their smaller counterparts. A rare cell type, the vesicular cell, was also observed and it possessed many vesicles but few or no granules. Using TEM, granulocytes were found to contain both electron-lucent and electron-dense granules of various sizes. These numerous granules were the only structures that took up the neutral red stain. Hyalinocytes had few of these granules relative to granulocytes. Large hyalinocytes had both various organelles and large vesicles in their abundant cytoplasm while small hyalinocytes had little room for organelles in their scant cytoplasm. Total hemocyte counts averaged 1.96×10(6) cells mL(-1) while differential hemocyte counts averaged 11% for small hyalinocytes, 12% for large hyalinocytes, 59% for small granulocytes, and 18% for large granulocytes. The results of this study provide a starting point for future studies on E. directus immune function. PMID:27015289

  4. Resolving the CaP-bone interface: a review of discoveries with light and electron microscopy.

    PubMed

    Grandfield, Kathryn; Palmquist, Anders; Engqvist, Håkan; Thomsen, Peter

    2012-01-01

    It has long been known that the interfacial relationship between synthetic materials and tissue is influential in the success of implant materials. Instability at the implant interface has been shown, in some cases, to lead to complete implant failure. Bioceramics, and in particular calcium phosphates, form a large fraction of the implantable devices on the market today due to the biocompatibility they exhibit in contact with bone and tooth-like tissues. The characterization of such bioceramic-tissue interfaces has played a crucial role in understanding the behavior of bioceramics in vivo. In this review, we shed light on the preparation methods, technological approaches and key advances in resolving the interface between calcium phosphate bioceramics and bone, and share a future outlook on this field. PMID:23507782

  5. An alternative means of retaining ocular structure and improving immunoreactivity for light microscopy studies

    PubMed Central

    Sun, Ning; Shibata, Brad; Hess, John F.

    2015-01-01

    Purpose Several properties of ocular tissue make fixation for light microscopy problematic. Because the eye is spherical, immersion fixation necessarily results in a temporal gradient of fixation, with surfaces fixing more rapidly and thoroughly than interior structures. The problem is compounded by the fact that the layers of the eye wall are compositionally quite different, resulting in different degrees of fixation-induced shrinkage and distortion. Collectively, these result in non-uniform preservation, as well as buckling and/or retinal detachment. This gradient problem is most acute for the lens, where the density of proteins can delay fixation of the central lens for days, and where the fixation gradient parallels the age gradient of lens cells, which complicates data interpretation. Our goal was to identify a simple method for minimizing some of the problems arising from immersion fixation, which avoided covalent modification of antigens, retained high quality structure, and maintained tissue in a state that is amenable to common cytochemical techniques. Methods A simple and inexpensive derivative of the freeze-substitution approach was developed and compared to fixation by immersion in formalin. Preservation of structure, immunoreactivity, GFP and tdTomato fluorescence, lectin reactivity, outer segment auto fluorescence, Click-iT chemistry, compatibility with in situ hybdrdization, and the ability to rehydrate eyes after fixation by freeze substitution for subsequent cryo sectioning were assessed. Results An inexpensive and simple variant of the freeze substitution approach provides excellent structural preservation for light microscopy, and essentially eliminates ocular buckling, retinal detachment, and outer segment auto-fluorescence, without covalent modification of tissue antigens. The approach shows a notable improvement in preservation of immunoreactivity. TdTomato intrinsic fluorescence is also preserved, as is compatibility with in situ

  6. Optical mapping of conduction in early embryonic quail hearts with light-sheet microscopy (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Ma, Pei; Gu, Shi; Wang, Yves T.; Jenkins, Michael W.; Rollins, Andrew M.

    2016-03-01

    Optical mapping (OM) using fluorescent voltage-sensitive dyes (VSD) to measure membrane potential is currently the most effective method for electrophysiology studies in early embryonic hearts due to its noninvasiveness and large field-of-view. Conventional OM acquires bright-field images, collecting signals that are integrated in depth and projected onto a 2D plane, not capturing the 3D structure of the sample. Early embryonic hearts, especially at looping stages, have a complicated, tubular geometry. Therefore, conventional OM cannot provide a full picture of the electrical conduction circumferentially around the heart, and may result in incomplete and inaccurate measurements. Here, we demonstrate OM of Hamburger and Hamilton stage 14 embryonic quail hearts using a new commercially-available VSD, Fluovolt, and depth sectioning using a custom built light-sheet microscopy system. Axial and lateral resolution of the system is 14µm and 8µm respectively. For OM imaging, the field-of-view was set to 900µm×900µm to cover the entire heart. 2D over time OM image sets at multiple cross-sections through the looping-stage heart were recorded. The shapes of both atrial and ventricular action potentials acquired were consistent with previous reports using conventional VSD (di-4-ANNEPS). With Fluovolt, signal-to-noise ratio (SNR) is improved significantly by a factor of 2-10 (compared with di-4-ANNEPS) enabling light-sheet OM, which intrinsically has lower SNR due to smaller sampling volumes. Electrophysiologic parameters are rate dependent. Optical pacing was successfully integrated into the system to ensure heart rate consistency. This will also enable accurately gated reconstruction of full four dimensional conduction maps and 3D conduction velocity measurements.

  7. Advanced electron microscopy of novel ferromagnetic materials and ferromagnet/oxide interfaces in magnetic tunnel junctions

    NASA Astrophysics Data System (ADS)

    Shi, Fengyuan

    We have studied novel ferromagnetic (FM) materials and FM electrode/tunnel barrier interfaces in magnetic tunnel junctions (MTJs) by advanced electron microscopy including scanning transmission electron microscopy (HRSTEM) and electron energy loss spectroscopy (EELS). MTJs are one of the prototypical spintronic devices, with applications in magnetic random access memory, sensors and read heads. The performance of MTJs depends on several factors, including the FM electrodes and the FM/tunnel barrier interfaces. Therefore, to realize the high performance of MTJs, we first need high quality ferromagnetic electrodes with high spin polarization. High-quality Fe3O4 and Fe4N electrodes with theoretically predicted -100% spin polarization were fabricated by various methods and investigated by HRSTEM and STEM EELS. The Fe3O4 and Fe4N thin films have low defect density and good crystallinity, but when integrated as electrodes in a MTJ, problems emerged. In a Fe4N/AlOx/Fe MTJ, the magnetoresistance was negative, but relatively small, due to a defective Fe 3O4 reaction layer formed at the Fe4N/tunnel barrier interface revealed by HRSTEM and EELS. The interfacial reaction layer was thin and discontinuous which made direct imaging difficult. Therefore, STEM EELS was used to map out the reaction layer. A Fe3O4 reaction layer was also found in a nominally symmetric CoFe/AlOx/CoFe MTJs after annealing, which also exhibited inverse TMR and a non-symmetric bias dependence. We also investigated the MTJs with the Heusler alloy Co2MnSi as one or both electrode and crystalline MgO as the tunnel barrier, which exhibit quite high TMR due to coherent tunneling. We showed that the Co2MnSi/MgO interface in these junctions is dominated by a configuration of a pure Mn plane bonded across the interface to O. This was the first observation of that interface termination. HRSTEM images also show that the fraction of MnMn/O interface termination increases with increasing Mn concentration in the CMS

  8. Estimation of age based on tooth cementum annulations: A comparative study using light, polarized, and phase contrast microscopy

    PubMed Central

    Kaur, Prabhpreet; Astekar, Madhusudan; Singh, Jappreet; Arora, Karandeep Singh; Bhalla, Gagandeep

    2015-01-01

    Context: The identification of living or deceased persons using unique traits and characteristics of the teeth and jaws is a cornerstone of forensic science. Teeth have been used to estimate age both in the young and old, as well as in the living and dead. Gradual structural changes in teeth throughout life are the basis for age estimation. Tooth cementum annulation (TCA) is a microscopic method for the determination of an individual's age based on the analysis of incremental lines of cementum. Aim: To compare ages estimated using incremental lines of cementum as visualized by bright field microscopy, polarized microscopy, and phase contrast microscopy with the actual age of subject and to determine accuracy and feasibility of the method used. Materials and Methods: Cementum annulations of 60 permanent teeth were analyzed after longitudinal ground sections were made in the mesiodistal plane. The incremental lines were counted manually using a light, polarized and phase contrast microscopy. Ages were estimated and then compared with the actual age of individual. Statistical Analysis: Analysis of variance (ANOVA), Student's t-test, the Pearson product-moment corre (PPMCC) and regression analysis were performed. Results: PPMCC value r = 0.347, 0.542 and 0.989 were obtained using light, polarized and phase contrast microscopy methods respectively. Conclusion: It was concluded that incremental lines of cementum were most clearly visible under a phase contrast microscope, followed by a polarized microscope, and then a light microscope when used for age estimation. PMID:26816462

  9. Effects of Pamidronate on Dental Enamel Formation Assessed by Light Microscopy, Energy-Dispersive X-Ray Analysis, Scanning Electron Microscopy, and Microhardness Testing.

    PubMed

    Soares, Ana P; do Espírito Santo, Renan F; Line, Sérgio R P; Pinto, Maria das G F; Santos, Pablo de M; Toralles, Maria Betania P; do Espírito Santo, Alexandre R

    2016-06-01

    The aim of the present work was to investigate birefringence and morphology of the secretory-stage enamel organic extracellular matrix (EOECM), and structural and mechanical properties of mature enamel of upper incisors from adult rats that had been treated with pamidronate disodium (0.5 mg/kg/week for 56 days), using transmitted polarizing and bright-field light microscopies (TPLM and BFLM), energy-dispersive X-ray (EDX) analysis, scanning electron microscopy (SEM) and microhardness testing. BFLM showed no morphological changes of the EOECM in pamidronate and control groups, but TPLM revealed a statistically significant reduction in optical retardation values of birefringence brightness of pamidronate-treated rats when compared with control animals (p0.05). The present study indicates that pamidronate can affect birefringence of the secretory-stage EOECM, which does not seem to be associated with significant changes in morphological and/or mechanical properties of mature enamel. PMID:27212049

  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. Single objective light-sheet microscopy for high-speed whole-cell 3D super-resolution

    DOE PAGESBeta

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; Edwards, Thayne L.; James, Conrad D.; Lidke, Keith A.

    2016-05-01

    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

  12. 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. PMID:27375939

  13. 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.

  14. Experimental study of the types of cavitation by air seeding using light microscopy.

    PubMed

    Shen, Fanyi; Cheng, Yanxia; Zhang, Li; Gao, Rongfu; Shao, Xuemeng

    2015-12-01

    Recently, three types of cavitation: (i) expanding gradually; (ii) expanding-exploding, becoming a long-shaped bubble-lengthening by degrees; (iii) suddenly exploding and fully filling the conduit instantly, were proposed. Directed by this theory, experiments were performed using light microscopy to study the natural drying processes of xylem sections of Platycladus orientalis (L.) Franco. Three different phenomena of gas filling process in conduits were captured by replaying recorded videos. The first phenomenon is that a bubble emerging in a conduit expands and elongates gradually to fill the conduit. The second phenomenon is that a bubble emerging in a conduit expands gradually, and then suddenly becomes long-shaped, and extends continuously. The third phenomenon is that a bubble instantly fully fills a conduit. This paper suggests in these experiments that after losing the bulk water of a section, as the water stress of that section became more severe, the water pressures of different conduits of the section were not necessarily the same, and as time went on, the water pressures decreased constantly. Considering some practical factors, the three phenomena captured in our experiment are explained by our theory. PMID:26338303

  15. Gradient light interference microscopy (GLIM) for imaging thick specimens (Conference Presentation)

    NASA Astrophysics Data System (ADS)

    Nguyen, Tan H.; Kandel, Mikhail E.; Popescu, Gabriel

    2016-03-01

    Compared to the Phase Contrast, Differential Interference Contrast (DIC) has been known to give higher depth sectioning as well as a halo-free images when investigating transparent specimens. Thanks to relying on generating two slightly shifted replicas with a small amount of shift, within the coherence area, DIC is able to operate with very low coherence light. More importantly, the method is able to work with very large numerical aperture of the illumination, which offer comparable sectioning capability to bright field microscopy. However, DIC is still a qualitative method, which limits potential applications of the technique. In this paper, we introduce a method that extends the capability of DIC by combining it with a phase shifting module to extract the phase gradient information. A theoretical model of the image formation is developed and the possibility of integrating the gradient function is analyzed.. Our method is benchmarked on imaging embryos during their 7-day development, HeLa cells during mitosis, and control samples.

  16. High-throughput spatial light modulation two-photon microscopy for fast functional imaging.

    PubMed

    Pozzi, Paolo; Gandolfi, Daniela; Tognolina, Marialuisa; Chirico, Giuseppe; Mapelli, Jonathan; D'Angelo, Egidio

    2015-01-01

    The optical monitoring of multiple single neuron activities requires high-throughput parallel acquisition of signals at millisecond temporal resolution. To this aim, holographic two-photon microscopy (2PM) based on spatial light modulators (SLMs) has been developed in combination with standard laser scanning microscopes. This requires complex coordinate transformations for the generation of holographic patterns illuminating the points of interest. We present a simpler and fully digital setup (SLM-2PM) which collects three-dimensional two-photon images by only exploiting the SLM. This configuration leads to an accurate placement of laser beamlets over small focal volumes, eliminating mechanically moving parts and making the system stable over long acquisition times. Fluorescence signals are diffraction limited and are acquired through a pixelated detector, setting the actual limit to the acquisition rate. High-resolution structural images were acquired by raster-scanning the sample with a regular grid of excitation focal volumes. These images allowed the selection of the structures to be further investigated through an interactive operator-guided selection process. Functional signals were collected by illuminating all the preselected points with a single hologram. This process is exemplified for high-speed (up to 1 kHz) two-photon calcium imaging on acute cerebellar slices. PMID:26157984

  17. 4-Dimensional light-sheet microscopy to elucidate shear stress modulation of cardiac trabeculation

    PubMed Central

    Lee, Juhyun; Fei, Peng; Packard, René R. Sevag; Kang, Hanul; Xu, Hao; Baek, Kyung In; Jen, Nelson; Chen, Junjie; Yen, Hilary; Chi, Neil C.; Ho, Chih-Ming; Hsiai, Tzung K.

    2016-01-01

    Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress–mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress–activated Notch signaling to modulate trabeculation. PMID:27018592

  18. High throughput imaging of blood smears using white light diffraction phase microscopy

    NASA Astrophysics Data System (ADS)

    Majeed, Hassaan; Kandel, Mikhail E.; Bhaduri, Basanta; Han, Kevin; Luo, Zelun; Tangella, Krishnarao; Popescu, Gabriel

    2015-03-01

    While automated blood cell counters have made great progress in detecting abnormalities in blood, the lack of specificity for a particular disease, limited information on single cell morphology and intrinsic uncertainly due to high throughput in these instruments often necessitates detailed inspection in the form of a peripheral blood smear. Such tests are relatively time consuming and frequently rely on medical professionals tally counting specific cell types. These assays rely on the contrast generated by chemical stains, with the signal intensity strongly related to staining and preparation techniques, frustrating machine learning algorithms that require consistent quantities to denote the features in question. Instead we opt to use quantitative phase imaging, understanding that the resulting image is entirely due to the structure (intrinsic contrast) rather than the complex interplay of stain and sample. We present here our first steps to automate peripheral blood smear scanning, in particular a method to generate the quantitative phase image of an entire blood smear at high throughput using white light diffraction phase microscopy (wDPM), a single shot and common path interferometric imaging technique.

  19. Morphology of the Lingual and Buccal Papillae in Alpaca (Vicugna pacos) - Light and Scanning Electron Microscopy.

    PubMed

    Goździewska-Harłajczuk, K; Klećkowska-Nawrot, J; Janeczek, M; Zawadzki, M

    2015-10-01

    The aim of this study was the description of the lingual and buccal papillae in adult alpaca (Vicugna pacos) by light and scanning electron microscopy (SEM). The tongue consisted of apex, body and root. Four types of lingual papillae (filiform, fungiform, conical and circumvallate) in addition to two types of buccal papillae were observed. The filiform papillae, some with secondary papillae, were distributed on both the corpus and apex of the tongue, with stratified epithelium, and layer of keratin coat were recognized. The short (small) cone papillae had pointed top, while bunoform papillae were wide with smooth apex. The much less numerous circumvallate papillae with pseudopapillae on the each rim of the caudal lingual body were present with weak layer of keratin and intra-epithelial taste buds. The small fungiform papillae were found on the dorsal lingual surface, while the large fungiform papillae were situated on the ventral surface of the tongue, especially, in rostral part and were round in shape with numerous gustatory pores and very thin keratin coat. Pseudopapillae were present on the buccal conical 'bunoform' papillae surface, while 'elongate' buccal papillae surface was rather softly folded with thin coat of keratin. Microridges were observed in the less keratinized parts of each type of papillae. The orientation of either lingual or buccal papillae into the throat side facilitates the emptying of oral cavity from nutrient and swallowing of food. In conclusion, the anatomical features of the alpaca tongue are an adaptation to the feeding habits. PMID:25223623

  20. A New Method for Automated Identification and Morphometry of Myelinated Fibers Through Light Microscopy Image Analysis.

    PubMed

    Novas, Romulo Bourget; Fazan, Valeria Paula Sassoli; Felipe, Joaquim Cezar

    2016-02-01

    Nerve morphometry is known to produce relevant information for the evaluation of several phenomena, such as nerve repair, regeneration, implant, transplant, aging, and different human neuropathies. Manual morphometry is laborious, tedious, time consuming, and subject to many sources of error. Therefore, in this paper, we propose a new method for the automated morphometry of myelinated fibers in cross-section light microscopy images. Images from the recurrent laryngeal nerve of adult rats and the vestibulocochlear nerve of adult guinea pigs were used herein. The proposed pipeline for fiber segmentation is based on the techniques of competitive clustering and concavity analysis. The evaluation of the proposed method for segmentation of images was done by comparing the automatic segmentation with the manual segmentation. To further evaluate the proposed method considering morphometric features extracted from the segmented images, the distributions of these features were tested for statistical significant difference. The method achieved a high overall sensitivity and very low false-positive rates per image. We detect no statistical difference between the distribution of the features extracted from the manual and the pipeline segmentations. The method presented a good overall performance, showing widespread potential in experimental and clinical settings allowing large-scale image analysis and, thus, leading to more reliable results. PMID:25986589

  1. Active intracellular transport in metastatic cells studied by spatial light interference microscopy

    NASA Astrophysics Data System (ADS)

    Ceballos, Silvia; Kandel, Mikhail; Sridharan, Shamira; Majeed, Hassaan; Monroy, Freddy; Popescu, Gabriel

    2015-11-01

    Spatiotemporal patterns of intracellular transport are very difficult to quantify and, consequently, continue to be insufficiently understood. While it is well documented that mass trafficking inside living cells consists of both random and deterministic motions, quantitative data over broad spatiotemporal scales are lacking. We studied the intracellular transport in live cells using spatial light interference microscopy, a high spatiotemporal resolution quantitative phase imaging tool. The results indicate that in the cytoplasm, the intracellular transport is mainly active (directed, deterministic), while inside the nucleus it is both active and passive (diffusive, random). Furthermore, we studied the behavior of the two-dimensional mass density over 30 h in HeLa cells and focused on the active component. We determined the standard deviation of the velocity distribution at the point of cell division for each cell and compared the standard deviation velocity inside the cytoplasm and the nucleus. We found that the velocity distribution in the cytoplasm is consistently broader than in the nucleus, suggesting mechanisms for faster transport in the cytosol versus the nucleus. Future studies will focus on improving phase measurements by applying a fluorescent tag to understand how particular proteins are transported inside the cell.

  2. Synchrotron radiation as a light source in confocal microscopy of biological processes

    NASA Astrophysics Data System (ADS)

    Gerritsen, Hans C.; van der Oord, C. J. R.; Levine, Yehudi K.; Munro, Ian H.; Myring, Wendy J.; Shaw, D. A.; Rommerts, Fokko F.

    1992-04-01

    A novel confocal microscope is presented using the Daresbury Synchrotron Radiation source as its light source. The broad spectrum of synchrotron radiation in combination with the UV compatible microscope allows the extension of confocal microscopy from the visible to the UV region down to about 200 nm. It is envisaged that structures separated by about 70 nm can be resolved at a wavelength of 200 nm. In addition, the tunability of synchrotron radiation affords the selective excitation of any specific fluorescent molecule at the maximum of the absorption band. This avoids the restriction of working at fixed laser lines. A further advantage of using synchrotron radiation is the realization of multiwavelength excitation. Test results using laser systems in the visible and in the UV are presented. Fluorescence images of test targets using UV excitation reveal the superior resolution of the microscope. Furthermore, images of Leydig cells incubated with a fluorescent cholesterol derivative whose maximum of absorption is at 325 nm are shown. These images cannot be produced by conventional confocal laser microscopes. Finally, promising preliminary results obtained with synchrotron radiation are presented.

  3. Reflection polarized light microscopy and its application to pyrolytic carbon deposits

    NASA Astrophysics Data System (ADS)

    Bortchagovsky, E. G.

    2004-05-01

    The methods for the measurement of extinction angles by reflection polarized light microscopy were analyzed with respect to the investigation of pyrolytic carbon deposits. It is demonstrated that measurements of the extinction angle for deposits with circular morphology (e.g., a pyrolytic carbon layer on a fiber) must be strictly distinguished from the standard measurements of optically uniaxial materials with spatially uniform optical properties, such as a single crystal (e.g., graphite or pyrolytic carbon on a planar substrate). On the basis of the mathematical description of the method, an expression of the extinction angle for materials with a circular morphology is derived. This expression differs from the equation describing measurements of the extinction angle for optically uniaxial materials with spatially uniform properties erroneously applied for circular morphology. It is demonstrated on the basis of the developed formalism that no discrepancy exists between measured extinction angles for circular pyrolytic carbon deposits and single crystalline graphite, which was discussed earlier due to the misinterpretation of data measured by two different methods. The physical meaning of extinction angles is discussed and approaches for the measurement of two meaningful ellipsometric parameters instead of one extinction angle are proposed.

  4. Bimodal endoscopic probe combining white-light microscopy and optical coherence tomography.

    PubMed

    Blattmann, M; Kretschmer, S; Thiele, S; Ataman, C; Zappe, H; Herkommer, A; Seifert, A

    2016-05-20

    We present a novel bimodal endoscopic imaging probe that can simultaneously provide full-field white-light video microscopy and confocal optical coherence tomography (OCT) depth scans. The two modalities rely on spectrally separated optical paths that run partially in parallel through a micro-optical bench system, which has a cross-section of only 2  mm×2.76  mm and is realized via standard silicon micromachining techniques. With a numerical aperture of 0.061, the video modality has a resolution and field of view of 9.3 and 1240  μm×1080  μm, respectively. The resolution is limited by the pixel spacing of the coherent fiber bundle, which relays the acquired image from the distal to the proximal end. A custom-designed diffractive optical element placed within the video imaging path significantly improves the image contrast by up to 45% in the medium frequency range. The OCT modality is optimized for 830 nm center wavelength, and works in a confocal arrangement with an NA of 0.018. It provides single-point depth probing at the center of the video image with a lateral resolution of 20 μm. Through its compact footprint and enhanced functionality, the probe can provide depth-resolved guiding capability for existing laparoscopes and represents a major step toward a new class of multimodal endoscopic imaging probes. PMID:27411158

  5. Correlative Light and Electron Microscopy Reveals the HAS3-Induced Dorsal Plasma Membrane Ruffles

    PubMed Central

    Rilla, Kirsi; Koistinen, Arto

    2015-01-01

    Hyaluronan is a linear sugar polymer synthesized by three isoforms of hyaluronan synthases (HAS1, 2, and 3) that forms a hydrated scaffold around cells and is an essential component of the extracellular matrix. The morphological changes of cells induced by active hyaluronan synthesis are well recognized but not studied in detail with high resolution before. We have previously found that overexpression of HAS3 induces growth of long plasma membrane protrusions that act as platforms for hyaluronan synthesis. The study of these thin and fragile protrusions is challenging, and they are difficult to preserve by fixation unless they are adherent to the substrate. Thus their structure and regulation are still partly unclear despite careful imaging with different microscopic methods in several cell types. In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression. Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation. Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis. PMID:26448759

  6. Correlative Light and Electron Microscopy Reveals the HAS3-Induced Dorsal Plasma Membrane Ruffles.

    PubMed

    Rilla, Kirsi; Koistinen, Arto

    2015-01-01

    Hyaluronan is a linear sugar polymer synthesized by three isoforms of hyaluronan synthases (HAS1, 2, and 3) that forms a hydrated scaffold around cells and is an essential component of the extracellular matrix. The morphological changes of cells induced by active hyaluronan synthesis are well recognized but not studied in detail with high resolution before. We have previously found that overexpression of HAS3 induces growth of long plasma membrane protrusions that act as platforms for hyaluronan synthesis. The study of these thin and fragile protrusions is challenging, and they are difficult to preserve by fixation unless they are adherent to the substrate. Thus their structure and regulation are still partly unclear despite careful imaging with different microscopic methods in several cell types. In this study, correlative light and electron microscopy (CLEM) was utilized to correlate the GFP-HAS3 signal and the surface ultrastructure of cells in order to study in detail the morphological changes induced by HAS3 overexpression. Surprisingly, this method revealed that GFP-HAS3 not only localizes to ruffles but in fact induces dorsal ruffle formation. Dorsal ruffles regulate diverse cellular functions, such as motility, regulation of glucose metabolism, spreading, adhesion, and matrix degradation, the same functions driven by active hyaluronan synthesis. PMID:26448759

  7. Active intracellular transport in metastatic cells studied by spatial light interference microscopy.

    PubMed

    Ceballos, Silvia; Kandel, Mikhail; Sridharan, Shamira; Majeed, Hassaan; Monroy, Freddy; Popescu, Gabriel

    2015-01-01

    Spatiotemporal patterns of intracellular transport are very difficult to quantify and, consequently, continue to be insufficiently understood. While it is well documented that mass trafficking inside living cells consists of both random and deterministic motions, quantitative data over broad spatiotemporal scales are lacking. We studied the intracellular transport in live cells using spatial light interference microscopy, a high spatiotemporal resolution quantitative phase imaging tool. The results indicate that in the cytoplasm, the intracellular transport is mainly active (directed, deterministic), while inside the nucleus it is both active and passive (diffusive, random). Furthermore, we studied the behavior of the two-dimensional mass density over 30 h in HeLa cells and focused on the active component. We determined the standard deviation of the velocity distribution at the point of cell division for each cell and compared the standard deviation velocity inside the cytoplasm and the nucleus. We found that the velocity distribution in the cytoplasm is consistently broader than in the nucleus, suggesting mechanisms for faster transport in the cytosol versus the nucleus. Future studies will focus on improving phase measurements by applying a fluorescent tag to understand how particular proteins are transported inside the cell. PMID:26271006

  8. Visualizing Collagen Network Within Human and Rhesus Monkey Vocal Folds Using Polarized Light Microscopy

    PubMed Central

    Julias, Margaret; Riede, Tobias; Cook, Douglas

    2014-01-01

    Objectives Collagen fiber content and orientation affect the viscoelastic properties of the vocal folds, determining oscillation characteristics during speech and other vocalization. The investigation and reconstruction of the collagen network in vocal folds remains a challenge, because the collagen network requires at least micron-scale resolution. In this study, we used polarized light microscopy to investigate the distribution and alignment of collagen fibers within the vocal folds. Methods Data were collected in sections of human and rhesus monkey (Macaca mulatta) vocal folds cut at 3 different angles and stained with picrosirius red. Results Statistically significant differences were found between different section angles, implying that more than one section angle is required to capture the network’s complexity. In the human vocal folds, the collagen fiber distribution continuously varied across the lamina propria (medial to lateral). Distinct differences in birefringence distribution were observed between the species. For the human vocal folds, high birefringence was observed near the thyroarytenoid muscle and near the epithelium. However, in the rhesus monkey vocal folds, high birefringence was observed near the epithelium, and lower birefringence was seen near the thyroarytenoid muscle. Conclusions The differences between the collagen networks in human and rhesus monkey vocal folds provide a morphological basis for differences in viscoelastic properties between species. PMID:23534129

  9. Endolymph movement visualized with light sheet fluorescence microscopy in an acute hydrops model.

    PubMed

    Brown, Daniel J; Pastras, Christopher J; Curthoys, Ian S; Southwell, Cassandra S; Van Roon, Lieke

    2016-09-01

    There are a variety of techniques available to investigate endolymph dynamics, primarily seeking to understand the cause of endolymphatic hydrops. Here we have taken the novel approach of injecting, via a glass micropipette, fluorescein isothiocyanate-dextran (FITC-dex) and artificial endolymph into scala media of anaesthetized guinea pigs, with subsequent imaging of the inner ear using Light Sheet Fluorescence Microscopy (LSFM) as a means to obtain highly resolved 3D visualization of fluid movements. Our results demonstrate endolymph movement into the utricle, semicircular canals and endolymphatic duct and sac when more than 2.5 μl of fluid had been injected into scala media, with no apparent movement of fluid into the perilymphatic compartments. There was no movement of endolymph into these compartments when less than 2.5 μl was injected. The remarkable uptake of the FITC-dex into the endolymphatic duct, including an absorption into the periductal channels surrounding the endolymphatic duct, highlights the functional role this structure plays in endolymph volume regulation. PMID:27377233

  10. Advances in white-light optical signal processing

    NASA Technical Reports Server (NTRS)

    Yu, F. T. S.

    1984-01-01

    A technique that permits signal processing operations which can be carried out by white light source is described. The method performs signal processing that obeys the concept of coherent light rather than incoherent optics. Since the white light source contains all the color wavelengths of the visible light, the technique is very suitable for color signal processing.

  11. Mesoscale Science with High Energy X-ray Diffraction Microscopy at the Advanced Photon Source

    NASA Astrophysics Data System (ADS)

    Suter, Robert

    2014-03-01

    Spatially resolved diffraction of monochromatic high energy (> 50 keV) x-rays is used to map microstructural quantities inside of bulk polycrystalline materials. The non-destructive nature of High Energy Diffraction Microscopy (HEDM) measurements allows tracking of responses as samples undergo thermo-mechanical or other treatments. Volumes of the order of a cubic millimeter are probed with micron scale spatial resolution. Data sets allow direct comparisons to computational models of responses that frequently involve long-ranged, multi-grain interactions; such direct comparisons have only become possible with the development of HEDM and other high energy x-ray methods. Near-field measurements map the crystallographic orientation field within and between grains using a computational reconstruction method that simulates the experimental geometry and matches orientations in micron sized volume elements to experimental data containing projected grain images in large numbers of Bragg peaks. Far-field measurements yield elastic strain tensors through indexing schemes that sort observed diffraction peaks into sets associated with individual crystals and detect small radial motions in large numbers of such peaks. Combined measurements, facilitated by a new end station hutch at Advanced Photon Source beamline 1-ID, are mutually beneficial and result in accelerated data reduction. Further, absorption tomography yields density contrast that locates secondary phases, void clusters, and cracks, and tracks sample shape during deformation. A collaboration led by the Air Force Research Laboratory and including the Advanced Photon Source, Lawrence Livermore National Laboratory, Carnegie Mellon University, Petra-III, and Cornell University and CHESS is developing software and hardware for combined measurements. Examples of these capabilities include tracking of grain boundary migrations during thermal annealing, tensile deformation of zirconium, and combined measurements of nickel

  12. Methods and apparatus of spatially resolved electroluminescence of operating organic light-emitting diodes using conductive atomic force microscopy

    NASA Technical Reports Server (NTRS)

    Hersam, Mark C. (Inventor); Pingree, Liam S. C. (Inventor)

    2008-01-01

    A conductive atomic force microscopy (cAFM) technique which can concurrently monitor topography, charge transport, and electroluminescence with nanometer spatial resolution. This cAFM approach is particularly well suited for probing the electroluminescent response characteristics of operating organic light-emitting diodes (OLEDs) over short length scales.

  13. [Experimental studies on the influence of selected preparations on secretion of tear fluid. II: Light and transmission electron microscopy].

    PubMed

    Oleszczyńska-Prost, E

    1994-01-01

    In the paper the influence of ambroxol, bethanechol, mesna and bromhexine hydrochloride on the morphologic appearance of the lacrimal glands in rabbits was studied with the use of light and transmission electron microscopy. The examination of the lacrimal glands in light microscopy has shown in comparison with the control group an increase in the number of secretory granules in rabbit glands after treatment with different drugs. In electron microscopy in animals, after treatment, much more prominent rough endoplasmic reticulum with a considerable distention of the cisternae and increase of the number of secretory granules in the cytoplasm of the cells were seen. The greatest stimulation of the production of tear fluid was observed after treatment with mesna. Nearly similar results were obtained after bethanechol and slightly worse after ambroxol. The least potent drug was bromhexine. PMID:7915766

  14. Grinding and polishing instead of sectioning for the tissue samples with a graft: Implications for light and electron microscopy.

    PubMed

    Mukhamadiyarov, Rinat A; Sevostyanova, Victoria V; Shishkova, Daria K; Nokhrin, Andrey V; Sidorova, Olga D; Kutikhin, Anton G

    2016-06-01

    A broad use of the graft replacement requires a detailed investigation of the host-graft interaction, including both histological examination and electron microscopy. A high quality sectioning of the host tissue with a graft seems to be complicated; in addition, it is difficult to examine the same tissue area by both of the mentioned microscopy techniques. To solve these problems, we developed a new technique of epoxy resin embedding with the further grinding, polishing, and staining. Graft-containing tissues prepared by grinding and polishing preserved their structure; however, sectioning frequently required the explantation of the graft and led to tissue disintegration. Moreover, stained samples prepared by grinding and polishing may then be assessed by both light microscopy and backscattered scanning electron microscopy. Therefore, grinding and polishing outperform sectioning when applied to the tissues with a graft. PMID:27023831

  15. Advanced electron microscopy characterization of tri-layer rare-earth oxide superlattices

    NASA Astrophysics Data System (ADS)

    Phillips, Patrick; Disa, Ankit; Ismail-Beigi, Sohrab; Klie, Robert; University of Illinois-Chicago Team; Yale University Team

    2015-03-01

    Rare-earth nickelates are known to display complex electronic and magnetic behaviors owed to a very localized and sensitive Ni-site atomic and electronic structure. Toward realizing the goal of manipulating of the energetic ordering of Ni d orbitals and 2D conduction, the present work focuses on the experimental characterization of thin film superlattice structures consisting of alternating layers of LaTiO3 and LaNiO3 sandwiched between a dull insulator, LaAlO3. Using advanced scanning transmission electron microscopy (STEM)-based methods, properties such as interfacial sharpness, electron transfer, O presence, and local electronic structure can be probed at the atomic scale, and will be discussed at length. By combining both energy dispersive X-ray (EDX) and electronic energy loss (EEL) spectroscopies in an aberration-corrected STEM, it is possible to attain energy and spatial resolutions of 0.35 eV and 100 pm, respectively. Focus of the talk will remain not only on the aforementioned properties, but will also include details and parameters of the acquisitions to facilitate future characterization at this level.

  16. Quantitative phase imaging of human red blood cells using phase-shifting white light interference microscopy with colour fringe analysis

    NASA Astrophysics Data System (ADS)

    Singh Mehta, Dalip; Srivastava, Vishal

    2012-11-01

    We report quantitative phase imaging of human red blood cells (RBCs) using phase-shifting interference microscopy. Five phase-shifted white light interferograms are recorded using colour charge coupled device camera. White light interferograms were decomposed into red, green, and blue colour components. The phase-shifted interferograms of each colour were then processed by phase-shifting analysis and phase maps for red, green, and blue colours were reconstructed. Wavelength dependent refractive index profiles of RBCs were computed from the single set of white light interferogram. The present technique has great potential for non-invasive determination of refractive index variation and morphological features of cells and tissues.

  17. Visualizing viral protein structures in cells using genetic probes for correlated light and electron microscopy.

    PubMed

    Ou, Horng D; Deerinck, Thomas J; Bushong, Eric; Ellisman, Mark H; O'Shea, Clodagh C

    2015-11-15

    Structural studies of viral proteins most often use high-resolution techniques such as X-ray crystallography, nuclear magnetic resonance, single particle negative stain, or cryo-electron microscopy (EM) to reveal atomic interactions of soluble, homogeneous viral proteins or viral protein complexes. Once viral proteins or complexes are separated from their host's cellular environment, their natural in situ structure and details of how they interact with other cellular components may be lost. EM has been an invaluable tool in virology since its introduction in the late 1940's and subsequent application to cells in the 1950's. EM studies have expanded our knowledge of viral entry, viral replication, alteration of cellular components, and viral lysis. Most of these early studies were focused on conspicuous morphological cellular changes, because classic EM metal stains were designed to highlight classes of cellular structures rather than specific molecular structures. Much later, to identify viral proteins inducing specific structural configurations at the cellular level, immunostaining with a primary antibody followed by colloidal gold secondary antibody was employed to mark the location of specific viral proteins. This technique can suffer from artifacts in cellular ultrastructure due to compromises required to provide access to the immuno-reagents. Immunolocalization methods also require the generation of highly specific antibodies, which may not be available for every viral protein. Here we discuss new methods to visualize viral proteins and structures at high resolutions in situ using correlated light and electron microscopy (CLEM). We discuss the use of genetically encoded protein fusions that oxidize diaminobenzidine (DAB) into an osmiophilic polymer that can be visualized by EM. Detailed protocols for applying the genetically encoded photo-oxidizing protein MiniSOG to a viral protein, photo-oxidation of the fusion protein to yield DAB polymer staining, and

  18. Advanced Light Source activity report 1996/97

    SciTech Connect

    1997-09-01

    Ten years ago, the Advanced Light Source (ALS) existed as a set of drawings, calculations, and ideas. Four years ago, it stored an electron beam for the first time. Today, the ALS has moved from those ideas and beginnings to a robust, third-generation synchrotron user facility, with eighteen beam lines in use, many more in planning or construction phases, and hundreds of users from around the world. Progress from concepts to realities is continuous as the scientific program, already strong in many diverse areas, moves in new directions to meet the needs of researchers into the next century. ALS staff members who develop and maintain the infrastructure for this research are similarly unwilling to rest on their laurels. As a result, the quality of the photon beams the authors deliver, as well as the support they provide to users, continues to improve. The ALS Activity Report is designed to share the results of these efforts in an accessible form for a broad audience. The Scientific Program section, while not comprehensive, shares the breadth, variety, and interest of recent research at the ALS. (The Compendium of User Abstracts and Technical Reports provides a more comprehensive and more technical view.) The Facility Report highlights progress in operations, ongoing accelerator research and development, and beamline instrumentation efforts. Although these Activity Report sections are separate, in practice the achievements of staff and users at the ALS are inseparable. User-staff collaboration is essential as they strive to meet the needs of the user community and to continue the ALS's success as a premier research facility.

  19. The U5. 0 Undulator for the Advanced Light Source

    SciTech Connect

    Hoyer, E.; Chin, J.; Halbach, K.; Hassenzahl, W.V.; Humphries, D.; Kincaid, B.; Lancaster, H.; Plate, D. )

    1992-01-01

    The U5.0 Undulator, an 89 period, 5 cm period length, 4.6 m long insertion device has been designed, is being fabricated, and is scheduled for completion in early 1992. This undulator will be the first high brightness source, in the 50 to 1,500 eV range, for the Advanced Light Source at the Lawrence Berkeley Laboratory. A hybrid magnetic configuration using Nd--Fe--B permanent magnet material and vanadium permendur poles has been selected to achieve the field quality needed to meet performance requirements. The magnetic structure is modular with each half consisting of five assembly sections, which provide the periodic structure, and end structures, for entrance and exit correction, mounted on a steel backing beam. Each assembly section consists of 35 half-period pole assemblies bolted to a mount. The required 0.837 T effective peak field at a 1.4 cm gap has been verified with model measurements. Vertical field integral correction is accomplished with the end structures, each having an arrangement of permanent magnet rotors which will be adjusted to minimize electron beam missteering over the undulator operating field range. To reduce the effect of environmental fields, the steel backing beams are connected through parallel, low-reluctance, Ni--Fe hinges. The magnetic structure is connected through four rollernuts to the drive system that provides gap adjustment with an arrangement of roller screws, chain drives, a gear reduction unit, and a stepper motor driven by a closed loop control system. Magnetic structure and drive system support are from a 2.4 m high structure which includes a support base with four vertical supports. The vacuum chamber design is a two-piece machined and welded 5083-H321 aluminum construction of 5.1 m length. Pumping is with a combination of ion, titanium sublimation pump and nonevaporable getter pumps. Magnetic design, subsystem design, and fabrication progress are presented.

  20. Lights Will Guide You : Sample Preparation and Applications for Integrated Laser and Electron Microscopy

    NASA Astrophysics Data System (ADS)

    Karreman, M. A.

    2013-03-01

    Correlative microscopy is the combined use of two different forms of microscopy in the study of a specimen, allowing for the exploitation of the advantages of both imaging tools. The integrated Laser and Electron Microscope (iLEM), developed at Utrecht University, combines a fluorescence microscope (FM) and a transmission electron microscope (TEM) in a single set-up. The region of interest in the specimen is labeled or tagged with a fluorescent probe and can easily be identified within a large field of view with the FM. Next, this same area is retraced in the TEM and can be studied at high resolution. The iLEM demands samples that can be imaged with both FM and TEM. Biological specimen, typically composed of light elements, generate low image contrast in the TEM. Therefore, these samples are often ‘contrasted’ with heavy metal stains. FM, on the other hand, images fluorescent samples. Sample preparation for correlative microscopy, and iLEM in particular, is complicated by the fact that the heavy metals stains employed for TEM quench the fluorescent signal of the probe that is imaged with FM. The first part of this thesis outlines preparation procedures for biological material yielding specimen that can be imaged with the iLEM. Here, approaches for the contrasting of thin sections of cells and tissue are introduced that do not affect the fluorescence signal of the probe that marks the region of interest. Furthermore, two novel procedures, VIS2FIXH and VIS2FIX­FS are described that allow for the chemical fixation of thin sections of cryo-immobilized material. These procedures greatly expedite the sample preparation process, and open up novel possibilities for the immuno-labeling of difficult antigens, eg. proteins and lipids that are challenging to preserve. The second part of this thesis describes applications of iLEM in research in the field of life and material science. The iLEM was employed in the study of UVC induced apoptosis (programmed cell death) of

  1. EDITORIAL: Special Issue on advanced and emerging light sources Special Issue on advanced and emerging light sources

    NASA Astrophysics Data System (ADS)

    Haverlag, Marco; Kroesen, Gerrit; Ferguson, Ian

    2011-06-01

    -based light sources. However, the progress in the last few years in LED and OLED sources has been even greater. In the editorial for the LS-11 conference by previous guest editor David Wharmby, it was stated that most LED lighting was still mostly used for signalling and decorative sources. In the three years that have passed, things have changed considerably and we now see LED light sources entering every application, ranging from street lighting and parking lots to shop lighting and even greenhouses. Currently LED prices for traditional lighting applications are high, but they are dropping rapidly. The papers published in this special issue give some indications of things to come. The paper by Jamil et al deals with the possibility of using silicon wafers as substrate material instead of the now commonly used (but more expensive) sapphire substrates. This is attractive from a cost price point of view, but leads to an increased lattice mismatch and therefore strain-induced defects. In this paper it is shown that when using intermediate matching layers it is possible to retain the same electrical and optical properties as with structures on sapphire. Another aspect that directly relates to cost is efficiency and droop in green InGaN devices, which is addressed in the paper by Lee et al. They show that by providing a flow of trymethylindium prior to the growth of the quantum wells it is possible to significantly increase the internal quantum efficiency of green LEDs. Improvement of the optical out-coupling of InGaN LEDs is discussed by Mak et al, and it is found that localized plasmon resonance of metallic nanoparticles (and especially silver) can help to increase the optical out-coupling in the wavelength region of interest. Nanoparticles in the form of ZnO nanorods are described by Willander et al as a possibility for phosphor-free wavelength conversion on polymer (O)LEDs. More advanced functions besides light emission can be achieved with OLEDs and this is demonstrated in

  2. Dynamics of orthodontic root resorption and repair in human premolars: a light microscopy study.

    PubMed

    Winter, Björn U; Stenvik, Arild; Vandevska-Radunovic, Vaska

    2009-08-01

    The purpose of the study was to investigate the relationship between root resorption and repair in human premolars that had been orthodontically intruded. The objective was to examine these processes related to time and root development. Seventy-six premolars were divided into subgroups: 33 teeth were intruded and then extracted (G1); 25 teeth were intruded and then left in situ for varying periods before extraction (G2); 18 teeth served as the controls (G3). All teeth were examined by light microscopy. Using non-parametric statistical analysis, differences between the groups were examined with the Pearson chi-square test. Teeth in G1 and G2 had significantly more resorptive lesions, 55 and 64 per cent, respectively, than the controls of 11 per cent. Resorption was observed over the whole root surface and increased with time. The occurrence increased to 100 per cent in both experimental groups after 36 days of intrusion. The appearance of lesions in relation to root development showed no differences between G1 and G2. In the apical part of the root, total resorption of the dentine was sometimes observed, but no resorptions extended into the predentine. Resorptive lesions undergoing repair were seen in both groups, with significantly more repair in G2 (58 per cent) than in G1 (32 per cent). Active resorption and repair were sometimes seen at the same resorption site. Deposition of cellular and acellular cementum was found to the same extent over the whole root when repair took place. With time, resorption appeared over the whole root surface. In some teeth, resorptive activity continued up to 10 days after removal of forces but on the other hand, repair of the resorbed area sometimes started during active movement. The individual variation in repair was much wider compared with resorption. The predentine layer in the apical area appeared not to be affected by the resorptive process. PMID:19465737

  3. Whole slide imaging diagnostic concordance with light microscopy for breast needle biopsies.

    PubMed

    Campbell, W Scott; Hinrichs, Steven H; Lele, Subodh M; Baker, John J; Lazenby, Audrey J; Talmon, Geoffrey A; Smith, Lynette M; West, William W

    2014-08-01

    This study investigated the diagnostic accuracy of whole slide imaging (WSI) in breast needle biopsy diagnosis in comparison with standard light microscopy (LM). The study examined the effects of image capture magnification and computer monitor quality on diagnostic concordance of WSI and LM. Four pathologists rendered diagnoses using WSI to examine 85 breast biopsies (92 parts; 786 slides) consisting of benign and malignant cases. Each WSI case was evaluated using images captured at either ×20 or ×40 magnifications and viewed using a Digital Imaging and Communication in Medicine (DICOM) grade, color-calibrated monitor or a standard, desktop liquid-crystal display (LCD) monitor. For each combination, the WSI result was compared with the original, LM diagnosis. The overall concordance rate observed between WSI and LM was 97.1% (95% confidence intervals [CI]: 94.3%-98.5%). After a washout period, all cases were reviewed a second time by each pathologist after using LM, and the second LM diagnosis was compared with the WSI diagnosis rendered by the same pathologist. Intraobserver concordance between WSI and LM was 95.4% (95% CI: 92.2%-97.4%). The second LM diagnoses were also compared with the original LM diagnoses, and the observed interobserver LM concordance rate was 97.3% (95% CI: 93.1%-99.0%). The study data demonstrated that breast needle biopsy diagnoses rendered by WSI were equivalent to diagnoses rendered by LM. No diagnostic differences were detected between the underlying viewing system parameters of monitor quality and image capture resolution. The results of this study demonstrated that WSI can be effectively used in subspecialty diagnostic cases where a minimum amount of tissue is available. PMID:24913758

  4. Modeling of Fibrin Gels Based on Confocal Microscopy and Light-Scattering Data

    PubMed Central

    Magatti, Davide; Molteni, Matteo; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

    2013-01-01

    Fibrin gels are biological networks that play a fundamental role in blood coagulation and other patho/physiological processes, such as thrombosis and cancer. Electron and confocal microscopies show a collection of fibers that are relatively monodisperse in diameter, not uniformly distributed, and connected at nodal points with a branching order of ∼3–4. Although in the confocal images the hydrated fibers appear to be quite straight (mass fractal dimension Dm = 1), for the overall system 1light-scattering measurements. We then derived an analytical form factor for accurately fitting the scattering data, which allowed us to directly recover the gels’ structural parameters. PMID:23473498

  5. Microgravity Emissions Laboratory Testing of the Light Microscopy Module Control Box Fan

    NASA Technical Reports Server (NTRS)

    McNelis, Anne M.; Samorezov, Sergey; Haecker, Anthony H.

    2003-01-01

    The Microgravity Emissions Laboratory (MEL) was developed at the NASA Glenn Research Center for the characterization, simulation, and verification of the International Space Station (ISS) microgravity environment. This Glenn lab was developed in support of the Fluids and Combustion Facility (FCF). The MEL is a six-degrees-of-freedom inertial measurement system that can characterize the inertial response forces (emissions) of components, subrack payloads, or rack-level payloads down to 10 7g. The inertial force output data generated from the steady-state or transient operations of the test article are used with finite element analysis, statistical energy analysis, and other analysis tools to predict the on-orbit environment at specific science or rack interface locations. Customers of the MEL have used benefits in isolation performance testing in defining available attenuation during the engineering hardware design phase of their experiment s development. The Light Microscopy Module (LMM) Control Box (LCB) fan was tested in the MEL in June and July of 2002. The LMM is planned as a remotely controllable on-orbit microscope subrack facility that will be accommodated in an FCF Fluids Integrated Rack on the ISS. The disturbances measured in the MEL test resulted from operation of the air-circulation fan within the LCB. The objectives of the testing were (1) to identify an isolator to be added to the LCB fan assembly to reduce fan-speed harmonics and (2) to identify the fan-disturbance forcing functions for use in rack-response analysis of the LMM and Fluids Integrated Rack facility. This report describes the MEL, the testing process, and the results from ground-based MEL LCB fan testing.

  6. Automatic identification and characterization of radial files in light microscopy images of wood

    PubMed Central

    Brunel, Guilhem; Borianne, Philippe; Subsol, Gérard; Jaeger, Marc; Caraglio, Yves

    2014-01-01

    Background and Aims Analysis of anatomical sections of wood provides important information for understanding the secondary growth and development of plants. This study reports on a new method for the automatic detection and characterization of cell files in wood images obtained by light microscopy. To facilitate interpretation of the results, reliability coefficients have been determined, which characterize the files, their cells and their respective measurements. Methods Histological sections and blocks of the gymnosperms Pinus canariensis, P. nigra and Abies alba were used, together with histological sections of the angiosperm mahogany (Swietenia spp.). Samples were scanned microscopically and mosaic images were built up. After initial processing to reduce noise and enhance contrast, cells were identified using a ‘watershed’ algorithm and then cell files were built up by the successive aggregation of cells taken from progressively enlarged neighbouring regions. Cell characteristics such as thickness and size were calculated, and a method was developed to determine the reliability of the measurements relative to manual methods. Key Results Image analysis using this method can be performed in less than 20 s, which compares with a time of approx. 40 min to produce the same results manually. The results are accompanied by a reliability indicator that can highlight specific configurations of cells and also potentially erroneous data. Conclusions The method provides a fast, economical and reliable tool for the identification of cell files. The reliability indicator characterizing the files permits quick filtering of data for statistical analysis while also highlighting particular biological configurations present in the wood sections. PMID:24989783

  7. Lens-free spectral light-field fusion microscopy for contrast- and resolution-enhanced imaging of biological specimens.

    PubMed

    Kazemzadeh, Farnoud; Jin, Chao; Molladavoodi, Sara; Mei, Yu; Emelko, Monica B; Gorbet, Maud B; Wong, Alexander

    2015-08-15

    A lens-free spectral light-field fusion microscopy (LSLFM) system is presented for enabling contrast- and resolution-enhanced imaging of biological specimens. LSLFM consists of a pulsed multispectral lens-free microscope for capturing interferometric light-field encodings at various wavelengths, and Bayesian-based fusion to reconstruct a fused object light-field from the encodings. By fusing unique object detail information captured at different wavelengths, LSLFM can achieve improved resolution, contrast, and signal-to-noise ratio (SNR) over a single-channel lens-free microscopy system. A five-channel LSLFM system was developed and quantitatively evaluated to validate the design. Experimental results demonstrated that the LSLFM system provided SNR improvements of 6-12 dB, as well as a six-fold improvement in the dispersion index (DI), over that achieved using a single-channel, resolution-enhancing lens-free deconvolution microscopy system or its multi-wavelength counterpart. Furthermore, the LSLFM system achieved an increase in numerical aperture (NA) of ∼16% over a single-channel resolution-enhancing lens-free deconvolution microscopy system at the highest resolution wavelength used in the study. Samples of Staurastrum paradoxum, a waterborne algae, and human corneal epithelial cells were imaged using the system to illustrate its potential for enhanced imaging of biological specimens. PMID:26274679

  8. In Depth Analyses of LEDs by a Combination of X-ray Computed Tomography (CT) and Light Microscopy (LM) Correlated with Scanning Electron Microscopy (SEM).

    PubMed

    Meyer, Jörg; Thomas, Christian; Tappe, Frank; Ogbazghi, Tekie

    2016-01-01

    In failure analysis, device characterization and reverse engineering of light emitting diodes (LEDs), and similar electronic components of micro-characterization, plays an important role. Commonly, different techniques like X-ray computed tomography (CT), light microscopy (LM) and scanning electron microscopy (SEM) are used separately. Similarly, the results have to be treated for each technique independently. Here a comprehensive study is shown which demonstrates the potentials leveraged by linking CT, LM and SEM. In depth characterization is performed on a white emitting LED, which can be operated throughout all characterization steps. Major advantages are: planned preparation of defined cross sections, correlation of optical properties to structural and compositional information, as well as reliable identification of different functional regions. This results from the breadth of information available from identical regions of interest (ROIs): polarization contrast, bright and dark-field LM images, as well as optical images of the LED cross section in operation. This is supplemented by SEM imaging techniques and micro-analysis using energy dispersive X-ray spectroscopy. PMID:27341190

  9. Artificial light at night advances avian reproductive physiology.

    PubMed

    Dominoni, Davide; Quetting, Michael; Partecke, Jesko

    2013-04-01

    Artificial light at night is a rapidly increasing phenomenon and it is presumed to have global implications. Light at night has been associated with health problems in humans as a consequence of altered biological rhythms. Effects on wild animals have been less investigated, but light at night has often been assumed to affect seasonal cycles of urban dwellers. Using light loggers attached to free-living European blackbirds (Turdus merula), we first measured light intensity at night which forest and city birds are subjected to in the wild. Then we used these measurements to test for the effect of light at night on timing of reproductive physiology. Captive city and forest blackbirds were exposed to either dark nights or very low light intensities at night (0.3 lux). Birds exposed to light at night developed their reproductive system up to one month earlier, and also moulted earlier, than birds kept under dark nights. Furthermore, city birds responded differently than forest individuals to the light at night treatment, suggesting that urbanization can alter the physiological phenotype of songbirds. Our results emphasize the impact of human-induced lighting on the ecology of millions of animals living in cities and call for an understanding of the fitness consequences of light pollution. PMID:23407836

  10. Artificial light at night advances avian reproductive physiology

    PubMed Central

    Dominoni, Davide; Quetting, Michael; Partecke, Jesko

    2013-01-01

    Artificial light at night is a rapidly increasing phenomenon and it is presumed to have global implications. Light at night has been associated with health problems in humans as a consequence of altered biological rhythms. Effects on wild animals have been less investigated, but light at night has often been assumed to affect seasonal cycles of urban dwellers. Using light loggers attached to free-living European blackbirds (Turdus merula), we first measured light intensity at night which forest and city birds are subjected to in the wild. Then we used these measurements to test for the effect of light at night on timing of reproductive physiology. Captive city and forest blackbirds were exposed to either dark nights or very low light intensities at night (0.3 lux). Birds exposed to light at night developed their reproductive system up to one month earlier, and also moulted earlier, than birds kept under dark nights. Furthermore, city birds responded differently than forest individuals to the light at night treatment, suggesting that urbanization can alter the physiological phenotype of songbirds. Our results emphasize the impact of human-induced lighting on the ecology of millions of animals living in cities and call for an understanding of the fitness consequences of light pollution. PMID:23407836

  11. Characterization of corrosion pit initiation in aluminum using advanced electron microscopy techniques

    NASA Astrophysics Data System (ADS)

    Elswick, Danielle S.

    The resistance to pitting corrosion in aluminum is due to the presence of a compact thin, approximately 5 nm, oxide. Certain conditions locally attack this protective oxide layer leading to its breakdown and resulting in the formation of corrosion pits. Numerous studies have investigated the growth and propagation stages of pitting corrosion yet the initiation stage remains not clearly defined nor well understood. The presence of aggressive chemical species, such as chloride, plays a critical role in the pitting phenomenon and is explored in this investigation. This dissertation focuses on the localization of pitting corrosion in high purity aluminum in order to accurately predict where and when the pit initiation process will occur so that microstructural changes associated with pit initiation can be easily identified and characterized using electron microscopy. A comprehensive investigation into the corrosion initiation process was attempted utilizing advanced characterization techniques in the transmission electron microscope (TEM) coupled with high-resolution microanalysis. Localization of pitting was successful through use of different sample geometries that reduced the length scale for which pitting events occurred. Three geometries were investigated, each with unique features for pitting corrosion. Electropolished Al needles localized pitting to a sharp tip due to a geometric field enhancement effect, while other experiments employed an Al wire micro-electrode geometry. Both geometries minimized the area where corrosion pits initiated and were electrochemically tested using a solution that contained the chloride species. A third geometry included electron beam evaporated Al films implanted with chloride, which induced pitting corrosion in an otherwise chloride-free environment. Localization of pitting was successfully achieved using novel sample geometries that isolated the desired stages of pitting corrosion, i.e. metastable pitting, through controlled

  12. Virtual-'Light-Sheet' Single-Molecule Localisation Microscopy Enables Quantitative Optical Sectioning for Super-Resolution Imaging

    PubMed Central

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T.; Herbert, Alex; Lando, David; Etheridge, Thomas J.; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M.; Klenerman, David; Lee, Steven F.

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated. PMID:25884495

  13. Virtual-'light-sheet' single-molecule localisation microscopy enables quantitative optical sectioning for super-resolution imaging.

    PubMed

    Palayret, Matthieu; Armes, Helen; Basu, Srinjan; Watson, Adam T; Herbert, Alex; Lando, David; Etheridge, Thomas J; Endesfelder, Ulrike; Heilemann, Mike; Laue, Ernest; Carr, Antony M; Klenerman, David; Lee, Steven F

    2015-01-01

    Single-molecule super-resolution microscopy allows imaging of fluorescently-tagged proteins in live cells with a precision well below that of the diffraction limit. Here, we demonstrate 3D sectioning with single-molecule super-resolution microscopy by making use of the fitting information that is usually discarded to reject fluorophores that emit from above or below a virtual-'light-sheet', a thin volume centred on the focal plane of the microscope. We describe an easy-to-use routine (implemented as an open-source ImageJ plug-in) to quickly analyse a calibration sample to define and use such a virtual light-sheet. In addition, the plug-in is easily usable on almost any existing 2D super-resolution instrumentation. This optical sectioning of super-resolution images is achieved by applying well-characterised width and amplitude thresholds to diffraction-limited spots that can be used to tune the thickness of the virtual light-sheet. This allows qualitative and quantitative imaging improvements: by rejecting out-of-focus fluorophores, the super-resolution image gains contrast and local features may be revealed; by retaining only fluorophores close to the focal plane, virtual-'light-sheet' single-molecule localisation microscopy improves the probability that all emitting fluorophores will be detected, fitted and quantitatively evaluated. PMID:25884495

  14. Mirage effect spectrometry and light profile microscopy: Two views of an optical depth profile (abstract)

    NASA Astrophysics Data System (ADS)

    Power, J. F.; Fu, S. W.; Nepotchatykh, O. V.

    2003-01-01

    Photothermal depth profiling techniques are well adapted for the inspection of optically absorbing features on the length scale of 1-100 μm in a variety of media. However, the depth profiling mechanism intrinsic to thermal wave imaging is inherently ill posed [J. F. Power, AIP Conf. Proc. 463, 3 (1999)], and suffers obvious disadvantages such as sensitivity to experimental errors (requiring regularization) and subsurface broadening of the regularized depth profiles. Recently, through the introduction of light profile microscopy (LPM) an alternate method of optical inspection was made available for depth profiling optically absorbing, scattering, and luminescent structures on this length scale [J. F. Power and S. W. Fu, Appl. Spectros. 53, 1507 (1999); J. F. Power and S. W. Fu, U.S. Patent Pending]. LPM inspects a thin film under test by directing a laser beam through the material along the depth axis, parallel to a polished cross-sectional viewing surface. Luminescence and elastic scatter excited in the beam volume is imaged by a microscope aligned orthogonal to the beam axis. The images obtained by this method showed striking depth contrast in a variety of materials with subsurface interfaces and depth variations of luminescence yield. When implemented in dual beam mode [J. F. Power and S. W. Fu, U.S. Patent Pending; J. F. Power and S. W. Fu, (unpublished)] with an associated mathematical method, LPM may be used to quantitatively resolve depth variable optical absorption from light scattering and luminescence efficiency. In contrast to photothermal methods, the LPM technique is well posed. LPM was evaluated in tandem with mirage effect spectrometry (in normal deflection mode with bicell detection) [J. F. Power, S. W. Fu, and M. A. Schweitzer, Appl. Spectros. 54, 110 (2000)], to determine the effective use of each technique in analysis problems on complex materials. This study used samples with known depth variations of optical properties including homogeneous

  15. Electrical and thermal injuries in pig skin--evaluated and compared by light microscopy.

    PubMed

    Danielsen, L; Thomsen, H K; Nielsen, O; Aalund, O; Nielsen, K G; Karlsmark, T; Genefke, I K

    1978-01-01

    The morphology of pig skin after electrical (el) and thermal exposure was studied in order to find methods applicable for disclosing electrical torture. Biopsies from pig skin exposed 24 hours earlier to either heat or electrical current under general anesthesia were studied by light microscopy. The amount of energy used of either type ranged from about 6 to about 100 joule deposited on two circular areas of the skin measuring 12 mm in diameter. In sections stained with hematoxylin and eosin alterations were found in heat-influenced specimens following the administration of moderate and large doses of energy. Changes were observed in el-influenced specimens also after the administration of low amounts of energy, which did not lead to macroscopical changes in the skin. The two types of energy induced different patterns of alterations inside the epidermis. The most characteristic change following heat exposure was the presence of a granular or fibrillar eosinophilic cytoplasm and subepidermal clefts, while el-expoure often produced white homogeneous cytoplasm and shadowy nuclei ("white necrosis") in the attached epidermis. The individual changes could be produced by both types of injury, but to a highly different degree. However, the alteration classified as "vesicular nuclei" was only observed observed in el-damaged skin. The dermis from the more severely injured skin of both groups showed a homogeneous appearance ("necrosis"). While the changes of epidermis and dermis in response to heat were diffusely distributed, the changes created by electrical injury were often present in segments. By electrical damage homogeneous areas with loss of stainability and shadowy nuclei were occasionally observed in sweat glands, hair sheaths and in vessel walls, structures which are suggested to serve as conductors for the electrical current. "Vesicular nuclei" were observed in similar structures. Some of the findings may be associated with differences in intensity of energy during

  16. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Oehring, Hartmut; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl-Otto

    1998-01-01

    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular

  17. Comet assay, cloning assay, and light and electron microscopy on one preselected cell

    NASA Astrophysics Data System (ADS)

    Koenig, Karsten; Oehring, H.; Halbhuber, Karl-Juergen; Fiedler, Ursula; Bauer, Eckhard; Greulich, Karl O.

    1997-12-01

    In order to perform long-term studies up to one week on a preselected single cell after micromanipulation (e.g. UVA and NIR microbeam exposure) in comparison with non-treated neighbor cells (control cells) we applied a variety of single cell diagnostic techniques and developed a special comet assay for single preselected cells. For that purpose adherent cells were grown in low concentrations and maintained in special sterile centimeter-sized glass cell chambers. After preselection, a single cell was marked by means of diamond-produced circles on the outer cell chamber window. During exposure to microbeams, NADH-attributed autofluorescence of the chosen cell was detected by fluorescence imaging and spectroscopy. In addition, cell morphology was video-monitored (formation of pseudopodia, membrane blebbing,...). Maintaining the microchamber in the incubator, the irradiated cell was examined 24 h later for cell division (clone formation) and modifications in autofluorescence and morphology (including daughter cells). In the case that no division occurred the vitality of the light-exposed cell and of the control cells were probed by intranuclear propidium iodide accumulation. After fixation, either electron microscopy or single cell gel electrophoresis (comet assay) was performed. To monitor comet formation indicating photoinduced DNA damage in the preselected single cell in comparison with the non-exposed neighbor cells the chamber was filled with low-melting gel and lysis solution and exposed to an electric field. In contrast to the conventional comet assay, where only randomly chosen cells of a suspension are investigated, the novel optimized electrophoresis technique should enhance the possibilities of DNA damage detection to a true single (preselected) cell level. The single cell techniques applied to UVA microexposed Chinese hamster ovary cells (364 nm, 1 mW, 3.5 W/cm2) revealed significant cell damage for J/cm2 fluences such as modifications of intracellular

  18. Observation platform for colloid science experiments using the ISS Light Microscopy Module

    NASA Astrophysics Data System (ADS)

    Kurk, Michael Andy; Todd, Paul; Vellinger, John C.

    2012-07-01

    The role of gravity in colloid self-assembly is a long-standing subject of inquiry. The International Space Station Light Microscopy Module (LMM) is a potentially powerful tool for implementing the observation of colloids in a high-quality low-gravity environment. The main requirements for making observations of colloid self-assembly include a small-volume, thermally stabilized environment, the addition and removal of small volumes of fluids (colloidal suspensions or reagents), and on-demand access to electrokinetic and/or magnetophoretic body forces. A modular device has been designed in which a custom electronics module is designed to mate with the existing LMM cold plate and LMM controlling power. All control features, electrical power, microscope illuminator, fluid pumps and valves are components of this module. This module lies under, mates with and serves an experiment module which houses the fluid containers that fit under the LMM objective lenses and fluid transfer tubing. Four versions of the experiment module have been designed: a hollow-slide stopped flow cell, a multiwell quiescent module, a magnetization module and an electrokinetic module. Interfaces can be established in the viewing field using the stopped-flow cell, which is also applicable to living systems such as microbial cultures, suspended blood cells, nematodes, etc. Multi-well modules can be equipped with in-line static mixers that allow the investigator to combine pairs of fluids or to re-homogenize settled samples. The maximum dimension of all modules is 16 cm, so the modules can be transported in large numbers on cargo or manned spacecraft to the ISS. A doubly-contained transfer tool can be used to transfer fluids in and out of the experiment module, which is equipped with a fluid coupling that mates to the transfer tool. Experiment-specific versions of these modules can be prepared for approved experimenters within a 1-year period. The research for these devices is supported by NASA

  19. High-resolution corneal topography and tomography of fish eye using wide-field white light interference microscopy

    NASA Astrophysics Data System (ADS)

    Srivastava, Vishal; Nandy, Sreyankar; Singh Mehta, Dalip

    2013-04-01

    Topography and tomography of fish cornea is reconstructed using high resolution white light interference microscopy. White light interferograms at different depths were recorded by moving the object axially. For each depth position, five phase shifted interferograms were recorded and analyzed. From the reconstructed phase maps, the corneal topography and hence the refractive index was determined and from amplitude images the cross-sectional image of fish cornea was reconstructed. In the present method, we utilize a nearly common-path interference microscope and wide field illumination and hence do not require any mechanical B-scan. Therefore, the phase stability of the recorded data is improved.

  20. NASA Laser Light Scattering Advanced Technology Development Workshop, 1988

    NASA Technical Reports Server (NTRS)

    Meyer, William V. (Editor)

    1989-01-01

    The major objective of the workshop was to explore the capabilities of existing and prospective laser light scattering hardware and to assess user requirements and needs for a laser light scattering instrument in a reduced gravity environment. The workshop addressed experimental needs and stressed hardware development.

  1. Analysis of acute brain slices by electron microscopy: a correlative light-electron microscopy workflow based on Tokuyasu cryo-sectioning.

    PubMed

    Loussert Fonta, Celine; Leis, Andrew; Mathisen, Cliff; Bouvier, David S; Blanchard, Willy; Volterra, Andrea; Lich, Ben; Humbel, Bruno M

    2015-01-01

    Acute brain slices are slices of brain tissue that are kept vital in vitro for further recordings and analyses. This tool is of major importance in neurobiology and allows the study of brain cells such as microglia, astrocytes, neurons and their inter/intracellular communications via ion channels or transporters. In combination with light/fluorescence microscopies, acute brain slices enable the ex vivo analysis of specific cells or groups of cells inside the slice, e.g. astrocytes. To bridge ex vivo knowledge of a cell with its ultrastructure, we developed a correlative microscopy approach for acute brain slices. The workflow begins with sampling of the tissue and precise trimming of a region of interest, which contains GFP-tagged astrocytes that can be visualised by fluorescence microscopy of ultrathin sections. The astrocytes and their surroundings are then analysed by high resolution scanning transmission electron microscopy (STEM). An important aspect of this workflow is the modification of a commercial cryo-ultramicrotome to observe the fluorescent GFP signal during the trimming process. It ensured that sections contained at least one GFP astrocyte. After cryo-sectioning, a map of the GFP-expressing astrocytes is established and transferred to correlation software installed on a focused ion beam scanning electron microscope equipped with a STEM detector. Next, the areas displaying fluorescence are selected for high resolution STEM imaging. An overview area (e.g. a whole mesh of the grid) is imaged with an automated tiling and stitching process. In the final stitched image, the local organisation of the brain tissue can be surveyed or areas of interest can be magnified to observe fine details, e.g. vesicles or gold labels on specific proteins. The robustness of this workflow is contingent on the quality of sample preparation, based on Tokuyasu's protocol. This method results in a reasonable compromise between preservation of morphology and maintenance of

  2. Imaging Structure and Composition Homogeneity of 300 mm SiGe Virtual Substrates for Advanced CMOS Applications by Scanning X-ray Diffraction Microscopy.

    PubMed

    Zoellner, Marvin H; Richard, Marie-Ingrid; Chahine, Gilbert A; Zaumseil, Peter; Reich, Christian; Capellini, Giovanni; Montalenti, Francesco; Marzegalli, Anna; Xie, Ya-Hong; Schülli, Tobias U; Häberlen, Maik; Storck, Peter; Schroeder, Thomas

    2015-05-01

    Advanced semiconductor heterostructures are at the very heart of many modern technologies, including aggressively scaled complementary metal oxide semiconductor transistors for high performance computing and laser diodes for low power solid state lighting applications. The control of structural and compositional homogeneity of these semiconductor heterostructures is the key to success to further develop these state-of-the-art technologies. In this article, we report on the lateral distribution of tilt, composition, and strain across step-graded SiGe strain relaxed buffer layers on 300 mm Si(001) wafers treated with and without chemical-mechanical polishing. By using the advanced synchrotron based scanning X-ray diffraction microscopy technique K-Map together with micro-Raman spectroscopy and Atomic Force Microscopy, we are able to establish a partial correlation between real space morphology and structural properties of the sample resolved at the micrometer scale. In particular, we demonstrate that the lattice plane bending of the commonly observed cross-hatch pattern is caused by dislocations. Our results show a strong local correlation between the strain field and composition distribution, indicating that the adatom surface diffusion during growth is driven by strain field fluctuations induced by the underlying dislocation network. Finally, it is revealed that a superficial chemical-mechanical polishing of cross-hatched surfaces does not lead to any significant change of tilt, composition, and strain variation compared to that of as-grown samples. PMID:25871429

  3. 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. PMID:25786567

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

    PubMed Central

    HÖHN, K.; FUCHS, J.; FRÖBER, A.; KIRMSE, R.; GLASS, B.; ANDERS‐ÖSSWEIN, M.; WALTHER, P.; KRÄUSSLICH, H.‐G.

    2015-01-01

    Summary 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. PMID:25786567

  5. Ti foil light in the ATA (Advanced Test Accelerator) beam

    SciTech Connect

    Slaughter, D.R.; Chong, Y.P.; Goosman, D.R.; Rule, D.W.; Fiorito, R.B.

    1987-09-01

    An experiment is in progress to characterize the visible light produced when a Ti foil is immersed in the ATA 2 kA, 43 MeV beam. Results obtained to date indicate that the optical condition of the foil surface is a critical determinant of these characteristics, with a very narrow angular distribution obtained when a highly polished and flat foil is used. These data are consistent with the present hypothesis that the light is produced by transition radiation. Incomplete experiments to determine the foil angle dependence of the detected light and its polarization are summarized and remaining experiments are described.

  6. Science at the Speed of Light: Advanced Photon Source

    ScienceCinema

    Murray Gibson

    2010-01-08

    An introduction and overview of the Advanced Photon Source at Argonne National Laboratory, the technology that produces the brightest x-ray beams in the Western Hemisphere, and the research carried out by scientists using those x-rays.

  7. Science at the Speed of Light: Advanced Photon Source

    SciTech Connect

    Murray Gibson

    2009-06-03

    An introduction and overview of the Advanced Photon Source at Argonne National Laboratory, the technology that produces the brightest x-ray beams in the Western Hemisphere, and the research carried out by scientists using those x-rays.

  8. Scanning Electron Microscopy of Pine Seedling Wood Tissue Sections Inoculated with the Pinewood Nematode Bursaphelenchus xylophilus Previously Prepared for Light Microscopy.

    PubMed

    Mamiya, Yasuharu

    2012-09-01

    Scanning electron microscopy (SEM) was applied to paraffin-embedded wood sections to study the histopathology of pine seedlings inoculated with the pinewood nematode (PWN), Bursaphelenchus xylophilus. The sections, which had been previously prepared and observed by light microscopy (LM) on glass slides, were originally obtained from experiments in which pine seedlings had been inoculated with PWN. The cover glass was removed by soaking the glass slide in xylene for 3 to 5 days. The glass slides were cut into small pieces so that each piece contained one wood section. Each piece of the glass slide was attached with double adhesive tape to an aluminum stub. The specimens were sputter-coated with gold and examined with a scanning electron microscope (JEOL-JSM 5200). Compared to LM (as documented in previous reports) SEM provided greater depth of focus and resolution of the damaged wood tissues, nematodes and associated bacteria. SEM made it possible to observe the relationship between bacterial distribution and nematode distribution in wood tissues. SEM observations also suggested the possibility of documenting the death of ray cells and other parenchyma cells in relation to disease development. Finally, the current study of PWN in pine seedlings demonstrated that glass slides prepared for LM observations more than 25 years earlier could be successfully processed for examination by SEM. PMID:23481606

  9. Advances in combined endoscopic fluorescence confocal microscopy and optical coherence tomography

    NASA Astrophysics Data System (ADS)

    Risi, Matthew D.

    Confocal microendoscopy provides real-time high resolution cellular level images via a minimally invasive procedure. Results from an ongoing clinical study to detect ovarian cancer with a novel confocal fluorescent microendoscope are presented. As an imaging modality, confocal fluorescence microendoscopy typically requires exogenous fluorophores, has a relatively limited penetration depth (100 μm), and often employs specialized aperture configurations to achieve real-time imaging in vivo. Two primary research directions designed to overcome these limitations and improve diagnostic capability are presented. Ideal confocal imaging performance is obtained with a scanning point illumination and confocal aperture, but this approach is often unsuitable for real-time, in vivo biomedical imaging. By scanning a slit aperture in one direction, image acquisition speeds are greatly increased, but at the cost of a reduction in image quality. The design, implementation, and experimental verification of a custom multi-point-scanning modification to a slit-scanning multi-spectral confocal microendoscope is presented. This new design improves the axial resolution while maintaining real-time imaging rates. In addition, the multi-point aperture geometry greatly reduces the effects of tissue scatter on imaging performance. Optical coherence tomography (OCT) has seen wide acceptance and FDA approval as a technique for ophthalmic retinal imaging, and has been adapted for endoscopic use. As a minimally invasive imaging technique, it provides morphological characteristics of tissues at a cellular level without requiring the use of exogenous fluorophores. OCT is capable of imaging deeper into biological tissue (˜1-2 mm) than confocal fluorescence microscopy. A theoretical analysis of the use of a fiber-bundle in spectral-domain OCT systems is presented. The fiber-bundle enables a flexible endoscopic design and provides fast, parallelized acquisition of the optical coherence tomography

  10. Advancing Human Circadian Rhythms with Afternoon Melatonin and Morning Intermittent Bright Light

    PubMed Central

    Revell, Victoria L.; Burgess, Helen J.; Gazda, Clifford J.; Smith, Mark R.; Fogg, Louis F.; Eastman, Charmane I.

    2013-01-01

    Context Both light and melatonin can be used to phase shift the human circadian clock, but the phase advancing effect of the combination has not been extensively investigated. Objective The objective of the study was to determine whether phase advances induced by morning intermittent bright light and a gradually advancing sleep schedule could be increased with afternoon melatonin. Participants Healthy adults (25 males, 19 females, between the ages of 19 and 45 yr) participated in the study. Design There were 3 d of a gradually advancing sleep/dark period (wake time 1 h earlier each morning), bright light on awakening [ four 30-min bright-light pulses (∼5000 lux) alternating with 30 min room light < 60 lux] and afternoon melatonin, either 0.5 or 3.0 mg melatonin timed to induce maximal phase advances, or matching placebo. The dim light melatonin onset was measured before and after the treatment to determine the phase advance. Results There were significantly larger phase advances with 0.5 mg (2.5 h, n = 16) and 3.0 mg melatonin (2.6 h, n = 13), compared with placebo (1.7 h, n = 15), but there was no difference between the two melatonin doses. Subjects did not experience jet lag-type symptoms during the 3-d treatment Conclusions Afternoon melatonin, morning intermittent bright light, and a gradually advancing sleep schedule advanced circadian rhythms almost 1 h/d and thus produced very little circadian misalignment. This treatment could be used in any situation in which people need to phase advance their circadian clock, such as before eastward jet travel or for delayed sleep phase syndrome. PMID:16263827

  11. Evaluation of advanced light scattering technology for microgravity experiments

    NASA Technical Reports Server (NTRS)

    Fredericks, W. J.; Rosenblum, W. M.

    1990-01-01

    The capabilities of modern light scattering equipment and the uses it might have in studying processes in microgravity are evaluated. Emphasis is on the resolution of polydisperse systems. This choice was made since a major use of light scattering was expected to be the study of crystal growth of macromolecules in low gravity environments. An evaluation of a modern photon correlation spectrometer and a Mie spectrometer is presented.

  12. Direct visualization method of the atomic structure of light and heavy atoms with double-detector C{sub s}-corrected scanning transmission electron microscopy

    SciTech Connect

    Kotaka, Yasutoshi

    2012-09-24

    The advent of C{sub s}-corrected scanning transmission electron microscopy (STEM) has advanced the observation of atomic structures in materials and nanotechnology devices. High-angle annular dark-field (HAADF)-STEM using an annular detector visualizes heavy elements as bright spots at atomic resolution that can be observed with the Z-contrast technique. In this study, the atomic column of light elements is directly observed as bright spots by middle-angle bright-field (MABF)-STEM imaging. Therefore, a double-detector STEM imaging method was developed, exploiting the advantage of both MABF-STEM and HAADF-STEM to maximum, which consists of multiple exposures of simultaneously observed MABF- and HAADF-STEM images in red-green-blue color.

  13. 77 FR 62270 - Proposed Revision Treatment of Non-Safety Systems for Passive Advanced Light Water Reactors

    Federal Register 2010, 2011, 2012, 2013, 2014

    2012-10-12

    ... COMMISSION Proposed Revision Treatment of Non-Safety Systems for Passive Advanced Light Water Reactors AGENCY... Treatment of Non-Safety Systems (RTNSS) for Passive Advanced Light Water Reactors.'' The current SRP does not contain guidance on the proposed RTNSS for Passive Advance Light Water Reactors. DATES:...

  14. Preflight Adjustment to Eastward Travel: 3 Days of Advancing Sleep with and without Morning Bright Light

    PubMed Central

    Burgess, Helen J.; Crowley, Stephanie J.; Gazda, Clifford J.; Fogg, Louis F.; Eastman, Charmane I.

    2005-01-01

    Jet lag is caused by a misalignment between circadian rhythms and local destination time. As humans typically take longer to re-entrain after a phase advance than a phase delay, eastward travel is often more difficult than westward travel. Previous strategies to reduce jet lag have focused on shaping the perceived light-dark cycle after arrival, in order to facilitate a phase shift in the appropriate direction. Here we tested treatments that travelers could use to phase advance their circadian rhythms prior to eastward flight. Thus, travelers would arrive with their circadian rhythms already partially re-entrained to local time. We determined how far the circadian rhythms phase advanced, and the associated side effects related to sleep and mood. Twenty-eight healthy young subjects participated in 1 of 3 different treatments, which all phase advanced each subject’s habitual sleep schedule by 1 h/day for 3 days. The 3 treatments differed in morning light exposure for the 1st 3.5 h after waking on each of the 3 days: continuous bright light (> 3000 lux), intermittent bright light (> 3000 lux, 0.5 h on, 0.5 off, etc.), or ordinary dim indoor light (< 60 lux). Aphase assessment in dim light (< 10 lux) was conducted before and after the treatments to determine the endogenous salivary dim light melatonin onset (DLMO). The mean DLMO phase advances in the dim, intermittent, and continuous light groups were 0.6, 1.5, and 2.1 h, respectively. The intermittent and continuous light groups advanced significantly more than the dim light group (p < 0.01) but were not significantly different from each other. The side effects as assessed with actigraphy and logs were small. A 2-h phase advance may seem small compared to a 6- to 9-h time zone change, as occurs with eastward travel from the USA to Europe. However, a small phase advance will not only reduce the degree of re-entrainment required after arrival, but may also increase postflight exposure to phase-advancing light relative

  15. Optimal experimental design for the detection of light atoms from high-resolution scanning transmission electron microscopy images

    SciTech Connect

    Gonnissen, J.; De Backer, A.; Martinez, G. T.; Van Aert, S.; Dekker, A. J. den; Rosenauer, A.; Sijbers, J.

    2014-08-11

    We report an innovative method to explore the optimal experimental settings to detect light atoms from scanning transmission electron microscopy (STEM) images. Since light elements play a key role in many technologically important materials, such as lithium-battery devices or hydrogen storage applications, much effort has been made to optimize the STEM technique in order to detect light elements. Therefore, classical performance criteria, such as contrast or signal-to-noise ratio, are often discussed hereby aiming at improvements of the direct visual interpretability. However, when images are interpreted quantitatively, one needs an alternative criterion, which we derive based on statistical detection theory. Using realistic simulations of technologically important materials, we demonstrate the benefits of the proposed method and compare the results with existing approaches.

  16. Analysis of micro-lens integrated flip-chip InGaN light-emitting diodes by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Li, K. H.; Feng, C.; Choi, H. W.

    2014-02-01

    A hexagonally close-packed microlens array has been integrated onto the sapphire face of a flip-chip bonded InGaN light-emitting diode (LED). The micro-optics is formed by etching a self-assembled monolayer of 1-μm silica microspheres coated on the sapphire substrate, producing hemispherical sapphire lenses. Without degrading electrical characteristic, the light output power of the lensed LED is increased by more than a quarter compared with the unlensed LED. Enhanced light extraction via micro-optics is verified by rigorous coupled wave analysis. The focusing behavior of the micro-lenses, as well as the emission characteristics of the lensed LED, is studied by confocal microscopy.

  17. Advances in Phosphors for Light-emitting Diodes.

    PubMed

    Lin, Chun Che; Liu, Ru-Shi

    2011-06-01

    Light-emitting diodes (LEDs) are excellent candidates for general lighting because of their rapidly improving efficiency, durability, and reliability, their usability in products of various sizes, and their environmentally friendly constituents. Effective lighting devices can be realized by combining one or more phosphor materials with chips. Accordingly, it is very important that the architecture of phosphors be developed. Although numerous phosphors have been proposed in the past several years, the range of phosphors that are suitable for LEDs is limited. This work describes recent progress in our understanding of the prescription, morphology, structure, spectrum, and packaging of such phosphors. It suggests avenues for further development and the scientific challenges that must be overcome before phosphors can be practically applied in LEDs. PMID:26295420

  18. Advances and prospects in nitrides based light-emitting-diodes

    NASA Astrophysics Data System (ADS)

    Jinmin, Li; Zhe, Liu; Zhiqiang, Liu; Jianchang, Yan; Tongbo, Wei; Xiaoyan, Yi; Junxi, Wang

    2016-06-01

    Due to their low power consumption, long lifetime and high efficiency, nitrides based white light-emitting-diodes (LEDs) have long been considered to be a promising technology for next generation illumination. In this work, we provide a brief review of the development of GaN based LEDs. Some pioneering and significant experiment results of our group and the overview of the recent progress in this field are presented. We hope it can provide some meaningful information for the development of high efficiency GaN based LEDs and solid-state-lighting. Project supported by the National High Technology Research and Development Program of China (No. 2013AA03A101).

  19. Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy

    PubMed Central

    Zhao, Ziqing W.; Roy, Rahul; Gebhardt, J. Christof M.; Suter, David M.; Chapman, Alec R.; Xie, X. Sunney

    2014-01-01

    Superresolution microscopy based on single-molecule centroid determination has been widely applied to cellular imaging in recent years. However, quantitative imaging of the mammalian nucleus has been challenging due to the lack of 3D optical sectioning methods for normal-sized cells, as well as the inability to accurately count the absolute copy numbers of biomolecules in highly dense structures. Here we report a reflected light-sheet superresolution microscopy method capable of imaging inside the mammalian nucleus with superior signal-to-background ratio as well as molecular counting with single-copy accuracy. Using reflected light-sheet superresolution microscopy, we probed the spatial organization of transcription by RNA polymerase II (RNAP II) molecules and quantified their global extent of clustering inside the mammalian nucleus. Spatiotemporal clustering analysis that leverages on the blinking photophysics of specific organic dyes showed that the majority (>70%) of the transcription foci originate from single RNAP II molecules, and no significant clustering between RNAP II molecules was detected within the length scale of the reported diameter of “transcription factories.” Colocalization measurements of RNAP II molecules equally labeled by two spectrally distinct dyes confirmed the primarily unclustered distribution, arguing against a prevalent existence of transcription factories in the mammalian nucleus as previously proposed. The methods developed in our study pave the way for quantitative mapping and stoichiometric characterization of key biomolecular species deep inside mammalian cells. PMID:24379392

  20. Light Microscopy Module: An On-Orbit Microscope Planned for the Fluids and Combustion Facility on the International Space Station

    NASA Technical Reports Server (NTRS)

    Doherty, Michael P.; Motil, Susan M.; Snead, John H.; Griffin, DeVon W.

    2001-01-01

    The Light Microscopy Module (LMM) is planned as a fully remotely controllable on-orbit microscope subrack facility, allowing flexible scheduling and control of fluids and biology experiments within NASA Glenn Research Center's Fluids and Combustion Facility on the International Space Station. Within the Fluids and Combustion Facility, four fluids physics experiments will utilize an instrument built around a light microscope. These experiments are the Constrained Vapor Bubble experiment (Peter C. Wayner of Rensselaer Polytechnic Institute), the Physics of Hard Spheres Experiment-2 (Paul M. Chaikin of Princeton University), the Physics of Colloids in Space-2 experiment (David A. Weitz of Harvard University), and the Low Volume Fraction Colloidal Assembly experiment (Arjun G. Yodh of the University of Pennsylvania). The first experiment investigates heat conductance in microgravity as a function of liquid volume and heat flow rate to determine, in detail, the transport process characteristics in a curved liquid film. The other three experiments investigate various complementary aspects of the nucleation, growth, structure, and properties of colloidal crystals in microgravity and the effects of micromanipulation upon their properties. Key diagnostic capabilities for meeting the science requirements of the four experiments include video microscopy to observe sample features including basic structures and dynamics, interferometry to measure vapor bubble thin film thickness, laser tweezers for colloidal particle manipulation and patterning, confocal microscopy to provide enhanced three-dimensional visualization of colloidal structures, and spectrophotometry to measure colloidal crystal photonic properties.

  1. Comparison of Partec Rapid Malaria Test with Conventional Light Microscopy for Diagnosis of Malaria in Northwest Ethiopia

    PubMed Central

    Birhanie, Meseret

    2016-01-01

    Background. Laboratory diagnosis of malaria is the key for effective disease management. Diagnosis of malaria infection requires rapid, sensitive, and specific test methods with an affordable cost. This study was aimed to assess the diagnostic performance of Partec rapid malaria test with reference to light microscopy for the diagnosis of malaria in Northwest Ethiopia. Methods. A total of 180 febrile patients were tested for malaria using Giemsa stain microscopy and Partec rapid malaria test from June to July 2013 at Gendewuha health centers, Metema district. Data were analyzed using SPSS version 20 statistical software. Odds ratio with 95% CI was calculated. Result. The sensitivity and specificity of Partec rapid malaria test were 93.8% (95% CI = 87.1%–100%) and 87.9% (95% CI = 79.7%–96.1%), respectively, while the positive predictive value and negative predictive value were 6.4% (95% CI = 77.2%–95.5%) and 94.6% (95% CI = 88.7%–100%), respectively. There was also an excellent agreement between two tests with Kappa value of 0.811 (95% CI = 0.625–0.996). Conclusion. Partec rapid malaria test showed good sensitivity and specificity with an excellent agreement to the reference light microscopy. Therefore PT can be considered as alternative diagnostic tools in malaria endemic areas. PMID:26881056

  2. Laser Light Scattering, from an Advanced Technology Development Program to Experiments in a Reduced Gravity Environment

    NASA Technical Reports Server (NTRS)

    Meyer, William V.; Tscharnuter, Walther W.; Macgregor, Andrew D.; Dautet, Henri; Deschamps, Pierre; Boucher, Francois; Zuh, Jixiang; Tin, Padetha; Rogers, Richard B.; Ansari, Rafat R.

    1994-01-01

    Recent advancements in laser light scattering hardware are described. These include intelligent single card correlators; active quench/active reset avalanche photodiodes; laser diodes; and fiber optics which were used by or developed for a NASA advanced technology development program. A space shuttle experiment which will employ aspects of these hardware developments is previewed.

  3. Light Scattering by Polymers: Two Experiments for Advanced Undergraduates.

    ERIC Educational Resources Information Center

    Matthews, G. P.

    1984-01-01

    Background information, procedures, equipment, and results for two experiments are presented. The first involves the measurement of the mass-average and degree of coiling of polystyrene and is interpreted by the full mathematical theory of light scattering. The second is the study of transitions in gelatin. (JN)

  4. Development of Advanced Light-Duty Powertrain and Hybrid Analysis Tool (SAE 2013-01-0808)

    EPA Science Inventory

    The Advanced Light-Duty Powertrain and Hybrid Analysis tool was created by Environmental Protection Agency to evaluate the Greenhouse gas emissions and fuel efficiency from light-duty vehicles. It is a physics-based, forward-looking, full vehicle computer simulator, which is cap...

  5. X-ray micro-Tomography at the Advanced Light Source

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The X-ray micro-Tomography Facility at the Advanced Light Source has been in operation since 2004. The source is a superconducting bend magnet of critical energy 10.5KeV; photon energy coverage is 8-45 KeV in monochromatic mode, and a filtered white light option yields useful photons up to 50 KeV. A...

  6. Progress and new advances in simulating electron microscopy datasets using MULTEM.

    PubMed

    Lobato, I; Van Aert, S; Verbeeck, J

    2016-09-01

    A new version of the open source program MULTEM is presented here. It includes a graphical user interface, tapering truncation of the atomic potential, CPU multithreading functionality, single/double precision calculations, scanning transmission electron microscopy (STEM) simulations using experimental detector sensitivities, imaging STEM (ISTEM) simulations, energy filtered transmission electron microscopy (EFTEM) simulations, STEM electron energy loss spectroscopy (EELS) simulations along with other improvements in the algorithms. We also present a mixed channeling approach for the calculation of inelastic excitations, which allows one to considerably speed up time consuming EFTEM/STEM-EELS calculations. PMID:27323350

  7. The microstructure of lingual papillae in the Egyptian fruit bat (Rousettus aegyptiacus) as observed by light microscopy and scanning electron microscopy.

    PubMed

    Jackowiak, Hanna; Trzcielińska-Lorych, Joanna; Godynicki, Szymon

    2009-03-01

    The microstructure of lingual papillae on the dorsal surface of the tongue of adult Egyptian fruit bats was examined by light microscopy (LM) and scanning electron microscopy (SEM). This elongated tongue with a rounded apex is approximately 3 cm long -- including the 1.7cm length of the anterior free part of the tongue -- which facilitates considerable freedom of movement. The surface of the tongue has four types of lingual papillae: two types of mechanical papillae -- filiform and conical papillae, and two types of gustatory papillae -- fungiform and vallate papillae. Most numerous are filiform papillae with well developed keratinized processes represented by four morphological subtypes -- small, giant, elongated, and bifid papillae. Our observations showed the small and giant filiform papillae to be present in the anterior part of the tongue and tilted to the back of the tongue. In the posterior part of the tongue, the filiform papillae with elongated processes were arranged on each side of the tongue and oriented perpendicularly to the median line of tongue. This arrangement of filiform papillae is considered to be useful for the efficient uptake of semiliquid food as it can be collected toward the median line of the tongue. Gustatory fungiform papillae were distributed among filiform papillae on the border of the apex and the anterior part of the body of the tongue and also on the posterior part of the tongue, while three vallate papillae surrounded by conical papillae were found on the root of the tongue. There were also taste buds along the ducts of the posterior lingual glands in the posterior-lateral part of the tongue. These morphological features are discussed in relation to adaptation to food uptake in the Egyptian fruit bat. PMID:19789409

  8. Optical coherence photoacoustic microscopy (OC-PAM) with an intensity-modulated continuous-wave broadband light source

    NASA Astrophysics Data System (ADS)

    Liu, Xiaojing; Wen, Rong; Li, Yiwen; Jiao, Shuliang

    2016-06-01

    We developed an optical coherence photoacoustic microscopy system using an intensity-modulated continuous-wave superluminescent diode with a center wavelength of 840 nm. The system can accomplish optical coherence tomography (OCT) and photoacoustic microscopy (PAM) simultaneously. Compared to the system with a pulsed light source, this system is able to achieve OCT imaging with quality as high as conventional spectral-domain OCT. Since both of the OCT and PAM images are generated from the same group of photons, they are intrinsically registered in the lateral directions. The system was tested for multimodal imaging the vasculature of mouse ear in vivo by using gold nanorods as contrast agent for PAM, as well as excised porcine eyes ex vivo. The OCT and PAM images showed complimentary information of the sample.

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

    NASA Astrophysics Data System (ADS)

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

    2010-09-01

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

  10. Lorentz microscopy sheds light on the role of dipolar interactions in magnetic hyperthermia

    NASA Astrophysics Data System (ADS)

    Campanini, M.; Ciprian, R.; Bedogni, E.; Mega, A.; Chiesi, V.; Casoli, F.; de Julián Fernández, C.; Rotunno, E.; Rossi, F.; Secchi, A.; Bigi, F.; Salviati, G.; Magén, C.; Grillo, V.; Albertini, F.

    2015-04-01

    Monodispersed Fe3O4 nanoparticles with comparable size distributions have been synthesized by two different synthesis routes, co-precipitation and thermal decomposition. Thanks to the different steric stabilizations, the described samples can be considered as a model system to investigate the effects of magnetic dipolar interactions on the aggregation states of the nanoparticles. Moreover, the presence of magnetic dipolar interactions can strongly affect the nanoparticle efficiency as a hyperthermic mediator. In this paper, we present a novel way to visualize and map the magnetic dipolar interactions in different kinds of nanoparticle aggregates by the use of Lorentz microscopy, an easy and reliable in-line electron holographic technique. By exploiting Lorentz microscopy, which is complementary to the magnetic measurements, it is possible to correlate the interaction degrees of magnetic nanoparticles with their magnetic behaviors. In particular, we demonstrate that Lorentz microscopy is successful in visualizing the magnetic configurations stabilized by dipolar interactions, thus paving the way to the comprehension of the power loss mechanisms for different nanoparticle aggregates.Monodispersed Fe3O4 nanoparticles with comparable size distributions have been synthesized by two different synthesis routes, co-precipitation and thermal decomposition. Thanks to the different steric stabilizations, the described samples can be considered as a model system to investigate the effects of magnetic dipolar interactions on the aggregation states of the nanoparticles. Moreover, the presence of magnetic dipolar interactions can strongly affect the nanoparticle efficiency as a hyperthermic mediator. In this paper, we present a novel way to visualize and map the magnetic dipolar interactions in different kinds of nanoparticle aggregates by the use of Lorentz microscopy, an easy and reliable in-line electron holographic technique. By exploiting Lorentz microscopy, which is

  11. Detection and characterization of stacking faults by light beam induced current mapping and scanning infrared microscopy in silicon

    NASA Astrophysics Data System (ADS)

    Vève-Fossati, C.; Martinuzzi, S.

    1998-08-01

    Non destructive techniques like scanning infrared microscopy and light beam induced current mapping are used to reveal the presence of stacking faults in heat treated Czochralski grown silicon wafers. In oxidized or contaminated samples, scanning infrared microscopy reveals that stacking faults grow around oxygen precipitates. This could be due to an aggregation of silicon self-interstitials emitted by the growing precipitates in the (111) plane. Light beam induced current maps show that the dislocations which surround the stacking faults are the main source of recombination centers, especially when they are decorated by a fast diffuser like copper. Des techniques non destructives telles que la microscopie infrarouge à balayage et la cartographie de photocourant induit par un spot lumineux ont été utilisées pour révéler la présence de fautes d'empilement après traitements thermiques, dans des plaquettes de silicium préparées par tirage Czochralski. Dans des échantillons oxydés ou contaminés, la microscopie infrarouge à balayage révèle des fautes d'empilement qui se développent autour des précipités d'oxygène. Cela peut être dû à la formation d'un agglomérat d'auto-interstitiels de silicium émis par la croissance des précipités dans les plans (111). Les cartographies de photocourant montrent que les dislocations qui entourent les fautes d'empilement sont la principale source de centres de recombinaison, et cela tout particulièrement quand ces fautes sont décorées par un diffuseur rapide tel que le cuivre.

  12. Lorentz microscopy sheds light on the role of dipolar interactions in magnetic hyperthermia.

    PubMed

    Campanini, M; Ciprian, R; Bedogni, E; Mega, A; Chiesi, V; Casoli, F; de Julián Fernández, C; Rotunno, E; Rossi, F; Secchi, A; Bigi, F; Salviati, G; Magén, C; Grillo, V; Albertini, F

    2015-05-01

    Monodispersed Fe3O4 nanoparticles with comparable size distributions have been synthesized by two different synthesis routes, co-precipitation and thermal decomposition. Thanks to the different steric stabilizations, the described samples can be considered as a model system to investigate the effects of magnetic dipolar interactions on the aggregation states of the nanoparticles. Moreover, the presence of magnetic dipolar interactions can strongly affect the nanoparticle efficiency as a hyperthermic mediator. In this paper, we present a novel way to visualize and map the magnetic dipolar interactions in different kinds of nanoparticle aggregates by the use of Lorentz microscopy, an easy and reliable in-line electron holographic technique. By exploiting Lorentz microscopy, which is complementary to the magnetic measurements, it is possible to correlate the interaction degrees of magnetic nanoparticles with their magnetic behaviors. In particular, we demonstrate that Lorentz microscopy is successful in visualizing the magnetic configurations stabilized by dipolar interactions, thus paving the way to the comprehension of the power loss mechanisms for different nanoparticle aggregates. PMID:25835488

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

    PubMed Central

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

    2015-01-01

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

  14. Programmable aperture microscopy: A computational method for multi-modal phase contrast and light field imaging

    NASA Astrophysics Data System (ADS)

    Zuo, Chao; Sun, Jiasong; Feng, Shijie; Zhang, Minliang; Chen, Qian

    2016-05-01

    We demonstrate a simple and cost-effective programmable aperture microscope to realize multi-modal computational imaging by integrating a programmable liquid crystal display (LCD) into a conventional wide-field microscope. The LCD selectively modulates the light distribution at the rear aperture of the microscope objective, allowing numerous imaging modalities, such as bright field, dark field, differential phase contrast, quantitative phase imaging, multi-perspective imaging, and full resolution light field imaging to be achieved and switched rapidly in the same setup, without requiring specialized hardwares and any moving parts. We experimentally demonstrate the success of our method by imaging unstained cheek cells, profiling microlens array, and changing perspective views of thick biological specimens. The post-exposure refocusing of a butterfly mouthpart and RFP-labeled dicot stem cross-section is also presented to demonstrate the full resolution light field imaging capability of our system for both translucent and fluorescent specimens.

  15. Improving signal-to-noise ratio of structured light microscopy based on photon reassignment

    PubMed Central

    Singh, Vijay Raj; Choi, Heejin; Yew, Elijah Y. S.; Bhattacharya, Dipanjan; Yuan, Luo; Sheppard, Colin J. R.; Rajapakse, Jagath C.; Barbastathis, George; So, Peter T. C.

    2011-01-01

    In this paper, we report a method for 3D visualization of a biological specimen utilizing a structured light wide-field microscopic imaging system. This method improves on existing structured light imaging modalities by reassigning fluorescence photons generated from off-focal plane excitation, improving in-focus signal strength. Utilizing a maximum likelihood approach, we identify the most likely fluorophore distribution in 3D that will produce the observed image stacks under structured and uniform illumination using an iterative maximization algorithm. Our results show the optical sectioning capability of tissue specimens while mostly preserving image stack photon count, which is usually not achievable with other existing structured light imaging methods. PMID:22254180

  16. Wide-field hyperspectral 3D imaging of functionalized gold nanoparticles targeting cancer cells by reflected light microscopy.

    PubMed

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

    2015-05-01

    We present a new hyperspectral reflected light microscopy system with a scanned broadband supercontinuum light source. This wide-field and low phototoxic hyperspectral imaging system has been successful for performing spectral three-dimensional (3D) localization and spectroscopic identification of CD44-targeted PEGylated AuNPs in fixed cell preparations. Such spatial and spectral information is essential for the improvement of nanoplasmonic-based imaging, disease detection and treatment in complex biological environment. The presented system can be used for real-time 3D NP tracking as spectral sensors, thus providing new avenues in the spatio-temporal characterization and detection of bioanalytes. 3D image of the distribution of functionalized AuNPs attached to CD44-expressing MDA-MB-231 human cancer cells. PMID:24961507

  17. Single-molecule super-resolution microscopy reveals how light couples to a plasmonic nanoantenna on the nanometer scale.

    PubMed

    Wertz, Esther; Isaacoff, Benjamin P; Flynn, Jessica D; Biteen, Julie S

    2015-04-01

    The greatly enhanced fields near metal nanoparticles have demonstrated remarkable optical properties and are promising for applications from solar energy to biosensing. However, direct experimental study of these light-matter interactions at the nanoscale has remained difficult due to the limitations of optical microscopy. Here, we use single-molecule fluorescence imaging to probe how a plasmonic nanoantenna modifies the fluorescence emission from a dipole emitter. We show that the apparent fluorophore emission position is strongly shifted upon coupling to an antenna and that the emission of dyes located up to 90 nm away is affected by this coupling. To predict this long-ranged effect, we present a framework based on a distance-dependent partial coupling of the dye emission to the antenna. Our direct interpretation of these light-matter interactions will enable more predictably optimized, designed, and controlled plasmonic devices and will permit reliable plasmon-enhanced single-molecule nanoscopy. PMID:25799002

  18. Possibilities and limitations of advanced transmission electron microscopy for carbon-based nanomaterials

    PubMed Central

    Bittencourt, Carla; Van Tendeloo, Gustaaf

    2015-01-01

    Summary A major revolution for electron microscopy in the past decade is the introduction of aberration correction, which enables one to increase both the spatial resolution and the energy resolution to the optical limit. Aberration correction has contributed significantly to the imaging at low operating voltages. This is crucial for carbon-based nanomaterials which are sensitive to electron irradiation. The research of carbon nanomaterials and nanohybrids, in particular the fundamental understanding of defects and interfaces, can now be carried out in unprecedented detail by aberration-corrected transmission electron microscopy (AC-TEM). This review discusses new possibilities and limits of AC-TEM at low voltage, including the structural imaging at atomic resolution, in three dimensions and spectroscopic investigation of chemistry and bonding. In situ TEM of carbon-based nanomaterials is discussed and illustrated through recent reports with particular emphasis on the underlying physics of interactions between electrons and carbon atoms. PMID:26425406

  19. Tip-enhanced Raman scattering microscopy: Recent advance in tip production

    NASA Astrophysics Data System (ADS)

    Fujita, Yasuhiko; Walke, Peter; De Feyter, Steven; Uji-i, Hiroshi

    2016-08-01

    Tip-enhanced Raman scattering (TERS) microscopy is a technique that combines the chemical sensitivity of Raman spectroscopy with the resolving power of scanning probe microscopy. The key component of any TERS setup is a plasmonically-active noble metal tip, which serves to couple far-field incident radiation with the near-field. Thus, the design and implementation of reproducible probes are crucial for the continued development of TERS as a tool for nanoscopic analysis. Here we discuss conventional methods for the fabrication of TERS-ready tips, highlighting the problems therein, as well as detailing more recent developments to improve reducibility. In addition, the idea of remote excitation-TERS is enlightened upon, whereby TERS sensitivity is further improved by using propagating surface plasmons to separate the incident radiation from the tip apex, as well as how this can be incorporated into the fabrication process.

  20. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells

    PubMed Central

    Bauluz, Blanca; Canudo, José Ignacio; Gasca, José Manuel; Torcida Fernández-Baldor, Fidel

    2016-01-01

    Abnormalities in the histo- and ultrastructure of the amniote eggshell are often related to diverse factors, such as ambient stress during egg formation, pathologies altering the physiology of the egg-laying females, or evolutionarily selected modifications of the eggshell structure that vary the physical properties of the egg, for example increasing its strength so as to avoid fracture during incubation. When dealing with fossil materials, all the above hypotheses are plausible, but a detailed taphonomical study has to be performed to rule out the possibility that secondary processes of recrystallization have occurred during fossilization. Traditional analyses, such as optical microscopy inspection and cathodoluminescence, have proven not to be enough to understand the taphonomic story of some eggshells. Recently, electron backscatter diffraction has been used, in combination with other techniques, to better understand the alteration of fossil eggshells. Here we present a combined study using scanning electron microscopy, optical microscopy, cathodoluminescence and electron backscatter diffraction of eggshell fragments assigned to Megaloolithus cf. siruguei from the Upper Cretaceous outcrops of the Cameros Basin. We focus our study on the presence of secondary shell units that mimic most aspects of the ultrastructure of the eggshell mammillae, but grow far from the inner surface of the eggshell. We call these structures extra-spherulites, describe their crystal structure and demonstrate their secondary origin. Our study has important implications for the interpretation of secondary shell units as biological or pathological structures. Thus, electron backscatter diffraction complements other microscope techniques as a useful tool for understanding taphonomical alterations in fossil eggshells. PMID:27144767

  1. Combined Use of Electron and Light Microscopy Techniques Reveals False Secondary Shell Units in Megaloolithidae Eggshells.

    PubMed

    Moreno-Azanza, Miguel; Bauluz, Blanca; Canudo, José Ignacio; Gasca, José Manuel; Torcida Fernández-Baldor, Fidel

    2016-01-01

    Abnormalities in the histo- and ultrastructure of the amniote eggshell are often related to diverse factors, such as ambient stress during egg formation, pathologies altering the physiology of the egg-laying females, or evolutionarily selected modifications of the eggshell structure that vary the physical properties of the egg, for example increasing its strength so as to avoid fracture during incubation. When dealing with fossil materials, all the above hypotheses are plausible, but a detailed taphonomical study has to be performed to rule out the possibility that secondary processes of recrystallization have occurred during fossilization. Traditional analyses, such as optical microscopy inspection and cathodoluminescence, have proven not to be enough to understand the taphonomic story of some eggshells. Recently, electron backscatter diffraction has been used, in combination with other techniques, to better understand the alteration of fossil eggshells. Here we present a combined study using scanning electron microscopy, optical microscopy, cathodoluminescence and electron backscatter diffraction of eggshell fragments assigned to Megaloolithus cf. siruguei from the Upper Cretaceous outcrops of the Cameros Basin. We focus our study on the presence of secondary shell units that mimic most aspects of the ultrastructure of the eggshell mammillae, but grow far from the inner surface of the eggshell. We call these structures extra-spherulites, describe their crystal structure and demonstrate their secondary origin. Our study has important implications for the interpretation of secondary shell units as biological or pathological structures. Thus, electron backscatter diffraction complements other microscope techniques as a useful tool for understanding taphonomical alterations in fossil eggshells. PMID:27144767

  2. Light sheet-based fluorescence microscopy (LSFM) reduces phototoxic effects and provides new means for the modern life sciences

    NASA Astrophysics Data System (ADS)

    Pampaloni, Francesco; Ansari, Nari; Girard, Philippe; Stelzer, Ernst H. K.

    2011-07-01

    Most optical technologies are applied to flat, basically two-dimensional cellular systems. However, physiological meaningful information relies on the morphology, the mechanical properties and the biochemistry of a cell's context. A cell requires the complex three-dimensional relationship to other cells. However, the observation of multi-cellular biological specimens remains a challenge. Specimens scatter and absorb light, thus, the delivery of the probing light and the collection of the signal light become inefficient; many endogenous biochemical compounds also absorb light and suffer degradation of some sort (photo-toxicity), which induces malfunction of a specimen. In conventional and confocal fluorescence microscopy, whenever a single plane, the entire specimen is illuminated. Recording stacks of images along the optical Z-axis thus illuminates the entire specimen once for each plane. Hence, cells are illuminated 10-20 and fish 100-300 times more often than they are observed. This can be avoided by changing the optical arrangement. The basic idea is to use light sheets, which are fed into the specimen from the side and overlap with the focal plane of a wide-field fluorescence microscope. In contrast to an epi-fluorescence arrangement, such an azimuthal fluorescence arrangement uses two independently operated lenses for illumination and detection. Optical sectioning and no photo-toxic damage or photo-bleaching outside a small volume close to the focal plane are intrinsic properties. Light sheet-based fluorescence microscopy (LSFM) takes advantage of modern camera technologies. LSFM can be operated with laser cutters and for fluorescence correlation spectroscopy. During the last few years, LSFM was used to record zebrafish development from the early 32-cell stage until late neurulation with sub-cellular resolution and short sampling periods (60-90 sec/stack). The recording speed was five 4-Megapixel large frames/sec with a dynamic range of 12-14 bit. We followed

  3. Advanced magneto-optical microscopy: Imaging from picoseconds to centimeters - imaging spin waves and temperature distributions (invited)

    NASA Astrophysics Data System (ADS)

    Urs, Necdet Onur; Mozooni, Babak; Mazalski, Piotr; Kustov, Mikhail; Hayes, Patrick; Deldar, Shayan; Quandt, Eckhard; McCord, Jeffrey

    2016-05-01

    Recent developments in the observation of magnetic domains and domain walls by wide-field optical microscopy based on the magneto-optical Kerr, Faraday, Voigt, and Gradient effect are reviewed. Emphasis is given to the existence of higher order magneto-optical effects for advanced magnetic imaging. Fundamental concepts and advances in methodology are discussed that allow for imaging of magnetic domains on various length and time scales. Time-resolved imaging of electric field induced domain wall rotation is shown. Visualization of magnetization dynamics down to picosecond temporal resolution for the imaging of spin-waves and magneto-optical multi-effect domain imaging techniques for obtaining vectorial information are demonstrated. Beyond conventional domain imaging, the use of a magneto-optical indicator technique for local temperature sensing is shown.

  4. Evolutionary/advanced light water reactor data report

    SciTech Connect

    1996-02-09

    The US DOE Office of Fissile Material Disposition is examining options for placing fissile materials that were produced for fabrication of weapons, and now are deemed to be surplus, into a condition that is substantially irreversible and makes its use in weapons inherently more difficult. The principal fissile materials subject to this disposition activity are plutonium and uranium containing substantial fractions of plutonium-239 uranium-235. The data in this report, prepared as technical input to the fissile material disposition Programmatic Environmental Impact Statement (PEIS) deal only with the disposition of plutonium that contains well over 80% plutonium-239. In fact, the data were developed on the basis of weapon-grade plutonium which contains, typically, 93.6% plutonium-239 and 5.9% plutonium-240 as the principal isotopes. One of the options for disposition of weapon-grade plutonium being considered is the power reactor alternative. Plutonium would be fabricated into mixed oxide (MOX) fuel and fissioned (``burned``) in a reactor to produce electric power. The MOX fuel will contain dioxides of uranium and plutonium with less than 7% weapon-grade plutonium and uranium that has about 0.2% uranium-235. The disposition mission could, for example, be carried out in existing power reactors, of which there are over 100 in the United States. Alternatively, new LWRs could be constructed especially for disposition of plutonium. These would be of the latest US design(s) incorporating numerous design simplifications and safety enhancements. These ``evolutionary`` or ``advanced`` designs would offer not only technological advances, but also flexibility in siting and the option of either government or private (e.g., utility) ownership. The new reactor designs can accommodate somewhat higher plutonium throughputs. This data report deals solely with the ``evolutionary`` LWR alternative.

  5. Angular and spectrally resolved investigations of yeast cells by light scattering microscopy and goniometric measurements

    NASA Astrophysics Data System (ADS)

    Stark, Julian; Müller, Dennis; Nothelfer, Steffen; Kienle, Alwin

    2015-07-01

    Spectrally and angular resolved light scattering from yeast cells was studied with a scattering microscope and a goniometer. Different cell models were investigated with help of analytical solutions of Maxwell's equations. It was found that extraction of precise morphological and optical cellular properties from the measured scattering patterns and phase functions requires more sophisticated cell models than standard Mie theory.

  6. Advanced Light Source report. Volume 7, No. 1

    SciTech Connect

    Not Available

    1994-09-01

    The exceptional science already emerging from the user program at the ALS shows that the promises of ``unique research opportunities`` and ``experiments not possible anywhere else`` made at the inception of the ALS are indeed coming true. In less than a year of beamline operations, the ALS has produced numerous high-quality results and achieved an enviable level of performance. Since the beginning of 1994, the ALS has operated for 92% of its scheduled hours, an outstanding achievement for a new machine. The ALS` ability to deliver the brightest light in the world in the ultraviolet and soft x-ray regions of the spectrum has attracted a who`s who of synchrotron research to the experiment floor. These users have produced a variety of scientifically significant results during the ALS` first year of operation, a few of which are highlighted in this article.

  7. Laboratory-Based Cryogenic Soft X-ray Tomography with Correlative Cryo-Light and Electron Microscopy

    SciTech Connect

    Carlson, David B.; Gelb, Jeff; Palshin, Vadim; Evans, James E.

    2013-02-01

    Here we present a novel laboratory-based cryogenic soft X-ray microscope for whole cell tomography of frozen hydrated samples. We demonstrate the capabilities of this compact cryogenic microscope by visualizing internal sub-cellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 micrometers thick. Structures as small as 90 nm can be detected in tomographic reconstructions at roughly 70 nm spatial resolution following a low cumulative radiation dose of only 7.2 MGy. Furthermore, the design of the specimen chamber utilizes a standard sample support that permits multimodal correlative imaging of the exact same unstained yeast cell via cryo-fluorescence light microscopy, cryo-soft x-ray microscopy and cryo-transmission electron microscopy. This completely laboratory-based cryogenic soft x-ray microscope will therefore enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

  8. Raman microscopy of phagocytosis: shedding light on macrophage foam cell formation

    NASA Astrophysics Data System (ADS)

    van Manen, Henk-Jan; van Apeldoorn, Aart A.; Roos, Dirk; Otto, Cees

    2006-02-01

    The phagocyte NADPH oxidase is a crucial enzyme in the innate immune response of leukocytes against invading microorganisms. The superoxide (O II -) that is generated by this enzyme upon infection is directly and indirectly used in bacterial killing. The catalytic subunit of NADPH oxidase, the membrane-bound protein heterodimer flavocytochrome b 558, contains two heme moieties. Here, we first briefly discuss our recent confocal resonant Raman (RR) spectroscopy and microscopy experiments on flavocytochrome b 558 in both resting and phagocytosing neutrophilic granulocytes. Such experiments allow the determination of the redox state of flavocytochrome b 558 inside the cell, which directly reflects the electron transporting activity of NADPH oxidase. Subsequently, we report that incubation of murine RAW 264.7 macrophages with PolyActive microspheres for 1 week in culture medium leads to morphological and biochemical changes in the macrophages that are characteristic for the generation of macrophage-derived foam cells. Lipid-laden foam cells are the hallmark of early atherosclerotic lesions. Using nonresonant Raman spectroscopy and microscopy, we demonstrate that the numerous intracellular droplets in macrophages exposed to microspheres are rich in cholesteryl esters. The finding that phagocytic processes may trigger foam cell formation reinforces the current belief that (chronic) infection and inflammation are linked to the initiation and progression of atherosclerotic lesions. The study of such a connection may reveal new therapeutic targets for atherosclerosis treatment or prevention.

  9. Visualizing non-lytic exocytosis of Cryptococcus neoformans from macrophages using digital light microscopy.

    PubMed

    Stukes, Sabriya; Casadevall, Arturo

    2014-01-01

    Many aspects of the infection of macrophages by Cryptococcus neoformans have been extensively studied and well defined. However, one particular interaction that is not clearly understood is non-lytic exocytosis. In this process, yeast cells are released into the extracellular space by a poorly understood mechanism that leaves both the macrophage and Cn viable. Here, we describe how to follow a large number of individually infected macrophages for a 24 hr infection period by time-lapsed microscopy. Infected macrophages are housed in a heating chamber with a CO2 atmosphere attached to a microscope that provides the same conditions as a cell-culture incubator. Live digital microscopy can provide information about the dynamic interactions between a host and pathogen that is not available from static images. Being able to visualize each infected cell can provide clues as to how macrophages handle fungal infections, and vice versa. This technique is a powerful tool in studying the dynamics that are behind a complex phenomenon. PMID:25350860

  10. Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy

    PubMed Central

    Manders, Erik M. M.; Jalink, Kees; Stallinga, Sjoerd; Rieger, Bernd

    2015-01-01

    Co-localization analysis is a widely used tool to seek evidence for functional interactions between molecules in different color channels in microscopic images. Here we extend the basic co-localization analysis by including the orientations of the structures on which the molecules reside. We refer to the combination of co-localization of molecules and orientational alignment of the structures on which they reside as co-orientation. Because the orientation varies with the length scale at which it is evaluated, we consider this scale as a separate informative dimension in the analysis. Additionally we introduce a data driven method for testing the statistical significance of the co-orientation and provide a method for visualizing the local co-orientation strength in images. We demonstrate our methods on simulated localization microscopy data of filamentous structures, as well as experimental images of similar structures acquired with localization microscopy in different color channels. We also show that in cultured primary HUVEC endothelial cells, filaments of the intermediate filament vimentin run close to and parallel with microtubuli. In contrast, no co-orientation was found between keratin and actin filaments. Co-orientation between vimentin and tubulin was also observed in an endothelial cell line, albeit to a lesser extent, but not in 3T3 fibroblasts. These data therefore suggest that microtubuli functionally interact with the vimentin network in a cell-type specific manner. PMID:26161965

  11. Qualitative study of young, adult, and aged Wistar rats temporomandibular synovial membrane employing light, scanning, and transmission electron microscopy.

    PubMed

    Duro, Christiano Cony; Ciena, Adriano Polican; De Almeida, Sonia Regina Yokomizo; Righetti, Marta Maria Da Silva; Grisolia, Débora De Freitas; Issa, João Paulo Mardegan; Da Silva, Marcelo Cavenaghi Pereira; Watanabe, Ii-Sei

    2012-11-01

    The aim of this study was to analyze the rat temporomandibular joint (TMJ) synovial membrane at different ages using light, scanning, and transmission electron microscopy. Under light microscopic analysis, the TMJ structures were observed such as condyle, capsule, disk, the synovial membrane collagen type, and cells distribution. In the scanning electron microscopy, the synovial membrane surface exhibited a smooth aspect in young animals and there was an increase with ageing in the number of folds. The transmission electron microscopic analysis showed more synoviocytes in the synovial layer in the young group and still a great number of vesicles and cisterns dilation of rough endoplasmic reticulum in the aged group. In the three groups, a dense layer of collagen fibers in the synovial layer and cytoplasmic extensions were clearly seen. It was possible to conclude that synovial membrane structures in aged group showed alterations contributing to the decrease in joint lubrication and in the sliding between disk and joint surfaces. These characteristic will reflect in biomechanics of chewing, and may cause the TMJ disorders, currently observed in clinical processes. PMID:22791633

  12. Hyperspectral reflected light microscopy of plasmonic Au/Ag alloy nanoparticles incubated as multiplex chromatic biomarkers with cancer cells.

    PubMed

    Patskovsky, Sergiy; Bergeron, Eric; Rioux, David; Simard, Mikaël; Meunier, Michel

    2014-10-21

    A hyperspectral microscopy system based on a reflected light method for plasmonic nanoparticle (NP) imaging was designed and compared with a conventional darkfield method for spatial localization and spectroscopic identification of single Au, Ag and Au/Ag alloy NPs incubated with fixed human cancer cell preparations. A new synthesis protocol based on co-reduction of Au and Ag salts combined with the seeded growth technique was used for the fabrication of monodispersed alloy NPs with sizes ranging from 30 to 100 nm in diameter. We validated theoretically and experimentally the performance of 60 nm Au, Ag and Au/Ag (50 : 50) NPs as multiplexed biological chromatic markers for biomedical diagnostics and optical biosensing. The advantages of the proposed reflected light microscopy method are presented for NP imaging in a complex and highly diffusing medium such as a cellular environment. The obtained information is essential for the development of a high throughput, selective and efficient strategy for cancer detection and treatment. PMID:25133743

  13. Computational Tissue Volume Reconstruction of a Peripheral Nerve Using High-Resolution Light-Microscopy and Reconstruct

    PubMed Central

    Gierthmuehlen, Mortimer; Freiman, Thomas M.; Haastert-Talini, Kirsten; Mueller, Alexandra; Kaminsky, Jan; Stieglitz, Thomas; Plachta, Dennis T. T.

    2013-01-01

    The development of neural cuff-electrodes requires several in vivo studies and revisions of the electrode design before the electrode is completely adapted to its target nerve. It is therefore favorable to simulate many of the steps involved in this process to reduce costs and animal testing. As the restoration of motor function is one of the most interesting applications of cuff-electrodes, the position and trajectories of myelinated fibers in the simulated nerve are important. In this paper, we investigate a method for building a precise neuroanatomical model of myelinated fibers in a peripheral nerve based on images obtained using high-resolution light microscopy. This anatomical model describes the first aim of our “Virtual workbench” project to establish a method for creating realistic neural simulation models based on image datasets. The imaging, processing, segmentation and technical limitations are described, and the steps involved in the transition into a simulation model are presented. The results showed that the position and trajectories of the myelinated axons were traced and virtualized using our technique, and small nerves could be reliably modeled based on of light microscopy images using low-cost OpenSource software and standard hardware. The anatomical model will be released to the scientific community. PMID:23785485

  14. High-resolution light-sheet microscopy: a simulation of an optical illumination system for oil immersion

    NASA Astrophysics Data System (ADS)

    Lu, Xiang; Heintzmann, Rainer; Leischner, Ulrich

    2015-09-01

    Light sheet microscopy is a microscopy technique characterized by an illumination from the side, perpendicular to the direction of observation. While this is often used and easy to implement for imaging samples with water-immersion, the application in combination with oil-immersion is less often used and requires a specific optimization. In this paper we present our design of a light-sheet illumination optical system with a ~1μm illumination thickness, a long working distance through the immersion oil, and including a focusing system allowing for moving the focus-spot of the lightsheet laterally through the field of view. This optical design allows for the acquisition of fluorescence images in 3D with isotropic resolution of below 1 micrometer of whole-mount samples with a size of ~1mm diameter. This technique enables high-resolution insights in the 3D structure of biological samples, e.g. for research of insect anatomy or for imaging of biopsies in medical diagnostics.

  15. Advanced Strategies for Outdoor LED Lighting Applications and Technologies to Curtail Regional Light Pollution Effects

    NASA Astrophysics Data System (ADS)

    Monrad, Christian Karl; Benya, James R.

    2015-08-01

    LED lighting systems for outdoor lighting applications continue to evolve as do strategies to mitigate related effects upon regional astronomical and ecological assets. The improving availability and relative lumen-per-watt efficiencies of blue-suppressed low correlated color temperature emitters, narrow band amber, phosphor converted amber, and various combinations of broadband emitters and sub-550NM and sub-500NM filters allow for a wide palette of choices to be assessed to suit site-specific and task-specific lighting needs. In addition to static spectral content options, readily available luminaire designs also include precise geometric beam shape selections and adaptive controls to include dimming, dynamic spectral shifting, motion detection, and dynamic beam shaping to minimize total environmental lumen emissions throughout the course of the nighttime hours.Regional and international light pollution mitigation regulations will also be briefly addressed in the context of luminaire shielding and spectral content control efforts to better protect human quality of life issues as well as astronomical and ecological interests.The presentation will include numerous spectral content graphs for various luminaire options as well as project-specific case studies to document comparisons of legacy lighting systems versus high-performance LED systems with regard to total lumen emissions, skyglow contributions, energy efficiency, and end-user satisfaction with the installed LED lighting systems. Physical samples of various luminaires will also be available for hands-on assessments.

  16. EDITORIAL: Special Issue on advanced and emerging light sources Special Issue on advanced and emerging light sources

    NASA Astrophysics Data System (ADS)

    Haverlag, Marco; Kroesen, Gerrit; Ferguson, Ian

    2011-06-01

    The papers in this special issue of Journal of Physics D: Applied Physics (JPhysD) originate from the 12th International Symposium on the Science and Technology of Light Sources and the 3rd International Conference on White LEDs and Solid State Lighting, held 11-16 July 2010 at Eindhoven University. Abstracts of all papers presented at this combined conference were published in the Conference Proceedings LS-WLED 2010 by FAST-LS, edited by M Haverlag, G M W Kroesen and T Taguchi. Special issues of the previous three LS conferences have been well-cited and have proven to be an important source of information for the lighting community. The 2010 LS-Symposium was a combined conference with the White LED Conference in order to enhance the scope of this conference series towards new light source technologies such as LEDs and OLEDs, and this co-operation will be continued in the future. Given the faster technology development in these areas it was also decided to shorten the interval between conferences from three to two years. Well over 200 invited presentations, landmark presentations and poster contributions were presented at the 2010 LS-Symposium. The organizing committee have selected from these a number of outstanding contributions with a high technological content and invited the authors to submit a full paper in JPhysD. The criteria were that the work should not be a repetition of the work already published in the Proceedings, but should be new, complete, within the scope of JPhysD, and meeting the normal quality standards of this journal. After peer review a combined set of 18 papers is published in this JPhysD special issue. In addition, a number of lighting-application-orientated papers will be published in a special issue of Journal of Light & Visual Environment later in 2011. The papers in this special issue of JPhysD show that research in the science and technology of light sources still covers a broad set of subject areas which includes both 'classical

  17. Elementary steps at the surface of ice crystals visualized by advanced optical microscopy

    PubMed Central

    Sazaki, Gen; Zepeda, Salvador; Nakatsubo, Shunichi; Yokoyama, Etsuro; Furukawa, Yoshinori

    2010-01-01

    Due to the abundance of ice on earth, the phase transition of ice plays crucially important roles in various phenomena in nature. Hence, the molecular-level understanding of ice crystal surfaces holds the key to unlocking the secrets of a number of fields. In this study we demonstrate, by laser confocal microscopy combined with differential interference contrast microscopy, that elementary steps (the growing ends of ubiquitous molecular layers with the minimum height) of ice crystals and their dynamic behavior can be visualized directly at air-ice interfaces. We observed the appearance and lateral growth of two-dimensional islands on ice crystal surfaces. When the steps of neighboring two-dimensional islands coalesced, the contrast of the steps always disappeared completely. We were able to discount the occurrence of steps too small to detect directly because we never observed the associated phenomena that would indicate their presence. In addition, classical two-dimensional nucleation theory does not support the appearance of multilayered two-dimensional islands. Hence, we concluded that two-dimensional islands with elementary height (0.37 and 0.39 nm on basal and prism faces, respectively) were visualized by our optical microscopy. On basal and prism faces, we also observed the spiral growth steps generated by screw dislocations. The distance between adjacent spiral steps on a prism face was about 1/20 of that on a basal face. Hence, the step ledge energy of a prism face was 1/20 of that on a basal face, in accord with the known lower-temperature roughening transition of the prism face. PMID:20974928

  18. New and unconventional approaches for advancing resolution in biological transmission electron microscopy by improving macromolecular specimen preparation and preservation.

    SciTech Connect

    Massover, W.; Materials Science Division

    2011-02-01

    Resolution in transmission electron microscopy (TEM) now is limited by the properties of specimens, rather than by those of instrumentation. The long-standing difficulties in obtaining truly high-resolution structure from biological macromolecules with TEM demand the development, testing, and application of new ideas and unconventional approaches. This review concisely describes some new concepts and innovative methodologies for TEM that deal with unsolved problems in the preparation and preservation of macromolecular specimens. The selected topics include use of better support films, a more protective multi-component matrix surrounding specimens for cryo-TEM and negative staining, and, several quite different changes in microscopy and micrography that should decrease the effects of electron radiation damage; all these practical approaches are non-traditional, but have promise to advance resolution for specimens of biological macromolecules beyond its present level of 3-10 {angstrom} (0.3-1.0 nm). The result of achieving truly high resolution will be a fulfillment of the still unrealized potential of transmission electron microscopy for directly revealing the structure of biological macromolecules down to the atomic level.

  19. Physically-based in silico light sheet microscopy for visualizing fluorescent brain models

    PubMed Central

    2015-01-01

    Background We present a physically-based computational model of the light sheet fluorescence microscope (LSFM). Based on Monte Carlo ray tracing and geometric optics, our method simulates the operational aspects and image formation process of the LSFM. This simulated, in silico LSFM creates synthetic images of digital fluorescent specimens that can resemble those generated by a real LSFM, as opposed to established visualization methods producing visually-plausible images. We also propose an accurate fluorescence rendering model which takes into account the intrinsic characteristics of fluorescent dyes to simulate the light interaction with fluorescent biological specimen. Results We demonstrate first results of our visualization pipeline to a simplified brain tissue model reconstructed from the somatosensory cortex of a young rat. The modeling aspects of the LSFM units are qualitatively analysed, and the results of the fluorescence model were quantitatively validated against the fluorescence brightness equation and characteristic emission spectra of different fluorescent dyes. AMS subject classification Modelling and simulation PMID:26329404

  20. Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

    PubMed Central

    Hoyer, Patrick; de Medeiros, Gustavo; Balázs, Bálint; Norlin, Nils; Besir, Christina; Hanne, Janina; Kräusslich, Hans-Georg; Engelhardt, Johann; Sahl, Steffen J.; Hell, Stefan W.; Hufnagel, Lars

    2016-01-01

    We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5–12-fold compared with their conventional diffraction-limited LS analogs. PMID:26984498

  1. Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT

    NASA Astrophysics Data System (ADS)

    Hoyer, Patrick; de Medeiros, Gustavo; Balázs, Bálint; Norlin, Nils; Besir, Christina; Hanne, Janina; Kräusslich, Hans-Georg; Engelhardt, Johann; Sahl, Steffen J.; Hell, Stefan W.; Hufnagel, Lars

    2016-03-01

    We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs.

  2. Breaking the diffraction limit of light-sheet fluorescence microscopy by RESOLFT.

    PubMed

    Hoyer, Patrick; de Medeiros, Gustavo; Balázs, Bálint; Norlin, Nils; Besir, Christina; Hanne, Janina; Kräusslich, Hans-Georg; Engelhardt, Johann; Sahl, Steffen J; Hell, Stefan W; Hufnagel, Lars

    2016-03-29

    We present a plane-scanning RESOLFT [reversible saturable/switchable optical (fluorescence) transitions] light-sheet (LS) nanoscope, which fundamentally overcomes the diffraction barrier in the axial direction via confinement of the fluorescent molecular state to a sheet of subdiffraction thickness around the focal plane. To this end, reversibly switchable fluorophores located right above and below the focal plane are transferred to a nonfluorescent state at each scanning step. LS-RESOLFT nanoscopy offers wide-field 3D imaging of living biological specimens with low light dose and axial resolution far beyond the diffraction barrier. We demonstrate optical sections that are thinner by 5-12-fold compared with their conventional diffraction-limited LS analogs. PMID:26984498

  3. Second-harmonic generation polarization microscopy by rotation of excitation light

    NASA Astrophysics Data System (ADS)

    Fwu, Peter Tramyeon; Chou, Chen-Kuan; Chen, Wei-Liang; Dong, Chen-Yuan

    2007-02-01

    When imaging anisotropic samples with a laser scanning optical microscope, the results are often affected by the polarization of the excitation light source. Quantifying the polarization dependence of biological fibrous material such as muscle and collagen allows us to gain molecular information at length scale below the resolution of optical microscopes. One problem associated with rotating the direction of linearly polarized excitation light for an epi-illuminated laser scanning microscope is due to the reflective properties of the main dichroic mirror. Depending on the direction of the incident polarization, the dichroic mirror can induce different amount of phase retardation, thus altering the desired output polarization. In this work, we theoretically determined the needed combination of wave plates and their angular positions to compensate for the effect of the dichroic mirror, thus achieving any arbitrary linear polarization angle for the excitation incident on sample.

  4. Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci.

    PubMed

    Lopez Perez, Ramon; Best, Gerrit; Nicolay, Nils H; Greubel, Christoph; Rossberger, Sabrina; Reindl, Judith; Dollinger, Günther; Weber, Klaus-Josef; Cremer, Christoph; Huber, Peter E

    2016-08-01

    Carbon ion radiation is a promising new form of radiotherapy for cancer, but the central question about the biologic effects of charged particle radiation is yet incompletely understood. Key to this question is the understanding of the interaction of ions with DNA in the cell's nucleus. Induction and repair of DNA lesions including double-strand breaks (DSBs) are decisive for the cell. Several DSB repair markers have been used to investigate these processes microscopically, but the limited resolution of conventional microscopy is insufficient to provide structural insights. We have applied superresolution microscopy to overcome these limitations and analyze the fine structure of DSB repair foci. We found that the conventionally detected foci of the widely used DSB marker γH2AX (Ø 700-1000 nm) were composed of elongated subfoci with a size of ∼100 nm consisting of even smaller subfocus elements (Ø 40-60 nm). The structural organization of the subfoci suggests that they could represent the local chromatin structure of elementary DSB repair units at the DSB damage sites. Subfocus clusters may indicate induction of densely spaced DSBs, which are thought to be associated with the high biologic effectiveness of carbon ions. Superresolution microscopy might emerge as a powerful tool to improve our knowledge of interactions of ionizing radiation with cells.-Lopez Perez, R., Best, G., Nicolay, N. H., Greubel, C., Rossberger, S., Reindl, J., Dollinger, G., Weber, K.-J., Cremer, C., Huber, P. E. Superresolution light microscopy shows nanostructure of carbon ion radiation-induced DNA double-strand break repair foci. PMID:27166088

  5. Vacuum system for the LBL Advanced Light Source (ALS)

    SciTech Connect

    Kennedy, K.; Henderson, T.; Meneghetti, J. )

    1989-03-01

    A 1.5 to 1.9 GeV synchrotron light source is being built at LBL. The vacuum system is designed to permit most synchrotron photons to escape the electron channel and be absorbed in an antechamber. The gas generated by the photons hitting the absorbers in the antechambers will be pumped by titanium sublimation pumps located directly under the absorbers. The electron channel and the antechamber are connected by a 10-mm-high slot that offers good electrodynamic isolation of the two chambers of frequencies affecting the store electron orbit. Twelve 10-meter-long vessels constitute the vacuum chambers for all the lattice magnets. Each chamber will be machined from two thick plates of 5083-H321 aluminum and welded at the perimeter. Machining both the inside and outside of the vacuum chamber permits the use of complex and accurate surfaces. The use of thick plates allows flanges to be machined directly into the wall of each chamber, thus avoiding much welding. 1 ref., 3 figs.

  6. Vacuum system for the LBL advanced light source (ALS)

    SciTech Connect

    Kennedy, K.

    1988-05-01

    A 1.5 to 1.9 GeV synchrotron light source is being built at LBL. The vacuum system is designed to permit all synchrotron photons on the median plane to escape the electron channel and go into an antechamber through a 10 mm high slot. This slot offers effective RF isolation between the electron duct and the antechamber. All unused synchrotron photons within a few mrad of the median plane will be stopped by 96 nearly horizontal absorbers located in the antechamber. The gas, generated by the photons hitting the absorbers, will be directed down to reactive titanium surfaces. Twelve 10 meter long vessels constitute the vacuum chambers for all the lattice magnets. Each chamber will be machined from two thick plates of 5083-H321 aluminum and welded at the perimeter. The nominal wall thickness of the vacuum chamber is 40 mm, which makes it possible to machine a flange into the chamber without the use of welding. 5 refs., 5 figs.

  7. Light scattering microscopy measurements of single nuclei compared with GPU-accelerated FDTD simulations

    NASA Astrophysics Data System (ADS)

    Stark, Julian; Rothe, Thomas; Kieß, Steffen; Simon, Sven; Kienle, Alwin

    2016-04-01

    Single cell nuclei were investigated using two-dimensional angularly and spectrally resolved scattering microscopy. We show that even for a qualitative comparison of experimental and theoretical data, the standard Mie model of a homogeneous sphere proves to be insufficient. Hence, an accelerated finite-difference time-domain method using a graphics processor unit and domain decomposition was implemented to analyze the experimental scattering patterns. The measured cell nuclei were modeled as single spheres with randomly distributed spherical inclusions of different size and refractive index representing the nucleoli and clumps of chromatin. Taking into account the nuclear heterogeneity of a large number of inclusions yields a qualitative agreement between experimental and theoretical spectra and illustrates the impact of the nuclear micro- and nanostructure on the scattering patterns.

  8. HIV detection by in-situ hybridization based on confocal reflected light microscopy

    NASA Astrophysics Data System (ADS)

    Smith, Louis C.; Jericevic, Zeljko; Cuellar, Roland; Paddock, Stephen W.; Lewis, Dorothy E.

    1991-05-01

    Elucidation of the pathogenesis of AIDS is confounded by the finding that few actively infected CD4+ cells (1 in 104-105) can be detected in the peripheral blood, even though there is dramatic depletion (often >90%) of CD4+ cells as the disease progresses. A sensitive, 35S-based human immunodeficiency virus (HIV) mRNA in situ hybridization technique was coupled with a new detection method, confocal laser scanning microscopy, to examine transcriptionally active HIV-infected cells from individuals at different disease stages. An algorithm for image segmentation and analysis has been developed to determine the proportion of HIV-positive cells. Data obtained using this improved detection method suggest that there are more HIV mRNA-producing cells in HIV-infected individuals than previously thought, based on other detection methods.

  9. Light scattering microscopy measurements of single nuclei compared with GPU-accelerated FDTD simulations.

    PubMed

    Stark, Julian; Rothe, Thomas; Kieß, Steffen; Simon, Sven; Kienle, Alwin

    2016-04-01

    Single cell nuclei were investigated using two-dimensional angularly and spectrally resolved scattering microscopy. We show that even for a qualitative comparison of experimental and theoretical data, the standard Mie model of a homogeneous sphere proves to be insufficient. Hence, an accelerated finite-difference time-domain method using a graphics processor unit and domain decomposition was implemented to analyze the experimental scattering patterns. The measured cell nuclei were modeled as single spheres with randomly distributed spherical inclusions of different size and refractive index representing the nucleoli and clumps of chromatin. Taking into account the nuclear heterogeneity of a large number of inclusions yields a qualitative agreement between experimental and theoretical spectra and illustrates the impact of the nuclear micro- and nanostructure on the scattering patterns. PMID:26976736

  10. Meeting Summary Advanced Light Water Reactor Fuels Industry Meeting Washington DC October 27 - 28, 2011

    SciTech Connect

    Not Listed

    2011-11-01

    The Advanced LWR Fuel Working Group first met in November of 2010 with the objective of looking 20 years ahead to the role that advanced fuels could play in improving light water reactor technology, such as waste reduction and economics. When the group met again in March 2011, the Fukushima incident was still unfolding. After the March meeting, the focus of the program changed to determining what we could do in the near term to improve fuel accident tolerance. Any discussion of fuels with enhanced accident tolerance will likely need to consider an advanced light water reactor with enhanced accident tolerance, along with the fuel. The Advanced LWR Fuel Working Group met in Washington D.C. on October 72-18, 2011 to continue discussions on this important topic.

  11. Lithographic measurement of EUV flare in the 0.3-NA Micro ExposureTool optic at the Advanced Light Source

    SciTech Connect

    Cain, Jason P.; Naulleau, Patrick; Spanos, Costas J.

    2005-01-01

    The level of flare present in a 0.3-NA EUV optic (the MET optic) at the Advanced Light Source at Lawrence Berkeley National Laboratory is measured using a lithographic method. Photoresist behavior at high exposure doses makes analysis difficult. Flare measurement analysis under scanning electron microscopy (SEM) and optical microscopy is compared, and optical microscopy is found to be a more reliable technique. In addition, the measured results are compared with predictions based on surface roughness measurement of the MET optical elements. When the fields in the exposure matrix are spaced far enough apart to avoid influence from surrounding fields and the data is corrected for imperfect mask contrast and aerial image proximity effects, the results match predicted values quite well. The amount of flare present in this optic ranges from 4.7% for 2 {micro}m features to 6.8% for 500 nm features.

  12. A Web Resource for Lab Activities Using SEM, EDX and Light Microscopy

    NASA Astrophysics Data System (ADS)

    Argast, A.; Tennis, C. F.

    2003-12-01

    A resource that facilitates undergraduate activities centered on the development of perthitic textures is available at http://www.geosci.ipfw.edu/sem/semedx.html. The web site provides a coherent set of data and images from a perthite and an anorthoclase sample. Backscatter and secondary electron images, plane- and cross-polarized light images, energy dispersive X-ray (EDX) data from 13 points on the perthite and 31 points on the anorthoclase (including spectra and results files with data expressed as wt. and atomic percents) and X-ray diffraction (XRD) data are available. Care has been taken to match the areas examined by light and electron methods, allowing students to directly compare the data available from each technique. Brief descriptions of the various methods are provided. The available information can be the basis for many different exercises. Those without easy access to mircoprobes or EDX spectrometers will find the spectra useful for discussions of microchemical techniques. Different phases can be identified in the light and SEM images. The chemical results can be used to calculate formulas. (In addition to the feldspar, the anorthoclase phenocryst contains olivine, clinopyroxene, ulvospinel, apatite and glass.) Feldspar compositions can be plotted to illustrate compositional differences in perthite and anorthoclase. Glass compositions from the interior and margins of the anorthoclase phenocrysts can be compared. Numerous other possibilities, of varying levels of complexity, exist. The perthite (from Perth, Ontario) and the anorthoclase (from Mt. Erebus, Antarctica) were purchased from Wards Natural Science. There are many students with little or no access to data derived from EDX, SEM, XRD and similar techniques. It is our responsibility to find ways to make this information more universally available to all students.

  13. An intercepted feedback mode for light sensitive spectroscopic measurements in atomic force microscopy.

    PubMed

    Smoliner, J; Brezna, W

    2007-10-01

    In most atomic force microscopes (AFMs), the motion of the tip is detected by the deflection of a laser beam shining onto the cantilever. AFM applications such as scanning capacitance spectroscopy or photocurrent spectroscopy, however, are severely disturbed by the intense stray light of the AFM laser. For this reason, an intercepted feedback method was developed, which allows to switch off the laser temporarily while the feedback loop keeps running. The versatility of this feedback method is demonstrated by measuring tip-force dependent Schottky barrier heights on GaAs samples. PMID:17979460

  14. Staining-free malaria diagnostics by multispectral and multimodality light-emitting-diode microscopy

    NASA Astrophysics Data System (ADS)

    Merdasa, Aboma; Brydegaard, Mikkel; Svanberg, Sune; Zoueu, Jeremie T.

    2013-03-01

    We report an accurate optical differentiation technique between healthy and malaria-infected erythrocytes by quasi-simultaneous measurements of transmittance, reflectance, and scattering properties of unstained blood smears using a multispectral and multimode light-emitting diode microscope. We propose a technique for automated imaging, identification, and counting of malaria-infected erythrocytes for real-time and cost-effective parasitaemia diagnosis as an effective alternative to the manual screening of stained blood smears, now considered to be the gold standard in malaria diagnosis. We evaluate the performance of our algorithm against manual estimations of an expert and show a spectrally resolved increased scattering from malaria-infected blood cells.

  15. Light Water Reactor Sustainability Program Advanced Seismic Soil Structure Modeling

    SciTech Connect

    Bolisetti, Chandrakanth; Coleman, Justin Leigh

    2015-06-01

    of interest. The specific nonlinear soil behavior included in the NLSSI calculation presented in this report is gapping and sliding. Other NLSSI effects are not included in the calculation. The results presented in this report document initial model runs in the linear and nonlinear analysis process. Final comparisons between traditional and advanced SPRA will be presented in the September 30th deliverable.

  16. Preparation and characterization of B4C coatings for advanced research light sources.

    PubMed

    Störmer, Michael; Siewert, Frank; Sinn, Harald

    2016-01-01

    X-ray optical elements are required for beam transport at the current and upcoming free-electron lasers and synchrotron sources. An X-ray mirror is a combination of a substrate and a coating. The demand for large mirrors with single layers consisting of light or heavy elements has increased during the last few decades; surface finishing technology is currently able to process mirror lengths up to 1 m with microroughness at the sub-nanometre level. Additionally, thin-film fabrication is able to deposit a suitable single-layer material, such as boron carbide (B4C), some tens of nanometres thick. After deposition, the mirror should provide excellent X-ray optical properties with respect to coating thickness errors, microroughness values and slope errors; thereby enabling the mirror to transport the X-ray beam with high reflectivity, high beam flux and an undistorted wavefront to an experimental station. At the European XFEL, the technical specifications of the future mirrors are extraordinarily challenging. The acceptable shape error of the mirrors is below 2 nm along the whole length of 1 m. At the Helmholtz-Zentrum Geesthacht (HZG), amorphous layers of boron carbide with thicknesses in the range 30-60 nm were fabricated using the HZG sputtering facility, which is able to cover areas up to 1500 mm long by 120 mm wide in one step using rectangular B4C sputtering targets. The available deposition area is suitable for the specified X-ray mirror dimensions of upcoming advanced research light sources such as the European XFEL. The coatings produced were investigated by means of X-ray reflectometry and interference microscopy. The experimental results for the B4C layers are discussed according to thickness uniformity, density, microroughness and thermal stability. The variation of layer thickness in the tangential and sagittal directions was investigated in order to estimate the achieved level of uniformity over the whole deposition area, which is considerably

  17. Preparation and characterization of B4C coatings for advanced research light sources

    PubMed Central

    Störmer, Michael; Siewert, Frank; Sinn, Harald

    2016-01-01

    X-ray optical elements are required for beam transport at the current and upcoming free-electron lasers and synchrotron sources. An X-ray mirror is a combination of a substrate and a coating. The demand for large mirrors with single layers consisting of light or heavy elements has increased during the last few decades; surface finishing technology is currently able to process mirror lengths up to 1 m with microroughness at the sub-nanometre level. Additionally, thin-film fabrication is able to deposit a suitable single-layer material, such as boron carbide (B4C), some tens of nanometres thick. After deposition, the mirror should provide excellent X-ray optical properties with respect to coating thickness errors, microroughness values and slope errors; thereby enabling the mirror to transport the X-ray beam with high reflectivity, high beam flux and an undistorted wavefront to an experimental station. At the European XFEL, the technical specifications of the future mirrors are extraordinarily challenging. The acceptable shape error of the mirrors is below 2 nm along the whole length of 1 m. At the Helmholtz-Zentrum Geesthacht (HZG), amorphous layers of boron carbide with thicknesses in the range 30–60 nm were fabricated using the HZG sputtering facility, which is able to cover areas up to 1500 mm long by 120 mm wide in one step using rectangular B4C sputtering targets. The available deposition area is suitable for the specified X-ray mirror dimensions of upcoming advanced research light sources such as the European XFEL. The coatings produced were investigated by means of X-ray reflectometry and interference microscopy. The experimental results for the B4C layers are discussed according to thickness uniformity, density, microroughness and thermal stability. The variation of layer thickness in the tangential and sagittal directions was investigated in order to estimate the achieved level of uniformity over the whole deposition area, which is

  18. Advanced Epi Tools for Gallium Nitride Light Emitting Diode Devices

    SciTech Connect

    Patibandla, Nag; Agrawal, Vivek

    2012-12-01

    Over the course of this program, Applied Materials, Inc., with generous support from the United States Department of Energy, developed a world-class three chamber III-Nitride epi cluster tool for low-cost, high volume GaN growth for the solid state lighting industry. One of the major achievements of the program was to design, build, and demonstrate the world’s largest wafer capacity HVPE chamber suitable for repeatable high volume III-Nitride template and device manufacturing. Applied Materials’ experience in developing deposition chambers for the silicon chip industry over many decades resulted in many orders of magnitude reductions in the price of transistors. That experience and understanding was used in developing this GaN epi deposition tool. The multi-chamber approach, which continues to be unique in the ability of the each chamber to deposit a section of the full device structure, unlike other cluster tools, allows for extreme flexibility in the manufacturing process. This robust architecture is suitable for not just the LED industry, but GaN power devices as well, both horizontal and vertical designs. The new HVPE technology developed allows GaN to be grown at a rate unheard of with MOCVD, up to 20x the typical MOCVD rates of 3{micro}m per hour, with bulk crystal quality better than the highest-quality commercial GaN films grown by MOCVD at a much cheaper overall cost. This is a unique development as the HVPE process has been known for decades, but never successfully commercially developed for high volume manufacturing. This research shows the potential of the first commercial-grade HVPE chamber, an elusive goal for III-V researchers and those wanting to capitalize on the promise of HVPE. Additionally, in the course of this program, Applied Materials built two MOCVD chambers, in addition to the HVPE chamber, and a robot that moves wafers between them. The MOCVD chambers demonstrated industry-leading wavelength yield for GaN based LED wafers and industry

  19. Advancements in quantum cascade laser-based infrared microscopy of aqueous media.

    PubMed

    Haase, K; Kröger-Lui, N; Pucci, A; Schönhals, A; Petrich, W

    2016-06-23

    The large mid-infrared absorption coefficient of water frequently hampers the rapid, label-free infrared microscopy of biological objects in their natural aqueous environment. However, the high spectral power density of quantum cascade lasers is shifting this limitation such that mid-infrared absorbance images can be acquired in situ within signal-to-noise ratios of up to 100. Even at sample thicknesses well above 50 μm, signal-to-noise ratios above 10 are readily achieved. The quantum cascade laser-based microspectroscopy of aqueous media is exemplified by imaging an aqueous yeast solution and quantifying glucose consumption, ethanol generation as well as the production of carbon dioxide gas during fermentation. PMID:27032367

  20. Advanced tip design for liquid phase vibration mode atomic force microscopy.

    PubMed

    Muramatsu, Hiroshi; Yamamoto, Yuji; Shigeno, Masatsugu; Shirakawabe, Yoshiharu; Inoue, Akira; Kim, Woo-Sik; Kim, Seung Jin; Chang, Sang-Mok; Kim, Jong Min

    2008-03-24

    We have fabricated polymer tips for atomic force microscopy in order to elucidate the effects of tip length and shape on cantilever vibration damping in liquids. The vibration damping is investigated by measuring the vibration amplitude of cantilevers as a function of tip-sample distance. The cantilever with a short tip provides a higher damping effect over long tip-sample distances. When the vibration amplitude was rescaled to show the effect of the cantilever width on oscillation damping, the vibration amplitude of cantilevers with various tip lengths was similarly obtained in a long distance range over 50 microm. This similarity is explained by an acoustic damping model in which an acoustic wave is generated by the cantilever. Finally, the results indicate a cantilever with a sufficiently long tip compared to the cantilever width can dramatically reduce the long-range damping effect in a liquid environment. PMID:18328326

  1. Traction force microscopy on soft elastic substrates: A guide to recent computational advances.

    PubMed

    Schwarz, Ulrich S; Soiné, Jérôme R D

    2015-11-01

    The measurement of cellular traction forces on soft elastic substrates has become a standard tool for many labs working on mechanobiology. Here we review the basic principles and different variants of this approach. In general, the extraction of the substrate displacement field from image data and the reconstruction procedure for the forces are closely linked to each other and limited by the presence of experimental noise. We discuss different strategies to reconstruct cellular forces as they follow from the foundations of elasticity theory, including two- versus three-dimensional, inverse versus direct and linear versus non-linear approaches. We also discuss how biophysical models can improve force reconstruction and comment on practical issues like substrate preparation, image processing and the availability of software for traction force microscopy. This article is part of a Special Issue entitled: Mechanobiology. PMID:26026889

  2. Advancement of X-Ray Microscopy Technology and its Application to Metal Solidification Studies

    NASA Technical Reports Server (NTRS)

    Kaukler, William F.; Curreri, Peter A.

    1996-01-01

    The technique of x-ray projection microscopy is being used to view, in real time, the structures and dynamics of the solid-liquid interface during solidification. By employing a hard x-ray source with sub-micron dimensions, resolutions of 2 micrometers can be obtained with magnifications of over 800 X. Specimen growth conditions need to be optimized and the best imaging technologies applied to maintain x-ray image resolution, contrast and sensitivity. It turns out that no single imaging technology offers the best solution and traditional methods like radiographic film cannot be used due to specimen motion (solidification). In addition, a special furnace design is required to permit controlled growth conditions and still offer maximum resolution and image contrast.

  3. Recent advances in light outcoupling from white organic light-emitting diodes

    NASA Astrophysics Data System (ADS)

    Gather, Malte C.; Reineke, Sebastian

    2015-01-01

    Organic light-emitting diodes (OLEDs) have been successfully introduced to the smartphone display market and have geared up to become contenders for applications in general illumination where they promise to combine efficient generation of white light with excellent color quality, glare-free illumination, and highly attractive designs. Device efficiency is the key requirement for such white OLEDs, not only from a sustainability perspective, but also because at the high brightness required for general illumination, losses lead to heating and may, thus, cause rapid device degradation. The efficiency of white OLEDs increased tremendously over the past two decades, and internal charge-to-photon conversion can now be achieved at ˜100% yield. However, the extraction of photons remains rather inefficient (typically <30%). Here, we provide an introduction to the underlying physics of outcoupling in white OLEDs and review recent progress toward making light extraction more efficient. We describe how structures that scatter, refract, or diffract light can be attached to the outside of white OLEDs (external outcoupling) or can be integrated close to the active layers of the device (internal outcoupling). Moreover, the prospects of using top-emitting metal-metal microcavity designs for white OLEDs and of tuning the average orientation of the emissive molecules within the OLED are discussed.

  4. New approach to study starch gelatinization applying a combination of hot-stage light microscopy and differential scanning calorimetry.

    PubMed

    Li, Qian; Xie, Qin; Yu, Shujuan; Gao, Qunyu

    2013-02-13

    To overcome the difficulty of the original polarizing microscope-based method in monitoring the gelatinization of starch, a new method for dynamically monitoring the gelatinization process, integral optical density (IOD), which was based on the digital image analysis technique, was proposed. Hot-stage light microscopy and differential scanning calorimetry (DSC) techniques were coupled to study the dynamic changes of three types of starches: type A (corn starch), type B (potato starch), and type C (pea starch), during the gelatinization process in an excess water system. A model of response difference change of crystallite could represent the responding intensity of crystallization changes in the process of starch gelatinization. Results demonstrated that three crystalline types of starch underwent a process of swelling, accompanied with gradual disappearing of the crystallite. This difference was mainly associated with the diversity and composition of the starch structure. The IOD method was of advantage compared to the previous traditional methods that are based on a polarization microscope, such as counting the particle number and calculating polarization area methods, because it was the product of two parameters: optical density and area, which would be a response of both light intensity and area of birefringence light. The single peak in DSC corresponded to the combination of crystalline helix-helix dissociation and the reduction of the molecule helix-coil transition, while the gelatinization degree measured by the IOD method mainly corresponded to the helix-helix dissociation. The gelatinization mechanism could be revealed clearer in this study. PMID:23339369

  5. Comprehensive optical and data management infrastructure for high-throughput light-sheet microscopy of whole mouse brains

    PubMed Central

    Müllenbroich, M. Caroline; Silvestri, Ludovico; Onofri, Leonardo; Costantini, Irene; Hoff, Marcel van’t; Sacconi, Leonardo; Iannello, Giulio; Pavone, Francesco S.

    2015-01-01

    Abstract. Comprehensive mapping and quantification of neuronal projections in the central nervous system requires high-throughput imaging of large volumes with microscopic resolution. To this end, we have developed a confocal light-sheet microscope that has been optimized for three-dimensional (3-D) imaging of structurally intact clarified whole-mount mouse brains. We describe the optical and electromechanical arrangement of the microscope and give details on the organization of the microscope management software. The software orchestrates all components of the microscope, coordinates critical timing and synchronization, and has been written in a versatile and modular structure using the LabVIEW language. It can easily be adapted and integrated to other microscope systems and has been made freely available to the light-sheet community. The tremendous amount of data routinely generated by light-sheet microscopy further requires novel strategies for data handling and storage. To complete the full imaging pipeline of our high-throughput microscope, we further elaborate on big data management from streaming of raw images up to stitching of 3-D datasets. The mesoscale neuroanatomy imaged at micron-scale resolution in those datasets allows characterization and quantification of neuronal projections in unsectioned mouse brains. PMID:26158018

  6. Axonal transport of organelles visualized by light microscopy: cinemicrographic and computer analysis.

    PubMed

    Forman, D S; Padjen, A L; Siggins, G R

    1977-11-11

    Rapid movements of intra-axonal organelles in acutely isolated single myelinated fibers from bullfrog sciatic nerve were visualized by dark-field microscopy. The movements were recorded by cinemicrography, and analyzed by computer-based methods. The movements are saltatory and bidirectional, but each particle moves mainly in a single direction. For more than 90% of the particles, the predominant movement direction is retrograde, i.e. toward the cell body. Quantitative measurements on a variety of parameters of the organelle movements are presented. Different particles in the same axon show a broad range of mean speeds. The average mean speed of movement in the retrograde direction at 28 degrees C was 1.08 micrometer/sec (S.D. - 0.41), equivalent to an axonal transport rate of 93 mm/day. Disperse distributions were also found for other parameters such as the instantaneous velocities of individual particles. Quantal velocities, periodic movement patterns, and specific 'channels' were not detected. When the data from a population of particles is treated statistically, the average mean speed, the distribution of velocities, and other statistical parameters are found to be similar in different axons studied at the same temperature. Direct microscopical observation of axonal organelle movement is a technique which provides information about axonal transport which is different from and complementary to that obtained from enzyme accumulation of radioactive tracer methods. PMID:72584

  7. Light and Electron Microscopy Methods for Examination of Cochlear Morphology in Mouse Models of Deafness.

    PubMed

    Parker, Andrew; Chessum, Lauren; Mburu, Philomena; Sanderson, Jeremy; Bowl, Michael R

    2016-01-01

    Mice are an invaluable model organism for the study of auditory function. Even though there are differences in size and frequency response, the anatomy and physiology of the mouse and human ear are remarkably similar. In addition, the tools available for genetic manipulation in the mouse have enabled the generation of models carrying mutations in orthologous human deafness-causing genes, helping to validate these lesions and assess their functional consequence. Reciprocally, novel gene mutations discovered to cause auditory deficits in the mouse highlight potential new loci for human hearing loss, and expand our basic knowledge of the mechanisms and pathways important for the function of the mammalian ear. Microscopy and imaging are invaluable techniques that allow detailed characterization of cochlear pathologies associated with particular gene mutations. However, the highly organized, delicate, and intricate structures responsible for transduction of sound waves into nerve impulses are encapsulated in one of the hardest bones in the body - the temporal bone. This makes sample preparation without damage to the soft tissue, be it from dissection or processing, somewhat challenging. Fortunately, there are numerous methods for achieving high-quality images of the mouse cochlea. Reported in this article are a selection of sample preparation and imaging techniques that can be used routinely to assess cochlear morphology. Several protocols are also described for immunodetection of proteins in the cochlea. In addition, the advantages and disadvantages between different imaging platforms and their suitability for different types of microscopic examination are highlighted. © 2016 by John Wiley & Sons, Inc. PMID:27584554

  8. Combined application of dynamic light scattering imaging and fluorescence intravital microscopy in vascular biology

    NASA Astrophysics Data System (ADS)

    Kalchenko, V.; Ziv, K.; Addadi, Y.; Madar-Balakirski, N.; Meglinski, I.; Neeman, M.; Harmelin, A.

    2010-08-01

    The dynamic light scattering imaging (DLSI) system combined with the conventional fluorescence intravital microscope (FIM) has been applied for the examination of blood and lymph vessels in the mouse ear in vivo. While the CCD camera can be shared by both techniques the combined application of DLSI and FIM allows rapid switching between the modalities. In current study temporal speckles fluctuations are used for rendering blood vessels structure and monitoring blood perfusion with the higher spatial resolution, whereas FIM provides the images of lymphatic vessels. The results clearly demonstrate that combined application of DLSI and FIM approaches provides synchronic in vivo images of blood and lymph vessels with higher contrast and specificity. The use of this new dual-modal diagnostic system is particularly important and has a great potential to significantly expand the capabilities of vascular diagnostics providing synchronic in vivo images of blood and lymph vessels.

  9. Correlative Light and Electron Microscopy to Study Microglial Interactions with β-Amyloid Plaques.

    PubMed

    Bisht, Kanchan; El Hajj, Hassan; Savage, Julie C; Sánchez, Maria G; Tremblay, Marie-Ève

    2016-01-01

    A detailed protocol is provided here to identify amyloid Aβ plaques in brain sections from Alzheimer's disease mouse models before pre-embedding immunostaining (specifically for ionized calcium-binding adapter molecule 1 (IBA1), a calcium binding protein expressed by microglia) and tissue processing for electron microscopy (EM). Methoxy-X04 is a fluorescent dye that crosses the blood-brain barrier and selectively binds to β-pleated sheets found in dense core Aβ plaques. Injection of the animals with methoxy-X04 prior to sacrifice and brain fixation allows pre-screening and selection of the plaque-containing brain sections for further processing with time-consuming manipulations. This is particularly helpful when studying early AD pathology within specific brain regions or layers that may contain very few plaques, present in only a small fraction of the sections. Post-mortem processing of tissue sections with Congo Red, Thioflavin S, and Thioflavin T (or even with methoxy-X04) can label β-pleated sheets, but requires extensive clearing with ethanol to remove excess dye and these procedures are incompatible with ultrastructural preservation. It would also be inefficient to perform labeling for Aβ (and other cellular markers such as IBA1) on all brain sections from the regions of interest, only to yield a small fraction containing Aβ plaques at the right location. Importantly, Aβ plaques are still visible after tissue processing for EM, allowing for a precise identification of the areas (generally down to a few square millimeters) to examine with the electron microscope. PMID:27286292

  10. Optomechanical properties of cancer cells revealed by light-induced deformation and quantitative phase microscopy

    NASA Astrophysics Data System (ADS)

    Kastl, Lena; Budde, Björn; Isbach, Michael; Rommel, Christina; Kemper, Björn; Schnekenburger, Jürgen

    2015-05-01

    There is a growing interest in cell biology and clinical diagnostics in label-free, optical techniques as the interaction with the sample is minimized and substances like dyes or fixatives do not affect the investigated cells. Such techniques include digital holographic microscopy (DHM) and the optical stretching by fiber optical two beam traps. DHM enables quantitative phase contrast imaging and thereby the determination of the cellular refractive index, dry mass and the volume, whereas optical cell stretching reveals the deformability of cells. Since optical stretching strongly depends on the optical properties and the shape of the investigated material we combined the usage of fiber optical stretching and DHM for the characterization of pancreatic tumor cells. The risk of tumors is their potential to metastasize, spread through the bloodstream and build distal tumors/metastases. The grade of dedifferentiation in which the cells lose their cell type specific properties is a measure for this metastatic potential. The less differentiated the cells are, the higher is their risk to metastasize. Our results demonstrate that pancreatic tumor cells, which are from the same tumor but vary in their grade of differentiation, show significant differences in their deformability. The retrieved data show that differentiated cells have a higher stiffness than less differentiated cells of the same tumor. Even cells that differ only in the expression of a single tumor suppressor gene which is responsible for cell-cell adhesions can be distinguished by their mechanical properties. Additionally, results from DHM measurements yield that the refractive index shows only few variations, indicating that it does not significantly influence optical cell stretching. The obtained results show a promising new approach for the phenotyping of different cell types, especially in tumor cell characterization and cancer diagnostics.

  11. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells.

    PubMed

    Peddie, Christopher J; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O'Toole, Peter; Larijani, Banafshe; Collinson, Lucy M

    2014-08-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. PMID:24637200

  12. Further description of Aspidodera raillieti (Nematoda: Aspidoderidae) from Didelphis marsupialis (Mammalia: Didelphidae) by light and scanning electron microscopy.

    PubMed

    Chagas-Moutinho, V A; Oliveira-Menezes, A; Cárdenas, M Q; Lanfredi, R M

    2007-10-01

    Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Freitas 1956 are widely distributed from Americas. The species of the genus Aspidodera Railliet and Henry 1912 are parasites of mammals of the orders Edentata, Marsupialia, and Rodentia. In the present work, Aspidodera raillieti (L. Travassos, Mem Inst Oswaldo Cruz 5(3):271-318, 1913), collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Valle del Cauca, Colombia, is redescribed. The association of light and scanning electron microscopy (SEM) allowed a detailed analysis of the morphology and ultrastructure of this nematode. Some taxonomic features, such as cephalic region, topography of the cuticle, sucker, spicules, posterior end of males, localization of vulva, the anus, and posterior end of females were observed. Important structures such as amphid, details of cephalic region, phasmid, and number and localization of caudal papillae are documented by SEM, for the first time adding characters to identify this species. Colombia is a new geographical record for A. raillieti. PMID:17622560

  13. Correlative and integrated light and electron microscopy of in-resin GFP fluorescence, used to localise diacylglycerol in mammalian cells

    PubMed Central

    Peddie, Christopher J.; Blight, Ken; Wilson, Emma; Melia, Charlotte; Marrison, Jo; Carzaniga, Raffaella; Domart, Marie-Charlotte; O׳Toole, Peter; Larijani, Banafshe; Collinson, Lucy M.

    2014-01-01

    Fluorescence microscopy of GFP-tagged proteins is a fundamental tool in cell biology, but without seeing the structure of the surrounding cellular space, functional information can be lost. Here we present a protocol that preserves GFP and mCherry fluorescence in mammalian cells embedded in resin with electron contrast to reveal cellular ultrastructure. Ultrathin in-resin fluorescence (IRF) sections were imaged simultaneously for fluorescence and electron signals in an integrated light and scanning electron microscope. We show, for the first time, that GFP is stable and active in resin sections in vacuo. We applied our protocol to study the subcellular localisation of diacylglycerol (DAG), a modulator of membrane morphology and membrane dynamics in nuclear envelope assembly. We show that DAG is localised to the nuclear envelope, nucleoplasmic reticulum and curved tips of the Golgi apparatus. With these developments, we demonstrate that integrated imaging is maturing into a powerful tool for accurate molecular localisation to structure. PMID:24637200

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

    NASA Technical Reports Server (NTRS)

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

    1992-01-01

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

  15. Light Microscopy Module: On-Orbit Microscope Planned for the Fluids Integrated Rack on the International Space Station

    NASA Technical Reports Server (NTRS)

    Motil, Susan M.

    2002-01-01

    The Light Microscopy Module (LMM) is planned as a remotely controllable, automated, on-orbit facility, allowing flexible scheduling and control of physical science and biological science experiments within the Fluids Integrated Rack (FIR) on the International Space Station. Initially four fluid physics experiments in the FIR will use the LMM the Constrained Vapor Bubble, the Physics of Hard Spheres Experiment-2, Physics of Colloids in Space-2, and Low Volume Fraction Entropically Driven Colloidal Assembly. The first experiment will investigate heat conductance in microgravity as a function of liquid volume and heat flow rate to determine, in detail, the transport process characteristics in a curved liquid film. The other three experiments will investigate various complementary aspects of the nucleation, growth, structure, and properties of colloidal crystals in microgravity and the effects of micromanipulation upon their properties.

  16. The bacterial rhizobiome of hyperaccumulators: future perspectives based on omics analysis and advanced microscopy

    PubMed Central

    Visioli, Giovanna; D'Egidio, Sara; Sanangelantoni, Anna M.

    2015-01-01

    Hyperaccumulators are plants that can extract heavy metal ions from the soil and translocate those ions to the shoots, where they are sequestered and detoxified. Hyperaccumulation depends not only on the availability of mobilized metal ions in the soil, but also on the enhanced activity of metal transporters and metal chelators which may be provided by the plant or its associated microbes. The rhizobiome is captured by plant root exudates from the complex microbial community in the soil, and may colonize the root surface or infiltrate the root cortex. This community can increase the root surface area by inducing hairy root proliferation. It may also increase the solubility of metals in the rhizosphere and promote the uptake of soluble metals by the plant. The bacterial rhizobiome, a subset of specialized microorganisms that colonize the plant rhizosphere and endosphere, makes an important contribution to the hyperaccumulator phenotype. In this review, we discuss classic and more recent tools that are used to study the interactions between hyperaccumulators and the bacterial rhizobiome, and consider future perspectives based on the use of omics analysis and microscopy to study plant metabolism in the context of metal accumulation. Recent data suggest that metal-resistant bacteria isolated from the hyperaccumulator rhizosphere and endosphere could be useful in applications such as phytoextraction and phytoremediation, although more research is required to determine whether such properties can be transferred successfully to non-accumulator species. PMID:25709609

  17. Recent advancements in the "water-window" microscopy with laser-plasma SXR source based on a double stream gas-puff target

    NASA Astrophysics Data System (ADS)

    Wachulak, P. W.

    2016-09-01

    An overview of our recent developments, regarding "water-window" soft X-ray (SXR) microscopy based on a laser-plasma double stream gas puff target sources is presented. The work, presented herein, describes two approaches to SXR microscopy. The first one is a low spatial resolution, achromatic SXR microscopy, employing Wolter type-I objective. The second one is a nanometer spatial resolution SXR microscopy, with the use of a Fresnel zone plate objective, for imaging various objects with quasimonochromatic light, emitted from a double stream gas puff target based short wavelength source. The developments regarding both systems are presented, as well as the possible applications, for which the SXR microscope was already employed. Such compact, table-top size, laboratory type microscopy setups may be employed in the near future for complementary-like studies to other, often used, microscopy techniques.

  18. Fluorescence light microscopy of pulmonary surfactant at the air-water interface of an air bubble of adjustable size.

    PubMed

    Knebel, D; Sieber, M; Reichelt, R; Galla, H-J; Amrein, M

    2002-07-01

    The structural dynamics of pulmonary surfactant was studied by epifluorescence light microscopy at the air-water interface of a bubble as a model close to nature for an alveolus. Small unilamellar vesicles of dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, a small amount of a fluorescent dipalmitoylphosphatidylcholine-analog, and surfactant-associated protein C were injected into the buffer solution. They aggregated to large clusters in the presence of Ca(2+) and adsorbed from these units to the interface. This gave rise to an interfacial film that eventually became fully condensed with dark, polygonal domains in a fluorescent matrix. When now the bubble size was increased or decreased, respectively, the film expanded or contracted. Upon expansion of the bubble, the dark areas became larger to the debit of the bright matrix and reversed upon contraction. We were able to observe single domains during the whole process. The film remained condensed, even when the interface was increased to twice its original size. From comparison with scanning force microscopy directly at the air-water interface, the fluorescent areas proved to be lipid bilayers associated with the (dark) monolayer. In the lung, such multilayer phase acts as a reservoir that guarantees a full molecular coverage of the alveolar interface during the breathing cycle and provides mechanical stability to the film. PMID:12080141

  19. Intracellular papillae of Actinocephalus (Eriocaulaceae-Poales) roots and their interaction with fungi: a light and transmission electron microscopy study.

    PubMed

    Oriani, Aline; Scatena, Vera L

    2007-01-01

    The genus Actinocephalus comprises 25 species and is restricted to Brazil, occurring mainly in the Espinhaço Mountains of Minas Gerais and Bahia States. Previous anatomical studies have reported the occurrence of intracellular papillae in the Actinocephalus roots, without dealing with their ultrastructure and function. The purpose of this paper is to investigate the structure, the composition and the probable function of the intracellular papillae of Actinocephalus roots, based on light microscopy, transmission electron microscopy and histochemical tests. The intracellular papillae occurred in all root tissues, from the rhizodermis to the vascular cylinder; they presented different forms and sizes and, ultrastructurally, they corresponded to material deposited between the cell wall and the plasma membrane. The histochemical tests carried out were positive for cellulose, pectin and callose. The intracellular papillae are responses of the plant cells to the interaction with fungi. They work as a physical barrier restricting fungal penetration, and they may also favor the supply of water and nutrients to the plant, since they increase root absorption surface. This might explain why the species of Actinocephalus are among the tallest Eriocaulaceae despite their reduced radicular system and the nutritional deficiency of the soil in which they grow. PMID:17067802

  20. Intense pulsed light therapy for superficial pigmented lesions evaluated by reflectance-mode confocal microscopy and optical coherence tomography.

    PubMed

    Yamashita, Toyonobu; Negishi, Kei; Hariya, Takeshi; Kunizawa, Naomi; Ikuta, Kaori; Yanai, Motohiro; Wakamatsu, Shingo

    2006-10-01

    Intense pulsed light (IPL) therapy is reported to be effective for pigment removal from pigmented lesions. However, the dynamic mechanism of pigment removal by IPL therapy is not completely understood. We investigated the mechanism of IPL therapy for the removal of pigmented skin lesions through non-invasive observation of the epidermis. Subjects with solar lentigines on the face were treated with three sessions of IPL therapy. The solar lentigines were observed on consecutive days after the treatments using reflectance-mode confocal microscopy (RCM) and optical coherence tomography (OCT). In addition, desquamated microcrusts that formed after the treatment were investigated by transmission electron microscopy (TEM). The images of RCM and OCT showed that the melanosomes in the epidermal basal layer rapidly migrated to the skin surface. The TEM images of the extruded microcrusts revealed numerous melanosomes together with cell debris. It was also found that the IPL irradiated melanocytes in the lesions seemed to be left intact and resumed their high activity after treatment. We conclude that IPL therapy effectively removed the dense melanosomes in the epidermal-basal layer. However, additional application of suppressive drugs such as hydroquinone or Q-switched laser irradiation is necessary to suppress the remaining active melanocytes. PMID:16741506

  1. Usefulness of combined light and electron microscopy: Evaluation of sputum samples for asbestos to determine past occupational exposure

    SciTech Connect

    Dodson, R.F.; Williams, M.G. Jr.; Corn, C.J.; Idell, S.; McLarty, J.W. )

    1989-07-01

    Ferruginous bodies (FB) in sputa are recognized as an indicator of past exposure to asbestos. However, a great variability exists in FB production, even in individuals with a history of occupational exposure. A further complication in interpreting the presence of FBs in sputa is that all individuals in modern society are exposed to asbestos and, in lung tissue studies, have been shown to harbor appreciable numbers of asbestos fibers. Thus, some of these individuals should occasionally produce FBs in their sputa. The present study was undertaken to determine if uncoated asbestos fiber content could be used to better discriminate occupationally exposed individuals from the general population. Randomly selected sputum samples from 12 former workers in an amosite asbestos plant and 12 controls were studied. The samples were prepared for the study by digesting the sputa in sodium hypochlorite. The digests were filtered through 0.2-microns polycarbonate filters for collection of particulates. The filters were screened for FBs by light microscopy at 200 X, and the presence or absence of uncoated asbestos fibers was determined at 5000 X in an AMRAY 1000A scanning electron microscope. The use of electron microscopy revealed the presence of commercial amphiboles in the sputa of the occupationally exposed individuals and enabled a differentiation of these samples from those of the general population.

  2. Investigating CNS synaptogenesis at single-synapse resolution by combining reverse genetics with correlative light and electron microscopy.

    PubMed

    Urwyler, Olivier; Izadifar, Azadeh; Dascenco, Dan; Petrovic, Milan; He, Haihuai; Ayaz, Derya; Kremer, Anna; Lippens, Saskia; Baatsen, Pieter; Guérin, Christopher J; Schmucker, Dietmar

    2015-01-15

    Determining direct synaptic connections of specific neurons in the central nervous system (CNS) is a major technical challenge in neuroscience. As a corollary, molecular pathways controlling developmental synaptogenesis in vivo remain difficult to address. Here, we present genetic tools for efficient and versatile labeling of organelles, cytoskeletal components and proteins at single-neuron and single-synapse resolution in Drosophila mechanosensory (ms) neurons. We extended the imaging analysis to the ultrastructural level by developing a protocol for correlative light and 3D electron microscopy (3D CLEM). We show that in ms neurons, synaptic puncta revealed by genetically encoded markers serve as a reliable indicator of individual active zones. Block-face scanning electron microscopy analysis of ms axons revealed T-bar-shaped dense bodies and other characteristic ultrastructural features of CNS synapses. For a mechanistic analysis, we directly combined the single-neuron labeling approach with cell-specific gene disruption techniques. In proof-of-principle experiments we found evidence for a highly similar requirement for the scaffolding molecule Liprin-α and its interactors Lar and DSyd-1 (RhoGAP100F) in synaptic vesicle recruitment. This suggests that these important synapse regulators might serve a shared role at presynaptic sites within the CNS. In principle, our CLEM approach is broadly applicable to the developmental and ultrastructural analysis of any cell type that can be targeted with genetically encoded markers. PMID:25503410

  3. Photosynthesis in a different light: spectro-microscopy for in vivo characterization of chloroplasts.

    PubMed

    Peter, Sébastien; Zell, Martina B; Blum, Christian; Stuhl, Alexander; Elgass, Kirstin; Sackrow, Marcus; Subramaniam, Vinod; Meixner, Alfred J; Harter, Klaus; Maurino, Veronica G; Schleifenbaum, Frank E

    2014-01-01

    During photosynthesis, energy conversion at the two photosystems is controlled by highly complex and dynamic adaptation processes triggered by external factors such as light quality, intensity, and duration, or internal cues such as carbon availability. These dynamics have remained largely concealed so far, because current analytical techniques are based on the investigation of isolated chloroplasts lacking full adaptation ability and are performed at non-physiologically low temperatures. Here, we use non-invasive in planta spectro-microscopic approaches to investigate living chloroplasts in their native environment at ambient temperatures. This is a valuable approach to study the complex function of these systems, because an intrinsic property-the fluorescence emission-is exploited and no additional external perturbations are introduced. Our analysis demonstrates a dynamic adjustment of not only the photosystemI/photosystemII (PSI/PSII) intensity ratio in the chloroplasts but also of the capacity of the LHCs for energy transfer in response to environmental and internal cues. PMID:25071790

  4. High resolution multimode light microscopy of cell migration: long-term imaging and analysis.

    PubMed

    Wöllert, Torsten; Langford, George M

    2009-01-01

    Cell migration is a multi-step process that involves sequential changes in the cytoskeleton, cell-substrate adhesion and components of the extracellular matrix. In multicellular organisms, directional cell migration is important for normal development, wound healing and immune responses and contributes to disease states such as tumor formation and metastasis. Many cells such as fibroblasts migrate as individuals while others, such as keratinocytes, move as groups or sheets of cells.In this chapter, we use human oral keratinocytes (OKF6/TERT-2) to illustrate the complex patterns of cell migration and its regulation. In culture, sheets of keratinocytes migrate and respond to human pathogens such as Candida albicans. The dynamic changes of the cytoskeleton, cell-cell and cell-substrate interactions that change during an infection for example require observation over long periods of time in order to identify the spatio-temporal coordinated regulation of the cytoskeleton and its associated components as well as the signaling pathways that control them.Microscopic techniques for long-term live cell observation and analysis of cell migration require high-resolution imaging systems that maintain perfect focus and optimal growth conditions (temperature, CO(2)) for cells. We describe two multimode digital imaging systems (VEC-DIC and BioStation IM), both with wide-field epifluorescence and transmitted light objectives for long-term time-lapse imaging and motion analysis. PMID:19768422

  5. Further evidence of centrophenoxine mediated protection in aluminium exposed rats by biochemical and light microscopy analysis.

    PubMed

    Nehru, Bimla; Bhalla, Punita; Garg, Aarti

    2007-12-01

    The environmental agent aluminium has been intensively investigated in the initiation and progression of various neurological disorders and the role of oxidative stress in these disorders is a widely discussed phenomenon. In this light, the present study is focused on the role of aluminium in mediating oxidative stress, which may help in better understanding its role in neuronal degeneration. Further, we have exploited a known anti-aging drug centrophenoxine to explore its potential in the conditions of metal induced oxidative damage. Aluminium was administered orally at a dose level of 100 mg/kg b.wt./day for a period of 6 weeks followed by a post treatment of centrophenoxine at a dose level of 100 mg/kg b.wt./day for another 6 weeks. Following aluminium exposure, a significant increase in lipid peroxidation levels (estimated by MDA) were observed which was accompanied by a decrease in reduced glutathione content in both cerebrum and cerebellum of rat brain. Post treatment of centrophenoxine significantly reduced the lipid peroxidation levels and also increased the reduced glutathione content in both the regions. Histologically observed marked deteriorations in the organization of various cellular layers in both cerebrum and cerebellum were observed after aluminium administration. Centrophenoxine treated animals showed an appreciable improvement in the histoarchitecture of the cellular layers. Our results indicate that centrophenoxine has an antioxidant potential and should be examined further in aluminium toxic conditions. PMID:17688990

  6. Robust incremental compensation of the light attenuation with depth in 3D fluorescence microscopy.

    PubMed

    Kervrann, C; Legland, D; Pardini, L

    2004-06-01

    Summary Fluorescent signal intensities from confocal laser scanning microscopes (CLSM) suffer from several distortions inherent to the method. Namely, layers which lie deeper within the specimen are relatively dark due to absorption and scattering of both excitation and fluorescent light, photobleaching and/or other factors. Because of these effects, a quantitative analysis of images is not always possible without correction. Under certain assumptions, the decay of intensities can be estimated and used for a partial depth intensity correction. In this paper we propose an original robust incremental method for compensating the attenuation of intensity signals. Most previous correction methods are more or less empirical and based on fitting a decreasing parametric function to the section mean intensity curve computed by summing all pixel values in each section. The fitted curve is then used for the calculation of correction factors for each section and a new compensated sections series is computed. However, these methods do not perfectly correct the images. Hence, the algorithm we propose for the automatic correction of intensities relies on robust estimation, which automatically ignores pixels where measurements deviate from the decay model. It is based on techniques adopted from the computer vision literature for image motion estimation. The resulting algorithm is used to correct volumes acquired in CLSM. An implementation of such a restoration filter is discussed and examples of successful restorations are given. PMID:15157197

  7. Combination of Light and Melatonin Time Cues for Phase Advancing the Human Circadian Clock

    PubMed Central

    Burke, Tina M.; Markwald, Rachel R.; Chinoy, Evan D.; Snider, Jesse A.; Bessman, Sara C.; Jung, Christopher M.; Wright, Kenneth P.

    2013-01-01

    Study Objectives: Photic and non-photic stimuli have been shown to shift the phase of the human circadian clock. We examined how photic and non-photic time cues may be combined by the human circadian system by assessing the phase advancing effects of one evening dose of exogenous melatonin, alone and in combination with one session of morning bright light exposure. Design: Randomized placebo-controlled double-blind circadian protocol. The effects of four conditions, dim light (∼1.9 lux, ∼0.6 Watts/m2)-placebo, dim light-melatonin (5 mg), bright light (∼3000 lux, ∼7 Watts/m2)-placebo, and bright light-melatonin on circadian phase was assessed by the change in the salivary dim light melatonin onset (DLMO) prior to and following treatment under constant routine conditions. Melatonin or placebo was administered 5.75 h prior to habitual bedtime and 3 h of bright light exposure started 1 h prior to habitual wake time. Setting: Sleep and chronobiology laboratory environment free of time cues. Participants: Thirty-six healthy participants (18 females) aged 22 ± 4 y (mean ± SD). Results: Morning bright light combined with early evening exogenous melatonin induced a greater phase advance of the DLMO than either treatment alone. Bright light alone and melatonin alone induced similar phase advances. Conclusion: Information from light and melatonin appear to be combined by the human circadian clock. The ability to combine circadian time cues has important implications for understanding fundamental physiological principles of the human circadian timing system. Knowledge of such principles is important for designing effective countermeasures for phase-shifting the human circadian clock to adapt to jet lag, shift work, and for designing effective treatments for circadian sleep-wakefulness disorders. Citation: Burke TM; Markwald RR; Chinoy ED; Snider JA; Bessman SC; Jung CM; Wright Jr KP. Combination of light and melatonin time cues for phase advancing the human circadian

  8. Advanced atomic force microscopy studies of ferroelectric domains and domain walls

    NASA Astrophysics Data System (ADS)

    Paruch, Patrycja

    2010-03-01

    The nanoscale resolution of atomic force microscopy (AFM) makes it a powerful tool for local studies of ferroelectric domain nucleation and growth. In particular, domain walls provide a useful model elastic disordered system: their behavior is governed by the competition between their elastic energy, which tends to minimize the domain wall surface, and the randomly varying potential landscape due to disorder present in the samples, which allows pinning. The domain walls present a characteristic static roughness, and a complex dynamic response when subjected to a driving force (electric field), with non-linear creep observed for small forces [1]. In addition, as a result of different symmetries and electronic structure, as well as possible defect migration, these intrinsically nanoscale interfaces often show additional properties, beyond those of their already multifunctional parent material, opening new perspectives for device applications. I will present results of our AFM studies of the static and dynamic behavior of domain walls in epitaxially grown thin films of Pb(Zr0.2Ti0.8)O3 focusing in particular on thermal effects, and on the observation of a lateral piezoresponse signal specifically due to the shear displacement of 180^o domain walls in this purely out-of-plane-polarized material [2], potentially useful for surface acoustic wave devices. I will also show how this same response can be more generally observed, necessitating care in the interpretation of lateral piezoresponse imaging in materials such as BiFeO3, where it is superimposed on signal due to the in-plane polarization components. Finally, I will present our studies of the switching mechanisms in this latter material under the influence of the electric field applied by the AFM tip. [4pt] [1] P. Paruch et al., Phys. Rev. Lett 94, 197601 (2005); J. Appl. Phys. 100, 051608 (2006); T. Tybell et al, Phys. Rev. Lett. 89, 097601 (2002)[0pt] [2] J. Guyonnet et al., Appl. Phys. Lett. 95 132902 (2009)

  9. LATEST LASER AND LIGHT-BASED ADVANCES FOR ETHNIC SKIN REJUVENATION

    PubMed Central

    Elsaie, Mohamed Lotfy; Lloyd, Heather Woolery

    2008-01-01

    Background: Advances in nonablative skin rejuvenation technologies have sparked a renewed interest in the cosmetic treatment of aging skin. More options exist now than ever before to reverse cutaneous changes caused by long-term exposure to sunlight. Although Caucasian skin is more prone to ultraviolet light injury, ethnic skin (typically classified as types IV to VI) also exhibits characteristic photoaging changes. Widespread belief that inevitable or irreversible textural changes or dyspigmentation occurs following laser- or light-based treatments, has been challenged in recent years by new classes of devices capable of protecting the epidermis from injury during treatment. Objective: The purpose of this article is to review recent clinical advances in the treatment of photoaging changes in ethnic skin. This article provides a basis for the classification of current advances in nonablative management of ethnic skin. PMID:19881986

  10. Simple 3D images from fossil and recent micromaterial using light microscopy.

    PubMed

    Haug, J T; Haug, C; Maas, A; Fayers, S R; Trewin, N H; Waloszek, D

    2009-01-01

    Abstract We present a technique for extracting 3D information from small-scale fossil and Recent material and give a summary of other contemporary techniques for 3D methods of investigation. The only hardware needed for the here-presented technique is a microscope that can perform dark field and/or differential interference contrast with a mounted digital camera and a computer. Serial images are taken while the focus is successively shifted from the uppermost end of the specimen to the lowermost end, resulting in about 200 photographs. The data are then processed almost completely automatically by successive use of three freely available programs. Firstly, the stack of images is aligned by the use of CombineZM, which is used to produce a combined image with a high depth of field. Secondly, the aligned images are cropped and sharp edges extracted with the aid of ImageJ. Thirdly, although ImageJ is also capable of producing 3D representations, we preferred to process the image stack further using osirix as it has the facility to export various formats. One of the interesting export formats is a virtual Quicktime movie file (QTVR), which can be used for documentation, and stereo images can also be produced from this Quicktime VR. This method is easy to apply and can be used for documenting specimens in 3D (at least some aspects) without having to prepare them. Therefore, it is particularly useful as a safe method for documenting limited material, before using methods that may destroy the specimen of interest, or to investigate type material that cannot be treated with any preparatory technique. As light microscopes are available in most labs and free computer programs are easily accessible, this method can be readily applied. PMID:19196416

  11. Comparison of tissue damage caused by various laser systems with tissue tolerable plasma by light and laser scan microscopy

    NASA Astrophysics Data System (ADS)

    Vandersee, Staffan; Lademann, Jürgen; Richter, Heike; Patzelt, Alexa; Lange-Asschenfeldt, Bernhard

    2013-10-01

    Tissue tolerable plasma (TTP) represents a novel therapeutic method with promising capabilities in the field of dermatological interventions, in particular disinfection but also wound antisepsis and regeneration. The energy transfer by plasma into living tissue is not easily educible, as a variety of features such as the medium’s actual molecule-stream, the ions, electrons and free radicals involved, as well as the emission of ultraviolet, visible and infrared light contribute to its increasingly well characterized effects. Thus, relating possible adversary effects, especially of prolonged exposure to a single component of the plasma’s mode of action, is difficult. Until now, severe adverse events connected to plasma exposure have not been reported when conducted according to existing therapeutic protocols. In this study, we have compared the tissue damage-potential of CO2 and dye lasers with TTP in a porcine model. After exposure of pig ear skin to the three treatment modalities, all specimens were examined histologically and by means of laser scan microscopy (LSM). Light microscopical tissue damage could only be shown in the case of the CO2 laser, whereas dye laser and plasma treatment resulted in no detectable impairment of the specimens. In the case of TTP, LSM examination revealed only an impairment of the uppermost corneal layers of the skin, thus stressing its safety when used in vivo.

  12. Application and development of advanced Lorentz microscopy techniques for the study of magnetic nanostructures

    NASA Astrophysics Data System (ADS)

    Beacham, Robert J.

    This PhD project presents an investigation into the development of magnetic imaging methods in the TEM and their application in imaging narrow domain walls in multilayer magnetic structures. Lorentz microscopy techniques are limited in quantitative magnetic imaging as this generally requires using scanning imaging modes which limits the capability of imaging dynamic processes. The first imaging method developed in this study is a phase gradient technique with the aim of producing quantitative magnetic contrast proportional to the magnetic induction of the sample whilst maintaining a live imaging mode. This method uses a specifically engineered, semi-electron-transparent graded wedge aperture to controllably perturb intensity in the back focal plane. The results of this study found that this method could produce magnetic contrast proportional to the sample induction, however the required gradient of the wedge aperture made this contrast close to the noise level with large associated errors. In the second part of this study we investigated the development of a technique aimed at gaining sub-microsecond temporal resolution within TEMs based on streak imaging. We are using ramped pulsed magnetic fields, applied across nanowire samples to both induce magnetic behaviour and detect the electron beam across the detector with respect to time. We are coupling this with a novel pixelated detector on the TEM in the form of a Medipix/Timepix chip capable of microsecond exposure times without adding noise. Running this detector in integral mode and allowing for practical limitations such as experiment time and aperture stability, the resultant streak images were taken in Fresnel, Foucault and low angle diffraction imaging modes. We found that while this method is theoretically viable, the limiting factor was the contrast of the magnetic signal in the streak and therefore the total image counts. Domain walls (DWs) in synthetic antiferromagnetically (SAF) coupled films patterned

  13. Advanced electrodynamic mechanisms for the nanoscale control of light by light

    NASA Astrophysics Data System (ADS)

    Andrews, David L.; Leeder, Jamie M.; Bradshaw, David S.

    2015-08-01

    A wide range of mechanisms is available for achieving rapid optical responsivity in material components. Amongst them, some of the most promising for potential device applications are those associated with an ultrafast response and a short cycle time. These twin criteria for photoresponsive action substantially favor optical, over most other, forms of response such as those fundamentally associated with photothermal, photochemical or optomechanical processes. The engagement of nonlinear mechanisms to actively control the characteristics of optical materials is not new. Indeed, it has been known for over fifty years that polarization effects of this nature occur in the optical Kerr effect - although in fluid media the involvement of a molecular reorientation mechanism leads to a significant response time. It has more recently emerged that there are other, less familiar forms of optical nonlinearity that can provide a means for one beam of light to instantly influence another. In particular, major material properties such as absorptivity or emissivity can be subjected to instant and highly localized control by the transmission of light with an off-resonant wavelength. This presentation introduces and compares the key electrodynamic mechanisms, discussing the features that suggest the most attractive possibilities for exploitation. The most significant of such mechanistic features include the off-resonant activation of optical emission, the control of excited-state lifetimes, the access of dark states, the inhibition or re-direction of exciton migration, and a coupling of stimulated emission with coherent scattering. It is shown that these offer a variety of new possibilities for ultrafast optical switching and transistor action, ultimately providing all-optical control with nanoscale precision.

  14. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy

    PubMed Central

    2015-01-01

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  15. A Bright Fluorescent Probe for H2S Enables Analyte-Responsive, 3D Imaging in Live Zebrafish Using Light Sheet Fluorescence Microscopy.

    PubMed

    Hammers, Matthew D; Taormina, Michael J; Cerda, Matthew M; Montoya, Leticia A; Seidenkranz, Daniel T; Parthasarathy, Raghuveer; Pluth, Michael D

    2015-08-19

    Hydrogen sulfide (H2S) is a critical gaseous signaling molecule emerging at the center of a rich field of chemical and biological research. As our understanding of the complexity of physiological H2S in signaling pathways evolves, advanced chemical and technological investigative tools are required to make sense of this interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which exhibits a rapid >1000-fold fluorescence response when treated with H2S, is selective for H2S over other biological analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than other commonly used azide-based systems, increasing its potential application in imaging experiments. To demonstrate the efficacy of this probe for H2S detection, we demonstrate the ability of MeRho-Az to detect differences in H2S levels in C6 cells and those treated with AOAA, a common inhibitor of enzymatic H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy (LSFM) to visualize H2S in the intestinal tract of live zebrafish. This application provides the first demonstration of analyte-responsive 3D imaging with LSFM, highlighting the utility of combining new probes and live imaging methods for investigating chemical signaling in complex multicellular systems. PMID:26061541

  16. Preliminary study of advanced turboprop and turboshaft engines for light aircraft. [cost effectiveness

    NASA Technical Reports Server (NTRS)

    Knip, G.; Plencner, R. M.; Eisenberg, J. D.

    1980-01-01

    The effects of engine configuration, advanced component technology, compressor pressure ratio and turbine rotor-inlet temperature on such figures of merit as vehicle gross weight, mission fuel, aircraft acquisition cost, operating, cost and life cycle cost are determined for three fixed- and two rotary-wing aircraft. Compared with a current production turboprop, an advanced technology (1988) engine results in a 23 percent decrease in specific fuel consumption. Depending on the figure of merit and the mission, turbine engine cost reductions required to achieve aircraft cost parity with a current spark ignition reciprocating (SIR) engine vary from 0 to 60 percent and from 6 to 74 percent with a hypothetical advanced SIR engine. Compared with a hypothetical turboshaft using currently available technology (1978), an advanced technology (1988) engine installed in a light twin-engine helicopter results in a 16 percent reduction in mission fuel and about 11 percent in most of the other figures of merit.

  17. Advanced methods for light trapping in optically thin silicon solar cells

    NASA Astrophysics Data System (ADS)

    Nagel, James Richard

    2011-12-01

    The field of light trapping is the study of how best to absorb light in a thin film of material when most light either reflects away at the surface or transmits straight through to the other side. This has tremendous application to the field of photovoltaics where thin silicon films can be manufactured cheaply, but also fail to capture all of the available photons in the solar spectrum. Advancements in light trapping therefore bring us closer to the day when photovoltaic devices may reach grid parity with traditional fossil fuels on the electrical energy market. This dissertation advances our understanding of light trapping by first modeling the effects of loss in planar dielectric waveguides. The mathematical framework developed here can be used to model any arbitrary three-layer structure with mixed gain or loss and then extract the total field solution for the guided modes. It is found that lossy waveguides possess a greater number of eigenmodes than their lossless counterparts, and that these "loss guided" modes attenuate much more rapidly than conventional modes. Another contribution from this dissertation is the exploration of light trapping through the use of dielectric nanospheres embedded directly within the active layer of a thin silicon film. The primary benefit to this approach is that the device can utilize a surface nitride layer serving as an antireflective coating while still retaining the benefits of light trapping within the film. The end result is that light trapping and light injection are effectively decoupled from each other and may be independently optimized within a single photovoltaic device. The final contribution from this work is a direct numerical comparison between multiple light trapping schemes. This allows us to quantify the relative performances of various design techniques against one another and objectively determine which ideas tend to capture the most light. Using numerical simulation, this work directly compares the absorption

  18. 78 FR 41436 - Proposed Revision to Treatment of Non-Safety Systems for Passive Advanced Light Water Reactors

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-10

    ... solicitation for public comment published in the Federal Register on October 12, 2012 (77 FR 62270), on the... COMMISSION Proposed Revision to Treatment of Non-Safety Systems for Passive Advanced Light Water Reactors... Treatment of Non-Safety Systems (RTNSS) for Passive Advanced Light Water Reactors.'' The NRC seeks...

  19. Multicatalytic proteinase is associated with characteristic oval structures in cortical Lewy bodies: an immunocytochemical study with light and electron microscopy.

    PubMed

    Masaki, T; Ishiura, S; Sugita, H; Kwak, S

    1994-04-01

    The ATP-ubiquitin-dependent proteolytic pathway (ubiquitin pathway) is believed to be involved in the formation of various neuronal inclusion bodies including Lewy bodies (LBs), a pathological hallmark of Parkinson disease and diffuse Lewy body disease (DLBD). Since multicatalytic proteinase (MCP) is involved in the ubiquitin pathway, an investigation of whether MCP is involved in neuronal inclusion bodies would provide a clue to the mechanism underlying the formation of neuronal inclusion bodies as well as to the pathogenesis of degenerative neurological disorders. In this study, we investigated detailed immunolocalization of MCP in LBs in DLBD brains using light and electron microscopy. We raised three different monoclonal antibodies against purified human MCP. Each of them recognized different sets of MCP subunits on Western blotting. Immunohistochemically, anti-MCP antibodies recognized all ubiquitin-positive cortical LBs in situ as well as those isolated from frozen DLBD cortices, suggesting that MCP is present in LBs as a whole molecule exhibiting protease activity. In electron microscopy, MCP immunoreactivity (MCP-IR) was exclusively localized on a characteristic oval structure with an approximate diameter of 100 nm. This structure was distributed throughout the LBs and was devoid of ubiquitin immunoreactivity. Treatment of isolated LBs with 2% SDS, but not with 0.5% Triton X-100, removed this structure from LBs in which fibrous materials predominated. Ubiquitin immunoreactivity was also decreased in isolated LBs treated with 2% SDS, suggesting that the fibrous structures in LBs were not ubiquitinated in situ. Thus, it is suggested that LBs are subjected to a proteolytic process in which MCP plays a role via processing of specific components of LBs. PMID:8021694

  20. High-resolution VUV spectroscopy: New results from the Advanced Light Source

    SciTech Connect

    Schlachter, F.; Bozek, J.

    1996-06-01

    Third-generation synchrotron light sources are providing photon beams of unprecedented brightness for researchers in atomic and molecular physics. Beamline 9.0.1, an undulator beamline at the Advanced Light Source (ALS), produces a beam in the vacuum-ultraviolet (VUV) region of the spectrum with exceptional flux and spectral resolution. Exciting new results from experiments in atomic and molecular VUV spectroscopy of doubly excited autoionizing states of helium, hollow lithium, and photoelectron spectroscopy of small molecules using Beamline 9.0.1 at the ALS are reported.