Science.gov

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.

    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

  3. 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.

  4. 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.

  5. 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.

  6. 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

  7. 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.

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

    PubMed Central

    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

    2016-01-01

    ABSTRACT 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

  9. Light sheet microscopy.

    PubMed

    Weber, Michael; Mickoleit, Michaela; Huisken, Jan

    2014-01-01

    This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over the last 10 years, we summarize the key concepts in light sheet microscopy, typical implementations, and successful applications. In particular, sample mounting for long time-lapse imaging and the resulting challenges in data processing are discussed in detail.

  10. Light microscopy digital imaging.

    PubMed

    Joubert, James; Sharma, Deepak

    2011-10-01

    This unit presents an overview of digital imaging hardware used in light microscopy. CMOS, CCD, and EMCCDs are the primary sensors used. The strengths and weaknesses of each define the primary applications for these sensors. Sensor architecture and formats are also reviewed. Color camera design strategies and sensor window cleaning are also described in the unit.

  11. 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

  12. 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.

  13. 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

  14. 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.

  15. 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

  16. Dynamic light scattering microscopy

    NASA Astrophysics Data System (ADS)

    Dzakpasu, Rhonda

    An optical microscope technique, dynamic light scattering microscopy (DLSM) that images dynamically scattered light fluctuation decay rates is introduced. Using physical optics we show theoretically that within the optical resolution of the microscope, relative motions between scattering centers are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The time scale for these intensity fluctuations is predicted. The spatial coherence distance defining the average distance between constructive and destructive interference in the image plane is calculated and compared with the pixel size. We experimentally tested DLSM on polystyrene latex nanospheres and living macrophage cells. In order to record these rapid fluctuations, on a slow progressive scan CCD camera, we used a thin laser line of illumination on the sample such that only a single column of pixels in the CCD camera is illuminated. This allowed the use of the rate of the column-by-column readout transfer process as the acquisition rate of the camera. This manipulation increased the data acquisition rate by at least an order of magnitude in comparison to conventional CCD cameras rates defined by frames/s. Analysis of the observed fluctuations provides information regarding the rates of motion of the scattering centers. These rates, acquired from each position on the sample are used to create a spatial map of the fluctuation decay rates. Our experiments show that with this technique, we are able to achieve a good signal-to-noise ratio and can monitor fast intensity fluctuations, on the order of milliseconds. DLSM appears to provide dynamic information about fast motions within cells at a sub-optical resolution scale and provides a new kind of spatial contrast.

  17. Confocal microscopy in transmitted light

    NASA Astrophysics Data System (ADS)

    Dodt, Hans-Ulrich; Becker, Klaus

    2003-10-01

    We developed a confocal microscope for transmitted light to visualize fine details in phase objects like unstained biological specimens. The main difficulty of confocal microscopy in transmission is the alignment of illumination and detector pinholes. This alignment was achieved by using "electronic pinholes" on the detector side. As a first step, we were able to image cells in onion skin at greater depths and with higher resolution than by using conventional microscopy.

  18. Advanced electron microscopy for advanced materials.

    PubMed

    Van Tendeloo, Gustaaf; Bals, Sara; Van Aert, Sandra; Verbeeck, Jo; Van Dyck, Dirk

    2012-11-01

    The idea of this Review is to introduce newly developed possibilities of advanced electron microscopy to the materials science community. Over the last decade, electron microscopy has evolved into a full analytical tool, able to provide atomic scale information on the position, nature, and even the valency atoms. This information is classically obtained in two dimensions (2D), but can now also be obtained in 3D. We show examples of applications in the field of nanoparticles and interfaces.

  19. The DIADEM Data Sets: Representative Light Microscopy Images of Neuronal Morphology to Advance Automation of Digital Reconstructions

    PubMed Central

    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.

    2015-01-01

    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 selecting 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

  20. 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

  1. Light Sheet Fluorescence Microscopy (LSFM).

    PubMed

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

    2015-01-05

    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 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.

  2. Fast Fluorescence Microscopy with Light Sheets.

    PubMed

    Daetwyler, Stephan; Huisken, Jan

    2016-08-01

    In light sheet microscopy, optical sectioning by selective fluorescence excitation with a sheet of light is combined with fast full-frame acquisition. This illumination scheme provides minimal photobleaching and phototoxicity. Complemented with remote focusing and multi-view acquisition, light sheet microscopy is the method of choice for acquisition of very fast biological processes, large samples, and high-throughput applications in areas such as neuroscience, plant biology, and developmental biology. This review explains why light sheet microscopes are much faster and gentler than other established fluorescence microscopy techniques. New volumetric imaging schemes and highlights of selected biological applications are also discussed. PMID:27638692

  3. 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.

  4. 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

  5. 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

  6. 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.

  7. 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.

  8. 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…

  9. 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.

  10. 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.

  11. 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

  12. 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

  13. Advanced Electron Microscopy in Materials Physics

    SciTech Connect

    Zhu, Y.; Jarausch, K.

    2009-06-01

    Aberration correction has opened a new frontier in electron microscopy by overcoming the limitations of conventional round lenses, providing sub-angstrom-sized probes and extending information limits. The imaging and analytical performance of these corrector-equipped microscopes affords an unprecedented opportunity to study structure-property relationships of matter at the atomic scale. This new generation of microscopes is able to retrieve high-quality structural information comparable to neutron and synchrotron x-ray experiments, but with local atomic resolution. These advances in instrumentation are accelerating the research and development of various functional materials ranging from those for energy generation, conversion, transportation and storage to those for catalysis and nano-device applications. The dramatic improvements in electron-beam illumination and detection also present a host of new challenges for the interpretation and optimization of experiments. During 7-9 November 2007, a workshop, entitled 'Aberration Corrected Electron Microscopy in Material Physics', was convened at the Center for Functional Nanomaterials, Brookhaven National Laboratories (BNL) to address these opportunities and challenges. The workshop was co-sponsored by Hitachi High Technologies, a leader in electron microscopy instrumentation, and BNL's Institute of Advanced Electron Microscopy, a leader in materials physics research using electron microscopy. The workshop featured presentations by internationally prominent scientists working at the frontiers of electron microscopy, both on developing instrumentation and applying it in materials physics. The meeting, structured to stimulate scientific exchanges and explore new capabilities, brought together {approx}100 people from over 10 countries. This special issue complies many of the advances in instrument performance and materials physics reported by the invited speakers and attendees at the workshop.

  14. Magnetic resonance microscopy versus light microscopy in human embryology teaching.

    PubMed

    Puerta-Fonollá, J; Vázquez-Osorio, T; Ruiz-Cabello, J; Murillo-González, J; Peña-Melián, A

    2004-07-01

    A study was carried out on the application of magnetic resonance microscopy (MRM) in teaching prenatal human development. Human embryos measuring 8 mm, 15 mm, 18.5 mm, and 22 mm were fixed in a 4% paraformaldehyde solution and sections obtained with magnetic resonance imaging (MRI) were compared to those prepared for light microscopy (LM), using the same embryos. The MRM and LM slices were of a similar quality. In the MRM sections, embryonic organs and systems were clearly visible, particularly the peripheral and central nervous systems, and the cardiovascular and digestive systems. The digitalization and clarity of the MRM images make them an ideal teaching aid that is suitable for students during the first years of a health-science degree, particularly medicine. As well as providing students with their first experience of MRM, these images allow students to access, at any time, all embryos used, to assess changes in the positions of different organs throughout their stages of development, and to acquire spatial vision, an absolute requirement in the study of human anatomy. We recommend that this technique be incorporated into the wealth of standard embryonic teaching methods already in use.

  15. 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

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Metallothioneins for correlative light and electron microscopy.

    PubMed

    Fernández de Castro, Isabel; Sanz-Sánchez, Laura; Risco, Cristina

    2014-01-01

    Structural biologists have been working for decades on new strategies to identify proteins in cells unambiguously. We recently explored the possibilities of using the small metal-binding protein, metallothionein (MT), as a tag to detect proteins in transmission electron microscopy. It had been reported that, when fused with a protein of interest and treated in vitro with gold salts, a single MT tag will build an electron-dense gold cluster ~1 nm in diameter; we provided proof of this principle by demonstrating that MT can be used to detect intracellular proteins in bacteria and eukaryotic cells. The method, which is compatible with a variety of sample processing techniques, allows specific detection of proteins in cells with exceptional sensitivity. We illustrated the applicability of the technique in a series of studies to visualize the intracellular distribution of bacterial and viral proteins. Immunogold labeling was fundamental to confirm the specificity of the MT-gold method. When proteins were double-tagged with green fluorescent protein and MT, direct correlative light and electron microscopy allowed visualization of the same macromolecular complexes with different spatial resolutions. MT-gold tagging might also become a useful tool for mapping proteins into the 3D-density maps produced by (cryo)-electron tomography. New protocols will be needed for double or multiple labeling of proteins, using different versions of MT with fluorophores of different colors. Further research is also necessary to render the MT-gold labeling procedure compatible with immunogold labeling on Tokuyasu cryosections and with cryo-electron microscopy of vitreous sections.

  1. Fourier phase microscopy with white light

    PubMed Central

    Bhaduri, Basanta; Tangella, Krishnarao; Popescu, Gabriel

    2013-01-01

    Laser-based Fourier phase microscopy (FPM) works on the principle of decomposition of an image field in two spatial components that can be controllably shifted in phase with respect to each other. However, due to the coherent illumination, the contrast in phase images is degraded by speckles. In this paper we present FPM with spatially coherent white light (wFPM), which offers high spatial phase sensitivity due to the low temporal coherence and high temporal phase stability due to common path geometry. Further, by using a fast spatial light modulator (SLM) and a fast scientific-grade complementary metal oxide semiconductor (sCMOS) camera, we report imaging at a maximum rate of 12.5 quantitative phase frames per second with 5.5 mega pixels image size. We illustrate the utility of wFPM as a contrast enhancement as well as dynamic phase measurement method by imaging section of benign colonic glands and red blood cell membrane fluctuation. PMID:24010005

  2. High-resolution light microscopy of nanoforms

    NASA Astrophysics Data System (ADS)

    Vodyanoy, Vitaly; Pustovyy, Oleg; Vainrub, Arnold

    2007-09-01

    We developed a high resolution light imaging system. Diffraction gratings with 100 nm width lines as well as less than 100 nm size features of different-shaped objects are clearly visible on a calibrated microscope test slide (Vainrub et al., Optics Letters, 2006, 31, 2855). The two-point resolution increase results from a known narrowing of the central diffraction peak for the annular aperture. Better visibility and advanced contrast of the smallest features in the image are due to enhancement of high spatial frequencies in the optical transfer function. The imaging system is portable, low energy, and battery operated. It has been adapted to use in both transmitting and reflecting light. It is particularly applicable for motile nanoform systems where structure and functions can be depicted in real time. We have isolated micrometer and submicrometer particles, termed proteons, from human and animal blood. Proteons form by reversible seeded aggregation of proteins around proteon nucleating centers (PNCs). PNCs are comprised of 1-2nm metallic nanoclusters containing 40-300 atoms. Proteons are capable of spontaneous assembling into higher nanoform systems assuming structure of complicated topology. The arrangement of complex proteon system mimics the structure of a small biological cell. It has structures that imitate membrane and nucleolus or nuclei. Some of these nanoforms are motile. They interact and divide. Complex nanoform systems can spontaneously reduce to simple proteons. The physical properties of these nanoforms could shed some light on the properties of early life forms or forms at extreme conditions.

  3. 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

  4. 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

  5. Low-Cost Cryo-Light Microscopy Stage Fabrication for Correlated Light/Electron Microscopy

    PubMed Central

    Carlson, David B.; Evans, James E.

    2011-01-01

    The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples. PMID:21673645

  6. Cryo-scanning electron microscopy and light microscopy for the study of fungi interactions.

    PubMed

    Sempere, F; Santamarina, M P

    2011-03-01

    The application of the cryo-scanning electron microscopy and light microscopy for the study of the interactions at different environmental conditions between Penicillium oxalicum and Fusarium verticillioides is described. A dual microculture was developed for the light microscopy analysis of the interaction. The microscope and macroscopic examinations were compared. Analysis of Petri plates revealed that F. verticillioides was a competitor for space and nutrients while P. oxalicum was a mycoparasite under the microscopic observations.

  7. 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.

  8. 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.

  9. The role of light microscopy in aerospace analytical laboratories

    NASA Technical Reports Server (NTRS)

    Crutcher, E. R.

    1977-01-01

    Light microscopy has greatly reduced analytical flow time and added new dimensions to laboratory capability. Aerospace analytical laboratories are often confronted with problems involving contamination, wear, or material inhomogeneity. The detection of potential problems and the solution of those that develop necessitate the most sensitive and selective applications of sophisticated analytical techniques and instrumentation. This inevitably involves light microscopy. The microscope can characterize and often identify the cause of a problem in 5-15 minutes with confirmatory tests generally less than one hour. Light microscopy has and will make a very significant contribution to the analytical capabilities of aerospace laboratories.

  10. Application of scanning acoustic microscopy to advanced structural ceramics

    NASA Technical Reports Server (NTRS)

    Vary, Alex; Klima, Stanley J.

    1987-01-01

    A review is presentod of research investigations of several acoustic microscopy techniques for application to structural ceramics for advanced heat engines. Results obtained with scanning acoustic microscopy (SAM), scanning laser acoustic microscopy (SLAM), scanning electron acoustic microscopy (SEAM), and photoacoustic microscopy (PAM) are compared. The techniques were evaluated on research samples of green and sintered monolithic silicon nitrides and silicon carbides in the form of modulus-of-rupture bars containing deliberately introduced flaws. Strengths and limitations of the techniques are described with emphasis on statistics of detectability of flaws that constitute potential fracture origins.

  11. 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

  12. Recent Advances in Fiber Lasers for Nonlinear Microscopy

    PubMed Central

    Xu, C.; Wise, F. W.

    2013-01-01

    Nonlinear microscopy techniques developed over the past two decades have provided dramatic new capabilities for biological imaging. The initial demonstrations of nonlinear microscopies coincided with the development of solid-state femtosecond lasers, which continue to dominate applications of nonlinear microscopy. Fiber lasers offer attractive features for biological and biomedical imaging, and recent advances are leading to high-performance sources with the potential for robust, inexpensive, integrated instruments. This article discusses recent advances, and identifies challenges and opportunities for fiber lasers in nonlinear bioimaging. PMID:24416074

  13. 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

  14. 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...

  15. 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.

  16. 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.

  17. Microscopy imaging device with advanced imaging properties

    DOEpatents

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

    2016-10-25

    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.

  18. 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.

  19. Confocal and Two-Photon Microscopy: Foundations, Applications and Advances

    NASA Astrophysics Data System (ADS)

    Diaspro, Alberto

    2001-11-01

    Confocal and Two-Photon Microscopy Foundations, Applications, and Advances Edited by Alberto Diaspro Confocal and two-photon fluorescence microscopy has provided researchers with unique possibilities of three-dimensional imaging of biological cells and tissues and of other structures such as semiconductor integrated circuits. Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances provides clear, comprehensive coverage of basic foundations, modern applications, and groundbreaking new research developments made in this important area of microscopy. Opening with a foreword by G. J. Brakenhoff, this reference gathers the work of an international group of renowned experts in chapters that are logically divided into balanced sections covering theory, techniques, applications, and advances, featuring: In-depth discussion of applications for biology, medicine, physics, engineering, and chemistry, including industrial applications Guidance on new and emerging imaging technology, developmental trends, and fluorescent molecules Uniform organization and review-style presentation of chapters, with an introduction, historical overview, methodology, practical tips, applications, future directions, chapter summary, and bibliographical references Companion FTP site with full-color photographs The significant experience of pioneers, leaders, and emerging scientists in the field of confocal and two-photon excitation microscopy Confocal and Two-Photon Microscopy: Foundations, Applications, and Advances is invaluable to researchers in the biological sciences, tissue and cellular engineering, biophysics, bioengineering, physics of matter, and medicine, who use these techniques or are involved in developing new commercial instruments.

  20. Near-infrared branding efficiently correlates light and electron microscopy.

    PubMed

    Bishop, Derron; Nikić, Ivana; Brinkoetter, Mary; Knecht, Sharmon; Potz, Stephanie; Kerschensteiner, Martin; Misgeld, Thomas

    2011-06-05

    The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

  1. 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

  2. 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

  3. Correlative video-light-electron microscopy: development, impact and perspectives.

    PubMed

    Rizzo, Riccardo; Parashuraman, Seetharaman; Luini, Alberto

    2014-08-01

    Green fluorescent protein (GFP)-based video microscopy can provide profound insight into biological processes by generating information on the 'history,' or dynamics, of the cellular structures involved in such processes in live cells. A crucial limitation of this approach, however, is that many such structures may not be resolved by light microscopy. Like more recent super-resolution techniques, correlative video-light-electron microscopy (CLEM) was developed to overcome this limitation. CLEM integrates GFP-based video microscopy and electron microscopy through a series of ancillary techniques, such as proper fixation, hybrid labeling and retracing, and so provides sufficient resolution as well as, crucially, cellular 'context' to the fluorescent dynamic structures of interest. CLEM 'multiplies' the power of video microscopy and is having an important impact in several areas cell and developmental biology. Here, we discuss potential, limitations and perspectives of correlative approaches aimed at integrating the unique insight generated by video microscopy with information from other forms of imaging. PMID:25030356

  4. Advanced Light Source control system

    SciTech Connect

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

    1989-03-01

    The Advanced Light Source (ALS) is a third generation 1--2 GeV synchrotron radiation source designed to provide ports for 60 beamlines. It uses a 50 MeV electron linac and 1.5 GeV, 1 Hz, booster synchrotron for injection into a 1--2 GeV storage ring. Interesting control problems are created because of the need for dynamic closed beam orbit control to eliminate interaction between the ring tuning requirements and to minimize orbit shifts due to ground vibrations. The extremely signal sensitive nature of the experiments requires special attention to the sources of electrical noise. These requirements have led to a control system design which emphasizes connectivity at the accelerator equipment end and a large I/O bandwidth for closed loop system response. Not overlooked are user friendliness, operator response time, modeling, and expert system provisions. Portable consoles are used for local operation of machine equipment. Our solution is a massively parallel system with >120 Mbits/sec I/O bandwidth and >1500 Mips computing power. At the equipment level connections are made using over 600 powerful Intelligent Local Controllers (ILC-s) mounted in 3U size Eurocard slots using fiber-optic cables between rack locations. In the control room, personal computers control and display all machine variables at a 10 Hz rate including the scope signals which are collected though the control system. Commercially available software and industry standards are used extensively. Particular attention is paid to reliability, maintainability and upgradeability. 10 refs., 11 figs.

  5. 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

  6. 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

  7. 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.

  8. 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

    2015-12-14

    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.

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

    PubMed Central

    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

  10. 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.

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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.

  16. Contributed review: Review of integrated correlative light and electron microscopy.

    PubMed

    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.

  17. 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.

  18. 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.

  19. 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.

  20. Light and electron microscopy of classical Ehlers-Danlos syndrome.

    PubMed

    de Almeida, Hiram L; Bicca, Eduardo; Rocha, Nara M; de Castro, Luis A S

    2013-02-01

    A 12-year-old boy with difficulty in wound healing and abnormal scars since early childhood was examined. Light microscopy showed loose and disperse dermal collagen with rare bundles, and fibroblasts show an irregular morphology. The fibrous sheath of hair presented a normal parallel distribution of the collagen fibers with normal spindle-shaped fibroblasts. Transmission electron microscopy also found disorganized collagen fibers, which were seen in a same field in longitudinal and cross sections. With high magnifications, an amorphous substance was seen near to loose collagen fibers, which showed variable diameters in cross sections. Scanning electron microscopy of the dermis showed disorganized collagen fibers and with higher magnification, important collagen disarrangement was observed with isolated and crossed-over fibers.

  1. Light Sheet Microscopy for Single Molecule Tracking in Living Tissue

    PubMed Central

    Ritter, Jörg Gerhard; Veith, Roman; Veenendaal, Andreas; Siebrasse, Jan Peter; Kubitscheck, Ulrich

    2010-01-01

    Single molecule observation in cells and tissue allows the analysis of physiological processes with molecular detail, but it still represents a major methodological challenge. Here we introduce a microscopic technique that combines light sheet optical sectioning microscopy and ultra sensitive high-speed imaging. By this approach it is possible to observe single fluorescent biomolecules in solution, living cells and even tissue with an unprecedented speed and signal-to-noise ratio deep within the sample. Thereby we could directly observe and track small and large tracer molecules in aqueous solution. Furthermore, we demonstrated the feasibility to visualize the dynamics of single tracer molecules and native messenger ribonucleoprotein particles (mRNPs) in salivary gland cell nuclei of Chironomus tentans larvae up to 200 µm within the specimen with an excellent signal quality. Thus single molecule light sheet based fluorescence microscopy allows analyzing molecular diffusion and interactions in complex biological systems. PMID:20668517

  2. 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.

  3. 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.

  4. Visualizing aquatic bacteria by light and transmission electron microscopy.

    PubMed

    Silva, Thiago P; Noyma, Natália P; Duque, Thabata L A; Gamalier, Juliana P; Vidal, Luciana O; Lobão, Lúcia M; Chiarini-Garcia, Hélio; Roland, Fábio; Melo, Rossana C N

    2014-01-01

    The understanding of the functional role of aquatic bacteria in microbial food webs is largely dependent on methods applied to the direct visualization and enumeration of these organisms. While the ultrastructure of aquatic bacteria is still poorly known, routine observation of aquatic bacteria by light microscopy requires staining with fluorochromes, followed by filtration and direct counting on filter surfaces. Here, we used a new strategy to visualize and enumerate aquatic bacteria by light microscopy. By spinning water samples from varied tropical ecosystems in a cytocentrifuge, we found that bacteria firmly adhere to regular slides, can be stained by fluorochoromes with no background formation and fast enumerated. Significant correlations were found between the cytocentrifugation and filter-based methods. Moreover, preparations through cytocentrifugation were more adequate for bacterial viability evaluation than filter-based preparations. Transmission electron microscopic analyses revealed a morphological diversity of bacteria with different internal and external structures, such as large variation in the cell envelope and capsule thickness, and presence or not of thylakoid membranes. Our results demonstrate that aquatic bacteria represent an ultrastructurally diverse population and open avenues for easy handling/quantification and better visualization of bacteria by light microscopy without the need of filter membranes.

  5. 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

  6. 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

  7. Design, assembly and calibration of white-light microscopy interferometer

    NASA Astrophysics Data System (ADS)

    Wang, Shuai; Gao, Zhishan; Li, Minjue; Ye, Jingfei; Cheng, Jinlong; Yang, Zhongming; Yuan, Qun

    2015-10-01

    A white-light microscopy interferometer was developed for measurement of the 3D profile and roughness.10X, 20X and 50X Mirau interference microscope objectives with the numerical aperture of 0.3, 0.4 and 0.55 were designed, manufactured and then provided as the accessories. Thickness deviation between beam splitter plate and reference mirror plate as well as the numerical aperture will both affect the contrast of interference fringe, according to optical modeling and image evaluation. The former would generate dispersion and then decrease the fringe contrast, while the latter would not produce dispersion separately but impact the amount of dispersion when thickness deviation exists, and their influence on fringe contrast was based on the expression of white-light interference intensity. Simulations for interference fringes from Mirau interference microscope objectives with different NA and thickness deviation were implemented, demonstrated that the fringe contrast will be falling with NA and thickness deviation increasing. A standard step with the nominal step value of 110 nm was used to calibrate the white-light microscopy interferometer, showing that less than1nm deviation can be reached.

  8. Optical coherence photoacoustic microscopy: accomplishing optical coherence tomography and photoacoustic microscopy with a single light source

    PubMed Central

    Zhang, Xiangyang; Zhang, Hao F.

    2012-01-01

    Abstract. We developed optical coherence photoacoustic microscopy (OC-PAM) to demonstrate that the functions of optical coherence tomography (OCT) and photoacoustic microscopy (PAM) can be achieved simultaneously by using a single illuminating light source. We used a pulsed broadband laser centered at 580 nm and detected the absorbed photons through photoacoustic detection and the back-scattered photons with an interferometer. In OC-PAM, each laser pulse generates both one OCT A-line and one PAM A-line simultaneously; as a result, the two imaging modalities are intrinsically co-registered in the lateral directions. In vivo images of the mouse ear were acquired to demonstrate the capabilities of OC-PAM. PMID:22502553

  9. Measuring Cell Death by Trypan Blue Uptake and Light Microscopy.

    PubMed

    Crowley, Lisa C; Marfell, Brooke J; Christensen, Melinda E; Waterhouse, Nigel J

    2016-01-01

    Trypan blue is a colorimetric dye that stains dead cells with a blue color easily observed using light microscopy at low resolution. The staining procedure is rapid and cells can be analyzed within minutes. The number of live (unstained) and dead (blue) cells can be counted using a hemocytometer on a basic upright microscope. Trypan blue staining is therefore a convenient assay for rapidly determining the overall viability of cells in a culture before commencing scientific experimentation, or for quantitating cell death following treatment with any cytotoxic stimuli. PMID:27371594

  10. Application of Light Microscopy to Direct Coal Liquefaction Research

    PubMed

    Irdi; Warzinski; Booher

    1998-01-01

    : Light microscopy was used to analyze the effects of added catalyst at different conditions (temperature and reaction times) in liquefaction testing of a low pyritic sulfur bituminous coal. Quantitative changes in vitrinite/vitroplast reflectance of coal and liquefaction residues were shown to be useful markers in analyzing and understanding the role of catalyst during the initial stage of coal particle hydrogenation. Lower reflectance values corresponded to increased conversions up to about the 60 min and 375 degreesC experimental conditions. Microscopical observation of liquefaction residues also revealed the presence of "wall scales" of varying width.

  11. 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.

  12. 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.

  13. White-light interferometric microscopy for wafer defect inspection

    NASA Astrophysics Data System (ADS)

    Zhou, Renjie; Edwards, Christopher; Bryniarski, Casey; Dallmann, Marjorie F.; Popescu, Gabriel; Goddard, Lynford L.

    2015-03-01

    White-light imaging systems are free of laser-speckle. Thus, they offer high sensitivity for optical defect metrology, especially when used with interferometry based quantitative phase imaging. This can be a potential solution for wafer inspection beyond the 9 nm node. Recently, we built a white-light epi-illumination diffraction phase microscopy (epi-wDPM) for wafer defect inspection. The system is also equipped with an XYZ scanning stage and real-time processing. Preliminary results have demonstrated detection of 15 nm by 90 nm in a 9 nm node densely patterned wafer with bright-field imaging. Currently, we are implementing phase imaging with epi-wDPM for additional sensitivity.

  14. 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

  15. 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

  16. 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

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

    PubMed

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

    2015-10-01

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

  18. The Light Microscopy Module Design and Performance Demonstrations

    NASA Astrophysics Data System (ADS)

    Motil, S. M.; Snead, J. H.; Griffin, D. W.; Hovenac, E. A.

    2002-01-01

    The Light Microscopy Module (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 Fluids and Combustion Facility (FCF) Fluids Integrated Rack (FIR) on the International Space Station (ISS). The LMM recently completed the preliminary design phase, and is progressing toward engineering model development. This paper will provide a description of the LMM preliminary design, LMM planned capabilities and key features, a summary of demonstrations for each of the diagnostics, and future plans. In addition, a brief description of the initial four experiments will be provided. Key diagnostic capabilities for meeting science requirements include video microscopy to observe microscopic phenonema 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. Sample cell design allows investigators to perform experiments and observations on a variety of sample materials, including potential biological specimens. The LMM features high resolution video microscopy, brightfield, darkfield, phase contrast, differential interference contrast (DIC), and spectrophotometry in the visible 400 nm to 700 nm range. Laser tweezers is a custom-built system, shown to be capable of trapping up to a 5x5 array of colloidal particles. The LMM confocal system includes a commercial-off-the-shelf unit which utilizes a 532 nm frequency doubled Nd:Yag laser. This instrument has been demonstrated to provide images up to 80 microns deep in collodial samples. Spectrophotometry is a custom-built system that utilizes existing optical components within the microscope in addition

  19. 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.

  20. The dento-gingival junction as seen with light microscopy and scanning electron microscopy.

    PubMed

    Garnick, J J; Ringle, R D

    1988-06-01

    The purpose of this paper is to review the anatomical relationship of the Dento-Gingival Junction as seen in the human dentition. The junction is described under light microscopy and then reviewed as seen in the SEM with the author's unpublished findings. The authors' material was derived from extracted human teeth with remaining marginal gingival tissue. The specimens were fixed with 2% glutaraldehyde in 0.15M sodium cacodylate buffer (pH 7.2) for 24 h. The specimens were then washed and freeze-fractured in Freon 113 using liquid nitrogen. Afterwards they were processed by freeze-drying or CPD methods, coated with gold, and placed in the scanning electron microscope (SEM) for viewing. These specimens demonstrated the presence of numerous Sharpey's fibers at the cemental surface. A large number of fibrils intermingled with the fibers to produce a dense mass of tissue. Junctional epithelium, with the adjacent homogeneous dental cuticle was demonstrated. Plaque deposits on the tooth surface extended to a cell-free zone. Morphological detail viewed with SEM and light microscopy are compared. PMID:3041569

  1. 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

  2. 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

  3. Quantitative polarized light microscopy of human cochlear sections

    PubMed Central

    Low, Jacob C. M.; Ober, Thomas J.; McKinley, Gareth H.; Stankovic, Konstantina M.

    2015-01-01

    Dysfunction of the inner ear is the most common cause of sensorineural hearing loss, which is the most common sensory deficit worldwide. Conventional imaging modalities are unable to depict the microanatomy of the human inner ear, hence the need to explore novel imaging modalities. We provide the first characterization of the polarization dependent optical properties of human cochlear sections using quantitative polarized light microscopy (qPLM). Eight pediatric cadaveric cochlear sections, aged 0 (term) to 24 months, were selected from the US National Temporal Bone Registry, imaged with qPLM and analyzed using Image J. Retardance of the bony otic capsule and basilar membrane were substantially higher than that of the stria vascularis, spiral ganglion neurons, organ of Corti and spiral ligament across the half turns of the spiraling cochlea. qPLM provides quantitative information about the human inner ear, and awaits future exploration in vivo. PMID:25780749

  4. 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.

  5. Rapid quantitative phase imaging for partially coherent light microscopy.

    PubMed

    Rodrigo, José A; Alieva, Tatiana

    2014-06-01

    Partially coherent light provides promising advantages for imaging applications. In contrast to its completely coherent counterpart, it prevents image degradation due to speckle noise and decreases cross-talk among the imaged objects. These facts make attractive the partially coherent illumination for accurate quantitative imaging in microscopy. In this work, we present a non-interferometric technique and system for quantitative phase imaging with simultaneous determination of the spatial coherence properties of the sample illumination. Its performance is experimentally demonstrated in several examples underlining the benefits of partial coherence for practical imagining applications. The programmable optical setup comprises an electrically tunable lens and sCMOS camera that allows for high-speed measurement in the millisecond range.

  6. Emission characteristics of light-emitting diodes by confocal microscopy

    NASA Astrophysics Data System (ADS)

    Cheung, W. S.; Choi, H. W.

    2016-03-01

    The emission profiles of light-emitting diodes have typically be measured by goniophotometry. However this technique suffers from several drawbacks, including the inability to generate three-dimensional intensity profiles as well as poor spatial resolution. These limitations are particularly pronounced when the technique is used to compared devices whose emission patterns have been modified through surface texturing at the micrometer and nanometer scales,. In view of such limitations, confocal microscopy has been adopted for the study of emission characteristics of LEDs. This enables three-dimensional emission maps to be collected, from which two-dimensional cross-sectional emission profiles can be generated. Of course, there are limitations associated with confocal microscopy, including the range of emission angles that can be measured due to the limited acceptance angle of the objective. As an illustration, the technique has been adopted to compare the emission profiles of LEDs with different divergence angles using an objective with a numerical aperture of 0.8. It is found that the results are consistent with those obtained by goniophotometry when the divergence angle is less that the acceptance angle of the objective.

  7. Advanced Light Source Linac subharmonic buncher cavities

    SciTech Connect

    Lo, C.C.; Taylor, B.; Lancaster, H.; Guigli, J.

    1989-03-01

    The Linear Accelerator (Linac) in the Advanced Light Source (ALS) is designed to provide either single or multiple bunches of 50 MeV electrons for the booster synchrotron. Three bunchers are used in the Linac. The 3 GHz S band buncher has been described elsewhere. This report deals with the two lower subharmonic bunchers. One operates at 124.914 MHz while the other operates at 499.654 MHz. 12 refs., 2 figs.

  8. Advanced Motion Compensation Methods for Intravital Optical Microscopy.

    PubMed

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

    2014-03-01

    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.

  9. 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

  10. Advanced analytical electron microscopy for alkali-ion batteries

    DOE PAGES

    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

  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. 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

  13. 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.

  14. 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

  15. Use of polarized light microscopy in porcine reproductive technologies.

    PubMed

    Caamaño, J N; Maside, C; Gil, M A; Muñoz, M; Cuello, C; Díez, C; Sánchez-Osorio, J R; Martín, D; Gomis, J; Vazquez, J M; Roca, J; Carrocera, S; Martinez, E A; Gómez, E

    2011-09-01

    The meiotic spindle in the oocyte is composed of microtubules and plays an important role during chromosome alignment and separation at meiosis. Polarized light microscopy (PLM) could be useful for a non-invasive evaluation of the meiotic spindle and may allow removal of nuclear structures without fluorochrome staining and ultraviolet exposure. In this study, PLM was used to assess its potential application in porcine reproductive technologies. The objectives of the present study were to assess the efficiency of PLM to detect microtubule-polymerized protein in in vitro-matured porcine oocytes; to examine its effects on the oocyte developmental competence; to select oocytes based on the presence of the meiotic spindle detected by PLM; and to assess the efficiency oocyte enucleation assisted with PLM. In the first experiment, the presence of microtubule-polymerized protein was assessed and confirmed in oocytes (n = 117) by immunostaining and chromatin detection. In the second experiment, oocytes (n = 160) were exposed or not (controls) to PLM for 10 minutes, and then parthenogenetically activated and cultured in vitro. In the third experiment, development competence of oocytes with a positive or negative signal to PLM was analyzed after in vitro fertilization. Finally, oocytes (n = 54) were enucleated using PLM as a tool to remove the meiotic spindle. A positive PLM signal was detected in 98.2 % of the oocytes, which strongly correlated (r = 1; p < 0.0001) with the presence of microtubule-polymerized protein as confirmed by immunostaining. Oocytes exposed to PLM did not differ significantly from controls on cleavage, total blastocyst, expanded blastocyst rates and total cell numbers. The percentage of oocytes at the MII stage and blastocyst formation rate in the negative PLM group significantly differed from control and PLM positive groups. Overall efficiency of spindle removal using the PLM-Oosight system was 92.6%. These results suggest that polarized light

  16. 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.

  17. The Advanced Light Source elliptically polarizing undulator

    SciTech Connect

    Marks, S.; Cortopassi, C.; DeVries, J.

    1997-05-01

    An elliptically polarizing undulator (EPU) for the Advanced Light Source (ALS) has been designed and is currently under construction. The magnetic design is a moveable quadrant pure permanent magnet structure featuring adjustable magnets to correct phase errors and on-axis field integrals. The device is designed with a 5.0 cm period and will produce variably polarized light of any ellipticity, including pure circular and linear. The spectral range at 1.9 GeV for typical elliptical polarization with a degree of circular polarization greater than 0.8 will be from 100 eV to 1,500 eV, using the first, third, and fifth harmonics. The device will be switchable between left and right circular modes at a frequency of up to 0.1 Hz. The 1.95 m long overall length will allow two such devices in a single ALS straight sector.

  18. 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

  19. Chemical Dynamics at the Advanced Light Source

    SciTech Connect

    Baer, T.; Berrah, N.; Fadley, C.; Moore, C.B.; Neumark, D.M.; Ng, C.Y.; Ruscic, B.; Smith, N.V.; Suits, A.G.; Wodtke, A.M.

    1999-02-02

    A day-long retreat was held January 15, 1999 to chart the future directions for chemical dynamics studies at the Advanced Light Source. This represents an important period for the Chemical Dynamics Beamline, as the hardware is well-developed, most of the initial experimental objectives have been realized and the mission is now to identify the future scientific priorities for the beamline and attract users of the highest caliber. To this end, we have developed a detailed scientific program for the near term; identified and prioritized the long range scientific opportunities, identified essential new hardware, and outlined an aggressive outreach program to involve the chemical physics community.

  20. Quantitative polarized light microscopy of unstained mammalian cochlear sections

    PubMed Central

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

    2013-01-01

    Abstract. 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

  1. Spermatozoa characteristics in six psittacine species using light microscopy.

    PubMed

    Stelzer, G; Schmidt, V; Sobiraj, A; Krautwald-Junghanns, M-E

    2009-12-01

    Even though breeding of companion birds has increased continuously for years, the fecundity assessment of birds has hardly been acknowledged. Knowledge of the structure of spermatozoa is crucial for evaluation of the basic reproductive biology of any species as well as for phylogenetic research and cladistic analyses of internal relationships. Spermatozoa of six different psittacine species (Nymphicus hollandicus, Myiopsitta monachus, Agapornis roseicollis, Melopsittacus undulatus, Tanygnathus lucionensis, Guarouba guarouba) were examined using light microscopy. Head length (nucleus including acrosome), head width, midpiece length and tail length were measured and documented. Significant differences were obvious among almost all of the species for almost all four parameters. However, in all the six species a significant moderate correlation between spermatozoa midpiece lengths and tail lengths (r=0.535, p

  2. [Light and electron microscopy of rhinoscleroma (author's transl)].

    PubMed

    Balázs, M; Elö, J; Juhász, J

    1975-02-01

    The authors report the case of a 50 year old male patient whose rhinoscleroma, localized to the upper respiratory tract, was demonstrated by the isolation of Klebsiella bacilli and histologically. Electron microscopically the Mikulicz cells were characterized by fused vacuoles occupying the largest portion of the cytoplasm and displacing the damaged cytoplasmic organelles. Phagosomes and dense bodies reminiscent of Russel bodies also occurred in the Mikulicz cells, in the vacuoles of which formations representing Klebsiella rhinoscleromatis were demonstrated. A light halo was visible around some of these formations. It could not be, however, decided whether these halos represented the mucous sheath of the bacillus or an artifact only. In the plasmacells the authors observed the bag-like dilatation of the ergastoplasm and the presence of Russel bodies. Transitory forms were not seen among the plasma and Mikulicz cells. As a result of the treatment, Klebsiella disappeared from the nasal mucosa of the patient. The authors wish to follow by means of electron microscopy the changes of the granulation tissue and pathogens following antibiotic therapy. PMID:1225879

  3. 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.

  4. 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

  5. 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

  6. Methods for studying tooth root cementum by light microscopy

    PubMed Central

    Foster, Brian L

    2012-01-01

    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

  7. 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.

  8. 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...

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

    PubMed

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

    2016-08-01

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

  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.

  11. 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.

  12. 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

  13. Seeing the forest tree by tree: super-resolution light microscopy meets the neurosciences.

    PubMed

    Maglione, Marta; Sigrist, Stephan J

    2013-07-01

    Light microscopy can be applied in vivo and can sample large tissue volumes, features crucial for the study of single neurons and neural circuits. However, light microscopy per se is diffraction-limited in resolution, and the substructure of core signaling compartments of neuronal circuits--axons, presynaptic active zones, postsynaptic densities and dendritic spines-can be only insufficiently characterized by standard light microscopy. Recently, several forms of super-resolution light microscopy breaking the diffraction-imposed resolution limit have started to allow highly resolved, dynamic imaging in the cell-biologically highly relevant 10-100 nanometer range ('mesoscale'). New, sometimes surprising answers concerning how protein mobility and protein architectures shape neuronal communication have already emerged. Here we start by briefly introducing super-resolution microscopy techniques, before we describe their use in the analysis of neuronal compartments. We conclude with long-term prospects for super-resolution light microscopy in the molecular and cellular neurosciences.

  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.

  15. 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

  16. 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

  17. 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

  18. Visualization of opiate receptor upregulation by light microscopy autoradiography.

    PubMed Central

    Tempel, A; Gardner, E L; Zukin, R S

    1984-01-01

    Light microscopy autoradiography has been used to visualize neuroanatomical patterns of brain opiate receptor upregulation in response to chronic naltrexone administration. Slide-mounted brain sections of frozen rat brain were labeled in vitro with dihydro[3H]morphine, a relatively selective mu opioid ligand. The greatest relative increases in opiate receptor density were observed in the nucleus accumbens, the amygdala, striatal patches, nuclei of the thalamus and hypothalamus, layers I and III of neocortex, substantia nigra compacta, midbrain periaqueductal gray regions, and the parabrachial nuclei of the brainstem. The substantia nigra reticulata, surrounding areas of striatal patches, and the locus ceruleus, were not affected by this drug treatment. These findings demonstrate that chronically administered naltrexone differentially regulates opiate receptors throughout the brain. In particular, three brain systems appear to be target areas of receptor upregulation : (i) the dopamine A9/A10 systems, (ii) the limbic system, and (iii) structures that receive input from afferent sensory pathways. Two possible mechanisms to account for this finding are (i) that the drug does not have uniform effects throughout the brain or (ii) that the receptors themselves may be associated with different functional systems. Receptor density changes are paralleled by increases in methionine-enkephalin content in the striatum, nucleus accumbens, periaqueductal gray, and hypothalamic areas of chronic naltrexone-treated rats relative to control rats. Thus opiate receptors and opioid peptides appear to be subject to regulatory mechanisms similar to those that modulate other neurotransmitters and their receptors. These results document in a visual manner brain patterns of opiate receptor upregulation . Images PMID:6328530

  19. 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

  20. 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

  1. 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.

  2. 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.

  3. 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)

  4. Characterization of Fatigue Fracture in Ni-20 Pct Cr Alloys Using White Light Interference Microscopy and Scanning Probe Microscopy

    NASA Astrophysics Data System (ADS)

    Nalladega, V.; Sathish, S.; Abburi, S.; Gigliotti, M. F. X.; Subramanian, P. R.

    2011-04-01

    Nanostructured and ultra-fine-grained metals and alloys are becoming of engineering interest. However, little is known about the influence of grain refinement on fatigue crack behavior. In this study, fatigue crack growth behavior and the key microstructural features controlling the fatigue fracture in nanocrystalline and ultra-fine-grain nickel alloys, processed using different techniques, were investigated. White light interference microscopy as well as the combination of atomic force microscopy (AFM) and ultrasonic force microscopy (UFM) were used to characterize the fractured surfaces of the metals. The role of grain size on the fatigue crack growth resistance and the effect of fracture surface roughness on the crack growth rate were evaluated. The combination of AFM and UFM is presented as a complementary tool to scanning electron microscopy in the fractography of metals.

  5. 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

  6. 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)

  7. 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

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

    PubMed

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

    2012-12-01

    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.

  9. Topographic contrast of ultrathin cryo-sections for correlative super-resolution light and electron microscopy

    PubMed Central

    Mateos, José María; Guhl, Bruno; Doehner, Jana; Barmettler, Gery; Kaech, Andres; Ziegler, Urs

    2016-01-01

    Fluorescence microscopy reveals molecular expression at nanometer resolution but lacks ultrastructural context information. This deficit often hinders a clear interpretation of results. Electron microscopy provides this contextual subcellular detail, but protein identification can often be problematic. Correlative light and electron microscopy produces complimentary information that expands our knowledge of protein expression in cells and tissue. Inherent methodological difficulties are however encountered when combining these two very different microscopy technologies. We present a quick, simple and reproducible method for protein localization by conventional and super-resolution light microscopy combined with platinum shadowing and scanning electron microscopy to obtain topographic contrast from the surface of ultrathin cryo-sections. We demonstrate protein distribution at nuclear pores and at mitochondrial and plasma membranes in the extended topographical landscape of tissue. PMID:27666401

  10. 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

  11. 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

  12. 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.

  13. Particle Shape Characterization of Lunar Regolith using Reflected Light Microscopy

    NASA Astrophysics Data System (ADS)

    McCarty, C. B.; Garcia, G. C.; Rickman, D.

    2014-12-01

    Automated identification of particles in lunar thin sections is necessary for practical measurement of particle shape, void characterization, and quantitative characterization of sediment fabric. This may be done using image analysis, but several aspects of the lunar regolith make such automations difficult. For example, many of the particles are shattered; others are aggregates of smaller particles. Sieve sizes of the particles span 5 orders of magnitude. The physical thickness of a thin section, at a nominal 30 microns, is large compared to the size of many of the particles. Image acquisition modes, such as SEM and reflected light, while superior to transmitted light, still have significant ambiguity as to the volume being sampled. It is also desirable to have a technique that is inexpensive, not resource intensive, and analytically robust. To this end, we have developed an image acquisition and processing protocol that identifies and delineates resolvable particles on the front surface of a lunar thin section using a petrographic microscope in reflected light. For a polished thin section, a grid is defined covering the entire thin section. The grid defines discrete images taken with 20% overlap, minimizing the number of particles that intersect image boundaries. In reflected light mode, two images are acquired at each grid location, with a closed aperture diaphragm. One image, A, is focused precisely on the front surface of the thin section. The second image, B, is made after the stage is brought toward the objective lens just slightly. A bright fringe line, analogous to a Becke line, appears inside all transparent particles at the front surface of the section in the second image. The added light in the bright line corresponds to a deficit around the particles. Particle identification is done using ImageJ and uses multiple steps. A hybrid 5x5 median filter is used to make images Af and Bf. This primarily removes very small particles just below the front surface

  14. Advanced microscopy techniques resolving complex precipitates in steels

    NASA Astrophysics Data System (ADS)

    Saikaly, W.; Soto, R.; Bano, X.; Issartel, C.; Rigaut, G.; Charaï, A.

    1999-06-01

    Scanning electron microscopy as well as analytical transmission electron microscopy techniques such as high resolution, electron diffraction, energy dispersive X-ray spectrometry (EDX), parallel electron energy loss spectroscopy (PEELS) and elemental mapping via a Gatan Imaging Filter (GIF) have been used to study complex precipitation in commercial dual phase steels microalloyed with titanium. Titanium nitrides, titanium carbosulfides, titanium carbonitrides and titanium carbides were characterized in this study. Both carbon extraction replicas and thin foils were used as sample preparation techniques. On both the microscopic and nanometric scales, it was found that a large amount of precipitation occurred heterogeneously on already existing inclusions/precipitates. CaS inclusions (1 to 2 μm), already present in liquid steel, acted as nucleation sites for TiN precipitating upon the steel's solidification. In addition, TiC nucleated on existing smaller TiN (around 30 to 50 nm). Despite the complexity of such alloys, the statistical analysis conducted on the non-equilibrium samples were found to be in rather good agreement with the theoretical equilibrium calculations. Heterogeneous precipitation must have played a role in bringing these results closer together.

  15. High-Speed Imaging of Amoeboid Movements Using Light-Sheet Microscopy

    PubMed Central

    Takeda, Takaaki; Sonobe, Seiji; Nonaka, Shigenori

    2012-01-01

    Light-sheet microscopy has been developed as a powerful tool for live imaging in biological studies. The efficient illumination of specimens using light-sheet microscopy makes it highly amenable to high-speed imaging. We therefore applied this technology to the observation of amoeboid movements, which are too rapid to capture with conventional microscopy. To simplify the setup of the optical system, we utilized the illumination optics from a conventional confocal laser scanning microscope. Using this set-up we achieved high-speed imaging of amoeboid movements. Three-dimensional images were captured at the recording rate of 40 frames/s and clearly outlined the fine structures of fluorescent-labeled amoeboid cellular membranes. The quality of images obtained by our system was sufficient for subsequent quantitative analysis for dynamics of amoeboid movements. This study demonstrates the application of light-sheet microscopy for high-speed imaging of biological specimens. PMID:23227214

  16. Consecutive light microscopy, scanning-transmission electron microscopy and transmission electron microscopy of traumatic human brain oedema and ischaemic brain damage.

    PubMed

    Castejon, O J; Castejon, H V; Diaz, M; Castellano, A

    2001-10-01

    Cortical biopsies of 11 patients with traumatic brain oedema were consecutively studied by light microscopy (LM) using thick plastic sections, scanning-transmission electron microscopy ((S)TEM) using semithin plastic sections and transmission electron microscopy (TEM) using ultrathin sections. Samples were glutaraldehyde-osmium fixed and embedded in Araldite or Epon. Thick sections were stained with toluidine-blue for light microscopy. Semithin sections were examined unstained and uncoated for (S)TEM. Ultrathin sections were stained with uranyl and lead. Perivascular haemorrhages and perivascular extravasation of proteinaceous oedema fluid were observed in both moderate and severe oedema. Ischaemic pyramidal and non-pyramidal nerve cells appeared shrunken, electron dense and with enlargement of intracytoplasmic membrane compartment. Notably swollen astrocytes were observed in all samples examined. Glycogen-rich and glycogen-depleted astrocytes were identified in anoxic-ischaemic regions. Dark and hydropic satellite, interfascicular and perivascular oligodendrocytes were also found. The status spongiosus of severely oedematous brain parenchyma observed by LM and (S)TEM was correlated with the enlarged extracellular space and disrupted neuropil observed by TEM. The (S)TEM is recommended as a suitable technique for studying pathological processes in the central nervous system and as an informative adjunct to LM and TEM.

  17. Advances in light-gas gun technology

    NASA Technical Reports Server (NTRS)

    Cowan, P. L.; Murphy, J. R.

    1968-01-01

    Constant-area accelerator used with light-gas guns increases the velocity of accelerating projectiles. A disposable accelerator on the muzzle of the gun uses the energy and momentum of a primary projectile, launched by the gun, to achieve high velocities of a light secondary projectile accelerated from rest in the accelerator.

  18. 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.

  19. 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

  20. 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

  1. 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

  2. Quantitative Analysis of Autophagy using Advanced 3D Fluorescence Microscopy

    PubMed Central

    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 arginine1. 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 starvation4,5. Although the essential components of this pathway are well-characterized6,7,8,9, 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 microscopy11,12. 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 stages of

  3. 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

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

    PubMed

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

    2013-05-03

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

  5. 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

  6. 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.

  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. 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.

  9. Thin Dielectric Film Thickness Determination by Advanced Transmission Electron Microscopy

    NASA Astrophysics Data System (ADS)

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

    2003-12-01

    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 nonspecialists. 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 has been steadily improved reaching now into the sub-Ångstrom 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 article, 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 article is the proposal of a reproducible method for film thickness determination.

  10. 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.

  11. 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.

  12. 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

  13. 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.

  14. 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.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. A primer on the fundamental principles of light microscopy: Optimizing magnification, resolution, and contrast.

    PubMed

    Goodwin, Paul C

    2015-01-01

    The light microscope is an indispensable tool in the study of living organisms. Most biologists are familiar with microscopes, perhaps being first introduced to the wonders of the world of small things at a very early age. Yet, few fully comprehend the nature of microscopy and the basis of its utility. This review (re)-introduces the concepts of magnification, resolution, and contrast, and explores how they are intimately related and necessary for effective microscopy.

  2. 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.

  3. 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

  4. 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

  5. Multilayer mounting for long-term light sheet microscopy of zebrafish.

    PubMed

    Weber, Michael; Mickoleit, Michaela; Huisken, Jan

    2014-02-27

    Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

  6. 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

  7. Probing structures of nanomaterials using advanced electron microscopy methods, including aberration-corrected electron microscopy at the Angstrom scale.

    PubMed

    Gai, Pratibha L; Yoshida, Kenta; Shute, Carla; Jia, Xiaoting; Walsh, Michael; Ward, Michael; Dresselhaus, Mildred S; Weertman, Julia R; Boyes, Edward D

    2011-07-01

    Structural and compositional studies of nanomaterials of technological importance have been carried out using advanced electron microscopy methods, including aberration-corrected transmission electron microscopy (AC-TEM), AC-high angle annular dark field scanning TEM (AC-HAADF-STEM), AC-energy filtered TEM, electron-stimulated energy dispersive spectroscopy in the AC-(S)TEM and high-resolution TEM (HRTEM) with scanning tunneling microscopy (STM) holder. The AC-EM data reveal improvements in resolution and minimization in image delocalization. A JEOL 2200FS double-AC field emission gun TEM/STEM operating at 200 kV in the Nanocentre at the University of York has been used to image single metal atoms on crystalline supports in catalysts, grain boundaries in nanotwinned metals, and nanostructures of tetrapods. Joule heating studies using HRTEM integrated with an STM holder reveal in situ crystallization and edge reconstruction in graphene. Real-time in situ AC-HAADF-STEM studies at elevated temperatures are described. Dynamic in-column energy filtering in an AC environment provides an integral new approach to perform dynamic in situ studies with aberration correction. The new results presented here open up striking new opportunities for atomic scale studies of nanomaterials and indicate future development directions.

  8. 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).

  9. Light sheet microscopy of cleared mouse brains: aberrations effects caused by refractive index mismatch

    NASA Astrophysics Data System (ADS)

    Silvestri, Ludovico; Sacconi, Leonardo; Pavone, Francesco S.

    2013-06-01

    Fluorescence light sheet microscopy has known a true renaissance in the last years. In fact, since optical sectioning is achieved in a wide-field detection scheme, this technique allows high resolution three-dimensional imaging with high frame rate. Light sheet microscopy is therefore an ideal candidate for reconstructing macroscopic specimens with micron resolution: coupled with clearing protocols based on refractive index matching it has been exploited to image entire mouse brains without physical sectioning. Use of clearing protocols poses several challenges to light sheet microscopy. First of all, residual light scattering inside the tissue expands the excitation light sheet, leading to the excitation of out-of-focus planes, and thus frustrating the very principle of light sheet illumination. To reject out-of-focus contributions we recently coupled light sheet illumination with confocal detection, achieving significant contrast enhancement in real time. Another issue which often arises when working with clearing agents is the refractive index mismatch between the clearing and the medium objective design medium. This introduces severe spherical aberration, which leads to broadening of the point spread function and to a strong reduction in its peak value: When imaging deep (several mm) inside macroscopic specimens, the signal can be reduced by more than an order of magnitude. We investigated the possibility of correcting such spherical aberration by introducing extra optical devices in the detection path.

  10. 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.

  11. 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

  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. Bridging microscopes: 3D correlative light and scanning electron microscopy of complex biological structures.

    PubMed

    Lucas, Miriam S; Günthert, Maja; Gasser, Philippe; Lucas, Falk; Wepf, Roger

    2012-01-01

    The rationale of correlative light and electron microscopy (CLEM) is to collect data on different information levels--ideally from an identical area on the same sample--with the aim of combining datasets at different levels of resolution to achieve a more holistic view of the hierarchical structural organization of cells and tissues. Modern three-dimensional (3D) imaging techniques in light and electron microscopy opened up new possibilities to expand morphological studies into the third dimension at the nanometer scale and over various volume dimensions. Here, we present two alternative approaches to correlate 3D light microscopy (LM) data with scanning electron microscopy (SEM) volume data. An adapted sample preparation method based on high-pressure freezing for structure preservation, followed by freeze-substitution for multimodal en-bloc imaging or serial-section imaging is described. The advantages and potential applications are exemplarily shown on various biological samples, such as cells, individual organisms, human tissue, as well as plant tissue. The two CLEM approaches presented here are per se not mutually exclusive, but have their distinct advantages. Confocal laser scanning microscopy (CLSM) and focused ion beam-SEM (FIB-SEM) is most suitable for targeted 3D correlation of small volumes, whereas serial-section LM and SEM imaging has its strength in large-area or -volume screening and correlation. The second method can be combined with immunocytochemical methods. Both methods, however, have the potential to extract statistically relevant data of structural details for systems biology.

  14. 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

  15. 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

  16. 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.

  17. 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

  18. Salmonella 3, 10:r: toxicity in rabbit ileum and liver by light and electron microscopy.

    PubMed

    Yashroy, R C

    2000-01-01

    Salmonella 3, 10:r:- (a monophasic variety of otherwise diphasic serotypes such as S. weltevreden and S. simi) Cell_free filtrate, when introduded into rabbit ligated ileal loops causes fluid exsorption, as studied 18-hr after treatment. Light microscopic histology of treated ileum shows denudation of the columnar epithelium at several places, thereby allowing the passage of the toxic principle into circulation. An important target organ, liver shows extensive centrilobular necrosis, as observed by light microscopy. Transmission electron microscopy of ileum reveals opening of membrane junctions between the adjacent cells of epithelial lining of the treated ileum at places, and focal devitalization including formation of intra-cellular membranous inclusinos. Electron microscopy of liver shows extensive damage and swelling of cytoplasmic membranes. However, the areas of darkly staining lamellae of granulated endoplasmic reticulum are also seen in stacks as will as dispersed. These studies stress that Salmonella toxic substances can cause extensive damage to intestine and liver both.

  19. 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.

  20. 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.

  1. 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

  2. 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

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

    PubMed Central

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

    2016-01-01

    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

  4. 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

  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. Correlated Light and Electron Microscopy/Electron Tomography of Mitochondria In Situ

    PubMed Central

    Perkins, Guy A.; Sun, Mei G.; Frey, Terrence G.

    2009-01-01

    Three-dimensional light microscopy and three-dimensional electron microscopy (electron tomography) separately provide very powerful tools to study cellular structure and physiology, including the structure and physiology of mitochondria. Fluorescence microscopy allows one to study processes in live cells with specific labels and stains that follow the movement of labeled proteins and changes within cellular compartments but does not have sufficient resolution to define the ultrastructure of intracellular organelles such as mitochondria. Electron microscopy and electron tomography provide the highest resolution currently available to study mitochondrial ultrastructure but cannot follow processes in living cells. We describe the combination of these two techniques in which fluorescence confocal microscopy is used to study structural and physiologic changes in mitochondria within apoptotic HeLa cells to define the apoptotic timeframe. Cells can then be selected at various stages of the apoptotic timeframe for examination at higher resolution by electron microscopy and electron tomography. This is a form of “virtual” 4-dimensional electron microscopy that has revealed interesting structural changes in the mitochondria of HeLa cells during apoptosis. The same techniques can be applied, with modification, to study other dynamic processes within cells in other experimental contexts. PMID:19348881

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

    PubMed

    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

  8. 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

  9. Generation of extended light-sheets for single and multi-photon fluorescence microscopy

    NASA Astrophysics Data System (ADS)

    Purnapatra, Subhajit B.; Pratim Mondal, Partha

    2013-07-01

    We theoretically propose and computationally demonstrate the generation of extended light-sheet for fluorescence microscopy. This is made possible by the introduction of a specially designed double-window spatial filter that allows the light to pass through the periphery and center of a cylindrical lens. When illuminated with a plane wave, the proposed filter results in an extended depth-of-focus along with side-lobes which are due to other interferences in the transverse focal plane. Computational studies show a maximum extension of light-sheet by 3.38 times for single photon excitation and 3.68 times for multiphoton excitation as compared to state-of-art single plane illumination microscopy system. This technique may facilitate the study of large biological specimens (such as Zebrafish embryo and tissue) with high spatial resolution and reduced photobleaching.

  10. Dynamic Light Scattering Microscopy. A Novel Optical Technique to Image Submicroscopic Motions. I: Theory

    PubMed Central

    Dzakpasu, Rhonda; Axelrod, Daniel

    2004-01-01

    The theoretical basis of an optical microscope technique to image dynamically scattered light fluctuation decay rates (dynamic light scattering microscopy) is developed. It is shown that relative motions between scattering centers even smaller than the optical resolution of the microscope are sufficient to produce significant phase variations resulting in interference intensity fluctuations in the image plane. The timescale and time dependence for the temporal autocorrelation function of these intensity fluctuations is derived. The spatial correlation distance, which reports the average distance between constructive and destructive interference in the image plane, is calculated and compared with the pixel size, and the distance dependence of the spatial correlation function is derived. The accompanying article in this issue describes an experimental implementation of dynamic light scattering microscopy. PMID:15298930

  11. Sildenafil accelerates reentrainment of circadian rhythms after advancing light schedules

    PubMed Central

    Agostino, Patricia V.; Plano, Santiago A.; Golombek, Diego A.

    2007-01-01

    Mammalian circadian rhythms are generated by a master clock located in the suprachiasmatic nuclei and entrained by light-activated signaling pathways. In hamsters, the mechanism responsible for light-induced phase advances involves the activation of guanylyl cyclase, cGMP and its related kinase (PKG). It is not completely known whether interference with this pathway affects entrainment of the clock, including adaptation to changing light schedules. Here we report that cGMP-specific phosphodiesterase 5 is present in the hamster suprachiasmatic nuclei, and administration of the inhibitor sildenafil (3.5 mg/kg, i.p.) enhances circadian responses to light and decreases the amount of time necessary for reentrainment after phase advances of the light–dark cycle. These results suggest that sildenafil may be useful for treatment of circadian adaptation to environmental changes, including transmeridian eastbound flight schedules. PMID:17519328

  12. 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.

  13. Shedding light on cutaneous innate immune responses: the intravital microscopy approach.

    PubMed

    Jain, Rohit; Weninger, Wolfgang

    2013-04-01

    The skin is under constant assault by environmental factors and microbes. Innate immune cells in epidermis and dermis regulate immune responses against pathogens while maintaining tolerance against commensal bacteria and autoantigens. The introduction of intravital imaging approaches, in particular multiphoton microscopy, has enabled studying the cellular and molecular regulation of cutaneous immunity in real time within intact skin. Here, we discuss recent advances in our understanding of innate immune cell behaviour in the skin, as unravelled by intravital microscopy, with emphasis on the function of myeloid cells, including dendritic cells, neutrophils and monocytes.

  14. Nonlinear Microscopy with Shaped Laser Pulses - Shedding New Light on Tissue

    NASA Astrophysics Data System (ADS)

    Fischer, Martin

    2010-10-01

    The advent of ultrafast pulsed lasers substantially advanced studies of nonlinear optical effects by providing high peak intensities at low average power. When applied to microscopy in highly scattering tissue, the localized nature of the nonlinear interaction leads to high spatial resolution, optical sectioning, and larger possible imaging depth than linear methods. However, nonlinear contrast (other than fluorescence) is generally difficult to measure because it is overwhelmed by the large background of detected illumination light. This background can be suppressed by using femtosecond pulse shaping to encode nonlinear interactions in background-free regions of the frequency spectrum. We will discuss two techniques aimed at measuring nonlinear absorptive and nonlinear dispersive contrast, respectively. Nonlinear absorption offers a dramatically expanded range of molecular contrast, because not all markers that absorb photons fluoresce. We will describe a technique that utilizes shaped pulse trains of multiple colors, where an amplitude modulation of the pump beam is transferred onto the probe beam of a different wavelength, thereby generating a new frequency in the probe beam. Using this technique we have been able to detect non-fluorescent metabolic markers in tissue (e.g. the imaging of different types of melanin in pigmented lesions and the mapping of oxygenation in blood vessels). We also have developed a technique that is able to measure nonlinear phase contrast (e.g. self-phase modulation) in tissue with very moderate laser power. The key concept of this technique is the fact that nonlinear processes can create new frequency components within the pulse spectrum. We can efficiently detect these spectral changes by appropriately pre-shaping the spectrum such that the changes show against a small background. Using these pulse shaping techniques we have been able to detect nonlinear optical signatures of neuronal activity in live neurons.

  15. 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

  16. 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.

  17. 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

  18. 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

  19. 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.

  20. 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

  1. 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.

  2. 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.

  3. Materials and light thermal structures research for advanced space exploration

    NASA Technical Reports Server (NTRS)

    Thornton, Earl A.; Starke, Edgar A., Jr.; Herakovich, Carl T.

    1991-01-01

    The Light Thermal Structures Center at the University of Virginia sponsors educational and research programs focused on the development of reliable, lightweight structures to function in hostile thermal environments. Technology advances in materials and design methodology for light thermal structures will contribute to improved space vehicle design concepts with attendant weight savings. This paper highlights current research activities in three areas relevant to space exploration: low density, high temperature aluminum alloys, composite materials, and structures with thermal gradients. Advances in the development of new aluminum-lithium alloys and mechanically alloyed aluminum alloys are described. Material properties and design features of advanced composites are highlighted. Research studies in thermal structures with temperature gradients include inelastic panel buckling and thermally induced unstable oscillations. Current and future research is focused on the integration of new materials with applications to structural components with thermal gradients.

  4. 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

  5. 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.

  6. 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

  7. 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

  8. 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.

  9. 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)

  10. 3D print customized sample holders for live light sheet microscopy.

    PubMed

    Jeandupeux, Emeric; Lobjois, Valérie; Ducommun, Bernard

    2015-08-01

    A major hurdle to the widespread application of light sheet microscopy is the lack of versatile and non-intrusive sample holders that are adaptable to a variety of biological samples for live imaging. To overcome this limitation, we present herein the application of 3D printing to the fabrication of a fully customizable casting kit. 3D printing enables facile preparation of hydrogel sample holders adaptable to any shape and number of specimen. As an example, we present the use of this device to produce a four-sample holder adapted to parallel live monitoring of multicellular tumor spheroid growth. To share our solution with the light sheet microscopy community, all files necessary to produce or customize sample holders are freely available online.

  11. 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.

  12. 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

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

    NASA Astrophysics Data System (ADS)

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

    2009-11-01

    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.

  14. 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.

  15. 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

  16. Plasmolysis of Pteridium protoplasts: A study using light and scanning-electron microscopy.

    PubMed

    Attree, S M; Sheffield, E

    1985-08-01

    A study was undertaken using gametophytes of the fern Pteridium aquilinum to examine the effects of plasmolysis on the topography of protoplasts. Methods are described whereby the surfaces of non-isolated protoplasts can be observed in the plasmolysed condition using scanning electron microscopy. Plasmolysed gametophytes were also examined in the light microscope using differential interference contrast and ultra-violet fluorescence microscopy after staining with fluorescein diacetate. With scanning electron microscopy, plasmolysed protoplast surfaces appeared smooth with no evidence of wrinkling or infolding of excess membrane. The formation of irregular-shaped protoplasts, protoplasmic threads, subprotoplasts, and protoplasmic networks covering internal wall surfaces all provided evidence for strong wall adhesion of the protoplasm. The availability of membrane for uptake into folds or vesicles is therefore thought to be minimal. Transmission electron microscopy showed some protoplasmic threads to be plasmodesmata, the remainder being cell-wall contact points. Remnants of these threads were occasionally observed on isolated protoplasts in both the light and electron microscopes.

  17. 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

  18. 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.

  19. 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

  20. In situ observation of elementary growth processes of protein crystals by advanced optical microscopy.

    PubMed

    Sazaki, Gen; Van Driessche, Alexander E S; Dai, Guoliang; Okada, Masashi; Matsui, Takuro; Otálora, Fermin; Tsukamoto, Katsuo; Nakajima, Kazuo

    2012-07-01

    To start systematically investigating the quality improvement of protein crystals, the elementary growth processes of protein crystals must be first clarified comprehensively. Atomic force microscopy (AFM) has made a tremendous contribution toward elucidating the elementary growth processes of protein crystals and has confirmed that protein crystals grow layer by layer utilizing kinks on steps, as in the case of inorganic and low-molecular-weight compound crystals. However, the scanning of the AFM cantilever greatly disturbs the concentration distribution and solution flow in the vicinity of growing protein crystals. AFM also cannot visualize the dynamic behavior of mobile solute and impurity molecules on protein crystal surfaces. To compensate for these disadvantages of AFM, in situ observation by two types of advanced optical microscopy has been recently performed. To observe the elementary steps of protein crystals noninvasively, laser confocal microscopy combined with differential interference contrast microscopy (LCM-DIM) was developed. To visualize individual mobile protein molecules, total internal reflection fluorescent (TIRF) microscopy, which is widely used in the field of biological physics, was applied to the visualization of protein crystal surfaces. In this review, recent progress in the noninvasive in situ observation of elementary steps and individual mobile protein molecules on protein crystal surfaces is outlined.

  1. 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.

  2. The Advanced Light Source (ALS) Radiation Safety System. Revised

    SciTech Connect

    Ritchie, A.L.; Oldfather, D.E.; Lindner, A.F.

    1993-08-01

    The Advanced Light Source (ALS) at the Lawrence Berkeley Laboratory (LBL) is a 1.5 Gev synchrotron light source facility consisting of a 120 kev electron gun, 50 Mev linear accelerator, 1.5 Gev booster synchrotron, 200 meter circumference electron storage ring, and many photon beamline transport systems for research. Figure 1. ALS floor plan. Pairs of neutron and gamma radiation monitors are shown as dots numbered from 1 to 12. The Radiation Safety System for the ALS has been designed and built with a primary goal of providing protection against inadvertent personnel exposure to gamma and neutron radiation and, secondarily, to enhance the electrical safety of select magnet power supplies.

  3. 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

  4. 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

  5. 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

  6. 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

  7. Polarization of excitation light influences molecule counting in single-molecule localization microscopy.

    PubMed

    Chen, Ye; Lin, Han; Ludford-Menting, Mandy J; Clayton, Andrew H; Gu, Min; Russell, Sarah M

    2015-01-01

    Single-molecule localization microscopy has been widely applied to count the number of biological molecules within a certain structure. The percentage of molecules that are detected significantly affects the interpretation of data. Among many factors that affect this percentage, the polarization state of the excitation light is often neglected or at least unstated in publications. We demonstrate by simulation and experiment that the number of molecules detected can be different from -40 up to 100% when using circularly or linearly polarized excitation light. This is determined mainly by the number of photons emitted by single fluorescent molecule, namely the choice of fluorescence proteins, and the background noise in the system, namely the illumination scheme. This difference can be further exaggerated or mitigated by various fixation methods, magnification, and camera settings We conclude that the final choice between circularly or linearly polarized excitation light should be made experimentally, based on the signal to noise ratio of the system.

  8. Interaction of light and surface plasmon polaritons in Ag islands studied by nonlinear photoemission microscopy.

    PubMed

    Buckanie, N M; Kirschbaum, P; Sindermann, S; Meyer zu Heringdorf, F-J

    2013-07-01

    Two photon photoemission microscopy was used to study the interaction of femtosecond laser pulses with Ag islands prepared using different strategies on Si(111) and SiO₂. The femtosecond laser pulses initiate surface plasmon polariton (SPP) waves at the edges of the island. The superposition of the electrical fields of the femtosecond laser pulses with the electrical fields of the SPP results in a moiré pattern that is comparable despite the rather different methods of preparation and that gives access to the wavelength and direction of the SPP waves. If the SPPs reach edges of the Ag islands, they can be converted back into light waves. The incident and refracted light waves result in an interference pattern that can again be described with a moiré pattern, demonstrating that Ag islands can be used as plasmonic beam deflectors for light.

  9. Enhanced light element imaging in atomic resolution scanning transmission electron microscopy.

    PubMed

    Findlay, S D; Kohno, Y; Cardamone, L A; Ikuhara, Y; Shibata, N

    2014-01-01

    We show that an imaging mode based on taking the difference between signals recorded from the bright field (forward scattering region) in atomic resolution scanning transmission electron microscopy provides an enhancement of the detectability of light elements over existing techniques. In some instances this is an enhancement of the visibility of the light element columns relative to heavy element columns. In all cases explored it is an enhancement in the signal-to-noise ratio of the image at the light column site. The image formation mechanisms are explained and the technique is compared with earlier approaches. Experimental data, supported by simulation, are presented for imaging the oxygen columns in LaAlO₃. Case studies looking at imaging hydrogen columns in YH₂ and lithium columns in Al₃Li are also explored through simulation, particularly with respect to the dependence on defocus, probe-forming aperture angle and detector collection aperture angles.

  10. 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.

  11. 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.

  12. Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

    PubMed

    Abe, Jun; Ozga, Aleksandra J; Swoger, Jim; Sharpe, James; Ripoll, Jorge; Stein, Jens V

    2016-04-01

    Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues.

  13. Light sheet fluorescence microscopy for in situ cell interaction analysis in mouse lymph nodes.

    PubMed

    Abe, Jun; Ozga, Aleksandra J; Swoger, Jim; Sharpe, James; Ripoll, Jorge; Stein, Jens V

    2016-04-01

    Reactive lymph nodes (LNs) are sites where pMHC-loaded dendritic cells (DCs) interact with rare cognate T cells, leading to their clonal expansion. While DC interactions with T cell subsets critically shape the ensuing immune response, surprisingly little is known on their spatial orchestration at physiologically T cell low precursor frequencies. Light sheet fluorescence microscopy and one of its implementations, selective plane illumination microscopy (SPIM), is a powerful method to obtain precise spatial information of entire organs of 0.5-10mm diameter, the size range of murine LNs. Yet, its usefulness for immunological research has thus far not been comprehensively explored. Here, we have tested and defined protocols that preserve fluorescent protein function during lymphoid tissue clearing required for SPIM. Reconstructions of SPIM-generated 3D data sets revealed that calibrated numbers of adoptively transferred T cells and DCs are successfully detected at a single cell level within optically cleared murine LNs. Finally, we define parameters to quantify specific interactions between antigen-specific T cells and pMHC-bearing DCs in murine LNs. In sum, our studies describe the successful application of light sheet fluorescence microscopy to immunologically relevant tissues. PMID:26844990

  14. 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

  15. Studying kinetochore-fiber ultrastructure using correlative light-electron microscopy

    PubMed Central

    Prior, Ian A.; Royle, Stephen J.

    2013-01-01

    Electron microscopy (EM) has dominated high-resolution cellular imaging for over 50 years thanks to its ability to resolve on a nanometer-scale intracellular structures such as the microtubules of the mitotic spindle. It is advantageous to view the cell of interest prior to processing the sample for EM. Correlative light electron microscopy (CLEM) is a technique that allows one to visualize cells of interest by light microscopy (LM) before being transferred to EM for ultra-structural examination. Here we describe how CLEM can be applied as an effective tool to study the spindle apparatus of mitotic cells. This approach allows transfected cells of interest, in desirable stages of mitosis, to be followed from LM through to EM. CLEM has often been considered as a technically challenging and laborious technique. In this chapter we provide step-by-step pictorial guides that allow successful CLEM to be achieved. In addition we explain how it is possible to vary the sectioning plane, allowing spindles and microtubules to be analyzed from different angles, and the outputs that can be obtained from these methods when applied to the study of kinetochore fiber (K-fiber) ultrastructure. PMID:23973081

  16. Spectral fingerprinting of individual cells visualized by cavity-reflection-enhanced light-absorption microscopy.

    PubMed

    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.

  17. 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

  18. 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

  19. 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.

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

    PubMed

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

    2014-08-01

    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. This article is part of a Special Issue entitled Tools to study lipid functions.

  1. 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.

  2. [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

  3. 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

  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. 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

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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.

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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

  17. Reflectance confocal microscopy: an overview of technology and advances in telepathology.

    PubMed

    Batta, Mari M; Kessler, Stephen E; White, Peter F; Zhu, Weijian; Fox, Christi A

    2015-05-01

    The value of in vivo reflectance confocal microscopy (RCM) as a noninvasive adjunctive tool in dermatology has steadily advanced since its inception. With RCM, dermatologists can view horizontal sections of lesions in a resolution comparable to histology, observe dynamic processes in living skin, and monitor lesion evolution longitudinally. This article will compare RCM to dermoscopy and histology, review the general principles of the microscope, describe the findings seen on confocal images, and discuss the clinical applications of this noninvasive tool. Additionally, we describe a telepathology network dedicated to the transfer of confocal images to remote dermatopathologists for interpretation. Finally, we will discuss the adoption of RCM and the telepathology network in clinical practice.

  18. Quantum dot immunocytochemical localization of somatostatin in somatostatinoma by Widefield Epifluorescence, super-resolution light, and immunoelectron microscopy.

    PubMed

    Killingsworth, Murray C; Lai, Ken; Wu, Xiaojuan; Yong, Jim L C; Lee, C Soon

    2012-11-01

    Quantum dot nanocrystal probes (QDs) have been used for detection of somatostatin hormone in secretory granules of somatostatinoma tumor cells by immunofluorescence light microscopy, super-resolution light microscopy, and immunoelectron microscopy. Immunostaining for all modalities was done using sections taken from an epoxy resin-embedded tissue specimen and a similar labeling protocol. This approach allowed assessment of labeling at light microscopy level before examination at super-resolution and electron microscopy level and was a significant aid in interpretation. Etching of ultrathin sections with saturated sodium metaperiodate was a critical step presumably able to retrieve some tissue antigenicity masked by processing in epoxy resin. Immunofluorescence microscopy of QD-immunolabeled sections showed somatostatin hormone localization in cytoplasmic granules. Some variable staining of tumor gland-like structures appeared related to granule maturity and dispersal of granule contents within the tumor cell cytoplasm. Super-resolution light microscopy demonstrated localization of somatostatin within individual secretory granules to be heterogeneous, and this staining pattern was confirmed by immunoelectron microscopy.

  19. Toward subdiffraction transmission microscopy of diffuse materials with silver nanoparticle white-light beacons.

    PubMed

    Chaudhuri, Debansu; Galusha, Jeremy W; Walter, Manfred J; Borys, Nicholas J; Bartl, Michael H; Lupton, John M

    2009-03-01

    We demonstrate high resolution transmission microscopy in a conventional two-photon wide-field fluorescence microscope by exploiting nonlinear white light generation from clusters of silver nanoparticles placed beneath the specimen. Surface-enhanced two-photon luminescence occurs at nanoparticle hot spots in the form of spectrally broad, spatially confined light which can be exploited to determine the transmission properties of a sample placed on the silver nanoparticles. We demonstrate the versatility of the technique by revealing individual crystalline domains formed in the diffuse biological photonic crystals of the scales of a beetle. We can identify submicron changes between photonic crystal facets as well as the occurrence of stacked domains invisible to surface-sensitive methods. Control over wavelength, polarization, and pulse shape promises selective addressing of hot spots in nanoparticle assemblies for motionless spatial scanning of the transmission properties with subdiffraction resolution.

  20. In situ transmission electron microscopy of light-induced photocatalytic reactions.

    PubMed

    Cavalca, F; Laursen, A B; Kardynal, B E; Dunin-Borkowski, R E; Dahl, S; Wagner, J B; Hansen, T W

    2012-02-24

    Transmission electron microscopy (TEM) makes it possible to obtain insight into the structure, composition and reactivity of photocatalysts, which are of fundamental interest for sustainable energy research. Such insight can be used for further material optimization. Here, we combine conventional TEM analysis of photocatalysts with environmental TEM (ETEM) and photoactivation using light. Two novel types of TEM specimen holder that enable in situ illumination are developed to study light-induced phenomena in photoactive materials, systems and photocatalysts at the nanoscale under working conditions. The technological development of the holders is described and two representative photo-induced phenomena are studied: the photodegradation of Cu₂O and the photodeposition of Pt onto a GaN:ZnO photocatalyst.

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

    PubMed

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

    2016-03-01

    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

  2. Deconvolution-free Subcellular Imaging with Axially Swept Light Sheet Microscopy

    PubMed Central

    Dean, Kevin M.; Roudot, Philippe; Welf, Erik S.; Danuser, Gaudenz; Fiolka, Reto

    2015-01-01

    The use of propagation invariant Bessel beams has enabled high-resolution subcellular light sheet fluorescence microscopy. However, the energy within the concentric side lobe structure of Bessel beams increases significantly with propagation length, generating unwanted out-of-focus fluorescence that enforces practical limits on the imaging field of view size. Here, we present a light sheet fluorescence microscope that achieves 390 nm isotropic resolution and high optical sectioning strength (i.e., out-of-focus blur is strongly suppressed) over large field of views, without the need for structured illumination or deconvolution-based postprocessing. We demonstrate simultaneous dual-color, high-contrast, and high-dynamic-range time-lapse imaging of migrating cells in complex three-dimensional microenvironments, three-dimensional tracking of clathrin-coated pits, and long-term imaging spanning >10 h and encompassing >2600 time points. PMID:26083920

  3. SLM-based off-axis Fourier filtering in microscopy with white light illumination.

    PubMed

    Steiger, Ruth; Bernet, Stefan; Ritsch-Marte, Monika

    2012-07-01

    In various microscopy applications spatial light modulators (SLMs) are used as programmable Fourier filters to realize different optical contrast enhancement methods. It is often advantageous to use the SLM in off-axis configuration, where the filtered image wave is sent into the first diffraction order of a blazed grating superposed to the phase mask on the SLM. Because of dispersion this approach is, however, typically limited to spectrally narrowband illumination. Here we suggest a method involving a grating for pre-compensation, which allows one to use spectrally broadband (even thermal) light in SLM-based Fourier filtering. The proposed approach is demonstrated by multicolor imaging of amplitude and phase objects, such as a resolution target, onion epidermal cells and human epithelial cheek cells. PMID:22772234

  4. 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.

  5. 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

  6. Bacteria-clay interactions investigated by light scattering and phase contrast microscopy

    NASA Astrophysics Data System (ADS)

    Alimova, Alexandra; Block, Karin; Rudolph, Elizabeth; Katz, A.; Steiner, J. C.; Gottlieb, P.; Alfano, R. R.

    2006-02-01

    Light scattering experiments and phase contrast microscopy are used to evaluate the aggregate-forming characteristics of simple clay-bacteria mixtures. Colloidal suspensions of negatively charged Pseudomonas syringae (Ps) and Mg 2+-, Li + - or Ca 2+ -exchanged smectite (and non-exchanged smectite) are flocculated in neutral (pH 7) aqueous media. Aggregate formation is monitored using changes in optical transmission. Clustering is observed in all the clay-bacteria preparations. The Li +-substituted clay aggregates average 50-300 microns in diameter, in contrast to the Ca 2+- substituted clay that produces aggregates of 10-50 microns in diameter. Light scattering measurements indicate that aggregates begin forming 3 hours after mixing and that the (larger sized) aggregates exhibit less scattering than a mixture with an equivalent concentration of unattached Ps and clay particles.

  7. 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.

  8. 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

  9. 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

  10. Recent advancements in nanoelectrodes and nanopipettes used in combined scanning electrochemical microscopy techniques.

    PubMed

    Kranz, Christine

    2014-01-21

    In recent years, major developments in scanning electrochemical microscopy (SECM) have significantly broadened the application range of this electroanalytical technique from high-resolution electrochemical imaging via nanoscale probes to large scale mapping using arrays of microelectrodes. A major driving force in advancing the SECM methodology is based on developing more sophisticated probes beyond conventional micro-disc electrodes usually based on noble metals or carbon microwires. This critical review focuses on the design and development of advanced electrochemical probes particularly enabling combinations of SECM with other analytical measurement techniques to provide information beyond exclusively measuring electrochemical sample properties. Consequently, this critical review will focus on recent progress and new developments towards multifunctional imaging.

  11. Application of Advanced Atomic Force Microscopy Techniques to Study Quantum Dots and Bio-materials

    NASA Astrophysics Data System (ADS)

    Guz, Nataliia

    In recent years, there has been an increase in research towards micro- and nanoscale devices as they have proliferated into diverse areas of scientific exploration. Many of the general fields of study that have greatly affected the advancement of these devices includes the investigation of their properties. The sensitivity of Atomic Force Microscopy (AFM) allows detecting charges up to the single electron value in quantum dots in ambient conditions, the measurement of steric forces on the surface of the human cell brush, determination of cell mechanics, magnetic forces, and other important properties. Utilizing AFM methods, the fast screening of quantum dot efficiency and the differences between cancer, normal (healthy) and precancer (immortalized) human cells has been investigated. The current research using AFM techniques can help to identify biophysical differences of cancer cells to advance our understanding of the resistance of the cells against the existing medicine.

  12. 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

  13. 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.

  14. Angle-resolved second harmonic light scattering from colloidal suspensions and second harmonic particle microscopy

    NASA Astrophysics Data System (ADS)

    Yang, Ningping

    2001-08-01

    We have carried out two nonlinear optical experiments with colloidal particles. Our first nonlinear optical experiment studied Second-Harmonic Generation (SHG) light scattering from colloidal suspension. In particular, we measured the angle-resolved second-harmonic generation light scattering from suspensions of centrosymmetric micron-size polystyrene spheres with surface-adsorbed dye (malachite green). The second-harmonic scattering angular profiles differ qualitatively from the linear light scattering angular profiles of the same particles. We have investigated these radiation patterns using several polarization configurations and particle diameters. We introduce a simple Rayleigh-Gans-Debye model to account for the SHG scattering anisotropy. The model compares favorably with our experimental data. Our measurements suggest scattering anisotropy may be used to isolate particle nonlinear optical effect from other bulk nonlinear optical effects in suspension. Our second nonlinear optical experiment studied the Second-Harmonic Generation (SHG) from single micron-size particles. We built a nonlinear optical microscope for this purpose. We report experimental observations of second harmonic generation from single micron-size polystyrene (PS), silica, and PolyMethylMethAcrylate (PMMA) spheres on flat substrates by SHG microscopy. At low input light intensities the SH signals depend quadratically on the intensity of the excitation beam, but at larger input intensities some of the SH signals increase exponentially with increasing input intensity. This exponential enhancement depends on particle size and sphere composition. We describe the experiments, report the observations and provide an approximate analytical framework for understanding our measurements.

  15. Two-photon microscopy reveals early rod photoreceptor cell damage in light-exposed mutant mice

    PubMed Central

    Maeda, Akiko; Palczewska, Grazyna; Golczak, Marcin; Kohno, Hideo; Dong, Zhiqian; Maeda, Tadao; Palczewski, Krzysztof

    2014-01-01

    Atrophic age-related and juvenile macular degeneration are especially devastating due to lack of an effective cure. Two retinal cell types, photoreceptor cells and the adjacent retinal pigmented epithelium (RPE), reportedly display the earliest pathological changes. Abca4−/−Rdh8−/− mice, which mimic many features of human retinal degeneration, allowed us to determine the sequence of light-induced events leading to retinal degeneration. Using two-photon microscopy with 3D reconstruction methodology, we observed an initial strong retinoid-derived fluorescence and expansion of Abca4−/−Rdh8−/− mouse rod cell outer segments accompanied by macrophage infiltration after brief exposure of the retina to bright light. Additionally, light-dependent fluorescent compounds produced in rod outer segments were not transferred to the RPE of mice genetically defective in RPE phagocytosis. Collectively, these findings suggest that for light-induced retinopathies in mice, rod photoreceptors are the primary site of toxic retinoid accumulation and degeneration, followed by secondary changes in the RPE. PMID:24706832

  16. Optically stabilized mercury short-arc lamp as UV light source for microscopy

    NASA Astrophysics Data System (ADS)

    Heynen, Susanne; Gough, David A.; Price, Jeffrey H.

    1997-05-01

    For certain applications in microscopy, mercury vapor short arc lamps are utilized as UV-light sources because of their high intensity and their road spectrum. Unfortunately, they are also very unstable. Especially for single wavelength fluorescence image cytometry, there is a need for a stable, high intensity light source. Substantially improved stability was achieved using optical feedback and fiberoptic scrambling. The system uses a photodiode to monitor the light intensity, and feeds the readout back to a controller. The controller compares this readout to a preset reference voltage and adjusts the lamp supply current accordingly. The optical fiber scrambles the light to correct the effects of arc wander. Preliminary results of performance tests of this system show a coefficient of variation (CV) of less than 0.1 percent over 20 hours at a sample frequency of 30 Hz. This CV is a factor of 30 better than a conventional current stabilized mercy vapor short arc lamp. Scrambled optical feedback is a necessary addition for systems with mercury short arc lamps, especially for image fluorometry applications.

  17. 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.

  18. 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

  19. Application of micro-PIXE, MRI and light microscopy for research in wood science and dendroecology

    NASA Astrophysics Data System (ADS)

    Merela, M.; Pelicon, P.; Vavpetič, P.; Regvar, M.; Vogel-Mikuš, K.; Serša, I.; Poličnik, H.; Pokorny, B.; Levanič, T.; Oven, P.

    2009-06-01

    Beech ( Fagus sylvatica L.) branches were topped and after five months the wound response was analyzed by PIXE, 3D-MRI and light microscopy. From freshly cut and deeply frozen sample 30 μm thick longitudinal-radial tissue sections were prepared for anatomical investigations and micro-PIXE analysis. Light microscopy revealed the structural response to wounding, i.e. occurrence of the reaction zone between the exposed and dehydrated dead tissue and healthy sound wood. The reaction zone was characterized by tylosis in vessels and accumulation of colored deposits in parenchyma cells, fibres and vessels. 3D MRI of a parallel sample showed that the moisture content in the reaction zone was three times higher than in normal healthy wood. Micro-PIXE mapping at margins of compromised wood in beech revealed an increased concentration of potassium in the reaction zone. The increase in the calcium concentration was associated with the dehydrated tissue adjacent to reaction zones. In addition, micro-PIXE was used to determine the elemental distribution in annual tree rings. This may be relevant for retrospective assessment of environmental pollution in wood by measuring yearly increments as a biomonitoring tool. The analysis of European larch ( Larix decidua Mill.) wood revealed a high similarity between optical characteristics (i.e. late versus earlywood) and elemental (e.g. Cl, K, Ca, Mn, Zn) distribution.

  20. Problems in polarized light microscopy observation of birefringence of calcium pyrophosphate dihydrate crystals.

    PubMed

    Omura, Yoko; Okamoto, Renzo; Konno, Minoru; Shiro, Motoo

    2010-12-01

    To reconsider the problems arising from the use of the phase plate as a test plate inserted into a polarized light microscope system for the analysis of triclinic calcium pyrophosphate dihydrate (t-CPPD) crystals, or Ca(2)P(2)O(7) x 2H(2)O in the synovial fluid of arthritis patients, we made the polarized light microscopy observations using a phase plate with a retardation of 530 nm for the synthesized t-CPPD crystals well-characterized by X-ray powder pattern indexing and single crystal X-ray diffraction measurements. The microscopy observations were made of crystals of different sizes, thicknesses and shapes. The retardation was assessed using the interference color chart at four extinction and diagonal positions both with and without the test plate. The addition and subtraction states produced by superimposing the retardations of two anisotropic materials, that is, the t-CPPD crystal and the 530 nm phase plate, were deduced from the interference color change by inserting the test plate at four diagonal positions. When the color change of a crystal at a diagonal position resulting from 90-degree rotation exhibits no clear birefringence, the interference color chart was shown to be useless. We suggested the use of a compensator whose retardation can be changed to obtain an accurate value for the retardation of the crystal.

  1. 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.

  2. A robust procedure for distinctively visualizing zebrafish retinal cell nuclei under bright field light microscopy.

    PubMed

    Fu, Jinling; Fang, Wei; Zou, Jian; Sun, Ming; Lathrop, Kira; Su, Guanfang; Wei, Xiangyun

    2013-03-01

    To simultaneously visualize individual cell nuclei and tissue morphologies of the zebrafish retina under bright field light microscopy, it is necessary to establish a procedure that specifically and sensitively stains the cell nuclei in thin tissue sections. This necessity arises from the high nuclear density of the retina and the highly decondensed chromatin of the cone photoreceptors, which significantly reduces their nuclear signals and makes nuclei difficult to distinguish from possible high cytoplasmic background staining. Here we optimized a procedure that integrates JB4 plastic embedding and Feulgen reaction for visualizing zebrafish retinal cell nuclei under bright field light microscopy. This method produced highly specific nuclear staining with minimal cytoplasmic background, allowing us to distinguish individual retinal nuclei despite their tight packaging. The nuclear staining is also sensitive enough to distinguish the euchromatin from heterochromatin in the zebrafish cone nuclei. In addition, this method could be combined with in situ hybridization to simultaneously visualize the cell nuclei and mRNA expression patterns. With its superb specificity and sensitivity, this method may be extended to quantify cell density and analyze global chromatin organization throughout the retina or other tissues.

  3. 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.

  4. 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

  5. Advanced light water reactor requirements document: Chapter 4, Reactor systems

    SciTech Connect

    Not Available

    1987-06-01

    The purpose of this chapter of the Advanced Light Water Reactor (ALWR) Plant Requirements Document is to establish utility requirements for the design of the Reactor Systems of Advanced LWR plants consistent with the objectives and principles of the ALWR program. The scope of this chapter covers the following for Pressurized Water Reactor (PWR) and Boiling Water Reactor (BWR): reactor pressure vessel, nozzles and safe-ends, reactor internals, in-vessel portions of fluid systems (including reactor internal pumps (Emergency Core Cooling System (ECCS) piping and spargers), nuclear fuel, and the control rods and control rod drive system (including hydraulic supply and accumulators). Special tools required for reactor system maintenance, inspection and testing are also covered.

  6. 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.

  7. 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.

  8. 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.

  9. The Advanced Light Source (ALS) Slicing Undulator Beamline

    SciTech Connect

    Heimann, P. A.; Glover, T. E.; Plate, D.; Brown, V. C.; Padmore, H. A.; Lee, H. J.; Schoenlein, R. W.

    2007-01-19

    A beamline optimized for the bunch slicing technique has been construction at the Advanced Light Source (ALS). This beamline includes an in-vacuum undulator, soft and hard x-ray beamlines and a femtosecond laser system. The soft x-ray beamline may operate in spectrometer mode, where an entire absorption spectrum is accumulated at one time, or in monochromator mode. The femtosecond laser system has a high repetition rate of 20 kHz to improve the average slicing flux. The performance of the soft x-ray branch of the ALS slicing undulator beamline will be presented.

  10. 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

  11. 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.

  12. 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

  13. 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.

  14. 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

  15. 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).

  16. 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.

  17. 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

  18. 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.

  19. 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

  20. 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

  1. 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%.

  2. 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.

  3. Quantitation of Cellular Dynamics in Growing Arabidopsis Roots with Light Sheet Microscopy

    PubMed Central

    Birnbaum, Kenneth D.; Leibler, Stanislas

    2011-01-01

    To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics. PMID:21731697

  4. Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours

    PubMed Central

    Glenn, D. R.; Zhang, H.; Kasthuri, N.; Schalek, R.; Lo, P. K.; Trifonov, A. S.; Park, H.; Lichtman, J. W.; Walsworth, R. L.

    2012-01-01

    Correlative light and electron microscopy promises to combine molecular specificity with nanoscale imaging resolution. However, there are substantial technical challenges including reliable co-registration of optical and electron images, and rapid optical signal degradation under electron beam irradiation. Here, we introduce a new approach to solve these problems: imaging of stable optical cathodoluminescence emitted in a scanning electron microscope by nanoparticles with controllable surface chemistry. We demonstrate well-correlated cathodoluminescence and secondary electron images using three species of semiconductor nanoparticles that contain defects providing stable, spectrally-distinguishable cathodoluminescence. We also demonstrate reliable surface functionalization of the particles. The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument. PMID:23155483

  5. 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.

  6. 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.

  7. Quantitation of cellular dynamics in growing Arabidopsis roots with light sheet microscopy.

    PubMed

    Sena, Giovanni; Frentz, Zak; Birnbaum, Kenneth D; Leibler, Stanislas

    2011-01-01

    To understand dynamic developmental processes, living tissues have to be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image, at cellular resolution, a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, to track cellular nuclei and to identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics.

  8. White light scattering spectroscopy and electron microscopy of laser induced melting in single gold nanorods.

    PubMed

    Zijlstra, Peter; Chon, James W M; Gu, Min

    2009-07-28

    We present the first measurements of laser induced melting and reshaping of single gold nanorods. Using a combination of white light scattering spectroscopy and electron microscopy we find a melting energy of 260 fJ for nanorods with an average size of 92 x 30 nm. Contrary to previous reports on ensembles of nanorods, this melting energy corresponds well to the theoretical prediction of 225 fJ. We observe a gradual shape change from a long and thin rod to a shorter and wider rod, which eventually collapses into a sphere when enough laser energy is deposited. We also observe that higher aspect ratio particles are thermodynamically less stable, leading to a greater reduction of the aspect ratio at lower laser pulse energy densities.

  9. 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.

  10. Light microscopy of basidia, basidiospores, and nuclei in spores and hyphae of Filobasidiella neoformans (Cryptococcus neoformans).

    PubMed Central

    Erke, K H

    1976-01-01

    Three hypha-forming strains of Cryptococcus neoformans were induced to form basidia and basidiospores. Light microscopy showed that basidia formed at the ends of terminal hyphal cells and were able to produce from a few to many basidiospores. The morphology of the sexual structures indicated that these strains belonged to the recently described perfect state of C. neoformans, Filobasidiella neoformans. The average dimensions of the basidiospores were 1.9 mum in width by 2.7 mum in length. Giemsa staining revealed that dikaryotic cells were formed in all three strains. Only one strain had both terminal and subterminal dikaryons, indicating functional clamp connections, whereas the two remaining strains had dikaryons restricted to the terminal cells. Basidiospores of two strains were mononucleate, and yeast cell clones derived from single basidiospores of these two strains were able to complete the sexual life cycle, thus indicating their primary homothallic nature. Images PMID:789347

  11. 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

  12. 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

  13. Nonequilibrium fluctuations in metaphase spindles: polarized light microscopy, image registration, and correlation functions

    NASA Astrophysics Data System (ADS)

    Brugués, Jan; Needleman, Daniel J.

    2010-02-01

    Metaphase spindles are highly dynamic, nonequilibrium, steady-state structures. We study the internal fluctuations of spindles by computing spatio-temporal correlation functions of movies obtained from quantitative polarized light microscopy. These correlation functions are only physically meaningful if corrections are made for the net motion of the spindle. We describe our image registration algorithm in detail and we explore its robustness. Finally, we discuss the expression used for the estimation of the correlation function in terms of the nematic order of the microtubules which make up the spindle. Ultimately, studying the form of these correlation functions will provide a quantitative test of the validity of coarse-grained models of spindle structure inspired from liquid crystal physics.

  14. Light-emitting diode fluorescence microscopy for tuberculosis diagnosis: a meta-analysis.

    PubMed

    Chang, Eva W; Page, Anne-Laure; Bonnet, Maryline

    2016-03-01

    Light-emitting diode fluorescence microscopy (LED-FM) is recommended by the World Health Organization to replace conventional Ziehl-Neelsen microscopy for pulmonary tuberculosis diagnosis. Uptake of LED-FM has been slow. One reason is its reported loss of specificity compared with Ziehl-Neelsen microscopy. We aimed to determine the diagnostic accuracy of LED-FM for tuberculosis detection and explore potential factors that might affect its performance.A comprehensive search strategy based on pre-specified criteria was employed to identify eligible studies between January 1, 2000 and April 1, 2014 in 11 databases. Standardised study selection, data extraction and quality assessment were conducted. Pooled sensitivity and specificity of LED-FM using culture as the reference standard were estimated through meta-analyses using a bivariate random-effects model. Investigation of heterogeneity was performed by subgroup analyses.We identified 12 unique studies, half of which were from peripheral healthcare facilities. LED-FM achieved a pooled sensitivity of 66.9% (95% CI 60.5-72.7%) and pooled specificity of 96.8% (95% CI 93.1-98.6%). A pooled sensitivity of 53.0% (95% CI 42.8-63.0%) and pooled specificity of 96.1% (95% CI 86.0-99.0%) were obtained by LED-FM among HIV-infected patients. Study methodology factors and differences in the LED-FM procedure or device could also affect the performance.LED-FM specificity is high and should not be a barrier to device introduction, particularly among peripheral healthcare settings where this technology is meant to be used. Sensitivity is reduced in HIV-infected patients.

  15. 3D Light-Sheet Fluorescence Microscopy of Cranial Neurons and Vasculature during Zebrafish Embryogenesis.

    PubMed

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

    2015-11-01

    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.

  16. Comparison of selective staining of fungi in paraffin sections by light microscopy, SEM and BEI

    SciTech Connect

    Berman, E.L.; Laudate, A.; Carter, H.W.

    1981-01-01

    Paraffin-embedded sections from human tissues with fungi or organisms classified with fungi were studied by light microscopy (LM), scanning electron microscopy (SEM), and the backscatter electron imaging (BEI) mode of the SEM. The fungal organisms selected for study were those familiar to the pathologist on the basis of their appearance in paraffin-embedded material stained with the Gomori-Grocott Chromic Acid Methenamine Silver Stain (GMS). The organisms were Actinomyces, Rhizopus, Cryptococcus, Histoplasma capsulatum, and Coccidia imitis. Sections were stained with the GMS Stain and/or the Becker modification of the GMS Stain (BGMS) and examined in the secondary electron imaging mode (SEI) and BEI mode with an annular backscatter electron detector. This silver staining technique accentuated the wall of fungal organisms, in the backscatter mode. Depending on the fungal organism and type of silver stain employed, the GMS seemed the preferable stain. The advantages of SEM over LM were greater depth of focus and potential range of magnifications. BEI may also be used in conjunction with LM stain for microorganisms to establish their presence.

  17. Two-photon microscopy: shedding light on the chemistry of vision.

    PubMed

    Imanishi, Yoshikazu; Lodowski, Kerrie H; Koutalos, Yiannis

    2007-08-28

    Two-photon microscopy (TPM) has come to occupy a prominent place in modern biological research with its ability to resolve the three-dimensional distribution of molecules deep inside living tissue. TPM can employ two different types of signals, fluorescence and second harmonic generation, to image biological structures with subcellular resolution. Two-photon excited fluorescence imaging is a powerful technique with which to monitor the dynamic behavior of the chemical components of tissues, whereas second harmonic imaging provides novel ways to study their spatial organization. Using TPM, great strides have been made toward understanding the metabolism, structure, signal transduction, and signal transmission in the eye. These include the characterization of the spatial distribution, transport, and metabolism of the endogenous retinoids, molecules essential for the detection of light, as well as the elucidation of the architecture of the living cornea. In this review, we present and discuss the current applications of TPM for the chemical and structural imaging of the eye. In addition, we address what we see as the future potential of TPM for eye research. This relatively new method of microscopy has been the subject of numerous technical improvements in terms of the optics and indicators used, improvements that should lead to more detailed biochemical characterizations of the eyes of live animals and even to imaging of the human eye in vivo. PMID:17676772

  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. Assessment of meiotic spindle configuration and post-warming bovine oocyte viability using polarized light microscopy.

    PubMed

    Caamaño, J N; Díez, C; Trigal, B; Muñoz, M; Morató, R; Martín, D; Carrocera, S; Mogas, T; Gómez, E

    2013-06-01

    The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule-polymerized protein in in vitro-matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro-matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule-polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(-) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post-warming viability in vitrified bovine oocytes.

  20. A false report of product tampering involving a rodent and soft drink can: light microscopy, image analysis and scanning electron microscopy/energy dispersive X-ray analysis.

    PubMed

    Platek, F; Ranieri, N; Wolnik, K A

    1997-11-01

    The "Pepsi Tamperings" of 1993 resulted in a large number of cases involving foreign objects reportedly found inside canned soft drinks. Although the majority of cases involved medical syringes and metallic objects, one case involved the report of a mouse found inside a can of Caffeine-Free Diet Pepsi. Using light and polarized light microscopy and computer-assisted image analysis, trace evidence and tooth structure from the suspect mouse were matched to scratches and indentions on the suspect can. Scanning electron microscopy and energy dispersive X-ray analysis were used to compare and match particles of gnawed metal from the lid of the suspect can to other particles recovered from the muzzle and stomach of the suspect mouse. The forensic analyses in this case proved the mouse could not have been canned in the soft drink product and refuted the defendant's sworn statements.

  1. Advances in quantitative nanoscale subsurface imaging by mode-synthesizing atomic force microscopy

    SciTech Connect

    Vitry, P.; Bourillot, E.; Plassard, C.; Lacroute, Y.; Lesniewska, E.; Tetard, L.

    2014-08-04

    This paper reports on advances toward quantitative non-destructive nanoscale subsurface investigation of a nanofabricated sample based on mode synthesizing atomic force microscopy with heterodyne detection, addressing the need to correlate the role of actuation frequencies of the probe f{sub p} and the sample f{sub s} with depth resolution for 3D tomography reconstruction. Here, by developing a simple model and validating the approach experimentally through the study of the nanofabricated calibration depth samples consisting of buried metallic patterns, we demonstrate avenues for quantitative nanoscale subsurface imaging. Our findings enable the reconstruction of the sample depth profile and allow high fidelity resolution of the buried nanostructures. Non-destructive quantitative nanoscale subsurface imaging offers great promise in the study of the structures and properties of complex systems at the nanoscale.

  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. 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.

  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. Performance of Advanced Light Source particle beam diagnostics

    SciTech Connect

    Hinkson, J.

    1993-05-01

    The Advanced Light Source (ALS), a third-generation synchrotron radiation facility, is complete. The particle beam diagnostics have been installed and tested. The beam injection systems have been running for two years. We have performance data on beam position monitors, beam intensity monitors, scintillators, beam collimators, a 50 {Omega} Faraday cup, and broad-band striplines and kickers used in the linac, transport lines, and the booster synchrotron. The single-turn monitoring capability of the booster beam position monitoring system has been particularly useful for studying beam dynamics. Beam diagnostics for the storage ring are being commissioned. In this paper we describe each instrument, show its performance, and outline how the instruments are controlled and their output data displayed.

  8. Applications of advanced aerodynamic technology to light aircraft.

    NASA Technical Reports Server (NTRS)

    Crane, H. L.; Mcghee, R. J.; Kohlman, D. L.

    1973-01-01

    This paper discusses a project for adapting advanced technology, much of it borrowed from the jet transport, to general aviation design practice. The NASA funded portion of the work began in 1969 at the University of Kansas and resulted in a smaller, experimental wing with spoilers and powerful flap systems for a Cessna Cardinal airplane. Some flight data and research pilot comments are presented. The project was expanded in 1972 to include a light twin-engine airplane. For the twin there was the added incentive of a potential increase in single-engine climb performance. The use of a new high-lift Whitcomb airfoil is planned for both the wing and the propellers. Preliminary data on the characteristics of the new airfoil are discussed. The configuration of an experimental wing for a Piper Seneca PA-34 and estimated airplane performance with this wing are discussed.

  9. Observations of collective effects at the Advanced Light Source

    SciTech Connect

    Byrd, J.M.; Barry, W.; Corlett, J.N.; Fox, J.; Teytelman, D.

    1995-10-01

    We present a summary of measurements of single beam collective effects in the Advanced Light Source (ALS). We describe measurements of coupled-bunch instabilities, including some recent results using the newly commissioned feedback systems and the results of an initial search for the fast ion instability. Single bunch effects include bunch lengthening, energy spread increase, HOM loss measurements, head-tail damping rates, current dependent tune shifts, and transverse mode coupling instability threshold. The longitudinal measurements are consistent with a broadband impedance {vert_bar}{Zeta}{sub {parallel}}/{eta}{vert_bar}{sub eff} = 0.22{plus_minus}0.07 {Omega} and transverse measurements indicate broadband impedances of {Zeta}{sub y,eff} = 155 k{Omega}/m and Z{sub x,eff} = 58 k{Omega}/m.

  10. 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.

  11. Swept source optical coherence microscopy using a 1310 nm VCSEL light source

    PubMed Central

    Ahsen, Osman O.; Tao, Yuankai K.; Potsaid, Benjamin M.; Sheikine, Yuri; Jiang, James; Grulkowski, Ireneusz; Tsai, Tsung-Han; Jayaraman, Vijaysekhar; Kraus, Martin F.; Connolly, James L.; Hornegger, Joachim; Cable, Alex; Fujimoto, James G.

    2013-01-01

    We demonstrate high speed, swept source optical coherence microscopy (OCM) using a MEMS tunable vertical cavity surface-emitting laser (VCSEL) light source. The light source had a sweep rate of 280 kHz, providing a bidirectional axial scan rate of 560 kHz. The sweep bandwidth was 117 nm centered at 1310 nm, corresponding to an axial resolution of 13.1 µm in air, corresponding to 8.1 µm (9.6 µm spectrally shaped) in tissue. Dispersion mismatch from different objectives was compensated numerically, enabling magnification and field of view to be easily changed. OCM images were acquired with transverse resolutions between 0.86 µm - 3.42 µm using interchangeable 40X, 20X and 10X objectives with ~600 µm x 600 µm, ~1 mm x 1 mm and ~2 mm x 2 mm field-of-view (FOV), respectively. Parasitic variations in path length with beam scanning were corrected numerically. These features enable swept source OCM to be integrated with a wide range of existing scanning microscopes. Large FOV mosaics were generated by serially acquiring adjacent overlapping microscopic fields and combining them in post-processing. Fresh human colon, thyroid and kidney specimens were imaged ex vivo and compared to matching histology sections, demonstrating the ability of OCM to image tissue specimens. PMID:23938673

  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. Automatic Evaluation of Collagen Fiber Directions from Polarized Light Microscopy Images.

    PubMed

    Novak, Kamil; Polzer, Stanislav; Tichy, Michal; Bursa, Jiri

    2015-08-01

    Mechanical properties of the arterial wall depend largely on orientation and density of collagen fiber bundles. Several methods have been developed for observation of collagen orientation and density; the most frequently applied collagen-specific manual approach is based on polarized light (PL). However, it is very time consuming and the results are operator dependent. We have proposed a new automated method for evaluation of collagen fiber direction from two-dimensional polarized light microscopy images (2D PLM). The algorithm has been verified against artificial images and validated against manual measurements. Finally the collagen content has been estimated. The proposed algorithm was capable of estimating orientation of some 35 k points in 15 min when applied to aortic tissue and over 500 k points in 35 min for Achilles tendon. The average angular disagreement between each operator and the algorithm was -9.3±8.6° and -3.8±8.6° in the case of aortic tissue and -1.6±6.4° and 2.6±7.8° for Achilles tendon. Estimated mean collagen content was 30.3±5.8% and 94.3±2.7% for aortic media and Achilles tendon, respectively. The proposed automated approach is operator independent and several orders faster than manual measurements and therefore has the potential to replace manual measurements of collagen orientation via PLM.

  14. 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

  15. 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

  16. Advanced microscopy of star-shaped gold nanoparticles and their adsorption-uptake by macrophages

    PubMed Central

    Plascencia-Villa, Germán; Bahena, Daniel; Rodríguez, Annette R.; Ponce, Arturo; José-Yacamán, Miguel

    2013-01-01

    Metallic nanoparticles have diverse applications in biomedicine, as diagnostics, image contrast agents, nanosensors and drug delivery systems. Anisotropic metallic nanoparticles possess potential applications in cell imaging and therapy+diagnostics (theranostics), but controlled synthesis and growth of these anisotropic or branched nanostructures has been challenging and usually require use of high concentrations of surfactants. Star-shaped gold nanoparticles were synthesized in high yield through a seed mediated route using HEPES as a precise shape-directing capping agent. Characterization was performed using advanced electron microscopy techniques including atomic resolution TEM, obtaining a detailed characterization of nanostructure and atomic arrangement. Spectroscopy techniques showed that particles have narrow size distribution, monodispersity and high colloidal stability, with absorbance into NIR region and high efficiency for SERS applications. Gold nanostars showed to be biocompatible and efficiently adsorbed and internalized by macrophages, as revealed by advanced FE-SEM and backscattered electron imaging techniques of complete unstained uncoated cells. Additionally, low voltage STEM and X-ray microanalysis revealed the ultra-structural location and confirmed stability of nanoparticles after endocytosis with high spatial resolution. PMID:23443314

  17. 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

  18. 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.

  19. The design and evaluation of three advanced daylighting systems: Light shelves, light pipes and skylights

    SciTech Connect

    Beltran, L.O.; Lee, E.S.; Papmichael, K.M.; Selkowitz, S.E.

    1994-03-01

    We present results from the design and evaluation of three advanced daylighting systems: a light shelf, a light pipe, and a skylight. These systems use optical films and an optimized geometry to passively intercept and redirect sunlight further into the building. The objectives of these designs are to increase daylighting illuminance levels at distances of 4.6-9.1 m (15--30 ft) from the window, and to improve the uniformity of the daylight distribution and the luminance gradient across the room under variable sun and sky conditions throughout the year. The designs were developed through a series of computer-assisted ray-tracing studies, photometric measurements, and observations using physical scale models. Comprehensive sets of laboratory measurements in combination with analytical routines were then used to simulate daylight performance for any solar position. Results show increased daylight levels and an improved luminance gradient throughout the year -- indicating that lighting energy consumption and cooling energy due of lighting can be substantially reduced with improvements to visual comfort. Future development of the designs may further improve the daylighting performance of these systems.

  20. Single-layer mirrors for advanced research light sources

    SciTech Connect

    Stoermer, M.; Horstmann, C.; Siewert, F.; Hertlein, F.; Matiaske, M.; Wiesmann, J.; Gaudin, J.

    2010-06-23

    X-ray mirrors are needed for beam guidance, beam alignment and monochromatisation at third-generation synchrotron light sources (PETRA III) and forthcoming Free-Electron Lasers (LCLS, European XFEL). Amorphous carbon coatings are currently used as total reflection mirrors at FLASH to guide the photon beam to the various beamlines. These coatings were prepared by means of magnetron sputtering. The new GKSS sputtering facility for the deposition of single and multilayer mirrors with a length of up to 1500 mm and a width of up to 120 mm is in operation. In this contribution we present the results of this new deposition system. A major advantage is that it is now possible to prepare one, two or more mirrors with similar properties over the whole deposition length. The mirror properties were investigated by means of X-ray reflectometry and interference microscopy. The performance of the mirrors is analyzed, considering X-ray reflectivity, film thickness and surface roughness. The uniformity of these properties over the whole deposition length of 1500 mm is demonstrated. The results obtained will be discussed and compared with former results.

  1. 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.

  2. 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

  3. Evanescent Light-Scattering Microscopy for Label-Free Interfacial Imaging: From Single Sub-100 nm Vesicles to Live Cells.

    PubMed

    Agnarsson, Björn; Lundgren, Anders; Gunnarsson, Anders; Rabe, Michael; Kunze, Angelika; Mapar, Mokhtar; Simonsson, Lisa; Bally, Marta; Zhdanov, Vladimir P; Höök, Fredrik

    2015-12-22

    Advancement in the understanding of biomolecular interactions has benefited greatly from the development of surface-sensitive bioanalytical sensors. To further increase their broad impact, significant efforts are presently being made to enable label-free and specific biomolecule detection with high sensitivity, allowing for quantitative interpretation and general applicability at low cost. In this work, we have addressed this challenge by developing a waveguide chip consisting of a flat silica core embedded in a symmetric organic cladding with a refractive index matching that of water. This is shown to reduce stray light (background) scattering and thereby allow for label-free detection of faint objects, such as individual sub-20 nm gold nanoparticles as well as sub-100 nm lipid vesicles. Measurements and theoretical analysis revealed that light-scattering signals originating from single surface-bound lipid vesicles enable characterization of their sizes without employing fluorescent lipids as labels. The concept is also demonstrated for label-free measurements of protein binding to and enzymatic (phospholipase A2) digestion of individual lipid vesicles, enabling an analysis of the influence on the measured kinetics of the dye-labeling of lipids required in previous assays. Further, diffraction-limited imaging of cells (platelets) binding to a silica surface showed that distinct subcellular features could be visualized and temporally resolved during attachment, activation, and spreading. Taken together, these results underscore the versatility and general applicability of the method, which due to its simplicity and compatibility with conventional microscopy setups may reach a widespread in life science and beyond. PMID:26517791

  4. 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

  5. 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.

  6. 3D structure tensor analysis of light microscopy data for validating diffusion MRI.

    PubMed

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

    2015-05-01

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

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

    PubMed Central

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

    2015-01-01

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

  8. 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

  9. [The neurocytes of the caudal part of the tuberomamillary nucleus in Ovis aries L. (light microscopy and electron microscopy studies)].

    PubMed

    Horáková, A; Horák, J; Tomajková, E

    1985-01-01

    The neurons of the pars caudalis nuclei tuberomammillaris (pc-NTM) were studed light-microscopically and electron-microscopically in sheep and rams of Merino breed. In our study we observed: In the regarded neural nucleus, there is the majority of the great neurons (up to 60 microns in diameter) rich in the NISSL's bodies. When stained with the cresyl violet, the NISSL's substance is apparently stored mainly in peripheral area of the cell body and in the distant parts of numerous protoplasmic processes, what evokes an impression of the "jagged" surface of these cells. After staining with paraldehyde fuchsin, we found purple coloured lumps of irregular shape stored outside the cell bodies, in the neuropil. The less extended cells, usually with lower content of NISSL bodies, are in pc-NTM less frequent. In the electron-microscopic study we identified 3 types of neurons: Cells rich in rough endoplasmic reticulum; "light" cells, "dark" cells. The cells of the 1st type were the most frequent ones. Cisterns of rough endoplasmic reticulum in the 1st type of cells are often dilated. The protoplasmic processes of these cells are frequently stepped over by flat tubuli of endoplasmic reticulum. The 2nd type of cells is characterized by the light cytoplasmic matrix, low quantity of endoplasmic reticulum and frequent occurrence of lipofuscin bodies. The 3rd type of cells are characterized by the high density of cytoplasmic matrix, well developed GOLGI complex, and very broad cisterns of endoplasmic reticulum, forming a labyrinth, and it is bound to a broad perinuclear space. PMID:4065505

  10. 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.

  11. Atomic Force Microscopy of Red-Light Photoreceptors Using PeakForce Quantitative Nanomechanical Property Mapping

    PubMed Central

    Kroeger, Marie E.; Sorenson, Blaire A.; Thomas, J. Santoro; Stojković, Emina A.; Tsonchev, Stefan; Nicholson, Kenneth T.

    2014-01-01

    Atomic force microscopy (AFM) uses a pyramidal tip attached to a cantilever to probe the force response of a surface. The deflections of the tip can be measured to ~10 pN by a laser and sectored detector, which can be converted to image topography. Amplitude modulation or “tapping mode” AFM involves the probe making intermittent contact with the surface while oscillating at its resonant frequency to produce an image. Used in conjunction with a fluid cell, tapping-mode AFM enables the imaging of biological macromolecules such as proteins in physiologically relevant conditions. Tapping-mode AFM requires manual tuning of the probe and frequent adjustments of a multitude of scanning parameters which can be challenging for inexperienced users. To obtain high-quality images, these adjustments are the most time consuming. PeakForce Quantitative Nanomechanical Property Mapping (PF-QNM) produces an image by measuring a force response curve for every point of contact with the sample. With ScanAsyst software, PF-QNM can be automated. This software adjusts the set-point, drive frequency, scan rate, gains, and other important scanning parameters automatically for a given sample. Not only does this process protect both fragile probes and samples, it significantly reduces the time required to obtain high resolution images. PF-QNM is compatible for AFM imaging in fluid; therefore, it has extensive application for imaging biologically relevant materials. The method presented in this paper describes the application of PF-QNM to obtain images of a bacterial red-light photoreceptor, RpBphP3 (P3), from photosynthetic R. palustris in its light-adapted state. Using this method, individual protein dimers of P3 and aggregates of dimers have been observed on a mica surface in the presence of an imaging buffer. With appropriate adjustments to surface and/or solution concentration, this method may be generally applied to other biologically relevant macromolecules and soft materials. PMID

  12. Three-dimensional reconstruction of specular reflecting technical surfaces using structured light microscopy

    NASA Astrophysics Data System (ADS)

    Kettel, Johannes; Müller, Claas; Reinecke, Holger

    2014-11-01

    In computer assisted quality control the three-dimensional reconstruction of technical surfaces is playing an ever more important role. Due to the demand on high measurement accuracy and data acquisition rates, structured light optical microscopy has become a valuable solution for the three-dimensional measurement of technical surfaces with high vertical and lateral resolution. However, the three-dimensional reconstruction of specular reflecting technical surfaces with very low surface-roughness and local slopes still remains a challenge to optical measurement principles. Furthermore the high data acquisition rates of current optical measurement systems depend on highly complex and expensive scanning-techniques making them impractical for inline quality control. In this paper we present a novel measurement principle based on a multi-pinhole structured light solution without moving parts which enables the threedimensional reconstruction of specular and diffuse reflecting technical surfaces. This measurement principle is based on multiple and parallel processed point-measurements. These point measurements are realized by spatially locating and analyzing the resulting Point Spread Function (PSF) in parallel for each point measurement. Analysis of the PSF is realized by pattern recognition and model-fitting algorithms accelerated by current Graphics-Processing-Unit (GPU) hardware to reach suitable measurement rates. Using the example of optical surfaces with very low surface-roughness we demonstrate the three-dimensional reconstruction of these surfaces by applying our measurement principle. Thereby we show that the resulting high measurement accuracy enables cost-efficient three-dimensional surface reconstruction suitable for inline quality control.

  13. 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

  14. 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.

  15. 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

  16. Structural characterization of the capybara (Hydrochaeris hydrochaeris) tongue by light, scanning, and transmission electron microscopy.

    PubMed

    Watanabe, Ii-Sei; Dos Santos Haemmerle, Carlos Alexandre; Dias, Fernando José; Cury, Diego Pulzatto; Da Silva, Marcelo Cavenaghi Pereira; Sosthines, Marcia Consentino Kronka; Dos Santos, Tatiana Carlesco; Guimarães, Juliana Plácido; Miglino, Maria Angélica

    2013-02-01

    Capybara is the largest rodent in the world and displays a seasonally dependent herbivore feeding behavior. Here, we present an anatomical contribution for understand this fact, by light, scanning, and transmission electron microscopy methodologies for tongue tissue analysis. The histological preparations revealed filiform, fungiform, vallate, and foliate papillae on the dorsal mucosa of the capybara tongue. The epithelial layer exhibited a lining of keratinized stratified squamous epithelial cells. The lamina propria was characterized by a dense connective tissue composed of the primary and secondary papillar projections. We also revealed the original aspects of the connective papillae. The shapes of the papillae varied by region of the tongue, and filiform, fungiform, vallate, and foliate papillae and subjacent layers of muscular fibers were observed. Pyriform taste buds occupying the epithelial layer of fungiform, vallate and foliate papillae were identified and the intracellular components of the taste buds and the intracorpuscular amyelinated nerve fibers were observed. The taste buds were characterized by the distribution of granular endoplasmic reticulum throughout the perinuclear area, the Golgi apparatus, and mitochondrial assemblies of various distinct diameters. Mitochondrial accumulation was also observed in the collagen bundle-surrounded amyelinated nerve fibers beside the basal cells. Therefore, these peculiar anatomical descriptions may contribute to understanding the adaptation of the feeding behavior of capybaras in a seasonally changing environment.

  17. 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

  18. 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

  19. 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.

  20. 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.

  1. Dual-slit confocal light sheet microscopy for in vivo whole-brain imaging of zebrafish

    PubMed Central

    Yang, Zhe; Mei, Li; Xia, Fei; Luo, Qingming; Fu, Ling; Gong, Hui

    2015-01-01

    In vivo functional imaging at single-neuron resolution is an important approach to visualize biological processes in neuroscience. Light sheet microscopy (LSM) is a cutting edge in vivo imaging technique that provides micron-scale spatial resolution at high frame rate. Due to the scattering and absorption of tissue, however, conventional LSM is inadequate to resolve cells because of the attenuated signal to noise ratio (SNR). Using dual-beam illumination and confocal dual-slit detection, here a dual-slit confocal LSM is demonstrated to obtain the SNR enhanced images with frame rate twice as high as line confocal LSM method. Through theoretical calculations and experiments, the correlation between the slit’s width and SNR was determined to optimize the image quality. In vivo whole brain structural imaging stacks and the functional imaging sequences of single slice were obtained for analysis of calcium activities at single-cell resolution. A two-fold increase in imaging speed of conventional confocal LSM makes it possible to capture the sequence of the neurons’ activities and help reveal the potential functional connections in the whole zebrafish’s brain. PMID:26137381

  2. New approach for the quantification of processed animal proteins in feed using light microscopy.

    PubMed

    Veys, P; Baeten, V

    2010-07-01

    A revision of European Union's total feed ban on animal proteins in feed will need robust quantification methods, especially for control analyses, if tolerance levels are to be introduced, as for fishmeal in ruminant feed. In 2006, a study conducted by the Community Reference Laboratory for Animal Proteins in feedstuffs (CRL-AP) demonstrated the deficiency of the official quantification method based on light microscopy. The study concluded that the method had to be revised. This paper puts forward an improved quantification method based on three elements: (1) the preparation of permanent slides with an optical adhesive preserving all morphological markers of bones necessary for accurate identification and precision counting; (2) the use of a counting grid eyepiece reticle; and (3) new definitions for correction factors for the estimated portions of animal particles in the sediment. This revised quantification method was tested on feeds adulterated at different levels with bovine meat and bone meal (MBM) and fishmeal, and it proved to be effortless to apply. The results obtained were very close to the expected values of contamination levels for both types of adulteration (MBM or fishmeal). Calculated values were not only replicable, but also reproducible. The advantages of the new approach, including the benefits of the optical adhesive used for permanent slide mounting and the experimental conditions that need to be met to implement the new method correctly, are discussed. PMID:20432096

  3. 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.

  4. 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.

  5. 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.

  6. 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.

  7. 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

  8. Prospects of anionic nanolipoplexes in nanotherapy: transmission electron microscopy and light scattering studies.

    PubMed

    Mozafari, M Reza; Reed, Celia J; Rostron, Christopher

    2007-01-01

    Currently nanosystems composed of polynucleotides and lipid vesicles (nanolipoplexes) are considered to be promising tools for gene therapeutics. Successful in vivo application of these vectors depends on their physicochemical, technological and biological characteristics including morphology, size distribution, molecular interactions and stability. Anionic nanoliposomes (DPPC:DCP:CHOL) were prepared by two different techniques, namely the conventional thin-film hydration method followed by extrusion, and the heating method (HM), in which no volatile solvent or detergent is used. A non-viral and non-cationic gene transfer vector was constructed by incorporating plasmid DNA (pcDNA3.1/His B/lacZ) to the HM-nanoliposomes by the electrostatic mediation of Ca(2+) ions. Transfection efficiency of the nanolipoplexes was evaluated using a human bronchial epithelial cell line (16HBE14o-) in the presence of serum. Particle characterisation, stability of the formulations and lipid-DNA interaction studies were performed using transmission electron microscopy (TEM) and light scattering. TEM pictures of nanolipoplexes showed presence of two to four closely packed vesicles with signs of fusion. Efficient delivery of plasmid DNA and subsequent beta-galactosidase expression was achieved using the anionic nanolipoplexes. Transfection efficiency increased with lipid:DNA ratio up to 7:1 (w/w), where transfection efficiency was 12-fold higher than in untreated cells. Further increase in lipid ratio decreased transfection. These nanolipoplexes appear to be safe, stable and efficient in the protection and delivery of DNA to different cells and tissues.

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

    DOE PAGES

    Meddens, Marjolein B. M.; Liu, Sheng; Finnegan, Patrick S.; 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

  10. 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.

  11. 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

  12. 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.

  13. 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

  14. 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

  15. Dictionary of Microscopy

    NASA Astrophysics Data System (ADS)

    Heath, Julian

    2005-10-01

    The past decade has seen huge advances in the application of microscopy in all areas of science. This welcome development in microscopy has been paralleled by an expansion of the vocabulary of technical terms used in microscopy: terms have been coined for new instruments and techniques and, as microscopes reach even higher resolution, the use of terms that relate to the optical and physical principles underpinning microscopy is now commonplace. The Dictionary of Microscopy was compiled to meet this challenge and provides concise definitions of over 2,500 terms used in the fields of light microscopy, electron microscopy, scanning probe microscopy, x-ray microscopy and related techniques. Written by Dr Julian P. Heath, Editor of Microscopy and Analysis, the dictionary is intended to provide easy navigation through the microscopy terminology and to be a first point of reference for definitions of new and established terms. The Dictionary of Microscopy is an essential, accessible resource for: students who are new to the field and are learning about microscopes equipment purchasers who want an explanation of the terms used in manufacturers' literature scientists who are considering using a new microscopical technique experienced microscopists as an aide mémoire or quick source of reference librarians, the press and marketing personnel who require definitions for technical reports.

  16. 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.

  17. 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.

  18. 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

  19. Light-sheet Bayesian microscopy enables deep-cell super-resolution imaging of heterochromatin in live human embryonic stem cells

    PubMed Central

    Hu, Ying S; Zhu, Quan; Elkins, Keri; Tse, Kevin; Li, Yu; Fitzpatrick, James A J; Verma, Inder M; Cang, Hu

    2016-01-01

    Background Heterochromatin in the nucleus of human embryonic cells plays an important role in the epigenetic regulation of gene expression. The architecture of heterochromatin and its dynamic organization remain elusive because of the lack of fast and high-resolution deep-cell imaging tools. We enable this task by advancing instrumental and algorithmic implementation of the localization-based super-resolution technique. Results We present light-sheet Bayesian super-resolution microscopy (LSBM). We adapt light-sheet illumination for super-resolution imaging by using a novel prism-coupled condenser design to illuminate a thin slice of the nucleus with high signal-to-noise ratio. Coupled with a Bayesian algorithm that resolves overlapping fluorophores from high-density areas, we show, for the first time, nanoscopic features of the heterochromatin structure in both fixed and live human embryonic stem cells. The enhanced temporal resolution allows capturing the dynamic change of heterochromatin with a lateral resolution of 50–60 nm on a time scale of 2.3 s. Conclusion Light-sheet Bayesian microscopy opens up broad new possibilities of probing nanometer-scale nuclear structures and real-time sub-cellular processes and other previously difficult-to-access intracellular regions of living cells at the single-molecule, and single cell level.

  20. Evanescent waves in high numerical aperture aplanatic solid immersion microscopy: effects of forbidden light on subsurface imaging.

    PubMed

    Yurt, Abdulkadir; Uyar, Aydan; Cilingiroglu, T Berkin; Goldberg, Bennett B; Ünlü, M Selim

    2014-04-01

    The collection of light at very high numerical aperture allows detection of evanescent waves above the critical angle of total internal reflection in solid immersion lens microscopy. We investigate the effect of such evanescent modes, so-called forbidden light, on the far-field imaging properties of an aplanatic solid immersion microscope by developing a dyadic Green's function formalism in the context of subsurface semiconductor integrated circuit imaging. We demonstrate that the collection of forbidden light allows for sub-diffraction spatial resolution and substantial enhancement of photon collection efficiency albeit inducing wave-front discontinuities and aberrations.

  1. 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.

  2. Nondestructive Characterization by Advanced Synchrotron Light Techniques: Spectromicroscopy and Coherent Radiology

    PubMed Central

    Margaritondo, Giorgio; Hwu, Yeukuang; Je, Jung Ho

    2008-01-01

    The advanced characteristics of synchrotron light has led in recent years to the development of a series of new experimental techniques to investigate chemical and physical properties on a microscopic scale. Although originally developed for materials science and biomedical research, such techniques find increasing applications in other domains – and could be quite useful for the study and conservation of cultural heritage. Specifically, they can nondestructively provide detailed chemical composition information that can be useful for the identification of specimens, for the discovery of historical links based on the sources of chemical raw materials and on chemical processes, for the analysis of damage, their causes and remedies and for many other issues. Likewise, morphological and structural information on a microscopic scale is useful for the identification, study and preservation of many different cultural and historical specimens. We concentrate here on two classes of techniques: in the first case, photoemission spectromicroscopy. This is the result of the advanced evolution of photoemission techniques like ESCA (Electron Microscopy for Chemical Analysis). By combining high lateral resolution to spectroscopy, photoemission spectromicroscopy can deliver fine chemical information on a microscopic scale in a nondestructive fashion. The second class of techniques exploits the high lateral coherence of modern synchrotron sources, a byproduct of the quest for high brightness or brilliance. We will see that such techniques now push radiology into the submicron scale and the submillisecond time domain. Furthermore, they can be implemented in a tomographic mode, increasing the information and becoming potentially quite useful for the analysis of cultural heritage specimens.

  3. 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

  4. Super resolution fluorescence microscopy

    PubMed Central

    Huang, Bo; Bates, Mark; Zhuang, Xiaowei

    2010-01-01

    Achieving a spatial resolution that is not limited by the diffraction of light, recent developments of super-resolution fluorescence microscopy techniques allow the observation of many biological structures not resolvable in conventional fluorescence microscopy. New advances in these techniques now give them the ability to image three-dimensional (3D) structures, measure interactions by multicolor colocalization, and record dynamic processes in living cells at the nanometer scale. It is anticipated that super-resolution fluorescence microscopy will become a widely used tool for cell and tissue imaging to provide previously unobserved details of biological structures and processes. PMID:19489737

  5. 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

  6. 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

    2016-06-16

    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.

  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. 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.

  9. 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

  10. 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.

  11. Micellar aggregates of saponins from Chenopodium quinoa: characterization by dynamic light scattering and transmission electron microscopy.

    PubMed

    Verza, S G; de Resende, P E; Kaiser, S; Quirici, L; Teixeira, H F; Gosmann, G; Ferreira, F; Ortega, G G

    2012-04-01

    Entire seeds of Chenopodium quinoa Willd are a rich protein source and are also well-known for their high saponin content. Due to their amphiphily quinoa saponins are able to form intricate micellar aggregates in aqueous media. In this paper we study the aggregates formed by self-association of these compounds from two quinoa saponin fractions (FQ70 and FQ90) as well as several distinctive nanostructures obtained after their complexation with different ratios of cholesterol (CHOL) and phosphatidylcholine (PC). The FQ70 and FQ90 fractions were obtained by reversed-phase preparative chromatography. The structural features of their resulting aggregates were determined by Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM). Novel nanosized spherical vesicles formed by self-association with mean diameter about 100-200 nm were observed in FQ70 aqueous solutions whereas worm-like micelles an approximate width of 20 nm were detected in FQ90 aqueous solutions. Under experimental conditions similar to those reported for the preparation of Quillaja saponaria ISCOM matrices, tubular and ring-like micelles arose from FQ70:CHOL:PC and FQ90:CHOL:PC formulations, respectively. However, under these conditions no cage-like ISCOM matrices were observed. The saponin composition of FQ70 and FQ90 seems to determine the nanosized structures viewed by TEM. Phytolaccagenic acid, predominant in FQ70 and FQ90 fractions, is accountable for the formation of the nanosized vesicles and tubular structures observed by TEM in the aqueous solutions of both samples. Conversely, ring-like micelles observed in FQ90:CHOL:PC complexes can be attributed to the presence of less polar saponins present in FQ90, in particular those derived from oleanolic acid.

  12. 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

  13. Modeling of fibrin gels based on confocal microscopy and light-scattering data.

    PubMed

    Magatti, Davide; Molteni, Matteo; Cardinali, Barbara; Rocco, Mattia; Ferri, Fabio

    2013-03-01

    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 D(m) = 1), for the overall system 1, joined at randomly distributed nodal points. The resulting 3D network strikingly resembles real fibrin gels and can be sketched as an assembly of densely packed fractal blobs, i.e., regions of size ξ, where the fiber concentration is higher than average. The blobs are placed at a distance ξ0 between their centers of mass so that they are overlapped by a factor η =ξ/ξ0 and have D(m) ∼1.2-1.6. The in silico gels' structure is quantitatively analyzed by its 3D spatial correlation function g(3D)(r) and corresponding power spectrum I(q) = FFT(3D[g3D(r)]), from which ρ, d, D(m), η, and ξ0 can be extracted. In particular, ξ0 provides an excellent estimate of the gel mesh size. The in silico gels' I(q) compares quite well with real gels' elastic light-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.

  14. 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.

  15. Pterygodermatites (Mesopectines) quentini (Nematoda, Rictulariidae), a parasite of Praomys rostratus (Rodentia, Muridae) in Mali: scanning electron and light microscopy

    PubMed Central

    2013-01-01

    Pterygodermatites (Mesopectines) quentini n. sp. (Nematoda, Rictulariidae) is described from the murine host Praomys rostratus in the south of the Republic of Mali. It differs from other species of the subgenus by the morphology of the head, which bears four simple cephalic papillae and a nearly axial oral opening, the number of caudal papillae, the number of precloacal cuticular formations, unequal spicules and the ratio of spicule lengths/body length. The use of scanning electron microscopy in combination with conventional light microscopy enabled us to give a detailed description of the morphological characters of this new species. PMID:24025692

  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

    -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

  17. 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

  18. 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

  19. 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

  20. Label-free intracellular transport measured by spatial light interference microscopy

    NASA Astrophysics Data System (ADS)

    Wang, Zhuo; Millet, Larry; Chan, Vincent; Ding, Huafeng; Gillette, Martha U.; Bashir, Rashid; Popescu, Gabriel

    2011-02-01

    We show that applying the Laplace operator to a speckle-free quantitative phase image reveals an unprecedented level of detail in cell structure, without the gradient artifacts associated with differential interference contrast microscopy, or photobleaching and phototoxicity limitations common in fluorescence microscopy. This method, referred to as Laplace phase microscopy, is an efficient tool for tracking vesicles and organelles in living cells. The principle is demonstrated by tracking organelles in cardiomyocytes and vesicles in neurites of hippocampal neurons, which to our knowledge are the first label-free diffusion measurements of the organelles in such cells.

  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. 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

  3. 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

  4. 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.

  5. 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

  6. 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.

  7. A correlative method for imaging identical regions of samples by micro-CT, light microscopy, and electron microscopy: imaging adipose tissue in a model system.

    PubMed

    Sengle, Gerhard; Tufa, Sara F; Sakai, Lynn Y; Zulliger, Martin A; Keene, Douglas R

    2013-04-01

    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.

  8. Modified technique to recover microsporidian spores in sodium acetate-acetic acid-formalin-fixed fecal samples by light microscopy and correlation with transmission electron microscopy.

    PubMed Central

    Carter, P L; MacPherson, D W; McKenzie, R A

    1996-01-01

    Microsporidia are an emerging cause of significant disease, particularly in the immunocompromised host. Until recently, the diagnosis of enteric infections has required invasive sampling, the use of expensive technology, and considerable technological expertise. The purpose of the present study was to examine three modifications to the processing of fecal specimens for light microscopy (LM) examination for microsporidian spores: the use of pretreatment with potassium hydroxide, modified centrifugation conditions, and a modified staining technique. A sodium acetate-acetic acid-formalin-fixed fecal sample containing numerous microsporidian spores confirmed to be positive by transmission electron microscopy (TEM) was used in all studies performed. A simulation of a heavy to lightly infected individual was used. The results of LM were correlated with those of TEM. Duplicate smears were stained with Weber's modified trichrome and Giemsa (GS) stains. The stained slides were randomized and examined blindly by LM at x 625 and x 1,250 magnifications. A portion of the dilutions after centrifugation were fixed for TEM. The Weber modified trichrome stain performance rating was higher than the Giemsa stain rating because of ease of interpretation, and material stained with Weber modified trichrome stain required less examination time at a lower magnification. The number of positive smears and the quantity of spores detected were significantly higher following pretreatment of the sample with KOH. TEM was positive only when numerous spores were present, but the quality of the photomicrographs was superior after pretreatment with KOH. Pretreatment of sodium acetate-acetic acid-formalin-fixed fecal samples with 10% KOH and then a 5-min centrifugation time and staining with Weber modified trichrome stain provide for the excellent recovery of microsporidia in the routine diagnostic parasitology laboratory. PMID:8897162

  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. Backscatter tolerant squeezed light source for advanced gravitational-wave detectors.

    PubMed

    Chua, Sheon S Y; Stefszky, Michael S; Mow-Lowry, Conor M; Buchler, Ben C; Dwyer, Sheila; Shaddock, Daniel A; Lam, Ping Koy; McClelland, David E

    2011-12-01

    We report on the performance of a dual-wavelength resonant, traveling-wave optical parametric oscillator to generate squeezed light for application in advanced gravitational-wave interferometers. Shot noise suppression of 8.6±0.8 dB was measured across the detection band of interest to Advanced LIGO, and controlled squeezing measured over 5900 s. Our results also demonstrate that the traveling-wave design has excellent intracavity backscattered light suppression of 47 dB and incident backscattered light suppression of 41 dB, which is a crucial design issue for application in advanced interferometers. PMID:22139282

  11. Direct visualization method of the atomic structure of light and heavy atoms with double-detector Cs-corrected scanning transmission electron microscopy

    NASA Astrophysics Data System (ADS)

    Kotaka, Yasutoshi

    2012-09-01

    The advent of Cs-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.

  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. Science at the Speed of Light: Advanced Photon Source

    ScienceCinema

    Murray Gibson

    2016-07-12

    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.

  14. 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.

  15. 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.

  16. 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.

  17. Role of myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability in vitro and in vivo.

    PubMed

    Wu, Fan; Guo, Xiaohua; Xu, Jing; Wang, Weiju; Li, Bingling; Huang, Qiaobing; Su, Lei; Xu, Qiulin

    2016-03-01

    We have previously reported that advanced glycation end products activated Rho-associated protein kinase and p38 mitogen-activated protein kinase, causing endothelial hyperpermeability. However, the mechanisms involved were not fully clarified. Here, we explored the role of myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability. Myosin light chain phosphorylation significantly increased by advanced glycation end products in endothelial cells in a time- and dose-dependent manner, indicating that myosin light chain phosphorylation is involved in the advanced glycation end product pathway. Advanced glycation end products also induced myosin phosphatase-targeting subunit 1 phosphorylation, and small interfering RNA knockdown of the receptor for advanced glycation end products, or blocking myosin light chain kinase with its inhibitor, ML-7, or small interfering RNA abated advanced glycation end product-induced myosin light chain phosphorylation. Advanced glycation end product-induced F-actin rearrangement and endothelial hyperpermeability were also diminished by inhibition of receptor for advanced glycation end product or myosin light chain kinase signalling. Moreover, inhibiting myosin light chain kinase with ML-7 or blocking receptor for advanced glycation end product with its neutralizing antibody attenuated advanced glycation end product-induced microvascular hyperpermeability. Our findings suggest a novel role for myosin light chain and myosin light chain kinase in advanced glycation end product-induced endothelial hyperpermeability.

  18. 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.

  19. 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:...

  20. Pulsed vs continuous light accelerated corneal collagen crosslinking: in vivo qualitative investigation by confocal microscopy and corneal OCT

    PubMed Central

    Mazzotta, C; Traversi, C; Caragiuli, S; Rechichi, M

    2014-01-01

    Purpose To assess qualitative corneal changes and penetration of pulsed and continuous light accelerated crosslinking by in vivo confocal microscopy and corneal OCT. Methods A total of 20 patients affected from progressive keratoconus were enrolled in the study. Ten eyes of 10 patients underwent an epithelium-off pulsed-light accelerated corneal collagen crosslinking (PL-ACXL) by the KXL UV-A source (Avedro Inc.) with 8 min (1 s on/1 s off) of UV-A exposure at 30 mW/cm2 and energy dose of 7.2 J/cm2; 10 eyes of 10 patients underwent an epithelium-off continuous-light accelerated corneal collagen crosslinking (CL-ACXL) at 30 mW/cm2 for 4 min. Riboflavin 0.1% dextran-free plus hydroxyl-propyl-methylcellulose solution (VibeX Rapid, Avedro Inc.) was used for a 10-min corneal soaking. Treated eyes were examined by in vivo scanning laser confocal analysis and spectral anterior segment OCT at 1, 3, and 6 months. Results Epithelial stratification and nerves regeneration improved in time, being complete at month 6 in both groups without endothelial damage. Keratocyte apoptosis in PL-ACXL was estimated at a mean depth of ∼200 μm, whereas an uneven demarcation line was detectable by confocal microscopy at a mean depth of 160 μm in CL-ACXL. Conclusion In vivo confocal microscopy and corneal OCT allowed a precise qualitative analysis of the cornea after epithelium-off PL-ACXL and CL-ACXL treatments. Apoptotic effect was higher in pulsed than in continuous light treatments, exceeding 200 μm in corneal stroma. According to different morphological data, the clinical efficacy of ACXL needs to be determined in a long-term follow-up and large cohort of patients. PMID:25060847

  1. 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

  2. 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.

  3. Spatial organization of RNA polymerase II inside a mammalian cell nucleus revealed by reflected light-sheet superresolution microscopy.

    PubMed

    Zhao, Ziqing W; Roy, Rahul; Gebhardt, J Christof M; Suter, David M; Chapman, Alec R; Xie, X Sunney

    2014-01-14

    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.

  4. 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.

  5. A simple procedure to analyze positions of interest in infectious cell cultures by correlative light and electron microscopy.

    PubMed

    Madela, Kazimierz; Banhart, Sebastian; Zimmermann, Anja; Piesker, Janett; Bannert, Norbert; Laue, Michael

    2014-01-01

    Plastic cell culture dishes that contain a thin bottom of highest optical quality including an imprinted finder grid (μ-Dish Grid-500) are optimally suited for routine correlative light and electron microscopy using chemical fixation. Such dishes allow high-resolution fluorescence and bright-field imaging using fixed and living cells and are compatible with standard protocols for scanning and transmission electron microscopy. Ease of use during cell culture and imaging, as well as a tight cover render the dishes particularly suitable for working with infectious organisms up to the highest biosafety level. Detailed protocols are provided and demonstrated by showing two examples: monitoring the production of virus-like particles of the Human Endogenous Retrovirus HERV-K(HML-2) by HeLa cells and investigation of Rab11-positive membrane-compartments of HeLa cells after infection with Chlamydia trachomatis.

  6. 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.

  7. Correlative fractography: combining scanning electron microscopy and light microscopes for qualitative and quantitative analysis of fracture surfaces.

    PubMed

    Hein, Luis Rogerio de Oliveira; de Oliveira, José Alberto; de Campos, Kamila Amato

    2013-04-01

    Correlative fractography is a new expression proposed here to describe a new method for the association between scanning electron microscopy (SEM) and light microscopy (LM) for the qualitative and quantitative analysis of fracture surfaces. This article presents a new method involving the fusion of one elevation map obtained by extended depth from focus reconstruction from LM with exactly the same area by SEM and associated techniques, as X-ray mapping. The true topographic information is perfectly associated to local fracture mechanisms with this new technique, presented here as an alternative to stereo-pair reconstruction for the investigation of fractured components. The great advantage of this technique resides in the possibility of combining any imaging methods associated with LM and SEM for the same observed field from fracture surface.

  8. Use of colloidal quantum dots as a digitally switched swept light source for gold nanoparticle based hyperspectral microscopy

    PubMed Central

    Hoshino, Kazunori; Joshi, Pratixa. P.; Bhave, Gauri.; Sokolov, Konstantin V.; Zhang, Xiaojing

    2014-01-01

    We propose a method to utilize colloidal quantum dots (QDs) as a swept light source for hyperspectral microscopy. The use of QD allows for uniform multicolor emission which covers visible-NIR wavelengths. We used 8 colors of CdSe/ZnS and CdTe/ZnS colloidal quantum dots with the peak emission wavelengths from 520 nm to 800 nm. The QDs are packed in a compact enclosure, composing a low-cost, solid-state swept light source that can be easily used in most microscopes. Multicolor emission from the QDs is simply controlled by digitally switching excitation UVLEDs, eliminating the use of mechanically-driven gratings or filters. We used gold nanoparticles as optical markers for hyperspectral microscopy. Due to the effect of localized surface plasmon resonance, gold nanoparticles demonstrate size and shape-dependent absorption spectra. Employed in a standard microscope, the QD light source enabled multispectral absorption imaging of macrophage cells labeled with gold nanorods and nanospheres. PMID:24877018

  9. Optical spectroscopy and microscopy of radiation-induced light-emitting point defects in lithium fluoride crystals and films

    NASA Astrophysics Data System (ADS)

    Montereali, R. M.; Bonfigli, F.; Menchini, F.; Vincenti, M. A.

    2012-08-01

    Broad-band light-emitting radiation-induced F2 and F3+ electronic point defects, which are stable and laser-active at room temperature in lithium fluoride crystals and films, are used in dosimeters, tuneable color-center lasers, broad-band miniaturized light sources and novel radiation imaging detectors. A brief review of their photoemission properties is presented, and their behavior at liquid nitrogen temperatures is discussed. Some experimental data from optical spectroscopy and fluorescence microscopy of these radiation-induced point defects in LiF crystals and thin films are used to obtain information about the coloration curves, the efficiency of point defect formation, the effects of photo-bleaching processes, etc. Control of the local formation, stabilization, and transformation of radiation-induced light-emitting defect centers is crucial for the development of optically active micro-components and nanostructures. Some of the advantages of low temperature measurements for novel confocal laser scanning fluorescence microscopy techniques, widely used for spatial mapping of these point defects through the optical reading of their visible photoluminescence, are highlighted.

  10. 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)

  11. 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...

  12. 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.

  13. Microscopy with microlens arrays: high throughput, high resolution and light-field imaging.

    PubMed

    Orth, Antony; Crozier, Kenneth

    2012-06-01

    We demonstrate highly parallelized fluorescence scanning microscopy using a refractive microlens array. Fluorescent beads and rat femur tissue are imaged over a 5.5 mm x 5.5 mm field of view at a pixel throughput of up to 4 megapixels/s and a resolution of 706 nm. We also demonstrate the ability to extract different perspective views of a pile of microspheres.

  14. 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.

  15. 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.

  16. 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

  17. A Genetically Encoded Tag for Correlated Light and Electron Microscopy of Intact Cells, Tissues, and Organisms

    PubMed Central

    Shu, Xiaokun; Lev-Ram, Varda; Deerinck, Thomas J.; Qi, Yingchuan; Ramko, Ericka B.; Davidson, Michael W.; Jin, Yishi; Ellisman, Mark H.; Tsien, Roger Y.

    2011-01-01

    Electron microscopy (EM) achieves the highest spatial resolution in protein localization, but specific protein EM labeling has lacked generally applicable genetically encoded tags for in situ visualization in cells and tissues. Here we introduce “miniSOG” (for mini Singlet Oxygen Generator), a fluorescent flavoprotein engineered from Arabidopsis phototropin 2. MiniSOG contains 106 amino acids, less than half the size of Green Fluorescent Protein. Illumination of miniSOG generates sufficient singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product resolvable by EM. MiniSOG fusions to many well-characterized proteins localize correctly in mammalian cells, intact nematodes, and rodents, enabling correlated fluorescence and EM from large volumes of tissue after strong aldehyde fixation, without the need for exogenous ligands, probes, or destructive permeabilizing detergents. MiniSOG permits high quality ultrastructural preservation and 3-dimensional protein localization via electron tomography or serial section block face scanning electron microscopy. EM shows that miniSOG-tagged SynCAM1 is presynaptic in cultured cortical neurons, whereas miniSOG-tagged SynCAM2 is postsynaptic in culture and in intact mice. Thus SynCAM1 and SynCAM2 could be heterophilic partners. MiniSOG may do for EM what Green Fluorescent Protein did for fluorescence microscopy. PMID:21483721

  18. 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

  19. Multiphoton light-sheet microscopy using wavelength mixing: fast multicolor imaging of the beating Zebrafish heart with low photobleaching

    NASA Astrophysics Data System (ADS)

    Mahou, Pierre; Vermot, Julien; Beaurepaire, Emmanuel; Supatto, Willy

    2015-03-01

    Two-photon laser scanning microscopy has become a standard to map thick and live tissues. However, its application for fast and multicolor imaging remains challenging. To address this issue, we report on the implementation of mixed wavelength excitation in a two-photon light-sheet microscope. We illustrate the potential of the technique by recording sustained multicolor two-photon movies of the beating heart in zebrafish embryos with negligible photobleaching at 28 million pixels/second. In particular, 3D reconstructions of the heart periodic motion are obtained with sufficient spatiotemporal resolution to track the fast movements of individual cells during a cardiac cycle.

  20. Advanced modeling of reaction cross sections for light nuclei

    SciTech Connect

    Resler, D.A.

    1991-01-01

    The shell model/R-matrix technique of calculating nuclear reaction cross sections for light projectiles incident on light nuclei is discussed, particularly in the application of the technique to thermonuclear reactions. Details are presented on the computational methods for the shell model which display how easily the calculations can be performed. Results of the shell model/R-matrix technique are discussed as are some of the problems encountered in picking an appropriate nucleon-nucleon interaction for the large model spaces which must be used for current problems. The status of our work on developing an effective nucleon-nucleon interaction for use in large-basis shell model calculations is presented. This new interaction is based on a combination of global constraints and microscopic nuclear data. 23 refs., 6 figs., 2 tabs.

  1. Discrepancies in quantitative assessment of normal and regenerated peripheral nerve fibers between light and electron microscopy.

    PubMed

    Ronchi, Giulia; Jager, Sara Buskbjerg; Vaegter, Christian Bjerggaard; Raimondo, Stefania; Giacobini-Robecchi, Maria Giuseppina; Geuna, Stefano

    2014-09-01

    Quantitative estimation of myelinated nerve fiber number, together with fiber size parameters, is one of the most important tools for nerve regeneration research. In this study we used a design-based stereological method to evaluate the regenerative process in two experimental paradigms: crush injury and autograft repair. Samples were embedded in resin and morphometric counting and measurements were performed using both light and electron microscopes. Results show a significant difference in myelinated fiber number estimation between light and electron microscopes, especially after autograft repair; light microscope significantly underestimates the number of fibers because of the large number of very small axons that can be detected only in electron microscope. The analysis of the size parameters also shows a higher number of small fibers in electron microscopic analysis, especially in regenerated nerves. This comparative study shows that the integration of data obtained in light microscope with those obtained in electron microscope is necessary in revealing very small myelinated fibers that cannot be detected otherwise. Moreover, the difference in the estimation of total number of myelinated fibers between light and electron microscopes must be considered in data analysis to ensure accurate interpretation of the results.

  2. 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.

  3. Recent advances in freeze-fracture electron microscopy: the replica immunolabeling technique

    PubMed Central

    2008-01-01

    Freeze-fracture electron microscopy is a technique for examining the ultrastructure of rapidly frozen biological samples by transmission electron microscopy. Of a range of approaches to freeze-fracture cytochemistry that have been developed and tried, the most successful is the technique termed freeze-fracture replica immunogold labeling (FRIL). In this technique, samples are frozen, fractured and replicated with platinum-carbon as in standard freeze fracture, and then carefully treated with sodium dodecylsulphate to remove all the biological material except a fine layer of molecules attached to the replica itself. Immunogold labeling of these molecules permits their distribution to be seen superimposed upon high resolution planar views of membrane structure. Examples of how this technique has contributed to our understanding of lipid droplet biogenesis and function are discussed. PMID:18385807

  4. Advances in electron microscopy: A qualitative view of instrumentation development for macromolecular imaging and tomography.

    PubMed

    Schröder, Rasmus R

    2015-09-01

    Macromolecular imaging and tomography of ice embedded samples has developed into a mature imaging technology, in structural biology today widely referred to simply as cryo electron microscopy.(1) While the pioneers of the technique struggled with ill-suited instruments, state-of-the-art cryo microscopes are now readily available and an increasing number of groups are producing excellent high-resolution structural data of macromolecular complexes, of cellular organelles, or the morphology of whole cells. Instrumentation developers, however, are offering yet more novel electron optical devices, such as energy filters and monochromators, aberration correctors or physical phase plates. Here we discuss how current instrumentation has already changed cryo EM, and how newly available instrumentation - often developed in other fields of electron microscopy - may further develop the use and applicability of cryo EM to the imaging of single isolated macromolecules of smaller size or molecules embedded in a crowded cellular environment.

  5. 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

  6. Assembly of Octopus dofleini hemocyanin. A study of the kinetics by sedimentation, light scattering and electron microscopy.

    PubMed

    van Holde, K E; Miller, K; Schabtach, E; Libertini, L

    1991-01-20

    The kinetics of association of Octopus dofleini hemocyanin subunits to form the native decameric molecule have been studied with a combination of sedimentation, light scattering and electron microscopy. The reaction, initiated by addition of magnesium, is relatively slow, requiring hours to reach completion, with monomer and decamer as predominant molecular species throughout. Analysis of the light-scattering data, including stopped-flow studies, reveals an initial lag period in the reaction, followed by a second-order process that is rate limiting. The lag period depends on both protein and magnesium ion concentration. Electron microscope studies reveal intermediates in the process, and support a model of assembly in which nucleation begins at the dimer level. Theoretical models for the process are compared. PMID:1992165

  7. 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

  8. Scanning capacitance microscopy investigation on InGaAs/InP avalanche photodiode structures: Light-induced polarity reversal

    NASA Astrophysics Data System (ADS)

    Yin, Hao; Li, Tianxin; Wang, Wenjuan; Hu, Weida; Lin, Le; Lu, Wei

    2009-08-01

    Cross-sectional scanning capacitance microscopy is applied to study the carrier distribution as well as its variation under irradiation in an InGaAs/InP avalanche photodiode. The photocarriers excited by the stray light of atomic force microscope laser beam lead to a dramatic deviation of the dC /dV profile in the unintended-doped absorption layer, and even cause the reversal of signal polarity. The existence of surface potential and its impact on the spreading of photocarriers near the cleaved face are demonstrated as the main origins of the light-induced dC /dV reversal. The effect provides experimental information on the distribution property of photoelectric process in devices.

  9. Integrated light and scanning electron microscopy of GFP-expressing cells.

    PubMed

    Peddie, Christopher J; Liv, Nalan; Hoogenboom, Jacob P; Collinson, Lucy M

    2014-01-01

    Integration of light and electron microscopes provides imaging tools in which fluorescent proteins can be localized to cellular structures with a high level of precision. However, until recently, there were few methods that could deliver specimens with sufficient fluorescent signal and electron contrast for dual imaging without intermediate staining steps. Here, we report protocols that preserve green fluorescent protein (GFP) in whole cells and in ultrathin sections of resin-embedded cells, with membrane contrast for integrated imaging. Critically, GFP is maintained in a stable and active state within the vacuum of an integrated light and scanning electron microscope. For light microscopists, additional structural information gives context to fluorescent protein expression in whole cells, illustrated here by analysis of filopodia and focal adhesions in Madin Darby canine kidney cells expressing GFP-Paxillin. For electron microscopists, GFP highlights the proteins of interest within the architectural space of the cell, illustrated here by localization of the conical lipid diacylglycerol to cellular membranes.

  10. 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.

  11. Elastic mid-infrared light scattering: A basis for microscopy of large-scale electrically active defects in semiconducting materials

    NASA Astrophysics Data System (ADS)

    Kalinushkin, V. P.; Yuryev, V. A.; Astafiev, O. V.

    1999-11-01

    A method of the mid-IR-laser microscopy has been recently proposed for the investigation of the large-scale electrically and recombination active defects in semiconductors and nondestructive inspection of semiconductor materials and structures in the industries of microelectronics and photovoltaics. The basis for this development was laid with a wide cycle of the investigations on the low-angle mid-IR-light scattering in semiconductors. The essence of the technical idea was to apply the dark-field method for spatial filtering of the scattered light in the scanning mid-IR-laser microscope. This approach enabled the visualization of large-scale electrically active defects which are the regions enriched with ionized electrically active centers. The photoexcitation of excess carriers within a small volume located in the probe mid-IR-laser beam enabled the visualization of the large-scale recombination-active defects like those revealed in the optical or electron beam induced current methods. Both these methods of the scanning mid-IR-laser microscopy are now introduced in detail in the present article as well as a summary of techniques used in the standard method of the low-angle mid-IR-light scattering itself. Besides the techniques for direct observations, methods for analyses of the defect composition associated with the mid-IR-laser microscopy are also discussed in the article. Special attention is paid upon potential applications of the above methods as characterization and testing techniques in the semiconductor science and industry. It is concluded that elastic midinfrared laser light scattering is a basis for the development of a variety of research techniques and instruments which could be useful in different branches of basic and applied research work in the field of defect engineered semiconductors as well as for the development of devices for quality inspections in the semiconductor industry. Being contactless, nondestructive and nonpolluting, techniques

  12. Real-time electron-holographic interference microscopy with a liquid-crystal spatial light modulator.

    PubMed

    Chen, J; Hirayama, T; Lai, G; Tanji, T; Ishizuka, K; Tonomura, A

    1993-11-15

    An electron-holographic interference microscope that produces a time-sequential interference micrograph at a TV rate is developed. In this system, the electron off-axis hologram detected with a TV camera is transferred to a liquid-crystal spatial light modulator as a video signal. The liquid-crystal spatial light modulator can function as a thin amplitude hologram or a thin phase hologram. Time-sequential interference micrograph is obtained at a TV rate by superimposition of a plane reference wave onto the reconstructed object wave. Experimental results for observing a dynamic domain-wall motion in thin Permalloy film are demonstrated.

  13. 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.

  14. Advanced Materials Research with 3RD Generation Synchrotron Light

    NASA Astrophysics Data System (ADS)

    Soukiassian, P.; D'angelo, M.; Enriquez, H.; Aristov, V. Yu.

    H and D surface nanochemistry on an advanced wide band gap semiconductor, silicon carbide is investigated by synchrotron radiation-based core level and valence band photoemission, infrared absorption and scanning tunneling spectroscopy, showing the 1st example of H/D-induced semiconductor surface metallization, that also occurs on a pre-oxidized surface. These results are compared to recent state-of-the-art ab-initio total energy calculations. Most interestingly, an amazing isotopic behavior is observed with a smaller charge transfer from D atoms suggesting the role of dynamical effects. Such findings are especially exciting in semiconductor physics and in interface with biology.

  15. 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

  16. 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

  17. In-vivo lymph dynamic monitoring using speckle-correlation technique and light microscopy

    NASA Astrophysics Data System (ADS)

    Galanzha, Ekateryna I.; Fedosov, Ivan V.; Solov'eva, Anastasiya V.; Stepanova, Tatyana V.; Tuchin, Valery V.; Brill, Gregory E.

    2002-05-01

    In this work we described the new modification of experimental setup designed on the basis of transmission microscopy and high-resolution speckle-correlation technique. This combined technique provides the simultaneous speckle and video registration of lymph dynamics that allows one to calibrate the speckle-correlation velocity sensor and to determine an absolute flow velocity and its direction. As a result many parameters of lymph dynamic were measured quickly, conveniently and simultaneously and a new data about the lymph flow velocity and other functions of microcirculation were received. The results of the experimental study of lymph microcirculation in small intestine mesentery of rat in vivo are presented.

  18. Action potential propagation imaged with high temporal resolution near-infrared video microscopy and polarized light

    PubMed Central

    Schei, Jennifer L.; McCluskey, Matthew D.; Foust, Amanda J.; Yao, Xin-Cheng; Rector, David M.

    2008-01-01

    To identify the neural constituents responsible for generating polarized light changes, we created spatially resolved movies of propagating action potentials from stimulated lobster leg nerves using both reflection and transmission imaging modalities. Changes in light polarization are associated with membrane depolarization and provide sub-millisecond temporal resolution. Typically, signals are detected using light transmitted through tissue; however, because we eventually would like to apply polarization techniques in-vivo, reflected light is required. In transmission mode, the optical signal was largest throughout the center of the nerve, suggesting that most of the optical signal arose from the inner nerve bundle. In reflection mode, polarization changes were largest near the edges, suggesting that most of the optical signal arose from the outer sheath. In support of these observations, an optical model of the tissue showed that the outer sheath is more reflective while the inner nerve bundle is more transmissive. In order to apply these techniques in-vivo, we must consider that brain tissue does not have a regular orientation of processes as in the lobster nerve. We tested the effect of randomizing cell orientation by tying the nerve in an overhand knot prior to imaging, producing polarization changes that can be imaged even without regular cell orientations. PMID:18272402

  19. 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.

  20. Laboratory-based cryogenic soft x-ray tomography with correlative cryo-light and electron microscopy.

    PubMed

    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 subcellular structures of Saccharomyces cerevisiae cells. The microscope is shown to achieve better than 50 nm half-pitch spatial resolution with a Siemens star test sample. For whole biological cells, the microscope can image specimens up to 5 μm thick. Structures as small as 90 nm can be detected in tomographic reconstructions 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 enable greater access to three-dimensional ultrastructure determination of biological whole cells without chemical fixation or physical sectioning.

  1. 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.

  2. Visualizing Quantum Dot Labeled ORAI1 Proteins in Intact Cells Via Correlative Light and Electron Microscopy.

    PubMed

    Peckys, Diana B; Alansary, Dalia; Niemeyer, Barbara A; de Jonge, Niels

    2016-08-01

    ORAI1 proteins are ion channel subunits and the essential pore-forming units of the calcium release-activated calcium channel complex essential for T-cell activation and many other cellular processes. In this study, we used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to image plasma membrane expressed ORAI1 proteins in whole Jurkat T cells in the liquid state. Utilizing a stably transfected Jurkat T cell clone expressing human ORAI1 with an extracellular human influenza hemagglutinin (HA) tag we investigated if liquid-phase STEM can be applied to detect recombinant surface expressed protein. Streptavidin coated quantum dots were coupled in a one-to-one stoichiometry to ORAI1 proteins detected by biotinylated anti-HA fragmented antibody fragments. High-resolution electron microscopic images revealed the individual label locations from which protein pair distances were determined. These data were analyzed using the pair correlation function and, in addition, an analysis of cluster size and frequency was performed. ORAI1 was found to be present in hexamers in a small fraction only, and ORAI1 resided mostly in monomers and dimers.

  3. 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.

  4. Visualizing Non-lytic Exocytosis of Cryptococcus neoformans from Macrophages Using Digital Light Microscopy

    PubMed Central

    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

  5. Characteristics of the polar assembly and disassembly of microtubules observed in vitro by darkfield light microscopy

    PubMed Central

    1979-01-01

    We describe here the continuous observations of the polymerization of individual microtubules in vitro by darkfield microscopy. In homogeneous preparations we verify that polymerization can occur onto both ends of microtubules. The assembly of microtubules is polar, with one end growing at three times the rate of the other. The differential rate of elongation can be used to determine the polarity of growth off cellular nucleating centers. We show that the microtubules grow off the proximal end of ciliary axonemes at a growth rate equal to that of the slow growing end of free microtubules, while growth off the distal end proceeds at the same rate as the fast growing end. Applying this technique to microtubule growth from metaphase chromosomes isolated from HeLa and CHO cells, we demonstrate that chromosomes initiate polymerization with the fast growing end facing away from the chromosome nucleation site. The opposite ends of free microtubules show different sensitivities to microtubule depolymerizing agents such as low temperature, Ca++ or colchicine as measured directly by darkfield microscopy. The differing rates of assembly and disassembly of each end of a microtubule suggest that a difference in polarity of growth off nucleating sites could serve as one basis for regulating the polymerization of different groups of microtubules in the same cell. PMID:511939

  6. 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

  7. Co-Orientation: Quantifying Simultaneous Co-Localization and Orientational Alignment of Filaments in Light Microscopy.

    PubMed

    Nieuwenhuizen, Robert P J; Nahidiazar, Leila; 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.

  8. Correlative light and electron microscopy of the frog adrenal gland cells using adjacent epon-embedded sections.

    PubMed

    Nakai, Y; Iwashita, T

    1976-07-01

    Correlative light and electron microscopy on the same cells of the adrenal gland of the frog, Rana nigromaculata, fixed in glutaraldehyde followed by osmium tetroxide, was done using the adjacent Epon embedded sections. Electron microscope observation revealed three different types of granule-filled secretory cells; the noradrenaline-storing cells (NA cells) filled with intensely dense and varying shaped granules, the adrenaline-strong cells (A cells) filled with relatively less dense granules and the summer cells (STILLING, 1898) containing very large, round or polygonal granules (0.2-1.3 mu in diameter). Light microscopically, an essential difference could be observed in the affinity to ammoniacal silver solution between NA and A cells. It was clarified that the granules of NA cells stained in black and were clearly distinguishable from the yellow- or brown-stained granules in both A cells and summer cells. This silver method can be applied for the light microscopic identification of the NA cells in the Epon-embedded sections. Furthermore, after immersing the thick sections in toluidine blue or methylene blue, the granules of NA cells showed much stronger affinity to both dyes than those of A cells and became dark blue and occasionally stained greenish blue in methylene blue, while the summer cells became blue and the granules of the A cells stained light blue.

  9. Validation of diffusion tensor MRI in the central nervous system using light microscopy: quantitative comparison of fiber properties.

    PubMed

    Choe, A S; Stepniewska, I; Colvin, D C; Ding, Z; Anderson, A W

    2012-07-01

    Diffusion tensor imaging (DTI) provides an indirect measure of tissue structure on a microscopic scale. To date, DTI is the only imaging method that provides such information in vivo, and has proven to be a valuable tool in both research and clinical settings. In this study, we investigated the relationship between white matter structure and diffusion parameters measured by DTI. We used micrographs from light microscopy of fixed, myelin-stained brain sections as a gold standard for direct comparison with data from DTI. Relationships between microscopic tissue properties observed with light microscopy (fiber orientation, density and coherence) and fiber properties observed by DTI (tensor orientation, diffusivities and fractional anisotropy) were investigated. Agreement between the major eigenvector of the tensor and myelinated fibers was excellent in voxels with high fiber coherence. In addition, increased fiber spread was strongly associated with increased radial diffusivity (p = 6 × 10(-6)) and decreased fractional anisotropy (p = 5 × 10(-8)), and was weakly associated with decreased axial diffusivity (p = 0.07). Increased fiber density was associated with increased fractional anisotropy (p = 0.03), and weakly associated with decreased radial diffusivity (p < 0.06), but not with axial diffusivity (p = 0.97). The mean diffusivity was largely independent of fiber spread (p = 0.24) and fiber density (p = 0.34).

  10. 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

  11. Human myoblasts from skeletal muscle biopsies: in vitro culture preparations for morphological and cytochemical analyses at light and electron microscopy.

    PubMed

    Malatesta, Manuela; Giagnacovo, Marzia; Cardani, Rosanna; Meola, Giovanni; Pellicciari, Carlo

    2013-01-01

    We describe protocols for the isolation of satellite cells from human muscle biopsies, for the in vitro culture of proliferating and differentiating myoblasts, and for the preparation of cell samples suitable for morphological and cytochemical analyses at light and electron microscopy. The procedures described are especially appropriate for processing small muscle biopsies, and allow obtaining myoblast/myotube monolayers on glass coverslips, thus preserving good cell morphology and immunoreactivity for protein markers of myoblast proliferation, differentiation, and senescence.These cell preparations are suitable for cytochemical, immunocytochemical, and FISH procedures at light microscopy, and can be observed not only in bright field, phase contrast, and differential interference contrast but also in fluorescence (which can hardly be used for cells grown on conventional plastic surfaces, which generally exhibit intense autofluorescence). In their ultrastructural cytochemical application, the protocols are intended for post-embedding techniques, by which ultrathin sections from a single sample may be used for detecting a wide variety of molecular markers. PMID:23400435

  12. Study on validity of a rapid diagnostic test kit versus light microscopy for malaria diagnosis in Ahmedabad city, India.

    PubMed

    Vyas, S; Puwar, B; Patel, V; Bhatt, G; Kulkarni, S; Fancy, M

    2014-04-01

    Light microscopy of blood smears for diagnosis of malaria in the field has several limitations, notably delays in diagnosis. This study in Ahmedabad in Gujarat State, India, evaluated the diagnostic performance of a rapid diagnostic test for malaria (SD Bioline Malaria Ag P.f/Pan) versus blood smear examination as the gold standard. All fever cases presenting at 13 urban health centres were subjected to rapid diagnostic testing and thick and thin blood smears. A total of 677 cases with fever were examined; 135 (20.0%) tested positive by rapid diagnostic test and 86 (12.7%) by blood smear. The sensitivity of the rapid diagnostic test for malaria was 98.8%, specificity was 91.5%, positive predictive value 63.0% and negative predictive value 99.8%. For detection of Plasmodium falciparum the sensitivity of rapid diagnostic test was 100% and specificity was 97.3%. The results show the acceptability of the rapid test as an alternative to light microscopy in the field setting.

  13. 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

  14. Distribution of adrenomedullin-like immunoreactivity in the rat central nervous system by light and electron microscopy.

    PubMed

    Serrano, J; Uttenthal, L O; Martínez, A; Fernández, A P; Martínez de Velasco, J; Alonso, D; Bentura, M L; Santacana, M; Gallardo, J R; Martínez-Murillo, R; Cuttitta, F; Rodrigo, J

    2000-01-24

    Adrenomedullin is a peptide of marked vasodilator activity first isolated from human pheochromocytoma and subsequently demonstrated in other mammalian tissues. Using a polyclonal antiserum against human adrenomedullin-(22-52) amide and the avidin-biotin peroxidase complex technique, we have demonstrated by light and electron microscopy that adrenomedullin-like immunoreactivity is widely distributed in the rat central nervous system. Western blotting of extracts of different brain regions demonstrated the fully processed peptide as the major form in the cerebellum, whereas a 14-kDa molecular species and a small amount of the 18-kDa propeptide were present in other brain regions. Immunoreactive neurons and processes were found in multipolar neurons and pyramidal cells of layers IV-VI of the cerebral cortex and their apical processes, as well as in a large number of telencephalic, diencephalic, mesencephalic, pontine and medullary nuclei. Cerebellar Purkinje cells and mossy terminal nerve fibers as well as neurons of the cerebellar nuclei were immunostained, as were neurons in area 9 of the anterior horn of the spinal cord. Immunoreactivity was also found in some vascular endothelial cells and surrounding processes that probably originated from perivascular glial cells. Electron microscopy confirmed the light microscopy findings and showed the reaction product in relation to neurofilaments and the external membrane of small mitochondria. Immunoreactive terminal boutons were occasionally seen. The distribution of adrenomedullin-like immunoreactivity in the central nervous system suggests that it has a significant role in neuronal function as well as in the regulation of regional blood flow.

  15. 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.

  16. 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.

  17. 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.

  18. Advanced Compton scattering light source R&D at LLNL

    SciTech Connect

    Albert, F; Anderson, S G; Anderson, G; Betts, S M; Chu, T S; Gibson, D J; Marsh, R A; Messerly, M; Shverdin, M Y; Wu, S; Hartemann, F V; Siders, C W; Barty, C P

    2010-02-16

    We report the design and current status of a monoenergetic laser-based Compton scattering 0.5-2.5 MeV {gamma}-ray source. Previous nuclear resonance fluorescence results and future linac and laser developments for the source are presented. At MeV photon energies relevant for nuclear processes, Compton scattering light sources are attractive because of their relative compactness and improved brightness above 100 keV, compared to typical 4th generation synchrotrons. Recent progress in accelerator physics and laser technology have enabled the development of a new class of tunable Mono-Energetic Gamma-Ray (MEGa-Ray) light sources based on Compton scattering between a high-brightness, relativistic electron beam and a high intensity laser pulse produced via chirped-pulse amplification (CPA). A new precision, tunable gamma-ray source driven by a compact, high-gradient X-band linac is currently under development and construction at LLNL. High-brightness, relativistic electron bunches produced by an X-band linac designed in collaboration with SLAC will interact with a Joule-class, 10 ps, diode-pumped CPA laser pulse to generate tunable {gamma}-rays in the 0.5-2.5 MeV photon energy range via Compton scattering. Based on the success of the previous Thomson-Radiated Extreme X-rays (T-REX) Compton scattering source at LLNL, the source will be used to excite nuclear resonance fluorescence lines in various isotopes; applications include homeland security, stockpile science and surveillance, nuclear fuel assay, and waste imaging and assay. After a brief presentation of successful nuclear resonance fluorescence (NRF) experiments done with T-REX, the new source design, key parameters, and current status are presented.

  19. 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

  20. Multispectral endoscopy and microscopy imaging system using a spectrally programmable light engine

    NASA Astrophysics Data System (ADS)

    MacKinnon, N.; Stange, Ulrich; Lane, Pierre M.; MacAulay, Calum E.

    2005-03-01

    We report a spectrally and temporally programmable light engine based on a spatial light modulator that can dynamically create any narrow or broadband spectral profile for hyperspectral, fluorescence, or principal component imaging. Most hyperspectral or multispectral imaging systems use wavelength selection devices such as acousto-optic tunable filters (AOTFs), tunable grating or prism-based monochromators, or filter wheels. While these devices can select wavelengths they cannot create arbitrary spectral profiles. This simple and economical system can be controlled at high speed (up to 5000 illumination profiles per second). Digitally controlled illumination is bit additive with image data providing high dynamic range imaging with monochrome or color imaging devices. This is especially advantageous for endoscopes employing small well CCD or CMOS sensors since the dynamic range now can extend beyond the limits of the sensor itself. In this report we show multispectral images of in vivo tissue and in vitro tissue samples using endoscopes, surgical microscopes and conventional microscopes.